TOXICOLOGICAL PROFILE FOR CHLOROFORM Prepared by: Research Triangle Institute Under Contract No. 205-93-0606 Prepared for: U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES Public Health Service Agency for Toxic Substances and Disease Registry September 1997 CHLOROFORM § (1 BL DISCLAIMER The use of company or product name(s) is for identification only and does not imply endorsement by the Agency for Toxic Substances and Disease Registry. CHLOROFORM iii UPDATE STATEMENT A Toxicological Profile for chloroform was released in April 1993. This edition supersedes any previously released draft or final profile. Toxicological profiles are revised and republished as necessary, but no less than once every three years. For information regarding the update status of previously released profiles, contact ATSDR at: Agency for Toxic Substances and Disease Registry Division of Toxicology/Toxicology Information Branch 1600 Clifton Road NE, E-29 [< A Atlanta, Georgia 30333 ) Zt 2 Ys TE87 1977 Peo FOREWORD This toxicological profile is prepared in accordance with guidelines* developed by the Agency for Toxic Substances and Disease Registry (ATSDR) and the Environmental Protection Agency (EPA). The original guidelines were published in the Federal Register on April 17, 1987. Each profile will be revised and republished as necessary. The ATSDR toxicological profile succinctly characterizes the toxicologic and adverse health effects information for the hazardous substance described therein. Each peer-reviewed profile identifies and reviews the key literature that describes a hazardous substance's toxicologic properties. Other pertinent literature is also presented, but is described in less detail than the key studies. The profile is not intended to be an exhaustive document; however, more comprehensive sources of specialty information are referenced. The focus of the profiles is on health and toxicologic information; therefore, each toxicological profile begins with a public health statement that describes, in nontechnical language, a substance's relevant toxicological properties. Following the public health statement is information concerning levels of significant human exposure and, where known, significant health effects. The adequacy of information to determine a substance's health effects is described in a health effects summary. Data needs that are of significance to protection of public health are identified by ATSDR and EPA. Each profile includes the following: (A) The examination, summary, and interpretation of available toxicologic information and epidemiologic evaluations on a hazardous substance to ascertain the levels of significant human exposure for the substance and the associated acute, subacute, and chronic health effects; (B) A determination of whether adequate information on the health effects of each substance is available or in the process of development to determine levels of exposure that present a significant risk to human health of acute, subacute, and chronic health effects; and (C) Where appropriate, identification of toxicologic testing needed to identify the types or levels of exposure that may present significant risk of adverse health effects in humans. The principal audiences for the toxicological profiles are health professionals at the Federal, State, and local levels; interested private sector organizations and groups; and members of the public. This profile reflects ATSDR’s assessment of all relevant toxicologic testing and information that has been peer-reviewed. Staff of the Centers for Disease Control and Prevention and other Federal scientists have also reviewed the profile. In addition, this profile has been peer-reviewed by a nongovernmental panel and was made available for public review. Final responsibility for the contents and views expressed in this toxicological profile resides with ATSDR. Gd Shh David Satcher, M.D., Ph.D. Administrator Agency for Toxic Substances and Disease Registry vi *L episiative Bac} The toxicological profiles are developed in response to the Superfund Amendments and Reauthorization Act (SARA) of 1986 (Public Law 99-499) which amended the Comprehensive Environmental Response, Compensation, and Liability Act of 1980 (CERCLA or Superfund). This public law directed ATSDR to prepare toxicological profiles for hazardous substances most commonly found at facilities on the CERCLA National Priorities List and that pose the most significant potential threat to human health, as determined by ATSDR and the EPA. The availability of the revised priority list of 275 hazardous substances was announced in the Federal Register on April 29, 1996 (61 FR 18744). For prior versions of the list of substances, see Federal Register notices dated April 17, 1987 (52 FR 12866); October 20, 1988 (53 FR 41280); October 26, 1989 (54 FR 43619); October 17,1990 (55 FR 42067); October 17, 1991 (56 FR 52166); October 28, 1992 (57 FR 48801); and February 28, 1994 (59 FR 9486). Section 104(i)(3) of CERCLA, as amended, directs the Administrator of ATSDR to prepare a toxicological profile for each substance on the list. CHLOROFORM vii CONTRIBUTORS CHEMICAL MANAGER(S)AUTHORS(S): Selene Chou, Ph.D. ATSDR, Division of Toxicology, Atlanta, GA Wayne Spoo, D.V.M,, D.AB.T. Research Triangle Institute, Research Triangle Park, NC THE PROFILE HAS UNDERGONE THE FOLLOWING ATSDR INTERNAL REVIEWS: 1. Green Border Review. Green Border review assures consistency with ATSDR policy. 2. Health Effects Review. The Health Effects Review Committee examines the health effects chapter of each profile for consistency and accuracy in interpreting health effects and classifying end points. 3. Minimal Risk Level Review. The Minimal Risk Level Workgroup considers issues relevant to substance-specific minimal risk levels (MRLs), reviews the health effects database of each profile, and makes recommendations for derivation of MRLs. CHLOROFORM ix PEER REVIEW A peer review panel was assembled for chloroform. The panel consisted of the following members: 1. Dr. Richard Bull, Senior Staff Scientist, Battelle Pacific Northwest Laboratories, Richland, Washington; 2. Dr. Derek Hodgson, Provost and Vice-president, Academic Affairs, Mississippi State University, Mississippi State, Mississippi; and 3. Dr. Nancy Reiches, Private Consultant, Bexley, Ohio. These experts collectively have knowledge of chloroform’s physical and chemical properties, toxico- kinetics, key health end points, mechanisms of action, human and animal exposure, and quantification of risk to humans. All reviewers were selected in conformity with the conditions for peer review specified in Section 104(i)(13) of the Comprehensive Environmental Response, Compensation, and Liability Act, as amended. Scientists from the Agency for Toxic Substances and Disease Registry (ATSDR) have reviewed the peer reviewers’ comments and determined which comments will be included in the profile. A listing of the peer reviewers’ comments not incorporated in the profile, with a brief explanation of the rationale for their exclusion, exists as part of the administrative record for this compound. A list of databases reviewed and a list of unpublished documents cited are also included in the administrative record. The citation of the peer review panel should not be understood to imply its approval of the profiles final content. The responsibility for the content of this profile lies with the ATSDR. CHLOROFORM Xi CONTENTS FOREWORD ..:.vv cron menmonsmni sa ss@sNennmincs $s 6MINImormsmespnsme nn sus v CONTRIBUTORS © oe vii PEER REVIEW ci: vuivinmnonsmenmrnmimusnsassatnssSsnnsmssenmenesmsuass ix LISTOF FIGURES ......coinintisinas Bi 5nmininasuesaenssmsssnmensnnswn mn XV LISTOF TABLES icin vnsninussmsnmsomemennnmemnsursmenssmshsmasmsmornss xvii i. PUBLIC HEALTH STATEMENT ..:::coiuiarsnsvsininssosnmomsmsamsnsvms ven 1 1.1 WHAT IS CHLOROFORM? . 1 1.2 WHAT HAPPENS TO CHLOROFORM WHEN IT ENTERS THE ENVIRONMENT? .. 2 1.3 HOWMIGHT IBEEXPOSED TOCHLOROFORM? .................vennvnn. 3 1.4 HOW CAN CHLOROFORM ENTER AND LEAVE MY BODY? ................. 4 1.5 HOW CAN CHLOROFORM AFFECT MY HEALTH? ......................... 4 1.6 IS THERE A MEDICAL TEST TO DETERMINE WHETHER I HAVE BEEN EXPOSED TOCHILOROFORM? . .....ocvismimnion:a isms aims ams £5 Has0 IDs mms wsomnn 6 1.7 WHAT RECOMMENDATIONS HAS THE FEDERAL GOVERNMENT MADE TO PROTECTHUMANHEALTH? ......... ci ii isis iii iin ssnnnnn 6 1.8 WHERE CANI1GET MORE INFORMATION? ......ccnvunsnsnnssnssssnsnnnn 8 2. HEALTH EFFECTS ... oii iti t iin ister steer rans sssnssnnsnnnrnns 9 21 INTRODUCTION ..ovvvmurmenn iain ion sMis Rs oui nsamsmonsions vsens mss 9 2.2 DISCUSSION OF HEALTH EFFECTS BY ROUTE OF EXPOSURE .............. 9 2.2.1 Inhalation Exposure . ......... 11 2211 Death o.ovvce vw vei sisssmismisaigesnsR is suse Benasnss 11 22.0.2 SYSIoMICEMEOS sus nintrssresmmsmemonmomennmmsansmsy 12 2.2.1.3 Immunological and Lymphoreticular Effects .................. 40 22.14 Newological Effects . . cc cion:vesminssnemmsnsnsvnr seems 41 22.1.3 Reproductive BffoRlS uv n:eomrsstmtnnrmemvenmsmenmmnmosms 42 2.2.1.6 Developmental Effects ................................. 43 221.7 GenotoxicEBiects . ....... cciciimisnesvensrusmrmnrnt ress 44 2.2.1.8 CANCE... oii 44 2.22 Oral EXposure . .......... 45 F331 Death .. ovis mcm tm nme mn bE AEA RE IS EH ENE 45 2222 SystemicEffects .....:u:vicurssnimesnonsnnsnsmosrmsmasa 46 2.2.2.3 Immunological and Lymphoreticular Effects .................. 90 2.2.2.4 Neurological Effects . ................. 91 22253 ReproductiveEBffects .......cvvuueninvensvssssmsnmninsin 92 2226 Developmental Effects ......cosncencnmsnrmossncusnnsns on 93 2227 GCenotOXICEHECIS on: nvs cs amanm sms nn rs moms vm sas wes me on 94 2.22.8 CANCE Lo. oti 95 2.2.3 Dermal EXposure . ....... 97 2931 Depth . vv vinmin mma mma asin t EIR IRAE RIESE WE EE 97 2232 SystemicEfects .......cc:asims msn senimsnasnenssns nn 97 CHLOROFORM Xii 3 2.2.3.3 Immunological and Lymphoreticular Effects ................. 100 2.23.4 Neurological Effects .................................. 100 2.2.3.5 Reproductive Effects .................... ............. 100 2.23.6 Developmental Effects ................................ 100 223.7 CenotoXICEHECl® oo. ci vv vnv sms rarer mm mtn 100 2238 COANCOT i vcontneomstn ns so 5o 6s sss be mes bm emm mms mmsmes 100 2.3 TOXICOKINETICS 100 23.1 Absorption... Lo... 101 2.3.1.1 Inhalation Exposure ....................... ........... 101 23:12 Oral EXpoSUIE o:cuivinmeoronvnrvonnssrmnscernmsrns wo. 102 23.0.3 Derm EXPOSUIS su: v:5ussinnissns ahs ninmevnnnrnmenss 104 23.2 Distribution... 105 2.3.2.1 Inhalation Exposure .................................. 105 23.22 OralEXposure .: vc :ocavoninsseonsmmsmenmonnenvnnsmes 107 2.3.23 Dermal Exposure .............. 107 2.3.3 Metabolism... 108 2.3.4 Elimination and Excretion ............................... ...... 112 2.3.4.1 Inhalation Exposure ........................ . .. ....... 112 23.42 Oral Exposure ................ 113 2343 Dermal Exposure .................. iii. 113 2.3.5 Physiologically Based Pharmacokinetic (PBPK)/Pharmacodynamic (PD) Models Ce 114 2.3.5.1 Summary of PBPK/PD Models .......................... 115 2.3.5.2 Chloroform PBPK Model Comparison ..................... 118 2.3.5.3 Discussion of Chloroform Models ........................ 118 24 MECHANISMS OF ACTION ..... i ie eee ee deen 128 2.4.1 Pharmacokinetic Mechanisms . . ................................. 128 2.42 Mechanisms of Toxicity .................. 131 2.43 Animal-to-Human Extrapolations ................................ 132 23 RELEVANCE TO PUBLIC HEALTH .cu:nsunosnsnn one smote ns sms nme omens 132 2.6 BIOMARKERS OF EXPOSURE AND EFFECT ........................... 156 2.6.1 Biomarkers Used to Identify or Quantify Exposure to Chloroform ......... 157 2.6.2 Biomarkers Used to Characterize Effects Caused by Chloroform .......... 159 2.7 INTERACTIONS WITH OTHER CHEMICALS ........................... 159 2.8 POPULATIONS THAT ARE UNUSUALLY SUSCEPTIBLE .................. 161 2.9. METHODS FOR REDUCING TOXIC EFFECTS .......................... 162 2.9.1 Reducing Peak Absorption Following Exposure ...................... 162 29.2 Reducing Body Burden ...................... ... .... .......... 163 2.9.3 Interfering with the Mechanism of Action for Toxic Effects ............. 163 2.10 ADEQUACY OF THE DATABASE . . . . . 165 2.10.1 Existing Information on Health Effects of Chloroform ................. 165 2.10.2 Identification of Data Needs . . . ................................. 166 2.10.3 Ongoing Studies . ........... 175 CHEMICAL AND PHYSICAL INFORMATION ............................... 177 3.1 CHEMICAL IDENTITY . .. 177 3.2 PHYSICAL AND CHEMICAL PROPERTIES ............................. 177 CHLOROFORM xiii 41 PRODUCTION ....vvvvemsuninrsemsrnasasmmsbinsiGsmaiasnisnenns ons 181 42 IMPORT/EXPORT ©. ct crs rim ama nit v tai sis Bs Am sm ARE IE a tows ww sans 183 4.3 USE 186 4.4 DISPOSAL oe 187 5. POTENTIAL FOR HUMAN EXPOSURE . ..... 189 S81 OVERVIEW cons vmome ms mn eds sd si bh sam sda Ms ma RE mes as mms mse 189 52 RELEASES TO THE ENVIRONMENT ........ 190 5.2.1 AIT oe ee 190 5.2.2 WALBE © ovo vi smn nossa sasnareansmmsmssmtorsnmeneatsonsansensss 194 B33 SOU oi tvrsninsnsmenm em aman cea rm AER AER INA ARs we 195 5.3 ENVIRONMENTAL FATE . . eee 196 53.1 Transport and Partitioning . : vo sccvmomsvnssvomcnunmsnsnnasmsves wo 196 5.3.2 Transformation and Degradation . ................................ 198 S321 AI vor mmm mma hi HA ARENAS EIR SRA VEE NE EE 198 5.3.2.2 Water . oo ott 199 5.3.2.3 Sediment and Soil ..... 201 5.4 LEVELS MONITORED OR ESTIMATED IN THE ENVIRONMENT ............ 201 5.4.1 BAIL vis ee mmm mE REE AE HEHE SHE SHIH ANAM NE hes 201 5.4.2 Water . oo oo 203 543 Sediment and Soil . . 205 5.4.4 Other Environmental Media . . ........... 206 5.5 GENERAL POPULATION AND OCCUPATIONAL EXPOSURE ............... 207 5.6 POPULATIONS WITH POTENTIALLY HIGH EXPOSURES ................. 209 57 ADEQUACY OF THE DATABASE. .......ccovvvvununvnvsnmrnanasnssnns 210 5.7.1 Identification of Data Needs . . . . . . 210 577 OngoMz SINAIES «ooo snr rrrrrsmsrasmsnmssmamomammensansnsibes 213 6. ANALYTICAL METHODS . ... chert insane miami ne nmsms mamas ms ene vs so 217 6.1 BIOLOGICAL SAMPLES . . . 217 6.2 ENVIRONMENTAL SAMPLES . eee 220 63 ADEQUACY OF THE DATABASE... cc: ccnmrnrsnenasonsmonsmusvmnmnnn 228 6.3.1 Identification of Data Needs . . . . .... ii 229 632 Ongoing Studies ............. iii 231 7. REGULATIONS AND ADVISORIES . . . eee 233 ZB. REFERENCES ©. civ vr time mmm sma noms mesic Mss Banu ens sms mamusnimes sess wo 247 0. GLOSSARY 291 APPENDICES A. ATSDR MINIMAL RISK LEVEL eee A-1 B. USERS GUIDE .. cv tems ims ts mms ms tds a i smi sms issn s Bow isianmaimnesms B-1 C. ACRONYMS, ABBREVIATIONS, AND SYMBOLS . ........................... C-1 CHLOROFORM XV 2-1 2-2 2-3 2-6 5-1 LIST OF FIGURES Levels of Significant Exposure to Chloroform—Inhalation ........................ 25 Levels of Significant Exposure to Chloroform—Oral ............................ 66 | Metabolic Pathways of Chloroform Biotransformation .......................... 109 Conceptual Representation of a Physiologically Based Pharmacokinetic (PBPK) Model for Hypothetical Chemical SUDSEINCE ...c.conivvsvisntvsunsnsmsnwsmnsnsns sma 116 Parameters used in the Corley PBPK Model ................................. 120 Existing Information on Health Effects of Chloroform .......................... 167 Frequency of NPL Sites With Chloroform Contamination . ....................... 191 CHLOROFORM XVii 2-1 2-2 2-3 2-5 2-6 3-1 3-2 4-1 4-2 5-1 6-1 6-2 7-1 LIST OF TABLES Levels of Significant Exposure to Chloroform—Inhalation ........................ 13 Levels of Significant Exposure to Chloroform—Oral ............................ 47 Levels of Significant Exposure to Chloroform—Dermal .......................... 99 Parameters Used in the Corley PBPK Model ................................. 121 Genotoxicity of Chloroform In Vitro ....................... i... 150 Genotoxicity of Chloroform In Vive . ...... «oo cvv iirc 151 Chemical Identity of Chloroform ......................... 178 Physical and Chemical Properties of Chloroform .............................. 179 U.S. Production of Chloroform... ....... 182 Facilities That Manufacture or Process Chloroform ............................ 184 Releases to the Environment From Facilities That Manufacture or Process Chloroform .... 192 Analytical Methods for Determining Chloroform in Biological Samples .............. 218 Analytical Methods for Determining Chloroform in Environmental Samples ........... 221 Regulations and Guidelines Applicable to Chloroform .......................... 235 CHLOROFORM 1 1. PUBLIC HEALTH STATEMENT This public health statement tells you about chloroform and the effects of exposure. The Environmental Protection Agency (EPA) identifies the most serious hazardous waste sites in the nation. These sites make up the National Priorities List (NPL) and the sites are targeted for long-term federal cleanup. Chloroform has been found in at least 717 of the 1.430 current or former NPL sites, including 6 in Puerto Rico and 1 in the Virgin Islands. However, it’s unknown how many NPL sites have been evaluated for this substance. As more sites are evaluated, the sites with chloroform may increase. This is important because exposure to this substance may harm you and because these sites may be sources of exposure. When a substance is released from a large area, such as an industrial plant, or from a container, such as a drum or bottle, it enters the environment. This release does not always lead to exposure. You are exposed to a substance only when you come in contact with it. You may be exposed by breathing, eating, or drinking the substance, or by skin contact. If you are exposed to chloroform, many factors determine whether you’ll be harmed. These factors include the dose (how much), the duration (how long), and how you come in contact with it. You must also consider the other chemicals you're exposed to and your age, sex, diet, family traits, lifestyle, and state of health. 1.1 WHAT IS CHLOROFORM? Chloroform is also known as trichloromethane or methyltrichloride. It is a colorless liquid with a pleasant, nonirritating odor and a slightly sweet taste. Most of the chloroform found in the environment comes from industry. It will only burn when it reaches very high temperatures. Chloroform was one of the first inhaled anesthetics to be used during surgery. but it is not used for anesthesia today. Nearly all the chloroform made in the United States CHLOROFORM 2 1. PUBLIC HEALTH STATEMENT today is used to make other chemicals, but some is sold or traded to other countries. We also import chloroform. Chloroform enters the environment from chemical companies and paper mills. It is also found in waste water from sewage treatment plants and drinking water to which chlorine has been added. Chlorine is added to most drinking water and many waste waters to destroy bacteria. Small amounts of chloroform are formed as an unwanted product during the process of adding chlorine to water. Chloroform can enter the air directly from factories that make or use it and by evaporating from water and soil that contain it. It can enter water and soil when waste water that contains chlorine is released into water or soil. It may enter water and soil from spills and by leaks from storage and waste sites. There are many ways for chloroform to enter the environment, so small amounts of it are likely to be found almost everywhere. You will find more information about what chloroform is, how it is used, and where it comes from in Chapters 3 and 4. 1.2 WHAT HAPPENS TO CHLOROFORM WHEN IT ENTERS THE ENVIRONMENT? Chloroform evaporates very quickly when exposed to air. Chloroform also dissolves easily in water, but does not stick to the soil very well. This means that it can travel down through soil to groundwater where it can enter a water supply. Chloroform lasts for a long time in both the air and in groundwater. Most chloroform in the air eventually breaks down, but this process is slow. The breakdown products in air include phosgene, which is more toxic than chloroform, and hydrogen chloride, which is also toxic. Some chloroform may break down in soil. Chloroform does not appear to build up in great amounts in plants and animals, but we may find some small amounts of chloroform in foods. You will find more information about where chloroform comes from, how it behaves, and how long it remains in the environment in Chapter 5. CHLOROFORM 3 1. PUBLIC HEALTH STATEMENT 1.3 HOW MIGHT | BE EXPOSED TO CHLOROFORM? You are probably exposed to small amounts of chloroform in your drinking water and in beverages (such as soft drinks) made using water that contains chloroform. You can also get chloroform in your body by eating food, by breathing air, and by skin contact with water that contains it. You are most likely to be exposed to chloroform by drinking water and breathing indoor or outdoor air containing it. The amount of chloroform normally expected to be in the air ranges from 0.02 to 0.05 parts of chloroform per billion parts (ppb) of air and from 2 to 44 ppb in treated drinking water. However, in some places, chloroform concentrations may be higher than 44 ppb. It is estimated that the concentration of chloroform in surface water is 0.1 ppb, the concentration in untreated groundwater is 0.1 ppb, and the amount in soil is 0.1 ppb. As much as 610 ppb was found in air at a municipal landfill and up to 88 ppb was found in treated municipal drinking water. Drinking water derived from well water near a hazardous waste site contained 1,900 ppb, and groundwater taken near a hazardous waste site also contained 1,900 ppb. Surface water containing 394 ppb has also been found near a hazardous waste site; however, no more than 0.13 ppb has been found in soil at hazardous waste sites. Chloroform has been found in the air from all areas of the United States and in nearly all of the public drinking water supplies. We do not know how many areas have surface water, groundwater, or soil that contains chloroform. The average amount of chloroform that you might be exposed to on a typical day by breathing air in various places ranges from 2 to 5 micrograms per day (ug/day) in rural areas, 6 to 200 pg/day in cities, and 80 to 2,200 pg/day in areas near major sources of the chemical. The estimated amount of chloroform you probably are exposed to in drinking water ranges from 4 to 88 pg/day. We cannot estimate the amounts that you may be exposed to by eating food and by coming into contact with water that has chloroform in it. People who swim in swimming pools absorbed chloroform through their skin. People who work at or near chemical plants and factories that make or use chloroform can be exposed to higher-than- normal amounts of chloroform. Higher exposures might occur in workers at drinking-water treatment plants, waste water treatment plants, and paper and pulp mills. People who operate waste-burning equipment may also be exposed to higher than normal levels. The National CHLOROFORM 4 1. PUBLIC HEALTH STATEMENT Institute for Occupational Safety and Health (NIOSH) estimated that 95,778 persons in the United States have occupational exposure to chloroform. You will find more information about how you can be exposed to chloroform in Chapter 5. 1.4 HOW CAN CHLOROFORM ENTER AND LEAVE MY BODY? Chloroform can enter your body if you breathe air, eat food, or drink water that contains chloroform. Chloroform easily enters your body through the skin. Therefore, chloroform may also enter your body if you take a bath or shower in water containing chloroform. In addition, you can breathe in chloroform if the shower water is hot enough for chloroform to evaporate. Studies in people and in animals show that after you breathe air or eat food that has chloroform in it, the chloroform can quickly enter your bloodstream from your lungs or intestines. Inside your body, chloroform is carried by the blood to all parts of your body, such as the fat, liver, and kidneys. Chloroform usually collects in body fat; however, its volatility ensures that it will eventually be removed once the exposure has been removed. Some of the chloroform that enters your body leaves unchanged in the air that you breathe out, and some chloroform in your body is broken down into other chemicals. These chemicals are known as breakdown products or metabolites, and some of them can attach to other chemicals inside the cells of your body and may cause harmful effects if they collect in high enough amounts in your body. Some of the metabolites also leave the body in the air you breathe out. Only a small amount of the breakdown products leaves the body in the urine and stool. You can find more information about the behavior of chloroform in the body in Chapter 2. 1.5 HOW CAN CHLOROFORM AFFECT MY HEALTH? To protect the public from the harmful effects of toxic chemicals and to find ways to treat people who have been harmed, scientists use many tests. CHLOROFORM . 5 1. PUBLIC HEALTH STATEMENT One way to see if a chemical will hurt people is to learn how the chemical is absorbed, used, and released by the body; for some chemicals, animal testing may be necessary. Animal testing may also be used to identify health effects such as cancer or birth defects. Without laboratory animals, scientists would lose a basic method to get information needed to make wise decisions to protect public health. Scientists have the responsibility to treat research animals with care and compassion. Laws today protect the welfare of research animals, and scientists must comply with strict animal care guidelines. In humans, chloroform affects the central nervous system (brain), liver, and kidneys after a person breathes air or drinks liquids that contain large amounts of chloroform. Chloroform was used as an anesthetic during surgery for many years before its harmful effects on the liver and kidneys were recognized. Breathing about 900 parts of chloroform in a million parts of air (900 ppm or 900,000 ppb) for a short time causes fatigue, dizziness, and headache. If you breathe air, eat food, or drink water containing elevated levels of chloroform, over a long period, the chloroform may damage your liver and kidneys. Large amounts of chloroform can cause sores when the chloroform touches your skin. We do not know whether chloroform causes harmful reproductive effects or birth defects in people. Miscarriages occurred in rats and mice that breathed air containing elevated levels (30 to 300 ppm) of chloroform during pregnancy and in rats that ate chloroform during pregnancy. Abnormal sperm were found in mice that breathed air containing elevated levels (400 ppm) of chloroform for a few days. Offspring of rats and mice that breathed chloroform during pregnancy had birth defects. Results of studies of people who drank chlorinated water showed a possible link between the chloroform in chlorinated water and the occurrence of cancer of the colon and urinary bladder. Cancer of the liver and kidneys developed in rats and mice that ate food or drank water that had large amounts of chloroform in it for a long time. We do not know whether liver and kidney cancer would develop in people after long-term exposure to chloroform in drinking water. Based on animal studies, the Department of Health and Human Services (DHHS) has determined that chloroform may reasonably be anticipated to be a carcinogen (a CHLOROFORM 6 1. PUBLIC HEALTH STATEMENT substance that causes cancer). The International Agency for Research on Cancer (IARC) has determined that chloroform is possibly carcinogenic to humans (2B). The EPA has determined that chloroform is a probable human carcinogen. You can find a more complete discussion about how chloroform affects your health in Chapter 2. 1.6 IS THERE A MEDICAL TEST TO DETERMINE WHETHER | HAVE BEEN EXPOSED TO CHLOROFORM? Although we can measure the amount of chloroform in the air that you breathe out, and in blood, urine, and body tissues, we have no reliable test to determine how much chloroform you have been exposed to or whether you will experience any harmful health effects. The measurement of chloroform in body fluids and tissues may help to determine if you have come into contact with large amounts of chloroform. However, these tests are useful only a short time after you are exposed to chloroform because it leaves the body quickly. Because it is a breakdown product of other chemicals (chlorinated hydrocarbons), chloroform in your body might also indicate that you have come into contact with those other chemicals. Therefore, small amounts of chloroform in the body may indicate exposure to these other chemicals and may not indicate low chloroform levels in the environment. From blood tests to determine the amount of liver enzymes, we can tell whether the liver has been damaged, but we cannot tell whether the liver damage was caused by chloroform. 1.7 WHAT RECOMMENDATIONS HAS THE FEDERAL GOVERNMENT MADE TO PROTECT HUMAN HEALTH? The federal government develops regulations and recommendations to protect public health. Regulations can be enforced by law. Federal agencies that develop regulations for toxic substances include EPA, the Occupational Safety and Health Administration (OSHA), and the Food and Drug Administration (FDA). Recommendations provide valuable guidelines to protect public health but cannot be enforced by law. Federal organizations that develop CHLOROFORM 1. PUBLIC HEALTH STATEMENT recommendations for toxic substances include the Agency for Toxic Substances and Disease Registry (ATSDR) and the National Institute for Occupational Safety and Health (NIOSH). Regulations and recommendations can be expressed in not-to-exceed levels in air, water, soil, or food that are usually based on levels that affect animals; then they are adjusted to help protect people. Sometimes these not-to-exceed levels differ among federal organizations because of different exposure times (an 8-hour workday or a 24-hour day), the use of different animal studies, or other factors. Recommendations and regulations are also periodically updated as more information becomes available. For the most current information, check with the federal agency or organization that provides it. Some regulations and recommendations for chloroform include the following: The EPA sets rules for the amount of chloroform allowed in water. The EPA limit for total trihalomethanes, a class of chemicals that includes chloroform, in drinking water is 100 micrograms per liter (ug/L, 1 ug/L = 1 ppb in water). Furthermore, EPA requires that spills of 10 pounds or more of chloroform into the environment be reported to the National Response Center. OSHA sets the levels of chloroform allowed in workplace air in the United States. A permissible occupational exposure limit is 50 ppm or 240 mg/m> (ceiling value) in air during an 8-hour workday, 40-hour workweek. CHLOROFORM 1. PUBLIC HEALTH STATEMENT 1.8 WHERE CAN | GET MORE INFORMATION? If you have any more questions or concerns, please contact your community or state health or environmental quality department or: Agency for Toxic Substances and Disease Registry Division of Toxicology 1600 Clifton Road NE, Mailstop E-29 Atlanta, GA 30333 * Information line and technical assistance Phone: (404) 639-6000 Fax: (404) 639-6315 or 6324 ATSDR can also tell you the location of occupational and environmental health clinics. These clinics specialize in recognizing, evaluating, and treating illnesses resulting from exposure to hazardous substances. * To order toxicological profiles. contact: National Technical Information Service 5285 Port Royal Road Springfield, VA 22161 Phone: (800) 553-6847 or (703) 487-4650 CHLOROFORM 9 2. HEALTH EFFECTS 2.1 INTRODUCTION The primary purpose of this chapter is to provide public health officials, physicians, toxicologists, and other interested individuals and groups with an overall perspective of the toxicology of chloroform. It contains descriptions and evaluations of toxicological studies and epidemiological investigations and provides conclusions, where possible, on the relevance of toxicity and toxicokinetic data to public health. A glossary and list of acronyms, abbreviations, and symbols can be found at the end of this profile. 2.2 DISCUSSION OF HEALTH EFFECTS BY ROUTE OF EXPOSURE To help public health professionals and others address the needs of persons living or working near hazardous waste sites, the information in this section is organized first by route of exposure —inhalation, oral, and dermal; and then by health effect—death, systemic, immunological, neurological, reproductive, developmental, genotoxic, and carcinogenic effects. These data are discussed in terms of three exposure periods—acute (14 days or less), intermediate (15-364 days), and chronic (365 days or more). Levels of significant exposure for each route and duration are presented in tables and illustrated in figures. The points in the figures showing no-observed-adverse-effect levels (NOAELSs) or lowest- observed-adverse-effect levels (LOAELSs) reflect the actual doses (levels of exposure) used in the studies. LOAELS have been classified into "less serious" or "serious" effects. "Serious" effects are those that evoke failure in a biological system and can lead to morbidity or mortality (e.g., acute respiratory distress or death). "Less serious" effects are those that are not expected to cause significant dysfunction or death, or those whose significance to the organism is not entirely clear. ATSDR acknowledges that a considerable amount of judgment may be required in establishing whether an end point should be classified as a NOAEL, "less serious" LOAEL, or "serious" LOAEL, and that in some cases, there will be insufficient data to decide whether the effect is indicative of significant dysfunction. However, the Agency has established guidelines and policies that are used to classify these end points. ATSDR believes that there is sufficient merit in this approach to warrant an attempt CHLOROFORM 10 2. HEALTH EFFECTS at distinguishing between "less serious" and "serious" effects. The distinction between "less serious” effects and "serious" effects is considered to be important because it helps the users of the profiles to identify levels of exposure at which major health effects start to appear. LOAELs or NOAELs should also help in determining whether or not the effects vary with dose and/or duration, and place into perspective the possible significance of these effects to human health. The significance of the exposure levels shown in the Levels of Significant Exposure (LSE) tables and figures may differ depending on the user’s perspective. Public health officials and others concerned with appropriate actions to take at hazardous waste sites may want information on levels of exposure associated with more subtle effects in humans or animals (LOAEL) or exposure levels below which no adverse effects (NOAELs) have been observed. Estimates of levels posing minimal risk to humans (Minimal Risk Levels or MRLs) may be of interest to health professionals and citizens alike. Levels of exposure associated with the carcinogenic effects (Cancer Effect Levels, CELs) of chloroform are indicated in Figure 2-2. Because cancer effects could occur at lower exposure levels, Figures 2-1 and 2-2 also show a range for the upper bound of estimated excess risks, ranging from a risk of 1 in 10,000 to 1 in 10,000,000 (10° to 107), as developed by EPA. Estimates of exposure levels posing minimal risk to humans (Minimal Risk Levels or MRLs) have been made for chloroform. An MRL is defined as an estimate of daily human exposure to a substance that is likely to be without an appreciable risk of adverse effects (noncarcinogenic) over a specified duration of exposure. MRLs are derived when reliable and sufficient data exist to identify the target organ(s) of effect or the most sensitive health effect(s) for a specific duration within a given route of exposure. MRLs are based on noncancerous health effects only and do not consider carcinogenic effects. MRLs can be derived for acute-, intermediate-, and chronic-duration exposures for inhalation and oral routes. Appropriate methodology does not exist to develop MRLs for dermal exposure. Although methods have been established to derive these levels (Barnes and Dourson 1988; EPA 1990b). uncertainties are associated with these techniques. Furthermore, ATSDR acknowledges additional uncertainties inherent in the application of the procedures to derive less than lifetime MRLs. As an example, acute inhalation MRLs may not be protective for health effects that are delayed in development or are acquired following repeated acute insults, such as hypersensitivity reactions, CHLOROFORM 1 2. HEALTH EFFECTS asthma, or chronic bronchitis. As these kinds of health effects data become available and methods to assess levels of significant human exposure improve, these MRLs will be revised. A User’s Guide has been provided at the end of this profile (see Appendix B). This guide should aid in the interpretation of the tables and figures for Levels of Significant Exposure and the MRLs. 2.2.1 Inhalation Exposure Most of the data regarding inhalation exposure to chloroform in humans were obtained from clinical reports describing health effects in patients under anesthesia. In some instances, the results may have been confounded by the concurrent administration of other drugs with chloroform or by artificial respiration of patients under chloroform anesthesia. Furthermore, most of the studies did not provide any information regarding actual exposure levels for observed effects. Nonetheless, chloroform- induced effects in humans are supported by those observed in animals under experimental conditions. The human studies cited in the profile provide qualitative information on chloroform toxicity in humans. 2.2.1.1 Death Information on the exposure levels of chloroform leading to death in humans was obtained from clinical reports of patients exposed to chloroform as a method of anesthesia. It should be noted that when examining the ability of chloroform to cause death, these clinical reports may be misleading, in that many of these patients had pre-existing health conditions that may have contributed to the cause of death and that chloroform toxicity may not have been the only factor involved in the death of the patient. Older clinical case reports suggested that concentrations of 40,000 ppm, if continued for several minutes, may be an overdose (Featherstone 1947). When a cohort of 1,502 patients, ranging in age from 1 to 80 years, exposed under anesthesia to less than 22,500 ppm chloroform was evaluated, no indication of increased mortality was found (Whitaker and Jones 1965). In most patients, the anesthesia did not last longer than 30 minutes; however, a few received chloroform for more than 2 hours. Several studies reported deaths in women after childbirth when chloroform anesthesia had been used (Royston 1924; Townsend 1939). No levels of actual exposure were provided in either study. Death was caused by acute hepatotoxicity. Prolonged labor with starvation, dehydration, and exhaustion contributed to the chloroform-induced hepatotoxicity. CHLOROFORM 12 2. HEALTH EFFECTS Levels of acute exposure resulting in animal deaths are generally lower than those reported for human patients under anesthesia; however, the exposure durations are generally longer in the animal studies. An inhalation LCs (lethal concentration, 50% kill) of 9,770 ppm for a 4-hour exposure was reported for female rats (Lundberg et al. 1986). One rat died after 6 exposures in one report where groups of 9-12 pregnant female rats exposed to chloroform at doses as high as 4,117 ppm for 1 hour a day for 8 days (Newell and Dilley 1978). However, exposure to 8,000 ppm for 4 hours was lethal to albino rats (Smyth et al. 1962). Male mice appear to be more sensitive than female mice. Following exposure to 1,024 ppm for 1-3 hours, 15 of 18 male mice died within 11 days; however, most of the female mice similarly exposed survived for several months (Deringer et al. 1953). Male mice that died had kidney and liver damage, while females did not. An exposure as low as 692 ppm for 1-3 hours resulted in the death of 3 of 6 male mice within 8 days. When exposed to 4,500 ppm chloroform for 9 hours, 10 of 20 female mice died (Gehring 1968). Increased mortality was observed in male rats exposed to 85 ppm chloroform for 6 months (Torkelson et al. 1976). The deaths were attributed to interstitial pneumonia. Rats of either sex exposed to 50 ppm survived. Exposure to 85 ppm for 6 months did not increase mortality in rabbits and guinea pigs. Similarly, no deaths were reported in dogs exposed to 25 ppm chloroform for the same time period. The LCs and all reliable LOAEL values for death in each species and duration category are recorded in Table 2-1 and plotted in Figure 2-1. 2.2.1.2 Systemic Effects No studies were located regarding dermal or ocular effects in humans or animals after inhalation exposure to chloroform. The highest NOAEL values and all reliable LOAEL values for each systemic effect in each species and duration category are recorded in Table 2-1 and plotted in Figure 2-1. Respiratory Effects. Changes in respiratory rate were observed in patients exposed to chloroform via anesthesia (exposure less than 22,500 ppm) (Whitaker and Jones 1965). Increased respiratory rates were observed in 44% of 1,502 patients who were exposed to light chloroform anesthesia. Respiratory rates were depressed, however, during deep and prolonged anesthesia when chloroform concentrations Table 2-1. Levels of Significant Exposure to Chloroform - Inhalation Exposure/ LOAEL a Key to Species/ duration/ NOAEL Less serious Serious figure (strain) frequency System (ppm) (ppm) (ppm) Reference ACUTE EXPOSURE Death 1 Rat 4 hr 8000 (5/6 died) Smyth et al. 1962 (Albino) 2 Rat 4 hr 9770 F (LCs) Lundberg et al. (Sprague- 1986 Dawley) 3 Rat 8d 4117 F (death; 1 animal on Newell and Dilley (Sprague- Gd 7-14 Gd 12) 1978 Dawley) 1 hr/d 4 Mouse 9 hr 4500 F (10/20 died) Gehring 1968 (Swiss- Webster) 5 Mouse 1-3 hr 692 M (3/6 died) Deringer et al. (C3H) 1953 Systemic 6 Human 113 min Cardio 8000 (arrhythmia) Smith et al. 1973 Gastro 8000 (vomiting) Hemato 8000 (increased prothrombin time) Hepatic 8000 (increased sulfobromophthalein sodium retention) S103443 H1TV3H °C WHO40HOTHO el Table 2-1. Levels of Significant Exposure to Chloroform - Inhalation (continued) . Exposure/ LOAEL Key to Species/ duration/ NOAEL Less serious figure (strain) frequency System (ppm) (ppm) Reference 7 Human 0.5-2 hr Resp 22,500 (changes in respiratory Whitaker and rate) Jones 1965 Cardio (cardiac arrhythmia, bradycardia) Gastro 22,500 (vomiting) Hepatic 22,500 (transient jaundice in 1 patient) 8 Rat 8d Bd Wt 2232 F 4117 F (60% decreased maternal Newell and Dilley (Sprague- Gd 7-14 body weight gain) 1978 Dawley) 1 hr/d 9 Rat 4 hr Hepatic 76 F 153 F (SDH-enzyme levels Lundberg et al. (Sprague- increased) 1986 Dawley) 10 Rat 10d Hepatic 300 F Baeder and (Wistar) Gd 7-16 Hofmann 1988 7hii Renal 300 F Bd Wt 30 F (18% decreased weight 100 F (24% decreased weight gain of dams) gain of dams) Other 30 F (decrease in feed consumption) 11 Rat 8 hr Hepatic 50 M (elevated liver Ikatsu and (Wistar) triglycerides and liver Nakajima 1992 GSH) 12 Rat 7d Resp 31 M 10.4 M (degeneration of Mery et al. 1994 (Fischer- 344) 6 hr/d Bowman's gland; new bone formation; increased number of S-phase nuclei) Bd Wt 100 M 271 M (unspecified decrease in body weight) S103443 HLTV3H °C WHO40HO THO vi Table 2-1. Levels of Significant Exposure to Chloroform - Inhalation (continued) » Exposure/ LOAEL Keyto Species/ duration/ NOAEL Less serious Serious figure (strain) frequency System (ppm) (PPM) pm) Reference 13 Rat 7d Resp 31 M 10.4 M (epithelial goblet cell Larson et al. 1994¢ (Fischer- 344) 6 hr/d hyperplasia and degeneration of Bowman's glands in olfactory mucosa) Hepatic 100 M 271 M (swelling and mild centrilobular vacuolation) Renal 104 M 29.3 M (increased number of S-phase nuclei for tubule cells in the cortex) Bd Wt 31M 10.4 M (decreased weight gain) 14 Rat 6 hr Hepatic 100 M 500 M (increased plasma GOT Wang et al. 1994 (Wistar) and GPT; decreased hepatic GSH) 15 Rat 6 hr Hepatic 100 500 (incr. plasma GOT Wang et al. 1995 (Wistar) activity) 16 Mouse 2 hr Hepatic 246 (fatty changes) Culliford and (CBA; WH.) Hewitt 1957 Renal 246 F 246 M (tubular necrosis in males) 17 Mouse 4 hr Hepatic 100 F (fatty changes) Kylin et al. 1963 (NS) 18 Mouse 1-3 hr Resp 1106 Deringer et al. (C3H) 1953 Hepatic 1106 F 942 M (liver necrosis in males that died) Renal 1106 F 692 M (tubular necrosis in males that died) S103443 H1TV3H 2 WHOJOHOTHO St Table 2-1. Levels of Significant Exposure to Chloroform - Inhalation (continued) a Exposure/ LOAEL Key to Species/ duration/ NOAEL Less serious Serious figure (strain) frequency System (ppm) (ppm) (ppm) Reference 19 Mouse 8d Hepatic 100 F (increased SGPT Murray et al. 1979 (CF1) Gd 1-7, activity; increased Gd 6-15, or absolute and relative Gd 8-15 liver weights) 7 hr/d Bd Wt 100 F (unspecified decreased weight gain in dams) 20 Mouse 9 hr Hepatic 4500 F (increased SGPT activity) Gehring 1968 (Swiss- Webster) 21 Mouse 7d Resp 3 F 10 F (increased number of Mery et al. 1994 (B6C3F1) 6 hr/d S-phase nuclei) Bd Wt 295 F 101 F (unspecified decr. in body weight) 22 Mouse 7d Resp 288 F Larson et al. 1994c (86C3F1) ~~ 6hr/d Hepatic 3b F 10 F (mild to moderate 101 F (centrilobular hepatocyte vacuolar changes in necrosis and severe centrilobular diffuse vacuolar hepatocytes) degeneration of midzonal and periportal hepatocytes) Renal 101 F 288 F (proximal tubules lined by regenerating epithelium) Bd Wt 288 F S103443 H1TV3H 2 WHO40HO THO 9l Table 2-1. Levels of Significant Exposure to Chloroform - Inhalation (continued) 2 Exposure/ LOAEL Key to Species/ duration/ NOAEL Less serious Serious figure (strain) frequency System (ppm) (ppm) (ppm) Reference 23 Mouse 4d Resp 2 F 10 F (mild, transient, Larson et al. 1996 (B6C3F1) 6 hr/d proliferative responses & bone thickening in the periosteum and posterior ventral areas) Musc/skel 88 F Hepatic 30 F 88 F (increased relative liver weights; hepatocyte lipid vacuolization & scattered individual hepatocyte necrosis; significant increase in hepatic LI) Renal 88 F Immunological/Lymphoreticular 24 Rat 10d 300 F Baeder and (Wistar) Gd 7-16 Hofmann 1988 7 hr/d 25 Mouse 1-3 hr 1106 Deringer et al. (C3H) 1953 Neurological 26 Human 3 min 920 (dizziness, vertigo) Lehman and Hasegawa 1910 27 Human 113 min 8000 (narcosis) Smith et al. 1973 28 Human 0.5-2 hr 22,500 (narcosis) Whitaker and Jones 1965 29 Mouse 0.5-2 hr 2500 3100 (slight narcosis) Lehmann and Flury (NS) 1943 30 Cat 5-93 min 7,200 (disturbed equilibrium) Lehmann and Flury (NS) 1943 S103443 H1V3H ¢ WHO4OHOTHO LL Table 2-1. Levels of Significant Exposure to Chloroform - Inhalation (continued) 5 Exposure/ LOAEL Keyto Species/ duration/ NOAEL Less serious Serious figure (strain) frequency System (ppm) (ppm) (ppm) Reference Reproductive 31 Rat 10d 100 F 300 F (73% decreased Schwetz et al. (Sprague- Gd 6-15 conception rate; increased 1974 Dawley) 7 hr/d incidence of fetal resorptions) 32 Rat 10d 30 F (empty implantations in Baeder and (Wistar) Gd 7-16 2/20 dams) Hofmann 1988 7 hr/d 33 Mouse 8d 100 F (increased incidence of Murray et al. 1979 (CF1) Gd 1-7, Gd resorptions; decrease in 6-15, or % pregnancies) Gd 8-15 7 hr/d 34 Mouse 5d 400 M (increased percentage of Land et al. 1979 (Cc57B1/ 4 hr/d abnormal sperm) C3H)F1 Developmental 35 Rat 10d 30 (delayed ossification and 100 (missing ribs; acaudate Schwetz et al. (Sprague- Gd 6-15 wavy ribs) fetuses with imperforate 1974 Dawley) 7 hr/d anus) 36 Rat 10d 30 (slight growth retardation) Baeder and (Wistar) Gd 7-16 Hofmann 1988 7 hr/d 37 Mouse 8d 100 (cleft palate, decreased Murray et al. 1979 (CF1) Gd 1-7, Gd ossification) 6-15, or Gd 8-15 7 hr/d S103443 H1V3H ¢ WHO40HOTHO 81 Table 2-1. Levels of Significant Exposure to Chloroform - Inhalation (continued) . Exposure/ LOAEL Key to Speclies/ duration/ NOAEL Less serious Serious figure (strain) frequency System (ppm) (ppm) (PPM) Reference INTERMEDIATE EXPOSURE Death 38 Rat 6 mo 85 M (increased mortality; 4/10 Torkelson et al. (NS) 5 d/wk died) 1976 7 hr/d Systemic 39 Human 1-6 mo Gastro 14 (nausea; vomiting) Phoon et al. 1983 Hepatic 14¢ (toxic hepatitis) 40 Rat 6 mo Resp 50 M 85 M (interstitial pneumonia) Torkelson et al. (NS) 5 d/wk 85 F 1976 7 hr/d Hemato 85 Hepatic 25 (degenerative changes) Renal 25 (cloudy swelling) Bd Wt 25 M 50 M (decreased body weight 85 F in males) 41 Mouse 3 wk Resp 88 Larson et al. 1996 (86C3F1) 7 d/wk 8 bid Musc/skel 88 Hepatic 10 29.6 (hepatocyte vacuolation and swelling; variations in nuclear size) Renal i0 M 29.6 M (nephropathy of epithelial 88 F cells of the proximal convoluted tubules) Bd Wt 88 S103443 H1TV3H °C WHO40HOTHO 61 Table 2-1. Levels of Significant Exposure to Chloroform - Inhalation (continued) 2 Exposure/ LOAEL Keyto Species/ duration/ NOAEL Less serious Serious figure (strain) frequency System (ppm) 0) (ppm) Reference 42 Mouse 6 wk Resp 88 Larson et al. 1996 (B6C3F1) 7 diwk 6 hid Musc/skel 88 Hepatic 10 29.6 F (16.1% increase in relative liver weights; mild degenerative changes in hepatocytes; increase in liver LI) Renal 88 Bd Wt 88 43 Mouse 13 wk Resp 88 Larson et al. 1996 (86C3F1) 7 d/wk 6 hid Musc/skel 88 Hepatic 10 296 (vacuolation and swelling of hepatocytes; variations in nuclear size) Renal 1.99 10 M (nephropathy & enlarged nuclei of the epithelial cells of the proximal convoluted tubules; mineralization in the cortex) 88 Bd Wt 88 S103443 H1TV3H ¢ WHO404dOTHO 0c Table 2-1. Levels of Significant Exposure to Chloroform - Inhalation (continued) a Exposure/ LOAEL Keyto Species/ duration/ NOAEL Less serious figure (strain) frequency System (ppm) TL) Reference 44 Mouse 13 wk Resp 88 Larson et al. 1996 (B6C3F1) 5 d/wk 6 hid Musc/skel 88 Hepatic 29.6 88 (increased relative liver weights; vacuolation & swelling of hepatocytes & enlarged nuclei; increased liver LI) Renal 1.99 10 M (nephropathies and enlarged nuclei in epithelial cells of the proximal convoluted tubules; mineralization within the cortex; increased cortical and medullary tissue LIs) 88 Bd Wt 29.6 45 Dog 6 mo Hemato 25 Torkelson et al. (NS) 5 d/wk 1976 2 Hepatic 25 Renal 25 25 F (cloudy swelling of tubular epithelium) 46 Rabbit 6 mo Resp 25 F (interstitial pneumonia) Torkelson et al. (NS) 5 d/wk 50 85 M (pneumonitis) 1976 7 hr/d Hemato 85 Hepatic 25 (centrilobular granular degeneration and necrosis) Renal 25 (interstitial nephritis) S103443 H1TV3H ¢ WHO40HO THO 12 Table 2-1. Levels of Significant Exposure to Chloroform - Inhalation (continued) . Exposure/ LOAEL Key to Species/ duration/ NOAEL Less serious Serious figure (strain) frequency System (ppm) (PPM) (PPM) Reference 47 Gn pig 6 mo Hemato 85 Torkelson et al. (NS) 5 d/wk 1976 7 hr/d H . . epatic 25 (centrilobular granular degeneration) Renal 25 (tubular and interstitial nephritis) Immunological/Lymphoreticular 48 Rat 6 mo 25 Torkelson et al. (NS) 5 d/wk 1976 7 hr/d Reproductive 49 Rat 6 mo 25 M Torkelson et al. (NS) 5 d/wk 1976 7 hr/d S103443 HLV3H 2 WHO40HO THO 2c Table 2-1. Levels of Significant Exposure to Chloroform - Inhalation (continued) » Exposure/ LOAEL Key to Species/ duration/ NOAEL Less serious Serious figure (strain) frequency System (PPM) (ppm) (ppm) Reference CHRONIC EXPOSURE Systemic 50 Human 10-24 mo Gastro 22 F (nausea) Challen et al. 1958 Hepatic 71 51 Human 1-4 yr Hepatic 29 (hepatomegaly) Bomski et al. 1967 52 Human 3-10 yr Gastro 77 F (nausea) Challen et al. 1958 Hepatic 237 F 53 Human 1-15 yr Hepatic 29.5 (elevated serum Li et al. 1993 prealbumin and transferrin) Renal 13.5 Immunological/Lymphoreticular 54 Human 1-4 yr 2 (splenomegaly) Bomski et al. 1967 Neurological 55 Human 3-10 yr 77 F (exhaustion, irritability, Challen et al. 1958 depression, lack of concentration) 56 Human 10-24 mo 22 F (exhaustion) Challen et al. 1958 S103443 H1TV3H 2 WHO40HO THO €2 Table 2-1. Levels of Significant Exposure to Chloroform - Inhalation (continued) » Exposure/ LOAEL Key to Species/ duration/ NOAEL Less serious Serious figure (strain) frequency System (ppm) (ppm) (ppm) Reference 57 Human 1-15 yr 13.49 (dizziness, fatigue, Li etal. 1993 somnolence, insomnia, increased dreaming, hypomnesia, anorexia, and palpitations) “The number corresponds to entries in Figure 2-1. PUsed to derive an acute-duration inhalation minimal risk level (MRL) of 0.1 ppm; concentration is converted to a human equivalent concentration and divided by an uncertainty factor of 30 (3 for extrapolation from animals to humans and 10 for human variability). “Used to derive an intermediate-duration inhalation MRL of 0.05 ppm; concentration is divided by an uncertainty factor of 100 (10 for use of a LOAEL and 10 for human variability). used to derive a chronic-duration inhalation MRL of 0.02 ppm; concentration is divided by an uncertainty factor 100 (10 for use of a LOAEL and 10 for human variability). Bd Wt = body weight; Cardio = cardiovascular; d = day(s); F = female; Gastro = gastrointestinal; Gd = gestational day; Gn pig = guinea pig; GOT = glutamic oxalotransaminase; GPT = glutamic pyruvic transaminase; GSH = glutathione; Hemato = hematological; hr = hour(s); LCso = lethal concentration, 50% kill; LOAEL = lowest-observed-adverse-effect level; LI = labeling index; M = male; min = minute(s); mo = moth(s); musc/skel = musculoskeletal; NOAEL = no-observed-adverse-effect level; NS = not specified; Resp = respiratory; SDH = sorbitol dehydrogenase; SGPT = serum glutamic pyruvic transaminase; wk = week; yr = year(s) S103443 H1TV3H 2 WHO40HO THO ve Figure 2-1. Levels of Significant Exposure to Chloroform - Inhalation (ppm) 100000 10000 100 10 0.1 0.01 Acute (<14 days) Systemic ~ ~ ¢ ££ 3 F $ F F & # Qo 9 = Oo 9 & F S$ 3 9° © 5 S © $ Fy 9 i $ & § & F & Q < O QO T S ZL 7 A A 2r ir =u 6 6 6 8 ® 3r ® A A A A zm ® 5m 3 18m 9 14r 15r ® 22m or 13x @@ 0 x So a “ 23 13r 14r 15r 17m 19m 22m ,3 0 6 wO0O0O0 a o Bn or o 23m Oo 12r 13r 21m 23m 2m > 00 > 12r 13r 21m Key 25m Oo O Oo 2 o m rouse LCu (animals) ' Minimal risk g guinea pig ® LOAEL for serious effects (animals) \ level for effects d dog ® LOAEL for less serious effects (animals) omer than : | ; pr O NOAEL (animals) | | A LOAEL for serious effects (humans) The number next to \ each point A LOAEL for less serious effects (humans) corresponds to A NOAEL (humans) entries in Table 2-1. * Doses represent the lowest dose tested per study that produced a tumorigenic response and do not imply the existence of a threshold for the cancer end point. S103443 H1TV3H ¢ WHO40dO THO Se Figure 2-1. Levels of Significant Exposure to Chloroform - Inhalation (cont.) Acute (<14 days) Systemic N&O > $ ge > & 2 > Sy $ & & $F SS S$ S Q (ppm) » $ « S$ S o v4 $s $ §¢ ££ & 8 ¢ 100000 < Q oO Sv oS < Q 28 A 27 10000 A 30¢ 8r 20m ® ® 3 ) $ m 26 29 1000 18m * ° A " @ 34m 10r 16m 22m 12r 22m 24r 31r @ ® o o Oo Oo @ 2m 53m 10r 12r 19m 21m 31r 33m 35r 100 0 ® O > 0 o e » We 13r 10r 21m 10r 32r 35r 36r o o Oo o > 0 13r 13r 10 o Key 1 : © ro se B | Cy (animals) Minimal risk g guinea pig ® LOAEL for serious effects (animals) | level for effects d dog ® LOAEL for less serious effects (animals) V other than 01 ¢ a O NOAEL (animals) rabbi A LOAEL for serious effects (humans) sl oni next to ; ea in A LOAEL for less serious effects (humans) 8 to 0.01 A NOAEL (humans) entries in Table 2-1. * Doses represent the lowest dose tested per study that produced a tumorigenic response and do not imply the existence of a threshold for the cancer end point. S103443 H1V3H ¢ WHO40HOTHO 92 Figure 2-1. Levels of Significant Exposure to Chloroform - Inhalation (cont.) Intermediate (15-364 days) 100 10 0.1} 0.01% Systemic Z T 3 3 $ 5 L g z S.3 @ § £5 § § oF § Qo 5 9 s 0 $ SE 5 5 5 £ & 3 5 3 s S§ § v «9 « £ 9 Io c S g £ a [}] Q [+] ()) > (1) Q oO > Q Q @ QO T S z 3 Q £3 38r 40r 42m 44m46h 40r 46h 47g 41m 43m 44m 42m 41m 43m ® 000000 O00 OOOO Qo Oo O00 of'm emo 42m 44m > 42m 40r 46h 45d 0 300 46h 40r > 45d 47g 4or or 48r 49r wg © 20 O00 O 200 0 Oo 41m 43m 45d 47g a6h A A 41m 42m 41m 43m 44m 0 0 O00 | | | | 43m 44m oo I I \ Key | | 7 ! fe LCs (animals) Minimal risk g guinea pig ®@ LOAEL for serious effects (animals) | level for effects ! d dog ® LOAEL for less serious effects (animals) other than Ww n ran O NOAEL (animals) A LOAEL for serious effects (humans) A LOAEL for less serious effects (humans) A NOAEL (humans) The number next to each point corresponds to entries in Table 2-1. S103443 HLTV3H 2 WHO40HOTHO LZ Figure 2-1. Levels of Significant Exposure to Chloroform - Inhalation (cont.) Chronic ( >365 days) Systemic N I > SE F & Ee S x (ppm) S & ” S$ $ x N > ® Q § & F & $ ££ JS & 1000 o I < Nv <> A 100 52 A A A A 52 50 A 55 A 50 53 A 56 A 10 53 57 51 A A 1 | 54 | I | 0.1 ! | I VV 0.01 Key 0.001 : 104 a use B LC, (animals) ' Minimal risk Estimated g guinea pig @® LOAEL for serious effects (animals) level for effects Upper-bound 0.0001 d dog ® LOAEL for less serious effects (animals) \V Other than 1054 Human Cancer © ca. O NOAEL (animals) cnet Risk Levels rabbi C0000) A LOAEL for serious effects (humans) The number next to i. . eacl In A LOAEL for less serious effects (humans) ro to A NOAEL (humans) entries in Table 2-1. 0.000001 * Doses represent the lowest dose tested per study that produced a tumorigenic response 107 and do not imply the existence of a threshold for the cancer end point. S103443 H1IV3H 2 WHO40HO THO 82 CHLOROFORM 29 2. HEALTH EFFECTS did not exceed 2.25%. No other studies were located regarding respiratory effects in humans after inhalation exposure to chloroform. Larson et al. (1996) investigated the ability of acute exposure to chloroform vapors to produce toxicity and regenerative cell proliferation in the liver, kidneys, and nasal passage of female B6C3F, mice. Groups of 5 animals were exposed to 0, 0.3, 2, 10, 30, or 90 ppm chloroform via inhalation for 6 hours a day for 4 consecutive days. Animals were administered bromodeoxyuridine (BrdU) via implanted osmotic pump for the last 3.5 days. At necropsy, livers and kidneys were removed, weighed, examined macroscopically, and prepared for microscopic evaluation. The nasal cavities were also removed and prepared for microscopic evaluation. Cell proliferation was quantitated as the percentage of cells in S-phase (labeling index = LI) measured by immunohistochemical detection of BrdU-labeled nuclei. This study found no overt clinical signs of toxicity in female mice exposed to chloroform for 4 days; however, some mild, transient changes occurred in the posterior ventral areas of nasal tissue in female mice exposed to the 10, 30, and 90 ppm concentrations of chloroform. The lesions were characterized by mild proliferative responses in the periosteum consisting of a thickening of the bone. The adjacent lamina also exhibited loss of acini of Bowman’s glands and vascular congestion. No microscopic changes were noted in nonnasal bones, nor were nonnasal bone Lls significantly different from those of controls. Another similar study by Larson et al. (1994c) using a wider range of inhaled doses investigated the ability of chloroform vapors to produce toxicity and regenerative cell proliferation in the liver and kidneys of female B6C3F, mice and male Fischer 344 rats, respectively. Nasal passages were also examined for toxic response. Groups of 5 animals were exposed to 0, 1, 3, 10, 30, 100, or 300 ppm chloroform via inhalation for 6 hours a day for 7 consecutive days. Actual exposure concentrations measured were 0, 1.2, 3, 10, 29.5, 101, and 288 ppm for mice; and 0, 1.5, 3.1, 10.4, 29.3, 100, and 271 ppm and for rats. Animals were administered BrdU via implanted osmotic pump for the last 3.5 days to quantitate S-phase cell proliferation using an LI method. Necropsies were performed on day 8. No histopathological lesions were observed in the nasal passages of female mice at any exposure concentration. In the nasal passages of rats, chloroform concentrations of 10 ppm and above induced histopathological changes that exhibited a clear concentration-related response. These lesions consisted of respiratory epithelial goblet cell hyperplasia and degeneration of Bowman's glands in olfactory mucosa with an associated osseous hyperplasia of the endo- and ectoturbinates in the periphery of the ethmoid region. CHLOROFORM 30 2. HEALTH EFFECTS Acute exposure to chloroform clearly can induce site-specific as well as biochemical changes in the nasal region of female B6C3F; mice and male Fischer 344 rats (Mery et al. 1994). To demonstrate the biochemical alterations, mice were exposed to 1.2, 3, 10, 29.5, 101, and 288 ppm chloroform and rats were exposed to 1.5, 3.1, 10.4, 29.3, 100, and 271 ppm for 6 hours a day for 7 days to determine the nasal cavity site-specific lesions and the occurrence of cell induction/proliferation associated with these varying concentrations of chloroform. In male rats, the respiratory epithelium of the nasopharyngeal meatus exhibited an increase in the size of goblet cells at 100 and 271 ppm chloroform, in addition to an increase in both neutral and acidic mucopolysaccharides. Affected epithelium was up to twice its normal thickness. New bone formation within the nasal region was prominently seen at 10.4 ppm and above, and followed a concentration response curve. At 29.3 and 100 ppm, new osseous spicules were present at the beginning of the first endoturbinate, while at 271 ppm, the width of the new bone was almost doubled compared to controls. The Bowman's glands were markedly reduced in size. Cytochrome P-450-2E1 staining was most prominent in the cytoplasm of olfactory epithelial sustentacular cells and in the acinar cells of Bowman's glands in control animals. In general, increasing the chloroform concentration tended to decrease the amount of P-450 staining in exposed animals. Exposure to chloroform resulted in a dramatic increase in the number of S-phase nuclei, with the proliferative response confined to activated periosteal cells, including both osteogenic (round) and preosteogenic (spindle) cells. The proximal and central regions of the first endoturbinate had the highest increase of cell proliferation. Interestingly, the only detectable treatment-related histologic change observed in female mice was a slight indication of new bone growth in the proximal part of the first endoturbinate in one mouse exposed to 288 ppm chloroform. The S-phase response was observed at chloroform concentrations of 10.4 ppm and higher. If similar nasal cavity changes occur in humans, the sense of smell could potentially be altered. In some animal species, the lung may be a target organ when inhalation exposure to chloroform is of intermediate duration. Interstitial pneumonitis was observed in male rats and rabbits exposed to 85 ppm and in female rabbits exposed to 25 ppm chloroform for 6 months (Torkelson et al. 1976). The NOAEL was 50 ppm for male rats and rabbits. No respiratory changes were reported in guinea pigs and dogs exposed to 85 and 25 ppm chloroform, respectively. Larson et al. (1996) investigated the ability of intermediate exposure to chloroform vapors to produce toxicity and regenerative cell proliferation in the nasal passage of male and female B6C3F | mice. Groups of 8 animals of each sex were exposed to 0, 0.3, 2, 10, 30, or 90 ppm chloroform via CHLOROFORM 31 2. HEALTH EFFECTS inhalation for 6 hours a day, 7 days a week for 3, 6, or 13 weeks; additional groups of 8 animals of each sex were exposed for 6 hours a day, 5 days a week for 13 weeks. Animals were administered BrdU via implanted osmotic pump and cell proliferation was quantitated as the percentage of cells in S-phase using LI (measuring by immunohistochemical detection of BrdU-labeled nuclei). At necropsy, the nasal cavities were removed and prepared for microscopic evaluation. No alterations in nasal tissues were noted at any exposure level in either sex after exposures of 3, 6, or 13 weeks. Cardiovascular Effects. Epidemiologic studies indicate that chloroform causes cardiac effects in patients under anesthesia. In a cohort of 1,502 patients (exposure less than 22,500 ppm), dose-related bradycardia developed in 8% of the cases, and cardiac arrhythmia developed in 1.3% of the cases (Whitaker and Jones 1965). Hypotension was observed in 27% of the patients and was related to the duration of the anesthesia and to pretreatment with thiopentone. Chloroform anesthesia (exposure 8,000—10,000 ppm) caused arrhythmia (nodal rhythm, first degree atrio-ventricular block, or complete heart block) in 50% of the cases from the cohort of 58 patients and hypotension in 12% (Smith et al. 1973). It should be noted that the effects seen may be secondary to surgical stress or the underlying disease which necessitated the surgical procedure. No studies were located regarding cardiovascular effects in animals after inhalation exposure to chloroform. Gastrointestinal Effects. Nausea and vomiting were frequently observed side effects in humans exposed to chloroform via anesthesia (exposure 8,000-22,500 ppm) (Royston 1924; Smith et al. 1973; Townsend 1939; Whitaker and Jones 1965). Nausea and vomiting were observed in male and female workers exposed solely to 14-400 ppm chloroform for 1-6 months (Phoon et al. 1983). Similarly, gastrointestinal symptoms (nausea, dry mouth, and fullness of the stomach) were reported in female workers occupationally exposed to 22-71 ppm chloroform for 10-24 months and 77-237 ppm chloroform for 3-10 years (Challen et al. 1958). No studies were located regarding gastrointestinal effects in animals after inhalation exposure to chloroform. Hematological Effects. The hematological system does not appear to be a significant target after inhalation exposure to chloroform. Except for increased prothrombin time in some individuals after CHLOROFORM 32 2. HEALTH EFFECTS anesthesia exposure to 8,000 ppm, no hematological effects were observed in humans after inhalation exposure to chloroform (Smith et al. 1973). This effect reflects the hepatotoxicity of chloroform because prothrombin is formed in the liver. No hematological effects were observed in rats, rabbits, and guinea pigs exposed to 85 ppm chloroform or in dogs exposed to 25 ppm chloroform for intermediate durations (Torkelson et al. 1976). Musculoskeletal Effects. Few musculoskeletal effects have been reported in the literature after an acute, intermediate, or chronic exposure to chloroform in humans or in laboratory animals. Larson et al. (1996) investigated the ability of acute- and intermediate-duration exposure to chloroform vapor to produce toxicity and regenerative cell proliferation in various tissues of female B6C3F | mice. Using the methods described in previous sections of this profile, Larson et al. (1996) found that, after acute exposure, no microscopic changes were noted in nonnasal bones, nor were non nasal bone LIs different from those of controls. In the intermediate duration studies, no alterations in nonnasal bone tissues were noted at any exposure level in either sex after exposures of 3, 6, or 13 weeks. Hepatic Effects. Chloroform-induced hepatotoxicity is one of the major toxic effects observed in both humans and animals after inhalation exposure. Increased sulfobromophthalein retention was observed in some patients exposed to chloroform via anesthesia (exposure 8,000—10,000 ppm), indicating impaired liver function (Smith et al. 1973). Serum transaminase, cholesterol, total bilirubin, and alkaline phosphatase levels were not affected. Transient jaundice has also been reported in one study (Whitaker and Jones 1965), while several earlier studies report acute hepatic necrosis in women exposed to chloroform via anesthesia (exact exposure not provided) during childbirth (Lunt 1953; Royston 1924; Townsend 1939). The effects observed in the women included jaundice, liver enlargement and tenderness, delirium, coma, and death. Centrilobular necrosis was found at autopsy in those who died. Workers exposed to 14-400 ppm chloroform for 1-6 months developed toxic hepatitis and other effects including jaundice, nausea, and vomiting, without fever (Phoon et al. 1983). The workers were originally diagnosed with viral hepatitis; however, in light of epidemiologic data, the diagnosis was changed to toxic hepatitis. No clinical evidence of liver injury was observed in workers exposed to as much as 71 and 237 ppm chloroform for intermediate and chronic durations, respectively; however, liver function was not well characterized (Challen et al. 1958). In contrast, toxic hepatitis (with hepatomegaly, enhanced serum glutamic pyruvic transaminase [SGPT] and serum CHLOROFORM 33 2. HEALTH EFFECTS glutamic oxaloacetic transaminase [SGOT] activities, and hypergammaglobulinemia) was observed in workers exposed to 2-205 ppm chloroform (Bomski et al. 1967). Co-exposure to trace amounts of other solvents was also detected, however. Elevated serum prealbumin and transferrin were noted in another study (Li et al. 1993); however, the data is questionable as the exposed individuals most likely received exposures to toxic substances other than just chloroform. An intermediate-duration inhalation MRL of 0.05 ppm was derived from the LOAEL of 14 ppm from the data presented by Phoon et al. (1983); a chronic-duration inhalation MRL of 0.02 ppm was derived from the LOAEL of 2 ppm from the data presented by Bomski et al. (1967). More information on these MRLs and how they were derived is located in the footnote to Table 2-1, Section 2.5 and in Appendix A of this profile. A study by Aiking et al. (1994) examined the possible hepatotoxicity of chloroform exposure in competitive swimmers who trained in indoor chlorinated swimming pools (n=10) compared to those who trained in outdoor chlorinated swimming pools (n=8). The actual amount of chloroform inhaled was not determined; however, the mean concentration of chloroform was determined to be 24 ug/L in the indoor pools and 18.4 ug/L in the outdoor pools. Mean blood chloroform concentration in the indoor pool swimmers was found to be 0.89 ug/L, while the control group and the outdoor pool swimmers had blood chloroform concentrations of less than 0.5 ug/L, suggesting that the chloroform could not be removed by environmental air currents (resulting in higher exposure dose) as it did in an outdoor pool environment. No significant differences in liver enzyme function was seen between any of the groups. Chloroform-induced hepatotoxicity in various animal species has been reported in several studies. No changes in SGPT activity were observed in rats exposed to 300 ppm chloroform during gestation days (Gd) 6-15 (Schwetz et al. 1974). No changes in liver weights were found in pregnant Wistar rats that were exposed to 0, 30, 100, or 300 ppm chloroform via inhalation during Gd 7-16, followed by termination on day 21 (Baeder and Hofmann 1988). In contrast, serum sorbitol dehydrogenase (SDH) activity was increased in rats exposed to 153 ppm and above for 4 hours (Lundberg et al. 1986), and SGPT levels were increased in mice exposed to 100 ppm, 7 hours a day for 8 days during various stages of pregnancy (Murray et al. 1979) and 4,500 ppm for 9 hours (Gehring 1968). These increased enzyme levels in serum indicate hepatocellular necrosis. Fatty changes were observed microscopically in male and female mice after acute exposure to chloroform concentrations >100 ppm (Culliford and Hewitt 1957; Kylin et al. 1963). Elevated liver triglycerides and liver glutathione (GSH) have also been reported (Ikatsu and Nakajima 1992). Liver necrosis was observed in female rats exposed to CHLOROFORM 34 2. HEALTH EFFECTS 4,885 ppm chloroform for 4 hours (Lundberg et al. 1986) and in male mice that died after acute exposure to 692—1,106 ppm chloroform (Deringer et al. 1953). Centrilobular granular degeneration was observed in rats, rabbits, and guinea pigs exposed to 25 ppm chloroform for 6 months, but not in dogs exposed to 25 ppm for the same time period (Torkelson et al. 1976); however, these pathological findings were not observed in the 50 ppm exposure group of rabbits and guinea pigs, or in the 85 ppm exposure group of guinea pigs. Although the liver effects in rabbits and guinea pigs were not dose- related, the small number of surviving animals in the higher exposure group may have biased the results of the study and may not fully describe the pathological effects of chloroform at the higher dose. In two separate studies, Wang et al. (1994, 1995) investigated the effect of ethanol consumption or fasting, respectively, on the metabolism and toxicity of chloroform administered by inhalation of an acute duration. In the first study, male Wistar rats were pretreated with either ethanol, 2,000 mg/kg, or water. Eighteen hours later, the animals were exposed to air containing chloroform at concentrations of 0, 50, 100, or 500 ppm for 6 hours (5 rats a group). At 24 hours postexposure, animals were anesthetized, blood samples were collected for determination of SGOT and SGPT levels; livers were harvested and processed for determination of GSH levels. Chloroform produced dose- dependent hepatotoxicity, and ethanol pretreatment enhanced this effect. In rats exposed by inhalation, hepatotoxicity was only evident at the highest dose (500 ppm); SGOT and SGPT values in treated rats were 47 and 24 international units per liter (IU/L) versus 30 and 16 IU/L in controls, respectively. GSH concentrations in rats exposed to chloroform were lowered in a dose-dependent manner. Significant (p<0.05) reductions were seen at the S00 ppm dose in rats exposed by inhalation. In the second study, rats were divided in two groups: those fasted over night and those allowed free access to food. The following day, 5 rats per group were exposed to chloroform once by inhalation at 0, 50, 100, or 500 ppm for 6 hours. Twenty-four hours after exposure, blood samples were collected for SGOT and SGPT determinations. Chloroform tended to produce hepatotoxicity in a dose-dependent manner, and fasting tended to potentiate the toxicity. Plasma SGOT activity was significantly elevated in the fasting group at 100 and 500 ppm and the fed group at 500 ppm as compared to controls. SGPT levels in the fasting group exposed to 500 ppm chloroform (212 IU/L) significantly exceeded those of the fasting control group (16 IU/L). GSH levels in the fasting group exposed to 500 ppm chloroform (2.22 mg/g) were significantly lower than those of the fasted control group (2.51 mg/g). CHLOROFORM 35 2. HEALTH EFFECTS Larson et al. (1996) investigated the ability of acute chloroform vapor exposure to produce toxicity and regenerative cell proliferation in the liver of female B6C3F mice. Groups of 5 animals were exposed to 0, 0.3, 2, 10, 30, or 90 ppm chloroform via inhalation for 6 hours a day for 4 consecutive days. Animals were administered BrdU via an implanted osmotic pump, and cell proliferation was quantitated as the percentage of cells in S-phase (LI) measured by immunohistochemical detection of BrdU-labeled nuclei. At necropsy, livers were removed, weighed, examined macroscopically, and prepared for microscopic evaluation. Exposure to 90 ppm chloroform resulted in increased relative liver weights. Female mice exposed to chloroform for 4 days experienced a dose-dependent mild response of uniform hepatocyte lipid vacuolization. Scattered individual hepatocyte necrosis also occurred in a dose-dependent manner. Hepatic LI was significantly elevated in female mice in the 90 ppm dose group after 4 days exposure (9-fold; p<0.05). In an earlier study, Larson et al. (1994c) investigated the ability of chloroform vapors to produce toxicity and regenerative cell proliferation in the liver and kidneys of female B6C3F | mice and male Fischer 344 rats, respectively. Groups of 5S animals were exposed to 0, 1, 3, 10, 30, 100, or 300 ppm chloroform via inhalation for 6 hours a day for 7 consecutive days. Actual exposure concentrations measured for mice were 0, 1.2, 3, 10, 29.5, 101, and 288 ppm and for rats were 0, 1.5, 3.1, 10.4, 29.3, 100, and 271 ppm. Necropsies were performed on day 8. Animals were administered BrdU via implanted osmotic pump for the last 3.5 days in order to measure S-phase cell proliferation using an LI method. Female mice exposed to 101 or 298 ppm exhibited centrilobular hepatocyte necrosis and severe diffuse vacuolar degeneration of mid-zonal and periportal hepatocytes, while exposure to 10 or 29.5 ppm resulted in mild-to-moderate vacuolar changes in centrilobular hepatocytes. Specifically, decreased eosinophilia of the centrilobular and mid-zonal hepatocyte cytoplasm relative to periportal hepatocytes was observed at 29.5 ppm. Livers of mice in the 1 and 3 ppm groups did not differ from controls. Slight, dose-related increases in the hepatocyte LIs were observed in the 10 and 30 ppm dose groups, while the LI was increased more than 30-fold in the 101 and 288 ppm groups. Relative liver weights were increased in a dose-dependent manner at exposures of 3 ppm and above. Livers from mice exposed to 101 or 288 ppm were enlarged and pale. In male rats, swelling and mild centrilobular vacuolation was observed only in the livers of rats exposed to 271 ppm. Necrosis was minimal and confined to individual hepatocytes immediately adjacent to the central vein; livers were dark red and congested. The hepatocyte LI in rats were increased only at 101 and 271 ppm, 3- and 7-fold over controls, respectively. An acute-duration inhalation MRL of 0.1 ppm was based on the CHLOROFORM 36 2. HEALTH EFFECTS NOAEL of 3 ppm for hepatic effects in mice. More information on this MRL and how it was derived is located in the footnote to Table 2-1, Section 2.5 in Appendix A this profile. Larson and coworkers (1996) investigated the ability of intermediate-duration chloroform vapor exposure to produce toxicity and regenerative cell proliferation in the liver of male and female B6C3F, mice. Groups of 8 animals of each sex were exposed to 0, 0.3, 2, 10, 30, or 90 ppm chloroform via inhalation for 6 hours a day, 7 days a week for 3, 6, or 13 weeks; additional groups of 8 animals of each sex were exposed for 6 hours a day, 5 days a week for 13 weeks. Animals were administered BrdU via implanted osmotic pump and cell proliferation was quantitated as the percentage of cells in S-phase. At necropsy, livers were removed, examined macroscopically, and then prepared for microscopic evaluation. In mice exposed for 3 weeks, no changes in relative liver weights occurred in males at any dose level, whereas females exposed to 90 ppm chloroform experienced a significant increase (10.7%; p<0.05) in relative liver weights. Liver lesions were noted in males and females at exposures of 30 and 90 ppm. Lesions were characterized by vacuolation and swelling of hepatocytes and variations in nuclear size. Cell proliferation was elevated in the livers of females and males at 30 and 90 ppm exposures, respectively. In mice exposed for 6 weeks, exposure to 90 ppm chloroform resulted in a significant increase (16.1%; p<0.05) in relative liver weights. Liver lesions were noted in females at exposures of 30 and 90 ppm; these lesions were characterized by mild degenerative changes in hepatocytes. An increase in liver LI was also noted in the females exposed to 30 and 90 ppm chloroform. In mice exposed 7 days a week for 13 weeks, relative liver weights increased in males exposed to 30 and 90 ppm chloroform and in females exposed to 90 ppm chloroform (p<0.05). Liver lesions were elevated above background in males and females at exposures of 30 and 90 ppm. Lesions were characterized by vacuolation and swelling of hepatocytes and variations in nuclear size. Cell proliferation was elevated in the livers of females and males at exposed to 90 ppm chloroform. Hepatic alterations in mice exposed 5 days a week for 13 weeks were similar to those of mice exposed 7 days a week; however, the severity of the lesions was diminished with significant effects seen only at the 90 ppm exposure level. Renal Effects. Several studies regarding kidney toxicity effects in humans after inhalation exposure to chloroform were found. No biochemical renal anomalies were reported in one study examining factory workers in China exposed to varying levels of chloroform (Li et al. 1993). One report was obtained from case reports of death among women exposed to chloroform via anesthesia during childbirth (Royston 1924). The fatty degeneration of kidneys observed at autopsy indicated CHLOROFORM 37 2. HEALTH EFFECTS chloroform-induced damage. A study by Aiking et al. (1994) examined the possible renal toxicity of chloroform exposure in competitive swimmers who trained in indoor and outdoor chlorinated swimming pools in the Netherlands. Although no significant differences in liver enzyme function were seen between any of the groups, the study did determine that 3-2-microglobulin was elevated in the indoor pool swimmers (after controlling for possible age bias using multiple regression analysis), suggesting some degree of renal damage due to higher inhaled air concentrations of chloroform present in the air of indoor swimming pools. In animals, the kidney is one of the target organs of inhalation exposure to chloroform. Groups of 20 female Wistar rats were exposed to 0, 30, 100, or 300 ppm chloroform via inhalation during Gd 7-16 and terminated on day 21 showed no changes in kidney weights compared to control animals (Baeder and Hofmann 1988). Larson et al. (1996) investigated the ability of acute chloroform vapor exposure to produce toxicity and regenerative cell proliferation in the kidneys of female B6C3F; mice. Groups of 5 animals were exposed to 0, 0.3, 2, 10, 30, or 90 ppm chloroform via inhalation for 6 hours a day for 4 consecutive days. At necropsy, kidneys were removed, weighed, examined macroscopically, and prepared for microscopic evaluation. Relative kidney weights were similar to controls at all chloroform exposure levels. Kidneys of female mice exposed to chloroform were not different from those of controls at any dose. Exposure to chloroform did not significantly affect the kidney cortex LI in females at any dose. In an earlier study, Larson et al. (1994c) examined the ability of chloroform vapors to produce toxicity and regenerative cell proliferation in the liver and kidneys of female B6C3F, mice and male Fischer 344 rats, respectively. Groups of 5 animals were exposed to 0, 1, 3, 10, 30, 100, or 300 ppm chloroform via inhalation for 6 hours a day for 7 consecutive days. Actual exposure concentrations measured for mice were 0, 1.2, 3, 10, 29.5, 101, and 288 ppm and for rats were 0, 1.5, 3.1, 10.4, 29.3, 100, and 271 ppm. Necropsies were performed on day 8. The kidneys of mice were affected only at the 300 ppm exposure, with approximately half of the proximal tubules lined by regenerating epithelium and an increased LI of tubule cells of 8-fold over controls. In the kidneys of male rats exposed to 300 ppm, about 25-50% of the proximal tubules were lined by regenerating epithelium. The LI for tubule cells in the cortex was increased at 30 ppm and above. CHLOROFORM 38 2. HEALTH EFFECTS Tubular necrosis was observed in male mice after acute exposure to chloroform concentrations >246 ppm (Culliford and Hewitt 1957; Deringer et al. 1953). Tubular calcifications were observed in mice that survived the exposure and were terminated after a 12-month recovery period. In a study of intermediate duration, increased kidney weight (both sexes) and cloudy swelling (males) were observed in rats exposed to chloroform concentrations >25 ppm chloroform (Torkelson et al. 1976). Results were not consistent in rabbits and guinea pigs under the same exposure conditions. Cloudy swelling, and tubular and interstitial nephritis were observed in groups of rats exposed to 25 ppm chloroform, but not in groups exposed to 50 ppm. The results in rabbits and guinea pigs, however, may be biased due to the low survival rate at the higher exposure level. Larson and coworkers (1996) also investigated the ability of intermediate-duration chloroform vapor exposure to produce toxicity and regenerative cell proliferation in the kidneys of male and female B6C3F, mice. Groups of 8 animals of each sex were exposed to 0, 0.3, 2, 10, 30, or 90 ppm chloroform via inhalation for 6 hours a day, 7 days a week for 3, 6, or 13 weeks; additional groups of 8 animals of each sex were exposed for 6 hours a day, 5 days a week for 13 weeks. At necropsy, kidneys were removed, examined macroscopically, and prepared for microscopic evaluation. In mice exposed for 3 weeks, no changes were noted in relative kidney weights in either sex at any exposure level. While kidneys of female mice did not differ from those of controls at any dose, those of males were significantly affected by chloroform exposures of 30 ppm or more. Lesions were mainly in the epithelial cells of the proximal convoluted tubules, with 25 and 50% of cells affected in the 30 and 90 ppm groups, respectively. Cell proliferation was elevated in males at the 30 ppm exposure level, while female kidney LIs were not affected at any exposure level. In mice exposed for 6 weeks, no changes were noted in relative kidney weights at any exposure level. Kidneys from exposed females were not histologically different from controls at any exposure level, and kidney LIs were similar to control values at all exposure levels. In mice exposed 7 days a week for 13 weeks, no changes were noted in relative kidney weights in either sex at any exposure level. While kidneys of female mice did not differ from those of controls at any dose, those of males were significantly affected by chloroform exposures of 10 ppm or more. Lesions were mainly in the epithelial cells of the proximal convoluted tubules, with 25 and 50% of cells affected in the 30 and 90 ppm groups, respectively. Mineralization within the cortex and enlarged nuclei in the epithelial cells were also noted. Cell proliferation was elevated in the cortical tissues of males at the 30 and 90 ppm exposure level; in contrast, female CHLOROFORM 39 2. HEALTH EFFECTS kidney LIs were not affected at any exposure level. In contrast, exposure to chloroform vapors 5 days a week for 13 weeks produced no adverse renal effects. Body Weight Effects. No studies were located regarding body weight effects in humans after inhalation exposure to chloroform. Larson et al. (1994c¢) noted that in female B6C3F, mice and male Fischer 344 rats exposed to 0, 1, 3, 10, 30, 100, or 300 ppm chloroform via inhalation for 6 hours a day for 7 consecutive days that body weight gains were significantly decreased relative to controls in mice exposed to 101 and 288 ppm (1% weight loss at 101 and 288 ppm). Body weight gain was significantly decreased in a concentration-dependent manner in rats exposed to 10 ppm of chloroform and above (2% weight loss at 271 ppm; weight gains of 9-12% at 10.4-101 ppm, as compared to 18% weight gain by controls). A dose-dependent reduction in feed consumption, resulting in decreased body weight gain, was observed in pregnant female rats exposed to 30 ppm chloroform (7 hours a day for 10 days) and above during gestation (Baeder and Hofmann 1988). Newell and Dilley (1978) report that maternal body weights decreased in Sprague-Dawley rats when the chloroform concentration reached 4,117 ppm when exposed for 1 hour a day during Gd 7-14. Similarly, decreased body weight was observed in pregnant mice exposed to 100 ppm chloroform during gestation (Murray et al. 1979). Decreased body weight was reported in male rats exposed to chloroform at 271 ppm for 6 hours a day for 7 days; however, no discernable decrease in body weight was noted at concentrations from 1.5 to 100 ppm. Decreases in body weight were also noted in female mice exposed to 101 ppm chloroform for the same duration (Mery et al. 1994). Decreased body weight also occurred in male rats exposed to 50 ppm for 6 months (Torkelson et al. 1976). Larson and coworkers (1996) also investigated the effect of intermediate exposure to chloroform vapor on body weight in male and female B6C3F | mice. Groups of 8 animals of each sex were exposed to 0, 0.3, 2, 10, 30, or 90 ppm chloroform via inhalation for 6 hours a day, 7 days a week for 3, 6, or 13 weeks; additional groups of 8 animals of each sex were exposed for 6 hours a day, 5 days a week for 13 weeks. In mice exposed 7 days a week for 3 weeks, body weights in females were unaffected, while those of males exposed to 90 ppm chloroform were significantly lower compared to controls (2% weight loss versus 6% weight gain). Exposure to chloroform 7 days a week for 6, or 13 weeks did not affect body weights in males or females; however, when exposed 5 days a week for 13 weeks, CHLOROFORM 40 2. HEALTH EFFECTS body weight gains in males and females exposed to 90 ppm chloroform were slightly reduced compared to their respective controls (93 and 91%, respectively). Other Systemic Effects. No studies were located regarding other systemic effects in humans after inhalation exposure to chloroform. Gearhart et al. (1993) studied the interactions of chloroform exposure with body temperature in mice. Male mice were exposed to chloroform concentrations up to 5,500 ppm chloroform for 6 hours and their core body temperature monitored. The largest decrease in core body temperature was observed in the 5,500 ppm exposure group, followed by the 2,000, 800, and 100 ppm groups. There was no significant decrease in in vitro cytochrome P-450 activity at any temperature tested. The data collected were used to develop a PBPK model, which is discussed in more detail in Section 2.3.5. Decreased feed consumption also been reported at chloroform doses as low as 30 ppm in rats (Baeder and Hofmann 1988). 2.2.1.3 Immunological and Lymphoreticular Effects No studies were located regarding in-depth immunological effects in humans after inhalation exposure to chloroform. Only one study (Bomski et al. 1967) described the health effects of a group of 68 workers occupationally exposed to chloroform for 1-4 years in a pharmaceutical plant. Chloroform air concentrations ranged from 0.01 to 1 mg/L, and other solvents were also reported in the air in trace amounts. Splenomegaly was the only immunologically detected health effect in a small percentage of these cases. Chloroform appears to have little effect on the spleen of laboratory animals. No histological changes were found in the spleen of mice exposed to chloroform concentrations as high as 1,106 ppm for 1-3 hours (Deringer et al. 1953) or in male rats receiving 25 ppm of chloroform for 6 months (Torkelson et al. 1976). Female Wistar rats exposed to 0, 30, 100, or 300 ppm chloroform for 10 days via inhalation during Gd 7-16 also failed to show a change in maternal spleen organ weights (Baeder and Hofmann 1988). Other information on the immunotoxicity of chloroform is limited to one study on effects of chloroform on host resistance in CD-1 mice. A single exposure to 10.6 ppm chloroform for 3 hours CHLOROFORM 41 2. HEALTH EFFECTS did not increase the mortality rate after streptococcal challenge and did not alter the ability of alveolar macrophages to destroy bacteria in these mice (Aranyi et al. 1986). After repeated chloroform exposure (3 hours a day for 5 days), the mortality rate significantly increased, but the bactericidal activity of macrophages was not suppressed compared to control animals. 2.2.1.4 Neurological Effects The central nervous system is a major target for chloroform toxicity in humans and in animals. Chloroform was once widely used as an anesthetic during surgery in humans, but is not currently used as a surgical inhalant anesthetic in modern-day medical practice. Levels of 3,000-30,000 ppm were used to induce anesthesia (Featherstone 1947; Smith et al. 1973; Whitaker and Jones 1965). Concentrations of =40,000 ppm, if continued for several minutes, could result in death (Featherstone 1947). To induce anesthesia, increasing the concentration of chloroform gradually to 25,000 or 30,000 ppm during the first 2 or 3 minutes with maintenance at much lower levels was recommended. Concentrations <1,500 ppm are insufficient to induce anesthesia; concentration of 1,500-2,000 ppm cause light anesthesia (Goodman and Gilman 1980). Dizziness and vertigo were observed in humans after exposure to 920 ppm chloroform for 3 minutes; headache and slight intoxication were observed at higher concentrations (Lehmann and Hasegawa 1910). Exhaustion was reported in 10 women exposed to >22 ppm chloroform during intermediate- and chronic-duration occupational exposures (Challen et al. 1958). Chronic exposure to chloroform concentrations >77 ppm caused exhaustion, lack of concentration, depression, or irritability in 9 of 10 occupationally exposed women. A case report of an individual addicted to chloroform inhalation for =12 years reported psychotic episodes, hallucinations and delusions, and convulsions (Heilbrunn et al. 1945). Withdrawal symptoms, consisting of pronounced ataxia and dysarthria, occurred following an abrupt discontinuation of chloroform use. Moderate, unspecified, degenerative changes were observed in the ganglion cells in the putamen and the cerebellum at autopsy. Death resulted from an unrelated disease. A study of 61 workers exposed for 1-15 years (average 7.8 years) attempted to delineate a possible exposure-effect relationship and to determine the toxicity of chloroform after long-term exposures at a low concentrations in factories in China (Li et al. 1993). Concentrations of chloroform ranged from 0.87 to 28.9 ppm. Dizziness, fatigue, somnolence, insomnia, increased dreaming, hypomnesia, CHLOROFORM 42 2. HEALTH EFFECTS anorexia, and palpitations were significantly elevated in these individuals. Depression, anger, and fatigue were also reported to be significantly elevated. Significant changes were found in neurologic testings of Simple Visual Reaction Time, Digital Symbol Substitution, Digit Span, Benten Retention and Aim Pursuiting in some workers. A limitation of this study was that the exposed group, based on information indicating where the exposed groups originated, indicated that these individuals probably inhaled much more than just chloroform (i.e., other solvents, drugs, pesticides, etc.) and all the effects attributed to chloroform may be attributable to other chemicals in addition to chloroform. Evidence of central nervous system toxicity in animals includes disturbed equilibrium in cats exposed to 7,200 ppm chloroform for 5 minutes, deep narcosis in cats exposed to 21,500 ppm for 13 minutes, deep narcosis in mice exposed to 4,000 ppm for 30 minutes, slight narcosis in mice exposed to 3,100 ppm for 1 hour, and no obvious effects in mice exposed to 2,500 ppm for 2 hours (Lehmann and Flury 1943). Memory retrieval was affected in mice exposed to chloroform via anesthesia (concentration not specified) (Valzelli et al. 1988). The amnesic effect was not long-lasting. The highest NOAEL value and all reliable LOAEL values for neurological effects in each species and duration category are recorded in Table 2-1 and plotted in Figure 2-1. 2.2.1.5 Reproductive Effects No studies were located regarding reproductive effects in humans after inhalation exposure to chloroform. Several studies indicate that inhalation exposure to chloroform may cause reproductive effects in animals. Rats exposed to chloroform during gestation had decreased conception rates after exposure to 300 ppm, but not after exposure to 100 ppm (Schwetz et al. 1974). Studies by Baeder and Hofmann (1988) indicated that exposure to as little as 30 ppm chloroform resulted in increased fetal resorptions. Similarly, a decreased ability to maintain pregnancy, characterized by an increased number of fetal resorptions and decreased conception rates, was observed in mice exposed to 100 ppm chloroform (Murray et al. 1979). In addition to the reproductive effects described above, a significant increase in the percentage of abnormal sperm was observed in mice exposed to 400 ppm chloroform for 5 days (Land et al. 1979). CHLOROFORM 43 2. HEALTH EFFECTS In males, groups of 10-12 male rats (strain not reported) were exposed to 0, 25, 50, or 85 ppm chloroform for 6 months. Adjusted testicular weights in the 50 and 85 ppm groups were greater than those of their respective controls, but were not different from those of other control groups within the same colony. Additionally, no histological changes were noted in testicular tissues of treated animals. The significant increase in testes weights reported in this study was considered to be spurious not likely a direct effect of chloroform exposure (Torkelson et al. 1976). The highest NOAEL value and all reliable LOAEL values for reproductive effects in each species in the acute-duration category are recorded in Table 2-1 and plotted in Figure 2-1. 2.2.1.6 Developmental Effects No studies were located regarding developmental effects in humans after inhalation exposure to chloroform. Chloroform-induced fetotoxicity and teratogenicity were observed in experimental animals. The offspring of rats exposed during gestation had delayed ossification and wavy ribs (30 ppm), acaudate fetuses with imperforate anus and missing ribs (100 ppm), and decreased fetal body weight and crown- rump length, and increased fetal resorptions (300 ppm) (Schwetz et al. 1974). Slight growth retardation of live fetuses at 30 ppm was observed in rats exposed during gestation; no major teratogenic effects were observed (Baeder and Hofmann 1988). The offspring of mice exposed to 100 ppm chloroform during gestation had increased incidences of cleft palate, decreased ossification, and decreased fetal crown-rump length (Murray et al. 1979). The observed malformations occurred in the fetuses that were exposed during organogenesis (days 8-15). Increased resorptions were observed in dams exposed during Gd 1-7. In another study using relatively higher doses, female Sprague-Dawley rats were exposed to 0, 942, 2,232, or 4,117 ppm chloroform 8 days during Gd 7-14, for 1 hour a day. The number of resorptions was enhanced (45% resorptions) and average fetal body weights declined in the highest exposure group only, with no adverse effects noted in the 2,232 ppm and lower doses. The average fetal weight was decreased at the highest dose. No gross teratologic effects or anomalies in ossification were observed in the offspring of exposed dams (Newell and Dilley 1978). CHLOROFORM 44 2. HEALTH EFFECTS All reliable LOAEL values for developmental effects in each species in the acute-duration category are recorded in Table 2-1 and plotted in Figure 2-1. 2.2.1.7 Genotoxic Effects No studies were located regarding genotoxic effects in humans after inhalation exposure to chloroform. Inhalation exposure to 400 ppm chloroform for 5 days increased the percentage of abnormal sperm in mice (Land et al. 1979). Other genotoxicity studies are discussed in Section 2.5. 2.2.1.8 Cancer No studies were located regarding cancer in humans or animals after inhalation exposure to chloroform. Studies in animals indicate that oral exposure to chloroform causes cancer (see Section 2.2.2.8). Because chloroform is carcinogenic in animals exposed orally and because chloroform has identical toxicological end points following oral or inhalation exposure, EPA (1985a) derived a q* for inhalation exposure to chloroform based on mouse liver tumor data from a chronic gavage study (NCI 1976). EPA considered the NCI (1976) study to be appropriate to use in the inhalation risk estimate because there are no inhalation cancer bioassays and no pharmacokinetic data to contraindicate the use of gavage data (IRIS 1995). The geometric mean of the estimates for male and female mice in the NCI (1976) study, 8x1072 (mg/kg/day)! was recommended as the inhalation q;* for chloroform. EPA (1985a) combined the estimates for both data sets because the data for males includes observations at a lower dose, which appear to be consistent with the female data. Expressed in terms of air concentration, the q,* is equal to 2.3x107 (ng/m>)! or 1.1x10 parts per billion (ppb)! The air concentrations associated with individual, lifetime upper-bound risks of 10% to 107 are 4.3x1073 to 4.3x10°° mg/m’ (8.8x107* to 8.8x107 ppm), assuming that a 70-kg human breathes 20 m’ air/day. The 10% to 1077 levels are indicated in Figure 2-1. CHLOROFORM 45 2. HEALTH EFFECTS 2.2.2 Oral Exposure 2.2.2.1 Death Information regarding mortality in humans after oral exposure to chloroform is limited. In one report, a man died of severe hepatic injury 9 days after reportedly drinking =6 ounces of chloroform (3,755 mg/kg) (Piersol et al. 1933). He was admitted to a hospital in a deep coma within 15 minutes of ingestion. This man was also noted to be a long-time user of chloroform in his occupation and a heavy drinker, suggesting that damage inflicted by previous use of chloroform and alcohol over a long period of time may have been contributing factors in his death. In contrast, a patient who ingested 4 ounces (= 2,410 mg/kg) recovered from toxic hepatitis (Schroeder 1965). The recovery may have been due to better therapeutic handling of the case. Fatal doses have been reported to be as low as 10 mL (14.8 grams) or 212 mg/kg (Schroeder 1965). Oral LD, (lethal dose, 50% kill) values in animals vary greatly. No deaths occurred in rats exposed to once 0.1-0.5 mL/kg chloroform by gavage in oil (Nakajima et al. 1995). Acute LD values of 2,000 mg/kg chloroform (Torkelson et al. 1976) and 2,180 mg/kg chloroform (Smyth et al. 1962) were reported for rats. LDs values in male rats varied with age: 446 mg/kg for 14-day-olds, 1,337 mg/kg for young adults, and 1,188 mg/kg for old adults (Kimura et al. 1971). LDs, values were different for male rats (908 mg/kg/day) and female rats (1,117 mg/kg/day) (Chu et al. 1982b). Similarly, the LDs for male mice was lower (1,120 mg/kg) than for female mice (1,400 mg/kg) (Bowman et al. 1978). In general, young adult males had lower survival rates. In another study, an acute oral LDs, value of 1,100 mg/kg/day was reported for male and female mice (Jones et al. 1958). Decreased survival rates were also observed in male mice exposed to 250 mg/kg/day chloroform for 14 days, but not in mice exposed to 100 mg/kg/day. Female mice, however, survived 500 mg/kg/day chloroform treatment (Gulati et al. 1988). Increased mortality was noted in 5 of 12 male mice exposed to 277 mg/kg/day in corn oil by gavage for 4 days (Larson et al. 1994d). Pregnant animals may be more susceptible to chloroform lethality. Increased mortality was observed in pregnant rats exposed to 516 mg/kg/day. Rabbits exposed to 63, 100, 159, 251, and 398 mg/kg/day chloroform during Gd 6-18 had increasing rates of mortality as the dose of chloroform increased (Thompson et al. 1974). There was a high rate of mortality in rats exposed to 142 mg/kg/day chloroform in drinking water for 90 days and during a 90-day observation period. Histopathological examination revealed atrophy of CHLOROFORM 46 2. HEALTH EFFECTS the liver and extensive squamous debris in the esophagus and gastric cardia. These changes suggested to the authors that the rats had died of starvation. Mortality was not increased in the 44 mg/kg/day group (Chu et al. 1982a). The vehicle and mode of administration may influence the lethality of chloroform in mice. In 90-day studies in mice, no effect on mortality was observed in groups treated by gavage with doses up to 250 mg/kg/day chloroform in oil (Munson et al. 1982) or with 435 mg/kg/day in drinking water (Jorgenson and Rushbrook 1980). The maximum tolerated dose of chloroform in drinking water was calculated as 306 mg/kg/day for mice (Klaunig et al. 1986). Survival was affected in mice exposed by gavage to 400 mg/kg/day chloroform in oil for 60 days, but not in those exposed to 100 mg/kg (Balster and Borzelleca 1982). Exposure to 150 mg/kg/day chloroform in toothpaste by gavage for 6 weeks caused death in 8 of 10 male mice (Roe et al. 1979). No death occurred in mice exposed to 149 mg/kg/day chloroform in oil for 30 days; there was an increased incidence of death in males exposed to 297 mg/kg/day (Eschenbrenner and Miller 1945a). No deaths occurred in dogs exposed to 120 mg/kg/day chloroform in toothpaste capsules for 12-18 weeks (Heywood et al. 1979). Decreased survival was observed in rats exposed by gavage to concentrations 290 mg/kg/day chloroform in oil for 78 weeks and in female mice exposed to 477 mg/kg/day, but not in male mice exposed to 277 mg/kg/day time-weighted average (TWA) during the same time period (NCI 1976). In addition, no increase in compound-related mortality was observed in mice exposed by gavage to 60 mg/kg/day chloroform in toothpaste (Roe et al. 1979) in rats or mice exposed to 2160 mg/kg/day chloroform in drinking water for chronic durations (Jorgenson et al. 1985; Klaunig et al. 1986). Similarly, mortality was not affected in dogs exposed to 30 mg/kg/day chloroform in toothpaste capsules for 7.5 years (Heywood et al. 1979). The LD4y, and all reliable LOAEL values for death in each species and duration category are recorded in Table 2-2 and plotted in Figure 2-2. 2.2.2.2 Systemic Effects No credible studies were located regarding ocular effects in humans or animals after oral exposure to chloroform. The other systemic effects of oral exposure to chloroform are discussed below. The highest NOAEL values and all reliable LOAEL values for each effect in each species and duration category are recorded in Table 2-2 and plotted in Figure 2-2. Key to" Species/ figure (strain) Exposure/ duration/ frequency (Specific route) System ACUTE EXPOSURE Death 1 Rat (Sprague- Dawley) 2 Rat (Sprague- Dawley) 3 Rat (Wistar) 4 Rat (Sprague- Dawley) 5 Rat (NS) 6 Mouse (ICR Swiss) 7 Mouse (CD-1) 8 Mouse (Swiss) 9 Mouse (B6C3F1) once (GO) once ©) once ©) 10d Gd 6-15 1-2 x/d (GO) once G) once (GO) 14d 1 x/d (GO) once (GO) 4d 1 x/d (GO) Table 2-2. Levels of Significant Exposure to Chloroform - Oral Less Serious Serious (mg/kg/day) 908 M (LDso) 1117 F (LDso) 1337 M (LDso for young adults) 1188 M (LDsofor old adults) 446 M (LDso for 14-day olds) 2180 F (LDso) 516 F (4/6 died) 2000 M (LDso) 1120 M (LDso) 1400 F (LDso) 250 M (5/8 died) 1100 (LDso) 277 M (Unscheduled deaths; 5/12 mice) Reference Chu et al. 1982b Kimura et al. 1971 Smyth et al. 1962 Thompson et al. 1974 Torkelson et al. 1976 Bowman et al. 1978 Gulati et al. 1988 Jones et al. 1958 Larson et al. 1994d S103443 HI1TV3H 2 WHO40HOTHO Ly Table 2-2. Levels of Significant Exposure to Chloroform - Oral (continued) Exposure/ LOAEL a duration/ Key to" Species/ frequency NOAEL Less Serious Serious figure (Strain) (specific route) SYSe™ (mgikg/day) (mg/kg/day) (mg/kg/day) Peferencs 10 Rabbit 13d 100 F (3/5 died) Thompson et al. (Dutch Belted) Gd 6-18 1974 2 x/d (GO) Systemic 11 Human once Resp 2410 M (respiratory tract Schroeder 1965 (IN) obstruction) Cardio 2410 M (arrhythmia) Gastro 2410 M (vomiting) Musc/skel 2410 M (muscle relaxation) Hepatic 2410 M (jaundice and toxic hepatitis) Renal 2410 M (oliguria) 12 Rat once Hemato 546 (reduced hemoglobin and Chu et al. 1982b (Sprague- (GO) hematocrit by 10-12%) Dawley) Renal 546 F (increased kidney weight) 13 Rat once Hepatic 34 M (elevated SDH, ALT and Larson et al. 1993 (Fischer- 344) (GO) AST; scattered necrotic foci) Renal 34 M (renal proximal tubule necrosis) Bd Wt 477M 14 Rat 4d Hepatic 10M 34 M (slight to mild Larson et al. 1995a (Fischer- 344) 1 x/d centrilobular sinusoidal (GO) leukostasis) Renal 10M 34 M (degeneration of renal proximal tubules) Bd Wt 90M 180 M (decreased body weight gain) S103443 H1TV3H 2 WHO40HO THO Table 2-2. Levels of Significant Exposure to Chloroform - Oral (continued) Exposure/ LOAEL a . duration/ Key to" Species/ frequency NOAEL Less Serious Serious figure (Sain) (specific route) SYStem (mgikgiday) (mg/kg/day) (mg/kg/day) Reference 15 Rat 4d Hepatic 332M 68.1 M (mild hepatocyte Larson et al. 1995a (Fischer- 344) 1 x/d vacuolation) WwW) Renal 575M Bd Wt 57.5 M (decreased body weight gain) 16 Rat 4d Resp 34 F (new bone formation; Larson et al. 1995b (Fischer- 344) (GO) periosteal hypercellularity; degeneration of the olfactory epithelium and superficial Bowman's glands) Hepatic 34 F 400 F (mild centrolobular vacuolization; sinusoidal leucostasis; mild to focally severe centrilobular hepatocyte degeneration and necrosis; diffuse centrolobular swelling) Renal 100 F 200 F (distal nephrons with hyaline casts; proximal tubules lined with degenerated, necrotic or regenerating epithelium) Bd Wt 34 F 400 F (weight loss approximately 14%) 17 Rat 10d Hemato 100 F (decreased hemoglobin Ruddick et al. 1983 (Sprague- Gd 6-15 and hematocrit) Dawley) 1 x/d (GO) Hepatic 100 F (increased liver weight) Renal 200 F 400 F (increased kidney weight) Bd Wt 100 F (32% decreased body weight gain) S103443 H1TV3H 2 WHO40"HOTHO 6v Table 2-2. Levels of Significant Exposure to Chloroform - Oral (continued) Exposure/ LOAEL " . duration/ Key to" Species/ frequency NOAEL Less Serious Serious figure (Strain) (specific route) SYS!EM (mglkgiday) (mg/kg/day) (mg/kg/day) Reference 18 Rat 10d Gastro 516 F (gastric erosions) Thompson et al. (Sprague- Gd 6-15 1974 Dawley) 1-2 x/d (GO) Hepatic 516 F (acute toxic hepatitis) Renal 516 F (acute toxic nephrosis) Bd Wt 79 F 126 F (decreased body weight gain) 19 Rat 10d Dermal 50 F 126 F (alopecia) Thompson et al. (Sprague- Gd 6-15 1974 Dawley) 2 x/d (GO) Bd Wit 20 F 50 F (decreased maternal body weight gain) 20 Rat once Hepatic 100 M (increased plasma GOT Wang et al. 1994 (Wistar) (GO) and GPT) 21 Rat once Hepatic 100 (increased plasma GOT Wang et al. 1995 (Wistar) (GO) & GPT activity ) 22 Mouse 14d Dermal 50 100 (rough hair coat) Gulati et al. 1988 (CD-1) 1x/d (GO) Bd Wt 100M 250 M (12% weight loss) 500 M (32% weight loss) 23 Mouse once Hepatic 35 (midzonal fatty changes) 350 (centrilobular necrosis) Jones et al. 1958 (Swiss- (GO) Webster) 24 Mouse once Hepatic 34 F 238 F (small randomly Larson et al. 1993 (B6C3F1) (GO) scattered foci of hepatocyte necrosis) 25 Mouse 4d Hepatic 90 F 238 F (centrilobular vacuolar Larson et al. 1994b (B6C3F1) 1 x/d degeneration; increased (GO) hepatic cell proliferation) S103443 H1V3H ¢C WHO40HOTHO Table 2-2. Levels of Significant Exposure to Chloroform - Oral (continued) Exposure/ LOAEL 2 . duration/ Key to" Species! frequency NOAEL Less Serious Serious figure (Strain) (specific route) SYSteM (mg/kg/day) (mg/kg/day) (mg/kg/day) Reference 26 Mouse 4d Hepatic 260 F 53 F (pale pink tinctorial Larson et al. 1994b (B6C3F1) WwW) changes in centrilobular hepatocytes) Bd Wt 81 F (approximately 20% decreased body weight) 27 Mouse 4d Hepatic 34 M (pale livers; mild 138 M (centrilobular hepatocyte Larson et al. 1994d (B6C3F1) 1 x/d centrilobular hepatocyte degeneration; scattered (GO) swelling, pale eosinophilc necrosis) staining; periportal hepatocyte vacuolation) Renal 34 M (extensive acute necrosis, proximal convoluted tubule) Bd Wt 277M 28 Mouse once Hepatic 59.2M 199 M (increased SGPT) Moore et al. 1982 (CFLP- (G) Swiss) Renal 592M 199 M (tubular necrosis; increased thymidine uptake) 29 Mouse once Hepatic 656M 273 M (increased thymidine Moore et al. 1982 (CFLP Swiss) (GO) uptake, increased SGOT) Renal 173M 65.6 M (tubular necrosis) 30 Mouse 14d Hemato 250 Munson et al. 1982 (CD-1) 1 x/d (GO) Hepatic 125 250 (increased SGPT and SGOT levels) Bd Wt 125M 250 M (16% decreased body weight) S103443 H1TV3H 2 WHO40HO THO IS Table 2-2. Levels of Significant Exposure to Chloroform - Oral (continued) Exposure/ LOAEL a ) duration/ Key to Species! frequency NOAEL Less Serious Serious figure (strain) (specific route) SYSeM (mgikg/day) (mg/kg/day) (mglkg/day) Reference 31 Mouse 5o0r12d Hepatic 263 F (necrotic, hydrotropic, Pereira 1994 (B6C3F1) 5 diwk swc'len, and rounded 1 x/d hepa*-cytes, macrophage (GO) and neutrophil infiltration) Bd Wt 263 F 32 Mouse 5d Hepatic 67.1 F (29% decreased relative Pereira 1994 (B6C3F1) 24 hr/d liver weight; smaller WwW) hepatocytes with dense nonvacuolated and basophilic cytoplasm) Bd Wt 67.1 F Other 67.1 F (decreased water consumption) 33 Mouse 12d Hepatic 625.4 F (vacuolated hepatocytes) Pereira 1994 (B6C3F1) 24 hr/d Ww) Bd Wt 625.4 F 34 Rabbit 13d Gastro 20 F (diarrhea) Thompson et al. (Dutch Belted) Gd 6-18 1974 1 x/d Bd Wt 35F 50 F (decreased maternal (GO) body weight gain) Immunological/Lymphoreticular 35 Rat once 765 F 1071 F (reduced lymphocytes) Chu et al. 1982b (Sprague- (GO) Dawley) 36 Mouse 14d 50 (suppressed humoral Munson et al. 1982 (CD-1) 1 x/d immunity) (GO) Neurological 37 Human once 2410 M (deep coma) Schroeder 1965 (IN) S103443 H1TV3H °C WHO40HOTHO 2s Table 2-2. Levels of Significant Exposure to Chloroform - Oral (continued) Exposure/ . LOAEL a ) duration/ Rey to Species frequency NOAEL Less Serious Serious figure (Strain) (specific route) SYSteM (mg/kg/day) (mg/kg/day) (mg/kg/day) Reference 38 Mouse 14d 311M Balster and (ICR) 1 x/d Borzelleca 1982 (GO) 39 Mouse once 484 M (calculated EDso for motor Balster and (ICR) (GO) performance) Borzelleca 1982 40 Mouse once 500 (ataxia, incoordination, and Bowman et al. (ICR Swiss) (GO) anesthesia; brain 1978 hemorrhage) 41 Mouse 14d 100M 250 M (hunched posture, Gulati et al. 1988 (CD-1) 1 x/d inactivity) (GO) 42 Mouse once 350 (calculated EDso for Jones et al. 1958 (Swiss) (GO) narcosis) 43 Mouse 10d 10M 30 M (taste aversion) Landauer et al. (CD-1) 1 x/d 1982 (GO) Reproductive 44 Rat 10d 300 F 316 F (increased resorptions) Thompson et al. (Sprague- Gd 6-15 1974 Dawley) 1-2 x/d (GO) 45 Rabbit 13d 25F 63 F (abortion; no viable Thompson et al. (Dutch Belted) Gd 6-18 concepti) 1974 2 x/d (GO) Developmental 46 Rat 10d 200 400 (19% decreased fetal Ruddick et al. 1983 (Sprague- Gd 6-15 weight) Dawley) 1 x/d (GO) S103443 HI1TV3H 2 WHO40H0THO Table 2-2. Levels of Significant Exposure to Chloroform - Oral (continued) Exposure/ LOAEL a . duration/ Key to" Species! frequency NOAEL Less Serious Serious figure (Strain) (specific route) SYSeM (mgikg/day) (mg/kg/day) (mg/kg/day) Reference 47 Rat 10d 300 316 (decreased fetal weight) Thompson et al. (Sprague- Gd 6-15 1974 Dawley) 1-2 x/d (GO) 48 Rat 10d 50 126 (decreased fetal weight) Thompson et al. (Sprague- Gd 6-15 1974 Dawley) 2 x/d (GO) 49 Rabbit 13d 100 Thompson et al. (Dutch Belted) Gd 6-18 1974 2 x/d (GO) S103443 H1TV3H 2 WHO40HO THO Table 2-2. Levels of Significant Exposure to Chloroform - Oral (continued) Exposure/ LOAEL a . duration/ Key to" Species! frequency NOAEL Less Serious Serious | figure (Strain) (specific route) SYSteM (mg/kg/day) (mg/kg/day) (mg/kg/day) Reference INTERMEDIATE EXPOSURE Death 50 Rat 90d 142.2 (high mortality during Chu et al. 1982a (Sprague- WwW) exposure and during Dawley) recovery period) 51 Mouse 6 wk 150 M (8/10 died) Roe et al. 1979 (Schofield) 6 d/wk (G) Systemic 52 Rat 90d Hemato 149.8 Chu et al. 1982a (Sprague- (W) Dawley) Bd Wt 449 142.2 (25% decreased body weight gain) 53 Rat 28d Hemato 228M 193 M (decreased neutrophils) Chu et al. 1982b (Sprague- (W) Dawley) Hepatic 193M Renal 193M 54 Rat 90d Resp 160M Jorgenson and (Osborne- W) Rushbrook 1980 Mendel) Gastro 160M Hemato 160M Hepatic 160M Renal 160M Bd Wt 81M 160 M (11-17% decreased body weight) S103443 H1TV3H 2 WHOJOHOTHO Table 2-2. Levels of Significant Exposure to Chloroform - Oral (continued) Exposure/ LOAEL a ) duration/ Key to" Species! frequency NOAEL Less Serious Serious figure (Strain) (specific route) SYSteM (mg/kg/day) (mg/kg/day) (mg/kg/day) Reference 55 Rat 3 wk Hepatic 90M 180 M (degeneration of Larson et al. 1995a (Fischer- 344) 5 d/wk centrilobular 1 x/d hepatocytes) (GO) Renal 90M 180 M (progressive degeneration of the proximal tubules) Bd Wt 180M 56 Rat 3 wk Hepatic 62.3 M 106 M (mild hepatocyte Larson et al. 1995a (Fischer- 344) 7 d/wk vacuolation) 1 x/d Renal 6.0M 17.4 M (increased numbers of Ww) focal areas of regenerating renal proximal tubular epithelium and cell proliferation) Bd Wt 62.3 M 106 M (25% decrease in weight gain - taken from graph) 57 Rat 3 wk Resp 34 F (new bone formation; Larson et al. 1995b (Fischer- 344) 5 d/wk periosteal (GO) hypercellularity) Hepatic 100 F 400 F (diffuse vacuolar change; focal centrilobular degeneration) Renal 34 F 100 F (proximal tubule epithelial regeneration, dilation and mineralized concretions) Bd Wt 100 F (significant decrease in weight gain) S103443 H1TV3H 2 WHOJOHOTHO 9S Table 2-2. Levels of Significant Exposure to Chloroform - Oral (continued) Exposure/ LOAEL » . duration/ Key to" Species/ froquency NOAEL Less Serious Serious figure (Strain) (specific route) SYSteM (mg/kg/day) (mg/kg/day) (mg/kg/day) Reference 58 Rat 13 wk Hemato 150 410 (increased cellular Palmer et al. 1979 (Sprague- 7 diwk proliferation in bone Dawley) 1 x/d marrow) (G) Hepatic 30 150 (increased relative liver 410 (fatty changes, necrosis) weight) Renal 30 150 (increased relative kidney weight) 59 Mouse 90d Hepatic 60 (fatty changes) 270 (cirrhosis) Bull et al. 1986 (B6C3F1) 1 x/d (GO) Bd Wt 130M 270 M (15% decreased body weight) 60 Mouse 30d Hepatic 297 594 (cirrhosis) Eschenbrenner and (Strain A) 1 x/d Miller 1945a (GO) 61 Mouse 105d Resp 41 Gulati et al. 1988 (CD-1) 1 x/d (GO) Hepatic 16 F 41 F (increased liver weight and hepatocellular degeneration) Renal 41 62 Mouse 90d Resp 435 F Jorgenson and (B6C3F1) WwW) Rushbrook 1980 Gastro 435 F Hemato 435 F Hepatic 32F 64 F (fatty changes) Renal 435 F S103443 H1TV3H 2 WHOJOHOTHO LS Table 2-2. Levels of Significant Exposure to Chloroform - Oral (continued) Exposure/ LOAEL a duration/ Key to" Species/ frequency NOAEL Less Serious Serious figure (Strain) gpecific route) SYSteM (mg/kg/day) (mg/kg/day) (mg/kg/day) Reference 63 Mouse 52 wk Resp 257M Klaunig et al. 1986 (B6C3F1) 7 diwk (W) Hepatic 86 M (focal necrosis) Renal 86 M (tubular necrosis) Bd Wt 86 M (15% decreased body weight gain) 64 Mouse 3 wk Hepatic 34 F (vacuolation of the Larson et al. 1994b (B6C3F1) 5 diwk centrilobular and 1 x/d midzonal hepatocytes; (GO) increased ALT and SDH) Renal 477 F 65 Mouse 3 wk Hepatic 82 F (increased liver weight) Larson et al. 1994b (B6C3F1) 7 diwk (W) Renal 329 F 66 Mouse 3 wk Hepatic 34M 90 M (centrilobular hepatocyte 138 M (centrilobular and periportal Larson et al. 1994d (B6C3F1) 5 diwk swelling; loss of hepatocyte degeneration 1 x/d eosinophilia) and necrosis) (GO) Renal 34 M (regenerating proximal 277 M (degeneration & necrosis of convoluted tubules) the proximal tubules) Bd Wt 138M 277 M (15-20% decrease in body weight) 67 Mouse 90 d Hepatic 50 (hydropic degeneration) Munson et al. 1982 (CD-1) 1 x/d (GO) Renal 50 (chronic inflammation of lymphocytes) Bd Wt 250 S103443 H1TV3H 2 WHO40HOTHO Table 2-2. Levels of Significant Exposure to Chloroform - Oral (continued) Exposure/ LOAEL a ) duration/ Key to Species) frequency NOAEL Less Serious Serious figure (SIrain) (specific route) SYSteM (mg/kg/day) (mg/kg/day) (mg/kg/day) Reference 68 Mouse 33 or Hepatic 263 F (30.1-38.2% increase in Pereira 1994 (B6C3F1) 159d relative liver weight; 5 d/wk focal areas of necrotic, 1 x/d swollen, rounded, and (GO) pale hepatocytes and a low number of mononuclear cells) Bd Wt 263 F 69 Mouse 33d Hepatic 4385 F Pereira 1994 (B6C3F1) 24 hr/d WwW) Bd Wt 4385 F 70 Mouse 159d Hepatic 363.5 F (31.4% increase in Pereira 1994 (B6C3F1) 24 hr/d relative liver weight) (W) Bd Wt 386 F 71 Dog 6 wk Hepatic 15¢ 30 (significantly increased Heywood et al. (Beagle) 6 d/wk SGPT activity) 1979 1 x/d ©) Immunological/Lymphoreticular 72 Mouse 90d 50 (depressed humoral Munson et al. 1982 (CD-1) 1 x/d immunity) (GO) Neurological 73 Mouse 90d 311M Balster and (ICR) 1x/d Borzelleca 1982 (GO) 74 Mouse 60d 100 M (operant behavior Balster and (ICR) 1x/d affected) Borzelleca 1982 (GO) S103443 H1TV3H 2 WHO30HOTHO 6S Table 2-2. Levels of Significant Exposure to Chloroform - Oral (continued) Exposure/ LOAEL 3 . duration/ Key to” Species! frequency NOAEL Less Serious Serious figure (strain) (Specific route) System (mg/kg/day) (mg/kg/day) (mg/kg/day) Reference Reproductive 75 Rat 90 d 160M Jorgenson and (Osborne- (WwW) Rushbrook 1980 Mendel) 76 Rat 13 wk 150 410 (gonadal atrophy) Palmer et al. 1979 (Sprague- 7 d/wk Dawley) 1 x/d (G) 77 Mouse 105d 41 Gulati et al. 1988 (CD-1) 1 x/d (GO) Developmental 78 Mouse 6-10 wk 311 Burkhalter and (ICR) 1 x/d Balster 1979 (GO) 79 Mouse 105d 41 M (increased epididymal Gulati et al. 1988 (CD-1) 1 x/d weights, degeneration of (GO) epididymal epithelium in Fi) 80 Mouse 105d 41 F (increased liver weight Gulati et al. 1988 (CD-1) 1 x/d and hepatocellular (GO) degeneration in F1 females) Cancer 81 Mouse 30d 594 (CEL: hepatomas) Eschenbrenner and (Strain A) 1 x/d Miller 1945a (GO) S103443 H1IV3H 2 WHOJOHOTHO Table 2-2. Levels of Significant Exposure to Chloroform - Oral (continued) Exposure/ LOAEL a duration/ Key to" Species! foquency NOAEL Less Serious Serious figure (Strain) (gi ecific route) SYSteM (mg/kg/day) (mg/kg/day) (mg/kg/day) Reference CHRONIC EXPOSURE Death 82 Rat 78 wk 90 M (decreased survival) NCI 1976 (Osborne- 5 diwk Mendel) 1 x/d (GO) 100 F 83 Mouse 78 wk 477 F (decreased survival) NCI 1976 (B6C3F1) 5 d/wk 1 x/d (GO) Systemic 84 Human 1-5yr Hepatic 0.96 De Salva et al. 1975 Renal 0.96 85 Human 10 yr Hemato 21 M (decreased erythrocytes) Wallace 1950 1 x/d Hepatic 21 M (increased (IN) sulfobromophthalein sodium retention) Renal 21 M (albuminuria) 86 Rat 104 wk Renal 160M Jorgenson et al. (Osborne- 7 d/wk 1985 Mendel) (W) Bd Wt 38M 81 M (decreased body weight) S103443 H1TV3H 2 WHO4JOHOTHO 19 Table 2-2. Levels of Significant Exposure to Chloroform - Oral (continued) Exposure/ LOAEL » duration/ Key to" Species/ frequency NOAEL Less Serious Serious figure (Strain) (gpecific route) SYSM (mg/kg/day) (mg/kg/day) (mg/kg/day) Reference 87 Rat 78 wk Resp 200 F NCI 1976 (Osborne- 5 d/iwk Mendel) 1 x/d (GO) Cardio 200 F Gastro 200 F Hemato 200 F Musc/skel 200 F Hepatic 100 F 200 F (necrosis of hepatic parenchyma) 180M Renal 200 F Bd Wt 90 M (15% decreased weight gain) 88 Rat 180 wk Hepatic 200 (adenofibrosis) Tumasonis et al. (Wistar) 7 d/wk 1985, 1987 WwW) Bd Wt 200 M (50% decreased body weight gain) 89 Mouse 104 wk Bd Wt 130 F 263 F (decreased body weight) Jorgenson et al. (B6C3F1) 7 diwk 1985 Ww) 90 Mouse 78 wk Resp 238 F (pulmonary inflammation) NCI 1976 (B6C3F1) 5 d/wk 1 x/d Cardio 238 F (cardiac thrombosis) (GO) Gastro 477F Hemato 477 F Musc/skel 477 F Hepatic 138 M (nodular hyperplasia of the liver) 238 F Renal 477 F Bd Wt 477 F S103443 HITV3H 2 WHOJ0HOTHO 29 Table 2-2. Levels of Significant Exposure to Chloroform - Oral (continued) Exposure/ LOAEL a . duration/ Key to” Species/ frequency NOAEL Less Serious Serious figure (Strain) (specific route) SYSteM (mg/kg/day) (mg/kg/day) (mg/kg/day) Reference 91 Mouse 80 wk Resp 60 Roe et al. 1979 (ICI) 6 d/wk (G) Hepatic 17 (fatty degeneration) Renal 60 Bd Wt 60 92 Dog 75yr Cardio 30 Heywood et al. (Beagle) 6 d/wk 1979 (C) Hemato 30 Hepatic 159 (increased SGPT activity) Renal 15 30 (fatty changes) Bd Wt 30 Neurological 93 Rat 78 wk 200 F NCI 1976 (Osborne- 5 d/wk Mendel) 1 x/d (GO) 94 Mouse 78 wk 477 F NCI 1976 (B6C3F1) 5 d/wk 1 x/d (GO) 95 Mouse 80 wk 60 Roe et al. 1979 (IC) 6 d/iwk (G) Reproductive 96 Rat 78 wk 200 F NCI 1976 (Osborne- 5 d/iwk Mendel) 1 x/d (GO) 180M S103443 H1TV3H 2 WHOJOHOTHO Table 2-2. Levels of Significant Exposure to Chloroform - Oral (continued) Exposure/ LOAEL a . duration/ Key to Species frequency NOAEL Less Serious Serious figure (strain) qo ific route) System (mg/kg/day) (mg/kg/day) (mg/kg/day) Reference 97 Mouse 78 wk 477 F NCI 1976 (B6C3F1) 5 diwk 1 x/d 277M (GO) 98 Dog 75yr 30 Heywood et al. (Beagle) 6 d/wk 1979 (€) Cancer 99 Rat 104 wk 160 M (CEL: tubular cell adenoma) Jorgenson et al. (Osborne- 7 diwk 1985 Mendel) (W) 100 Rat 78 wk 90 M (CEL: tubular cell adenoma NCI 1976; (Osborne- 5 d/iwk and carcinoma; tubular cell Dunnick and Mendel) 1 x/d neoplasms in 4/50) Melnick 1993 (GO) 200 F (CEL: kidney tubular cell neoplasms in 2/48) 101 Rat 180 wk 200 F (CEL: hepatic neoplastic Tumasonis et al. (Wistar) 7 diwk nodules) 1985, 1987 Ww) 102 Mouse 78 wk 138 M (CEL: hepatocellular NCI 1976, (B6C3F1) 5 d/wk adenomas or carcinomas ~~ Dunnick and 1 x/d in 18/50 mice) Melnick 1993 (GO) 238 F (CEL: hepatocellular adenomas or carcinomas in 36/45 mice) S103443 H1TV3H 2 WHOJ0HOTHO Table 2-2. Levels of Significant Exposure to Chloroform - Oral (continued) Exposure/ LOAEL a . duration/ Key to Specissf frequency NOAEL Less Serious Serious figure (Strain) (specific route) SYSteM (mgikg/day) (mg/kg/day) (mglkg/day) Reference 103 Mouse 80 wk 60 M (CEL: epithelial tumors of ~~ Roe et al. 1979 (ICl) 6 d/wk the kidney) (G) 3The number corresponds to entries in Figure 2-2 PUsed to derive an acute oral minimal risk level (MRL) of 0 3 mg/kg/day, dose divided by an uncertainty factor of 100 (10 for extrapolation from animals to humans, and 10 for human variability). “Used to derive an intermediate oral MRL of 0.1 mg/kg/day, dose adjusted for intermittent exposure and divided by an uncertainty factor of 100 (10 for extrapolation from animals to humans, and 10 for human variability). dUsed to derive an chronic oral MRL of 0.01 mg/kg/day; dose adjusted for intermittent exposure and divided by an uncertainty factor of 1000 (10 for use of a LOAEL, 10 for extrapolation from animals to humans, and 10 for human variability). ALT = alanine amino transferase; ST = aspartate amino transferase, Bd Wt = body weight; (C) = capsule; Cardio = cardiovascular, CEL = cancer effect level, d = day(s); EDso = effective dose for a given effect in 50% of animals; F = female; F, = first filial generation; (G) = gavage, Gastro = gastrointestinal, Gd = gestational day; (GO) = gavage in oil, GOT = glutamic oxaloacetic transaminase; GPT = glutamic pyruvic transaminase; Hemato = hematological; hr = hour(s); (IN) = ingestion; LDso = lethal dose, 50% kill, LOAEL = lowest-observed-adverse-effect level, M = male; Musc/skel = musculoskeletal; NOAEL= no-observed-adverse-effect level, NS = not specified; Resp = respiratory; SDH = sorbitol dehydrogenase; SGOT = serum glutamic oxaloacetic transaminase; SGPT = serum glutamic pyruvic transaminase; (W) = drinking water; wk = week; x = time(s); yr = year(s) S103443 H1TV3H 2 WHOJOHOTHO S9 Figure 2-2. Levels of Significant Exposure to Chloroform - Oral Acute (<14 days) Systemic sg Q 5 3 S Ss O I) > 8 & S x 5 &§ § S $ © S fF § £ 5 3 5 (mg/kg/day) § £5 4 £& 8 & 10000 Q x O O T Ss T 3r 5r 11 1 11 1 1 , B Hom sm A A A A 1000 | > « WH HR 18r 12 or 18 ) o> me 7m 9M ® D 30m ® ® ® 24m 25m 28m 3 30m 31m ® 17:0 170 200 21r 0 27m () o © 100 + ® 151 0 26m ® 29m O 32m 161 13 14r Der 2m 4m = @ 2m O O 3m OQ Qo 5 0 OO dO O O 2m 10 L oO 15r 26m 14r I 1 Tr 1 W 0.1 Key ro rat ® | D,, (animals) i Minimal risk 0.01 } m mouse . . level for effect d dog @® LOAEL for serious effects (animals) , She ri eCis } other than h rabbit ® LOAEL for less serious effects (animals) cancer 0.001 } . O NOAEL (animals) @ CEL: cancer effect level (animals) The number next to 0.0001 } . each point A LOAEL for serious efects (humans) SOHGSPONsID A LOAEL for less serious effects (humans) entries in Table 2-2. 0.00001 } A NOAEL (humans) * Doses represent the lowest dose tested per study that produced a tumorigenic response and do 0.000001 L not imply the existence of a threshold for the cancer end point. S103443 H1TV3H 2 WHO30HOTHO Figure 2-2. Levels of Significant Exposure to Chloroform - Oral (cont.) Acute (<14 days) Systemic ~ XN 0 ~ % 33 ~ 2 3 83 3g S08 5 & oS & 5 $ 28 $ 5 & 5 SQ o o (mg/kg/day) g § 3 Sg § £ 3 & é hd £ > 10000 @ Q Q Oo ~ Q@ 1 37 a 35r A 1000 17 18r 13r 16 22m 33m 40m 461 o od ® Oo. Oo @®. 27m 30m 31m O 3 20g” & ON 12r 14r 32%" 0 0 O 3am Q 48 19r 18r r ® 0 & 0 22m Qo 17r 22m 32m 44r ar 100 } 15r OQ 17r 28m 29m 0 Or @ a 19rQ 0 Oo 34h 36m Oo 45h 4g¢ Oo 130 141 O tar 0® 00 rar @ ler op 30m O o Q Bm 4m am @ O 4h 0 @ 29M or 22m Oo ‘ 32m oO o Oo 48r 14r 27m OQ Oo 34h 45h 10 F 19r Oo 43m 1} 01} Key r rat ® | D,, (animals) 1 Minimal risk m use . i 0.01 + d dog @ LOAEL for serious effects (animals) } ost fo effects . . ] other than h rabbit Q LOAEL for less serious effects (animals) V cancer 0.001 + O NOAEL (animals) @ CEL: cancer effect level (animals) The number next to . each point 0.0001 + A LOAEL for serious effects (humans) corresponds to A LOAEL for less serious effects (humans) entries in Table 2-2. 0.00001 - A NOAEL (humans) * Doses represent the lowest dose tested per study that produced a tumorigenic response and do not imply the existence of a threshold for the cancer end point. 0.000001 *“ S103443 H1TV3H C WHO40HOTHO 49 Figure 2-2. Levels of Significant Exposure to Chloroform - Oral (cont.) Intermediate (15-364 days) Systemic g ® = oO S LO § £ & o 5 2 < (mgkg/day) & 8 z £ I I 10000 ~ g Z 2 E 1000 | 62m os 62m 58r 62m 57r 58r 5 ® 48m 69m m m 50r 51m 54r O O 54r O 52r > 54r oO oS 54r &> tr ® ® O 66m © 9 100 | @ ® O © O Co O oO g9 som 60M 62m > 3 Beem mn 57 61m 58r os 58r Qo 5 Qo 64m 66m Qo 71d o O & "sO oO > OO go. Qo 58r O 62m oO 10 } 61m 71d I 1 1 1F 1 I 1 01 } Key WY ro rat ® | D,, (animals) 1 Minimal risk m mouse P . 0.01 | d dog @® LOAEL for serious effects (animals) | eve for effects : ; y other than h rabbit Q LOAEL for less serious effects (animals) “YY _incer 0.001 F O NOAEL (animals) @ CEL: cancer effect level (animals) The number next to . each point 0.0001 F A LOAEL for serious effects (humans) corresponds to A LOAEL for less serious effects (humans) entries in Table 2-2. 0.00001 | A NOAEL (humans) * Doses represent the lowest dose tested per study that produced a tumorigenic response and do not imply the existence of a threshold for the cancer end point. 0.000001 + S103443 H1TV3H 2 WHOJOHOTHO Figure 2-2. Levels of Significant Exposure to Chloroform - Oral (cont.) Intermediate (15-364 days) Systemic NI ~ ZF S or Q SS S = $< S 3d kN SQ O S (mg/kg/day) 5 S § £ 3g & 10000 g 2 — 1000 62m 64m 69M 70m 76¢ 65m 53r 54r 551 581 © O O ® 52r 54r > SD oO rol Oo 750 ® 57r 63m Q O ser 63m am 0) Oo ao o oO O em 100 hm 67m O ss Som 72m 0 76r 77m 55¢ 58r Oo 66m Qo oO 57r 59m Qo 73m Oo ser O O Qo 54r sy oO 0 0 57r 52r oO 56r 1 0.1 Key r rat . oH LDs, (animals) + Minimal risk m use 0.01 d dog @® LOAEL for serious effects (animals) jovel for effects 0 a h rabbit ® LOAEL for less serious effects (animals) “Y _ancer 0.001 O NOAEL (animals) @ CEL. cancer effect level (animals) The number next to 0.0001 A LOAEL for serious effects (humans) cach nds o A LOAEL for less serious effects (humans) entries in Table 2-2. 0.00001 A NOAEL (humans) * Doses represent the lowest dose tested per study that produced a tumorigenic response and do not imply the existence of a threshold for the cancer end point. 0.000001 WHOJ0HOTHO S103443 H1TV3H 2 69 Figure 2-2. Levels of Significant Exposure to Chloroform - Oral (cont.) Chronic (>365 days) Systemic ~ o> > 3 S & T 0 o > s & > 2 5 8 § 0 o = 9° 9 oO S 5 * O S -£ Q LQ 0 = 9° ° O s X ge ££ F023 5 5 8 g g (mg/kg/day) 5 & 5 z § $ g s s > 8 5 @ O ZT S IT @ < @ 10000 | —2 © & 8 1000 + % 2 2 9 2 z 94m 20m om 87r 87r gy 23 In 101r 86r 99r 2 00 00 oOo oOo © 00 g" 80 or ® 2 o * 4" 100 @ 87r 87r 87r 87r QO ssr 87r 91m Qo 91m 95m gg, + 92d 92d 87r 92d a7r 89m O 92d Oo 98d 100r 91m Oo 8% O 85 92d 8 o ©O 103m A A 0 A oO ser 10 F 1 om 92d I 84 i 84 1F A A I 104 I 5 ! Estimated = | : . 10% Upper-Bound 1 Human ! Cancer Risk 0.01 F v Levels Key 106 0.001 ro rat MB | D,, (animals) 1 Minimal risk level m mouse - d dog @ LOAEL for serious effects (animals) | foreiisnts thes 107 h rabbit (J LOAEL for less serious effects (animals) Ww 0.0004 [- O NOAEL (animals) } : The number next to 4@ CEL: cancer effect level (animals) each point 0.00001 F A LOAEL for serious effects (humans) corresponds to A LOAEL for less serious effects (humans) entries in Table 2-2. A NOAEL (humans 0.000001 + : ) * Doses represent the lowest dose tested per study that produced a tumorigenic response and do not imply the existence of a threshold for the cancer end point. S103443 H1TV3H 2 WHO404HO THO 0L CHLOROFORM 71 2. HEALTH EFFECTS Respiratory Effects. Information regarding respiratory effects in humans after oral exposure to chloroform is limited. Upper respiratory tract obstruction due to muscular relaxation was observed in a patient who accidentally ingested =2,410 mg/kg chloroform (Schroeder 1965). Congested lungs and scattered patches of pneumonic consolidation were found at autopsy in a man who committed suicide by drinking =6 ounces (3,755 mg/kg) of chloroform (Piersol et al. 1933). In both of these case studies, very large doses of chloroform were consumed. Thus, the respiratory effects noted may not be characteristic of those seen after ingestion of more moderate doses. The majority of animal data suggest that the respiratory system is not a target of chloroform-induced toxicity after oral exposure. However, in one study, female Fisher 344 rats administered chloroform by gavage in corn oil at doses of 34, 100, 200, or 400 mg/kg/day for 4 days or 3 weeks did exhibit dose-dependent nasal lesions consisting of early phases of new bone formation, periosteal hypercellularity, and degeneration of the olfactory epithelium and superficial Bowman’s glands (Larson et al. 1995b). In other studies, no treatment-related histopathological changes were found in the lungs of rats exposed to 160 mg/kg/day or mice exposed to 435 mg/kg/day chloroform in drinking water in a 90-day study (Jorgenson and Rushbrook 1980), in mice exposed by gavage to 41 mg/kg/day chloroform in oil for 105 days (Gulati et al. 1988), or in mice exposed to 257 mg/kg/day in drinking water for 1 year (Klaunig et al. 1986). Following chronic exposure, no histopathological changes were observed in rats exposed by gavage to 200 mg/kg/day TWA chloroform in oil (NCI 1976). Respiratory disease was observed in all chloroform-exposed groups of rats (=15 mg/kg/day); however, no histopathological changes were observed in a 60 mg/kg/day exposure group during another experiment by the same investigators (Palmer et al. 1979). No histopathological changes were observed in the lungs of male mice exposed by gavage to 277 mg/kg/day TWA chloroform in oil for 78 weeks (NCI 1976) or to 60 mg/kg/day in toothpaste for 80 weeks (Roe et al. 1979). Cardiovascular Effects. Information regarding cardiovascular effects after oral exposure to chloroform is limited to case report studies. On admission to the hospital, the blood pressure was 140/90 mm Hg and pulse was 70 beats per minute (bpm) in a patient who accidentally ingested =2,410 mg/kg chloroform (Schroeder 1965). Electrocardiography showed occasional extra systoles and a slight S-T segment depression. The patient recovered with no persistent cardiovascular change. In another individual, blood pressure was 100/40 mm Hg and pulse was 108 bpm after ingestion of an CHLOROFORM 72 2. HEALTH EFFECTS unknown quantity of chloroform and alcohol (Storms 1973). In both of these case studies, other factors (e.g., age, consumption of alcohol, suicidal/agitated state) may have contributed to the cardiovascular effects seen. Thus, it would appear that no studies exist which reliably indicate the presence of cardiovascular effects after oral chloroform exposure in humans. Information regarding cardiovascular effects in animals after oral exposure to chloroform is limited; the data suggest that, at the doses utilized, the cardiovascular system was not a target for chloroform toxicity. No histopathological changes were observed in rats and mice chronically exposed by gavage to 200 and 477 mg/kg/day TWA chloroform, respectively, for 78 weeks (NCI 1976). In this study, cardiac thrombosis was observed in low-dose female mice, but was not seen in high-dose female or male mice or either sex of rat at any dose. Similarly, no cardiovascular changes were observed in dogs exposed to 30 mg/kg/day chloroform in toothpaste capsules for 7.5 years (Heywood et al. 1979). Gastrointestinal Effects. Retrosternal soreness, pain on swallowing, and gastric distress with vomiting were reported in cases of intentional and accidental ingestion of chloroform (Piersol et al. 1933; Schroeder 1965). At autopsy, congestion with patchy necrosis of the mucosa was observed in the stomach and duodenum of a man who died after drinking =3,755 mg/kg chloroform (Piersol et al. 1933). The colonic mucosa was edematous, and the rectosigmoid junction was hemorrhagic. A 16-year-old female who ingested an unknown of amount of chloroform arrived at a hospital semiconscious and with repeated vomiting. She was treated with gastric lavage, antacids, intravenous glucose, and antiemetics. She had apparently recovered and was released. Seven days later, she presented with hepatomegaly, slightly depressed hemoglobin, and an abnormal liver sonogram, but no gastric side-effects (Hakim et al. 1992). The effects of chronic oral exposure to chloroform, as a by-product of the chlorination of drinking water, were evaluated in four epidemiology studies (Alavanja et al. 1978; Cantor et al. 1978; Saurez- Varela et al. 1994; Young et al. 1981). The association between the incidence of gastrointestinal cancer in humans and the chlorination of drinking water is discussed in Section 2.2.2.8. The data from these studies should be viewed with caution as many other known or suspected carcinogens are known to exist in chlorinated drinking water. Gastrointestinal irritation has been observed in some animals after oral exposure to chloroform. Gastric erosions were observed in pregnant rats gavaged with 516 mg/kg chloroform in oil during CHLOROFORM 73 2. HEALTH EFFECTS gestation (Thompson et al. 1974). Rabbits exposed by gavage to 20 mg/kg/day chloroform in oil during gestation had diarrhea; no histological results were provided. In a 90-day drinking-water study, no histopathological changes were observed in rats and mice exposed to 160 and 435 mg/kg/day chloroform, respectively (Jorgenson and Rushbrook 1980). Vomiting was observed in dogs exposed to 30 mg/kg/day chloroform in toothpaste capsules for 12-18 weeks (Heywood et al. 1979). In a chronic exposure study, no histopathological changes in gastrointestinal tissue were observed in rats and mice exposed by gavage to 200 and 477 mg/kg/day TWA chloroform, respectively (NCI 1976). Hematological Effects. The only information regarding hematological effects in humans after chronic oral exposure to chloroform was reported in a case study. Decreased erythrocytes and hemoglobin were observed in a subject who ingested =21 mg/kg/day chloroform in a cough medicine for 10 years (Wallace 1950). The lack of detail and the potential for confounding factors in this study does not allow a firm conclusion regarding the hematological effects of oral exposure to chloroform in humans. Hematological effects have been observed in some animals after oral exposure to chloroform. Hemoglobin and hematocrit decreased in male and female rats after a single oral dose of 546 mg/kg chloroform in oil (Chu et al. 1982b) and in female rats exposed to 100 mg/kg/day chloroform during gestation (Ruddick et al. 1983). However, no hematological changes were observed in mice exposed to 250 mg/kg/day for 14 days (Munson et al. 1982). In an intermediate-duration study, decreased neutrophils were observed in rats exposed to 192.98 mg/kg/day in drinking water (Chu et al. 1982b); however, no hematological changes were observed in rats and mice exposed to 160 and 435 mg/kg/day chloroform, respectively, for 90 days in drinking water (Chu et al. 1982a; Jorgenson and Rushbrook 1980). Increased cellular proliferation in the bone marrow was observed in rats exposed by gavage for 13 weeks to 410 mg/kg chloroform in toothpaste (Palmer et al. 1979). No hematological changes were observed, however, in rats similarly exposed to 165 and 60 mg/kg/day chloroform by gavage for 52 and 80 weeks, respectively. Moreover, no histopathological changes in hematopoietic tissues were observed in rats and mice after chronic exposure to 200 and 477 mg/kg/day TWA chloroform in oil, respectively (NCI 1976). No hematological effects were observed in dogs exposed to 30 mg/kg/day chloroform in toothpaste in capsules for 7.5 years (Heywood et al. 1979). In conclusion, no consistent hematological effects were noted in human or animal studies of oral exposure to chloroform. CHLOROFORM 74 2. HEALTH EFFECTS Musculoskeletal Effects. The only information regarding musculoskeletal effects in humans after oral exposure to chloroform was reported in a case study. Muscular relaxation of the jaw caused upper respiratory obstruction in a man who accidentally ingested approximately 2,410 mg/kg chloroform (Schroeder 1965), reflecting the neurological effects of chloroform exposure. Only one report detailing musculoskeletal effects in animals after oral exposure to chloroform was located. In that study, no histopathological changes were observed in the musculoskeletal system of rats and mice after chronic gavage exposure to 200 and 477 mg/kg/day TWA chloroform in oil, respectively (NCI 1976). Hepatic Effects. The liver is a primary target of chloroform toxicity in humans, with some evidence that suggests that the damage may be reversible (Wallace 1950). Hepatic injury occurred in patients within 1-3 days following chloroform ingestion (Piersol et al. 1933; Schroeder 1965; Storms 1973). Jaundice and liver enlargement and tenderness developed in all patients. The clinical observations were supported by blood biochemistry results with increased SGOT, SGPT, and lactate dehydrogenase (LDH) activities and increased bilirubin levels. At autopsy, fatty degeneration and extensive centrilobular necrosis were observed in one fatal case (Piersol et al. 1933). A 16-year-old female who ingested an unknown of amount of chloroform and arrived at a hospital semiconscious and with repeated vomiting was reported by Hakim et al. (1992). She was treated with gastric lavage, antacids, intravenous glucose, and antiemetics. She had apparently recovered and was released. Seven days later, she presented with hepatomegaly, slightly depressed hemoglobin, and an abnormal liver sonogram, suggesting toxic hepatic disease due to chloroform toxicosis. A 33-year-old female had injected herself intravenously with 0.5 mL of chloroform and then became unconscious. When she awoke approximately 12 hours later, she then drank another 120 mL of chloroform. She was treated with hyperbaric oxygen, cimetidine (to inhibit cytochrome P-450 and formation of phosgene), and N-acetylcystine (to replenish GSH stores). Liver serum enzymes alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and LDH were elevated in a pattern that suggested liver cell necrosis. Generally, these enzymes were noted to peak by day 4 and decrease by day 11. Total bilirubin and direct bilirubin did not change appreciably. GGT (gamma glutamyltransferase, also known as gamma glutamyl transpeptidase), alpha-feto protein and retinol binding protein showed increases between 6 and 8 days after ingestion, but still within normal ranges for humans (Rao et al. 1993). CHLOROFORM 75 2. HEALTH EFFECTS Increased sulfobromophthalein retention indicated impaired liver function in an individual who ingested 21 mg/kg/day chloroform in a cough medicine for 10 years (Wallace 1950). The changes reversed to normal after exposure was discontinued. Biochemical tests indicate that liver function in male and female humans was not affected by the use of mouthwash providing 0.96 mg/kg/day chloroform for <5 years (De Salva et al. 1975). The liver is also a target organ for chloroform toxicity in animals. In acute studies, hepatitis was observed in pregnant rats exposed by gavage to 516 mg/kg/day chloroform in oil (Thompson et al. 1974), while increased maternal liver weight without any histopathological changes was observed in pregnant rats similarly exposed to 100 mg/kg/day chloroform (Ruddick et al. 1983). Increased serum levels of transaminases, indicative of liver necrosis, were observed in mice treated with a single gavage dose of 199 mg/kg chloroform in toothpaste, 273 mg/kg in oil (Moore et al. 1982), or 250 mg/kg/day in oil for 14 days (Munson et al. 1982). Similar results were reported for rats treated with a single gavage dose of 100 mg/kg (Wang et al. 1994, 1995) or 0.1 mL/kg (Nakajima et al. 1995) Centrilobular necrosis of the liver with massive fatty changes was also observed in mice after a single dose of 350 mg/kg chloroform in oil (Jones et al. 1958). At a dose of 35 mg/kg, minimal lesions consisting of mid-zonal fatty changes were observed in mice. Similar results were reported by Larson et al. (1993) in male rats in order to identify target tissues for the acute effects of chloroform in rats and mice and to establish the time-course of chloroform-induced histopathologic and proliferative responses. Rats were given 34, 180, or 477 mg/kg once in corn oil by gavage and sacrificed 24 hours after administration {acute-duration study). In a related time-course study (which focused on histologic changes in tissues over time), rats received 180 mg/kg chloroform in corn oil by gavage and were sacrificed at 0.5, 1, 2, 4, and 8 days after treatment, or received 477 mg/kg in corn oil by gavage and were sacrificed either 1 or 2 days after administration. In the acute study, gross liver to body weight ratios were unaffected at all doses. Histologically, chloroform caused hepatic injury, in a dose-related manner, producing morphologic changes generally limited to the centrilobular hepatocytes. Liver enzymes (SDH, ALT, and AST) were slightly elevated above controls in the 34 and 180 mg/kg group, but were significantly higher in the 477 mg/kg group for all three enzymes. In the time-course study, 1 day after dosing, about 50% of the hepatocytes adjacent to the central veins were degenerated or necrotic in the 180 mg/kg treatment group. Larger vessels had perivascular edema, influx of neutrophils and eosinophils. Only scattered hepatocyte necrosis was seen CHLOROFORM 76 2. HEALTH EFFECTS by day 2 after treatment. By eight days however, the livers were not histologically different from controls. In a similar study, mice were administered 34, 238, or 477 mg/kg once in corn oil by gavage and sacrificed 24 hours after administration (acute study) or 350 mg/kg chloroform in corn oil by gavage and sacrificed at 0.5, 1, 2, 4, and 8 days after treatment (time-course study). Livers of female mice were much more sensitive than the kidneys to the toxic effects of chloroform. In the acute study, livers of mice receiving 34 mg/kg chloroform were not histologically different from controls; however, those treated with 238 mg/kg had few small randomly scattered foci of hepatocyte necrosis. Livers from the 477 mg/kg group had centrilobular coagulative necrosis of 50% of the lobule. In the time- course study, a significant increase in liver weights and liver to body-weight ratios was observed in mice at 2 and 4 days after treatment with the 350 mg/kg dose of chloroform. At 12 hours after treatment, mice had marked swelling of the centrilobular hepatocytes, affecting about 50% of the lobule. One day after treatment, the hepatocytes adjacent to the central vein were necrotic. Two days after chloroform treatment, centrilobular sinusoids were dilated with inflammatory cells associated with centrilobular necrosis. At eight days after treatment, the livers from the treated mice were not histologically different from those of control animals. Serum liver enzymes (SDH and ALT) were elevated in the groups sacrificed at 0.5, 1, 2, and 4 days after treatment, but not in controls or in those animals sacrificed 8 days after treatment. Another study by Larson et al. (1994d) identified the relationships among chloroform-induced cytolethality, regenerative cell proliferation, and tumor induction in male B6C3F| mice dosed with 0, 34, 90, 138, or 277 mg/kg/day chloroform by gavage in corn oil. Mice exposed to 90 mg/kg/day experienced prominent centrilobular hepatocyte swelling with loss of cytoplasmic eosinophilia. Mice exposed to 138 and 277 mg/kg/day experienced increasing levels of centrilobular hepatocyte swelling and degeneration, as well as scattered necrosis and inflammatory cell accumulation. Dose-dependent increases in hepatocyte proliferation were seen in all dose groups after exposure to chloroform for 4 days. Differences in chloroform toxicity have been noted in female mice when chloroform was administered in different vehicles and by different dosing regimes (Larson et al. 1994b). Mice were treated orally with 3, 10, 34, 90, 238, or 477 mg/kg/day of chloroform in corn oil, or with 16, 26, 53, 81, or 105 mg/kg/day in the drinking water, for 4 days. Chloroform treatment resulted in significant CHLOROFORM 77 2. HEALTH EFFECTS increases in liver weights of mice at the 238 and 477 mg/kg/day dose levels. Mice treated with 238 mg/kg chloroform had moderate centrilobular vacuolar degeneration of hepatocytes and scattered centrilobular and subcapsular hepatocyte necrosis. At the 477 mg/kg dose, severe centrilobular coagulative necrosis with small number of inflammatory cells in the necrotic areas was also observed. Dose-dependent increases in both ALT and SDH were also observed. At daily doses of 90 mg/kg or less, no increase in hepatic cell proliferation was noted. Dose-dependent increases in hepatic cell proliferation and cells observed to be in S-phase occurred in the 238 and 477 mg/kg/day doses. For mice dosed with 16, 26, 53, 81, or 105 mg/kg/day in the drinking water, serum ALT or SDH were not different from controls at any dose. In the 53, 81, and 105 mg/kg/day treatment groups, the livers had changes that were characterized by pale cytoplasmic eosinophilic staining of centrilobular hepatocytes compared to the periportal hepatocytes and controls. Livers from mice treated with 26 mg/kg/day chloroform or less failed to showed significant histologic changes when compared to controls. Cell proliferations in the liver were not found at any dose or duration. An acute oral MRL of 0.3 mg/kg/day was calculated using the 26.4 mg/kg/day NOAEL based on the hepatic effects in these animals from this study. More information on this MRL and how it was derived is located in the footnote to Table 2-2, Section 2.5, and in Appendix A of this profile. Larson et al. (1995a) examined the dose-response relationships for the induction of cytolethality and regenerative cell proliferation in the livers of male Fischer 344 rats given chloroform by gavage. Groups of 12 rats were administered oral doses of 0, 3, 10, 34, 90, and 180 mg/kg/day chloroform in corn oil by gavage for 4 days. BrdU was administered via an implanted osmotic pump to label cells in S-phase. Cells having incorporated BrdU were visualized in tissue sections immunohistochemically and the LI evaluated as the percentage of S-phase cells. Necropsies and histopathological examinations were performed at death. The relative liver weights were increased at doses of 10 mg/kg/day and above at 4 days posttreatment. Rats treated with 34, 90, or 180 mg/kg/day by gavage for 4 days had mild-to-moderate degeneration of centrilobular hepatocytes. The livers of rats given 90 mg/kg/day for 4 days had a slight increase in centrilobular pallor and necrosis of hepatocytes surrounding the central vein; the remaining central and some mid-zonal hepatocytes were swollen and displayed a cytoplasmic granularity. In the 180 mg/kg/day dose group, the livers had scattered individual cell necrosis throughout the central and mid-zonal regions. The cytoplasm of the centrilobular hepatocytes were pale, eosinophilic and mildly vacuolated. Dose-dependent increases in both ALT and SDH were observed at 4 days in the 90 and 180 mg/kg/day dose groups and at 3 weeks CHLOROFORM 78 2. HEALTH EFFECTS in the 180 mg/kg/day dose group only. A dose-dependent increase in LI was seen in rat liver after 4 days of treatment with 90 and 180 mg/kg/day by gavage. Larson et al. (1995a) also examined the toxicological effects of chloroform administered in the drinking water in rats. Groups of 12 rats were administered chloroform ad libitum in drinking water at concentrations of 0, 60, 200, 400, 900, and 1,800 ppm for 4 days. BrdU was administered via an implanted osmotic pump to label cells in S-phase. Cells having incorporated BrdU were visualized in tissue sections immunohistochemically and the LI evaluated as the percentage of S-phase cells. Necropsies and histopathological examinations were performed at death. Average daily doses of chloroform ingested from drinking water were: 0, 6.6, 19.3, 33.2, 68.1, and 57.5 mg/kg/day for 0, 60, 200, 400, 900, and 1,800 ppm concentration levels, respectively. Only mild hepatocyte vacuolation was observed in rats given 900 or 1,800 ppm in water for 4 days; no increase in the hepatic LI was observed at any time point. In another study by Larson et al. (1995b), female Fisher 344 rats administered 400 mg/kg chloroform by gavage in corn oil for 4 days exhibited hepatic lesions consisting of mild centrolobular vacuolization, scattered necrotic hepatocytes, sinusoidal leucostasis, mild-to-focally severe centrilobular hepatocyte degeneration and necrosis, and diffuse centrolobular swelling. Rats in the 100 and 200 mg/kg groups had only slight centrolobular changes, while those in the 34 mg/kg group did not differ from controls. Pereira (1994) investigated the effects of chloroform exposure in different vehicles and by different dosing regimes on hepatic cell proliferation in female B6C3F; mice. Animals received either 263 mg/kg/day chloroform by gavage in corn oil or 1,800 ppm chloroform in drinking water, 24 hours a day and were sacrificed at 5 or 12 days. When administered by gavage, chloroform exposure resulted in significantly increased relative liver weights at 5 days (53%; p<0.05), but not at 12 days. The livers of mice exposed to chloroform for 5 days exhibited toxicity consisting of necrotic hepatocytes, infiltration of macrophages and neutrophils in the central zone, and hydrotropic, swollen, and rounded hepatocytes of a pale ground glass appearance in the midzone. Hepatotoxicity was less severe at 12 days. Cell proliferation was significantly increased at both 5 and 12 days. In contrast, chloroform administered in drinking water for 5 days reduced absolute and relative liver weight while exposure for 12 days had no effect on relative liver weights. The livers of mice exposed to chloroform in drinking water exhibited limited toxicity after 5 days consisting of smaller hepatocytes CHLOROFORM 79 2. HEALTH EFFECTS with dense nonvacuolated and basophilic cytoplasm; hepatotoxicity after 12 days was limited to vacuolated hepatocytes. Cell proliferation, was significantly reduced (p<0.05) at 5 and 12 days as compared to controls. Liver effects in animals have been reported in numerous oral studies of intermediate duration (Chu et al. 1982b; Eschenbrenner and Miller 1945a; Larson et al. 1995b). Larson et al. (1994b) exposed female mice to 3, 10, 34, 90, 238, and 477 mg/kg/day of chloroform in corn oil via gavage 5 days a week for 3 weeks. Chloroform treatment resulted in significant increases in liver weights of mice at the 90, 238, and 477 mg/kg/day doses. Doses of 34 mg/kg/day resulted in pale cytoplasmic eosinophilia of the centrilobular hepatocytes and mild vacuolation of the centrilobular and mid-zonal hepatocytes relative the periportal hepatocytes and livers from control mice. At the 238 mg/kg/day dose, the livers were characterized by a severe centrilobular hepatocyte necrosis. At 477 mg/kg/day, the central zone of the liver was populated by degenerate vacuolated hepatocytes and regenerating hepatocytes with markedly basophilic cytoplasm and small round nuclei with clumped chromatin and prominent nucleoli. Significant dose-dependent increases in ALT and SDH were observed at doses of 34 mg/kg/day and greater. Cell proliferation was markedly increased in the liver at the 238 and 477 mg/kg/day doses. Mice dosed with 16, 43, 82, 184, or 329 mg/kg/day of chloroform in the drinking water for 7 days a week for 3 weeks resulted in no histological changes in livers at all doses studied. Liver weights were significantly increased at 82, 184, and 329 mg/kg/day. Larson et al. (1995a) examined the dose-response relationships for the induction of cytolethality and regenerative cell proliferation in the livers of male Fischer 344 rats given chloroform by gavage. Groups of 12 rats were administered oral doses of 0, 3, 10, 34, 90, and 180 mg/kg/day chloroform in corn oil by gavage for 5 days a week for 3 weeks. BrdU was administered via an implanted osmotic pump to label cells in S-phase. Cells having incorporated BrdU were visualized in tissue sections immunohistochemically and the LI evaluated as the percentage of S-phase cells. Necropsies and histopathological examinations were performed at death. The relative liver weights were increased at doses of 90 mg/kg/day and greater at 3 weeks. After 3 weeks of exposure, livers of rats in the 34 or 90 mg/kg/day dose groups did not differ from controls. In the 180 mg/kg/day dose group, effects were similar to those seen at 4 days after exposure. Dose-dependent increases in both ALT and SDH were observed after 3 weeks in the 180 mg/kg/day dose group only. CHLOROFORM 80 2. HEALTH EFFECTS Larson et al. (1995a) also examined the toxicological effects of chloroform administered in the drinking water in rats. Groups of 12 rats were administered chloroform ad libitum in drinking water at concentrations of 0, 60, 200, 400, 900, and 1,800 ppm for 7 days a week for 3 weeks. BrdU was administered via an implanted osmotic pump to label cells in S-phase. Cells having incorporated BrdU were visualized in tissue sections immunohistochemically and the LI evaluated as the percentage of S-phase cells. Necropsies and histopathological examinations were performed at death. Average daily doses of chloroform ingested from drinking water were: 0, 6, 17.4, 32, 62.3, and 106 mg/kg/day for 3 weeks exposure for 0, 60, 200, 400, 900, and 1,800 ppm concentration levels, respectively. Only mild hepatocyte vacuolation was observed in rats given 1,800 ppm in water for 3 weeks. No increase in the hepatic LI was observed at any time point. Larson et al. (1995b) further examined the dose-response relationship for chloroform-induced cytotoxicity and cell proliferation in the liver of female Fisher 344 rats via gavage. Animals received chloroform in corn oil at doses of 0, 34, 100, 200, or 400 mg/kg/day for 3 weeks (5 days a week). At completion of dosing, animals were sacrificed, the livers were evaluated microscopically and cell proliferation was quantitated. Exposure to 400 mg/kg chloroform resulted in hepatic lesions consisting of slight to mild diffuse vacuolar change and focal centrilobular degeneration. Rats in the 200 mg/kg groups had only slight centrilobular vacuolation, while those in the 100 and 34 mg/kg dose groups did not differ from controls. Pereira (1994) provided further evidence of the effect of dosing method (gavage versus drinking water) and vehicle (corn oil versus water) on hepatic cell proliferation in female B6C3F, mice. Animals received either 263 mg/kg/day chloroform by gavage in corn oil or 1,800 ppm chloroform in drinking water, 24 hours a day and were sacrificed at 33 or 159 days. When administered by corn oil gavage, chloroform exposure resulted in increased relative liver weights at 33 and 159 days (30.1 and 38.2% increases, respectively). The livers of mice exposed to chloroform for 33 or 159 days exhibited limited toxicity consisting of focal areas of necrotic hepatocytes and a limited number of mononuclear cells and swollen, rounded, pale hepatocytes. Cell proliferation was significantly increased at both 33 and 159 days as compared to controls. In contrast, administration of chloroform in drinking water had no effect on absolute or relative liver weights after 33 days, while exposure for 159 days resulted in significantly increased relative liver weights (31.4%; p<0.05). However, the livers of mice exposed for either 33 or 159 days exhibited no signs of toxicity. In addition, cell proliferation was not significantly affected by exposure to chloroform in drinking water for either 33 or 159 days. CHLOROFORM 81 2. HEALTH EFFECTS Fatty changes, necrosis, increased liver weight, and hyperplasia have been observed in rats exposed to 2150 mg/kg/day chloroform in a toothpaste vehicle via gavage for 13 weeks (Palmer et al. 1979). An increased incidence of sporadic, mild, reversible, liver changes occurred in rats exposed to chloroform in the drinking water at doses of 0.64—150 mg/kg/day for 90 days, but the incidences were not significantly higher than the incidences in controls (Chu et al. 1982a). The effect- and no-effect-levels in the study are clearly defined. Fatty and hydropic changes, necrosis, and cirrhosis were observed in mice treated by gavage with 250 mg/kg/day chloroform in oil for 90 days (Bull et al. 1986; Munson et al. 1982) or 86 mg/kg/day in drinking water for 1 year (Klaunig et al. 1986). In contrast, centrilobular fatty changes observed in mice at 64 mg/kg/day chloroform in drinking water for 90 days appeared to be reversible (Jorgenson and Rushbrook 1980), and no liver effects were found in mice treated with 250 mg/kg/day chloroform in aqueous vehicles (Bull et al. 1986). In addition, hepatocellular degeneration was induced in F; females in a 2-generation study in which mice were treated by gavage with 41 mg/kg/day chloroform in oil (Gulati et al. 1988). Significantly increased (p<0.05) SGPT activity occurred in dogs beginning at 6 weeks of exposure to chloroform in toothpaste in capsules at a dose of 30 mg/kg/day in a 7.5-year study (Heywood et al. 1979). SGPT activity was not increased at 15 mg/kg/day until week 130. Therefore, 15 mg/kg/day was the NOAEL for intermediate-duration exposure. This NOAEL was used to derive an intermediate-duration oral MRL of 0.1 mg/kg/day. More information on this MRL and how it was derived is located in the footnote to Table 2-2, Section 2.5, and in Appendix A of this profile. The relationships among chloroform-induced cytolethality, regenerative cell proliferation, and tumor induction in male B6C3F, mice dosed with chloroform by gavage in corn oil has also been studied. Mice received chloroform at doses of 0, 34, 90, 138, or 277 mg/kg for 4 consecutive days or 5 days a week for 3 weeks. To monitor cell proliferation, mice were administered BrdU via implanted osmotic pump for the last 3.5 days. Chloroform treatment for 3 weeks also resulted in a small (<10%) but significant increase in relative liver weight of mice at the highest dose level. Macroscopically, pale livers and kidneys were noted at all dose levels after 3 weeks of chloroform exposure; treatment with 138 or 277 mg/kg for 3 weeks resulted in the formation of white subcapsular foci. After 3 weeks of chloroform exposure, the livers of all mice in the 34 mg/kg/day group and 3 of 5 mice in the 90 mg/kg/day group were histologically similar to those of controls. Livers of 2 mice in the 90 mg/kg/day group exhibited centrilobular hepatocyte swelling with loss of eosinophilia. Mice dosed with 138 mg/kg/day experienced marked centrilobular hepatocyte swelling, mild to moderate periportal vacuolation, and scattered centrilobular and periportal degeneration and necrosis. Mice dosed with CHLOROFORM 82 2. HEALTH EFFECTS 277 mg/kg/day experienced marked centrilobular hepatocyte degeneration and necrosis. Cell proliferation was significantly elevated in the 138 and 277 mg/kg dose groups only. In chronic-duration exposure studies, liver effects have been observed in rats, mice, and dogs after oral exposure to chloroform. Male and female ICI mice were exposed to 17 or 60 mg/kg/day chloroform by gavage using toothpaste as a vehicle for 80 weeks followed by 13-24-week observation period. No significant histopathological findings (noncancerous) were recorded in the kidneys or lung tissues. Moderate or severe fatty degeneration of the liver was slightly more prevalent among treated animals than controls (statistical significance not provided) beginning at the 17 mg/kg/day. Necrosis was observed in female rats treated by gavage with 200 mg/kg/day chloroform in oil for 78 weeks (NCI 1976). Nodular hyperplasia occurred in all groups of male and female mice similarly treated at 138 and 238 mg/kg/day, respectively. Fibrosis of the liver was observed in both sexes of rats exposed to 200 mg/kg/day chloroform in the drinking water for <180 weeks (Tumasonis et al. 1985, 1987). Increased SGPT was observed in dogs given chloroform in toothpaste capsules for 7.5 years (Heywood et al. 1979). The lowest oral dose administered to animals in chronic studies was 15 mg/kg/day, which increased SGPT in dogs. This LOAEL was used to derive a chronic oral MRL of 0.01 mg/kg/day. More information on this MRL and how it was derived is located in the footnote to Table 2.2, Section 2.5, and in Appendix A of this profile. Renal Effects. The kidney is also a major target of chloroform-induced toxicity in humans. Oliguria was observed 1 day after the ingestion of =3,755 or 2,410 mg/kg chloroform (Piersol et al. 1933; Schroeder 1965). Increased blood urea nitrogen (BUN) and creatinine levels also indicated renal injury. Albuminuria and casts were detected in the urine. Histopathological examination at autopsy revealed epithelial swelling and hyaline and fatty degeneration in the convoluted tubules of kidneys in one fatal case of oral exposure to chloroform (Piersol et al. 1933). Numerous hyaline and granular casts and the presence of albumin were observed in the urine of one subject who ingested 21 mg/kg/day chloroform in cough medicine for 10 years (Wallace 1950). The urinalysis results reversed to normal after discontinuation of chloroform exposure. No indications of renal effects were observed in humans who ingested estimated doses of 0.34-0.96 mg/kg/day chloroform in mouthwash for 5 years (De Salva et al. 1975). The renal toxicity of chloroform in animals has been reported in many studies of acute duration. Larson et al. (1993) studied the effects of dose and time after chloroform administration on the renal CHLOROFORM 83 2. HEALTH EFFECTS toxicology of chloroform in male rats. Rats were given 34, 180, or 477 mg/kg once in corn oil by gavage and sacrificed 24 hours after administration. In a related time-course study (which focused on histologic changes in tissues over time), rats received 180 mg/kg chloroform in corn oil by gavage and were sacrificed at 0.5, 1, 2, 4, and 8 days after treatment; others received 477 mg/kg in corn oil by gavage and were sacrificed either 1 or 2 days after administration. Histologically, chloroform caused extensive renal damage and, to a much lesser extent, hepatic injury, in a dose-related manner. One day after treatment with a single dose of chloroform of 34 mg/kg or greater, the kidneys of male rats developed tubular necrosis that was restricted to the proximal convoluted tubules. The severity of these lesions occurred in a dose-dependent manner. Rats given 34 mg/kg had scattered necrotic tubules affecting less than 10% of the midcortical nephrons. In the 180 mg/kg group, 25% of the proximal convoluted tubules were necrotic. Nearly all segments of the proximal tubules had necrosis in the rats receiving 477 mg/kg chloroform. Despite extensive renal injury, increases in BUN or in urinary protein or glucose were not observed. In the time-course study, the kidneys, after 12 hours of treatment, had a diffuse granularity of cytoplasm of the epithelium lining of the proximal convoluted tubules in the 180 mg/kg group. Damage was severe after 1 day, and after 2 days, 100% of the proximal tubules were lined by necrotic epithelium. After 8 days, the kidneys had returned to normal appearance. No increases in BUN or urinary protein or glucose were noted at any time after treatment. Larson et al. (1995a) also examined the toxicological effects of chloroform administered in the drinking water in rats. Groups of 12 rats were administered chloroform ad libitum in drinking water at concentrations of 0, 60, 200, 400, 900, and 1,800 ppm for 4 days. BrdU was administered via an implanted osmotic pump to label cells in S-phase. Cells having incorporated BrdU were visualized in tissue sections immunohistochemically and the LI evaluated as the percentage of S-phase cells. Necropsies and histopathological examinations were performed at death. Average daily doses of chloroform ingested from drinking water were: 0, 6.6, 19.3, 33.2, 68.1, and 57.5 mg/kg/day for 4 days exposure for the 0, 60, 200, 400, 900, and 1,800 ppm concentration levels, respectively. When chloroform was administered in the drinking water, no microscopic alterations were seen in the kidneys after 4 days of treatment. The overall renal LI was not increased at any dose. The same study (Larson et al. 1995a) examined the dose-response relationships for the induction of cytolethality and regenerative cell proliferation in the kidneys of male Fischer 344 rats given chloroform by gavage. Groups of 12 rats were administered oral doses of 0, 3, 10, 34, 90, and CHLOROFORM 84 2. HEALTH EFFECTS 180 mg/kg/day chloroform in corn oil by gavage for 4 days. BrdU was administered via an implanted osmotic pump to label cells in S-phase. Cells having incorporated BrdU were visualized in tissue sections immunohistochemically and the LI evaluated as the percentage of S-phase cells. Necropsies and histopathological examinations were performed at death. Rats treated with 34, 90, or 180 mg/kg/day by gavage for 4 days had mild-to-moderate degeneration of renal proximal tubules. After 4 days of dosing with 34 mg/kg/day, the proximal convoluted tubule epithelial cells had increased numbers and prominence of apical cytoplasmic vacuoles. Likewise, rats given 90 mg/kg/day for 4 days displayed swelling and vacuolation of 25-50% of the proximal tubules. Progressive degeneration of the proximal tubules was observed in rats exposed to 180 mg/kg/day. At 4 days, swollen and vacuolated cytoplasm in approximately 10-20% of proximal tubule epithelium was observed. LI were increased in the kidney cortex only in the rats treated with 180 mg/kg/day for 4 days. The dose-response relationship for chloroform-induced cytotoxicity and cell proliferation in the kidneys of female Fisher 344 rats has also been elucidated. Animals received 34, 100, 200, or 400 mg/kg chloroform by gavage in corn oil for 4 days. At completion of dosing, kidneys were prepared for microscopic evaluation, and cell proliferation was quantitated. Rats in the high dose group had 50-75% of proximal tubules lined with necrotic or attenuated regenerating epithelium, as well as distal nephrons containing hyaline casts. Rats in the 200 mg/kg group had kidneys with 25-50% of proximal tubules lined with degenerated, necrotic or regenerating epithelium. Kidneys from rats in the 2 lowest dose groups were similar to those of controls (Larson et al. 1995b). Acute toxic nephrosis was observed in female rats exposed to 516 mg/kg/day chloroform by gavage in oil during Gd 6-15, with maternal lesions characterized by tubular swelling, hydropic or fatty degeneration and necrosis (Thompson et al. 1974). Increased kidney weight was observed in female rats after a single gavage dose of 546 mg/kg chloroform (Chu et al. 1982b). Similarly, rats exposed to 400 mg/kg/day by gavage during gestation had increased kidney weight (Ruddick et al. 1983). No increase in kidney weight was found in the rats treated with 200 mg/kg/day during gestation. Renal necrosis in convoluted tubules was observed in male mice after a single dose of 199 mg/kg chloroform in toothpaste or 65.6 mg/kg chloroform in oil (Moore et al. 1982). The relationships among chloroform-induced cytolethality, regenerative cell proliferation, and tumor induction in male B6C3F, mice dosed with chloroform by gavage in corn oil of acute duration has CHLOROFORM 85 2. HEALTH EFFECTS also been reported. Mice received chloroform at doses of 0, 34, 90, 138, or 277 mg/kg/day by gavage for 4 consecutive days. To monitor cell proliferation, mice were administered BrdU via implanted osmotic pump for the last 3.5 days. After four days of exposure, 2 of 4 mice dosed with 34 mg/kg/day and all mice dosed with 90 mg/kg/day or more experienced extensive (>75% of tissue) acute necrosis of the proximal convoluted tubule, characterized by a thin layer of eosinophilic necrotic cellular debris lining the tubular basement membrane. Distal tubules and collecting ducts were filled with hyaline casts. The remaining 2 mice in the 34 mg/kg/day dose group experienced scattered necrosis in proximal convoluted tubules. In the kidneys, significant increases in cell proliferation were noted in all dose groups given chloroform for 4 days. The authors concluded that the results of this study confirmed the sensitivity of the male mouse, relative to the female mouse, to the nephrotoxic effects of chloroform (Larson et al. 1994d). In intermediate-duration exposures, several studies suggest that mice appeared to be more sensitive than rats to the nephrotoxic effects of chloroform. Rats exposed to 193 mg/kg/day for 28 days (Chu et al. 1982b) or to 160 mg/kg/day chloroform for 90 days (Jorgenson and Rushbrook 1980) in drinking water had no kidney effects. Increased relative kidney weight was observed in rats exposed by gavage to 150 mg/kg/day for 13 weeks, but not in rats exposed to 30 mg/kg/day (Palmer et al. 1979). Chronic inflammatory changes were observed in the kidneys of mice exposed to 50 mg/kg/day chloroform (dissolved in an emulsion prepared with emulphor in water) by gavage (Munson et al. 1982); however, no changes were observed in mice exposed to 41 mg/kg/day by gavage (Gulati et al. 1988) or in mice exposed to 435 mg/kg/day chloroform in drinking water (Jorgenson and Rushbrook 1980). Nonetheless, exposure to 86 mg/kg/day in drinking water for 1 year caused tubular necrosis in mice (Klaunig et al. 1986). Larson et al. (1995b) examined the dose-response relationship for chloroform-induced cytotoxicity and cell proliferation in the kidneys of female Fisher 344 rats using a wide range of doses. Animals received 34, 100, 200, or 400 mg/kg chloroform by gavage in corn oil for 3 weeks (5 days a week). At completion of dosing, the kidneys were prepared for microscopic evaluation, and cell proliferation was quantitated. Rats in the 100, 200, and 400 mg/kg dose groups had 50-75% of proximal tubules lined with regenerating epithelium; many of the tubules were dilated and contained mineralized concretions. Kidneys from rats in the lowest dose group were similar to those of controls. CHLOROFORM 86 2. HEALTH EFFECTS Lipsky et al. (1993) studied groups of male Fischer 344 rats gavaged with either 90 or 180 mg/kg/day of chloroform in corn oil or water for 5 days a week for 4 weeks. Rats exposed to chloroform by gavage in corn oil displayed acute cell injury and necrosis, primarily in the epithelial cells lining the S2 segment of the proximal tubule, with some apparent damage/necrosis occurring in the S1 segment as well. This injury was present in all rats exposed to the 180 mg/kg/day dose and in less than half of the animals exposed to the 90 mg/kg/day dose. There was also a dose-dependent increase in the total BrdU labeling of nuclei in renal cells of the chloroform-treated oil-gavaged animals compared to controls. The largest increase in DNA BrdU labeling was in the cells of the S2 segment. The 90 mg/kg/day dose of chloroform also produced increase in DNA labeling in the S3 segment, but not for the 180 mg/kg/day dose of chloroform. Animals exposed to chloroform in water showed minimal histopathologic alterations in the kidneys. Mild injury and necrosis was seen in cells of the S2 segment in 1 of 6 animals in the 180 mg/kg/day group, while none were seen in the 90 mg/kg/day dose group. Little to no change in DNA labeling of renal cells was seen in the water-gavaged rats. Larson et al. (1995a) examined the dose-response relationships for the induction of cytolethality and regenerative cell proliferation in the kidneys of male Fischer 344 rats given chloroform by gavage. Groups of 12 rats were administered oral doses of 0, 3, 10, 34, 90, and 180 mg/kg/day chloroform for 5 days a week for 3 weeks. BrdU was administered via an implanted osmotic pump to label cells in S-phase. Cells having incorporated BrdU were visualized in tissue sections immunohistochemically and the LI evaluated as the percentage of S-phase cells. Necropsies and histopathological examinations were performed at death. Relative kidney weights were increased after 3 weeks in the 180 mg/kg/day dose group only. Rats treated with 34, 90, or 180 mg/kg/day by gavage for 4 days had mild-to-moderate degeneration of renal proximal tubules. These alterations were absent or slight after 3 weeks of treatment, except at the highest dose level. After 4 days of dosing with 34 mg/kg/day, the proximal convoluted tubule epithelial cells had increased numbers and prominence of apical cytoplasmic vacuoles, but these changes were not observed at 3 weeks. Likewise, rats given 90 mg/kg/day for 4 days displayed swelling and vacuolation of 25-50% of the proximal tubules, at 3 weeks only 1 of 3 rats had vacuolated and degenerated epithelium. Progressive degeneration of the proximal tubules was observed in rats exposed to 180 mg/kg/day. At 4 days, swollen and vacuolated cytoplasm in approximately 10-20% of proximal tubule epithelium was observed, while at 3 weeks the percentage was 25-50%. At 3 weeks, scattered tubules also had mineral concretions that appeared subepithelial. LI was increased in the kidney cortex only in the rats treated with 180 mg/kg/day for 4 days. CHLOROFORM 87 2. HEALTH EFFECTS Larson et al. (1995a) also examined the toxicological effects of chloroform administered in the drinking water in rats. Groups of 12 rats were administered chloroform ad libitum in drinking water at concentrations of 0, 60, 200, 400, 900, and 1,800 ppm for 7 days a week for 3 weeks. BrdU was administered via an implanted osmotic pump to label cells in S-phase. Cells having incorporated BrdU were visualized in tissue sections immunohistochemically and the LI evaluated as the percentage of S-phase cells. Necropsies and histopathological examinations were performed at death. Average daily doses of chloroform ingested via drinking water were 0, 6, 17.4, 32, 62.3, and 106 mg/kg/day for 3 weeks exposure for 0, 60, 200, 400, 900, and 1,800 ppm concentration levels, respectively. As a general observation, rats treated for 3 weeks with 200 ppm chloroform and greater had slightly increased numbers of focal areas of regenerating renal proximal tubular epithelium and cell proliferation than were noted in controls, but no clear dose-response relationship was evident. The overall renal LI was not increased at any dose or time point. Larson et al. (1994b) exposed female mice to 3, 10, 34, 90, 238, and 477 mg/kg/day of chloroform in corn oil via gavage for 5 days a week for 3 weeks. Mice were also dosed with 16, 43, 82, 184, or 329 mg/kg/day of chloroform in the drinking water for 7 day a week for 3 weeks. In both studies, no increases in cell proliferation were noted and no significant changes in renal histopathology were reported. In another study by Larson et al. (1994d) the possible relationships among chloroform-induced cytolethality, regenerative cell proliferation, and tumor induction were identified in male B6C3F, mice dosed with chloroform by gavage in corn oil. Mice received chloroform at doses of 0, 34, 90, 138, or 277 mg/kg/day for 5 days a week for 3 weeks. To monitor cell proliferation, mice were administered BrdU via implanted osmotic pump for the last 3.5 days. Renal lesions were similar to, but less severe than, lesions seen in mice exposed for 4 days and were characterized by extensive tubular regeneration. The kidneys of mice dosed with 34-138 mg/kg/day chloroform exhibited dose- dependent increases in regenerating proximal convoluted tubules. The kidneys of mice dosed with 277 mg/kg/day chloroform exhibited severe nephropathy characterized by degeneration, necrosis, and regeneration of the proximal tubules. The renal interstitium was swollen due to fibroplasia, edema, and inflammatory cell infiltration. After 3 weeks of exposure, renal cell proliferation was still elevated relative to controls at doses of >90 mg/kg/day, but LI values declined from levels seen after 4 days of exposure. CHLOROFORM 88 2. HEALTH EFFECTS In chronic oral studies, no definite renal effects were observed in rats exposed to <200 mg/kg/day or mice exposed to <477 mg/kg/day TWA (Heindel et al. 1995; Jorgenson et al. 1985; NCI 1976; Roe et al. 1979). In dogs, however, fat deposition in renal glomeruli was observed at a dose of 30 mg/kg/day chloroform for 7.5 years, but not at 15 mg/kg/day (Heywood et al. 1979). Dermal Effects. No studies were located regarding dermal effects in humans after oral exposure to chloroform. Alopecia was observed in pregnant rats exposed to 126 mg/kg/day chloroform in oil (Thompson et al. 1974). Rough coats were observed in mice exposed to 100 mg/kg/day chloroform in oil for 14 days (Gulati et al. 1988). Ocular Effects. Only one reference was located that discussed the ocular effects of chloroform after oral ingestion. Li et al. (1994) examined the effects of chloroform administered in drinking water to guinea pigs with cedar pollen-induced allergic conjunctivitis, prepared by passive cutaneous anaphylaxis. Groups of 5 male Hartley guinea pigs were given drinking water with chloroform concentrations of 0.01, 0.1, 1, 10, 100, or 1,000 ppm 48 hours before applying an antigen eye drop (starting on the 8th day after antiserum administration). One control group was not administered chloroform and another control group was not administered the antiserum (chloroform alone) for every dose level. The light absorption rate of Evans blue extracted from conjunctiva was used as an index of the relative intensity of allergic conjunctivitis. In a separate experiment, using the dose level which caused the most intense aggravating effect in the above testing, groups of 3 male guinea pigs were given 1 ppm chloroform in drinking water for 48 hours and the residual effect on the allergic conjunctivitis was examined. Animals were examined immediately after, and 1, 2, 4, 7, and 14 days after exposure; antigen eye drops were applied 10 days after the antiserum administration. Water intake was monitored and blood chloroform concentrations were measured. At 0.1 ppm chloroform, significant aggravation of allergic conjunctivitis was observed. Allergic conjunctivitis was most intensely aggravated at 1 ppm chloroform. At higher doses (10 and 100 ppm) the aggravation was still noticeable, yet less significant. At 1,000 ppm chloroform, the aggravating effect was not present. Blood chloroform concentrations increased as the concentration in drinking water increased from 0.01 to 1,000 ppm. CHLOROFORM 89 2. HEALTH EFFECTS Body Weight Effects. No studies were located regarding body weight effects in humans after oral exposure to chloroform. Several studies were located regarding body weight changes in animals after oral exposure to chloroform; however, the effect of chloroform on body weight is variable and depends somewhat on the dose and dosing method. Body weight was unaffected in male Wistar rats receiving single doses of chloroform ranging from 0.1 to 0.5 mL/kg (Nakajima et al. 1995), in female B6C3F, mice exposed to 263 mg/kg/day chloroform by gavage in corn oil for 5 or 12 days (Pereira 1994), and in male rats exposed once to 477 mg/kg day of chloroform (Larson et al. 1993), or for 4 days in male mice dosed at 277 mg/kg/day by gavage (Larson et al. 1994d). When female mice were exposed to 1,800 ppm chloroform in drinking water (24 hours a day) for 5 or 12 days, body weight initially declined; however, this was attributed to decreased water consumption (Pereira 1994). A dose-related decrease in body weight gain was observed in rats exposed to 100 mg/kg/day and in rabbits exposed to 50 mg/kg/day chloroform by gavage in oil during gestation (Ruddick et al. 1983; Thompson et al. 1974). In addition, decreased body weight was observed in male mice after acute exposure to 250 mg/kg/day chloroform by gavage in oil (Gulati et al. 1988; Munson et al. 1982). Others have reported similar reductions in body weight after oral dosing with chloroform (Davis and Berndt 1992, 1994b, 1995a, 1995b; Reddy et al. 1992). In studies of intermediate duration, dose-related decreases in body weight or body weight gain were observed in rats exposed to >81 mg/kg/day in water (Jorgenson and Rushbrook 1980) or in oil (Larson et al. 1994b, 1995b; NCI 1976) and in mice (Bull et al. 1986; Klaunig et al. 1986; Roe et al. 1979). Similar effects were found in rats exposed to 260 mg/kg/day regardless of the vehicle (Jorgenson et al. 1985; Larson 1995a; NCI 1976; Palmer et al. 1979; Tumasonis et al. 1985) and mice exposed to 263 mg/kg/day in water (Jorgenson et al. 1985) in studies of chronic exposure; taste aversion may have been a complicating factor in these studies. In contrast, no effect on body weight was observed in mice treated with 477 mg/kg/day by gavage in oil (NCI 1976) or dogs treated with 30 mg/kg/day chloroform (Heywood et al. 1979) in studies of chronic duration. Food and/or water consumption were decreased in chloroform-exposed animals in some studies (Chu et al. 1982a; Jorgenson and Rushbrook 1980), but others reported fluctuating food intake unrelated to chloroform exposure (Palmer et al. 1979) or no significantly depressed food consumption at the lowest LOAEL level for body weight effects (Thompson et al. 1974). The effects chloroform has on changes in body weight and water consumption when administered orally in different vehicles and varying doses have also been CHLOROFORM 90 2. HEALTH EFFECTS reported in female mice (Pereira 1994). In this study, female B6C3F| mice received 263 mg/kg/day chloroform by gavage in corn oil or 1,800 ppm/day chloroform in their drinking water, and animals were sacrificed at 5, 12, 33, or 159 days. Chloroform administered by gavage did not affect body weight; however, when administered in drinking water, body weights in exposed animals initially declined, but increased by day 33 to control levels. This was attributed to changes in drinking-water consumption, which was suppressed during the first 5 days but was greater than that of controls from days 6-12. 2.2.2.3 Immunological and Lymphoreticular Effects No studies were located regarding immunological effects in humans after oral exposure to chloroform. Information regarding immunological effects in animals after oral exposure to chloroform is limited to three studies. Reduced lymphocyte counts were observed in female rats after a single gavage dose of 1,071 mg/kg chloroform (Chu et al. 1982b); no effects were observed in the 765 mg/kg group. Humoral immunity, defined as antibody-forming cells (AFC)/spleen x 100,000, was depressed in both sexes of mice after oral dosing with 50 mg/kg/day chloroform for 14 days (Munson et al. 1982). In contrast, hemagglutination titer was not significantly influenced, and no changes in cell-mediated immunity were recorded. Similar results were obtained in a 90-day experiment (Munson et al. 1982). Depressed humoral immunity was observed in mice exposed to 50 mg/kg/day chloroform. Cell- mediated immunity (delayed-type hypersensitivity) was affected in the high-dose (250 mg/kg/day) group of females. The chloroform-induced changes were more marked in the 14-day study than in the 90-day study. Although the data are limited, there are indications that the immune system is a target of chloroform-induced toxicity after oral exposure. The data also indicate that humoral immunity may be more severely affected than cell-mediated immunity. These conclusions, however, should be viewed with caution due to the small number of studies. Li et al. (1994) examined the effects of chloroform administered in drinking water to guinea pigs with cedar pollen-induced allergic conjunctivitis, prepared by passive cutaneous anaphylaxis. Groups of 5 male Hartley guinea pigs were given drinking water with chloroform concentrations of 0.01, 0.1, 1, 10, 100, or 1,000 ppm 48 hours before applying an antigen eye drop (starting on the 8th day after antiserum administration). At 0.1 ppm chloroform, significant aggravation of allergic conjunctivitis was observed. Allergic conjunctivitis was most intensely aggravated at 1 ppm chloroform. At higher CHLOROFORM 91 2. HEALTH EFFECTS doses (10 and 100 ppm) the aggravation was still noticeable, yet less significant. At 1,000 ppm chloroform, the aggravating effect was not present. The highest NOAEL value and all reliable LOAEL values for immunological effects in each species and duration category are recorded in Table 2-2 and plotted in Figure 2-2. 2.2.2.4 Neurological Effects The data regarding neurological effects in humans after oral exposure to chloroform were obtained from clinical case reports. Deep coma occurred immediately after exposure to 2,410 or 3,755 mg/kg in all cases of intentional or accidental ingestion of chloroform (Piersol et al. 1933; Schroeder 1965; Storms 1973). All reflexes were abolished, and pupil size varied. All patients survived the first coma and became fully conscious; however, one patient died in coma several days later due to extensive liver necrosis (Piersol et al. 1933). Mild cerebellar damage (instability of gait, intentional tremor) was observed in one patient, but reversed to normal in two weeks (Storms 1973). The central nervous system in animals is a target of chloroform toxicity after oral exposure to chloroform. High single doses of chloroform caused ataxia, incoordination, and anesthesia in mice (Balster and Borzelleca 1982; Bowman et al. 1978). Sprague-Dawley rats administered a single 200 mg/kg dose of chloroform gavage experienced significant decreases in midbrain 5-hydroxy- indolacetic acid (5-HIAA) levels and significant increases in hypothalamic dopamine concentrations 2 hours after dosing (p<0.05) (Kanada et al. 1994). The calculated EDs, (dose is effective on 50% of animals) for motor performance was 484 mg/kg chloroform (Balster and Borzelleca 1982). The effects disappeared within 90 minutes postexposure. A minimal narcotic dose for 50% of the treated mice was calculated to be 350 mg/kg (Jones et al. 1958). Hunched posture and inactivity were observed in male mice exposed by gavage to 250 mg/kg chloroform in oil for 14 days (Gulati et al. 1988). No effects were observed after exposure to 100 mg/kg day. Hemorrhaging in the brain was observed during gross pathological examinations of mice that died under chloroform anesthesia following doses >500 mg/kg/day (Bowman et al. 1978). Lower concentrations of chloroform-induced taste aversion to a saccharin solution in mice exposed by gavage for 10 days to 30 mg/kg/day in oil, but not in mice exposed to 10 mg/kg/day (Landauer et al. 1982). No signs of behavioral toxicity were observed in mice exposed to 31.1 mg/kg/day chloroform for 14, 60, or 90 days, or in mice exposed to 100 mg/kg for 30 days (Balster and Borzelleca 1982). Operant behavior in mice was affected after exposure to CHLOROFORM 92 2. HEALTH EFFECTS 100 mg/kg/day for 60 days (Balster and Borzelleca 1982). The most severe effects were observed early in the experiment; partial tolerance was observed later. No histopathological changes were observed in the brains of rats after chronic exposure to 200 mg/kg/day, in the brains of mice after chronic exposure to 477 mg/kg/day (NCI 1976), or in the brains of mice after chronic exposure to 60 mg/kg/day (Roe et al. 1979). The highest NOAEL values and all reliable LOAEL values for neurological effects in each species and duration category are recorded in Table 2-2 and plotted in Figure 2-2. 2.2.2.5 Reproductive Effects No definitive studies were located regarding reproductive effects in humans after oral exposure to chloroform. However, in a study by Bove et al. (1995), the effects of drinking-water consumption on birth outcomes were evaluated in women giving birth in northern New Jersey during the period of Jan 1, 1985 to Dec 31, 1988. A total of 80,938 live births and 594 fetal deaths were studied. Exposure to total trihalomethane (TTHM) levels >0.1 ppm resulted in a 70.4 g reduction in mean birth weight among term babies, increased odds ratio (OR) for low birth weight among term births (1.42), an increased OR for reduced size at gestational age birth (1.50), and an increased OR for oral cleft defects (3.17). In addition, exposure to TTHM of >0.08 ppm resulted in an increased OR for central nervous system defects (2.59) and neural tube defects (2.96). The results of this study should be viewed with caution since the outcomes data were not correlated directly with chloroform concentrations, but rather with TTHM concentrations; hence, the effects observed may be due to exposure to other THMs. The authors of this study also acknowledged the presence of other non-THM contaminants, and that some or all of these contaminants may have contributed to the observed effects as well. In rats, increased resorptions were observed at a dose of 316 mg/kg/day chloroform during gestation but not at 300 mg/kg/day; increased resorptions were also observed in rabbits exposed to 100 mg/kg/day during gestation (Thompson et al. 1974). Furthermore, abortions (not otherwise specified) were observed in rabbits exposed to 63 mg/kg/day chloroform during gestation (Thompson et al. 1974). No histopathological changes were observed in the testes of rats exposed to 160 mg/kg/day chloroform in drinking water for intermediate durations (Jorgenson and Rushbrook 1980). Gonadal atrophy was observed in both sexes of rats treated by gavage with 410 mg/kg/day CHLOROFORM 93 2. HEALTH EFFECTS chloroform in toothpaste (but not with 150 mg/kg/day) (Palmer et al. 1979). In a 2-generation reproductive study in mice, exposure to 41 mg/kg/day of chloroform by gavage in oil did not affect the fertility in either generation (Gulati et al. 1988). No remarkable histopathological differences regarding the reproductive system were observed in dogs receiving up to 30 mg/kg/day of chloroform delivered in toothpaste capsules for 7.5 years (Heywood et al. 1979). No histopathological changes were observed in the reproductive organs of male and female rats and mice chronically exposed to 200 and 477 mg/kg/day chloroform via gavage (NCI 1976). The highest NOAEL values and all reliable LOAEL values for reproductive effects in each species and duration category are recorded in Table 2-2 and plotted in Figure 2-2. 2.2.2.6 Developmental Effects One study (Kramer et al. 1992) was located regarding developmental effects in humans after oral exposure to chloroform via the drinking water. The study was conducted to determine whether water supplies containing relatively high levels of chloroform and other THMs within the state of Iowa are associated with low birth weight, prematurity, or intrauterine growth retardation (the most sensitive end point). Subjects selected include 159 low-birth-weight infants, 342 premature infants, and 187 growth- retarded infants; however, case definitions were not mutually exclusive. Infants studied were divided into three groups: those who lived in areas where the water supply had undetectable amounts of chloroform, those who lived in areas where the water supply had 1-9 pg/L chloroform, and those who lived in areas where the water supply had more than 10 pg/L. The estimated relative risk of low birth weight associated with drinking-water sources having chloroform levels of greater than or equal to 10 pg/L was 30% higher than the risk for sources with undetectable levels of chloroform. Prematurity was not associated with chloroform/THM exposure. The estimated relative risk of intrauterine growth retardation associated with drinking-water supplies with chloroform concentration of >10 pg/L was 80% more than the risk for those sources with undetectable levels of chloroform. Sources with intermediate chloroform levels (1-9 pg/L) had an elevated risk of 30%. The authors concluded that there is an increased risk of intrauterine growth retardation associated with higher concentrations of waterborne chloroform and dichlorobromomethane; however, it also should be noted that other organic halides that can co-occur in chlorinated drinking water (haloacetic acids and haloacetonitriles) produce developmental effects in animals. CHLOROFORM 94 2. HEALTH EFFECTS No teratological effects or skeletal anomalies in rats or rabbits after oral exposure to chloroform were reported in developmental studies (Ruddick et al. 1983; Thompson et al. 1974). Decreased fetal weight was observed in the offspring of rats exposed by gavage to 400 mg/kg/day chloroform during gestation, but not in those exposed to 200 mg/kg/day (Ruddick et al. 1983). In a preliminary dose- finding study, decreased fetal weight and increased resorptions were observed in rats exposed to 316 mg/kg/day chloroform during gestation (Thompson et al. 1974). In the principal study, reduced birth weight of the offspring was reported in the 126 mg/kg/day group; no effects were observed in the 50 mg/kg/day exposure group. No behavioral effects were observed in the offspring of the Fy generation mice treated for 6-10 weeks with 31.1 mg/kg chloroform (Burkhalter and Balster 1979). In a 2-generation reproductive study, increased epididymal weights and degeneration of epididymal ductal epithelium were observed in mice in the F, generation dosed with 41 mg/kg/day in oil (Gulati et al. 1988). The production and viability of sperm was not affected, however. Swiss mice given drinking water containing a mixture of contaminants including 7 ppm chloroform experienced no significant developmental effects. In the same study, Sprague-Dawley rat pups of the F, generation had lower body weights from birth through mating; however, this was likely an artifact of decreased water intake. No other developmental effects were noted (Heindel et al. 1995). These data are limited by the possible interactions caused by the concurrent exposure to other water contaminants. The highest NOAEL values and all reliable LOAEL values for developmental effects in each species and duration category are recorded in Table 2-2 and plotted in Figure 2-2. 2.2.2.7 Genotoxic Effects No studies were located regarding genotoxic effects in humans after oral exposure to chloroform. Unscheduled DNA synthesis (UDS) in hepatocytes was not increased in rats exposed to chloroform at gavage doses 2400 mg/kg in oil (Mirsalis et al. 1982). Exposure to 200 mg/kg/day chloroform in oil by gavage for 4 days increased sister chromatid exchange frequency in bone marrow cells of mice (Morimoto and Koizumi 1983). Other genotoxicity studies are discussed in Section 2.5. CHLOROFORM 95 2. HEALTH EFFECTS 2.2.2.8 Cancer Epidemiology studies suggest an association between cancer in humans and the consumption of chlorinated drinking water, but the results are not conclusive at this time (Alavanja et al. 1978; Cantor et al. 1978; Ijsselmuiden et al. 1992; McGeehin et al. 1993; Young et al. 1981; Zierler et al. 1988). Such an association implicates chloroform because chloroform is a known animal carcinogen (see below) and is the predominant THM in chlorinated drinking water (see Chapter 5); however, it is important to note that some of the many chemicals produced in the process of water chlorination are highly mutagenic and/or carcinogenic. Although attempts were made to control for various demographic variables in all of these studies (e.g., social class, ethnic group, marital status, occupation, urban or rural, etc.), many confounding effects remained unaccounted for, most notably the likelihood that numerous chemicals other than chloroform were present in the drinking water, as stated above. Furthermore, the studies differed regarding the type of cancer associated with consumption of chlorinated water. Bladder cancer was reported to have the strongest association with chlorinated water in several studies (Cantor et al. 1978; McGeehin et al. 1993; Zierler et al. 1988), but only colon cancer had an elevated OR (3.6) in another study (Young et al. 1981). In addition, Ijsselmuiden et al. (1992) found the use of municipal water to be associated with pancreatic cancer. All these studies superficially suggest that low-level oral chloroform consumption may increase the risk of some cancers in humans; however, it is equally important to note that most of these studies had confounding factors that make it difficult to definitively state that chloroform is the chemical chiefly responsible for the induction of these specific types of cancer. Confounding factors, such as the presence of other THMs (i.e., brominated THMs), haloacetic acids, haloacetonitriles, halogenated aldehydes, ketones and furanones, and chlorine content (both free and total), all of which may vary widely from one chlorinated drinking-water source to another, may have a large influence on the incidence of these cancers. In addition, many of these studies did not account for migration and historical exposures to any THMs, the wide ranges of potential exposure doses, occupational exposures to other chemicals, and the lack of a direct measurement of chloroform (or other THM) consumption in the drinking-water source (Cantor et al. 1978; Ijsselmuiden et al. 1992; Young et al. 1981; Zierler et al. 1988). Overall, the human data are insufficient to support any conclusion regarding the carcinogenic potential of orally consumed chloroform in humans. Differing results on the carcinogenic capabilities of chloroform have been demonstrated in laboratory animals. Chloroform is carcinogenic in laboratory animals after oral exposure in some studies of CHLOROFORM 96 2. HEALTH EFFECTS intermediate durations. An increased incidence of hepatomas was observed in mice exposed by gavage for 30 days to 594 mg/kg/day chloroform in oil, but not in mice exposed to 297 mg/kg/day (Eschenbrenner and Miller 1945a). An 8-week exposure to 1,800 mg/kg/day chloroform in oil by gavage did not induce lung tumors in mice (Stoner et al. 1986). Chloroform in corn oil acted as a promoter rather than an initiator of preneoplastic foci in a rat liver bioassay (Deml and Oesterle 1985). In addition, no increase in tumors was found in mice exposed to 257 mg/kg/day chloroform in drinking water for 52 weeks (Klaunig et al. 1986). Interestingly, Reddy et al. (1992) dosed male rats with 14, 25, 52, and 98 mg/kg/day of chloroform in the drinking water for 12 weeks. The study conclusively showed that chloroform, at the doses administered and routes studied in the rat, reduced the number of preneoplastic enzyme-altered foci (gamma-glutamyltranspeptidase-positive and GSH S-transferase-positive) in the liver of male rats after induction of foci with diethylnitrosamine in a dose-related fashion. The exact mechanism behind this effect was not determined. Chloroform was found to be carcinogenic in several chronic animal studies of oral exposure. Renal tumors (tubular cell adenoma and carcinoma) were observed in male Osborne-Mendel rats after a 78-week exposure to 90 mg/kg/day chloroform by gavage in corn oil (NCI 1976). Dunnick and Melnick (1993) demonstrated the incidence of liver and kidney tumors in rats and mice dosed by gavage in corn oil for 5 days a week for 78 weeks. In rats, kidney tubular cell neoplasms did not occur in controls but were observed at 90 mg/kg/day (4 of 50) and at 180 mg/kg/day (12 of 50) in males, and at 200 mg/kg/day (2 of 48) in females. In male mice, hepatocellular neoplasms were rarely seen in controls (1 of 18), but were frequently observed in the 138 mg/kg group (18 of 50) and 277 mg/kg group (44 of 45). In female mice, no hepatocellular neoplasms were recorded in controls but were observed in the 238 mg/kg group (36 of 45) and 477 mg/kg group (39 of 41). The incidence of hepatic neoplastic nodules was increased in female Wistar rats chronically exposed to 200 mg/kg/day chloroform in drinking water (Tumasonis et al. 1987). An increased incidence of tubular cell adenoma and carcinoma was observed in the kidneys of Osborne-Mendel rats chronically exposed to 160 mg/kg/day chloroform in drinking water but not in those exposed to 81 mg/kg/day (Jorgenson et al. 1985). The 160 mg/kg/day dose in this study also resulted in decreased water consumption (taste aversion). In contrast, no increase in the incidence of tumors was observed in Sprague-Dawley rats exposed by gavage to 60 and 165 mg/kg/day chloroform in toothpaste for 80 and 52 weeks, respectively (Palmer et al. 1979). Hepatocellular carcinoma was observed in all groups of male B6C3F, mice exposed to gavage doses >138 mg/kg/day chloroform in oil for 78 weeks (NCI 1976). An increased incidence of kidney tumors was observed in ICI mice chronically exposed to CHLOROFORM 97 2. HEALTH EFFECTS 60 mg/kg/day chloroform by gavage, but not in those exposed to 17 mg/kg/day (Roe et al. 1979). Under the same experimental conditions, chloroform exposure had no effect on the frequency of tumors in C57BL, CBA, and CF-1 mice. Moreover, no increase in tumor incidence was observed in B6C3F| mice exposed to 263 mg/kg/day chloroform in drinking water for 2 years (Jorgenson et al. 1985). Cancer was not observed in dogs exposed to 30 mg/kg/day chloroform in toothpaste capsules for 7.5 years (Heywood et al. 1979). From these data it would appear that the method of dosing (e.g. gavage versus drinking water) and the vehicle utilized may influence outcomes in chronic trials. The CELs (cancer effect levels) are recorded in Table 2-2 and plotted in Figure 2-2. EPA (IRIS 1995) selected the study by Jorgenson et al. (1985) as the basis for the a for oral exposure to chloroform because administration via drinking water better approximates oral exposure in humans than does administration in corn oil by gavage as used in the NCI (1976) study. Based on the incidence of renal tumors in male Osborne-Mendel rats, the a was calculated to be 6.1x107 (mg/kg/day). The oral doses associated with individual lifetime upper-bound risks of 10% to 1077 are 1.6x1072 to 1.6x107 mg/kg/day, respectively, and are plotted in Figure 2-2. 2.2.3 Dermal Exposure 2.2.3.1 Death No studies were located regarding death in humans after dermal exposure to chloroform. No deaths resulted from dermal exposure of rabbits exposed to doses of up to 3,980 mg/kg chloroform for 24 hours (Torkelson et al. 1976). 2.2.3.2 Systemic Effects No studies were located regarding respiratory, cardiovascular, gastrointestinal, hematological, musculoskeletal, or ocular effects in humans or animals after dermal exposure to chloroform. Hepatic Effects. No studies were located regarding hepatic effects in humans after dermal exposure to chloroform. CHLOROFORM 98 2. HEALTH EFFECTS No hepatic effects were observed in rabbits when 3,980 mg/kg chloroform was applied to the abdominal skin for 24 hours (Torkelson et al. 1976). The NOAEL for hepatic effects is recorded in Table 2-3. Renal Effects. No studies were located regarding renal effects in humans after dermal exposure to chloroform. Renal degenerative tubular changes were observed in rabbits when 1,000 mg/kg chloroform was applied to the abdominal skin for 24 hours (Table 2-3) (Torkelson et al. 1976). Dermal Effects. Completely destroyed stratum corneum was observed in the skin of 2 young volunteers exposed to chloroform for 15 minutes on 6 consecutive days (Malten et al. 1968). Milder changes were observed in two older individuals. Chloroform was applied in a glass cylinder (exact exposure was not specified). A clinical study of 21 females and 21 males used to determine the efficacy of using aspirin dissolved in chloroform which was then applied topically to patients infected with herpes zoster and post-- therapeutic neuralgia with painful skin lesions has been reported. When an aspirin/chloroform combination (approximately 43.3 mg/mL) was applied, the only reported side-effect was an occasional burning sensation on the skin as the chloroform evaporated from the skin surface; however, the possible impact on other major body organs (liver, kidney, etc.) was not investigated (King 1993). Application of 0.01 mL chloroform for 24 hours to the skin of rabbits caused only slight irritation (Smyth et al. 1962). Skin necrosis was observed in rabbits dermally exposed to 1,000 mg/kg chloroform for 24 hours (Torkelson et al. 1976). These LOAEL values are recorded in Table 2-3. Body Weight Effects. No studies were located regarding body weight effects in humans after dermal exposure to chloroform. Dermal exposure to 1,000 mg/kg chloroform for 24 hours caused weight loss in rabbits (Table 2-3) (Torkelson et al. 1976). Table 2-3. Levels of Significant Exposure to Chloroform - Dermal LOAEL Exposure/ Species/ duration/ System NOAEL Less Serious Serious Reference (strain) frequency ACUTE EXPOSURE Systemic Rabbit 24 hr Dermal 0.01mL M (slight skin irritation) Smyth et al. 1962 (New Zealand) Rabbit 24 hr Hepatic 3980 Torkelson et al. (NS) mg/kg 1976 Renal 1000 (degenerative tubular mg/kg changes) Dermal 1000 (necrosis) mg/kg Bd Wt 1000 (unspecified weight loss) mg/kg Bd Wt = body weight; hr = hour(s); LOAEL = lowest-observed-adverse-effect-level; M = male; NOAEL = no-observed-adverse-effect-level; NS = not specified S103443 H1TV3H °C WHO40HO THO CHLOROFORM 100 2. HEALTH EFFECTS No credible studies were located regarding the following health effects in humans or animals after dermal exposure to chloroform: 2.2.3.3 Immunological and Lymphoreticular Effects 2.2.3.4 Neurological Effects 2.2.3.5 Reproductive Effects 2.2.3.6 Developmental Effects 2.2.3.7 Genotoxic Effects Genotoxicity studies are discussed in Section 2.5. 2.2.3.8 Cancer No studies were located regarding cancer in humans or animals after dermal exposure to chloroform. 2.3 TOXICOKINETICS Overview. Sufficient information exists on the absorption, distribution, metabolism, and excretion of chloroform, with most information on the pharmacokinetics being derived from animal data. Generally, chloroform is absorbed easily into the blood from the lungs after inhalation exposures. Following oral exposure, peak blood levels are achieved within 5-6 minutes, depending on the dosing vehicle and dosing frequency used. The chemical properties of chloroform also permit percutaneous absorption without difficulty. After absorption, chloroform has been reported to distribute to adipose tissues, brain, liver, kidneys, blood, adrenals, and embryonic neural tissues. Higher levels of chloroform can be found in the renal cortex of male animals than in female animals, a finding apparently mediated by the presence of testosterone. Approximately 50% of a dose of chloroform is eventually metabolized to carbon dioxide in humans; however, an intermediate toxic metabolite, phosgene, is formed in the process in the liver. Chloroform undergoes metabolism primarily in the liver and may undergo covalent binding to both lipid and microsomal protein. Chloroform is excreted from the body either unchanged by pulmonary desorption or in the form of carbon dioxide, with small amounts of either detectable in the urine and feces. CHLOROFORM 101 2. HEALTH EFFECTS 2.3.1 Absorption 2.3.1.1 Inhalation Exposure Chloroform absorption depends on the concentration in inhaled air, the duration of exposure, the blood/air partition coefficient, the solubility in various tissues, and the state of physical activity which influences the ventilation rate and cardiac output. Pulmonary absorption of chloroform is also influenced by total body weight and total fat content, with uptake and storage in adipose tissue increasing with excess body weight and obesity. In inhalation exposures, the arterial blood concentration of chloroform is directly proportional to the concentration in inhaled air. At anesthetic concentrations (8,000-10,000 ppm), steady-state arterial blood concentrations of chloroform were 7-16.2 mg/mL (Smith et al. 1973). Total body equilibrium with inspired chloroform concentration required at least two hours in normal humans at resting ventilation and cardiac output (Smith et al. 1973). The amount of chloroform absorbed and exhaled from the body in alveolar air from male and female swimmers in indoor swimming pools in Italy was measured by Aggazzotti et al. (1993). Alveolar air samples were collected from both swimmers and observers present in indoor chlorinated swimming pools. Of all the nonexposed subjects, 47% had chloroform concentrations below the detection limit of the assay, and the remainder of this control group had low concentrations (75.39 nmol/m?) of chloroform present in their alveolar air. Median alveolar chloroform concentrations for persons exposed to the indoor swimming pools (swimmers and observers), were significantly higher than those of nonexposed subjects (median=695.02 nmol/m°). No differences were found between males and females in any exposure group. Cammann and Huebner (1995) attempted to correlate chloroform exposure with blood and urine chloroform concentrations in persons using indoor swimming pools. Water and air samples were collected from three swimming pools in Germany, with blood and urine samples collected from attendants, normal swimmers, and agonistic swimmers before and after environmental exposure. Pool water chloroform levels ranged from 3.04 to 27.8 pg/L, while air concentrations ranged from 7.77 to 191 pg/m3. In general, blood chloroform levels increased with exposure. Blood levels were lowest in attendants (0.13-2.45 pg/L), followed by normal swimmers (0.56—1.65 pg/L) and agonistic swimmers CHLOROFORM 102 2. HEALTH EFFECTS (1.14-5.23 pg/L). Based upon the differences seen in the two swimming groups, the authors concluded that increased physical activity leads to increased absorption and/or ingestion of chloroform. In a similar study, Levesque et al. (1994) attempted to quantitate the body burden of chloroform following exposure in an indoor pool. Scuba divers were exposed to chloroform-laden water and air on each of seven days. On each exposure day, the subjects exercised for a 55-minute period; alveolar air samples were collected before exercise and after 35 or 55 minutes of exercise. Pre-exercise alveolar levels of chloroform averaged 52.6 ppb; this was attributed to air contamination in the locker room. Alveolar air concentrations of chloroform after 35 and 55 minutes of exercise increased steadily through day 5, averaging 100-950 and 104-1,093 ppb, respectively. On day 6, when scuba gear was worn by the subjects, alveolar air concentrations after 35 and 55 minutes of exercise were 196 and 209 ppb, respectively. The authors concluded from this data that the average proportion of body burden due to inhalation after 35 and 55 minutes exercise was 76 and 78%, respectively. Nashelsky et al. (1995) described one non-fatal assault and three deaths in which chloroform was utilized. Blood and/or tissue concentrations of chloroform were determined in the assault victim and one decedent within 24 hours, within 10 days in another decedent who was frozen for the majority of that period, and after 5 months without preservation in the last decedent. Blood concentrations in 2 decedents were 2 and 3 pg/mL; fat concentrations were 10 and 42 pg/mL; brain concentrations were 3 and 46 pg/mL; and the liver concentration in one decedent was 24 pg/mL. Due to the nature of the tissues analyzed, these data should be regarded as qualitative indicators of chloroform absorption only. No studies were located regarding absorption in animals after inhalation exposure to chloroform. Evidence that chloroform is absorbed after inhalation exposure is provided in toxicity studies (see Section 2.2.1), but the rate and extent cannot be determined from the toxicity data. 2.3.1.2 Oral Exposure In one case report, a 33-year-old female (weight not reported) injected herself intravenously with 0.5 mL of chloroform and became unconscious. She awoke approximately 12 hours later and drank another 120 mL of chloroform. Plasma chloroform levels were determined 18 hours after ingestion by gas chromatography (GC) and showed a blood chloroform level of 0.66 mg/dL. Subsequent serum CHLOROFORM 103 2. HEALTH EFFECTS samples were analyzed for chloroform content and were reported to have been less than this level, steadily declining over time (Rao et al. 1993). Absorption of an oral dose of 13C_labeled chloroform (0.5 grams in a gelatin capsule) was rapid in volunteers, reaching peak blood levels in 1 hour (Fry et al. 1972). Almost 100% of the dose was absorbed from the gastrointestinal tract. Experiments in mice, rats, and monkeys indicate that oral doses (60 mg/kg) of 14C-1abeled chloroform in olive oil were almost completely absorbed as indicated by a 80-96% recovery of radioactivity in expired air, urine, and carcass (Brown et al. 1974a; Taylor et al. 1974). Absorption in mice and monkeys was rapid; the peak blood levels were reached 1 hour after oral administration of 60 mg/kg chloroform in olive oil. Intestinal absorption of chloroform in either water or corn oil administered intragastrically to rats was rapid with both vehicles, but the rate and extent of absorption varied greatly (Withey et al. 1983). The peak concentration of chloroform in blood was 39.3 pg/mL when administered in water and 5.9 pg/mL when administered in corn oil. The greater degree of absorption following administration in water can be explained by the faster partitioning of a lipophilic compound such as chloroform with mucosal lipids from an aqueous vehicle. Peak blood concentrations were reached somewhat more rapidly with the water vehicle (5.6 minutes as opposed to 6 minutes for corn oil). The uptake from a corn oil solution was more complex (pulsed) than from aqueous solution. A possible explanation for this behavior is that the chloroform in corn oil was broken up into immiscible globules, some of which did not come into contact with the gastric mucosa. Another possible explanation was that intragastric motility may have separated the doses into aliquots that were differentially absorbed from the gastrointestinal tract. In a similar study, Pereira (1994) investigated the uptake and protein binding of chloroform in the liver and kidney in female B6C3F, mice. Animals received single doses of chloroform by gavage in either water or corn oil. Uptake of chloroform from water into the liver peaked in 1.5 minutes, and hepatic uptake during the first 20 minutes exceeded that of chloroform delivered in oil. During the first 20 minutes after dosing, binding of chloroform to macromolecules in the liver was greater when water vehicle was utilized; beyond 20 minutes, the amount of binding was equivalent between the 2 vehicle groups. Renal uptake of chloroform from water exceeded uptake of chloroform from oil over the entire 4-hour period. The extent of binding to macromolecules in CHLOROFORM 104 2. HEALTH EFFECTS kidneys was consistently greater in the group given chloroform in water. Differences in chloroform toxicity based on the vehicle have also been recently reported elsewhere (Larson et al. 1994b, 1995a) 2.3.1.3 Dermal Exposure A limited number of experimental studies were located regarding dermal absorption of chloroform in humans. Levesque et al. (1994) attempted to quantitate the body burden of chloroform following dermal and inhalation exposure in an indoor swimming pool. Male scuba divers were exposed to chloroform-laden water and air on each of seven days. On each exposure day the subjects exercised for a 55-minute period. On day 6 of the experiment, subjects wore scuba gear so as to determine the percentage body burden due to dermal exposure. On day 6, when scuba gear was worn by the subjects, alveolar air concentrations after 35 and 55 minutes of exercise were 196 and 209 ppb, respectively. From this data it would appear that the average proportion of body burden due to dermal exposure after 35 and 55 minutes exercise was 24 and 22%, respectively. Cammann and Huebner (1995) attempted to correlate chloroform exposure with blood and urine chloroform concentrations in persons using indoor swimming pools. Water and air samples were collected from three pools in Germany, and blood and urine samples were collected from attendants, normal swimmers and agonistic swimmers before and after exposure. Pool water chloroform levels ranged from 3.04 to 27.8 pg/L, while air concentrations ranged from 7.77 to 191 g/m’. Blood chloroform levels generally increased with higher chloroform exposure levels. Blood levels were lowest in attendants (0.13-2.45 pg/L), followed by normal swimmers (0.56-1.65 pg/L) and agonistic swimmers (1.14-5.23 pg/L). Based upon the differences seen in the two swimming groups, the authors concluded that increased physical activity leads to increased absorption and/or ingestion. With the exception of the inclusion of attendants, the authors did not attempt to differentiate between inhalation and dermal absorption of chloroform. However, the increased blood concentrations seen in the swimmers seems to indicate that dermal absorption did indeed occur Dick et al. (1995) examined the absorption of chloroform through human skin in vivo using volunteers and in vitro using fresh, excised abdominal skin. In the in vivo study, the ventral forearm skin of four male volunteers was dosed with a solution of chloroform in either water or ethanol. The solution remained on the skin for eight hours. When administered in water, the total absorbed dose was 8.2%. In contrast, the total absorbed dose was only 1.68% when chloroform was administered in ethanol. In CHLOROFORM 108 2. HEALTH EFFECTS the in vitro study, two doses were applied to the skin and remained there for four hours. At study termination, the percentages of the low and high doses of chloroform absorbed were 5.6 and 7.1%, respectively. According to dermal absorption studies with solvents other than chloroform, the absorption of such solvents in guinea pigs is more rapid than the metabolism or pulmonary excretion (Jakobson et al. 1982). A dermal absorption rate of 329 nmol/minute/cm? (£60 nmol/minute/cm?) was calculated for the shaved abdominal skin of mice (Tsuruta 1975). This is equivalent to a human absorption rate of 29.7 mg/minute, assuming that a pair of hands are immersed in liquid chloroform (Tsuruta 1975). However, this calculation was based on the assumptions that the rate of chloroform penetration is uniform for all kinds of skin and that the total surface area of a pair of human hands is 800 cm?; the former assumption is especially dubious. Islam et al. (1995) investigated the fate of topically applied chloroform in male hairless rats. For exposures under 4 minutes, chloroform-laden water was applied to shaved back skin; for exposures of 4-30 minutes, rats were submerged in baths containing chloroform-laden water. Selected skin areas were tape-stripped a various number of times after various delay periods. It appeared that there was an incremental build-up of chloroform in the skin over the first four minutes. When compared to uptake measured by bath concentration differences, approximately 88% of lost chloroform was not accounted for in the stratum corneum and was assumed to be systemically absorbed. 2.3.2 Distribution 2.3.2.1 Inhalation Exposure Chloroform is lipid soluble and readily passes through cell membranes, causing narcosis at high concentrations. Blood chloroform concentrations during anesthesia (presumed concentrations 8,000-10,000 ppm) were 7-16.2 mg/mL in 10 patients (Smith et al. 1973). An arterial chloroform concentration of 0.24 mg/mL during anesthesia corresponded to the following partition coefficients: blood/gas, 8; blood/vessel rich compartment, 1.9; blood/muscle compartment, 1.9; blood/fat compartment, 31; blood/vessel poor compartment, 1; and blood/liver, 2 (Feingold and Holaday 1977). Recently, partition coefficients were calculated for humans based on results in mice and rats, and in human tissues in vitro: blood/air, 7.4; liver/air, 17; kidney/air, 11; and fat/air, 280 (Corley et al. 1990). CHLOROFORM 106 2. HEALTH EFFECTS The chloroform levels in 7 patients who died after excessive administration during anesthesia were: brain, 372-480 mg/kg; lungs, 355-485 mg/kg; and liver, 190-275 mg/kg (Gettler and Blume 1931). The chloroform levels in patients under anesthesia who died from other causes were: brain, 120-182 mg/kg; lungs, 92-145 mg/kg; and liver, 65-88 mg/kg tissue wet weight. Nashelsky et al. (1995) describe one nonfatal assault and three deaths in which chloroform was utilized. Blood and/or tissue concentrations of chloroform were determined in the assault victim and one decedent within 24 hours, within 10 days in another decedent who was frozen for the majority of that period, and after 5 months without preservation in the last decedent. Blood concentrations in 2 decedents were 2 and 3 pg/mL; fat concentrations were 10 and 42 pg/mL; brain concentrations were 3 and 46 pg/mL; and the liver concentration in one decedent was 24 pg/mL. After whole-body autoradiography to study the distribution of 14C.1abeled chloroform in mice, most of the radioactivity was found in fat immediately after exposure, while the concentration of radioactivity in the liver increased during the postanesthetic period, most likely due to covalent binding to lipid and protein in the liver (Cohen and Hood 1969). Partition coefficients (tissue/air) for mice and rats were 21.3 and 20.8 for blood; 19.1 and 21.1 for liver; 11 and 11 for kidney; and 242 and 203 for fat, respectively (Corley et al. 1990). Arterial levels of chloroform in mongrel dogs reached 0.35-0.40 mg/mL by the time animals were in deep anesthesia (Chenoweth et al. 1962). Chloroform concentrations in the inhaled stream were not measured, however. After 2.5 hours of deep anesthesia, there were 392 mg/kg chloroform in brain tissue, 1,305 mg/kg in adrenals, 2,820 mg/kg in omental fat, and 290 mg/kg in the liver. Radioactivity from 14C labeled chloroform was detected in the placenta and fetuses of mice shortly after inhalation exposure (Danielsson et al. 1986). In early gestation, accumulation of radioactivity was observed in the embryonic neural tissues, while the respiratory epithelium was more involved in chloroform metabolism in the late fetal period. Due to its lipophilic character, chloroform accumulates to a greater extent in tissues of high lipid content. As shown by the results presented above, the relative concentrations of chloroform in various tissues decreased as follows: adipose tissue > brain > liver > kidney > blood. CHLOROFORM 107 2. HEALTH EFFECTS 2.3.2.2 Oral Exposure No studies were located regarding distribution in humans after oral exposure to chloroform. High concentrations of radioactivity were observed in body fat and livers of rats, mice, and squirrel monkeys given oral doses of 60 mg/kg 14C-labeled chloroform (Brown et al. 1974a). The maximum levels of radioactivity in the blood appeared within 1 hour and were 3 pg equivalents chloroform/mL for mice and 10 pg equivalents chloroform/mL for monkeys, which represented =0.35 and 1%, respectively, of the total radioactivity. In monkeys, bile concentrations peaked within 6 hours. The distribution of radioactively labeled chloroform was studied in three strains of mice (Taylor et al. 1974). No strain-related differences were observed; however, higher levels of radioactivity were found in the renal cortex of males and in the liver of females. The renal binding of radioactive metabolites may have been altered by variations in the testosterone levels as a result of hormonal pretreatment in females or castration in males. Sex-linked differences in chloroform distribution were not observed in rats or monkeys (Brown et al. 1974a). Chloroform accumulates in the adipose tissue of rats after oral exposure of intermediate duration (Pfaffenberger et al. 1980). 2.3.2.3 Dermal Exposure A limited number of studies were located regarding distribution in humans or animals after dermal exposure to chloroform. Dick et al. (1995) examined the absorption of chloroform through human skin in vivo using volunteers and in vitro using fresh, excised abdominal skin. In the in vivo study, the ventral forearm skin of four male volunteers was dosed with a solution of chloroform in either water or ethanol. The solution remained on the skin for eight hours. When administered in water, urinary excretion was 0.42%, while excretion from the lungs over the first 48 hours postexposure averaged 7.8%. Tape-stripping data indicated that only 0.01% of the dose remained in the skin after 3 days. When chloroform was administered in ethanol, urinary excretion was 0.07% while excretion from the lungs over the first 48 hours postexposure averaged 0.83%. Tape-stripping data indicated that the percentage of the dose remaining in the skin after three days was non detectable. In the in vitro study, two doses were applied to the skin and remained there for four hours. At study termination, the majority of the CHLOROFORM 108 2. HEALTH EFFECTS absorbed dose was found in the perfusate (7.0%), with only minor amounts remaining in the skin (0.14%). Islam et al. (1995) investigated the fate of topically applied chloroform in male hairless rats. For exposures under 4 minutes, chloroform-laden water was applied to shaved back skin; for exposures of 4-30 minutes, rats were submerged in baths containing chloroform-laden water. Selected skin areas were tape-stripped a various number of times after various delay periods. The authors found that the accumulated amount of chloroform declined rapidly with depth of stratum corneum. As the time of exposure decreased, smaller amounts of chloroform were found in the deeper layers of stratum corneum; by five minutes postexposure, the amount of chloroform at the first tape strip (skin surface) dropped to negligible levels. It appeared that there was an incremental build-up of chloroform in the skin over the first four minutes. When compared to uptake measured by bath concentration differences, approximately 88% of the chloroform dose was not accounted for in the stratum corneum and was assumed to be systemically absorbed. 2.3.3 Metabolism The metabolism of chloroform is well understood. Approximately 50% of an oral dose of 0.5 grams of chloroform was metabolized to carbon dioxide in humans (Fry et al. 1972). Metabolism was dose- dependent, decreasing with higher exposure. A first-pass effect was observed after oral exposure (Chiou 1975). Approximately 38% of the dose was converted in the liver, and <17% was exhaled unchanged from the lungs before reaching the systemic circulation. On the basis of pharmacokinetic results obtained in rats and mice exposed to chloroform by inhalation, and of enzymatic studies in human tissues in vitro, in vivo metabolic rate constants (VC = 15.7 mg/hour/kg, K, = 0.448 mg/L) were defined for humans (Corley et al. 1990). The metabolic activation of chloroform to its toxic intermediate, phosgene, was slower in humans than in rodents. Metabolic pathways of chloroform biotransformation are shown in Figure 2-3. Metabolism studies indicated that chloroform was, in part, exhaled from the lungs or was converted by oxidative dehydrochlorination of its carbon-hydrogen bond to form phosgene (Pohl et al. 1981; Stevens and Anders 1981). This reaction was mediated by cytochrome P-450 and was observed in the liver and kidneys (Ade et al. 1994; Branchflower et al. 1984; Smith et al. 1984). In renal cortex microsomes of CHLOROFORM 109 2. HEALTH EFFECTS Figure 2-3. Metabolic Pathways of Chloroform Biotransformation MAJOR AEROBIC PATHWAY P450 O, H Gly oer Moms HoCCl, | -HCI Acceptor Protein H,0 nes. (YZ2C Cy rent. Z HCY + CO, CO Phosgene Cellular Protein Binding CO.H Glutathione Conjugates? / A 2-Oxothiazolidine Ne 4-Carboxylic Acid oO MINOR ANAEROBIC PATHWAY Phospholipid Binding P450 Anaerobic NADPH TS CHCl, > | cro) Reduced Microsomes Jue CO + 2 HCI CH,Cl, CHLOROFORM 110 2. HEALTH EFFECTS DBA/2J mice, the majority of chloroform metabolism was oxidative under ambient oxygen conditions, while anoxic conditions resulted in reductive metabolism (Ade et al. 1994). Phosgene may react with two molecules of GSH to form diglutathionyl dithiocarbonate, which is further metabolized in the kidneys, or it may react with other cellular elements and induce cytotoxicity (Pohl and Gillette 1984). In vitro studies indicate that phosgene and other reactive chloroform metabolites bind to lipids and proteins of the endoplasmic reticulum proximate to the cytochrome P-450 (Sipes et al. 1977; Wolf et al. 1977). The metabolism of chloroform to reactive metabolites occurs not only in microsomes but also in nuclear preparations (Gomez and Castro 1980). Covalent binding of chloroform to lipids can occur under anaerobic and aerobic conditions, while binding to the protein occurs only under aerobic conditions (Testai et al. 1987). It was further demonstrated that chloroform can induce lipid peroxidation and inactivation of cytochrome P-450 in rat liver microsomes under anaerobic conditions (De Groot and Noll 1989). Covalent binding of chloroform metabolites to microsomal protein in vitro was intensified by microsomal enzyme inducers and prevented by GSH (Brown et al. 1974b). It was proposed that the reaction of chloroform metabolites with GSH may act as a detoxifying mechanism. When GSH is depleted, however, the metabolites react with microsomal protein, and may cause necrosis. This is supported by observations that chloroform doses that caused liver GSH depletion produced liver necrosis (Docks and Krishna 1976). In fasted animals, chloroform has been found to be more hepatotoxic (Brown et al. 1974b; Docks and Krishna 1976) even though animals were found to have lower blood chloroform concentrations (Wang et al. 1995); this phenomenon would apparently be explained by a decreased GSH content and resultant inability to bind toxic metabolites. This may explain the clinical finding of severe acute hepatotoxicity in women exposed to chloroform via anesthesia during prolonged parturition. Evidence that chloroform is metabolized at its carbon- hydrogen bond is provided by experiments using the deuterated derivative of chloroform (Branchflower et al. 1984; McCarty et al. 1979: Pohl et al. 1980a). Deuterated chloroform was one- half to one-third as cytotoxic as chloroform, and its conversion to phosgene was much slower. The results confirmed that the toxicity of chloroform is primarily due to its metabolites. A recent in vitro study of mice hepatic microsomes indicated that a reductive pathway may also play an important role in chloroform hepatotoxicity (Testai et al. 1990). It was demonstrated that radical chloroform metabolites bind to macromolecules (proteins, lipids) and the process can be inhibited by reduced GSH. CHLOROFORM 191 2. HEALTH EFFECTS The final product of the aerobic metabolic pathway of chloroform is carbon dioxide (Brown et al. 1974a; Fry et al. 1972). This carbon dioxide is mostly eliminated through the lungs, but some is incorporated into endogenous metabolites and excreted as bicarbonate, urea, methionine, and other amino acids (Brown et al. 1974a). Chloride ions are an end product of chloroform metabolism found in the urine (Van Dyke et al. 1964). Carbon monoxide was a minor product of the anaerobic metabolism of chloroform in vitro (Ahmed et al. 1977) and in vivo in rats (Anders et al. 1978). A sex-related difference in chloroform metabolism was observed in mice (Taylor et al. 1974). Chloroform accumulated and metabolized in the renal cortex of males to a greater extent than in females, while liver chloroform concentrations were greater in females than in males; the results may have been influenced by testosterone levels. This effect was not observed in any other species and may explain why male mice were more susceptible to the lethal and renal effects of chloroform than were females (Deringer et al. 1953). Wang et al. (1994) found that, in male Wistar rats, pretreatment with ethanol increased chloroform metabolism about 1.5-fold but did not affect hepatic microsomal protein of cytochrome P-450 content. In addition, intraperitoneal administration of chloroform resulted in greater blood concentrations, peak values, and area of the curves (AUCs), as compared to oral administration. AUCs in rats administered chloroform orally ranged from 0.34 to 6.45 versus 0.58 to 8.78 in rats administered chloroform intraperitoneally. The authors concluded that differences between route groups in hepatotoxicity were due to differences in the proportion of dose exposed to first-pass metabolism. Since oral dosing results in the greatest first-pass exposure, this route resulted in the greatest hepatotoxicity. The degree of hepatic exposure also influenced the enhancing effect of ethanol; the group receiving chloroform orally was affected the most by ethanol pretreatment. The authors also concluded that intraperitoneal exposure produced data which most like that of inhalation exposure, presumably due to the smaller proportion of dose going through first-pass metabolism. Interspecies differences in the rate of chloroform conversion were observed in mice, rats, and squirrel monkeys, with species differences in metabolism being highly dose-dependant. The conversion of chloroform to carbon dioxide was highest in mice (80%) and lowest in squirrel monkeys (18%) (Brown et al. 1974a). Similarly, chloroform metabolism was calculated to be slower in humans than in rodents. Therefore, it was estimated that the exposure to equivalent concentrations of chloroform would lead to a much lower delivered dose in humans (Corley et al. 1990). CHLOROFORM 112 2. HEALTH EFFECTS A study by Gearhart et al. (1993) was conducted to determine the interactions of chloroform exposure with body temperature, gas uptake, and tissue solubility in mice as possible explanations for the difficulty in fitting a physiologically based pharmacokinetic/pharmacodynamic (PBPK) model to chloroform gas-uptake data to derive in vivo metabolic constants. Male mice were exposed to air concentrations of 100, 800, 2,000, or 5,500 ppm chloroform for 6 hours and their core body temperatures monitored frequently over the exposure period. After exposure, blood, liver, thigh muscle, and fat tissues were removed for tissue/air and tissue/blood partition coefficient analysis at 3 temperatures (25, 31, and 37 °C). For all tissues, tissue/air partition coefficients exhibited temperature-dependent decreases with increasing temperature. The rate of decrease was greatest for the blood/air partition coefficient. Average body temperatures for each exposure group decreased as the exposure concentrations increased. Temperature dependent decreases in core body temperature were hypothesized to decrease overall metabolism of chloroform in mice. The data collected were also used to develop a PBPK model for chloroform disposition. 2.3.4 Elimination and Excretion 2.3.4.1 Inhalation Exposure Chloroform was detected in the exhaled air of volunteers exposed to a normal environment, to heavy automobile traffic, or to air in a dry cleaning establishment (Gordon et al. 1988). Higher chloroform levels in the breath corresponded to higher exposure levels. The calculated biological half-time for chloroform was 7.9 hours. Excretion of radioactivity in mice and rats was monitored for 48 hours following exposure to 14 labeled chloroform (Corley et al. 1990). In general, 92-99% of the total radioactivity was recovered in mice, and 58-98% was recovered in rats; percentage of recovery decreased with increasing exposure. With increasing concentration, mice exhaled 80-85% of the total radioactivity recovered as C-labeled carbon dioxide, 0.4-8% as 14C.1abeled chloroform, and 8-11 and 0.6-1.4% as urinary and fecal metabolites, respectively. Rats exhaled 48-85% of the total radioactivity as 14C_Jabeled carbon dioxide, 2-42% as '4C-labeled chloroform, and 8-11 and 0.1-0.6% in the urine and feces, respectively. A 4-fold increase in exposure concentration was followed by a 50- and 20-fold increase in the amount of exhaled, unmetabolized chloroform in mice and rats, respectively. CHLOROFORM 113 2. HEALTH EFFECTS 2.3.4.2 Oral Exposure Following a single, oral exposure, most of the 0.5 grams of radioactively labeled chloroform administered to volunteers was exhaled during the first 8 hours after exposure (Fry et al. 1972). A slower rate of pulmonary excretion was observed during the first eight hours in volunteers who had more adipose tissue than the other volunteers. Up to 68.3% of the dose was excreted unchanged, and up to 50.6% was excreted as carbon dioxide. A positive correlation was made between pulmonary excretion and blood concentration. Less than 1% of the radioactivity was detected in the urine. Approximately 80% of a single dose of 60 mg/kg 14C labeled chloroform was converted within 24 hours to 14C-labeled carbon dioxide in mice (Brown et al. 1974a; Taylor et al. 1974), while only =66% of the dose was converted to 14C.1abeled carbon dioxide in rats (Brown et al. 1974a). Eight hours after administration of 100-150 mg/kg of “C-labeled chloroform, 49.6 and 6.5% of radioactivity was converted to carbon dioxide, 26.1 and 64.8% was expired as unmetabolized parent compound, and 4.9 and 3.6% was detected in the urine in mice and rats, respectively (Mink et al. 1986). These results indicate that mice metabolize high doses of chloroform to a greater degree than rats do. Only 18% of a chloroform dose was metabolized to C-labeled carbon dioxide in monkeys, and =79% was detected as unchanged parent compound or toluene soluble metabolites (Brown et al. 1974a). Within 48 hours after exposure, =2, 8, and 3% of the administered radioactivity was detected in the urine and feces of monkeys, rats, and mice, respectively. 2.3.4.3 Dermal Exposure One study was located regarding excretion in humans after dermal exposure to chloroform. Dick et al. (1995) examined the fate of chloroform applied to human skin in vivo using volunteers and in vitro using fresh, excised abdominal skin. In the in vivo study, the ventral forearm skin of four male volunteers was dosed with a solution of chloroform in either water or ethanol. The solution remained on the skin for eight hours. When administered in water, urinary excretion was 0.42%, while excretion from the lungs over the first 48 hours postexposure averaged 7.8%. Tape-stripping data indicated that only 0.01% of the dose remained in the skin after three days. When chloroform was administered in ethanol, urinary excretion was 0.07% while excretion from the lungs over the first 48 hours postexposure averaged 0.83%. Tape-stripping data indicated that the percentage of the dose remaining in the skin after three days was non detectable. In the in vitro study, two doses were CHLOROFORM +44 2. HEALTH EFFECTS applied to the skin and remained there for four hours. At study termination, the majority of the absorbed dose was found in the perfusate (7%), with only minor amounts remaining in the skin (0.14%). No animal studies were located regarding the excretion of chloroform after dermal exposure to chloroform. 2.3.5 Physiologically Based Pharmacokinetic (PBPK)/Pharmacodynamic (PD) Models Physiologically based pharmacokinetic (PBPK) models use mathematical descriptions of the uptake and disposition of chemical substances to quantitatively describe the relationships among critical biological processes (Krishnan et al. 1994). PBPK models are also called biologically based tissue dosimetry models. PBPK models are increasingly used in risk assessments, primarily to predict the concentration of potentially toxic moieties of a chemical that will be delivered to any given target tissue following various combinations of route, dose level, and test species (Clewell and Andersen 1985). Physiologically based pharmacodynamic (PBPD) models use mathematical descriptions of the dose-response function to quantitatively describe the relationship between target tissue dose and toxic end points. PBPK/PD models refine our understanding of complex quantitative dose behaviors by helping to delineate and characterize the relationships between: (1) the external/exposure concentration and target tissue dose of the toxic moiety, and (2) the target tissue dose and observed responses (Andersen et al. 1987: Andersen and Krishnan 1994). These models are biologically and mechanistically based and can be used to extrapolate the pharmacokinetic behavior of chemical substances from high to low dose, from route to route, between species, and between subpopulations within a species. The biological basis of PBPK models results in more meaningful extrapolations than those generated with the more conventional use of uncertainty factors. The PBPK model for a chemical substance is developed in four interconnected steps: model representation, model parameterization, model simulation, and (4) model validation (Krishnan and Andersen 1994). In the early 1990s, validated PBPK models were developed for a number of toxicologically important chemical substances, both volatile and nonvolatile (Krishnan and Andersen 1994; Leung 1993). PBPK models for a particular chemical substance require estimates of the CHLOROFORM 115 2. HEALTH EFFECTS chemical substance-specific physicochemical parameters, and species-specific physiological and biological parameters. The numerical estimates of these model parameters are incorporated within a set of differential and algebraic equations that describe the pharmacokinetic processes. Solving these differential and algebraic equations provides the predictions of tissue dose. Computers then provide process simulations based on these solutions. The structure and mathematical expressions used in PBPK models significantly simplify the true. complexities of biological systems. This simplification, however, is desirable if the uptake and disposition of the chemical substance(s) is adequately described because data are often unavailable for many biological processes and using a simplified scheme reduces the magnitude of cumulative uncertainty. The adequacy of the model is therefore of great importance and thus model validation must be critically considered. PBPK models improve the pharmacokinetic extrapolation aspects of the risk assessment process, which seeks to identify the maximal (i.e., safe) levels for human exposure to chemical substances (Andersen and Krishnan 1994). PBPK models provide a scientifically sound means to predict the target tissue dose of chemicals in humans who are exposed to environmental levels (for example, levels that might occur at hazardous waste sites) based upon the results of studies where doses were higher or were administered in different species. Figure 2-4 shows a conceptualized representation of a PBPK model. If PBPK models for chloroform exist, the overall results and individual models are discussed in this section in terms of their use in risk assessment, tissue dosimetry, and dose, route, and species extrapolations. 2.3.5.1 Summary of PBPK/PD Models Several rodent and human models have been used to predict the absorption (oral, inhalation, and dermal) from water and air, distribution, metabolism, and excretion of chloroform. In a PBPK model that used simulations with mice, rats, and humans (Corley et al. 1990), the tissue delivered dose from equivalent concentrations of chloroform was highest in the mouse, followed by rats and then humans. The authors suggest that this behavior is predicted by the model because of the lower relative rates of metabolism, ventilation, and cardiac output (per kg of body weight) in the CHLOROFORM 116 2. HEALTH EFFECTS Figure 2-4. Conceptual Representation of a Physiologically Based Pharmacokinetic (PBPK) Model for a Hypothetical Chemical Substance Inhaled chemical — — —4 © —> Exhaled chemical | y Ingestion Lungs |! TS | Liver < Vv \ ¥ A E | R N Vmax Km Gl [| T Oo Tract E uU < Fat R S A Slowly L ee] perfused « B tissues L 2 < Richly B D = perfused L tissues % D < Kidney Urine + Skin < A L — — Chemicals in air contacting skin Source: adapted from Krishnan et al. 1992 Note: This is a conceptual representation of a physiologically based pharmacokinetic (PBPK) model for a hypothetical chemical substance. The chemical substance is shown to be absorbed via the skin, by inhalation, or by ingestion, metabolized in the liver, and excreted in the urine or by exhalation. CHLOROFORM 117 2. HEALTH EFFECTS larger species. Assuming that equivalent target doses produce equivalent toxicities in target tissues, the relative sensitivities of the three species used in the study (mouse > rat > human) predicted by the model under identical exposure conditions are quite different from the relative sensitivity to chloroform assumed by the "uncertainty factor." In a PBPK/PD model based closely on the Corley model, Reitz et al. (1990) described a pharmaco- dynamic end point (cytotoxicity) in the livers of chloroform-exposed animals produced by phosgene, the reactive metabolite of chloroform. In gas-uptake experiments, Gearhart et al. (1993) demonstrated a dose-dependent decrease in core body temperature with increased inhaled concentrations of chloroform. The decrease in body temperature could account for decreased in vivo chloroform metabolism, partition coefficients, pulmonary ventilation, and cardiac output rates in mice. Chinery and Gleason (1993) used a shower model for chloroform-contaminated water to predict breath concentration (as a quantifiable function of tissue dose) and actual absarbed dose from a measured water supply concentration following exposure while showering. The models predictions demonstrated that dose information based only on dermal absorption (without considering an inhalation component) may underestimate actual dose to target organs in dosimetric assessment for chloroform in water supplies during shower. The model also predicted a steady-state stratum corneum permeability of chloroform in human skin in the range of 0.16-3.6 cm/hour with the most likely value being 0.2 cm/hour. The authors suggest that the results predicted by this model could be used to estimate household exposures to chloroform or other exposures which include dermal absorption. McKone (1993) demonstrated that chloroform in shower water had an average effective dermal permeability between 0.16 and 0.42 cm/hour for a 10-minute shower. The model predicted that the ratio of chloroform dermally absorbed in the shower (relative to chloroform-contaminated water concentration) ranged between 0.25 and 0.66 mg per mg/L. In addition, the McKone model demonstrated that chloroform metabolism by the liver was not linear across all dermal/inhalation exposure concentrations and became nonlinear at higher (60-100 mg/L) dose concentrations. CHLOROFORM 118 2. HEALTH EFFECTS 2.3.5.2 Chloroform PBPK Model Comparison Five chloroform PBPK models that describe the disposition of chloroform in animals and humans have been identified from the recent open literature (early 1980s—1994). Based on the information presented in these five models, there appears to be sufficient evidence to suggest that PBPK models for chloroform are fairly refined and have a strong potential for use in human risk assessments. The PBPK model developed by Corley et al. (1990) has provided a basic model for the fate of chloroform in humans and laboratory animals. Using this model as a template, other more sophisticated and refined models have been developed that can be used in human risk assessment work. The models of Corley et al. (1990) and Reitz et al. (1990) have described several aspects of chloroform metabolism and disposition in laboratory animals and humans; however, they do not address the dermal routes of exposure. The models of McKone (1993) and Chinery and Gleason (1993) address both the inhalation and dermal exposure routes in humans the Chinery and Gleason model uses a 3-compartment skin component which may more accurately reflect the flux of chloroform through the skin after dermal only or dermal plus inhalation exposure scenarios, while the McKone model uses a single compartment within the skin to describe chloroform flux. Further discussion of each model and its application in human risk assessments is presented below. 2.3.5.3 Discussion of Chloroform Models The Corley Model The Corley model (Corley et al. 1990) was the first chloroform PBPK model to describe and ultimately predict the fate of chloroform in several species (including humans) under a variety of exposure conditions. Many subsequent PBPK models for chloroform (Chinery and Gleason 1993; McKone 1993) are based on the Corley model. The Corley model has been used for cancer risk assessment (Reitz et al. 1990). Risk Assessment. This model successfully described the disposition of chloroform in rats, mice and humans following various exposure scenarios and developed dose surrogates more closely related to toxicity response. With regard to target tissue dosimetry, the Corley model predicts the relative order of susceptibility to chloroform toxicity consequent to binding to macromolecules (MMB) to be mouse > rat > human. Linking the pharmacokinetic parameters of this model to the pharmacodynamic CHLOROFORM 119 2. HEALTH EFFECTS cancer model of Reitz et al. (1990) provides a biologically based risk assessment model for chloroform. Description of the Model. The Corley chloroform PBPK model was based on an earlier PBPK model developed by Ramsey and Andersen (1984) to describe the disposition of styrene exposure in rats, mice, and humans. A schematic representation of the Corley model (taken from Corley et al. 1990) is shown in Figure 2-5 with oral, inhalation, and intraperitoneal routes represented. The dermal route of exposure is not represented in this model; however, others have modified the Corley model to include this route of exposure (see below). Liver and kidney are represented as separate compartments since both are target organs for chloroform. The physiologic, biochemical constants and partition coefficients required for the model are shown in Table 2-4. Physiologic constants (organ weight, blood flows, etc) were similar to those used by Andersen et al. (1987) or were taken from other literature sources. Tissue and blood partition coefficients were determined in tissues by vial equilibration techniques in the rat and human, with extrapolated values used for the mouse. All metabolism of chloroform was assumed to occur only in the liver and kidneys through a single metabolic pathway (mixed function oxidase) that followed simple Michaelis-Menten kinetic parameters. Metabolic rate constants were obtained from the gas- uptake experiments. Human metabolic rate constants were obtained from in vitro human microsomal fractions of liver and kidney samples using 14Cchcl, as the substrate. Binding of chloroform metabolites (phosgene) to MMBs was assumed to occur in bioactivating tissues (liver and kidney) in a non-enzymatic, nonspecific, and dose-independent fashion. Macromolecular binding constants for the liver and kidney were estimated from in vivo MMB data obtained from rats and mice exposed to 14CCHCL, via inhalation. The gas-uptake data for rats were well described using a single Michaelis-Menten equation to describe metabolism. For the mouse inhalation studies, a simple Michaelis-Menten equation failed to adequately describe the chloroform-metabolizing capacity based on the data collected and model constants. The authors suspected that, following the administration of chloroform (particularly at higher concentrations), destruction of microsomal enzymes and subsequent resynthesis of microsomal enzymes was important in the mouse. This phenomenon has been documented in phenobarbital- induced but not naive rats. To account for this phenomenon, a first-order rate constant for the loss and subsequent regeneration of metabolic capacity was incorporated into the model for mice only. CHLOROFORM 2. PUBLIC HEALTH 120 Figure 2-5. Parameters Used in the Corley PBPK Model QP Hy Cl Qc cv Qc CVR Qs CVs QF CVF QK CVK QL CVL Physiological model used to describe the pharmacokinetics of chloroform in rats, mice, and humans during RAPIDLY PERFUSED ~ ) ALVEOLAR SPACE — A a 1) LUNG BLOOD \ _/ ~ 7) J SLOWLY PERFUSED \ J a ) FAT \_ _/ r 3 KIDNEY \_ J ¥ Kk, V.K AMK Kk loss’ "resym LIVER ¥ Ko Youd AML Kiss’ resym inhalation, oral, and intraperitoneal exposures. QP an. CX QC ———————y CA QR «—] CA Qs «—— CA QF «—] CA QK Cee) CA QL CA k.S Oral AMK = amount metabolized in kidney; AML = amount metabolized in liver IP CHLOROFORM 121 2. HEALTH EFFECTS Table 2-4. Parameters Used in the Corley PBPK Model Parameters Mouse Rat Human Weights (kg): Body 0.02858 0.230 70.0 Percentage of Body Weight Liver 5.86 2.53 3.14 Kidney 1.70 0.71 0.44 Fat 6.00 6.30 23.10 Rapidly perfused tissues 3.30 4.39 3.27 Slowly perfused tissues 74.14 77.07 61.05 Flows (L/hr/kg) Alveolar ventilation 2.01 5.06 347.9 Cardiac output 2.01 5.06 347.9 Percentage of Cardiac Oulput Liver 25.0 25.0 25.0 Kidney 25.0 25.0 25.0 Fat 2.0 5.0 5.0 Rapidly perfused tissues 29.0 26.0 26.0 Slowly perfused tissues 19.0 19.0 19.0 Partition Coefficients Blood/air 21.3 20.8 7.43 Liver/air 19.1 21.1 17.00 Kidney/air 11.0 11.0 11.00 Fat/air 242.0 203.0 280.00 Rapidly perfused/air 19.1 21.1 17.0 Slowly perfused/air 13.0 13.9 12.0 Metabolic and macromolecular binding constants Vmax © (mg/hr/kg) 22.8 6.8 15.7 Ky, (mg/L) 0.352 0.543 0.448 Koss (/MQ) 572x104 0 0 Kresyn (71) 0.125 0 0 A (kidney/liver) 0.153 0.052 0.033 fMMB (hr), liver 0.003 0.00104 0.00202 fMMB (hr), kidney 0.010 0.0086 0.00931 Gavage Absorption Rate Constants kag (hr), com oil 0.6 0.6 0.6 Kas (hr), water 5.0 5.0 5.0 Intraperitoneal Injection Absorption Rate Constant k. (hr) 1.0 1.0 1.0 —a CHLOROFORM 122 2. HEALTH EFFECTS The model also provided a good description of the in vivo levels of MMB in both rats and mice, with good agreement between observed and predicted values. Validation of the Model. The Corley model was validated using chloroform data sets from oral (Brown et al. 1974a) and intraperitoneal (Ilett et al. 1973) routes of administration and from human pharmacokinetic studies (Fry et al. 1972). Metabolic rate constants obtained from the gas-uptake experiments were validated by modeling the disposition of radiolabeled chloroform in mice and rats following inhalation of chloroform at much lower doses. For the oral data set, the model accurately predicted the total amounts of chloroform metabolized for both rats and mice. Target Tissues. The model provided excellent predictions of MMB in both the target tissues of chloroform (liver and kidney) after intraperitoneal administration in mice (rat data was not generated). The model adequately predicted the amount of unchanged material exhaled at infinite time and the total amount metabolized by groups of male and female humans of widely varying age and weight. Species Extrapolation. The Corley model used species-specific information to outline the model parameters; little extrapolation of information among mice, rats and humans was required. Certain parameters previously reported in the scientific literature were assumed, however, such as body weight, percentage of body weight, and percentage of blood from the heart (i.e., percentage of cardiac output of body organs, see Table 2-4). High-low Dose Extrapolation. The Corley model was designed to facilitate extrapolations from high doses (similar to those used for chronic rodent studies) to low doses that humans may potentially be exposed to at home or in the workplace. Interroute Extrapolation. The Corley model used three routes of administration, intraperitoneal, oral and inhalation, in rats and mice to describe the disposition of chloroform. This data was validated for humans by comparing the model output using the animal data with actual human data from human oral chloroform pharmacokinetic studies. Using the human pharmacokinetic constants from the in vitro studies conducted by Corley, the model made adequate predictions of the amount of chloroform metabolized and exhaled in both males and females. CHLOROFORM 123 2. HEALTH EFFECTS The Reitz Model Risk Assessment. The Reitz model is based on the assumption that cytotoxicity and reparative hyperplasia are responsible for liver neoplasia. Dose-surrogates, a more sophisticated and more accurate measure of target tissue dose derived from measuring a pharmacodynamic effect, were used. Description of the Model. The Reitz PBPK model was largely based on the Corley et al. (1990) model, but differed in the use of a pharmacodynamic end point, cytotoxicity in the livers of chloroform-exposed animals (mice) produced by phosgene (the reactive metabolite of chloroform). The Reitz model focused on the liver as the target organ for chloroform, hence the kidney compartment toxicity was not addressed. The kidney compartment was combined with the rapidly perfused tissue group. The Reitz model used two types of dose measurement, referred to as dose surrogates. One type of dose surrogate used was covalent binding to MMBs (average daily macromolecular binding, AVEMMB), a rate independent parameter. The second type of dose surrogate was cytotoxicity (PTDEAD), a rate dependent parameter that measured cell death (by histopathological analysis and 3Hihymidine uptake) due to the formation of reactive chloroform metabolites (i.e., phosgene). Model calculations of PTDEAD were based on several assumptions: that liver cells have a finite capability for repairing damage caused by CHCl; metabolites; that liver cells differ from cell to cell in their capabilities to repair this damage; and that induction of cytotoxicity in liver cells does not occur instantaneously. Validation of the Model. The model simulations of PTDEAD were compared with two experimental measures of cytotoxicity: the percentage of nonviable cells observed microscopically in mice gavaged with solutions of chloroform in corn oil, and the rate of incorporation of 3 Hthymidine into normal DNA during compensatory cell replication (CCR). CCR was measured following exposure of mice to chloroform vapor for 5-6 hours. Model predictions were in good agreement (within 10%) with observed percentages of dead liver cells evaluated microscopically. Agreement between predicted and observed values of cell killing based on CCR was less satisfactory. Target Tissues. The Reitz model only applies to the metabolism of chloroform and the induction of cytotoxicity in liver tissue following exposure by inhalation, drinking water, and gavage routes using rat and mouse data. CHLOROFORM 124 2. HEALTH EFFECTS Species Extrapolation. The Reitz model used the same species and physiologic parameters that the Corley model utilized (average body weights, organ percentage of body weight, blood flow, etc.) for model predictions. See Table 2-4 for these parameters. However, the model assumed equivalent intrinsic sensitivity of mouse and human hepatocytes. High-low Dose Extrapolation. The Reitz model was designed to facilitate extrapolations from high doses (similar to those used for chronic rodent studies) to low doses that humans may potentially be exposed to at home or in the workplace. Interroute Extrapolation. Inhalation and oral routes of administration were examined in the Reitz model; however, interroute extrapolations were not specifically addressed in the Reitz model. The Gearhart Model Risk Assessment. The Gearhart model provided strong evidence that temperature changes play an important role in predicting chloroform metabolism in mice and also provided a testable hypothesis for the lack of fit of the Corley model prediction with respect to the mouse data. These data strengthen the Corley model and its implications for human risk assessment (see the Corley model description above). Description of the Model. Gearhart et al. (1993) developed a PBPK model that described the effects of decreased core body temperature on the analysis of chloroform metabolic data. Experimental data showed that when male B6C3F; mice were exposed for 6 hours to chloroform vapor concentrations of 100-5,500 ppm, a dose-dependent drop in core body temperature occurred, with the least amount of temperature drop occurring at the 100 ppm concentration and the most dramatic drop in temperature occurring at the 5,500 ppm level. The Gearhart model incorporated a model previously used by Ramsey and Andersen (1984) (the same model and parameters the Corley model was based on) in conjunction with a separate model reflecting changes in body core temperature to drive equations accounting for changes in partition coefficients, cardiac output, minute ventilation volumes, and rate of chloroform metabolism. The model predicted that the V for chloroform metabolism without correcting for core temperature effects was 14.2 mg/hour/kg (2/3 of that reported in the Corley model) and the K, was 0.25 mg/L. CHLOROFORM 125 2. HEALTH EFFECTS Without body temperature corrections, the model underpredicted the rate of metabolism at the 5,500 ppm vapor concentration. Addition of a first-order kinetic rate constant (kf=1.86 hour!) to account for liver metabolism of chloroform at high doses of chloroform did provide a small improvement in model predictions at 5,500 ppm, but was still considered inadequate for predicting metabolism at high concentrations. Validation of the Model. The Gearhart model was not validated against a comparable data set. Corrections for the temperature effects (V increased to 15.1 mg/hour/kg) and inclusion of a first- max order metabolism correction equation provided an accurate prediction of chloroform metabolism across all concentrations tested. Target Tissues. The liver was the target tissue for this model. Species Extrapolation. No species extrapolation was specifically addressed by the Gearhart model. High-low Dose Extrapolation. No high-low dose extrapolation was specifically addressed by the Gearhart model. Interroute Extrapolation. No interroute extrapolation was specifically addressed by the Gearhart model. The Chinery-Gleason Model Risk Assessment. The Chinery-Gleason model has the greatest potential for use in estimating exposures to chloroform in a household environment as well as for occupational exposures that result from dermal exposure. Description of the Model. The Chinery and Gleason (1993) PBPK model is a combination of the Corley et al. (1990) model and other existing models that includes a multicompartment skin component similar to that of Shatkin and Szejnwald-Brown (1991). This compartment is used to simulate penetration of chloroform into the skin while showering for 10 minutes with water containing chloroform. The skin module for this new model assumed a physiologic skin compartment consisting CHLOROFORM 126 2. HEALTH EFFECTS of three linear compartments: the dilute aqueous solution compartment; the stratum corneum (the primary barrier to the absorption of most chemicals, including chloroform); and the viable epidermis. Validation of the Model. The model was validated using published data on experimentally derived exhaled breath concentrations of chloroform following exposure in a shower stall (Jo et al. 1990a). Target Tissues. Based on the data set of Jo et al. (1990a), the Chinery-Gleason model predicted the stratum corneum permeability coefficient for chloroform to be 0.2 cm/hour (range, 0.6 and 2.2) and the estimated ratio of the dermally and inhaled absorbed doses to be 0.75 (range, 0.6 and 2.2) cm/hour. This new model showed that a simple steady-state model can be used to predict the degree of dermal absorption for chloroform. It was also shown that the model would be useful in predicting the concentrations of chloroform in shower air and in the exhaled breath of individuals exposed both dermally and by inhalation routes while showering with water containing low amounts (20 pg/L) of chloroform. At this concentration, the model predicted a dermal absorption dose of 0.0047 mg and inhalation of 0.0062 mg. In addition, the model also demonstrated that as the concentration of chloroform rises due to increases in chloroform vapor, the absorbed inhalation dose increases faster and becomes larger than the absorbed dermal dose. Species Extrapolation. No species extrapolation was specifically addressed by the this model. High-low Dose Extrapolation. No high-low dose extrapolation was specifically addressed by this model. Interroute Extrapolation. The Chinery-Gleason model examined two routes of exposure, inhalation-only exposure and inhalation/dermal exposure. The model was useful in predicting the concentration of chloroform in shower air and in the exhaled breath of individuals exposed by the dermal and inhalation routes. The McKone Model Risk Assessment. The McKone model has some use in human chloroform risk assessments, in that the model defined the relationship between the dermal and inhalation exposure to measures of dose and the amounts that can be metabolized by the liver by each route. The model also provided CHLOROFORM 127 2. HEALTH EFFECTS information about the inhalation and dermal exposure concentrations at which chloroform metabolism becomes nonlinear in humans. Description of the Model. The McKone (1993) PBPK model addressed potential exposure to chloroform by the inhalation and dermal routes. McKone revised existing shower-compartment, dermal uptake and PBPK models to produce a revised PBPK model for simulating chloroform breath levels in persons exposed in showers by the inhalation route only and by the inhalation and dermal routes combined. Parameters used by this model were taken primarily from two main sources, Jo et al. (1990a) and Corley et al. (1990). The model was also used to assess the relationship of dermal and inhalation exposure to metabolized dose in the liver, as well as to determine the tap-water concentrations at which hepatic metabolism of dermal and inhalation doses of chloroform become nonlinear. This information is especially useful for risk assessment on persons exposed to a wide range of chloroform concentrations. Experimentally measured ratios of chloroform concentrations in air and breath to tap water concentration (Jo et al. 1990a) were compared with the model predictions. Validation of the Model. The McKone model used one data set to evaluate the model results (Jo et al. 1990a). The McKone model results were also compared to other existing chloroform models, with an in-depth discussion of similarities an differences between those models. Target Tissues. The skin and lung were the target tissues studied in this model. Based on the information presented, the McKone model is appropriate for simulating chloroform breath levels in persons exposed in showers by both exposure routes. A major difference between the McKone model and the Chinery-Gleason model is that the McKone model assumes the skin to be a one compartment organ, whereas the Chinery-Gleason model assumed three compartments within the skin. The McKone model indicated that the ratio of chloroform dermally absorbed in the shower to the concentration in tap water ranges from 0.25 to 0.66 mg/L, and that chloroform can effectively permeate through the skin at a rate of 0.16-0.42 cm/hour during a 10-minute shower. Species Extrapolation. The human was the only species addressed by the McKone model. No extrapolation between species was addressed in this model. CHLOROFORM 128 2. HEALTH EFFECTS High-low Dose Extrapolation. For tap-water concentrations below 100 mg/L, the model predicted a linear relationship between potential dose (i.e., amounts present in the drinking water, inhaled in a shower, or skin surface contact) and the cumulative metabolized dose. At tap-water concentrations greater than 100 mg/dL for inhalation-only showers and 60 mg/L or normal showers, however, the relationship was no longer linear and modifications to this model may be required. Interroute Extrapolation. The dermal and inhalation routes were addressed in this model. The McKone model did not specifically address interroute extrapolations for chloroform. 2.4 MECHANISMS OF ACTION 2.4.1 Pharmacokinetic Mechanisms Absorption. In humans and laboratory animals, chloroform is generally absorbed quickly. Primarily because of its high blood/air partition coefficient, it passes with some ease through most tissue and cellular barriers in the body. Chloroform can be absorbed by inhalation and ingestion, and by dermal routes of exposure. Inhalation studies were performed by Corley et al. (1990) on groups of mice exposed to various concentrations of chloroform for 6 hours and sacrificed 48 hours after the last exposure. Chloroform absorption by the lungs varied by concentration and was generally 34-46%. An earlier study by Von Oettingen (1964) found that when dogs were exposed to 15,000 ppm chloroform, the concentration of chloroform in the blood rose quickly and leveled off, apparently establishing a steady-state concentration in the blood at 80-100 minutes after inhalation exposures began. The average steady-state concentration in the blood was 0.4 mg/mL. Less information is available on the absorption of chloroform by inhalation in humans. Humans exposed to 10,000 ppm of chloroform during surgical anesthesia showed a rapid absorption of chloroform detected in arterial blood samples, with peak concentrations occurring within 2 hours after initiation of anesthesia. The average arterial blood concentration of chloroform was reported to be about 0.1 mg/mL (Smith et al. 1973). Dick et al. (1995) examined the absorption of chloroform through human skin in vivo using volunteers and in vitro using fresh, excised abdominal skin. In the in vivo study, the ventral forearm skin of four male volunteers was dosed with a solution of chloroform in either water or ethanol. The solution remained on the skin for eight hours. When administered in water, the total absorbed dose was 8.2%. In contrast, the total absorbed dose was only 1.68% when chloroform was administered in ethanol. In the in vitro study, two doses were applied to the skin and remained there for four hours. CHLOROFORM 129 2. HEALTH EFFECTS At study termination, the percentages of the low and high doses of chloroform absorbed were 5.6 and 7.1%, respectively. Rats and mice exposed to 60 mg/kg 14¢_chloroform orally demonstrated that absorption was practically complete within 48 hours for mice and within 96 hours in rats. Peak blood levels occurred within 1 hour after the oral dose (Brown et al. 1974a). Humans dosed orally with 0.5 grams of 13C_chloroform delivered as a capsule containing olive oil showed near complete absorption of chloroform within 8 hours after administration. Peak blood levels generally occurred at approximately 1 hour after dosing, with 13C-chloroform concentrations in blood ranging from 1 to 5 pg/mL (Fry et al. 1972). Chloroform can also permeate the stratum corneum of rabbit skin (Torkelson et al. 1976) and mouse skin (Tsuruta 1975). Percutaneous absorption of chloroform across mouse skin was calculated to be approximately 38 pg/min/cm? , indicating that the dermal absorption of chloroform occurs fairly rapidly in mice. No reliable studies report the percutaneous absorption of chloroform in humans; however, a few clinical reports indicate that chloroform is used as a vehicle for drug delivery (King 1993). Islam et al. (1995) investigated the fate of topically applied chloroform in male hairless rats. For exposures under 4 minutes, chloroform-laden water was applied to shaved back skin; for exposures of 4-30 minutes, rats were submerged in baths containing chloroform-laden water. Selected skin areas were tape-stripped a various number of times after various delay periods. It appeared that there was an incremental build-up of chloroform in the skin over the first four minutes. When compared to uptake measured by bath concentration differences, approximately 88% of lost chloroform was not accounted for in the stratum corneum and was assumed to be systemically absorbed. Distribution. Radiolabeled chloroform in mice, once absorbed, is widely distributed to most organs and tissues, specifically the liver, kidney, lungs, spleen, body fat, muscle, and nervous tissue, as reported by Cohen and Hood (1969) and Bergman (1979). Significant accumulations were noted 48 hours after inhalation exposure in the central nervous system, particularly in the cerebellar cortex, spinal nerves, and meninges. When administered orally (Brown et al. 1974a), rats and squirrel monkeys showed significant accumulations of 14¢_chloroform in the brain, lung, muscle, and kidney in both species, with an unusual accumulation of chloroform in the gall bladder of the monkey. When administered orally to mice, similar accumulations of chloroform occurred in the liver, kidney, lung, CHLOROFORM 130 2. HEALTH EFFECTS muscle, blood, intestines, and gall bladder (Taylor et al. 1974). Little current information on the distribution of chloroform in humans was available for review. Chloroform (or phosgene) tends to accumulate to a significantly higher degree in the kidneys of male mice than in those of female mice given equivalent doses, which leads to a higher degree of chloroform nephrotoxicity in male mice. The sex differences seen with the renal cortical accumulation of chloroform or phosgene can be halted if chloroform is administered to castrated males; the sex difference can be reversed if chloroform is administered to females pretreated with testosterone prior to dosing with chloroform. This difference in chloroform accumulation is obviously dependent on the presence of testosterone and is very consistent with a body of evidence that indicates chloroform is more nephrotoxic to male mice than to female mice (Ilett et al. 1973; Pohl et al. 1984; Smith et al. 1973). Although this sex-related toxic effect is known to occur in mice, it is not known at present if a similar effect occurs in humans. Excretion. Chloroform is largely excreted either in the parent form or as the end metabolite (carbon dioxide, CO,) in the bodies of both laboratory animals and humans. Corley et al. (1990) demonstrated that mice exposed to 10 or 89 ppm of chloroform by inhalation excreted 99% of the chloroform body burden as CO, in exhaled air. As the chloroform concentrations in the air rose however, the amount of chloroform metabolized to CO, decreased and the amount of unchanged chloroform rose in the exhaled air, indicating that chloroform metabolism in mice is a saturable process. Rats exposed in a similar manner to 93, 356, and 1,041 ppm chloroform excreted 2, 20, and 42.5%, respectively, of the total body burden of chloroform as unchanged parent compound, indicating that chloroform is metabolized to CO, in rats but to a lesser degree than in mice. In humans, Fry et al. (1972) administered 500 mg of chloroform orally in olive oil in capsular form and found that 17-67% of the total dose of chloroform was exhaled as unchanged parent compound, and that the extent of pulmonary elimination of chloroform was governed inversely by the amount of adipose tissue on the individual ingesting the chloroform. The study also found that most of the chloroform tended to be exhaled between 40 minutes and 2 hours after dosing, which coincided with peak blood levels of chloroform produced at approximately 1 hour after dosing. CHLOROFORM 131 2. HEALTH EFFECTS Chloroform in humans tends to be eliminated in a biphasic manner. After ingesting 500 mg of chloroform orally, an initial (0) half-life in the blood of 9-21 minutes was reported, with the second (B) half-life ranging from 86 to 96 hours. 2.4.2 Mechanisms of Toxicity Chloroform is widely distributed to many tissues of the body in laboratory animals and, presumably, in humans; however, many studies have demonstrated that chloroform does not tend to accumulate in the body for extended periods. Chloroform may accumulate to some degree in the body fat stores; however, it quickly partitions out the fat and is excreted by the normal routes and mechanisms. The liver (primary) and kidneys (secondary) are considered to be the target organs for chloroform toxicity in both humans and laboratory animals. Thus, humans (and animals) with existing hepatic or renal disease who are exposed to chloroform, particularly by the oral or inhalation routes, are more likely to be at risk to the toxic effects of chloroform. Reproductive/developmental effects due to chloroform's presence in the drinking water of both humans and laboratory animals has been reported, thereby placing women of childbearing age at a potentially higher risk of reproductive organ anomalies than those women past menopausal age. Chloroform is largely metabolized in many tissues (particularly the liver and kidney) to CO, in humans and animals (Brown et al. 1974a; Corley et al. 1990; Fry et al. 1972). Chloroform metabolism is catalyzed by cytochrome P-450, initiating an oxidative cleavage of the C-H bond producing trichloromethanol. Trichloromethanol is unstable and is rapidly transformed to phosgene (COCl,). Phosgene may react with water to form CO,, which can be exhaled by the lung or excreted in the urine as carbonate or bicarbonate, and hydrochloric acid. Phosgene can also react with other molecules such as cysteine, deplete hepatic GSH (Docks and Krishna 1976; Pohl et al. 1981) and form adducts with microsomal proteins (Corley et al. 1990). Chloroform toxicity can be attributed to the presence of both the parent compound and the formation of phosgene in most instances of toxicosis. High doses of inhaled chloroform have been reported to cause death (due to respiratory depression), ataxia, narcosis, and central nervous system depression, and are due to the direct effects of the parent compound. Lower doses of chloroform in the air, feed, or water, or administered by gavage, with variable exposure times, may induce toxicity due to the presence of the parent compound or to production of phosgene during metabolism. It appears that the CHLOROFORM 132 2. HEALTH EFFECTS metabolite is responsible for hepatocellular damage, resulting in the ultimate elevation of hepatic enzymes (SGPT, SGOT, GGT, etc.) and cell damage/necrosis. The accumulation of chloroform in the renal cortex of mice with the subsequent metabolism to phosgene most likely contributes to the renal toxicity of chloroform seen in male mice. Tubular necrosis, calcification, nephritis, increased kidney weight, alterations in Na/K excretion, and other cellular anomalies were observed in response to one or both of these toxicants. Although the sex-related nephrotoxic effect is known to occur in mice, it is not known at present if a similar effect occurs in humans or other laboratory animals. 2.4.3 Animal-to-Human Extrapolations Many laboratory animal models have been used to describe the toxicity and pharmacology of chloroform. By far, the most commonly used laboratory animal species are the rat and mouse models. Generally, the pharmacokinetic and toxicokinetic data gathered from rats and mice compare favorably with the limited information available from human studies. PBPK models have been developed using pharmacokinetic and toxicokinetic data for use in risk assessment work for the human. The models are discussed in depth in Section 2.3.5. As mentioned previously, male mice have a sex-related tendency to develop severe renal disease when exposed to chloroform, particularly by the inhalation and oral exposure routes. This effect appears to be species-related as well, since experiments in rabbits and guinea pigs found no sex-related differences in renal toxicity. 2.5 RELEVANCE TO PUBLIC HEALTH Overview. Data are available regarding health effects in humans and animals after inhalation, oral, and dermal exposure to chloroform; however, data regarding dermal exposure are quite limited. Chloroform was used as a general anesthetic, pain reliever, and antispasmodic for more than a century before its toxic effects were fully recognized. High levels of chloroform (23-400 ppm) (Challen et al. 1958; Phoon et al. 1983) in the air are found specifically in highly industrialized areas. Exposure of the general population to chloroform can also occur via the drinking water as a result of the chlorination process. Occupational exposure is another source of inhalation and/or dermal exposure for humans. Most of the presented information regarding chloroform toxicity following inhalation exposure in humans was obtained from clinical case reports of patients undergoing anesthesia. In some instances, CHLOROFORM 133 2. HEALTH EFFECTS the results in these studies may have been confounded by unreported data, such as the intake of other drugs or the use of artificial respiration during anesthesia. The target organs of chloroform toxicity in humans and animals are the central nervous system, liver, and kidneys. There is a great deal of similarity between chloroform-induced effects following inhalation and oral exposure. No studies were located regarding reproductive effects in humans after exposure to chloroform alone; however, Bove et al. (1995) studied the effects of drinking-water consumption on birth outcomes and found that exposure to TTHM at levels >0.1 ppm resulted in reduced birth weight and size as well as an increased risk of oral cleft, central nervous system, and neural tube defects. Since the authors did not specifically monitor chloroform levels, the effects seen may be due to exposure to other THMs. In addition, non-THM contaminants in the drinking water may have contributed to the observed effects as well. Only one study was located regarding the developmental effects of chloroform in humans. Animal studies indicate that chloroform can cross the placenta and cause fetotoxic and teratogenic effects. Chloroform exposure has also caused increased resorptions in animals. Epidemiology studies suggest a possible risk of colon and bladder cancer in humans that is associated with chloroform in drinking water. In animals, chloroform was carcinogenic after oral exposure. Minimal Risk Levels for Chloroform. Inhalation MRLs. * An MRL of 0.1 ppm has been derived for acute-duration inhalation exposure (14 days or less) to chloroform. The MRL was based on a hepatic NOAEL of 3 ppm chloroform administered for 6 hours a day for 7 consecutive days to mice (Larson et al. 1994c). Female mice exposed to 100 or 300 ppm exhibited centrilobular hepatocyte necrosis and severe diffuse vacuolar degeneration of midzonal and periportal hepatocytes, while exposure to 10 or 30 ppm resulted in mild-to-moderate vacuolar changes in centrilobular hepatocytes. Decreased eosinophilia of the centrilobular and midzonal hepatocyte cytoplasm relative to periportal hepatocytes was observed at 30 ppm. Livers of mice in the 1 and 3 ppm groups did not differ significantly from control animals and were considered to be NOAELSs for liver effects. The NOAEL of 3 ppm was converted to the Human Equivalent Concentration (HEC) as described in Equation 4-10 in Interim Methods for Development of Inhalation Reference CHLOROFORM 134 2. HEALTH EFFECTS Concentrations (EPA 1990b). This calculation resulted in a NOAELyg() of 3 ppm. An uncertainty factor of 30 (3 for extrapolation from animals to humans and 10 for human variability) was applied to the NOAEL yg) value, which resulted in an MRL of 0.1 ppm. Reports regarding chloroform hepatotoxicity in animals are numerous (Larson et al. 1993, 1994a, 1994b, 1994c¢). Liver damage has been reported in several other studies, and was usually indicated by liver biochemical/enzyme alterations in rats (Lundberg et al. 1986) and mice (Gehring 1968; Murray et al. 1979) after acute inhalation exposure. Fatty changes (Culliford and Hewitt 1957; Kylin et al. 1963) and liver necrosis (Deringer et al. 1953) were observed histologically in mice after acute inhalation exposure. Histological findings indicative of liver toxicity were also observed in other laboratory animals following inhalation exposure of intermediate duration, but the findings were not dose-related (Torkelson et al. 1976). The 17.3 mg/kg dose was also a NOAEL for kidney effects, but tubular necrosis occurred at 65.6 mg/kg/day. « An MRL of 0.05 ppm has been derived for intermediate-duration inhalation exposure (15 days to 364 days) to chloroform. The MRL was based on a LOAEL of 14 ppm in workers exposed to concentrations of chloroform of up to 400 ppm for less than 6 months (Phoon et al. 1983). Vomiting and toxic hepatitis were noted to occur at an inhaled chloroform concentration of 14 ppm. The LOAEL of 14 ppm was divided by an uncertainty factor of 100 (10 for the use of a LOAEL and 10 for human variability) and a modifying factor of 3 (insufficient diagnostic data to determine the seriousness of hepatotoxic effects) to arrive at the MRL of 0.05 ppm. Alterations in liver functions have been reported in several studies in both humans and animals, and is discussed in more detail in the chronic-duration inhalation MRL section immediately below. « An MRL of 0.02 ppm has been derived for chronic-duration inhalation exposure (365 days or more) to chloroform. The MRL was based on a LOAEL of 2 ppm in workers exposed to concentrations of chloroform ranging from 2 to 205 ppm for 1-4 years (Bomski et al. 1967). The LOAEL of 2 ppm was divided by an uncertainty factor of 100 (10 for use of a LOAEL and 10 for human variability) to arrive at the MRL of 0.02 ppm. Hepatomegaly was found in 25% of chloroform-exposed workers. Toxic hepatitis was found in 5.6% of the liver enlargement cases. Hepatosteatosis (fatty liver) was detected in 20.6% CHLOROFORM 135 2. HEALTH EFFECTS of liver-enlargement cases. Chloroform-exposed workers had a higher frequency of jaundice over the years than the control group. Alterations in liver functions have been reported in several studies in both humans and animals. In humans, impaired liver function was indicated by increased sulfobromo- phthalein retention in some patients exposed to chloroform via anesthesia (Smith et al. 1973), in addition to acute toxic hepatitis developing after childbirth in several women exposed to chloroform via anesthesia (Lunt 1953; Royston 1924; Townsend 1939). In contrast, no clinical evidence of liver toxicity was found in another study among chloroform workers exposed to <237 ppm (Challen et al. 1958). Oral MRLs. * An MRL of 0.3 mg/kg/day has been derived for acute-duration oral exposure (14 days or less) to chloroform. The MRL was based on a NOAEL of 26 mg/kg/day in the drinking water for 4 days for hepatic effects in mice (Larson et al. 1994b). The NOAEL of 26.4 mg/kg/day was divided by an uncertainty factor of 100 (10 for extrapolation from animals to humans and 10 for human variability) to arrive at the MRL of 0.3 mg/kg/day. A study performed by Moore et al. (1982) found renal effects in CFLP Swiss mice dosed at 65.5 mg/kg/day by gavage in oil. Another study by Larson et al. (1993) found both hepatic (elevated SDH, ALT and AST, hepatocyte necrosis) and renal (proximal tubule necrosis) lesions in Fischer 344 rats and hepatic lesions only in B6C3F, mice induced by chloroform administered at 34 mg/kg/day once by gavage in oil. Lesions in the Larson et al. (1993) study were ranked as less serious LOAELSs. * An MRL of 0.1 mg/kg/day has been derived for intermediate-duration oral exposure (15-364 days) to chloroform. This MRL is based on a NOAEL of 15 mg/kg/day for hepatic effects in dogs dosed with chloroform in a capsule 1 time a day, 6 days a week for 6 weeks (Heywood et al. 1979). The NOAEL of 15 mg/kg/day was divided by an uncertainty factor of 100 (10 for extrapolation from animals to humans and 10 for human variability) to arrive at the MRL of 0.1 mg/kg/day. Clinical chemistry parameters showed significantly increase SGPT in the 30 mg/kg/day group beginning at 6 weeks. SGPT activity was not increased in the 15 mg/kg/day group until week 130. Liver effects in animals have been reported in numerous oral studies of intermediate duration. Fatty changes, necrosis, CHLOROFORM 136 2. HEALTH EFFECTS increased liver weight, and hyperplasia have been observed in rats exposed to 2150 mg/kg/day chloroform in drinking water for 90 days (Palmer et al. 1979). An increased incidence of sporadic, mild, reversible liver changes occurred in mice exposed to chloroform in drinking water at doses of 0.3-114 mg/kg/day for 90 days, but the incidences were not significantly higher than the incidences in controls (Chu et al. 1982a). Fatty and hydropic changes, necrosis, and cirrhosis were observed in mice treated by gavage with >50 mg/kg/day chloroform in oil for 90 days (Bull et al. 1986; Munson et al. 1982) or at 86 mg/kg/day in drinking water for 1 year (Klaunig et al. 1986). In contrast, centrilobular fatty changes observed in mice at 64 mg/kg/day chloroform in drinking water for 90 days appeared to be reversible (Jorgenson and Rushbrook 1980), and no liver effects were found in mice treated with >50 mg/kg/day in aqueous vehicles (Bull et al. 1986). In addition, hepatocellular degeneration was induced in F| females in a 2-generation study in which mice were treated by gavage with 41 mg/kg/day chloroform in oil (Gulati et al. 1988). + An MRL of 0.01 mg/kg/day has been derived for chronic-duration oral exposure (365 days or more) to chloroform. This MRL is based on a LOAEL of 15 mg/kg/day for hepatic effects in dogs dosed with chloroform 6 days a week for 7.5 years (Heywood et al. 1979). The LOAEL of 15 mg/kg/day was divided by an uncertainty factor of 1,000 (10 for use of a LOAEL, 10 for extrapolation from animals to humans and 10 for human variability) to arrive at the MRL of 0.01 mg/kg/day. SGPT activity was not increased in the 15 mg/kg/day group until week 130, providing the LOAEL on which this MRL was based. Numerous chronic oral studies examined hepatic and renal end points as well as neurological and cancer effects. Serious effects occurred at higher doses; 15 mg/kg/day was the lowest dose used in available animals studies. A NOAEL of 2.46 mg/kg/day for liver and kidney effects (SGPT, SGOT, BUN and SAP) was found in humans who used a dentifrice containing 0.34% or a mouthwash containing 0.43% chloroform for 1-5 years (DeSalva et al. 1975). The reader is advised to exercise caution in the extrapolation of toxicity data from animals to humans. Species-related differences in sensitivity must be accounted for. Some studies utilized to derive MRLs or otherwise extrapolate data, is dated; however, they do represent the body of knowledge regarding chloroform toxicity. In addition, many of the human studies quoted involved clinical case reports in which chloroform was utilized either as an anesthetic or as an agent of suicide. Such doses are clearly CHLOROFORM 137 2. HEALTH EFFECTS excessive and would not be encountered by the general population. These and other issues are addressed in Section 2.10. Death. Chloroform levels of =40,000 ppm cause death in patients under chloroform anesthesia (Featherstone 1947; Whitaker and Jones 1965). Death is usually due to severe respiratory depression/ failure or disturbances in cardiac rhythm. Accidental or intentional ingestion of large doses of chloroform may lead to death (Piersol et al. 1933). Death in humans after oral exposure to chloroform is usually caused by respiratory obstruction by the tongue due to jaw relaxation, central respiratory paralysis, acute cardiac failure, or severe hepatic injury (Piersol et al. 1933; Schroeder 1965). The levels of chloroform exposure that cause death in animals are usually lower than those administered to patients to induce anesthesia; however, the duration of exposure in animals is generally longer. Following acute exposure to high concentrations of chloroform, all male mice died; however, most females survived the exposure for several months (Deringer et al. 1953). Survival was associated with lower testosterone levels, as suggested by the higher mortality rate in noncastrated adult males. This conclusion is supported by similar observations of higher survival rates in female rats, compared to male rats, after intermediate-duration exposure to chloroform (Torkelson et al. 1976). In regard to LCs, values in rats, survival rates were highest among females and lowest among young adult males. The correlation between mortality rates and male hormone levels is evident. Deaths were apparently potentiated by starvation, dehydration, and exhaustion (Ekstrom et al. 1986, 1988; Royston 1924; Townsend 1939). Increased mortality was also observed in rats and mice after oral exposure of intermediate and chronic duration (Balster and Borzelleca 1982; Chu et al. 1982a; Jorgenson et al. 1985; Klaunig et al. 1986; NCI 1976; Palmer et al. 1979; Roe et al. 1979). Deaths were caused by toxic liver and kidney effects, and tumors. Deaths after dermal exposures in either humans or laboratory animals have not been reported. Chloroform concentrations in air and drinking water in the general environment or near hazardous waste sites are not likely to be high enough to cause death in humans after acute exposure. Whether chronic exposure to low levels of chloroform in the environment, drinking water, or hazardous wastes could shorten the life span of humans is not currently known. Currently available epidemiologic findings about the chronic exposure to chloroform are inconsistent at best which, in large part, may be due to study design issues. CHLOROFORM 138 2. HEALTH EFFECTS Systemic Effects. Respiratory Effects. The respiratory failure observed in patients under chloroform anesthesia was probably due to a direct effect of chloroform on the respiratory center of the central nervous system. A decline of the systolic pressure in the cerebral vessels may also contribute to respiratory failure, as demonstrated in animals: when respiration had stopped under chloroform anesthesia, the animals (species not specified) breathed again if positioned head down (Featherstone 1947). Upper respiratory tract obstruction can occur in patients after inhalation exposure to chloroform via anesthesia (Featherstone 1947) and after chloroform ingestion (Schroeder 1965). Few autopsy reports were located in the literature. Hemorrhage into the lungs, without any signs of consolidation, was reported in a case study involving death after inhalation exposure (Royston 1924); however, congested lungs with pneumonic consolidation were observed in a man who died after drinking chloroform (Piersol et al. 1933). In addition to lower respiratory tract effects, chloroform has been demonstrated to induce changes in the nasal region of rats and mice after inhalation and oral exposure. Increased sizes of goblet cells and nasal epithelium, degeneration of the nasal epithelium and Bowman's glands, changes in the proliferation rates of cells, new bone formation, and changes in biochemical parameters (especially cytochrome P-450-2E1) have been reported (Larson et al. 1995b, 1996; Mery et al. 1994), indicating that chloroform can adversely affect the upper as well as the lower respiratory tract at low concentrations. The incidence of respiratory lesions after oral (gavage) administration also indicates a systemic mechanism of action for chloroform-induced toxicity. Interstitial pneumonitis was observed in male rats and rabbits after inhalation exposure to 85 or 50 ppm chloroform, respectively, for 6 months (Torkelson et al. 1976). In most oral studies, no exposure-related histopathological changes were observed in the lungs of exposed animals (Gulati et al. 1988; Jorgenson and Rushbrook 1980; NCI 1976; Palmer et al. 1979; Roe et al. 1979). Respiratory effects are more likely to occur after inhalation exposure to high concentrations of chloroform. It has been demonstrated that chloroform has a destructive influence on the pulmonary surfactant (Enhorning et al. 1986). This effect is probably due to the solubility of phospholipids in the surfactant monolayer and can cause collapse of the respiratory bronchiole due to the sudden increase in inhalation tension. Immediate death after chloroform inhalation may be due principally to this effect CHLOROFORM 139 2. HEALTH EFFECTS in the lungs (Fagan et al. 1977). It is unlikely that exposure levels of chloroform in the general environment or at hazardous waste sites would be high enough to cause these severe respiratory effects. Cardiovascular Effects. Chloroform induces cardiac arrhythmia in patients exposed to chloroform via anesthesia (Smith et al. 1973; Whitaker and Jones 1965). Similarly, heart effects were observed upon electrocardiography of an individual who accidentally ingested chloroform (Schroeder 1965). Hypotension was observed in 12-27% of patients exposed to chloroform via anesthesia (Smith et al. 1973; Whitaker and Jones 1965) and also was observed in a patient who ingested chloroform (Storms 1973). No studies were located regarding cardiovascular effects in animals after inhalation exposure to chloroform. No histopathological changes were observed in the heart of rats, mice (NCI 1976), or dogs (Heywood et al. 1979) chronically exposed to chloroform; however, cardiovascular function was not assessed in these studies. It has been demonstrated in an in vitro study on heart-lung preparations of guinea pigs that chloroform may cause a permanent contractile failure of the heart (Doring 1975). The effect is due to structural damage of the transverse tubular system and is accompanied by increased storage of adenosine triphosphate (ATP) and phosphocreatine. The in vitro induction of changes showed that contractile failure is a direct effect on the cardiovascular system rather than an indirect cardiovascular effect on the central nervous system. This mechanism may operate in humans exposed to high vapor concentrations such as those used in anesthesia or in humans exposed to high oral doses from accidental or intentional ingestion. It is unlikely, however, that concentrations of chloroform in the environment would be high enough to cause overt cardiovascular effects. Cytotoxicity of chloroform (1,000 ppm) in male Sprague-Dawley rat cardiac myocytes has been examined in vitro. Cell viability was measured using the criterion of Trypan blue exclusion as well as counting the number of rod and spherical cells in the media. Creatinine phosphokinase (CPK) leakage was measured as an indirect measurement of heart cell function. Myocytes treated with chloroform showed statistically significant decreases in cell viability and significant decreases in rod-shaped cells compared to controls. Significant increases in enzyme leakage of CPK from myocytes were noted (El-Shenawy and Abdel-Rahman 1993b). The effects of various concentrations of chloroform on the in vitro transfer of dyes between cardiac myocytes from Sprague-Dawley rats has also been examined (Toraason et al. 1992). The cells were exposed to one of 11 concentrations of chloroform in dimethyl CHLOROFORM 140 2. HEALTH EFFECTS sulfoxide (DMSO), and Lucifer yellow CH dye injected the cells, and the rate of transfer of dye from the injected myocyte to the non-injected myocyte recorded. As the cells were exposed to increasing concentrations of chloroform, the number of cells that transferred the dye decreased to zero. Heart cells also tended to beat slower or stop beating completely when exposed to chloroform but resumed normal spontaneous beating when chloroform was washed out. This in vitro data suggest that incorporation of halocarbons in the membrane may block intercellular communication through modification of the immediate environment of gap junctions. The data from these two studies indicate that chloroform exposure may induce reversible toxicity in the heart. Gastrointestinal Effects. Nausea and vomiting were not only frequently observed side effects in patients exposed to chloroform via anesthesia (Hakim et al. 1992; Royston 1924; Smith et al. 1973; Townsend 1939; Whitaker and Jones 1965), but also occurred in humans exposed to lower chloroform concentrations (22-237 ppm) in occupational settings (Challen et al. 1958; Phoon et al. 1983). Vomiting, gastric distress, and pain were observed in individuals who intentionally or accidentally ingested high doses of chloroform (Piersol et al. 1933; Schroeder 1965; Storms 1973). No studies were located regarding gastrointestinal effects in animals after inhalation exposure to chloroform. Vomiting in dogs (Heywood et al. 1979) and gastric erosions in rats (Thompson et al. 1974) were observed in oral studies of intermediate duration. These results suggest that severe gastrointestinal irritation in humans and animals is due to direct damage of the gastrointestinal mucosa caused by ingesting high concentrations of chloroform (Piersol et al. 1933; Schroeder 1965; Thompson et al. 1974). Nausea and vomiting experienced by occupationally exposed individuals is likely due to neurotoxicity. Since toxic hepatitis may occur at occupational levels as low as 2 ppm (Bomski et al. 1967), it is possible that levels of chloroform in the air at hazardous waste sites may be high enough to cause some liver effects with secondary gastrointestinal effects, if exposure is prolonged. Hematological Effects. Information regarding hematological effects in humans exposed to chloroform is limited. Increased prothrombin time was observed in some patients, following exposure to chloroform via anesthesia (Smith et al. 1973). This effect, however, reflects chloroform hepato- toxicity, because prothrombin is formed in the liver. Decreased erythrocytes and hemoglobin were observed in a patient who was chronically exposed to chloroform in a cough medicine (Wallace 1950). CHLOROFORM 141 2. HEALTH EFFECTS No hematological effects were observed in rats, rabbits, guinea pigs, or dogs after inhalation exposure to chloroform for intermediate durations (Torkelson et al. 1976). Studies report conflicting results regarding hematological effects in animals after oral exposure to chloroform. No conclusion about hematological effects in humans after exposure to chloroform can be made on the basis of one case study in humans. From the experimental data in animals, it is evident that all hematological effects observed in rats were due to oral exposure of acute, intermediate, or chronic duration. It is possible that the hematological effects observed in rats are transient. Human exposure to chloroform in the environment, drinking water, or at hazardous waste sites is likely to cause few or no hematological effects. Musculoskeletal Effects. Little data is available that examines the effects of chloroform toxicity on the musculoskeletal system; however, it appears that chloroform has few significant toxic effects on this system. Hepatic Effects. The liver is a primary target organ of chloroform toxicity in humans and animals after inhalation and oral exposure, with some evidence that suggests that the damage may be reversible (Wallace 1950). Impaired liver function was indicated by increased sulfobromophthalein retention in some patients exposed to chloroform via anesthesia (Smith et al. 1973). Acute toxic hepatitis developed after childbirth in several women exposed to chloroform via anesthesia (Lunt 1953; Royston 1924; Townsend 1939). Upon autopsy, centrilobular necrosis was observed in the women who died; however, the hepatotoxicity was associated with exhaustion from prolonged delivery, starvation, and dehydration, indicating improper handling of the delivery procedure by an obstetrician. Toxic hepatic disease, characterized by hepatomegaly and abnormal liver sonograms as late as seven days after an unknown amount of oral chloroform, has been reported (Hakim et al. 1992). Elevated liver enzymes and changes in GGT, alpha-feto protein and retinol binding protein were reported in a female who injected herself intravenously and also consumed chloroform orally during a 12-hour period (Rao et al. 1993). During occupational exposure to concentrations ranging from 14 to 400 ppm, chloroform hepatotoxicity was characterized by jaundice (Phoon et al. 1983), hepatomegaly, enhanced SGPT and SGOT activities, and hypergammaglobulinemia following exposure to concentrations ranging from 2 to 205 ppm (Bomski et al. 1967). In contrast, no clinical evidence of liver toxicity was found in another study among chloroform workers exposed to <237 ppm (Challen et al. 1958). Case reports of intentional and accidental ingestion of high doses (>2,410 mg/kg) of chloroform indicate severe liver CHLOROFORM 142 2. HEALTH EFFECTS injury (Piersol et al. 1933; Schroeder 1965; Storms 1973). The diagnosis was supported by clinical and biochemical results: fatty degeneration and extensive centrilobular necrosis were observed in one patient who died (Piersol et al. 1933). Liver damage was induced by chronic use of a cough medicine containing chloroform (Wallace 1950), but not by chronic exposure to chloroform in mouthwash (De Salva et al. 1975). An intermediate-duration inhalation MRL of 0.05 ppm was derived from the LOAEL of 14 ppm from the data presented by Phoon et al. (1983); a chronic-duration inhalation MRL of 0.02 ppm was derived from the LOAEL of 2 ppm from the data presented by Bomski et al. (1967). Reports regarding chloroform hepatotoxicity in animals are numerous (Larson et al. 1993, 1994a, 1994b, 1994c, 1995b, 1996; Nakajima et al. 1995; Pereira 1994; Wang et al. 1994, 1995). An acute-duration inhalation MRL of 0.1 ppm was based on a NOAEL for hepatic effects in mice exposed to 3 ppm chloroform for 6 hours a day for 7 days (Larson et al. 1994c). Liver damage was indicated by biochemical changes in rats (Lundberg et al. 1986; Nakajima et al. 1995; Wang et al. 1994, 1995) and mice (Gehring 1968; Murray et al. 1979) after acute inhalation exposure. Fatty changes (Culliford and Hewitt 1957; Kylin et al. 1963) and liver necrosis (Deringer et al. 1953: Larson et al. 1995b, 1996; Pereira 1994) were observed histologically in mice and rats after acute inhalation exposure. Histological findings indicative of liver toxicity were also observed in rabbits and guinea pigs following inhalation exposure of intermediate duration, but the findings were not dose-related (Torkelson et al. 1976). Liver effects have been observed in many species (rats, mice, and dogs) that were tested by the oral route by various methods of administration (gavage or drinking water) and durations (acute, intermediate, or chronic). Observed effects include increased liver weight, increased serum levels of transaminases indicative of liver necrosis, and histological evidence of swelling, fatty changes, hydropic changes, vacuolation, necrosis, hyperplasia, cirrhosis, macrophage and neutrophil infiltration, and toxic hepatitis (Bull et al. 1986; Chu et al. 1982b; Heindel et al. 1995; Heywood et al. 1979; Jones et al. 1958; Jorgenson and Rushbrook 1980; Klaunig et al. 1986; Larson et al. 1993, 1994b, 1995b, 1996; Nakajima et al. 1995; NCI 1976; Pereira 1994; Tumasonis 1985, 1987). Two acute oral studies define a LOAEL and a NOAEL for liver effects in mice. Fatty infiltration was observed in mice given a single gavage dose of 35 mg/kg/day chloroform in oil (Jones et al. 1958). No toxic effects on the livers of mice occurred after a single dose of 17.3 or 59.2 mg/kg chloroform in oil, but increased SGPT occurred at 199 mg/kg (Moore et al. 1982). The 17.3 mg/kg dose was also a NOAEL for kidney effects, but tubular necrosis occurred at 65.6 mg/kg/day. Ina 7.5-year study in which dogs were administered chloroform in toothpaste, SGPT activity was CHLOROFORM 143 2. HEALTH EFFECTS significantly increased at 30 mg/kg/day beginning at 6 weeks (Heywood et al. 1979). SGPT activity was not increased at 15 mg/kg/day until 130 weeks. Therefore, 15 mg/kg/day was a NOAEL for intermediate-duration exposure and a LOAEL for chronic-duration exposure. The 15 mg/kg/day dose was used to derive MRL values of 0.1 and 0.01 mg/kg/day for intermediate- and chronic-duration oral exposure, respectively. Data regarding chloroform-induced hepatotoxicity were also supported by results obtained after acute intraperitoneal exposure in rats (Bai et al. 1992; Ebel et al. 1987; El-Shenawy and Abdel-Rahman 1993a; Lundberg et al. 1986; Wang et al. 1994), mice (Klaassen and Plaa 1966), dogs (Klaassen and Plaa 1967), and gerbils (Ebel et al. 1987). No hepatic effects were observed in rabbits when chloroform was applied to their skin for 24 hours (Torkelson et al. 1976). The toxicity of chloroform on laboratory animal hepatocytes in vitro has been reported (Azri-Meehan et al. 1992, 1994: Bai and Stacey 1993; El-Shenawy and Abdel-Rahman 1993a; Suzuki et al. 1994). As discussed in Section 2.3.3, the mechanism of chloroform-induced liver toxicity may involve metabolism to the reactive intermediate, phosgene, which binds to lipids and proteins of the endoplasmic reticulum, lipid peroxidation, or depletion of GSH by reactive intermediates. Because liver toxicity has been observed in humans exposed to chloroform levels as low as 2 ppm in the workplace and in several animal species after inhalation and oral exposure, it is possible that liver effects could occur in humans exposed to environmental levels, to levels in drinking water, or to levels found at hazardous waste sites. Endocrine Effects. No reports of chloroform toxicity to endocrine organs have been reported. Renal Effects. Clinical reports indicate that the renal damage observed in women exposed to chloroform via anesthesia during prolonged parturition most likely occurs when chloroform anesthesia is associated with anoxia. Competitive swimmers who swim in indoor pools have been reported to have elevated 3-2-microglobin, suggesting some degree of renal damage (Aiking et al. 1994). Case studies of individuals who intentionally or accidentally ingested high doses of chloroform report biochemical changes indicative of kidney damage, as well as fatty degeneration at autopsy (Piersol et al. 1933; Schroeder 1965). Albuminuria and casts were also reported in a case of chronic use of a cough medicine containing chloroform (Wallace 1950); however, no renal effects were observed in individuals chronically exposed to chloroform in a mouthwash (De Salva et al. 1975). CHLOROFORM 144 2. HEALTH EFFECTS Renal effects of chloroform after inhalation have also been examined in animals (Larson et al. 1994b, 1994c, 1996). Kidney effects in animals after inhalation exposure to chloroform include tubular necrosis, tubular calcification, increased kidney weight, cloudy swelling, mineralization of the cortex, and interstitial nephritis. Animal studies regarding renal toxicity after oral exposure are numerous. Effects include acute toxic nephrosis, tubular dilation, necrosis and regeneration, chronic inflammation, mineralized concretions, hyaline cast formation, and fatty degeneration. The effects of dose and vehicle have been examined (Heindel et al. 1995; Larson et al. 1993, 1995b; Lipsky et al. 1993). Mice seem to be more sensitive to chloroform-induced renal toxicity than other experimental animals. Certain strains of male mice are susceptible to chloroform-induced nephrotoxicity, while female mice appear to be somewhat resistant (Culliford and Hewitt 1957; Eschenbrenner and Miller 1945b; Larson et al. 1996). Castrated mice were no longer susceptible to the effect, and testosterone treatment increased the severity of kidney damage in females, suggesting the role of hormones in chloroform- induced nephrotoxicity. It has been demonstrated that sensitivity to kidney damage is related to the capacity of the kidney to metabolize chloroform to phosgene (Pohl et al. 1984). The activation of chloroform to its reactive metabolites appeared to be cytochrome P-450-dependent: the covalent binding to microsomal protein required nicotinamide adenine dinucleotide phosphate (NADPH) and oxygen, and could be inhibited by carbon monoxide (Hook and Smith 1985; Smith and Hook 1983, 1984: Smith et al. 1984). Furthermore, administration of chloroform to male mice caused a depletion of renal GSH, indicating that GSH can react with reactive intermediates, thereby reducing the extent of the reaction with tissue MMBs and kidney damage. The renal toxicity of chloroform in rats after intraperitoneal dosing has also been reported (Kroll et al. 1994a, 1994b). It is likely that kidney effects may occur in humans after inhalation or oral exposure to high levels of chloroform; however, it is not known whether such effects would occur at the levels of chloroform found in the environment, in drinking water, or at hazardous waste sites. Dermal Effects. No reports are available on the toxicity of chloroform to skin after inhalation and oral exposures in humans. Stratum corneum damage was reported after a topical exposure of chloroform of 15 minutes duration for 6 consecutive days (Malten et al. 1968). Chloroform was used as a vehicle for the topical application of aspirin for the treatment of painful herpes zoster lesions in CHLOROFORM 145 2. HEALTH EFFECTS male and female humans. The only reported side-effect was an occasional burning sensation to the skin as the chloroform evaporated after application (King 1993). Few reports exist on the dermal effects of chloroform in animals after inhalation or oral exposures. Alopecia has been observed in pregnant rats (Thompson et al. 1974) and in mice (Gulati et al. 1988). Skin irritation and necrosis and been reported in rabbits after topical application of chloroform (Smyth et al. 1962; Torkelson et al. 1976). Ocular Effects. No studies were located regarding the ocular effects of chloroform in humans or animals. Body Weight Effects. Decreased body weight has been observed frequently in animals after inhalation or oral exposure to chloroform, although the degree of body weight changes are somewhat variable and may be linked to taste aversion (in oral studies) (Chu et al. 1982b; Larson et al. 1995b, 1996; Munson et al. 1982; Newell and Dilley 1978; Torkelson et al. 1976; Tumasonis et al. 1985, 1987). The degree of decreased weight gain was often dose-related and was caused by chloroform toxicity. Decreased weight gain generally occurred at exposure levels similar to or lower than those that induced liver and kidney effects in animals. The possibility of effects on body weight in humans exposed to ambient or elevated levels of chloroform cannot be dismissed. Immunological and Lymphoreticular Effects. No studies were located regarding immunological effects in humans after inhalation, oral, or dermal exposure to chloroform. Information about immunological effects in animals is limited. After repeated inhalation exposure to chloroform, mortality was increased in mice challenged with streptococcus infection, suggesting increased susceptibility (Aranyi et al. 1986). However, the bacterial activity of alveolar macrophages was not suppressed in this study. After acute oral exposure, reduced lymphocyte counts were observed in rats (Chu et al. 1982b). Furthermore, humoral immunity was depressed in mice exposed to 50 mg/kg/day chloroform for acute or intermediate durations (Munson et al. 1982). In contrast, cell-mediated immunity was influenced only at high chloroform concentrations administered orally for intermediate durations; however, the chloroform-induced immunological changes appeared to be more severe following acute exposure. In vitro treatment of serum with chloroform resulted in a loss of complement activity (Stefanovic et al. 1987). Immunological effects may result from the ability of CHLOROFORM 146 2. HEALTH EFFECTS chloroform to dissociate antigen-antibody complexes, since it can cause dissociation of certain enzyme- inhibitor complexes (Berger et al. 1983). Every day, humans are exposed to very low levels of chloroform in the environment, mainly via inhalation and oral exposure (Hajimiragha et al. 1986; Peoples et al. 1979; Wallace et al. 1987a, 1989). There is a risk of chloroform exposure at or near hazardous waste sites. Although no evidence that chloroform can cause immunological effects in humans was located in the literature, the possibility remains that these effects may result from exposure to chloroform. Neurological Effects. Neurological effects in humans after acute inhalation exposure to chloroform are well documented because chloroform has been used as an anesthetic for surgery. Inhaled chloroform acts as a depressant on the central nervous system. Chronic inhalation exposure to chloroform resulted in exhaustion, lack of concentration, depression, and irritability in occupationally exposed people (Challen et al. 1958). In a case study, chloroform inhalation for 12 years resulted in psychotic episodes, hallucinations, and convulsions (Heilbrunn et al. 1945). Central nervous system toxicity was observed in humans after oral exposure to chloroform, which suggests that the effects of inhalation and oral exposure are similar. In case reports of patients who intentionally or accidentally ingested several ounces of chloroform, deep coma with abolished reflexes occurred within a few minutes (Piersol et al. 1933; Schroeder 1965; Storms 1973). Inhalation exposure to high chloroform concentrations induced narcosis (Lehmann and Flury 1943; Sax 1979) and reversible impairment of memory retrieval in animals. High, single, oral doses of chloroform caused ataxia, incoordination, anesthesia, and brain hemorrhage in mice (Balster and Borzelleca 1982; Bowman et al. 1978). Behavioral effects were observed at lower oral doses. Chloroform concentrations from 1.5 to 6 mmol chloroform were used to determine how chloroform may modify glutamate receptor agonist responses in mouse brain cortical wedges. The two agonists examined were N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA). Responses were determined by measuring electrical responses within the cortical slices. Three mmol of chloroform selectively inhibited AMPA but did not affect NMDA responses. Higher concentrations of chloroform failed to inhibit the AMPA or NMDA content in the wedges (Carla and Moroni 1992). Male Sprague-Dawley rats administered a single 200 mg/kg dose of chloroform experienced a significant decrease in midbrain 5-HIAA levels and a significant increase in hypothalamic dopamine concentrations (Kanada et al. 1994). CHLOROFORM 147 2. HEALTH EFFECTS The clinical effects of chloroform toxicity on the central nervous system are well documented. However, the molecular mechanism of action is not well understood. It has been postulated that anesthetics induce their action at a cell-membrane level due to lipid solubility. The lipid-disordering effect of chloroform and other anesthetics on membrane lipids was increased by gangliosides (Harris and Groh 1985), which may explain why the outer leaflet of the lipid bilayer of neuronal membranes, which has a large ganglioside content, is unusually sensitive to anesthetic agents. Anesthetics may affect calcium-dependent potassium conductance in the central nervous system (Caldwell and Harris 1985). The blockage of potassium conductance by chloroform and other anesthetics resulted in depolarization of squid axon (Haydon et al. 1988). Based upon existing data, the potential for neurological and behavioral effects in humans exposed to chloroform at levels found in the environment, in drinking water, or at hazardous waste sites is very minimal. Reproductive Effects. It has not been definitively determined whether chloroform exposure induces reproductive effects in humans. No studies were located regarding reproductive effects in humans after inhalation or dermal exposure to chloroform. Only one study was located regarding reproductive effects in humans after oral exposure to chloroform. Bove et al. (1995) studied the effects of drinking-water consumption on birth outcomes and found that exposure to TTHM at levels >0.1 ppm resulted in reduced birth weight and size as well as an increased risk of oral cleft, central nervous system, and neural tube defects. These results should be viewed with caution since the authors did not specifically monitor chloroform levels. The effects seen may be due to exposure to other THMs or non-THM contaminants in the drinking water. Studies indicate that exposure to chloroform causes reproductive effects in animals. Dose-related increases of embryonal resorptions were observed in rats and mice after inhalation or oral exposure to chloroform during gestation. A significant increase in the incidence of abnormal sperm was observed in mice after acute inhalation exposure (Land et al. 1979, 1981). Gonadal atrophy was observed in male and female rats treated by gavage (Palmer et al. 1979). Fertility was not affected in either generation of mice exposed orally to chloroform in a 2-generation study (Gulati et al. 1988). In contrast, a 2-generation study in Sprague-Dawley rats and Swiss mice demonstrated significant decreases in combined live pup weights and in the proportion of male pups born live (rats), a significant reduction in sperm concentrations and sperm head counts, and increased numbers of unclear CHLOROFORM 148 2. HEALTH EFFECTS or no estrous cycles (mice). However, the animals in this study were administered drinking water containing a mixture of 25 contaminants; thus, other toxicants may have elicited these effects. Oral exposure to chloroform did not induce histopathological changes in the reproductive organs of rats exposed for intermediate durations (Jorgenson and Rushbrook 1980) or in rats and mice (NCI 1976) and dogs (Heywood et al. 1979) exposed for chronic durations. Developmental Effects. One study regarding developmental effects in humans after oral exposure to chloroform has been reported (Kramer et al. 1992). The estimated relative risk of low birth weight associated with drinking-water sources having chloroform levels of 210 ng/L was 30% higher than sources with undetectable levels of chloroform. Prematurity was not associated with chloroform/THM exposure. The estimated relative risk of intrauterine growth retardation associated with drinking-water supplies with chloroform concentrations of >10 pg/L was 80% higher than the risk for sources with undetectable levels of chloroform. Sources with intermediate chloroform levels (1-9 pg/L) had an elevated risk of 30%. There seems to be reasonable evidence to suggest that some correlation with an increased risk of intrauterine growth retardation associated with higher concentrations of waterborne chloroform and dichlorobromomethane does exist. Inhalation exposure to chloroform during gestation induced fetotoxicity and teratogenicity in rats (Schwetz et al. 1974) and mice (Murray et al. 1979). Decreased fetal crown-rump length, decreased ossifications, imperforate anus (rats), and cleft palate (mice) were observed in the offspring of exposed dams. In contrast, fetotoxicity (decreased fetal weight), but not teratogenicity, was observed in rats after oral exposure to chloroform (Ruddick et al. 1983; Thompson et al. 1974). Increased resorptions were observed in rats and rabbits (Thompson et al. 1974). In a 2-generation oral study, degeneration of the epididymal ductal epithelium was observed in mice of the F, generation (Gulati et al. 1988). Due to its chemical nature, chloroform can cross the placenta easily, as demonstrated by its detection in the placenta and fetuses of mice a short time after inhalation exposure (Danielsson et al. 1986). Chloroform may accumulate in the amniotic fluid and fetal tissues. Various developmental effects may result from exposure, depending on the period of in utero exposure. Although no studies have conclusively reported developmental effects in humans, chloroform (or in tandem with other organic halomethanes) may have the potential to cause developmental effects in humans. Whether such effects could occur from exposure to levels in the environment, in drinking water, or at hazardous waste sites is not known. CHLOROFORM 149 2. HEALTH EFFECTS Genotoxic Effects. In vivo and in vitro studies of the genotoxic effects of chloroform are summarized in Tables 2-5 and 2-6. Information regarding genotoxic effects after in vivo exposure to chloroform is limited. Mice exposed to chloroform by gavage had an increase in sister chromatid exchange frequency in bone marrow cells (Morimoto and Koizumi 1983). No sperm-head abnormalities were noted in mice after receiving 5 daily intraperitoneal injections of chloroform in concentrations up to 0.25 mg/kg/day in corn oil (Topham 1980). Oral exposure to chloroform did not increase UDS in rat hepatocytes (Mirsalis et al. 1982). Chloroform exposure caused mitotic arrest in grasshopper embryos (Liang et al. 1983) and a nonsignificant increase in the recessive lethals in Drosophila melanogaster (Gocke et al. 1981). In general, most of the assays for chloroform genotoxicity are negative. Therefore, it seems that chloroform is a weak mutagen and that its potential to interact with DNA is low. In in vitro experiments, chloroform did not cause reverse mutations in Salmonella typhimurium (Gocke et al. 1981; San Augustin and Lim-Sylianco 1978; Simmon et al. 1977; Uehleke et al. 1977; Van Abbe et al. 1982; Varma et al. 1988) or in Escherichia coli (Kirkland et al. 1981) with or without metabolic activation. Inconclusive results were obtained in Saccharomyces cerevisiae and Schisto- zosaccharomyces pombe (Callen et al. 1980; De Serres et al. 1981). Chloroform, however, induced Aneuploidia in Aspergillus nidulans (Crebelli et al. 1988). Chloroform caused forward mutations in L5178Y mouse lymphoma cells after metabolic activation (Mitchell et al. 1988), but did not cause mutations at 8-azaguanine locus in Chinese hamster lung fibroblasts (Sturrock 1977) or sister chromatid exchange in Chinese hamster ovary cells (White et al. 1979). A study performed in mice examined the ability of chloroform to induce UDS in hepatocytes in vitro from 15-week-old female B6C3F | mice. Chloroform concentrations ranged from 0.01 to 10 mmol. Mice were sacrificed at 2 and 12 hours postdosing to determine if and when UDS began to occur. Dimethylnitrosamine, a known inducer of UDS, was used as a positive control and did induce UDS in these hepatic cells. No induction of DNA repair was observed at any concentration of chloroform at either the 2-hour or 12-hour posttreatment groups. All concentrations of chloroform added to the cell cultures of mouse hepatocytes proved to be toxic. The study showed that chloroform is not directly genotoxic in hepatocytes of female mice, either in vivo or in vitro, despite the fact that it is the target organ of chloroform carcinogenesis (Larson et al. 1994a). In human lymphocytes, chloroform did not induce UDS (Peroccio and Prodi 1981) and did not increase the frequency of sister chromatid exchange and chromosome aberrations (Kirkland et al. 1981). In contrast, increases in sister chromatid exchange were reported after metabolic activation in another study (Morimoto and Koizumi 1983). Table 2-5. Genotoxicity of Chloroform /n Vivo Species (test system) End point Results Reference Mammalian cells: Rat hepatocytes Mouse bone marrow Mouse Mouse Grasshopper embryo Drosophila melanogaster Host-mediated assays: Salmonella typhimurium TA1535 (mouse host-mediated assay) S. typhimurium TA1537 (mouse host-mediated assay) (males only) Unscheduled DNA synthesis - Sister chromatid exchange - Sperm-head abnormalities - Sperm abnormalities + Mitotic arrest + Recessive lethals = Reverse mutation - Reverse mutation + Mirsalis et al. 1982 Morimoto and Koizumi 1983 Topham 1980 Land et al. 1981 Liang et al. 1983 Gocke et al. 1981 San Agustin and Lim-Sylianco 1978 San Agustin and Lim-Sylianco 1978 - = negative result; + = positive result; DNA = Deoxyribonucleic acid S103443 H1TV3aH 2 WHO40HOTHO 0st Table 2-6. Genotoxicity of Chloroform /n Vitro Results Species (test system) End point With Without Reference activation activation Prokaryotic organism: Salmonella typhimurium TAS8 Reverse mutation - = Gocke et al. 1981 S. typhimurium TA100 Reverse mutation - ~- Gocke et al. 1981 S. typhimurium TA1535 Reverse mutation - - Gocke et al. 1981 S. typhimurium TA1535 Reverse mutation - = Uehleke et al. 1977 S. typhimurium TA1538 Reverse mutation = = Uehleke et al. 1977 S. typhimurium TA98 Reverse mutation = - Simmon et al. 1977 S. typhimurium TA100 Reverse mutation = - Simmon et al. 1977 S. typhimurium TA1535 Reverse mutation - - Simmon et al. 1977 S. typhimurium TA1537 Reverse mutation - = Simmon et al. 1977 S. typhimurium TA98 Reverse mutation ~ - Van Abbe et al. 1982 S. typhimurium TA100 Reverse mutation - ~ Van Abbe et al. 1982 S. typhimurium TA1535 Reverse mutation ~ ~ Van Abbe et al. 1982 S. typhimurium TA1537 Reverse mutation - ~ Van Abbe et al. 1982 S. typhimurium TA1538 Reverse mutation S - Van Abbe et al. 1982 S. typhimurium TA98 Reverse mutation - (+) Varma et al. 1988 S. typhimurium TA100 Reverse mutation + (+) Varma et al. 1988 S. typhimurium TA1535 Reverse mutation - (+) Varma et al. 1988 S. typhimurium TA1537 Reverse mutation - (+) Varma et al. 1988 S. typhimurium TA98 Reverse mutation Not tested - San Augustin and Lim-Sylianco 1978 S. typhimurium TA1535 Reverse mutation Not tested ~ San Augustin and Lim-Sylianco 1978 S. typhimurium TA1537 Reverse mutation Not tested - San Augustin and Lim-Sylianco 1978 WHO40HOTHO S103443 HLTV3H ¢ ISH Table 2-6. Genotoxicity of Chloroform /n Vitro (continued) WHO40HO THO Results Species (test system) End point With Without Reference activation activation Escherichia coli Reverse mutation - - Kirkland et al. 1981 Aspergillus nidulans Aneuploidia + Not tested Crebelli et al. 1988 Saccharomyces cerevisiae Reverse mutation = (+) De Serres et al. 1981 Schizosaccharomyces pombe Recombinations - (+) Callen et al. 1980 Eukaryotic organisms: Mammalian cells: L5178Y mouse lymphoma cells Forward mutation + - Mitchell et al. 1988 Chinese hamster lung fibroblasts Mutation at 8-azaquonine - Sturrock 1977 Chinese hamster ovary cells Human lymphocytes Human lymphocytes Human lymphocytes Human lymphocytes Sister chromatid exchange Unscheduled DNA synthesis Sister chromatid exchange Sister chromatid exchange Chromosome aberrations White et al. 1979 Perocco and Prodi 1981 Morimoto and Koizumi 1983 Kirkland et al. 1981 Kirkland et al. 1981 - = negative result; + = positive result; (+) = weakly positive; DNA = Deoxyribonucleic acid S103443 H1V3H 2 ast CHLOROFORM 153 2. HEALTH EFFECTS Cancer. No studies were available regarding cancer in humans or animals after inhalation exposure to chloroform. Epidemiology studies suggest an association between chronic exposure to chlorinated drinking-water sources and increased incidences of colon cancer (Young et al. 1981), pancreatic cancer (Ijsselmuiden et al. 1982) and bladder cancer (Cantor et al. 1978; McGeehin et al. 1993; Zierler et al. 1988). However, numerous other potential toxicants known to exist in chlorinated drinking water may easily account for these effects. The carcinogenic potential of chloroform has been tested in animal studies. A dose-related increase in the incidence of hepatomas was observed in mice exposed to chloroform for intermediate durations (Eschenbrenner and Miller 1945a). Chronic-duration exposure induced an increased incidence of renal adenoma and carcinoma in rats exposed to chloroform in drinking water (Jorgenson et al. 1985). Increased incidence of neoplastic nodules in the liver was observed in female Wistar rats ingesting chloroform in drinking water (Tumasonis et al. 1987). In addition, hepatocellular carcinoma was observed in B6C3F | mice given chloroform in oil by gavage (NCI 1976), and kidney tumors were observed in male ICI mice exposed by gavage to chloroform in toothpaste (Roe et al. 1979). The incidence of liver and kidney tumors in male and female rats given chloroform in a chronic-duration study has been reported (Dunnick and Melnick 1993; NCI 1976). While no hepatocellular or large intestine neoplasms were noted in either sex of rat, kidney tubular cell neoplasms were observed at 90 mg/kg/day and 180 mg/kg/day in male rats and at 200 mg/kg/day in female rats. In a another study by the same authors, using male and female mice dosed with similar amounts of chloroform, no kidney tubular cell neoplasms or large intestine neoplasms were reported in either sex of mice, while hepatocellular neoplasms were recorded in both sexes. In a similar study, Jorgenson et al. (1985) examined the carcinogenic effects of chloroform administered chronically (104 weeks) in drinking water to male Osborne-Mendel rats and female B6C3F | mice. While no treatment related enhancement of tumor formation was observed in mice, kidney tubular cell neoplasms were observed in male rats exposed to 160 mg/kg/day chloroform. The data concerning mouse liver tumors are conflicting. In contrast to the increased incidence of liver tumors observed in B6C3F | mice exposed by gavage to chloroform in oil (NCI 1976), no increased incidence of liver tumors was observed in female B6C3F | mice exposed to chloroform in drinking water (Jorgenson et al. 1985). This result is consistent with the absence of liver tumor effects in four other strains of mice exposed by gavage to chloroform in toothpaste (Roe et al. 1979). In a pharmacokinetic study, chloroform was absorbed more slowly and to a lesser extent from corn oil than CHLOROFORM 154 2. HEALTH EFFECTS water (Withey et al. 1983), suggesting that pharmacokinetic effects are not responsible for the differences in liver tumor responses. Nevertheless, data from historical controls indicate that corn oil alone 1s not responsible for the increased incidence of liver tumors (Jorgenson et al. 1985). The corn oil vehicle effect on the induction of mouse liver neoplasms may be due to an interaction between the vehicle and chloroform (Bull et al. 1986; Jorgenson et al. 1985), possibly resulting in altered pharmacokinetics. Larson et al. (1994b) demonstrated that female B6C3F | mice developed increased hepatocyte toxicity after gavage dosing in oil with chloroform concentrations of 238 and 477 mg/kg/day for 4 days or 3 weeks (as determined by BrdU-labelling of hepatocytes). However, there was no increase in LI in the liver of the same strain of female mice administered up to 1,800 ppm in the drinking water for 4 days or 3 weeks. The actual doses for the mice administered at 1,800 ppm were 105 and 329 mg/kg/day, respectively. Other studies have shown similar intake dose difference due to vehicle effect (Jorgenson et al. 1985; NCI 1976). The difference in results are most likely due to the method of dosing and the vehicle used, both having effects on the pharmacokinetics of chloroform and hence the degree of hepatotoxicity (and perhaps the renal toxicity in males) that chloroform may induce in these mice. Gavaged animals typically receive a large dose of chloroform all at one time over a period of several days, while the animals in the drinking-water studies consume somewhat equal amounts of chloroform; however, it is consumed in small sips (Larson et al. 1994b). It seems clear that the design of the gavage studies inherently results in repeated and relatively massive doses of chloroform to the liver (and other susceptible cells) over a short period of time that likely overwhelm the liver defense mechanisms for chloroform detoxification, resulting in hepato- toxicity, cell death, or both. Conversely, drinking-water studies expose the liver to continuous, low doses of chloroform, resulting in detoxification, elimination, and few apparent signs of hepatocellular damage. Clearly, further studies that describe the differences in pharmacokinetics between dosing method (gavage as opposed to drinking water) and vehicle effects (oil as opposed to water) need to be performed to correctly estimate human risk to orally consumed chloroform. The possible association between cell proliferation as a result of cytotoxicity and chemical carcinogenesis has been the subject of considerable debate (Melnick et al. 1996). Chemically induced cell proliferation does play an important role in the carcinogenic process; however, the relationship between induced cell proliferation and tumorigenesis is not a direct cause-and-effect relationship (Farber 1995). Based on short-term exposures studies (4, 7, or 21 days), Larson et al. (1994b) suggest that chloroform causes cancer by inducing cytotoxicity, followed by cell regeneration. However, a CHLOROFORM 155 2. HEALTH EFFECTS review by Chiu et al. (1996), correlating chloroform-induced short-term and long-term cytotoxicity studies with cancer in target tissues, suggests that the mode of action of chloroform carcinogenesis can not be concluded with the currently available data. There is a qualitative correlation between short-term toxicological end points of cytotoxicity and the occurrence of neoplasia in the liver of B6C3F; mice exposed to chloroform either by corn oil gavage or in the drinking water. However, the association between cytotoxicity and cancer is lacking in other test systems. For example, short-term cytotoxicity (4 days) was observed in the kidneys of B6C3F, mice (Larson et al. 1994d) without an observable increase in neoplasia in male B6C3F, mice exposed to chloroform in corn oil by gavage for 78 weeks at similar doses (NCI 1976). These data indicate that increased cell proliferation may not always be sufficient to cause increased tumor incidence. Some studies suggest that the carcinogenic response in male rat kidneys (Jorgensen and Rushbrook 1980; Jorgensen et al. 1985) may not be mediated by a mechanism involving necrosis and regenerative cell proliferation. This observation is supported by the fact that chloroform exposure, by either drinking water or corn oil gavage, induced kidney neoplasia in male Osborne-Mendel rats without any reported short-term and long-term cytotoxicity. There was no treatment-related biochemical and microscopic/gross histopathological changes in the kidneys of the rats at 30, 60, or 90 days after receiving chloroform in the drinking water (Jorgensen and Rushbrook 1980). Neither necrosis nor tubular cell hyperplasia was found in the kidneys of male Osborne-Mendel rats treated with chloroform by gavage or drinking water in the 2-year bioassays (Jorgensen et al. 1985; NCI 1986). In the liver bioassay for GGTase positive foci, chloroform had neither an initiating effect nor a promoting effect when administered in drinking water (Herren-Freund and Pereira 1987), but had a promoting effect of these loci initiated by diethylnitrosamine if given in a corn oil vehicle (Deml and Oesterle 1985); both studies were performed in rats. Moreover, chloroform enhanced the growth of experimentally inoculated tumors in mice (Capel et al. 1979). In contrast, chloroform had an inhibiting effect on the growth of tumors induced by known carcinogens (1,2-dimethylhydrazine and ethylnitrosurea) in rats (Daniel et al. 1989; Herren-Freund and Pereira 1987). In epidemiologic studies, chloroform is not identified as the sole or primary cause of excess cancer rates, but it is one of many organic contaminants found in chlorinated drinking water, many of which are considered to have carcinogenic potential. These studies are often flawed by a lack of measured CHLOROFORM 156 2. HEALTH EFFECTS chloroform concentrations in drinking water; lack of data concerning concentrations of other organics, limited information concerning personal drinking-water consumption, long latency periods, and effects of migration, making it difficult to quantify exposure. Although human data suggest a possible increased risk of cancer from exposure to chloroform in chlorinated drinking water, the data are too weak to draw a conclusion about the carcinogenic potential of chloroform in humans. Based on animal studies, chloroform has been classified as a probable human carcinogen by EPA (IRIS 1995), as a possible human carcinogen by IARC (1987), and as a substance that may reasonably be anticipated to be carcinogenic in humans (NTP 1989). 2.6 BIOMARKERS OF EXPOSURE AND EFFECT Biomarkers are broadly defined as indicators signaling events in biologic systems or samples. They have been classified as markers of exposure, markers of effect, and markers of susceptibility (NAS/NRC 1989). Due to a nascent understanding of the use and interpretation of biomarkers, implementation of biomarkers as tools of exposure in the general population is very limited. A biomarker of exposure is a xenobiotic substance or its metabolite(s), or the product of an interaction between a xenobiotic agent and some target molecule(s) or cell(s) that is measured within a compartment of an organism (NAS/NRC 1989). The preferred biomarkers of exposure are generally the substance itself or substance-specific metabolites in readily obtainable body fluid(s) or excreta. However, several factors can confound the use and interpretation of biomarkers of exposure. The body burden of a substance may be the result of exposures from more than one source. The substance being measured may be a metabolite of another xenobiotic substance (e.g., high urinary levels of phenol can result from exposure to several different aromatic compounds). Depending on the properties of the substance (e.g., biologic half-life) and environmental conditions (e.g., duration and route of exposure), the substance and all of its metabolites may have left the body by the time samples can be taken. It may be difficult to identify individuals exposed to hazardous substances that are commonly found in body tissues and fluids (e.g., essential mineral nutrients such as copper, zinc, and selenium). Biomarkers of exposure to chloroform are discussed in Section 2.6.1. Biomarkers of effect are defined as any measurable biochemical, physiologic, or other alteration within an organism that, depending on magnitude, can be recognized as an established or potential health CHLOROFORM 157 2. HEALTH EFFECTS impairment or disease (NAS/NRC 1989). This definition encompasses biochemical or cellular signals of tissue dysfunction (e.g., increased liver enzyme activity or pathologic changes in female genital epithelial cells), as well as physiologic signs of dysfunction such as increased blood pressure or decreased lung capacity. Note that these markers are often not substance specific. They also may not be directly adverse, but can indicate potential health impairment (e.g., DNA adducts). Biomarkers of effects caused by chloroform are discussed in Section 2.6.2. A biomarker of susceptibility is an indicator of an inherent or acquired limitation of an organism's ability to respond to the challenge of exposure to a specific xenobiotic substance. It can be an intrinsic genetic or other characteristic or a pre-existing disease that results in an increase in absorbed dose, biologically effective dose, or target tissue response. If biomarkers of susceptibility exist, they are discussed in Section 2.7, Populations That Are Unusually Susceptible. 2.6.1 Biomarkers Used to Identify or Quantify Exposure to Chloroform Chloroform concentrations measured in tissue and/or air samples can not be currently be used as specific biomarkers for chloroform exposure; however, they may indicate exposure to chloroform or other halogenated compounds that have undergone metabolism to chloroform. Methods for measuring chloroform in biological fluids, tissues, and exhaled breath are available; however, there is relatively little quantitative information relating monitored chloroform levels in tissues or fluids to exposure. The presence of chloroform or its metabolites in biological fluids and tissues may result from the metabolism of other chlorinated hydrocarbons; thus, elevated tissue levels of chloroform or its metabolites may reflect exposure to other compounds. The relationship between chloroform concentration in inspired air and resulting blood chloroform levels is the most well defined measure of exposure due to the extensive use of chloroform as a surgical anesthetic. A mean arterial blood concentration of 9.8 mg/dL (range 7-16.6 mg/dL) was observed among 10 patients receiving chloroform anesthesia at an inspired air concentration of 8,000-10,000 ppm (Smith et al. 1973). Monitoring of blood levels in workers experiencing toxic jaundice due to chloroform exposure revealed that when workroom air concentrations were estimated to be >400 ppm, the blood samples of 13 workers with jaundice were 0.10-0.3 ug/100 mL blood (Phoon et al. 1983). In another group of 18 workers with toxic hepatitis, blood samples revealed chloroform in some but not all workers, and workroom air contained 14.4-50.4 ppm on various days. These data suggest an association between CHLOROFORM 158 2. HEALTH EFFECTS increased blood concentrations and increased exposure concentrations, but the blood levels varied too greatly to establish a direct quantitative relationship. Environmental exposure to chloroform in humans probably represents a combination of inhalation exposure (from the air polluted with volatile halogenated hydrocarbons) and oral exposure (from chlorinated water sources), in addition to a dermal absorption route for chloroform from contaminated water sources (from showering, bathing, or swimming). The chloroform levels detected in human blood varied according to geographical areas. Chloroform levels ranged from 13 to 49 pg/L in serum samples taken from 10 individuals in Florida (Peoples et al. 1979). The level of environmental exposure was not reported. The mean blood chloroform concentration was 1.5 pg/L in blood samples taken from 250 individuals in Louisiana; exposure levels were not reported (Antoine et al. 1986). Chloroform was found in breath samples from large cohorts of people from New Jersey, North Carolina, and North Dakota (Wallace et al. 1987a). The levels of chloroform in breathing zone (personal) air were consistently higher than outdoor concentrations and correlated with chloroform concentrations in the exhaled breath samples. Some activities such as visiting the dry cleaners (an industry associated with high chloroform levels) or showering were associated with increased chloroform breath levels (Jo et al. 1990a, 1990b; Wallace et al. 1989). Chloroform was detected in 7 of 42 samples of human milk collected in 4 geographical areas in the United States (Pellizzari et al. 1982). Tissue levels of chloroform obtained at autopsy reflected environmental exposure levels in other studies. The levels ranged from 20 to 49 ng/kg of chloroform from adipose tissue extracted into hexane from samples taken from 10 individuals in Florida (Peoples et al. 1979). In 30 autopsy cases in Germany, the adipose tissue contained a mean of 23.4 pg/kg wet tissue; 24.8 pg/kg perinephric fat; 10.8 pg/kg liver tissue; 9.9 pg/kg lung tissue; and 10 pg/kg muscle tissue (Alles et al. 1988). The maximum chloroform content increased with age and was not dependent on the volume of fat in the tissues. No correlation has been made between the exact environmental levels of chloroform and the amount of chloroform in the exhaled breath or in the blood. Furthermore, chloroform also can be detected in the breath after exposure to carbon tetrachloride and other chlorinated hydrocarbons (Butler 1961). Therefore, chloroform levels cannot be used as reliable biomarkers of exposure to this chemical. CHLOROFORM 159 2. HEALTH EFFECTS 2.6.2 Biomarkers Used to Characterize Effects Caused by Chloroform The primary targets of chloroform toxicity are the central nervous system, liver, and kidney. The signs and symptoms of central nervous system effects (e.g., dizziness, fatigue, headache) are easily recognized. Monitoring liver and kidney effects induced by exposure to low levels of chloroform requires the testing of organ functions. Liver effects are commonly detected by monitoring for elevated levels of liver enzymes in the serum or testing for sulfobromophthalein retention. Urinalysis and measurements of BUN and B-2-microglobin are used to detect abnormalities in kidney function. Because many toxic chemicals can cause adverse liver and kidney effects, these tests are not specific for chloroform. No specific biomarkers used to characterize effects caused specifically by chloroform were located. For more information on biomarkers for renal and hepatic effects of chemicals see ATSDR/CDC Subcommittee Report on Biological Indicators of Organ Damage (1990) and for information on biomarkers for neurological effects see OTA (1990). 2.7 INTERACTIONS WITH OTHER CHEMICALS The interactions of chloroform with other chemicals are an issue of great importance; as with many chemicals, exposure to chloroform alone seldom occurs. This is especially true when considering exposure to chlorinated water, which usually contains other trihalomethanes and may contain other potential toxicants. Clinical reports of patients who underwent chloroform anesthesia indicated that premedication with morphine caused serious respiratory depression when chloroform was co-administered. Thiopentone (thiopental Na, an ultra-short-acting barbiturate anesthetic) was associated with increased incidences of hypotension in chloroform-anesthetized patients (Whitaker and Jones 1965). Several animal studies indicate that chloroform interacts with other chemicals within the organism. The lethal and hepatotoxic effects of chloroform were increased by dicophane (DDT) (McLean 1970) and phenobarbital (a long-acting barbiturate) in rats (Ekstrom et al. 1988; McLean 1970; Scholler 1970). Increased hepatotoxic and nephrotoxic effects were observed after interaction with ketonic solvents and ketonic chemicals in rats (Hewitt and Brown 1984; Hewitt et al. 1990) and in mice CHLOROFORM 160 2. HEALTH EFFECTS (Cianflone et al. 1980; Hewitt et al. 1979). The hepatotoxicity of chloroform was also enhanced by co-exposure to carbon tetrachloride in rats (Harris et al. 1982) and by co-exposure to ethanol in mice (Kutob and Plaa 1962). Furthermore, ethanol pretreatment in rats enhanced chloroform-induced hepatotoxicity (Wang et al. 1994) and increased the in vitro metabolism of chloroform (Sato et al. 1981). A mixture of cadmium and chloroform potentiated the cytotoxicity of each in in vitro experiments in rat hepatocytes (Stacey 1987a, 1987b). In contrast, mirex did not increase chloroform toxicity in mice (Hewitt et al. 1979). Disulfiram, an inhibitor of microsomal enzymes, decreases the hepatotoxicity of chloroform (Masuda and Nakayama 1982; Scholler 1970). Diethyldithiocarbamate and carbon disulfide pretreatment also protect against chloroform hepatotoxicity (Gopinath and Ford 1975; Masuda and Nakayama 1982, 1983), presumably by inhibiting microsomal enzymes. In general, chloroform toxicity can be influenced by chemicals that alter microsomal enzyme activity or hepatic GSH levels. The role that dichloroacetate (DCA) and trichloroacetate (TCA) play in chloroform toxicity was studied in rats (Davis 1992). TCA and DCA are formed in conjunction with chloroform during the chlorination of drinking water; therefore, animals drinking chlorinated water may be exposed to all three compounds simultaneously. It was found that DCA increases the hepatotoxicity and nephrotoxicity of chloroform in rats, that TCA increases the nephrotoxicity of chloroform, and that these effects were gender-specific, occurring mainly in females. The effects of monochloroacetate (MCA) on chloroform toxicity has also been investigated, with the combination (MCA + chloroform) shown to have toxic effects on the liver and kidneys of rats (Davis and Berndt 1992). The effect of chloroform and other organic halides (i.e., dichlorobromomethane) on intrauterine growth retardation has also been explored (Kramer et al. 1992). Ikatsu and Nakajima (1992) studied the effect of low-dose inhalation of chloroform with or without co-exposure to carbon tetrachloride on hepatotoxicity when rats were or were not previously exposed to ethanol. Groups of control or ethanol-pretreated rats inhaled 0, 50, or 100 ppm chloroform alone; 0, 25, or 50 ppm chloroform with 5 ppm carbon tetrachloride; or 0, 10, 25, or 50 ppm chloroform with 10 ppm carbon tetrachloride. Exposures to either 50 or 100 ppm of chloroform alone did not significantly change SGOT, SGPT, liver, or serum malondialdehyde (MDA) concentrations. In the rats pretreated with ethanol, SGOT and SGPT levels were significantly elevated above control animals at 100 ppm chloroform and SGOT levels were increased at 50 ppm chloroform as well. There was no CHLOROFORM 161 2. HEALTH EFFECTS change in either serum or liver concentrations of MDA in either exposure group. Liver triglycerides and GSH levels were significantly elevated above those of control animals for both exposure levels for animals not pretreated with ethanol; however, overall liver weights were elevated at only 100 ppm chloroform. In rats pretreated with ethanol, there was no significant change in liver triglyceride concentrations at either dose; however, liver GSH and liver weights were significantly elevated above control at both exposure concentrations. In chloroform and carbon tetrachloride treated rats not pretreated with ethanol, elevations in SGOT (10 ppm CCl, plus 10 and 50 ppm CHCl;), SGPT (5 ppm CCl, plus 50 ppm CHClj, 10 ppm CCl, plus 25 ppm CHCl), and plasma MDA (10 ppm CCl, plus 10, 25, and 50 ppm CHCl3) were observed. In chloroform and carbon tetrachloride-treated rats pretreated with ethanol, elevations in SGOT (all doses), SGPT (all doses except 5S ppm CCl, + 25 ppm CHCly), liver MDA (all doses), and plasma MDA (5 and 10 ppm CCl, + 50 ppm CHClj, 10 ppm CCl, + 25 ppm CHCl3) were observed. In chloroform and carbon tetrachloride treated rats not pretreated with ethanol, elevations in liver triglyceride (all doses) and GSH (5 ppm CCl, + 50 ppm CHCl5, 10 ppm CCl, + 10 and 50 ppm CHCl3) were observed. In chloroform and carbon tetrachloride treated rats pretreated with ethanol, elevations in liver triglyceride (all chloroform doses at 10 ppm CCl,) and GSH (all doses except 5 ppm CCl, + 25 ppm CHCl3) were observed. The results suggest that chloroform enhances carbon tetrachloride-induced hepatotoxicity. 2.8 POPULATIONS THAT ARE UNUSUALLY SUSCEPTIBLE A susceptible population will exhibit a different or enhanced response to chloroform than will most persons exposed to the same level of chloroform in the environment. Reasons may include genetic makeup, age, health and nutritional status, and exposure to other toxic substances (e.g., cigarette smoke). These parameters may result in reduced detoxification or excretion of chloroform, or compromised function of target organs affected by chloroform. Populations who are at greater risk due to their unusually high exposure to chloroform are discussed in Section 5.6, Populations With Potentially High Exposure. Since the liver and kidney are the two main organs responsible for chloroform metabolism, individuals who have hepatic or renal impairment may be more susceptible to chloroform toxicity; one such population would be those who abuse alcohol (Wang et al. 1994; Kutob and Plaa 1962). Also, exhaustion and starvation may potentiate chloroform hepatotoxicity, as indicated in some human clinical reports (Royston 1924; Townsend 1939) and in animal studies (Ekstrom et al. 1988; McMartin CHLOROFORM 162 2. HEALTH EFFECTS et al. 1981). Animal studies indicate that male mice and rats may be more susceptible to the lethal and renal effects of chloroform than female mice and rats (Deringer et al. 1953; Torkelson et al. 1976). The greater susceptibility of adult male animals is associated with testosterone levels in the animals (Deringer et al. 1953). Evidence also exists for age-related effects; young male mice were less susceptible to the lethal effects of chloroform compared to adult males (Deringer et al. 1953). Kimura et al. (1971) noted similar differences between young and old adult rats, but also found that chloroform was significantly more toxic in 14-day-old rats than in adult rats. Whether or not these subpopulations in humans would be more susceptible than their respective counterparts is not presently known. 2.9. METHODS FOR REDUCING TOXIC EFFECTS This section will describe clinical practice and research concerning methods for reducing toxic effects of exposure to chloroform. However, because some of the treatments discussed may be experimental and unproven, this section should not be used as a guide for treatment of exposures to chloroform. When specific exposures have occurred, poison control centers and medical toxicologists should be consulted for medical advice. The following texts provide specific information about treatment following exposures to chloroform: Ellenhorn, MJ and Barceloux, DG, (eds.) (1988). Medical Toxicology: Diagnosis and Treatment of Human Poisoning. Elsevier Publishing, New York, NY., pp. 972-974. Dreisback, RH, (ed.) (1987). Handbook of Poisoning. Appleton and Lange, Norwalk, CT. Haddad, LM and Winchester, JF, (eds.) (1990). Clinical Management of Poisoning and Drug Overdose. 2nd edition, WB Saunders, Philadelphia, PA. Aaron, CK and Howland, MA (eds.) (1994). Goldfrank's Toxicologic Emergencies. Appleton and Lange, Norwalk, CT. 2.9.1 Reducing Peak Absorption Following Exposure Human exposure to chloroform may occur by inhalation, ingestion, or by dermal contact. General recommendations for reducing absorption of chloroform include removing the exposed individual from the contaminated area and removing the contaminated clothing. If the eyes and skin were exposed, they should be flushed with water. In order to reduce absorption of ingested chloroform, emesis may CHLOROFORM 163 2. HEALTH EFFECTS be considered unless the patient is comatose, is convulsing, or has lost the gag reflex. Controversy exists concerning use of emesis because of the rapid onset of central nervous system depression, the risk of aspiration of stomach contents into the lungs, and the relative ineffectiveness of this method. In comatose patients with absent gag reflexes, an endotracheal intubation may be performed in advance to reduce the risk of aspiration pneumonia. Gastric lavage may also be used. 2.9.2 Reducing Body Burden Chloroform is not stored to any appreciable extent in the human body and is mostly metabolized to phosgene and eventually CO, (see Section 2.3); however, some chloroform may be stored in fat depots in the body. The half-life of chloroform in humans has been calculated to be 7.9 hours following inhalation exposure (Gordon et al. 1988). Furthermore, an oral-exposure study found most of the chloroform dose being eliminated within 8 hours postexposure (Fry et al. 1972). Hepatic and pulmonary first-pass effect was reported in humans (Chiou 1975). Despite a relatively fast clearance of chloroform from the body, toxic effects may develop in exposed individuals. No method is commonly practiced to enhance the elimination of the absorbed dose of chloroform. Although there is evidence that ethanol pretreatment of rats can increase the in vitro metabolism of chloroform (Sato et al. 1981), such treatment would not be recommended (Kutob and Plaa 1962) because it would increase the toxicity of chloroform and it is a very poor practice generally. 2.9.3 Interfering with the Mechanism of Action for Toxic Effects Target organs of chloroform toxicity are the central nervous system, liver, and kidneys (see Section 2.2). Respiratory, cardiovascular, and gastrointestinal toxic effects have also been reported. Studies in animals also indicated that chloroform exposure may induce reproductive and developmental effects and cause cancer. Several studies investigated the possible mechanism for chloroform-induced toxicity (see Section 2.5). Proposed mechanisms of chloroform toxicity and potential mitigations based on these mechanisms are discussed below. The potential mitigation techniques mentioned are all experimental. CHLOROFORM 164 2. HEALTH EFFECTS One of the possible mechanisms of chloroform toxicity is thought to be linked to its high lipid solubility and its ability to bind covalently to lipids (Testai et al. 1987). For example, neurotoxic and respiratory effects of chloroform may be due to the interaction of chloroform with gangliosides in neuronal membranes (Harris and Groh 1985) and phospholipids in the surfactant monolayer of the lower respiratory tract (Enhorning et al. 1986), respectively. Another proposed reaction of chloroform and lipids would result in the formation of conjugated dienes which are indicative of lipid peroxidation (De Groot and Noll 1989). Some authors reported that conjugated dienes may play a key role in the hepatotoxicity induced by haloalkanes (Comporti 1985; Recknagel et al. 1982). Others, however, argue that lipid peroxidation alone is not responsible for all changes found in the liver following chloroform exposure (Brown et al. 1974b; Lavigne and Marchand 1974). Instead, it was proposed that the mechanism of chloroform-induced liver and kidney toxicity involved metabolism to the reactive intermediate, phosgene, which binds to proteins of the endoplasmatic reticulum (Pohl et al. 1980a, 1980b). While this is true in the rat, it has not been established in other species, including humans. The toxicity of chloroform is increased by inducers of cytochrome P-450 such as phenobarbital (Scholler 1970). The involvement of cytochrome P-450 is further supported by the finding that disulfiram (Scholler 1970) and methoxsalen (Letteron et al. 1987), both inhibitors of microsomal enzymes, decreased the liver injury caused by chloroform in rats and mice, respectively. In addition, pretreatment with diethyldithiocarbamate and carbon disulfide protected mice against chloroform hepatotoxicity as indicated by biochemical and histopathological results (Gopinath and Ford 1975; Masuda and Nakayama 1982, 1983). Similarly, pretreatment of mice with methoxsalen (Letteron et al. 1987) and piperonyl butoxide (Kluwe and Hook 1981) reduced the chloroform-induced nephrotoxicity. Further research to determine which isozymes of P-450 are involved in metabolism to the more harmful metabolite, phosgene, as well as which isozymes are involved in enhancing the elimination of chloroform, could lead to the development of strategies designed to selectively inhibit specific P-450 isozymes, and thus reduce the toxic effects of chloroform. Administration of chloroform to laboratory animals resulted in the depletion of renal GSH, indicating that GSH reacts with reactive intermediates, thus reducing the kidney damage otherwise caused by the reaction of these intermediates with tissue MMBs (Hook and Smith 1985; Smith and Hook 1983, 1984; Smith et al. 1984). Similarly, chloroform treatment resulted in the depletion of hepatic GSH and alkylation of MMBs (Docks and Krishna 1976). Other studies demonstrated that sulfhydryl compounds such as L-cysteine (Bailie et al. 1984) and reduced GSH (Kluwe and Hook 1981) may CHLOROFORM 165 2. HEALTH EFFECTS provide protection against nephrotoxicity induced by chloroform. The sulfhydryl compound N-acetyl- cysteine is an effective antidote for poisoning by acetaminophen, which, like chloroform, depletes GSH and produces toxicity by reactive intermediates. All mitigations of the chloroform-induced toxicity cited above are experimental. Further studies would be needed for implications of any of these methods to humans. 2.10 ADEQUACY OF THE DATABASE Section 104(1)(5) of CERCLA directs the Administrator of ATSDR (in consultation with the Administrator of EPA and agencies and programs of the Public Health Service) to assess whether adequate information on the health effects of chloroform is available. Where adequate information is not available, ATSDR, in conjunction with the National Toxicology Program (NTP), is required to assure the initiation of a program of research designed to determine the health effects (and techniques for developing methods to determine such health effects) of chloroform. The following categories of possible data needs have been identified by a joint team of scientists from ATSDR, NTP, and EPA. They are defined as substance-specific informational needs that if met would reduce or eliminate the uncertainties of human health assessment. This definition should not be interpreted to mean that all data needs discussed in this section must be filled. In the future, the identified data needs will be evaluated and prioritized, and a substance-specific research agenda will be proposed. 2.10.1 Existing Information on Health Effects of Chloroform The existing data on health effects of inhalation, oral, and dermal exposure of humans and animals to chloroform are summarized in Figure 2-6. The purpose of this figure is to illustrate the existing information concerning the health effects of chloroform. Each dot in the figure indicates that one or more studies provide information associated with that particular effect. The dot does not necessarily imply anything about the quality of the study or studies, nor should missing information in this figure be interpreted as a "data need." A data need, as defined in ATSDR's Decision Guide for Identifying Substance-Specific Data Needs Related to Toxicological Profiles (ATSDR 1989), is substance-specific information necessary to conduct comprehensive public health assessments. Generally, ATSDR CHLOROFORM 166 2. HEALTH EFFECTS defines a data gap more broadly as any substance-specific information missing from the scientific literature. As seen from Figure 2-6, information is available regarding death, systemic effects, and neurological effects in humans after inhalation and oral exposure to chloroform. In addition, information is available regarding carcinogenic effects in humans after oral exposure to chlorinated drinking water. Limited information is available regarding dermal effects in humans after exposure to chloroform. Inhalation and oral studies in animals provide data on death, systemic effects after acute- and intermediate-duration exposure, immunological effects, neurological effects, developmental effects, reproductive effects, and genotoxic effects. Information is available regarding systemic effects and carcinogenic effects in animals after oral exposure to chloroform. The carcinogenic effects after oral exposure is inconsistent and not totally conclusive. In addition, data regarding death and acute systemic effects in animals after dermal exposure to chloroform were located in the available literature. 2.10.2 Identification of Data Needs Acute-Duration Exposure. Clinical reports indicate that the central nervous system, cardiovascular system, stomach, liver, and kidneys in humans are target organs of chloroform toxicity after inhalation and oral exposure to chloroform (Schroeder 1965; Smith et al. 1973; Whitaker and Jones 1965). These findings are supported by results obtained from acute inhalation and oral-exposure studies in animals in which target organs identical to those observed in human studies (central nervous CHLOROFORM 167 Figure 2-6. Existing Information on Health Effects of Chloroform © . @ Systemic © K WD & 0) 2) Q QO TY QO 5& > & & Ng WO eC /° » RSS XP SSESS SSCS SES LSS SE L/L SELF ES RSS Inhalation ° ° . ° * Oral @ ® ® ® ® Dermal Human OO & Systemic © Q © > 2 &F / © & 5° FS ESSE &/ SLES SS RST L/L SES LXER SRR Inhalation ® * ° oo o eo Oral ® ® ® @ @® & ® ® ® ® Dermal ® Animal ® Existing Studies CHLOROFORM 168 2. HEALTH EFFECTS system, liver, and kidney) were identified (Culliford and Hewitt 1957; Jones et al. 1958; Lehmann and Flury 1943; Lundberg et al. 1986; Moore et al. 1982). The data are sufficient to derive an MRL for acute oral exposure. An acute inhalation MRL was derived based on a NOAEL for hepatic effects in mice (Larson et al. 1994c). Lethality studies were conducted in rats and mice after acute inhalation exposure (Deringer et al. 1953; Gehring 1968; Lundberg et al. 1986; Smyth et al. 1962). Similarly, lethal doses were identified after single oral exposure in rats and mice (Bowman et al. 1978; Chu et al. 1982b: Jones et al. 1958; Kimura et al. 1971; Smyth et al. 1962). Information regarding dermal effects in humans and animals after exposure to chloroform is limited. Degenerative changes in the kidney tubules of rabbits were reported in one dermal study (Torkelson et al. 1976). Toxicokinetic data regarding dermal exposure are very limited; however, there is evidence that chloroform can be absorbed through the skin (Tsuruta 1975). Due to its lipophilic quality after dermal exposure, chloroform is likely to be distributed in the organism in patterns similar to those for inhalation and oral exposure. Information regarding acute dermal exposure in rodents would be useful to identify target organs and threshold levels of chloroform toxicity. Several in vitro skin models are available that would be adequate for describing the absorption of chloroform through the skin and the effects that differing concentrations of chloroform would have on skin histology. Intermediate-Duration Exposure. An occupational study suggests that the liver is a target organ of chloroform toxicity after inhalation exposure of intermediate duration (Phoon et al. 1983). No data were located regarding intermediate-duration oral and dermal exposure in humans. Several studies were located regarding chloroform toxicity in animals after oral exposure (including 3 90-day studies in rats, 3 90-day studies in mice, and a >6-week oral study in dogs) (Bull et al. 1986; Chu et al. 1982a, 1982b; Heywood et al. 1979; Jorgenson and Rushbrook 1980; Klaunig et al. 1986; Munson et al. 1982: Palmer et al. 1979); fewer data were located regarding inhalation exposure (Torkelson et al. 1976), and no data were located regarding dermal exposure. In animals, the target organs for chloroform toxicity were identified as the central nervous system, liver, and kidneys. An intermediate-duration oral MRL was derived based on liver effects in dogs (Heywood et al. 1979). An intermediate-duration inhalation MRL was derived based on toxic hepatitis which occurred in humans (Phoon et al. 1983). Pharmacokinetic data regarding dermal exposure to chloroform are limited, but it is known that chloroform can be absorbed through the skin. Intermediate-duration dermal studies in animals would provide information about chloroform toxicity via this exposure route. CHLOROFORM 169 2. HEALTH EFFECTS The information would be useful for populations living at or near hazardous waste sites, who may be exposed to chloroform for intermediate durations. Chronic-Duration Exposure and Cancer. Information regarding chronic-exposure inhalation exposure to chloroform in humans is limited to occupational studies (Bomski et al. 1967; Challen et al. 1958). The liver and central nervous system are target organs of chloroform toxicity. Regarding chronic-duration oral exposure in humans, limited information is available from a case study reporting hematological, hepatic, and renal effects in an individual who used a cough medicine containing chloroform for 10 years (Wallace 1950) and from a follow-up study of individuals who used a mouthwash containing chloroform for <5 years (De Salva et al. 1975). Animal data indicate that the central nervous system, liver, and kidneys are target organs of chloroform toxicity after chronic oral exposure (Heywood et al. 1979; Jorgenson et al. 1985; NCI 1976; Roe et al. 1979; Tumasonis et al. 1985, 1987). The data are sufficient to derive a chronic oral MRL. No studies were located regarding chloroform toxicity in humans and animals after dermal exposure to chloroform and in animals after inhalation exposure to chloroform. Considering the similar pattern of chloroform toxicity after inhalation and oral exposures for acute and intermediate durations, similar target organs in animals after chronic inhalation exposure to chloroform may be predicted. Nonetheless, studies designed to assess the chronic toxicity of chloroform in animals after inhalation and dermal exposure would be useful to establish dose-response relationships. This information is important to humans occupationally exposed or exposed to contaminated air, water, or soil at or near hazardous waste sites. Epidemiology studies suggest a possible association between chloroform in drinking water and cancer risk. Increased incidences of colon and bladder cancer were identified in separate populations exposed to chlorinated water. However, as mentioned before, other toxic compounds have also been identified in chlorinated drinking water, making the role of chloroform in cancer induction questionable. Studies in rats and mice indicate that oral exposure to chloroform causes cancer (Jorgenson et al. 1985; NCI 1976; Roe et al. 1979; Tumasonis et al. 1985, 1987); however, some of these studies utilized gavage dosing instead of drinking water. No data were located regarding carcinogenicity in humans and animals following inhalation and dermal exposure to chloroform. Nonetheless, pharmacokinetic data indicate similar toxicokinetics of chloroform after inhalation and oral exposure; therefore, similar targets for carcinogenic effects may be predicted. Data were located suggesting different effects of chloroform depending on the vehicle and method of oral administration. Chloroform in corn oil administered by gavage caused an increased incidence of liver tumors (NCI 1976), while CHLOROFORM 170 2. HEALTH EFFECTS administration of the same dose in drinking water did not (Jorgenson et al. 1985). It was demonstrated, however, that chloroform uptake is much slower from the oil vehicle (Withey et al. 1983). Therefore, the higher cancer incidence cannot be explained merely by the levels of chloroform in tissues. Furthermore, chloroform acted as a promoter rather than an initiator of preneoplastic foci in a rat liver bioassay (Deml and Oesterle 1985). In contrast, some studies indicate that chloroform inhibits the growth of tumors induced by known carcinogens (Daniel et al. 1989; Herren-Freund and Pereira 1987). Animal studies also suggest an epigenetic mechanism for the carcinogenicity of chloroform. Because of these differences, further studies on the possible mechanism of chloroform carcinogenicity would be useful. Genotoxicity. Chloroform has been tested for genotoxicity in several in vitro and in vivo experiments. Its potency to induce mutations seems to be weak. No induction of reverse mutations was observed in prokaryotic systems (Gocke et al. 1981; Kirkland et al. 1981; San Augustin and Lim- Sylianco 1978; Simmon et al. 1977; Uehleke et al. 1977; Van Abbe et al. 1982; Varma et al. 1988). Mixed results were obtained in the induction of mutations in human lymphocytes and Chinese hamster cells in vitro (Kirkland et al. 1981; Mitchell et al. 1988; Peroccio and Prodi 1981; White et al. 1979). Nonetheless, an increase in sperm anomalies and sister chromatid exchanges in the bone marrow of rodents was observed after in vivo exposure (Land et al. 1979, 1981; Morimoto and Koizumi 1983). Cytogenetic analysis of peripheral lymphocytes from exposed individuals would provide useful information about the ability of chloroform to induce mutations in-humans if a suitable population can be identified. Reproductive Toxicity. No information was located regarding reproductive effects in humans exposed to chloroform via any route or in animals exposed by the dermal route. Increased resorptions were observed in rats and mice after inhalation exposure to chloroform during gestation (Murray et al. 1979: Schwetz et al. 1974) and in rats and rabbits after oral exposure (Thompson et al. 1974). In addition to effects in dams, abnormal sperm were found in mice after inhalation exposure (Land et al. 1979, 1981). Furthermore, exposure-related gonadal atrophy was observed in both sexes of rats following oral exposure to chloroform (Palmer et al. 1979). The results suggest that reproductive organs are a target of chloroform toxicity in animals; however, some inhalation and oral studies in animals do not report any effects. More studies assessing the reproductive function in animals would be useful for the purpose of extrapolating the data to human exposure. CHLOROFORM 171 2. HEALTH EFFECTS Developmental Toxicity. Only one study was located regarding developmental effects in humans exposed to chloroform via an oral route. Animal data indicate that chloroform can cross the placenta. Fetotoxicity effects (decreased birth weight, decreased fetal crown-rump length, increased resorptions) and teratogenicity (acaudate fetuses with imperforate anus, cleft palates) were observed in rats and mice after inhalation exposure to chloroform (Murray et al. 1979; Schwetz et al. 1974). Oral exposure to chloroform-induced fetotoxicity in rats and rabbits (Ruddick et al. 1983; Thompson et al. 1974). Degeneration of the epididymal ductal epithelium (not affecting the fertility) was observed in mice in the F, generation in a 2-generation oral reproductive study (Gulati et al. 1988). No information is available regarding the developmental toxicity of chloroform after dermal exposure. More data regarding developmental toxicity both in humans and in experimental animals (especially after oral and dermal exposure) would be useful to identify the possible risk for humans. Immunotoxicity. No data were located regarding immunological effects in humans after inhalation, oral, or dermal exposure to chloroform. The data obtained from animal studies are limited to one inhalation study in mice and three oral studies in rats and mice (Aranyi et al. 1986; Chu et al. 1982b:; Munson et al. 1982). Depressed humoral and cell-mediated immunity were detected; however, the chloroform-induced changes were more serious in the acute exposure study than in the intermediate- duration study, indicating that the changes may be transient. Studies regarding skin sensitization with chloroform were not performed. A battery of immune function tests has not been performed in humans or in animals, but would provide helpful information to support or refute the limited evidence for chloroform immunotoxicity. Neurotoxicity. The central nervous system is a target organ for chloroform toxicity in humans after inhalation and oral exposure. The neurotoxic effect is well documented in studies of patients exposed to chloroform via anesthesia (Featherstone 1947; Smith et al. 1973; Whitaker and Jones 1965) or of individuals who intentionally and accidentally ingested the chemical (Piersol et al. 1933; Schroeder 1965; Storms 1973). Lower chloroform doses produced neurological effects during occupational exposure (Challen et al. 1958). Similarly, neurotoxicity is reported in animal studies involving inhalation and oral exposure to chloroform (Bowman et al. 1978; Jones et al. 1958; Lehmann and Flury 1943). A battery of neurobehavioral tests was conducted in mice after oral exposure to chloroform (Balster and Borzelleca 1982). No data were located regarding chloroform neurotoxicity in humans or animals after dermal exposure to chloroform. Animal studies involving dermal exposure to chloroform would be useful for risk assessment of occupational exposure. Continued research on the CHLOROFORM 172 2. HEALTH EFFECTS toxicity of inhaled and dermally absorbed chloroform in humans when exposed to water sources containing elevated concentrations of chloroform during showering would also be useful. More information regarding the mechanism of chloroform-induced neurotoxicity and structural alterations produced in the central nervous system would be helpful. Epidemiological and Human Dosimetry Studies. Populations may be exposed to chloroform in the workplace, near hazardous waste sites containing chloroform, from chlorinated water, and from various consumer products that contain chloroform. Limited information was obtained from occupational studies reporting central nervous system and liver effects in exposed workers (Bomski et al. 1967; Challen et al. 1958; Phoon et al. 1983). Reliable dosimetry data correlating occupational exposure with signs of toxic effects would be useful. Epidemiology studies suggest an association between elevated chloroform levels in drinking water and colon, rectal, and bladder cancer in humans (Alavanja et al. 1978; Cantor et al. 1978; Young et al. 1981). All of these studies were limited by a lack of attention to important details (e.g., migration, exposure to other carcinogens). Better designed and better conducted epidemiology studies of occupational exposure would be helpful. The information can be useful to populations living near hazardous waste sites where chloroform is present. In addition, further refining of the PBPK/PD models would further advance our understanding of chloroform tissue dosimetry in humans and animals. Biomarkers of Exposure and Effect. Exposure. Methods for detecting chloroform in exhaled breath, blood, urine, and tissues are available. Nevertheless, it is difficult to correlate chloroform levels in biological samples with exposure, because of the volatility and short half-life of chloroform in biological tissues. Several studies monitored chloroform levels in environmentally exposed populations (Antoine et al. 1986; Hajimiragha et al. 1986; Peoples et al. 1979); however, the measured levels probably reflect both inhalation and oral exposure. Moreover, increased tissue levels of chloroform or its metabolites may reflect exposure to other chlorinated hydrocarbons. Studies to better quantitate chloroform exposure would enhance the database. Effect. No biomarkers were identified that are particularly useful in characterizing the effects induced by exposure to chloroform. The target organs of chloroform toxicity are the central nervous system, the liver, and kidneys; however, damage to these organs may result from exposure to other chemicals. CHLOROFORM 173 2. HEALTH EFFECTS More effort to identify subtle biochemical changes to serve as biomarkers of effects of chloroform exposure would be useful in detecting early, subtle signs of chloroform-induced damage. Absorption, Distribution, Metabolism, and Excretion. Human data indicate that chloroform absorption from the lungs is rapid and fairly complete (Smith et al. 1973). The data also indicate that absorption after oral exposure is fairly complete for both animals and humans (Brown et al. 1974a: Fry et al. 1972; Taylor et al. 1974). Although there are no experimental data regarding dermal absorption in humans, some data have been extrapolated from mouse studies (Tsuruta 1975). The rate of absorption following oral or inhalation exposure is rapid (within 1-2 hours). Additional animal studies investigating the rate of dermal absorption would be useful to quantitate dermal absorption and to compare information from oral and inhalation studies. Data are available regarding the distribution of chloroform in animals after inhalation and oral exposure to chloroform (Brown et al. 1974a; Chenoweth et al. 1962; Cohen and Hood 1969: Corley et al. 1990; Danielsson et al. 1986; Taylor et al. 1974); however, data regarding the distribution of chloroform in humans is very limited (Feingold and Holaday 1977) and warrants further investigation. It appears that distribution following oral exposure is similar to that following inhalation exposure. Another well conducted animal study focusing on distribution and excretion after dermal exposure would be useful to assess exposure via this route. The metabolic pathways of chloroform metabolites are well understood. It appears that both the mode of oral administration and the vehicle affect metabolism. Additional data investigating the mode and vehicle of administration would be useful in order to understand the role of these factors in the mechanism of chloroform's toxicity. The co-administration of other compounds (e.g., ethanol) has been shown to alter chloroform metabolism and toxicity. Further investigations of the hazards associated with exposure to complex mixtures containing chloroform would be useful. The excretion of chloroform and its metabolites is understood, based on human and animal data derived from oral and inhalation studies (Brown et al. 1974a; Corley et al. 1990; Fry et al. 1972: Taylor et al. 1974). The major route of chloroform elimination is pulmonary, but minor pathways are through enterohepatic circulation, urine, and feces as parent compound or metabolites. There are no human or animal data regarding excretion of dermally applied chloroform. CHLOROFORM 174 2. HEALTH EFFECTS Comparative Toxicokinetics. Target organs for chloroform distribution appear to be similar in humans and animals, according to inhalation studies (Corley et al. 1990; Feingold and Holaday 1977). Nonetheless, human and animal studies indicate that there are large interspecies differences in chloroform metabolism and tissue partition coefficients (Brown et al. 1974a; Corley et al. 1990). Marked sex-related differences in tissue distribution and covalent binding to tissue MMBs in mice also have been observed (Taylor et al. 1974). Excretion data indicate that humans and nonhuman primates excrete chloroform in the breath primarily as unchanged chloroform; mice eliminated almost 80% of an oral chloroform dose as CO, (Brown et al. 1974a). Thus, toxicokinetic data indicate that it may be difficult to compare the toxicokinetics of chloroform in animals with that in humans. There are a large number of oral studies, relatively few inhalation studies, and almost no dermal studies regarding the toxicokinetics of chloroform. Quantitative toxicokinetic studies in several animal species involving exposure to chloroform via all three routes, especially inhalation and dermal, would help complete the database. Methods for Reducing Toxic Effects. Protective clothing and protective breathing devices may be used to prevent exposure to large amounts of chloroform, although for everyday low exposures to chloroform, these methods are obviously impractical. General procedures such as flushing the skin with water following dermal exposure and emesis or gastric lavage following oral exposure may be used to reduce absorption of chloroform. However, specific medical treatments that prevent absorption of chloroform have not been identified. Such mechanisms might be beneficial because they might be more effective than general procedures and might involve less risk than procedures such as emesis. Ways to enhance elimination of chloroform from the body are not known. Although chloroform is eliminated fairly rapidly, methods to accelerate elimination without producing toxic metabolites would be helpful in reducing toxicity. The mechanism by which chloroform produces toxicity appears to involve metabolism by phenobarbital-inducible isozymes of cytochrome P-450 to phosgene (Pohl et al. 1980a, 1980b). Development of methods to selectively inhibit the P-450 isozymes responsible for this reaction might reduce chloroform toxicity. There is also evidence that GSH conjugates with reactive products of chloroform metabolism, providing protection from damaging effects (Docks and Krishna 1976: Hook and Smith 1985). Development of a method to maintain high tissue GSH levels following exposure to chloroform might have a mitigating effect on toxicity. Therefore, although no treatments are currently available to block the toxic action of chloroform or repair damage caused by this chemical, there are indications that further research in this area would enable identification of such treatments. CHLOROFORM 175 2. HEALTH EFFECTS 2.10.3 Ongoing Studies A few ongoing studies involving chloroform have been identified. The effects of volatile anesthetics on the N-methyl-D-aspartate (NMDA) receptor-channel complex are being studied. Specific aims are to determine the effects of several volatile anesthetics (halothane, enflurane, isoflurane, diethyl ether, cyclopropane, nitrous oxide, and chloroform) on ligand binding to glutamate binding sites on the NMDA receptor complex; to study ligand binding to the glycine modulatory site, glutamate, glycine, divalent cation, and spermidine activation of NMDA receptor ion channels; and to examine NMDA- receptor mediated changes in calcium content of rat brain microvesicles (Aronstam 1994). Mechanistic work on the hepatotoxicity and toxigenic sequence will be studied in vitro with suspensions of hepatocytes exposed to carbon tetrachloride and other agents known to alter calcium homeostasis and stimulate phospholipase A, (bromotrichloromethane and chloroform) (Glende 1994). Mechanisms of toxic chemical interactions in the liver resulting in hepatotoxicity will utilize the hepatotoxic effects of chloroform to explore interactions among metals and organics in the induction of stress response proteins such as metallothionein, heme oxygenase, and nitric oxide synthase, and the induction of inflammatory cytokines (i.e., tumor necrosis factor-alpha, interleukin-1, and interleukin-6). Work by Swenburg will determine if exposure to environmental carcinogens, including 1,1,2-trichloro- ethylene, 1,1,2,2-tetrachloroethylene, 1,1,2-trichloroethane, chloroform, and carbon tetrachloride. induces or modulates the formation and/or repair of cyclic DNA adducts. Work by Benjamin (1995) will also continue to study the phenomenon of hepatic cell proliferation, and the effect that this response has on the initiation and promotion of cancer. Other research will investigate the toxicity and bioaccumulation of a mixture of sediment contaminants (trichlorethylene, lead, benzene, chloroform, phenol, chromium, and arsenic) in several species of invertebrates and fish. Uptake and depuration will be measured in chironomids (Chironomus riparius), and pharmacokinetic models will be developed to describe bioaccumulation of these sediment contaminants (Clements 1994). Studies by Yang (1994) will continue to evaluate age- and dosing-related changes in pharmacokinetics, biochemical markers, liver cell proliferation, and histopathology in male Fischer 344 rats chronically exposed (up to 2 years) to low levels of a chemical mixture of 7 organic and inorganic groundwater pollutants (including arsenic, benzene, chloroform, chromium, lead, phenol, trichloroethylene). Also, this research will further explore the pharmacokinetic modeling of chemical mixtures and incorporate time-course information on biochemical markers, cell proliferation and histopathology into pharmaco- kinetic and pharmacodynamic modeling. A study by the Japan Industrial Safety and Health Association (Japan Bioassay Lab) that explores the toxicity of chloroform inhaled over a 2-year period CHLOROFORM 176 2. HEALTH EFFECTS (6 hours a day, 5 days a week) in male and female Fischer 344 rats and BDF, mice is reportedly close to completion (Matsushima 1994). Efforts are also being made to develop a high-efficiency activated carbon granule for drinking-water treatment that can remove water contaminants, including chloroform (Mieville 1992). CHLOROFORM 177 3. CHEMICAL AND PHYSICAL INFORMATION 3.1 CHEMICAL IDENTITY Information regarding the chemical identity of chloroform is located in Table 3-1. 3.2 PHYSICAL AND CHEMICAL PROPERTIES Information regarding the physical and chemical properties of chloroform is located in Table 3-2. CHLOROFORM 178 3. CHEMICAL AND PHYSICAL INFORMATION Table 3-1. Chemical Identity of Chloroform Characteristic Information Reference Chemical name Trichloromethane SANSS 1990 Synonym(s) Metheny! chloride, IARC 1979 methane trichloride, methyl trichloride, formyl trichloride Registered trade name(s) Freon 20, R 20, IARC 1979 R 20 refrigerant Chemical formula CHCl, Weast 1988 Chemical structure Cl IARC 1979 H—C —Cl Cl Identification numbers: CAS registry 67-66-3 Weast 1988 NIOSH RTECS FS 9100000 HSDB 1996 EPA hazardous waste uo44 HSDB 1996 OHM/TADS 7216639 HSDB 1996 DOT/UN/NA/IMCO shipping Chloroform; UN 1888; IMO 6.1 HSDB 1996 HSDB 56 HSDB 1996 NCI C02686 HSDB 1996 CAS = Chemical Abstracts Services; DOT/UN/NA/IMCO = Department of Transportation/United Nations/ North America/International Maritime Dangerous Goods Code; EPA = Environmental Protection Agency; HSDB = Hazardous Substances Data Bank; NCI = National Cancer Institute; NIOSH = National Institute for Occupational Safety and Health; OHM/TADS = Oil and Hazardous Materials/Technical Assistance Data System; RTECS = Registry of Toxic Effects of Chemical Substances CHLOROFORM 179 3. CHEMICAL AND PHYSICAL INFORMATION Table 3-2. Physical and Chemical Properties of Chloroform Property Information Reference Molecular weight 119.38 Deshon 1979 Color Colorless Hawley 1981 Physical state Liquid Deshon 1979 Melting point -63.2 °C Deshon 1979 -64 °C Verschueren 1983 -63.5 °C Weast 1988 Boiling point 61.3°C Deshon 1979 62 °C Verschueren 1983 61.7 °C Weast 1988 Density: at 20 °C 1.485 g/cm? Hawley 1981 1.4832 g/cm’ Weast 1988 Odor Pleasant, ethereal, nonirritating Deshon 1979 Pleasant, sweet NFPA 1994 Odor threshold: Water 2.4 ppm (w/v) Amoore and Hautala 1983 Air 85 ppm (v/v) Amoore and Hautala 1983 Solubility: Water at 25 °C Organic solvent(s) Partition coefficients: Log Kyw Log Kc Vapor pressure at 20 °C Henry's law constant: at 20 °C at24.8 °C at25 °C 7.22x10° mg/L 9.3x10° mg/L 7.43 x 10° mg/L Miscible with principal organic solvents Miscible with alcohol, benzene, ether, petroleum ether, carbon tetrachloride, carbon disulfide, oils 1.97 1.65 2.40 159 mm Hg 160 mm Hg 160 mm Hg 3.0x103 atm-mS/mol 3.67x102 atm-m3/mol 4.06x1073 atm-m3/mol Banerjee et al. 1980 Verschueren 1983 Merck 1989 Deshon 1979 Merck 1989 Hansch and Leo 1985, Verschueren 1983 Sabljic 1984 Aster 1996 Boublik et al. 1984 Verschueren 1983 NFPA 1994 Nicholson et al. 1984 Gossett 1987 SRC 1994a CHLOROFORM 180 3. CHEMICAL AND PHYSICAL INFORMATION Table 3-2. Physical and Chemical Properties of Chloroform (continued) Property Information Reference Decomposition rates Hydrolysis rate constant at 25 °C for pH>8 Autoignition temperature Flashpoint Flammability limits Conversion factors in air (20 °C) in air (20 °C) Other Explosive limits Negligible rate of hydrolysis Half-life of 80 days in air with photochemically produced hydroxyl radicals Residence time in air 116 days 6.44x10° L/mol-sec >1,000 °C None No data 1 ppm (v/v)=4.96 mg/m°3 1 mg/m==0.20 ppm (V/V) Reacts with strong alkalies and aluminum Oxidized by strong oxidizing agents such as chromic acid, with formation of phosgene and chlorine gas No data Mabey and Mill 1978 Hampson 1980 Singh et al. 1981 SRC 1994b Deshon 1979 Deshon 1979 No data Calculated Calculated NFPA 1994 HSDB 1996 No data v/v = volume per volume; w/v = weight per volume CHLOROFORM 181 4. PRODUCTION, IMPORT/EXPORT, USE, AND DISPOSAL Chloroform is used primarily in the production of chlorodifluoromethane (hydrochlorofluorocarbon-22 or HCFC-22) used as a refrigerant for home air conditioners or large supermarket freezers and in the production of fluoropolymers (CMR 1995). Chloroform has also been used as a solvent, a heat transfer medium in fire extinguishers, an intermediate in the preparation of dyes and pesticides, and other applications highlighted below. Its use as an anesthetic has been largely discontinued. It has limited medical uses in some dental procedures and in the administration of drugs for the treatment of some diseases. 4.1 PRODUCTION The chlorination of methane and the chlorination of methyl chloride produced by the reaction of methanol and hydrogen chloride are the two common methods for commercial chloroform production (Ahlstrom and Steele 1979; Deshon 1979). The Vulcan Materials Co., Wichita, Kansas, was documented as still using the methanol production process during the late 1980s, with all other facilities in the United States at that time using the methyl chloride chlorination process (SRI 1990). One U.S. manufacturer began chloroform production in 1903, but significant commercial production was not reported until 1922 (IARC 1979). Since the early 1980s, chloroform production increased by 20-25%, due primarily to a higher demand for HCFC-22, the major chemical produced from chloroform. The Montreal Protocol established goals for phasing out the use of a variety of ozone- depleting chemicals, including most chlorofluorocarbons (CFCs). HCFC-22 was one of the few fluorocarbons not restricted by the international agreement. Chloroform is used in the manufacture of HCFC-22, and an increase in the production of this refrigerant has led to a modest increase in the demand for chloroform (CMR 1989). These increasing trends in U.S. production, based on information compiled in the trade journal Chemical & Engineering News for period from 1983 though 1994 (CEN 1995), are summarized in Table 4-1. The manufacturers and sites of major chloroform production facilities identified for 1993 (SRI 1993) include the following: Dow Chemical U.S.A., Freeport, Texas, and Plaquemine, Louisiana; Occidental Petroleum Corp., Belle, West Virginia; and Vulcan Materials Co., Geismar, Louisiana, and Wichita, Kansas. Estimated annual production capacity (SRI 1993) from these facilities as of January 1, 1993, CHLOROFORM 182 4. PRODUCTION, IMPORT/EXPORT, USE, AND DISPOSAL Table 4-1. U.S. Production of Chloroform U.S. production U.S. production Year (in millions of pounds) (in millions of kg) 1983 362 164 1984 405 183 1985 275 124 1986 422 191 1987 462 209 1988 524 237 1989 588 266 1990 484 219 1991 505 229 1992 na na 1993 476 215 1994 565 254 Taken from Chemical & Engineering News 1995 CHLOROFORM 183 4. PRODUCTION, IMPORT/EXPORT, USE, AND DISPOSAL was 460 million pounds (208 million kg). By early 1995, the Occidental Petroleum Corporation facility in West Virginia had ceased its chloroform production operations (CMR 1995; SRI 1995). While precise production figures for the 4 remaining sizable U.S.-based facilities are not available, the annual production capacity estimate for the Dow plant in Freeport, Texas, was 135 million pounds (61.8 million kg); capacity for the Dow plant at Plaquemine, Louisiana, was 150 million pounds (67.5 million kg); capacity at the Vulcan plant at Geismar, Louisiana, was 90 million pounds (40.5 million kg); and capacity at the Vulcan plant at Wichita, Kansas, was 145 million pounds (62.3 million kg); for a total United States annual production capacity of around 520 million pounds (234 million kg) from these 4 large plants (CMR 1995). An estimate of actual production (CEN 1995) in the United States during 1994 was 565 million pounds (254 million kg). Table 4-2 lists the facilities in each state that manufacture or process chloroform, the intended use, and the range of maximum amounts of chloroform that are stored on site. The data listed in Table 4-2 are derived from the Toxics Release Inventory (TRI93 1995). Only plants from 3 states (associated with the 4 plants noted above) actually generate chloroform as an end-product for sale or distribution. In most cases, chloroform is a chemical intermediary, impurity, or waste by-product at the 172 facilities included in the TRI survey. Only certain types of facilities were required to report; therefore, this is not an exhaustive list. In some cases, facility names are not available or numeric values for amounts of chloroform produced, stored, transferred, or released are missing. This complicates making comparisons between the TRI listings and information from other information sources. 4.2 IMPORT/EXPORT In 1985, the United States imported 27.6 million pounds (12.5 million kg) of chloroform; 24 million pounds (10.8 million kg) of chloroform were imported into the United States in 1988 (CMR 1989; HSDB 1996). More recent U.S. import figures from the National Trade Data Bank (NTDB 1995) are: U.S. Imports for Year Quantity (kg) 1990 10,624,006 1991 9,460,747 1992 6,038,483 1993 8,467,294 1994 2,398,668 CHLOROFORM 184 4. PRODUCTION, IMPORT/EXPORT, USE, AND DISPOSAL Table 4-2. Facilities That Manufacture or Process Chloroform Range of maximum amounts on site Number of in thousands State" facilities of pounds b Activities and uses © AK 2 0-1 1,5,6 AL 12 0-1000 1,5,6,7,12 AR 6 0-1000 1,5,6,7,13 AZ 1 0-1 1,5 CA 2 0-0 1,6,13 co 1 10-100 2,6, 11 CT 1 100-1000 1 FL 7 0-100 1,5,6,13 GA 6 0-10 1,5,6 ID 1 1-10 1.5 IL 2 10-1000 1 IN 3 1-100 11,13 KS 2 100-10000 1,3,4,7,13 KY 7 0-10000 1,3,5,6,7,10,13 LA 16 0-50000 1,2,3,4,56,7,11,13 MA 1 10-100 12 MD 1 0-1 1,5 ME 7 0-1 1,5,6 Mi 5 0-10000 1,5,6,7,11,12,13 MN 3 0-100 1,5,8,12 MO 2 10-100 8,10, 11 MS 2 0-1 1,6 MT 1 0-1 1,5 NC 4 0-1000 1,5, 6, 11 NH 1 0-1 1,5 NJ 4 0-1000 1,5,7,8, 11 NY 3 0-100 1,5,6, 11,13 OH 3 0-100 1,6,10, 11 OK 1 0-0 1,5 OR 3 0-10 1,6, 11 PA 7 0-1000 1,5,6, 11 PR 6 0-1000 11,12,13 SC 5 0-10 1,5,6,8,10 TN 2 0-1 1,5,6 hRd 17 0-50000 1,2,4,5,6,7,9,12,13 VA 4 0-1000 1,5, 11 vT 1 10-100 8 WA 7 0-10 1,5,6 CHLOROFORM 185 4. PRODUCTION, IMPORT/EXPORT, USE, AND DISPOSAL Table 4-2. Facilities That Manufacture or Process Chloroform (continued) Range of maximum amounts on site Number of in thousands c State® facilities of pounds © Activities and uses wi 11 0-100 1,5,6,7,11,13 wv 3 10-10000 1,4,5,9, 11,13 Source: TRI93 1995 2 Post office state abbreviations used b Data in TRI are maximum amounts on site at each facility ¢ Activities/Uses: 1. Produce 8. As a formulation component 2. Import 9. As a product component 3. For on-site use/processing 10. For repackaging only 4. For sale/distribution 11. As a chemical processing aid 5. As a by-product 12. As a manufacturing aid 6. As an impurity 13. Ancillary or other uses 7. As a reactant CHLOROFORM 186 4. PRODUCTION, IMPORT/EXPORT, USE, AND DISPOSAL These import statistics suggest a slight decrease in chloroform imports from the late 1980s into the 1990s. Additional data would be needed to evaluate properly the drastic decrease in imports for 1994. In 1985, 33.5 million pounds (15.2 million kg) of chloroform were exported (HSDB 1994); for 1988, exports of 40 million pounds (18.1 million kg) were estimated (CMR 1989). More recent export figures from the National Trade Data Bank (NTDB 1995) are: U.S. Exports for Year Quantity (kg) 1989 26,756,412 1990 21,897,011 1991 23,709,482 1992 20,133,535 1993 31,620,815 1994 42.320,259 These statistics suggest an overall increase in export levels from the mid-1980s through the mid-1990s. 4.3 USE The major use for chloroform (CMR 1989) is in the manufacture of the refrigerant HCFC-22. Chloroform has been used in the past as a solvent or an extraction solvent for fats, oils, greases, resins, lacquers, rubber, alkaloids, gums, waxes, gutta-percha, penicillin, vitamins, flavors, floor polishes, and adhesives in artificial silk manufacture. It is also used as a dry cleaning spot remover, in fire extinguishers, as an intermediate in the manufacture of dyes and pesticides, and as a fumigant (Deshon 1979). Chloroform was previously used as an anesthetic, but it has been replaced by safer and more versatile materials (Deshon 1979). The U.S. Food and Drug Administration (FDA) banned chloroform use in drug, cosmetic, and food packaging products in 1976 (see Chapter 7). This ruling did not include drug products that contain chloroform in residual amounts resulting from its use as a processing solvent in manufacturing or its presence as a by-product from the synthesis of drug ingredients (IARC 1979). Chloroform can still apparently be used as a local anesthetic and solvent in certain dental endodontic (gutta-percha root canal) surgery procedures (McDonald and Vire 1992). CHLOROFORM 187 4. PRODUCTION, IMPORT/EXPORT, USE, AND DISPOSAL Topically applied aspirin-chloroform mixtures are also used to relieve pain from severe cases of herpes zoster (shingles) or posttherapeutic neuralgia (King 1993). A recent summary of major domestic uses for chloroform noted the following percentage breakdowns typical of the early to mid-1990s (CMR 1995): use for manufacture of HCFC-22, 98% (refrigerants, 70%; fluoropolymers, 30%); other miscellaneous uses, including laboratory reagents and extraction solvents for pharmaceuticals, 2%. As discussed in Chapter 5, the most common chloroform exposure opportunities for members of the general population are related less to any commercially produced form of the chemical than to chloroform generated when organic materials come in contact with chlorinated oxidants (e.g., chlorine or hypochlorous acid) widely used to purify water or remove pathogens from waste materials. 4.4 DISPOSAL According to the 1993 TRI, the amount of chloroform released to land is only a small fraction (less than 1%) of the total amount of chloroform released to the environment by facilities that produce and process the chemical (see Section 5.2.3) (TRI93 1995). TRI also documents 2,386,285 pounds (1,073,828 kg) transferred to off-site waste handling sites in 1993, a level amounting to around 17% the total releases to environmental media and larger than any releases to environmental media other than air (TRI93 1995). The TRI data should be used with caution since only certain types of facilities are required to report. This is not an exhaustive list. Chloroform has been identified as a hazardous waste by EPA, and disposal of this waste is regulated under the Federal Resource Conservation and Recovery Act (RCRA) (EPA 1988a, 1989b). Specific information regarding federal regulations on chloroform disposal on land is available in the Code of Federal Regulations (EPA 1988a, 1989b). Ultimate disposal of chloroform, preferably mixed with another combustible fuel, can be accomplished by controlled incineration. Complete combustion must be ensured to prevent phosgene formation, and an acid scrubber should be used to remove the haloacids produced. Chloroform also is a potential candidate for liquid injection incineration. Because chloroform has been used in some pesticides, the disposal of containers for these pesticides may be relevant. Combustible containers from organic or many metallo-organic pesticides could be disposed of in pesticide incinerators or in specified landfill sites. Noncombustible containers could be disposed CHLOROFORM 188 4. PRODUCTION, IMPORT/EXPORT, USE, AND DISPOSAL of in a designated landfill or recycled (HSDB 1996). Except for the TRI statistics, no data were located regarding the approximate amounts of chloroform disposed or released to environmental media. Chapter 7 provides more details on federal or state regulations governing the disposal of chloroform. CHLOROFORM 189 5. POTENTIAL FOR HUMAN EXPOSURE 5.1 OVERVIEW Chloroform is both a synthetic and naturally occurring compound, although anthropogenic sources are responsible for most of the chloroform in the environment. Chloroform is released into the environment as a result of its manufacture and use; its formation in the chlorination of drinking water, municipal and industrial waste water, and swimming pool and spa water; and from other water treatment processes involving chlorination. Under anaerobic conditions, some bacteria can dehalogenate carbon tetrachloride to release chloroform. Most of the chloroform released into the environment will eventually enter the atmosphere. In the atmosphere, chloroform may be transported long distances before ultimately being degraded by indirect photochemical reactions with such free radicals as hydroxyl. The compound has been detected in ambient air in locations that are remote from anthropogenic sources. Chemical hydrolysis is not a significant removal process. While microbial biodegradation can take place, such reactions are generally possible only at fairly low concentration levels due to chloroform's toxicity. Microbial biodegradation of chloroform may also be inhibited due to high levels of other aromatics (e.g., toluene), chlorinated hydrocarbons (e.g., trichloro- ethylene [TCE]), or heavy metals (e.g., zinc). Because of its low soil adsorption and slight, but significant, water solubility, chloroform will readily leach from soil into groundwater. In groundwater, chloroform is expected to persist for a long time. The general population is exposed to chloroform by ingesting water and food, inhaling contaminated air, and possibly through dermal contact with chloroform-containing water. Generalizations can be made concerning the chloroform concentrations in the environment. Background air concentrations appear to be in the sub-ppb range, but certain urban, indoor, and source-dominated areas may show elevated concentrations when compared to background concentrations. Drinking water levels as high as 311 ppb have been reported in public water supplies, although most of the reported concentrations are less than 50 ppb, typically ranging between 2 and 44 ppb. Levels in drinking water derived from groundwater contaminated with leachate from landfills and hazardous waste sites can sometimes be much higher. Except for a few special surveys, regular testing for chloroform or other trihalomethanes (THMSs) has focused on larger community water treatment systems serving at least 10,000 people. Very limited information was located regarding the concentrations found in ambient soil. Chloroform has also been detected in the ppb range in certain foods. CHLOROFORM 190 5. POTENTIAL FOR HUMAN EXPOSURE Occupational exposure to higher than background levels of chloroform can be expected to occur in some occupations although few quantitative exposure data were located. Populations with the highest potential exposures appear to be workers employed in or persons living near industries and facilities that manufacture or use chloroform; operators and individuals who live near municipal and industrial waste water treatment plants and incinerators, and paper and pulp plants; and persons who derive their drinking water from groundwater sources contaminated with leachate from hazardous waste sites. Chloroform has been identified in at least 717 of 1,428 current or past EPA National Priorities List (NPL) sites (HazDat 1996). However, the number of sites evaluated for chloroform is not known. Figure 5-1 shows the distribution of sites in the continental United States; there are 710 such sites. In addition, there are 6 sites in the Commonwealth of Puerto Rico and one site in the Virgin Islands (not shown in Figure 5-1). 5.2 RELEASES TO THE ENVIRONMENT 5.2.1 Air According to the 1991 Toxic Chemical Release Inventory (TRI91 1993), releases of chloroform to the air from 182 large processing facilities were 17,034,926 pounds (8,413,971 kg). This represented about 95% of the total releases to environmental media. For TRI information from 1993 (TRI93 1995), 13,485,992 pounds (6,068,696.4 kg) were reported as released to the air from 172 facilities, which represents around 97.1% of the total releases to environmental media. Since there was a slight decrease in overall chloroform production in 1993 as compared with 1991 (CEN 1995), it is difficult to attribute this decrease in releases to the air to improved pollution prevention measures as opposed to short-term fluctuations related to production capacity utilization factors. The releases of chloroform to air from facilities that manufactured and processed it in the United States during 1993 are reported in Table 5-1 (TRI93 1995). The TRI data should be used with caution because only certain types of facilities are required to report. This is not an exhaustive list. Current comprehensive quantitative data or estimates of chloroform releases to the atmosphere are lacking. Some direct releases to the atmosphere are expected to occur during the manufacture, loading, and transport of chloroform (EPA 1985a, 1985b). Indirect chloroform releases have resulted from its use in the manufacture of hydrochlorofluorocarbon-22, fluoropolymers, pharmaceuticals, Derived from HazDat 1996 Figure 5-1. Frequency of NPL Sites with Chloroform Contamination JHNSOdX3 NVINNH HOH TVILN3ILOd 'S WHO40HO THO 161 Table 5-1. Releases to the Environment from Facilities That Manufacture or Process Chloroform Number of Range of reported amounts released in pounds per year * State® facilities : Underground Total POTW Off-site Air Water Land injection environment transfer waste transfer AK 2 140697-240000 19000-40869 0 0 181566-259000 0 0 AL 12 250-620000 0-16000 0-2000 0 250-636000 0 0-132346 AR 6 1-229000 0-3600 0-5 0 1.229960 0 0-250 AZ 1 23000 0 22400 0 45400 0 0 CA 2 0-3150 0-3600 0 0 0-6750 0 0 co 1 500 0 0 0 500 250 67000 cT 1 2250 14400 0 0 16650 0 242520 FL 7 62-235000 0-11100 0-3700 0 62-239120 0-53580 0-18500 GA 6 21199-329000 04500 0-750 0 21949333273 0 0-130 ID 1 88000 750 5 0 88755 0 0 IL 2 108-2150 0 0 0 108-2150 0-500 12242-160130 IN 3 250-9700 0-30 0 0 250-9730 0 5-99000 KS 2 40000-44662 0 0 0-37950 40000-82612 0 781-7726 KY 7 0-150000 0-7200 0-5 0 0-157205 0-849 0-89630 LA 16 3.280000 0-16600 0-250 0-15 34-284950 0 0-72280 MA 1 17650 0 0 0 17650 0 975 MD 1 17000 0 0 0 17000 32000 0 ME 7 28933441000 650-2200 0-130 0 30377441756 0 0-370 MI 5 2475-55000 2-570 0-79 0 2500-55600 0-15000 0-596 MN 3 8400-140000 04000 0-7 0 12407-140000 0-18417 0-1600 MO 2 8750-105000 0 0 0 8750-105000 250-3400 32100-114500 MS 2 25510-292000 1269-15000 1-5 0 26780-307005 0 0 MT 1 49800 700 0 0 50500 0 0 NC 4 98000-730000 0-3200 0 0 98000-730650 0-5 0-730 NH 1 36953 79 18 0 37050 0 0 NJ 4 150-18200 0-439 0 0 150-18639 0-13350 044300 NY 3 10-11000 0-250 0 0 10-11250 0-12 250-28729 OH 3 406-22100 0-820 0 0 406-22920 0 04037 3HNSOdX3 NVIWNH HOH TVILN3LOd 'S WHO40HOTHO 261 Table 5-1. Releases to the Environment from Facilities That Manufacture or Process Chloroform (continued) Number of Range of reported amounts released in pounds per year * State® facilities Water Land ile greta ot hit oo wansfer OK 1 93200 700 550 0 94450 0 0 OR 3 1000-143800 0-3000 0 0 1000-146800 0-100000 250-16000 PA 7 10700-231000 0-2900 0-15 0 10700-232705 0-70000 0-55700 PR 6 0-92072 0 0 0 0-92072 0-161519 0-306374 SC 5 500-136540 0-5649 0 0 500-138440 0-5 0-500 TN 2 38000-47763 1900-7441 0-160 0 39900-55364 0 0-3 ™ 17 5-600000 0-5350 0-900 0-74 5-600000 0-112000 0-377935 VA 4 55950-187000 31-2600 0-170 0 57979-187063 0 0 vT 1 4 0 0 0 4 0 168700 WA 7 5036-196000 0-15000 0-5 0 12275-200500 0 0 wi 1 1000- 150000 0-4900 0-250 0 1000-152500 0-13 0-51000 wv 3 870-22276 0-654 0-200 0 870-23130 0-1 0-1328 Source: TRI93 1995 ® Data in TRI are maximum amounts released by each facility. ® Post office state abbreviations used © The sum of all releases of the chemical to air, land, water, and underground injection wells by a given facility POTW = publicly owned treatment works 3HNSOdX3 NYANH HOS TVILN3L1Od 'S WHO40HOTHO £61 CHLOROFORM 194 5. POTENTIAL FOR HUMAN EXPOSURE ethylene dichloride, dyes, and fumigants (Deshon 1979; EPA 1985a, 1985b; HSDB 1996). Chloroform releases result from its formation and subsequent volatilization from chlorinated waters including drinking water, municipal and industrial waste waters, process waters and effluent from the bleaching of pulp in pulp and paper mills, cooling-tower water, and swimming-pool and whirlpool-spa water (Benoit and Jackson 1987; EPA 1985a, 1985b; Hoigne and Bader 1988). Increased release rates of the chloroform in waters can be expected from chloroform-containing waters that are heated (e.g., water used for cooking, showers, swimming pools, and spas). Aeration and use of groundwater contaminated with chloroform are a potential source of emission to the atmosphere (Crume et al. 1990). Chloroform is released as a result of hazardous and municipal waste treatment processes. The chloroform released may have initially been present in the waste or possibly formed during chlorination treatment (Corsi et al. 1987; EPA 1990b; Namkung and Rittmann 1987). Releases may also occur from hazardous waste sites and sanitary landfills where chloroform was disposed, and from municipal and hazardous waste incinerators that burn chloroform-containing wastes or produce chloroform during the combustion process (LaRegina et al. 1986; Travis et al. 1986). In the past, minor releases may have resulted from the use of consumer products (e.g., certain air deodorizers and cleaning products) that contained chloroform as a component or residual product (Bayer et al. 1988; Wallace et al. 1987a). Chloroform is widely used in laboratory work as an extractant. It is also still used in certain medical procedures, such as dental root canal surgeries (McDonald and Vire 1992), and in combination with aspirin as an experimental treatment for serious cases of herpes zoster (King 1993). These medical uses are extremely limited and would contribute very minor amounts of chloroform as releases to the air. 5.2.2 Water In 1991, releases of chloroform to the water from as many as 167 large processing facilities were 654,452 pounds (323,250 kg) (TRI91 1993), amounting to about 3.6% of total releases to all environmental media. TRI information from 1993 (TRI93 1995) indicates releases of chloroform to water from as many as 165 facilities were 335,032 pounds (150,764.4 kg) (TRI93 1995), or about 2.4% of the total releases to all environmental media. The releases of chloroform to water from facilities that manufactured and processed it in the United States during 1993 are reported in Table 5-1 (TRI93 1995). The TRI data indicate that only a small fraction of the chloroform released to the CHLOROFORM 195 5. POTENTIAL FOR HUMAN EXPOSURE environment is released to water. The TRI data should be used with caution because only certain types of facilities are required to report. This is not an exhaustive list. Current, more comprehensive quantitative data or estimates of chloroform releases to natural waters are lacking. Direct releases to water are expected via waste waters generated during chloroform manufacture and its use in the manufacture of other chemicals and materials (EPA 1985a). Direct discharge sources are expected to be relatively minor contributors to total chloroform emissions to water relative to the formation of chloroform resulting from the chlorination of drinking water or chlorination to eliminate pathogens in discharged wastes or other process waters (EPA 1985a). Since chlorination to disinfect water supplies is nearly universal, chloroform contamination resulting from chlorination will also be widespread (see discussion on levels monitored or estimated in water in Section 5.4.2). Other chloroform emission sources tend to be relatively isolated point sources. Chlorination of municipal and industrial waste waters at municipal and industrial waste water-treatment plants, process waters and effluent from the bleaching of pulp in pulp and paper mills, cooling-tower water, and swimming-pool and whirlpool-spa water will also result in chloroform formation (Benoit and Jackson 1987; Comba et al. 1994; EPA 1985a, 1985b, 1990a; Hoigne and Bader 1988). The use of modern treatment facilities may reduce the amounts of chloroform released to environmental waters. This has been demonstrated at a modern kraft pulp mill (Paasivirta et al. 1988); however, much of the chloroform removed from the waste water may be released to the atmosphere by volatilization. Release of chloroform to groundwater has resulted from improper disposal of chloroform-containing waste at hazardous waste sites (Clark et al. 1982; Dewalle and Chian 1981; Harris et al. 1984; Sawhney 1989). An additional minor source of water contamination may be atmospheric rainout since chloroform has been found in rainwater (Kawamura and Kaplan 1983). Other sources of chloroform release to surface water include breweries, thermal combustion of plastics, reaction of dissolved chlorine with sediment and other materials in water, biological production by marine algae, and the reaction of chlorinated pollutants with humic materials in natural waters (EPA 1985a). 5.2.3 Soil In 1991, releases of chloroform to the land from as many as 137 large processing facilities were 28,582 pounds (14,117 kg), which amounts to less than 1% of the total releases to all environmental CHLOROFORM 196 5. POTENTIAL FOR HUMAN EXPOSURE media (TRI91 1993). TRI information from 1993 (TRI93 1995) indicates releases to the land from as many as 124 facilities was 32,926 pounds (14,816.7 kg), which is less than 1% of the total releases to all environmental media. The releases of chloroform to soil from facilities that manufactured and processed it in the United States during 1993 are reported in Table 5-1 (TRI93 1995). The TRI data indicate that only a very small fraction of the chloroform released to the environment is released to land. The TRI data should be used with caution since only certain types of facilities are required to report. This is not an exhaustive list. Current comprehensive quantitative data or estimates of chloroform releases to soil are lacking. Chloroform releases to soil have occurred at hazardous waste sites containing improperly disposed wastes where chloroform has leached through soil to groundwater (Clark et al. 1982; Dewalle and Chian 1981; Harris et al. 1984; Sawhney 1989). Land disposal of sludge from municipal and industrial waste water-treatment plants may also result in chloroform releases to soil (EPA 1990a). Direct land disposal of chloroform-containing wastes may have occurred in the past, but land disposal of chloroform wastes is currently subject to restrictive regulations (EPA 1988a, 1989b). An additional minor source of soil contamination may be atmospheric rainout since chloroform has been found in rainwater (Kawamura and Kaplan 1983). Chloroform has been used as a carrier or solvent for some pesticides (HSDB 1996). It is still used as a carrier for at least one pesticide formulation with dichlorvos as the active ingredient (Petrelli et al. 1993). Application of pesticides using chloroform in the carriers could have resulted in releases of chloroform to the land. It is impossible to quantify the magnitude of such releases, and the chloroform could be expected to be transported to either the atmosphere through volatilization or, if dissolved in water, carried into surface waters or groundwater. 5.3 ENVIRONMENTAL FATE 5.3.1 Transport and Partitioning Based upon a vapor pressure of 159 mm Hg at 20 °C, chloroform is expected to exist almost entirely in the vapor phase in the atmosphere (Boublik et al. 1984; Eisenreich et al. 1981). Large amounts of chloroform in the atmosphere may be removed by wet deposition since chloroform has significant solubility in water. This is confirmed by its detection in rainwater (Kawamura and Kaplan 1983). CHLOROFORM 197 5. POTENTIAL FOR HUMAN EXPOSURE Most of the chloroform removed in precipitation, however, is likely to reenter the atmosphere by volatilization. Trace amounts of chloroform have been documented in air samples from remote, often relatively pristine, areas of the world (Class and Ballschmidter 1986). Since chloroform is relatively nonreactive in the atmosphere, long-range transport within the atmosphere is possible. The detections in remote areas may also mean that the chloroform is produced as the result of more localized transformation processes, possibly including the reaction of naturally generated chlorinated oxidants with organic matter. The dominant fate process for chloroform in surface waters is volatilization. Chloroform present in surface water is expected to volatilize rapidly to the atmosphere. An experimental half-disappearance range of 18-25 minutes has been measured for volatilization of chloroform from a 1 ppm solution with a depth of 6.5 cm that was stirred with a shallow pitch propeller at 200 rpm at 25 °C under still air (=0.2 mph air currents) (Dilling 1977; Dilling et al. 1975). Using the Henry's law constant, a half- life of 3.5 hours was calculated for volatilization from a model river 1 meter deep flowing at 1 meter/second, with a wind velocity of 3 m/second, and neglecting adsorption to sediment (Lyman et al. 1982). A half-life of 44 hours was estimated for volatilization from a model pond using EXAMS (1988). Based on a measured soil organic carbon sorption coefficient (Kye) of 45 (or a log [K] of 1.65), chloroform is not expected to adsorb significantly to sediment or suspended organic matter in surface water (Sabljic 1984). This prediction is supported by sediment monitoring data that indicate that this compound has not been detected (or was detected at very low concentrations) in sediment samples (Bean et al. 1985; Ferrario et al. 1985; Helz and Hsu 1978). Little or no chloroform concentration was observed on peat moss, clay, dolomite limestone, or sand added to water (Dilling et al. 1975). Chloroform slightly adsorbed to aquifer solids in laboratory studies utilizing different amounts of two different aquifer materials with K |. values ranging from 63.4 to 398. The authors reported higher adsorption with increasing organic content of the solids (Uchrin and Mangels 1986). K. values ranging from 45 to 80 in soil have been experimentally determined for chloroform (Sabljic 1984; Wilson et al. 1981). Chloroform does not appear to bioconcentrate in higher aquatic organisms, based upon measured bioconcentration factors (BCF) of 6 and 8 for bluegill sunfish (Lepomis macrochirus) (Barrows et al. 1980; Veith et al. 1980). Information from EPA’s ASTER (1996) database document a calculated CHLOROFORM 198 5. POTENTIAL FOR HUMAN EXPOSURE BCF for the fathead minnow of 14, a low value suggesting little potential for bioconcentration in fish. A BCF of 690 experimentally determined for the bioconcentration of chloroform in the green algae Selenastrum capricornutum suggests that the compound has a moderate tendency to concentrate in nonvascular aquatic plants (Mailhot 1987). No data regarding the biomagnification potential of chloroform were found. Based upon the observed BCF, however, significant biomagnification of chloroform is apparently unlikely. In soil, the dominant transport mechanism for chloroform near the surface will probably be volatilization because of its high volatility and low soil adsorption. Volatilization rates seem relatively constant over a wide variety of soil types (Park et al. 1988). In other laboratory studies, 75% of the chloroform initially present in water volatilized when applied to a fine sandy soil, and 54% of the chloroform volatilized from a soil column during a percolation study utilizing a sandy soil (Piwoni et al. 1986; Wilson et al. 1981). All or nearly all of the remaining chloroform traveled through the soil because of its low adsorption onto soil. Another laboratory study of 15 common volatile or semi- volatile organic chemicals reported a disappearance half-life for chloroform of 4.1 days, which assumed first-order kinetic decay (Anderson et al. 1991). The leaching potential of chloroform is further confirmed by the detection of chloroform in groundwater, especially at hazardous waste sites (Clark et al. 1982; Dewalle and Chian 1981; Harris et al. 1984; Sawhney 1989). 5.3.2 Transformation and Degradation For air, the major degradation process involves reactions with free radicals such as hydroxyl groups (Atkinson 1985). For other media, it is clear that chloroform can be mineralized through both abiotic and biotic processes. Information in the available literature (Bouer and McCarty 1983; Rhee and Speece 1992) documents the disappearance of chloroform in water and soil media under both aerobic and anaerobic conditions as well as identification of the end products. 5.3.2.1 Air The vapor-phase reaction of chloroform with photochemically generated hydroxyl radicals is the dominant degradation process in the atmosphere. The rate constant for this process at 25 °C has been experimentally determined as 1.0x10"13 cm?/molecule-second, which corresponds to a half-life of =80 days based upon a 12-hour sunlit day in a typical atmosphere containing 1x10° hydroxyl CHLOROFORM 199 5. POTENTIAL FOR HUMAN EXPOSURE radicals/cm’ (Hampson 1980; Singh et al. 1981). Breakdown products from reaction with hydroxyl radicals probably include phosgene and hydrogen chloride (Atkinson 1985). Chloroform is more reactive in photochemical smog conditions where the approximate half-life is 11 days (Dimitriades and Joshi 1977). Direct photolysis of chloroform will not be a significant degradation process in the atmosphere. Chloroform solutions sealed in quartz tubes and exposed to sunlight for one year degraded at almost the same rate as solutions in sealed tubes stored in the dark, which indicated that very little or no photodegradation of the compound had occurred (Dilling et al. 1975). This is expected because chloroform does not show significant light absorbance at wavelengths >290 nm (Hubrich and Stuhl 1980). 5.3.2.2 Water Hydrolysis will not be a significant degradation process in water based upon rate constants experimentally determined at 25 °C that correspond to half-lives ranging from 1,850 to 3,650 years at pH 7, and from 25 to 37 years at pH 9 (Jeffers et al. 1989; Mabey and Mill 1978). Direct photolysis of chloroform will not be a significant degradation process in surface waters because, as noted above, the compound does not absorb light at wavelengths >290 nm (Hubrich and Stuhl 1980). The reaction rate of chloroform with hydrated electrons photochemically produced from dissolved organic matter has been predicted to correspond to a near-surface half-life of =44 days based upon an experimentally determined rate constant and a hydrated electron concentration of 1.2x10°!7 mol of hydrolyzed electrons/L (Zepp et al. 1987). This latter process is probably too slow to effectively compete with volatilization as a removal process from surface waters. Biological degradation of chloroform has been studied primarily with an eye to batch process operation at waste water treatment plants or remediation possibilities at hazardous waste disposal sites. Above certain dosage levels, chloroform becomes toxic to anaerobic and aerobic microorganisms. This is especially noticeable for biological treatment facilities that use anaerobic digestion systems, where sustained inputs with chloroform concentrations approaching 100 mg/L can all but eliminate methanogenic (methane-fermenting) bacteria (Rhee and Speece 1992). Other studies have shown appreciable inhibition of methanogenesis with levels of chloroform in the range of 1 mg/L (Hickey et al. 1987). Other chlorinated hydrocarbons, and particularly such common 2-carbon chlorinated aliphatics as TCE, can similarly inhibit bacteria found in sewage sludges (Long et al. 1993; Rhee and Speece 1992). Similar inhibition effects can be the result of heavy metal toxics, zinc being CHLOROFORM 200 5. POTENTIAL FOR HUMAN EXPOSURE particularly stressful to methanogenic bacteria (van Beelen et al. 1994; van Vlaardingen and van Beelen 1992). Studies of actual natural waters or waste waters, where it is difficult to control the levels of specific chemicals or preclude inputs of other toxicants, yield a wide variety of results on the efficiencies of chloroform biodegradation. For instance, little or no degradation was observed during 25 weeks in aqueous aerobic screening tests utilizing primary sewage effluent inocula (Bouwer et al. 1981a). No chloroform degradation was observed in aerobic biofilm column studies (Bouwer et al. 1981b). Significant degradation of chloroform (46-49% loss in 7 days, at least some of which was apparently due to volatilization) in aerobic screening tests utilizing settled domestic waste water as inoculum was reported (Tabak et al. 1981). Under the proper conditions, chloroform appears to be much more susceptible to anaerobic biodegradation. Degradation of chloroform under anaerobic conditions was more rapid at lower chloroform concentrations (81 and 99% degradation after 2 and 16 weeks, respectively, at 16 ppb); a more gradual degradation was observed at higher concentrations and 78% degradation after 2 and 16 weeks, respectively, at 157 ppb) (Bouwer et al. 1981a). No degradation was observed, however, when chloroform was incubated with aquifer material under anaerobic conditions for 27 weeks (Wilson et al. 1981). In the absence of toxicity from other solvents, chlorinated hydrocarbons, or heavy metals, and where chloroform concentrations can be held below approximately 100 ppb, both aerobic and anaerobic bacteria can biodegrade chloroform, with removal rates well over 80% in a period of 10 days (Long et al. 1993). Deviations from these ideal conditions can lead to lower removal efficiencies. These biodegradation reactions generally lead to the mineralization of the chloroform to chlorides and carbon dioxide (Bouwer and McCarty 1983; Rhee and Speece 1992). One study, however, documents the production of the toxicant methylene chloride (dichloromethane) from the breakdown of chloroform- containing wastes in a mixed culture of bacteria from sewage sludge (Rhee and Speece 1992 citing results from work at Tyndall AFB, Florida). Caution should be exercised in making generalizations without site-specific evidence, however, since commercial grades of chloroform will often contain methylene chloride as an impurity (HSDB 1996). In waters containing mixtures of different chlorinated aliphatics, biodegradation may produce new chloroform, at least as a temporary by-product, the breakdown of carbon tetrachloride into chloroform having been confirmed in laboratory studies (ATSDR 1994; Long et al. 1993; Picardal et al. 1993). CHLOROFORM 201 5. POTENTIAL FOR HUMAN EXPOSURE 5.3.2.3 Sediment and Soil Little information was located regarding the degradation of chloroform in soil. Based upon data for degradation in water, chemical degradation in soil is not expected to be significant. The available soil data suggest that chloroform biodegradation rates in soil may vary, depending upon conditions. In soil column studies, the chloroform present in the influent secondary waste water appeared to pass through the column nearly unchanged even though some of the other organic compounds present were apparently biodegraded, which indicated that the waste water was not too toxic to the microorganisms in the soil (Bouwer et al. 1981b). In contrast to these studies, significant degradation of chloroform (33% removed in 6 days) was observed in fine sandy soil in sealed bottles; however, the chloroform may have been co-metabolized by methylotropic bacteria already present in the soil. The aerobic degradation was even faster in methane-enriched soil (Henson et al. 1988). Such bio-oxidation of chloroform was also observed under methanogenic conditions in batch experiments using an inoculum derived from activated sludge and in a continuous-flow laboratory scale column, using a methanogenic fixed film derived from primary sewage effluent (Bouwer and McCarty 1983). Overall, biodegradation in soil is not expected to compete with the predicted rapid rate of volatilization from soil (Park et al. 1988). As with biodegradation in water, concentrations of chloroform above certain threshold levels may inhibit many bacteria, especially methane-fermenting bacteria under anaerobic or near-anaerobic conditions (Hickey et al. 1987). 5.4 LEVELS MONITORED OR ESTIMATED IN THE ENVIRONMENT 5.4.1 Air Data from the most recent study located (1982-85 air samples) reported that the background level of chloroform concentrations over the northern Atlantic ocean ranges from 2x10 to 5x107 ppm (Class and Ballschmidter 1986). This range does not differ significantly from the range reported for 1976-79 (1.4-4x107 ppm) and the range reported from the 1987 update of the National Ambient Volatile Organic Compounds Database (NAVOCDB), which was 2x10 ppm (Brodzinsky and Singh 1982; EPA 1988b; Singh 1977; Singh et al. 1979). The maximum and background levels found in 7 U.S. cities between 1980 and 1981 were 5.1x103 and 2x10 ppm, respectively (Singh et al. 1982). Average atmospheric levels in U.S. cities ranged from 2x10 to 2x1073 ppm between 1980 and 1981. The median concentration reported between 1977 and 1980 was 7.2x10™ ppm, and the median CHLOROFORM a 202 5. POTENTIAL FOR HUMAN EXPOSURE reported in the 1987 update of the NAVOCDB was 6x107 ppm (Brodzinsky and Singh 1982; EPA 1988b; Singh et al. 1981, 1982; Wallace et al. 1986a, 1986b, 1988). A recent update to EPA's volatile organic compounds (VOC) databases on background ambient air concentrations for hazardous air pollutants (Kelly et al. 1994) estimated a chloroform background ambient level for chloroform in the United States as 4x10 ppm. The median concentration for source-dominated areas in the United States is 8.2x1074 ppm for data reported between 1977 and 1980, and this figure does not differ significantly from the 5.1x107# ppm values reported in the 1987 update of the NAVOCDB (Brodzinsky and Singh 1982; EPA 1988b). Certain source-dominated areas contained much higher chloroform levels. The ambient air concentrations outside homes in Love Canal, New York, in 1978, ranged from 2x10% to 2.2x1072 ppm, and the maximum concentration found in ambient air at 20 California municipal landfills was 0.61 ppm (Barkley et al. 1980; Wood and Porter 1987). Concentrations ranging from 2.9x104 to 6x1073 ppm were found in air samples taken from 5 hazardous waste sites in New Jersey (LaRegina et al. 1986). Ambient air samples measured near a hazardous waste landfill contained <1x1073 ppm chloroform. All these data indicate that chloroform levels in air can be much higher in areas near hazardous waste sites (Stephens et al. 1986). Other source-dominated areas that may have ambient air chloroform concentrations significantly higher than background levels include areas near facilities that treat hazardous and municipal waste, as well as areas near contaminated groundwater, and municipal- and hazardous-waste incinerators (Corsi et al. 1987; EPA 1990a; LaRegina et al. 1986; Namkung and Rittmann 1987; Travis et al. 1986). Typical median indoor air concentrations of chloroform range from =2x10 to 4x1073 ppm (Barkley et al. 1980; Pellizzari et al. 1986; Wallace et al. 1987c, 1989). Chloroform concentration ratios of indoor air to outdoor air range from <1 to 25 (Pellizzari et al. 1986). One of the most significant indoor sources of chloroform is chlorinated tap water, and taking showers (and breathing air where chloroform has been released from the shower water) is expected to contribute a substantial amount to the indoor chloroform levels (Andelman 1985a, 1985b; Wallace 1987). A recent study investigating typical levels of various VOCs in the air in Canadian homes noted median chloroform concentrations of 4x10 ppm (Otson et al. 1994). The air around swimming pools may also contain chloroform. This is especially likely in heated, indoor pools, which can approximate the conditions found in shower stalls. Concentrations ranging CHLOROFORM 203 5. POTENTIAL FOR HUMAN EXPOSURE from 3.5x10°2 to 19.9x10°2 ppm (440-2,335 nmol/m°) have been reported for environmental air at an indoor swimming pool (Aggazzotti et al. 1990, 1993). Chloroform has been identified in at least 717 of 1,428 current or past NPL sites (HazDat 1996). Chloroform has been detected in air samples taken at an estimated 57 of the 717 NPL hazardous waste sites where chloroform was detected. 5.4.2 Water Recent monitoring data regarding the presence of chloroform in surface water, sediments, and groundwater were not located. The most recent monitoring data that were obtained involved chloroform levels in drinking water. Finished drinking water collected in 1988 from 35 sites across the United States contained median concentrations of chloroform ranging from 9.6 to 15 pg/L (Krasner et al. 1989). In an analysis of available monitoring data from raw water supplies, the maximum detected value was 136 ppb (EPA/AMWA 1989). Data from earlier studies indicate a wide range of concentrations have been found in drinking-water supplies. The reported chloroform concentrations that were detected ranged from trace levels to 311 ppb, with one study reporting a median concentration of 16.7 ppb and another study reporting a geometric mean concentration of 1.81 ppb. Most of the concentrations ranged between 22 and 68 ppb (Brass et al. 1977; EPA/AMWA 1989; Furlong and D'Itri 1986; Kasso and Wells 1981; Krasner et al. 1989: Rogers et al. 1987; Symons et al. 1975). Chloroform can be expected to exist in virtually all chlorinated drinking-water supplies. The main source of chloroform found in municipal drinking water is the chlorination of naturally occurring humic materials found in raw-water supplies (Bellar et al. 1974; Cech et al. 1982). Factors that can increase the amount of chloroform in drinking water include seasonal effects (high summer values) and increased contact time between chlorine and humic material. Sources of water with high humic material content will contain higher levels of chloroform. The chloroform concentration increases with time, indicating that concentrations of the compound increase as the water moves through the distribution system (Kasso and Wells 1981). Drinking water derived from groundwater, especially groundwater at or near some hazardous waste sites and landfills, may contain higher levels of chloroform than normally encountered in drinking water derived from surface water. Chloroform levels ranging from 2.1 to 1,890 ppb have been observed in drinking water derived from wells near a hazardous waste dump (Clark et al. 1982). The leachate from one solid waste landfill contained 21,800 ppb chloroform; drinking water obtained from wells in the vicinity of the landfill had CHLOROFORM 204 5. POTENTIAL FOR HUMAN EXPOSURE chloroform levels of 0.3-1.6 ppb (Dewalle and Chian 1981). Data from the most recent study of Kansas groundwater sampled in 1986 indicate concentrations ranging from =0.3 to 91 ppb in both raw and treated groundwater; the average and median concentrations in the treated water were 7.6 and 0.5 ppb, respectively (Miller et al. 1990). Of the sample sites in a national groundwater supply survey, 45% had detectable levels of chloroform, and median and maximum concentrations were 1.5 and 300 ppb, respectively (Westrick et al. 1989). In addition to drinking water, chlorinated oxidants reacting with organic materials will lead to the formation of chloroform in swimming pools. Since swimming pools are not routinely analyzed for their chloroform content, data are limited and derived from special studies. Such studies will often cover the broader family of THMs, and while chloroform levels in blood or alveolar air samples will be reported, the environmental agents will often simply be recorded in terms of THMs. A rule of thumb (Copaken 1990) is that up to 90% of the THMs in chlorinated water samples will be chloroform. Recorded concentrations in samples from public pools fall in a range of 25-137 ppb (Aiking et al. 1994; Barnes et al. 1989; Copaken 1990). In poorly tended or very crowded public or private pools, where there are large inputs of organic materials or heavy use of chlorinating agents, chloroform levels as high as 163 ppb have been documented (Barnes et al. 1989). Chloroform production in swimming pools can be increased where the pools are treated with copper-containing algicides. In tests on chlorinated water using various doses of chlorine, copper (cupric valence form) from different copper-containing salts, and varying levels of humic acid (Barnes et al. 1989), chloroform concentrations after a given reaction time were generally 50% or more higher in samples treated with copper, which acts as a catalyst in the reactions with the humic acids. Current reviews of surface water monitoring data in the peer reviewed literature are lacking. The highest concentrations observed in surface waters of the United States sampled before 1984 were 394 and 120 ppb. These concentrations were observed in rivers in highly industrialized cities (Ewing et al. 1977; Pellizzari et al. 1979). Typical concentrations for most sites that are not heavily industrialized appear to range from trace levels to =22 ppb (Ohio River Valley Sanitation Commission 1980, 1982). Data from EPA's STORET database indicate that chloroform was detected in 64% of 11,928 surface water sample data points at a median concentration of 0.30 ppb (Staples et al. 1985). Chloroform at 0.25 ppb has been found in rainwater collected in Los Angeles, California, during 1982 (Kawamura and Kaplan 1983). CHLOROFORM 205 5. POTENTIAL FOR HUMAN EXPOSURE Chloroform has been detected in surface-water samples taken at an estimated 139 of 717 current or past NPL sites where chloroform was detected (HazDat 1996). Detections in groundwater are documented at 552 sites. From the available information found in HazDat (1996), groundwater appears to be the most common environmental medium at NPL sites where chloroform might be encountered. 5.4.3 Sediment and Soil Chloroform has been found in sediment samples. Chloroform was found in sediment samples taken in 1980 from the 3 passes of Lake Pontchartrain, Louisiana, at concentrations ranging from 1.7 to 18 ng/kg (w/w [weight per weight] basis) (Ferrario et al. 1985). Chloroform was found at concentrations ranging from 30 to 80 ng/kg (dry weight basis) in sediment samples exposed to chlorinated electrical power plant cooling water; the control samples that were not exposed to cooling water contained nearly the same amounts of chloroform (Bean et al. 1985). Data from EPA's STORET database indicate that chloroform was detected in 8% of 425 sediment sample data points at a median concentration of <5.0 ng/kg (Staples et al. 1985). Routine sediment sampling for chloroform does not seem common in the United States, and sampling at relatively unpolluted ambient monitoring sites may overlook the levels possible in more restricted hotspots. Anaerobic biodegradation of chlorinated hydrocarbons may generate chloroform, especially in harbors, shipping canals, or areas receiving large amounts of industrial discharges. Where the sediments contain large concentrations of zinc, which is widely used to galvanize metal or as an ingredient in common industrial rust inhibitors, methanogenic bacteria populations may be adversely affected, thus preventing the mineralization of chloroform. Chloroform in interstitial water may then build up to levels as high as 50 ppb (van Beelen et al. 1994; van Vlaardingen and van Beelen 1992). Soil monitoring data in the peer reviewed literature could not be located. It can be predicted that chloroform contamination occurs at hazardous waste sites where chloroform-containing leachate moves through the soil to groundwater. An explanation of the lack of data results from the fact that any chloroform in the soil is expected to either rapidly volatilize or leach. Laboratory studies using a variety of different soil types document the effectiveness of volatilization in removing chloroform from soils (Park et al. 1988). CHLOROFORM a z0b 5. POTENTIAL FOR HUMAN EXPOSURE Chloroform has been found in soil samples taken at an estimated 213 of the 717 current or past NPL where chloroform was detected (HazDat 1996). Detections in sediment samples are documented for 79 current or past NPL sites. Detections on soil gas obtained from soil samples were documented at 18 sites. 5.4.4 Other Environmental Media Chloroform has been detected in various foods at the following concentrations: soft drinks and beverages (2.7-178 pg/kg), dairy products (7-1,110 pg/kg), oils and fats (traces <12 pg/kg), dried legumes (6.1-57.2 pg/kg), and grains and milled grain products (1.4-3,000 pg/kg) (Abdel-Rahman 1982: Entz et al. 1982; Graham and Robertson 1988; Heikes 1987; Heikes and Hopper 1986; Lovegren et al. 1979). In a study of various foods, 41% of 231 samples contained chloroform at levels ranging from 4 to 312 pg/kg; the average level was 52 pg/kg (Daft 1988a). In another broad study, 55% of 549 samples contained between 2 and 830 pg/kg. The average level in this study was 71 pg/kg (Daft 1989). The chloroform concentration observed in other foods ranged from 6.1 to 1,110 pg/kg. The highest amounts were found in butter (1,110 pg/kg), mixed cereal (220 pg/kg), infant/junior food (230 pg/kg), and cheddar cheese (83 pg/kg) (Heikes 1987). Chloroform has been detected in the air above outdoor and indoor pools and in spas at maximum concentrations of 2.8x1072, 5.0x1072, and 5.2x1072 ppm, respectively; water concentrations ranged between 4 and 402, 3 and 580, and <0.1 and 530 ppb, respectively (Armstrong and Golden 1986). In another study, air samples above whirlpool spas treated with chlorine disinfectant contained chloroform at concentrations ranging from 8x10 to 1.5x107! ppm; the concentration in the water ranged from 15 to 674 ppb (Benoit and Jackson 1987). Chloroform has been detected at <37 ppb in the cooling water of a nuclear reactor; a concentration of 50 ppb was detected 0.75 miles downstream from the reactor cooling tower in one study (Hollod and Wilde 1982). Since chloroform is highly volatile and shows little tendency to bioconcentrate or bioaccumulate in higher life forms such as fishes, it is not ordinarily included in the types of persistent pollutants that are the focus of state fish consumption advisory programs. Information from HazDat (1996) does document detections of chloroform in tissues from fishes from at least 3 current or past NPL sites. CHLOROFORM 207 5. POTENTIAL FOR HUMAN EXPOSURE 5.5 GENERAL POPULATION AND OCCUPATIONAL EXPOSURE The general population is probably exposed to chloroform through drinking water and beverages, eating food, inhaling contaminated air, and through dermal contact with water (e.g., while showering, bathing, cleaning, washing, swimming). All humans are expected to be exposed to at least low levels of chloroform. Accurate, current estimates of the daily intake of chloroform by various exposure routes are not available, or possible, due to the lack of appropriate current monitoring data. Typical levels of atmospheric exposure in remote, urban, and source-dominated areas range from 2x10 to 5x10, 6x107 to 2x1073, and 8.2x107 to 2.2x10°2 ppm, respectively (Barkley et al. 1980; Brodzinsky and Singh 1982; Class and Ballschmidter 1986; EPA 1988a; Singh et al. 1981, 1982; Wallace et al. 1986a, 1986b, 1988; Wood and Porter 1987). Exposure via ingestion of contaminated drinking water is expected to be extensive since most U.S. community drinking-water supplies are chlorinated (see Singer 1994 for an overview of the entire topic of disinfection by-products in drinking water). Typical levels in drinking water range from 2 to 68 ppb (Brass et al. 1977; EPA/AMWA 1989: Furlong and D'Ttri 1986; Kasso and Wells 1981; Krasner et al. 1989; Rogers et al. 1987; Symons et al. 1975). Although data regarding levels in food are rather scant, typical average chloroform levels in certain foods are estimated to range from 52 to 71 pg/kg (Daft 1988a, 1988b, 1989). Although data are available from various studies regarding concentrations of chloroform found in human tissues, blood, and expired air, only limited data are available that compare these concentrations to measured or estimated environmental exposure levels. Furthermore, no correlation has been made between these measured human tissue concentrations and the corresponding environmental exposure levels (see Section 2.5.1 for a discussion of the relationship between chloroform exposure levels and concentrations found in humans). Much of the data available is from the Total Exposure Assessment Methodology (TEAM) studies (see Andelman 1990 or Wallace 1995 for succinct overviews) in which the concentration of chloroform was measured in personal air samples and exhaled human breath (Wallace 1987; Wallace et al. 1984, 1986a, 1986b, 1988). For example, in one TEAM study, the ratios of the concentrations of chloroform detected in personal air samples to those found in human exhaled breath air varied from 0.66:1 to 13.3:1 (Wallace 1987) (see Section 2.5.1 for more data regarding concentrations found in humans, including data obtained during autopsies). Recent studies supported through IARC on swimmers using indoor pools in Europe showed good correlations between the chloroform concentrations in alveolar air and blood plasma concentrations (Aggazzotti et al. 1990, 1993). Plasma levels ranging from 0.8 to 25.1 nmol/L were observed. Statistical analysis CHLOROFORM 208 5. POTENTIAL FOR HUMAN EXPOSURE showed the plasma levels to be significantly correlated with concentrations of chloroform in the pool water, time spent swimming, the number of swimmers in the pool, and the chloroform concentration in the environmental air. Limited current data were located regarding occupational exposure to chloroform. Although some of the exposure levels encountered in workplaces may be comparable to exposure the worker receives in his own home, there are probably many specific jobs that expose the workers to significantly higher levels of chloroform. These occupations include work at or near source-dominated areas such as chemical plants and other facilities that manufacture or use chloroform, operation of chlorination processes in drinking-water plants, work at or near waste water-treatment plants and paper and pulp plants, and other facilities where large amounts of chloroform are released (e.g., hazardous and municipal-waste incinerators). Persons working at waste water and other treatment plants can be exposed to significant levels of chloroform. A maximum level of 3.8x1073 ppm was found in the air at an activated sludge waste-water treatment plant (Lurker et al. 1983). Maintenance workers, attendants, and life guards at indoor pools and spas may encounter maximum concentrations of 5.0x1072 and 1.5x10°! ppm, respectively (Armstrong and Golden 1986; Benoit and Jackson 1987). Persons who use tap water often, especially if it is heated and/or sprayed (e.g., water used for cleaning, washing clothes and dishes, showering, and cooking), may be exposed to higher than background levels. For example, levels in personal air samples as high as 2.2x1072 and 1.1x1072 ppm have been measured during household cleaning activities and showering (Wallace et al. 1987d). While the use of activated carbon filters may provide some reduction in the tap water levels for cold water feeds, such filters are not effective with hot water where the elevated temperatures will induce volatilization from the filter media. Persons using certain cleaning agents and pesticides in enclosed spaces with poor ventilation or persons working where these materials are used may be exposed to relatively high levels of chloroform. A National Occupational Exposure Survey (NOES) conducted by NIOSH from 1981 to 1983 estimated that 95,778 workers in the United States are potentially exposed to chloroform (NOES 1991). In the absence of more recent national occupational exposure data, the NOES information still has some value. The NOES database does not contain information on the frequency, concentration, or exposure duration of workers; it only provides estimates of the number of workers potentially exposed to chemicals in the workplace. Of the 151 different occupational groups with potential chloroform exposure risks listed in the NOES database, approximately 50% of the potentially exposed workers fall CHLOROFORM 209 5. POTENTIAL FOR HUMAN EXPOSURE into the following Standard Industrial Classification (SIC) categories: Funeral Service and Crematory Technicians from SIC category 7261; Biological, Engineering or Chemical Technicians and Chemists from the Research and Development Laboratory SIC category 7391; Adhesive and Sealant Chemical Technicians from SIC category 2891; Assemblers from SIC category 3679 (Electronic Components) and SIC Category 3622 (Industrial Controls); Petroleum Refining Machine Operators and other Workers from SIC category 2911; and General Medical and Surgical Hospital Clinical Laboratory Technologists and Technicians from SIC category 8062. 5.6 POPULATIONS WITH POTENTIALLY HIGH EXPOSURES All humans are exposed to low concentrations of chloroform. Those with potentially high exposures are workers employed in chloroform manufacturing and use industries. Persons living in certain source-dominated areas may be at risk for higher than background exposures to chloroform. These may include persons living near industries and facilities that manufacture and use chloroform, municipal and industrial waste water-treatment plants and incinerators, paper and pulp plants, and persons who derive their drinking water from groundwater sources contaminated with chloroform- containing leachate from hazardous waste sites. Previously reported air monitoring data from landfills and other waste sites (see Section 5.4.1) suggest that potentially high exposure may occur via inhalation of contaminated air near hazardous waste sites. Other possibilities include drinking water from wells contaminated with chloroform that leached from the sites and, perhaps, living in homes built directly on top of former waste sites. Although some of the drinking-water supplies contaminated solely by leached chloroform (e.g., levels <1.6 ppb from a water well near a site with documented chloroform contamination) (Dewalle and Chian 1981) have shown levels lower or comparable to that in normal chlorinated drinking water, where chloroform levels in the range of 2-44 ppb are common (EPA/AMWA 1989), much higher levels (1,890 ppb) have been found in water from wells near a waste dump (Clark et al. 1982). HazDat (1996) documents 239 current or past NPL sites where specific concentration levels for groundwater are contained in the database. At least 97 current or past NPL sites show groundwater samples where values are 100 ppb or higher. A drinking-water standard of 100 ppb for total trihalomethanes (TTHMs) (where chloroform is usually the predominant constituent) is EPA's current requirement for treated water systems serving 10,000 or more consumers (see Chapter 7 below). CHLOROFORM 210 5. POTENTIAL FOR HUMAN EXPOSURE 5.7 ADEQUACY OF THE DATABASE Section 104(i)(5) of CERCLA, as amended, directs the Administrator of ATSDR (in consultation with the Administrator of EPA and agencies and programs of the Public Health Service) to assess whether adequate information on the health effects of chloroform is available. Where adequate information is not available, ATSDR, in conjunction with the NTP, is required to assure the initiation of a program of research designed to determine the health effects (and techniques for developing methods to determine such health effects) of chloroform. The following categories of possible data needs have been identified by a joint team of scientists from ATSDR, NTP, and EPA. They are defined as substance-specific informational needs that if met would reduce the uncertainties of human health assessment. This definition should not be interpreted to mean that all data needs discussed in this section must be filled. In the future, the identified data needs will be evaluated and prioritized, and a substance-specific research agenda will be proposed. 5.7.1 Identification of Data Needs Physical and Chemical Properties. As reported in Table 3-2, the physical and chemical properties of chloroform have been characterized sufficiently to permit estimation of its environmental fate. Production, Import/Export, Use, Release, and Disposal. Data regarding the production methods and current, past, projected future production capacity volumes, and current import and export volumes are available (Ahlstrom and Steele 1979; CEN 1995; Deshon 1979; NTDB 1994; SRI 1993, 1994: TRI92 1994). However, these statistics will generally not include all instances where chloroform is generated as a chemical intermediate or waste product. With the exception of the partial coverage provided in the Toxics Release Inventory (TRI91 1993; TRI93 1995) comprehensive information regarding current release and disposal patterns, are lacking. General disposal information is adequately detailed in the literature, and information regarding disposal regulations of chloroform is available (EPA 1988a, 1988b). Production, release, and disposal data are useful to determine where environmental exposure to chloroform may be high. A major data need is to achieve a better understanding of why a substantial number of NPL sites show chloroform levels in groundwater at or above concentrations of 100 ppb. There are 239 sites in HazDat (1996) where groundwater CHLOROFORM 211 5. POTENTIAL FOR HUMAN EXPOSURE concentration levels are documented. At least 36 sites show signs of groundwater contamination with levels of 1,000 ppb (or 1 ppm) or higher, levels high enough to suggest disposal or spills of wastes containing very high concentrations of chloroform. Sixty-one (61) sites show groundwater levels in a range from 100 to 1,000 ppb, and the remaining 142 sites show levels below 100 pp. While some sites show very low levels comparable to the concentrations associated with many chlorinated public drinking-water supplies, the substantial number of sites with values at or slightly above the community drinking-water standard for TTHMs of 100 ppb deserves some scrutiny. Is the chloroform at such sites the result of past disposal of chloroform-laden wastes, or are some other sources (e.g., in situ generation of chloroform as the result of chemical or biochemical transformation of other on-site wastes) for the chloroform contamination involved? Present data sources are lacking to explain adequately the large number of NPL sites showing elevated levels of chloroform in such media as groundwater that pose major off-site exposure risks. Environmental Fate. Experimental data are available regarding the transport and partitioning properties of chloroform in surface waters (Bean et al. 1985; Clark et al. 1982: Class and Ballschmidter 1986; Dilling 1977; Ferrario et al. 1985; Piwoni et al. 1986; Sawhney 1989). Chloroform partitions mainly into the atmosphere and into groundwater. Empirical measurements or model predictions on half-disappearance times in such media as soil could not be identified in the literature. Chloroform can be transported long distances in air. Data are available regarding the degradation of chloroform in the atmosphere, but less is known about degradation rates in water and soil (Anderson et al. 1991; Bouwer et al. 1981a, 1981b; Dilling et al. 1975; Hampson 1980; Henson et al. 1988; Jeffers et al. 1989; Park et al. 1988; Singh et al. 1981; Tabak et al. 1981: Wilson et al. 1981). Hydrolysis and direct photodegradation are not significant removal processes. Although data regarding biodegradation rates in natural media are lacking, volatilization is expected to dominate over biodegradation as a removal process from surface water and near-surface soil. Chloroform seems relatively persistent in the atmosphere and groundwater. The environmental fate of chloroform releases related to most common anthropogenic sources appears to be sufficiently determined by the available data. In light of the documented occurrence (Class and Ballschmidter 1986) of chloroform in remote, often pristine, areas, however, further study is warranted to help quantify the relative role of long-range transport processes as opposed to a variety of more localized potential chemical transformation processes. These more localized processes could include the reaction of naturally generated chlorinated oxidants with organic materials to yield chloroform. More data would be useful on the half-lives of chloroform in such media as soils. CHLOROFORM 212 5. POTENTIAL FOR HUMAN EXPOSURE Bioavailability from Environmental Media. Chloroform is absorbed following inhalation, oral, and dermal contact. Toxicity studies of exposure to chloroform in air, water, and food demonstrated the bioavailability of chloroform by these routes. Data regarding its bioavailability from soil are lacking, but near-surface soil concentrations can be expected to be low due to volatilization (Piwoni et al. 1986; Wilson et al. 1981). Food Chain Bioaccumulation. Data are available that indicate that chloroform does not bioconcentrate in aquatic organisms (Barrows et al. 1980; Veith et al. 1980); however, data are lacking for plants and other animals (e.g., vacuolar plants, shellfish, or macroinvertebrates) as well as for the biomagnification potential of chloroform in terrestrial and aquatic food chains. Additional information on bioconcentration and biomagnification could be useful in establishing the significance of food chain bioaccumulation as a route of human exposure. Exposure Levels in Environmental Media. All humans are exposed to at least low levels of chloroform via inhalation of contaminated air, and most humans are exposed by drinking contaminated water. Estimates from intake via inhalation and ingestion of drinking water, based on limited data, are available (see Section 5.5). Exposure from foods cannot be estimated, due to the lack of data. Current information on exposure to chloroform from water, air, and foods, especially for workers or people who live near manufacturing and use facilities, water and waste water-treatment plants, municipal and industrial incinerators, hazardous waste sites, and other sources of significant release, in addition to data regarding exposure levels in indoor air would be useful. Exposure Levels in Humans. Data regarding exposure levels in humans are incomplete and are usually the result of limited, special studies. Chloroform has been found in human blood and expired air of both occupationally and nonoccupationally exposed groups, and in breast milk of nonoccupationally exposed groups (Hajimiragha et al. 1986; Pellizzari et al. 1982; Wallace et al. 1987a). A detailed recent database of exposure would be helpful in determining the current exposure levels, thus allowing an estimation of the average daily dose associated with various scenarios, such as living near a point source of release, drinking contaminated water, or working in a contaminated place. This information is necessary for assessing the need to conduct health studies on these populations. Exposure Registries. No exposure registries for chloroform were located. This substance is not currently one of the compounds for which a subregistry has been established in the National Exposure CHLOROFORM 213 5. POTENTIAL FOR HUMAN EXPOSURE Registry. The substance will be considered in the future when chemical selection is made for subregistries to be established. The information that is amassed in the National Exposure Registry facilitates the epidemiological research needed to assess adverse health outcomes that may be related to exposure to this substance. 5.7.2 Ongoing Studies As part of the Third National Health and Nutrition Evaluation Survey (NHANES III), the Environment Health Laboratory Sciences Division of the National Center for Environment Health, Centers for Disease Control and Prevention, will be analyzing human urine samples for chloroform (Needham et al. 1990). These data will give an indication of the frequency of occurrence and background levels of these compounds in the general population. Research at Colorado State University (FEDRIP 1994) will investigate toxicity and bioaccumulation potential of chloroform and other organic and inorganic chemicals. The laboratory studies will use a simple food chain containing larval invertebrates and fish. The results would be applicable to many natural systems with fish foraging on insect larvae in contaminated sediments. Another research project at Colorado State University (FEDRIP 1994) will study the microbial degradation kinetics of pollutant mixtures, which will include chloroform. One aspect of this research will focus on both the degradation of chloroform as well as its inhibitory effects when present above certain threshold concentrations. A project at the University of Arizona (FEDRIP 1996) will study microbial dehalogenation of several compounds, including chloroform. A major part of the study will focus on the facultative anaerobic bacteria Shewanella putrefaciens sp., which is known to catalyze the transformation of carbon tetrachloride to chloroform and other as yet unidentified products. The organic substrates will also contain metals. It is hoped that the end-products from the biochemical treatment can be subjected to a photolytic finishing process that will completely mineralize any remaining halogenated compounds. A project at the University of Idaho (FEDRIP 1996) will study the biodegradation of several halogenated hydrocarbons in soils amended with plant residues from various Brassica cultivars (e.g. rape seed). These residues contain chemicals that may help catalyze the degradation of some CHLOROFORM 214 5. POTENTIAL FOR HUMAN EXPOSURE chlorinated hydrocarbons. Work to date has shown the potential for phrophyrins metallated with cobalt or nickel to cause the breakdown of carbon tetrachloride into such products as chloroform, dichlormethane, and carbon monoxide. A project at the University of California at Berkeley (FEDRIP 1996) will study factors affecting the rates of degradation for several chlorinated hydrocarbons by soil microbial populations. Chloroform will be included as one of the organic chemicals studied. A project conducted by the USDA (FEDRIP 1996) at its Western Regional Research Center in Albany, California, will study the use of ozone or peroxide treatment as an alternative to chlorinated bleaching in the preparation of walnuts intended for in-the-shell sales. There are concerns that the current use of hypochlorite bleaching agents can adversely impact taste as well as leaving behind THM residues containing chloroform. A project at the University of California at Riverside (FEDRIP 1996) will study factors affecting the biodegradation in soils of several pesticides and halogenated organics by such microbes as Methano- bacterium thermoautotrophicum. This anaerobic bacterium shows the potential for very rapid oxidation of several organics, including chloroform. In practice, however, one or more limiting factors dramatically reduces the expected degradation Kinetics. A project at the USDA’s Western Research Center in Albany, California, will study ways to reduce exposures to chloroform for workers at poultry processing plants (FEDRIP 1994). At various points in the processing of poultry, the carcasses are rinsed in chiller-water baths that kill pathogens. Currently, chlorine is the only USDA-approved sanitizing agent. This study will investigate the potential of such alternative agents as chlorine dioxide to achieve comparable germicidal effects while reducing the levels of chloroform generated. The United States Geological Survey (FEDRIP 1996) is conducting a study on the Mississippi River and its major tributaries focused on the transport and degradation of organic substances. Experiments will be conducted at 12 stations to determine the THM and organic halide formation potentials. The results will be summarized in a chapter on water purification by-products in a Report to Congress on the Mississippi River. CHLOROFORM 215 5. POTENTIAL FOR HUMAN EXPOSURE Remedial investigations and feasibility studies at NPL sites that contain chloroform will also provide further information on environmental concentrations and human exposure levels near waste sites. CHLOROFORM 217 6. ANALYTICAL METHODS The purpose of this chapter is to describe the analytical methods that are available for detecting and/or measuring and monitoring chloroform in environmental media and in biological samples. The intent is not to provide an exhaustive list of analytical methods that could be used to detect and quantify chloroform. Rather, the intention is to identify well established methods that are used as the standard methods of analysis. Many of the analytical methods used to detect chloroform in environmental samples are the methods approved by federal organizations such as EPA and the National Institute for Occupational Safety and Health (NIOSH). Other methods presented in this chapter are those that are approved by groups such as the Association of Official Analytical Chemists (AOAC) and the American Public Health Association (APHA). Additionally, analytical methods are included that refine previously used methods to obtain lower detection limits, and/or to improve accuracy and precision. 6.1 BIOLOGICAL SAMPLES Methods for analyzing chloroform in the biological matrices (breath, blood, urine, and tissues) are listed in Table 6-1. None of these methods has been standardized by an organization or federal agency, although the blood method of Ashley et al. (1992) was developed at the Centers for Disease Control and Prevention (CDC). Sample preparation methods are based on headspace analysis, purge- and-trap, or solvent extraction. Sample preparation for breath samples typically utilizes an adsorbent followed by thermal desorption or direct analysis of an aliquot of breath. These methods all use gas chromatography (GC) with various detection methods as an analytical technique. Cardinali et al. (1994) describe a procedure for the production of blank water for use with analysis of organic compounds in human blood at the parts per trillion (ppt) level; the availability of such blank samples is very important if reliable results are to be obtained. With limits of detection (LODs) in the low-ppt range, these methods are sufficiently sensitive to measure background levels of chloroform in the general population as well as chloroform levels at which health effects might occur after short-term or long-term exposure. However, many studies do not report the method detection limit and/or the recovery percentage for the method. For more information regarding the use of GC methods and detectors, see Section 6.2. Table 6-1. Analytical Methods for Determining Chloroform in Biological Samples Sample Sample Analytical detection Percent matrix Preparation method method limit recovery Reference Exhaled air Collection in Tedlar bag; HRGC/FID and No data No data Krotoszynski et al. 1979 (breath) preconcentration by Tenax-GC; thermal HRGC/MS desorption Alveolar air Collection of exhaled air using a GC/MS 0.5 pg/m3 112 (8% RSD) Raymer et al. 1990 specially-designed device to provide (0.5 ppt, w/v) at5.1 pg/m3 pure air for inhalation. Breath collected into duplicate evacuated canisters. Alveolar air Collection directly into an evacuated GC/MS No data No data Pleil and Lindstrom 1995 canister Exhaled air Collection of whole breath into a GC/Electrolytic ~~ 13 ng on 63 + 13 Jo et al. 1990 sampling bag; collected sample pulled conductivity cartridge through a Tenax cartridge Alveolar air Collection onto carbonaceous sorbent; GC/ITMS No data No data Phillips and Greenberg 1992 thermal desorption Alveolar air Collection of end-expired air into glass GC/ECD, 8.4 nmo/m® No data Aggazzotti et al. 1993 tube GC/MS (1 pg/m3; 1 ppt, Wiv) Whole blood Purge-and-trap pre-concentration (with ~~ GC/Isotope 0.040 ppb 105 at 0.054 Ashley et al. 1992 isotopically-labelled internal standard) dilution MS ppb Whole blood Headspace analysis GC/FID 0.02 pg/mL No data Seto et al. 1993 (0.02 ppm, w/v) Whole blood Purge-and-trap preconcentration; GC/MS <0.5 pg/L No data Antoine et al. 1986 thermal desorption Adipose Purge-and-trap pre-concentration; GC/Hall with No data 100 at 0.6 pg/L Peoples et al. 1979 tissue and thermal desorption GC/MS (serum); No data serum confirmation (tissue) SAOHL3IW TVOILATVYNY 9 WNHO40HOTHO 81¢ Table 6-1. Analytical Methods for Determining Chloroform in Biological Samples (continued) Sample Sample Analytical detection Percent matrix Preparation method method limit recovery Reference Whole blood Solvent extraction HRGC/ECD with No data 99 at 50 pg/L Kroneld 1986 HRGC/MS confirmation Serum and Purge-and-trap pre-concentration; GC/Hall with 0.05 pg/L for No data Pfaffenberger et al. 1980 adipose thermal desorption GC/MS serum tissue confirmation Water, Solvent extraction HRGC/ECD 1 pg/L for 100.3 at 50 pg/L Reunanen and Kroneld 1982 serum, and serum and (water); 103 at urine urine 2.5 pg/L (serum); 141 at 2.3 pg/L (urine) Blood, urine Blood collected into vacutainers Headspace No data No data Cammann and Hiibner 1995 containing EDTA. Two-mL aliquots of GC/ECD blood or urine dispensed into headspace vials for analysis Blood, urine, Addition of sample to internal standard; GC/ECD At least No data Streete et al. 1992 and tissues addition of proteolytic enzyme; 1 ppm equilibration at elevated temperature; analysis of headspace gas Blood, urine, Purge-and-trap pre-concentration; GC/HSD or 0.10 pg/L No data EPA 1985a and tissue thermal desorption GC/MS (blood and urine) ECD = electron capture detector; FID = flame ionization detector; GC = gas chromatography; Hall = Hall electrolytic conductivity detector; HRGC = high-resolution gas chromatography; HSD = Halogen-specific electrolytic conductivity conductor; MS = mass spectrometry; RSD = relative standard deviation; w/v = weight:volume SAOHL3IN TVOILATVYNY 9 WHO40HO THO 6lC CHLOROFORM 220 6. ANALYTICAL METHODS Chloroform is transformed by mammalian P-450 enzymes in vivo to trichloromethanol which undergoes spontaneous dechlorination to yield phosgene (COCIl,) (Pohl et al. 1977), a highly reactive electrophile (Mansuy et al. 1977). Phosgene can react with cysteine to form 2-oxothiazolidine- 4-carboxylic acid (Pohl et al. 1977, 1980b), with two molecules of glutathione to form diglutathionyl dithiocarbonate (Pohl et al. 1981), or with water to produce chloride ion and carbon dioxide (Pohl et al. 1980b). Although 2-oxothiazolidine-4-carboxylic acid and diglutathionyl dithiocarbonate have been measured in liver microsomal preparations (Pohl et al. 1977, 1980b, 1981), the applicability of these methods to human tissues is unknown. Phosgene can also be formed from bromotrichloromethane and carbon tetrachloride (Pohl et al. 1981), so the formation of phosgene and any subsequent products cannot be related exclusively to exposure to chloroform. 6.2 ENVIRONMENTAL SAMPLES Analytical methods for determining chloroform in environmental samples are presented in Table 6-2. As with all extremely volatile chemicals, it is essential to take precautions during sampling, storage, and analysis to avoid loss of chloroform. Methods commonly used for the determination of chloroform concentrations in air are based on either adsorption onto a sorbent column followed by thermal or solvent desorption with subsequent analysis using GC (EPA 1988f; NIOSH 1994; OSHA 1979) or on cryogenic concentration of chloroform directly from a parcel of air (Bureau International Technique des Solvants Chlores 1976; EPA 1988g, 1988i) followed by GC. The disadvantages of the sorption tubes are that sorption and desorption efficiencies may not be 100%, and that the background impurities in the sorbent tubes may limit the detection limit for samples at low concentrations (Cox 1983). In addition, storage of sorbent tubes before desorption and analysis can result in losses of chloroform (OSHA 1979), and poor retention of chloroform by the sorbent can result in poor LODs (EPA 1988h). Determination of chloroform using isolation methods based on cryogenic trapping can be limited by moisture condensation in the trap (EPA 1988g). Evacuated canisters used to collect air samples in the field for transport to the laboratory must be carefully cleaned to avoid contamination of the sample (EPA 19881). Solid phase microextraction (SPME) has been shown to be useful for the determination of chloroform in air (Chai and Pawliszyn 1995). This technique is based upon the absorption of chloroform into a polymer coated on a silica fiber. Following equilibration of the fiber with the atmosphere, chloroform is released via thermal desorption in the injection port of a gas chromatograph. Sample preparation is Table 6-2. Analytical Methods for Determining Chloroform in Environmental Samples Sample Analytical detection Sample matrix Preparation method method limit Percent recovery Reference Air Adsorption onto carbon GC/MS (EPA No data 91 (15% RSD) at EPA 1988f molecular sieve; thermal Method TO2) 89 ng/L desorption Air Cryogenic preconcentration; GC/ECD (EPA No data 100 (5.8% RSD) at EPA 1988g thermal transfer to GC Method TO3) 3.5 ppb Air Collection into SUMMA GC/FID/ECD or No data No data EPA 1988i polished canister GC/MS (EPA Method TO14) Air Adsorption onto charcoal; GC/FID (OSHA 0.11 ppm for 10 L 96 (8.5% RSD) OSHA 1979 desorption with carbon disulfide Method 05) sample Air Adsorption onto charcoal; GC/FID 0.7 mg/m°3 for15L 97 at 120-493 mg/m3 NIOSH 1994 desorption with carbon (NIOSH sample disulfide, containing internal method 1003) standard if desired Ambient air Adsorption onto Tenax; thermal GC/FID or ECD No data No data Parsons and Mitzner and stacks desorption 1975 Air Collection in flask; transfer to GC/ECD 1.5 pg/md No data Bureau International GC by syringe Technique des Solvants Chlores 1976 Air Solid Phase Microextraction; GClion trap MS 2 ppb (v/v); 6% RSD No data Chai and Pawliszyn transfer to GC via thermal 1995 desorption of fiber. Air Exposure of passive sampler GC/ECD/FID 4 ng/m3 98 (10.0% inter-day Begerow et al. 1995 (3M 3500 OVM) for four weeks. Extraction with carbon disulfide. RSD) SAOHLIW TVOILATVYNY 9 WHO40HOTHO Lee Table 6-2. Analytical Methods for Determining Chloroform in Environmental Samples (continued) Sample matrix Preparation method Analytical method Sample detection limit Percent recovery Reference Air (phosgene) Air (phosgene) Air (phosgene) Air, water Drinking water Drinking water Drinking water Phosgene collection in an impinger containing a solution of aniline (forming carbanilide); evaporation of solvent; dissolution of residue in acetonitrile Phosgene collection onto Chromosorb coated with 1-(2-pyridyl)-piperazine; elution of derivative with acetonitrile Passage of air through a tube filled with magnesium perchlorate (for water removal); cryogenic trapping Solid phase microextraction (from air, water, or headspace over water) Direct injection or purge-and- trap pre-concentration Purge-and-trap pre- concentration; thermal desorption Direct injection HPLC/UV (EPA Method TOB) HPLC/UV GC/ECD GC/ECD GC/ECD, GC/Hall GC/MS GC/MS 0.1 ppb (v/v) 0.005 ppm (w/v) 7 ppt (V/V) 0.9 ppb (v/v) gas phase; 30 ng/L (30 ppt, w/v) liquid phase 1 pg/L (1 ppb, wiv direct); 0.1 pg/L (0.1 ppb, w/v with purge-and-trap) 0.1 pg/L (0.1 ppb, w/v) 0.1 pg/L (0.1 ppb, w/v) 100 at 3 ppb (v/v); 15% RSD 100 over range 0.02-1 ppm (w/v) No data No data 103-126 at 35-70 pg/L (direct); 91-106 at 35-70 pg/L (purge- and-trap) No data No data EPA 1988n Rando et al. 1993 Bachmann and Polzer 1989 Chai et al. 1993 Nicholson et al. 1977 Coleman et al. 1976 Fujii 1977 SAOHL3W TVOILATVYNY 9 WHO40HO THO [444 Table 6-2. Analytical Methods for Determining Chloroform in Environmental Samples (continued) Sample Analytical detection Sample matrix Preparation method method limit Percent recovery Reference Seawater and ~~ Solvent extraction with pentane; GC/ECD 80 ng/L (0.08 ppb, No data Bureau International freshwater extract dried with sodium w/v) Technique des sulfate Solvants Chlores 1976 Water Permeation through a silicon GC/FID 74.0 pg/L (74 ppb, No data Blanchard and Hardy polycarbonate membrane into w/v) 1986 an inert gas stream and into GC port Drinking water Acidification and dechlorination; GC/ECD or 0.002 pg/L 100 (13% RSD) at EPA 1990g extraction with methyl-t-butyl GC/MS (EPA (0.002 ppm, w/v) 0.005 pg/L ether; direct injection of extract ~~ Method 551) Drinking Purge-and-trap pre- GC/MS < 0.1 pg/L (reagent 105 (3% RSD) Greenberg et al. 1992 water, waste concentration; thermal (Standard water) using narrow narrow bore column water desorption Method 6210) bore capillary column Tap water Solvent extraction HRGC/ECD No data No data Kroneld 1986 with HRGC/MS confirmation Water Solvent extraction HRGC/ECD 1 pg/L (ppb, wiv) 100.3 at 50 pg/L Reunanen and Kroneld 1982 Drinking water ~~ Purge-and-trap pre- GC with PID 0.02 pg/L 98 (2.5% RSD) Ho 1989 concentration onto Tenax/ and Hall in silica/charcoal; thermal series desorption Finished Purge-and-trap pre- GC/Hall (EPA No data 0.90C+3.44 where EPA 1991a drinking/raw concentration onto Tenax/ method 502.1) C = true source water silica/charcoal; thermal desorption concentration (pg/L) SAOHL3NW TVOILATVYNY 9 WHO40HOTHO £22 Table 6-2. Analytical Methods for Determining Chloroform in Environmental Samples (continued) Sample Analytical detection Sample matrix Preparation method method limit Percent recovery Reference Finished Purge-and-trap pre- GC with PID 0.10 pg/L (0.1 ppb, 98 (2% RSD) EPA 1991b drinking/raw concentration onto and HSD in w/v) source water Tenax/silica/charcoal; thermal series (EPA desorption method 502.2) Finished Purge-and-trap pre- Subambient 0.2 pg/L (0.2 ppb, 103 at 1.0 pg/L EPA 1991c drinking/raw concentration onto Tenax/ programmable ~~ w/v) source water silica/charcoal; thermal HRGC/MS desorption (EPA method 524.1) Finished Purge-and-trap pre- Cryofocusing 0.03 pg/L (0.03 ppb, 90 (6.1% RSD) at EPA 1992a drinking/raw concentration onto Tenax/ (wide or narrow w/v) with wide bore 0.5-10 pg/L (wide source water silica/charcoal; thermal bore); column; 0.02 pg/L bore); 95 (3.2% RSD) desorption HRGC/MS (0.02 ppb, w/v) with at 0.1 pg/L with (EPA method narrow bore column narrow bore column 524.2) Groundwater, Direct injection of head-space GC/HSD (EPA 0.5 pg/L (ppb, w/v) Water: 0.93C -0.39 EPA 1986a liquid, and gas (EPA method 5020) or method 8010) for groundwater, where C = true solid matrices ~~ purge-and-trap pre- 0.5 pg/g (ppm, w/w) concentration in concentration and thermal for low-level soil, pg/L. desorption (EPA method 5030) 500 pg/L (ppb, w/v) for water-miscible liquid waste, 1,250 pg/g (ppm, w/w) for soil, sludge, and non-water- miscible waste Waste water Purge-and-trap pre- GC/HSD or MS 0.05 pg/L (for HSD); 102 at 0.44-50 ug/L EPA 19982a concentration; thermal desorption (EPA methods 601 and 624) 1.6 pg/L (for MS) (for HSD): 101 at 10-100 pg/L (for MS) SAOHL3IW TVOILATVYNY 9 WHO40HO THO vee Table 6-2. Analytical Methods for Determining Chloroform in Environmental Samples (continued) Sample Analytical detection Sample matrix Preparation method method limit Percent recovery Reference Solid and Dispersion in glycol; purge-and- GC/ECD and No data 105 at 5 pg/L Lopez-Avila et al. liquid waste, trap pre-concentration onto FID in series 1987 soil Tenax/ silica/charcoal; thermal desorption Sediment Extraction into methanol; GC/FID/ECD 0.1 ug/g (0.1 ppm, 99 (4% RSD) at Amaral et al. 1994 dilution with water; purge-and- w/w) 0.8 pg/mL (0.8 ppm, trap pre-concentration w/v) Bulk oils Purge-and-trap pre- GC/MS 1 ppb (w/v) 88 (6.7% RSD) at Thompson 1994 concentration (with deuterated 93 ppb internal standards) onto Tenax; thermal desorption Food Extraction of composited, table- GC-ECD/Hall 5 ng/g (ECD); 5 ng/g 15-161 Daft 1988a, 1989 ready food with isooctane or (Hall) acetone-isooctane; micro- Florisil clean-up (if fat content 21-70%); direct injection into GC Volatile food Direct injection of head-space GC/ECD or MS 4.2 ng/kg (4.2 ppb, No data Entz et al. 1982 components gas w/w in beverages); No data 12.5 pg/kg (12.5 ppb, w/w in No data dairy products); No data 18 pg/kg (meats); 28 pg/kg (fats/oils) ECD = electron capture detector; EPA = Environmental Protection Agency; FID = flame ionization detector; GC = gas chromatography; Hall = Hall electrolytic conductivity detector; HPLC/UV = high performance liquid chromatography with ultraviolet absorbance detection; HRGC = high-resolution gas chromatography; HSD = halogen-specific electrolytic conductivity detector; MS = mass spectrometry; NIOSH = National Institute for Occupational Safety and Health; OSHA = Occupational Safety and Health Administration; PID = photo-ionization detector; RSD = relative standard deviation; v/v = volume/volume; w/v = weight/volume SAOHL3IN TVOILATVYNY 9 WHO40HOTHO Gace CHLOROFORM 226 6. ANALYTICAL METHODS very easy and fast (90 seconds for chloroform) with this technique although sample collection conditions and thermal desorption conditions must be carefully controlled for the best precision. Samples containing very volatile analytes, like chloroform, must be analyzed quickly after sample collection to avoid analyte loss with storage. Phosgene has been identified as an atmospheric decomposition product of several chlorinated compounds, including chloroform (Bichmann and Polzer 1989). Phosgene can be determined in air samples using, for example, capillary GC with electron capture detection (ECD) (Bichmann and Polzer 1989), high-performance liquid chromatography (HPLC) after the conversion of phosgene to carbanilide (EPA 1988n), and HPLC after the reaction of phosgene with 1-(2-pyridyl)-piperazine (Rando et al. 1993). Chloroformates can interfere in the carbanilide analysis (EPA 1988n). In the method of Rando et al. (1993), the apparent recovery of phosgene was nearly quantitative from air at up to 25% relative humidity; this decreased to about 65% at 95% humidity. The most common method for the determination of chloroform levels in water, sediment, soil, and foods is the purging of the vapor from the sample, or its suspension in a solvent with an inert gas and trapping (purge-and-trap) the desorbed vapors onto a sorbent trap (EPA 1991a, 1991b, 1991c, 1992; Greenberg et al. 1992; Ho 1989; Lopez-Avila et al. 1987). SPME is a method that combines the ease of headspace analysis with some of the concentration benefits of purge-and-trap (Chai et al. 1993). Subsequent thermal desorption is used for the quantification of chloroform concentrations. Solvent extraction is also used in a number of methods (Amaral et al. 1994; Daft 1988a, 1989; EPA 1990g; Kroneld 1986; Reunanen and Kroneld 1982). No methods were found for phosgene in water, sediment, soil, and foods. All of the methods listed above for the analysis of environmental samples use GC with various detection methods. The two methods that provide the lowest detection limits are halide-specific detectors (e.g., Hall electrolytic conductivity detector or electron capture detector) and the mass spectrometer (EPA 1986a; Ho 1989; Lopez-Avila et al. 1987; Ramus et al. 1984). The advantage of halide specific detectors is they are not only very sensitive, but are also specific for halide compounds. The mass spectrometer, on the other hand, provides additional confirmation of the presence of a compound through its ionization pattern and is desirable when a variety of compounds are required to be identified and quantified. The disadvantage of halide-specific detectors is their inability to detect and quantify nonhalogen compounds, if nonhalogenated compounds are of interest also; this can be CHLOROFORM 227 6. ANALYTICAL METHODS greatly overcome by using other detectors (e.g., photoionization detector) in series (Lopez-Avila et al. 1987). High-resolution gas chromatography (HRGC) with capillary columns coupled with mass spectrometry (MS) provides better resolution and increased sensitivity for volatile compounds than packed columns. In methods such as EPA method TO14 (EPA 1988i), desorbed compounds are cryogenically trapped onto the head of the capillary column. Such HRGC/MS methods overcome some common problems involved in analyses of excessively complex samples, samples with large ranges of concentrations, and samples that also contain high-boiling compounds (Dreisch and Munson 1983; EPA 1986a). LODs in the sub-parts per billion (ppb) range are routinely possible in both air and liquid/solid matrices. Numerous standard methods exist. Methods for rapid sample introduction to an ion trap mass spectrometer have been developed for the determination of organic compounds, including chloroform, in aqueous samples. In the method of Bauer and Solyom (1994) a polymeric membrane is placed into contact with the sample, the organics dissolve in the membrane and diffuse to the other side where they are swept directly into the mass spectrometer. This technique, known as membrane introduction mass spectrometry (MIMS) was shown to be sensitive to chloroform in water to 0.025 ppb. Another sample introduction technique known as inertial spray extraction nebulizes an aqueous sample (water, blood) into a small chamber where a countercurrent stream of helium sweeps any released volatile organic compounds (VOCs) into a jet separator at the inlet of an ion trap mass spectrometer (St-Germain et al. 1995). Up to 1 mL of sample can be introduced and the released VOCs are detected by the MS. Although both of these techniques provide for high throughput, no separation step is employed and this can result in interferences from ions formed by compounds other than the target analytes. These methods have great utility in selected applications. A fiber-optic device has been described that can monitor chlorinated hydrocarbons in water (Gobel et al. 1994). The sensor is based on the diffusion of chlorinated hydrocarbons into a polymeric layer surrounding a silver halide optical fiber through which is passed broad-band mid-infrared radiation. The chlorinated compounds concentrated in the polymer absorb some of the radiation that escapes the fiber (evanescent wave); this technique is a variant of attenuated total reflection (ATR) spectroscopy. A LOD for chloroform was stated to be 5 mg/L (5 ppm). This sensor does not have a high degree of selectivity for chloroform over other chlorinated aliphatic hydrocarbons, but appears to be useful for continuous monitoring purposes. CHLOROFORM 228 6. ANALYTICAL METHODS The reproducibilities of the methods listed in Table 6-2 are generally acceptable, but will vary, depending on the laboratories doing the analyses. Probable interferences for the methods of analysis include contamination from chloroform vapors in the laboratory. For this reason, it is often recommended that the laboratories doing the analysis should not contain chloroform or any other solvent to be measured in the sample (EPA 1986a). Plastic or rubber system components should be avoided as they can contaminate a sample or result in carryover from one analysis to the next (EPA 1992). The formation of aerosols and foam during purge-and-trap of liquid samples can contaminate the analytical system, so precautions must be taken (Thompson 1994; Vallejo-Cordoba and Nakai 1993). The use of field blanks is extremely important to correct for chloroform that might have diffused into the sample during shipping and storage (EPA 1986a). Other interferences include those volatile compounds that have similar retention times in the various GC columns used. This problem is often eliminated by analyzing the samples with two different types of GC columns such that the retention times will not be coincidental in both columns. Mass spectrometric detection can also help to overcome interferences resulting from incomplete chromatographic resolution. Refer to the references cited in Table 6-2 and the text for specific information regarding reproducibility and potential interferences. 6.3 ADEQUACY OF THE DATABASE Section 104(i)(5) of CERCLA directs the Administrator of ATSDR (in consultation with the Administrator of EPA and agencies and programs of the Public Health Service) to assess whether adequate information on the health effects of chloroform is available. Where adequate information is not available, ATSDR, in conjunction with NTP, is required to assure the initiation of a program of research designed to determine the health effects (and techniques for developing methods to determine such health effects) of chloroform. The following categories of possible data needs have been identified by a joint team of scientists from ATSDR, NTP, and EPA. They are defined as substance-specific informational needs that if met would reduce or eliminate the uncertainties of human health assessment. This definition should not be interpreted to mean that all data needs discussed in this section must be filled. In the future, the identified data needs will be evaluated and prioritized, and a substance-specific research agenda will be proposed. CHLOROFORM 229 6. ANALYTICAL METHODS 6.3.1 Identification of Data Needs Methods for Determining Biomarkers of Exposure and Effect. Methods are available for the determination of chloroform in breath (Aggazzotti et al. 1993; Jo et al. 1990; Krotoszynski et al. 1979; Phillips and Greenberg 1992; Pleil and Lindstrom 1995; Raymer et al. 1990), blood (Antoine et al. 1986; Ashley et al. 1992; Cammann and Hiibner 1995; EPA 1985a; Kroneld 1986; Peoples et al. 1979; Pfaffenberger et al. 1980; Reunanen and Kroneld 1982; Seto et al. 1993; Streete et al. 1992), and other fluids and tissues such as urine and adipose (Cammann and Hiibner 1995; EPA 1985a; Peoples et al. 1979; Pfaffenberger et al. 1980; Reunanen and Kroneld 1982; Streete et al. 1992). Sub-ppb limits of detection have been shown (e.g., Ashley et al. 1992; Pfaffenberger et al. 1980) and the methods are adequate for the determination of chloroform concentrations in samples from the general population. No biomarker that can be associated quantitatively with chloroform exposure has been identified (see Sections 2.5.1 and 6.1). Although chloroform levels can be determined in biological samples, the relationship between these levels and the exposure levels has not been adequately studied. In one study, the concentrations of chloroform in alveolar air of people attending activities at an indoor swimming pool were found to be proportional to the concentrations in air (Aggazzotti et al. 1993). Such proportionality was observed, in part, because the alveolar air samples were taken soon after exposure termination. Good correlations have also been measured between chloroform concentrations measured in blood and breath with those in air or water after exposure to water/air during showering (Jo et al. 1990) and swimming (Cammann and Hiibner 1995; Lévesque et al. 1994). The studies of Jo et al. (1990) and Lévesque et al. (1994) demonstrated and quantified the uptake of chloroform via dermal absorption. Correlations of alveolar air concentrations with exposure air concentrations based on breath samples at unknown postexposure times will be complicated by metabolism and other factors, such as activity, important in the elimination of chloroform from the body (see PBPK discussion, Chapter 2). Furthermore, the presence of chloroform, or a transformation product of chloroform such as phosgene and reaction products of phosgene, in a biological sample may have resulted from the metabolism of another chlorinated hydrocarbon. If a biomarker of exposure for this compound in a human tissue or fluid was available and a correlation between the level of the biomarker and exposure existed, it could be used as an indication of the extent of chloroform exposure. Further information regarding the accuracy of sample recovery for the methods of chloroform analysis would be useful in interpreting monitoring data. CHLOROFORM 230 6. ANALYTICAL METHODS No biomarker of effect that can be associated quantitatively and directly to chloroform exposure has been identified (see Section 2.5.2). If biomarkers of effect were available for this compound and a correlation between the level or intensity of the biomarker of effect and the exposure level existed, it could be used as an indication of the levels and extent of chloroform exposure. However, in cases where an exposure to chloroform has been known to occur, measurements of chloroform in breath or blood can indicate body burden. Methods for Determining Parent Compounds and Degradation Products in Environmental Media. Methods for determining chloroform in the environment are available. These include methods for drinking water (Blanchard and Hardy 1986; EPA 1990g, 1991a, 1991b, 1991¢, 1992; Greenberg et al. 1992; Ho 1989; Kroneld 1986; Nicholson et al. 1977), air (Bergerow et al. 1995, Chai and Pawliszyn 1995, EPA 1988f, 1988g, 1988h, 1988i; NIOSH 1994; OSHA 1979; Parsons and Mitzner 1975), and foods (Daft 1988a, 1989; Entz et al. 1982; Thompson 1994). These three media are of most concern for human exposure. The precision, accuracy, reliability, and specificity of the methods are well documented and well suited for the determination of low levels of chloroform and levels at which health effects occur. For example, the MRL for acute-duration inhalation is 0.1 ppm (weight per volume [w/v] or 0.0099 mg/m’) so any method used must have a limit of detection equal to or less than this. The methods of Chai et al. (1993), Chai and Pawliszyn (1995), and Bergerow et al. (1995) report limits of detection of 0.9 ppb volume per volume (v/v), 2 ppb, and 4 ng/m>, respectively, and are adequate for the measurement of chloroform in air. Although no limits of detection were reported for EPA methods TO2 and TO3 (EPA 1988f, 1988g), recoveries were acceptable for low-ppb concentrations of chloroform in air and thus these methods should certainly be applicable to concentrations at the acute-duration inhalation MRL. Similarly, the chronic-duration oral MRL is 0.01 mg/kg/day, which converts to 0.7 mg/day for a 70-kg person. For a 2 L/day water consumption, this translates into a required method limit of detection of 0.35 mg/L. This concentration is easily measured by the methods of EPA (1990g) (limit of detection 0.002 pg/L), Greenberg et al. (1992) (limit of detection 0.1 pg/L), and Chai et al. (1993) (limit of detection 30 ng/L). Assuming a food intake of 2 kg/day, this oral MRL translates to a needed method limit of detection in food of 0.35 mg/kg. The methods of Entz et al. (1982) and Daft (1988a, 1989) provide limits of detection of less than 0.028 mg/kg and are adequate. Method sensitivities are clearly adequate for matrices in which the higher acute-duration oral MRL is of concern. There is not much information regarding the degradation products of chloroform in the environment. Although phosgene can be produced in the environment and its high reactivity suggests that it would not persist, several CHLOROFORM 231 6. ANALYTICAL METHODS methods were found for the quantification of phosgene in ambient air (Bichmann and Polzer 1989: EPA 1988n; Rando et al. 1993). No methods were found for phosgene in other environmental matrices and it is not likely that it would be found in matrices other than air. 6.3.2 Ongoing Studies The Environmental Health Laboratory Sciences Division of the Center for Environmental Health and Injury Control, Centers for Disease Control and Prevention, is developing methods for the analysis of chloroform and other VOCs in blood. These methods use purge-and-trap methodology, HRGC, and magnetic sector mass spectrometry which gives detection limits in the low-ppt range (see Ashley et al. 1992). The following information was obtained form a search of Federal Research in Progress (FEDRIP, 1996). Researchers at Physical Sciences, Inc. are developing an imaging infrared spectrometer that can rapidly screen field sites to detect the presence of VOCs, including chloroform, from remote locations (either in the air or on the ground). The following research projects were identified as having objectives that might require the development or modification of methods to measure chloroform. Researchers at Colorado State University are studying the biodegradation of pollutants, including chloroform, in bioreactors. Researchers at the University of California at Berkeley and Riverside are conducting studies on the biodegradation of organic compounds, including chloroform, in soil. CHLOROFORM 233 7. REGULATIONS AND ADVISORIES The international, national, and state regulations and guidelines regarding chloroform in air, water and other media are summarized in Table 7-1. An MRL of 0.1 ppm has been derived for acute-duration inhalation exposure to chloroform. The MRL is based on a NOAEL of 3 ppm for hepatic effects in mice (Larson et al. 1994c). An MRL of 0.05 ppm has been derived for intermediate-duration inhalation exposure to chloroform. The MRL is based on a LOAEL of 14 ppm for toxic hepatitis in workers exposed to up to 400 ppm for less than 6 months (Phoon et al. 1983). An MRL of 0.02 ppm has been derived for chronic-duration inhalation exposure to chloroform. The MRL was based on a LOAEL of 2 ppm for hepatic effects in workers exposed to concentrations of chloroform ranging from 2 to 205 ppm for 1-4 years (Bomski et al. 1967). An MRL of 0.3 mg/kg/day has been derived for acute-duration oral exposure to chloroform. The MRL is based on a NOAEL of 26.4 mg/kg/day for hepatic effects in mice (Larson et al. 1994b). An MRL of 0.1 mg/kg/day has been derived for intermediate-duration oral exposure to chloroform. The MRL is based on a NOAEL of 15 mg/kg/day for liver effects (increased SGPT) in dogs exposed to chloroform in toothpaste for 26 weeks (Heywood et al. 1979). An MRL of 0.01 mg/kg/day has been derived for chronic-duration oral exposure to chloroform based on a LOAEL for liver effects (increased SGPT) in dogs administered 15 mg/kg/day chloroform in toothpaste in capsules for 7.5 years (Heywood et al. 1979). The chronic-duration oral reference dose (RfD) for chloroform is also 0.01 mg/kg/day, based on the LOAEL for liver effects in dogs administered 15 mg/kg/day chloroform (Heywood et al. 1979; IRIS 1996). No reference concentration (RfC) exists for the compound. CHLOROFORM 234 7. REGULATIONS AND ADVISORIES The EPA has determined that chloroform is a probable human carcinogen; corresponding group B2 (IRIS 1996). Gavage studies conducted by the NTP were positive for carcinogenicity in female mice and male mice and rats, but negative for female rats (NTP 1995). The International Agency for Research on Cancer (IARC) has determined that chloroform is possibly carcinogenic to humans; Group 2B classification (IARC 1987). Chloroform is regulated by the Clean Water Act Effluent Guidelines for the following industrial point sources: electroplating; organic chemicals; steam electric, asbestos, and timber products processing; paving and roofing; paint and formulating; formulating; gum, wood and carbon black; metal molding, casting, and finishing; coil coating; copper forming; and electrical and electronic components (EPA 1981a). CHLOROFORM 7. REGULATIONS AND ADVISORIES 235 Table 7-1. Regulations and Guidelines Applicable to Chloroform Agency Description Information References INTERNATIONAL Guidelines: IARC Carcinogenic classification Group 2B? IARC 1987 WHO Drinking water guidelines 30 ug/L WHO 1984 NATIONAL Regulations: a. Air: EPA OAQPS Intent to list under Section 112 of Clean Air Yes 40 CFR 61.01 Act EPA 1985d New Source Performance Standards Chemicals Produced at SOCMI Facilities Yes 40 CFR 60.489 EPA 1983b Chemical Affected by Standards for SOCMI Yes 40 CFR 60.667 Distillation Operations EPA 1990e National Emission Standards for Hazardous Air Pollutants Hazardous Air Pollutants Yes 40 CFR 61.01 EPA 1985d Proposed Rule: Identification of "More Yes 59 FR 15504 Hazardous" Emission Decreases 40 CFR 63.48 EPA 1994a Proposed Rule: National Emission Standards Yes 58 FR 62566 for Halogenated Solvent Clearing-Applicability 40 CFR 63.460 EPA 1993a OSHA PEL-TWA 50 ppm 29 CFR 1910 OSHA 1974 Permissible Exposure Limit (Ceiling) 240 mg/m? OSHA 1974 Occupational Exposure to Hazardous Yes OSHA 1974 Chemicals in Laboratories b. Water: EPA PQL 0.5 ug/L 40 CFR 264 and 270 EPA 1987a Effluent Guidelines and Standards List of Toxic Pollutants Yes 40 CFR 401.15 EPA 1979b List of Toxic Pollutants Subject to Yes 40 CFR 403, App. B Pretreatment Standards EPA 1981a Definition of Total Toxic Organics (TTO) for Yes 40 CFR 413.02 Electroplating Point Source Category EPA 1981b Bulk Organic Chemicals in Wastewater from Yes 40 CFR 414.70 Sources of Organic Chemicals, Plastics and Synthetic Fibers EPA 1987e CHLOROFORM 7. REGULATIONS AND ADVISORIES 236 Table 7-1. Regulations and Guidelines Applicable to Chloroform (continued) Agency Description Information References NATIONAL (cont.) Organic Chemicals, Plastics, and Synthetic 40 CFR 414.91 Fibers - Effluent Limitations for Direct EPA 1987e Discharge Point Sources Using End-of-Pipe Biological Treatment One-day maximum 46 ug/L Maximum for monthly average 21 ug/L Organic Chemicals, Plastics, and Synthetic 40 CFR 414.101 Fibers - Effluent Limitations for Direct EPA 1987e Discharge Point Sources That Do Not Use End-of-Pipe Biological Treatment One-day maximum 325 pg/L Maximum for monthly average 111 ug/l Priority Pollutants, Associated With Steam Yes 40 CFR 423, App. A Electric Power Generating Sources EPA 1982b Proposed Rule: Effluent Limitations for Pupl, 58 FR 66078 Paper, and Paperboard - Dissolving Kraft 40 CFR 430 (bleach plant effluent) EPA 1993d 1-Day Max. Monthly Avg. Existing BAT New Source Pretreatment 10.1 g/kkg 7.06 10.1 g/kkg g/kkg 7.07 g/kkg Bleached paper grade kraft and soda 5.06 g/kkg 2.01 (bleach plant effluent - existing BAT and g/kkg pretreatment for existing Dissolving sulfite - pretreatment for new 23.2 g/kkg 74.4 sources g/kkg Proposed Rule: Monitoring Requirements for Yes 59 FR 12567 Pulp, Paper, and Paperboard Category 40 CFR 430.02 EPA 1994c Definition of TTO for Metal Finishing Sources Yes 40 CFR 433.11 EPA 1983d Definition of TTO for Metal Molding and Yes 40 CFR 464.21 Casting Sources EPA 1985e Definition of TTO for Ferrous Casting Sources 40 CFR 464.31 EPA 1985e Definition of TTO for Coil Coating Sources Yes 40 CFR 465.02 EPA 1982c EPA ODW National Primary Drinking Water Regulations Definition of Trihalomethanes (THM) Yes 40 CFR 141.2 EPA 1975 Maximum Contaminant Levels for THM 0.10 mg/L 40 CFR 141.12 EPA 1991f Method of Analysis for THM Yes 40 CFR 141, App. C EPA 1979a CHLOROFORM 7. REGULATIONS AND ADVISORIES 237 Table 7-1. Regulations and Guidelines Applicable to Chloroform (continued) Agency Description Information References NATIONAL (cont.) Monitoring for Inorganic and Organic Yes 40 CFR 141.40 Chemicals EPA 1987d Proposed Rule: Total Trihalomethane Yes 58 FR 65622 Sampling and Analysis 40 CFR 141.30 EPA 1993c Proposed Rule: Maximum Contaminant Levels Yes 59 FR 38668 for Total THM 40 CFR 141.12 EPA 1994b Proposed Rule: Total THM Sampling, Yes 59 FR 38668 Analytical and Other Requirements 40 CFR 141.30 EPA 1994b Proposed Rule: Best Available Technology for ~~ Yes 59 FR 38668 Achieving Compliance with MCLs for Total 40 CFR 141.64 THM EPA 1994b Proposed Rule: Disinfection By-Product Yes 59 FR 38668 Compliance Monitoring for Total THM 40 CFR 141.133 EPA 1994b EPA OW Designation of Hazardous Substances Yes 40 CFR 116.4 EPA 1978 Reportable Quantities for Hazardous 10 pounds 40 CFR 117.3 Substances EPA 1986e National Pollutants Charge Elimination Yes 40 CFR 122, App. D System: Permit Application Testing EPA 1983c Requirements Proposed Rule: Application of Part 132 Yes 58 FR 20802 Requirements to Great Lake States and Tribes 40 CFR 132.6 - Pollutants of Initial Focus EPA 1993b Test Procedures for the Analysis of Pollutants Yes 40 CFR 136.3 EPA 1973 Methods of Analysis of Municipal and Yes 40 CFR 136, App. A Industrial Wastewater EPA 1973 c. Food: FDA Substances used only as components of Yes 21 CFR 175.105 adhesives FDA 1977 d. Other: EPA OERR RQ (Ruled) 10 pounds 54 FR 155 EPA 1989c Threshold planning quantity 10,000 pounds EPA 1987b Constituents for Solid Waste Detection Monitoring Hazardous Constituents Subject to Regulatory Requirements Maximum Concentration Contaminants for the Toxicity Characteristic Yes Yes Yes 40 CFR 258, App. | EPA 1991g 40 CFR 258, App. II EPA 1991g 40 CFR 261.24 EPA 1990d CHLOROFORM 7. REGULATIONS AND ADVISORIES 238 Table 7-1. Regulations and Guidelines Applicable to Chloroform (continued) Agency Description Information References NATIONAL (cont.) Discarded commercial chemical products, off- Yes 40 CFR 261.33 specification species, container residues, and EPA 1990d spill residues Basis for Listing Hazardous Waste Yes 40 CFR 261, App. VII EPA 1981c Proposed Rule: Basis Listing Hazardous Yes 59 FR 9808 Waste 40 CFR 261, App. VII EPA 1994d Hazardous Constituents Yes 40 CFR 261, App. VIII EPA 1988) Groundwater Monitoring List for Hazardous Yes 40 CFR 264, App. IX Waste TSDF EPA 1991d Health Based Limits for Exclusion of Waste- 6x102 40 CFR 266, App. VII Derived Residues EPA 1991d Potential PICs For Determination of Exclusion Yes 40 CFR 266, App. VIII of Waste-Derived Residues EPA 1991d EPA Land Disposal Restrictions Identification of Waste to Be Evaluated by Yes 40 CFR 268.10 August 8, 1988 EPA 1986f Constituent Concentrations Which May Not be 40 CFR 268.43 Exceeded for Land Disposal EPA 1988k Waste Code: Waste Non- water waste water F025 0.046 mg/L 6.2 mg/kg F039, K117, U044 0.046 mg/L 5.6 mg/kg Halogenated organic compounds regulated Yes 40 CFR 268, App. Ill under 40 CFR 268.32 EPA 1987g Proposed Rule: Treatment Standards for Land 58 FR 48092 Disposed Waste 40 CFR 268.40 & 268.48 EPA 1993e Waste Code: Waste water ~~ Non- waste water D022, K019, K029, K150, K151, universal 0.046 mg/L 6/9 mg/kg treatment standard F025, KO73 0.046 mg/L 6.2 mg/kg F038, K117, K118, K136, U044 0.046 mg/L 5.6 mg/kg K009, K010 0.1 mg/L 6.0 mg/kg CHLOROFORM 7. REGULATIONS AND ADVISORIES 239 Table 7-1. Regulations and Guidelines Applicable to Chloroform (continued) Agency Description Information References NATIONAL (cont.) List of Hazardous Substances and Reportable 10 pounds 40 CFR 302.4 Quantities EPA 1989d Proposed Rule: Reportable Quantity Yes 58 FR 54836 Adjustments - Designation of Hazardous 40 CFR 302.4 Waste EPA 1993f Guidelines: a. Air: ACGIH NIOSH b. Water: EPA EPA ODW NAS Extremely Hazardous Substances and Threshold Planning Quantities Chemicals Subject to Toxic Chemical Release Reporting: Community Right-to-Know Chemicals Subject to the Health and Safety Data Reporting Requirements Under TSCA TLV-TWA Threshold Limit Value for Occupational Exposure STEL (60 minutes) Recommended Exposure Limit for Occupational Exposure (STEL-60 min) Immediately Dangerous to Life & Health 1-d Health Advisory 10-d Health Advisory (child & adult) Longer-term Health Advisory (child & adult) Maximum Contaminant Level Chloroform Total THM (community and non-transcient, non-community water systems) Total THM (serve more than 10,000 people) Maximum Contaminant Level Goal qt cancer slope factor (oral exposure) RfD (oral) Individual lifetime cancer risk 107° Suggested No-Adverse-Response Level 24-hour 7-day 10,000 pounds Yes Yes 10 ppm (49 mg/m3) 49 mg/m? 2 ppm (9.78 mg/m3) 9.78 mg/m3 1,000 ppm 4 mg/L (child) 4 mg/L (child) 0.1 mg/L (child 0.4 mg/L (adult) 0.1 mg/L 0.080 mg/L 0.040 mg/L zero 6.1x10°3 per mg/(kg/day) 1.102 mg/kg/day) (UF 1,000) 60 ug/L 22 mg/L 3.2 mg/L 40 CFR 355, App. A EPA 1987h 40 CFR 372.65 EPA 1988| 40 CFR 716.120 EPA 1988m ACGIH 1992 ACGIH 1994 NIOSH 1990 NIOSH 1992 NIOSH 1990 EPA 1994e EPA 1994e EPA 1994e EPA 1994e 59 FR 38668 40 CFR 141.64 EPA 1994b 59 FR 38668 40 CFR 141.53 EPA 1994b IRIS 1996 NAS 1980 CHLOROFORM 7. REGULATIONS AND ADVISORIES 240 Table 7-1. Regulations and Guidelines Applicable to Chloroform (continued) Agency Description Information References NATIONAL (cont.) c. Non-specific media EPA q4" (oral) 6.1103 (mg/kg/day)! IRIS 1996 gy" (inhalation) 8.1102 (mg/kg/day)! (2.3x103 (g/m) RfD (chronic oral) 0.01 mg/kg/day Cancer classification B2P d. Other EPA Cancer classification B2 NIOSH Cancer classification Yes NIOSH 1990 NIOSH 1992 Regulations and Guidelines: a. Air: AZ Acceptable ambient air concentrations 60 g/m’ NATICH 1992 1 hour 16 ug/m> 24 hours 0.043 ug/m® 1 year CT 8 hours 250 g/m FL (Ft. 8 hours 0.5 mg/m3 Lauderdale) FL (Pinellas 8 hours 97.8 ug/m® Co.) 24 hours 23.5 ug/im3 1 year 0.043 g/m IN 8 hours 48.9 ug/m> Annual 0.043 ug/m> IN (Innap) 8 hours 1200 g/m’ KS 1 year 0.0435 ug/m3 KS-KC Annual 0.0435 ug/m® State of Kentucky 1986 LA Annual 4.3 ug/m® NATICH 1992 MA 24 hours 133 pg/m® 1 year 0.04 pg/m® ME 1 year 0.043 ug/m® MI 1 year 0.40 ug/m® NC 1 year 0.0043 mg/m® NC 1 year 0.0043 mg/m? ND BACT NV 8 hours 1.19 mg/m> NY 1 year 167 ug/md OK 24 hours 97 g/m’ CHLOROFORM 7. REGULATIONS AND ADVISORIES 241 Table 7-1. Regulations and Guidelines Applicable to Chloroform (continued) Agency Description Information References STATE (cont.) PA (Phil.) 1 year 120 ug/m3 RI 1 year 0.04 1g/m3 8G 24 hours 250 ug/md TX 30 minutes 98 ug/m3 1 year 10 pg/m3 VA 24 hours 490 ug/m3 VT 1 year 0.043 g/m3 WA/SWEST 1 year 0.043 g/m? b. Water: Water Quality: Human Health FSTRAC 1990 AZ Drinking water quality standards 0.49 ng/L Domestic/ Drinking 5.7pg/L Sittig 1994 FL Domestic/ Drinking 100 pg/L IL 1 ug/L CELDs 1994 MA 5 ug/L Mi Domestic/ Drinking 5.6 pg/L Sittig 1994 MN 57 ug/L CELDS 1994 NJ Domestic/ Drinking 6 pg/L Sittig 1994 NY Domestic/ Drinking 0.2-100pg/L OR Domestic /Drinking 10 pg/L RI Drinking water standard 6-100 pg/L CELDS 1994 TN Domestic/ Drinking 100 ng/L Sittig 1994 TX Domestic/ Drinking 100 ng/L VT Domestic/ Drinking 6pg/L Water Quality: Human Health CELDs 1994 AZ Domestic Water Source (DWS) TTHM Fish Consumption (FC) 590 pg/L Full Body Contact (FBC) 230 ug/L Partial Body Contact (PBC) 1400 ug/L CO MCL 0.10 mg/L CT MCL 2.0 ug/L DC Water Quality Criteria Cc 3,000 mg/L D 0.2 mg/L FL MCL 0.10 mg/L CHLOROFORM 242 7. REGULATIONS AND ADVISORIES Table 7-1. Regulations and Guidelines Applicable to Chloroform (continued) Agency Description Information References STATE (cont.) all Maximum organic contaminant level: Total trihalomethane (TTHM) (TTHM = the sum of the concentration of bromodichloromethane, dibromochloromethane and chloroform) Acute Chronic Fresh water 9,600 ng/L ns Salt water ns ns Fish consumption 5.1 IN Outside of mixing zone 157 ug/L Point of water intake 1.9 ug/L MCL (TTHM) 0.10 mg/L KY Consumption of fish 15.7 ug/L MCL 0.19 pg/L KS MCL (TTHM) 0.10 mg/L LA Drinking water 5.30 ug/L Non-drinking water 70.00 ug/L ME Maximum level (TTHM) 0.10 mg/L NH MCL (TTHM) 0.10 mg/L NM Groundwater levels 0.1 mg/L NY Groundwater effluent standard (max.) 7 ug/L ND MCL (TTHM) 0.10 mg/L NC MCL (TTHM) 0.10 mg/L Water quality standard for class GS 0.00019 mg/L Groundwater OG Groundwater quality reference level (TTHM) 0.100 mg/L MCL (TTHM) 0.10 mg/L OK Groundwater water quailty criteria 10.0 (no units specified) MCL (TTHM) 0.10 mg/L OH MCL (TTHM) 0.10 mg/L SC MCL (TTHM) 0.10 mg/L VT Class A or B waters 0.19 mg/L Class C water 15.7 mg/L WV MCL (TTHM) 0.10 mg/L WI Human cancer criteria: Public Water Supplier Warm water sport fish communities 1.9 mg/L Cold water communities 1.8 mg/L Great Lakes communities 1.8 mg/L Non-Public Water Supplies Warm water sport fish communities 87 mg/L Cold water communities 31 mg/L Warm water forage and limited fish 380 mg/L communities and limited aquatic life Groundwater Enforcement standard Prevention action limit 6 ug/L CHLOROFORM 243 7. REGULATIONS AND ADVISORIES Table 7-1. Regulations and Guidelines Applicable to Chloroform (continued) Agency Description Information References STATE (cont.) WV All water use categories, maximum criteria B1, B2, B3 waters 15.7 ug/L A 0.19 pg/L Water Quality: Aquatic Life CELDs 1994 AZ Acute Criteria for Aquatic and Wildlife Uses Acute Chronic Cold water fishery (A/W) 14,000 ug/L 900 ng/L Warm water fishery (A/W) 14,000 ng/L 900 ug/L Effluent dominated water (A/W) 14,000 ng/L 900 ug/L Ephemeral (A/W) NNS NNS CO Aquatic Life Segments Acute 28,900 ng/L Chronic 1,240 pg/L FL MCL for mixing zone pollutants 1.57 mg/L LA Acute Criteria Freshwater 2,890 ug/L-acute 1,445 pg/L-chronic Marine water 8,150 ng/L-acute 4,075 ng/L-chronic OH Outside mixing zone: maximum 1,800 ng/L Inside mixing zone: maximum 3,600 ng/L Cold water mixing zone: 30-day average 79 ug/L Groundwater Monitoring CELDs AL Yes CA Yes CO Yes IL Yes KY Yes LA Yes MN Yes MT Yes NY Yes OH Yes SC Yes TN Yes VA Yes WI Yes wv Yes CHLOROFORM Table 7-1. Regulations and Guidelines Applicable to Chloroform (continued) 7. REGULATIONS AND ADVISORIES 244 Agency Description Information References STATE (cont.) Hazardous Constituents CELDs AL Maxixmum Concentration of Contaminants for 60 mg/L the Toxicity Characteristics CA Concentration in waste; non-RCRA solvent 2.0 mg/kg waste Maximum concentration for the toxicity 6.0 mg/L characteristics CO Maximum concentration for the toxicity 6.0 mg/L characteristics IL Maximum concentration for the toxicity 6.0 mg/L characteristics KY Maximum concentration for the toxicity 6.0 mg/L characteristics LA Maximum concentration for the toxicity 6.0 mg/L characteristics MA Maximum concentration for the toxicity 6.0 mg/L characteristics MD Maximum concentration for the toxicity 6.0 mg/L characteristics ME Maximum concentration for the toxicity Yes characteristics MN Maximum concentration for the toxicity 6.0 mg/L characteristics MT Maximum concentration for the toxicity 6.0 mg/L characteristics ND Maximum concentration for the toxicity 6.0 mg/L characteristics NH Maximum concentration for the toxicity Yes characteristics NJ Maximum concentration for the toxicity 6.0 mg/L characteristics NY Maximum concentration for the toxicity Yes characteristics PA Maximum concentration for the toxicity 6.0 mg/L characteristics OH Maximum concentration for the toxicity 6.0 mg/L characteristics SC Maximum concentration for the toxicity 6.0 mg/L characteristics TX Maximum leachable concentration 6.0 mg/L VA Maximum concentration for the toxicity 6.0 mg/L characteristics CHLOROFORM 245 7. REGULATIONS AND ADVISORIES Table 7-1. Regulations and Guidelines Applicable to Chloroform (continued) Agency Description Information References STATE (cont.) VT Maximum concentration for the toxicity 6.0 mg/L characteristics WI Maximum concentration for the toxicity 6.0 mg/L characteristics WV Maximum concentration for the toxicity Yes characteristics wy Maximum concentration for the toxicity 6.0 mg/L characteristics 8Group 2B: Possible human carcinogen bGroup B2: Probable human carcinogen ACGIH = American Conference of Governmental Industrial Hygienists; AW = Aquatic and Wildlife; BAT = Best Available Technology Economically Achievable; CELDs = Computer-aided Environmental Legislative Database; CFR = Code of Federal Regulations; EPA = Environmental Protection Agency; FR = Federal Register; FSTRAC = Federal State Toxicology and Regulatory Alliance Committee; IARC = International Agency for Research on Cancer; NATICH = National Air Toxics Information Clearinghouse; NIOSH = National Institute for Occupational Safety and Health; ns = No Standard Has Been Developed; NNS = No Numerical Standard; OAQPS = Office of Air Quality Planning and Standards; ODW = Office of Drinking Water; OERR = Office of Emergency and Remedial Response; OSHA = Occupational Safety and Health Administration; OW = Office of Water; PEL = Permissible Exposure Limit; PICs = Product of Incomplete Combustion; PQL = Permissible Quantity Limit; RfD = Reference Dose; RQ = Reportable Quantity; SOCMI = Synthetic Organic Chemical Manufacturing Industry; STEL = Short-Term Exposure Limit; THM = Trihalomethanes; TLV = Threshold Limit Value; TSCA = Toxic Substance Control Act; TSDF = Treatment, Storage and Disposal Facility; TTO = Total Toxic Organics; TWA = Time-Weighted Average for 8-hour exposure; WHO=World Health Organization CHLOROFORM 247 8. 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Ecotoxicol Environ Safety 9:233-240. *Tumasonis CF, McMartin DN, Bush B. 1987. Toxicity of chloroform and bromodichloromethane when administered over a lifetime in rats. J Environ Pathol Toxicol Oncol 7:55-64. CHLOROFORM 287 8. REFERENCES *Uchrin CG, Mangels G. 1986. Chloroform sorption to New Jersey coastal plain groundwater aquifer solids. Environ Toxicol Chem 5:339-343. Uehleke H, Werner T. 1975. A comparative study on the irreversible binding of labeled halothane, trichlorofluoromethane, chloroform, and carbon tetrachloride to hepatic protein and lipids in vitro and in vivo. Arch Toxicol 34:289-308. *Uehleke H, Werner T, Greim H, et al. 1977. Metabolic activation of haloalkanes and tests in vitro for mutagenicity. Xenobiotica 7:393-400. *USITC. 1989. Synthetic organic chemicals, United States production and sales, 1988. USITC publication no 2219. Washington, DC: U.S. International Trade Commission. Utidijan HMD. 1976. Criteria Documents. 1. Recommendations for a chloroform standard. J Occup Med 18:253-257. *Vallejo-Cordoba B, Nakai S. 1993. Using a simultaneous factor optimization approach for detection of volatiles in milk by dynamic headspace gas chromatographic analysis. J Agric Food Chem 41(12):2378-2384. *Valzelli L, Kozak W, Skorupska M. 1988. Effect of some anesthetics on memory and exploration. Methods and Findings in Experimental and Clinical Pharmacology 10(4):239-242. *Van Abbe NJ et al. 1982. Bacterial mutagenicity studies on chloroform in vitro. Food Chem Toxicol 20:557-561. *van Beelen P, van Vlaardingen PLA, Fleuren-Kemila AK. 1994. Toxic effects of pollutants on the mineralization of chloroform in river sediments. Ecotoxicol Environ Safety 27(2):158-167. *Van Dyke RA, Chenoweth MB, Poznak AV. 1964. Metabolism of volatile anesthetics - I. Conversion in vivo of several anesthetics to 14CO2 and chloride. Biochem Pharmacol 13:1239-1247. *van Vlaardigen PLA, van Beelen P. 1992. 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REFERENCES Yang RSH, Rauckman EJ. 1987. Toxicological studies of chemical mixtures of environmental concern at the National Toxicology Program: Health effects of groundwater contaminants. Toxicology 47:15-34. *Young TB, Kanarek MS, Tsiatis AA. 1981. Epidemiologic study of drinking water chlorination and Wisconsin female cancer mortality. J Natl Cancer Inst 67:1191-1198. *Zepp RG, Braun AM, Hoigne J, et al. 1987. Photoproduction of hydrated electrons from natural organic solutes in aquatic environments. Environ Sci Technol 21:485-490. *Zierler S, Feingold L, Danley RA, et al. 1988. Bladder cancer in Massachusetts related to chlorinated chloraminated drinking water: A case control study. Archives of Environmental Health 43(2):195-200. Zogorski JS. 1984. Experience in monitoring domestic water sources and process waters for trace organics. J Environ Sci Health A19:233-249. CHLOROFORM 291 9. GLOSSARY Acute Exposure—Exposure to a chemical for a duration of 14 days or less, as specified in the Toxicological Profiles. Adsorption Coefficient (K .)—The ratio of the amount of a chemical adsorbed per unit weight of organic carbon in the soil or sediment to the concentration of the chemical in solution at equilibrium. Adsorption Ratio (Kd)—The amount of a chemical adsorbed by a sediment or soil (i.e., the solid phase) divided by the amount of chemical in the solution phase, which is in equilibrium with the solid phase, at a fixed solid/solution ratio. It is generally expressed in micrograms of chemical sorbed per gram of soil or sediment. Bioconcentration Factor (BCF)—The quotient of the concentration of a chemical in aquatic organisms at a specific time or during a discrete time period of exposure divided by the concentration in the surrounding water at the same time or during the same period. Cancer Effect Level (CEL)—The lowest dose of chemical in a study, or group of studies, that produces significant increases in the incidence of cancer (or tumors) between the exposed population and its appropriate control. Carcinogen—A chemical capable of inducing cancer. Ceiling Value—A concentration of a substance that should not be exceeded, even instantaneously. Chronic Exposure—Exposure to a chemical for 365 days or more, as specified in the Toxicological Profiles. Developmental Toxicity—The occurrence of adverse effects on the developing organism that may result from exposure to a chemical prior to conception (either parent), during prenatal development, or postnatally to the time of sexual maturation. Adverse developmental effects may be detected at any point in the life span of the organism. Embryotoxicity and Fetotoxicity—Any toxic effect on the conceptus as a result of prenatal exposure to a chemical; the distinguishing feature between the two terms is the stage of development during which the insult occurred. The terms, as used here, include malformations and variations, altered growth, and in utero death. EPA Health Advisory—An estimate of acceptable drinking water levels for a chemical substance based on health effects information. A health advisory is not a legally enforceable federal standard, but serves as technical guidance to assist federal, state, and local officials. Immediately Dangerous to Life or Health (IDLH)—The maximum environmental concentration of a contaminant from which one could escape within 30 minutes without any escape-impairing symptoms or irreversible health effects. Intermediate Exposure—Exposure to a chemical for a duration of 15-364 days, as specified in the Toxicological Profiles. CHLOROFORM 292 9. GLOSSARY Immunologic Toxicity—The occurrence of adverse effects on the immune system that may result from exposure to environmental agents such as chemicals. In Vitro—Isolated from the living organism and artificially maintained, as in a test tube. In Vivo—Occurring within the living organism. Lethal Concentration LO) (LCy,g)—The lowest concentration of a chemical in air which has been reported to have caused death in humans or animals. Lethal Concentration gg, (LCgp)—A calculated concentration of a chemical in air to which exposure for a specific length of time is expected to cause death in 50% of a defined experimental animal population. Lethal Dosey (LDy g)—The lowest dose of a chemical introduced by a route other than inhalation that is expected to have caused death in humans or animals. Lethal Doses) (LDgg)—The dose of a chemical which has been calculated to cause death in 50% of a defined experimental animal population. Lethal Time 5 (LTgp)—A calculated period of time within which a specific concentration of a chemical is expected to cause death in 50% of a defined experimental animal population. Lowest-Observed-Adverse-Effect Level (LOAEL)—The lowest dose of chemical in a study, or group of studies, that produces statistically or biologically significant increases in frequency or severity of adverse effects between the exposed population and its appropriate control. Malformations—Permanent structural changes that may adversely affect survival, development, or function. Minimal Risk Level—An estimate of daily human exposure to a dose of a chemical that is likely to be without an appreciable risk of adverse noncancerous effects over a specified duration of exposure. Mutagen—A substance that causes mutations. A mutation is a change in the genetic material in a body cell. Mutations can lead to birth defects, miscarriages, or cancer. Neurotoxicity—The occurrence of adverse effects on the nervous system following exposure to chemical. No-Observed-Adverse-Effect Level (NOAEL)—The dose of chemical at which there were no statistically or biologically significant increases in frequency or severity of adverse effects seen between the exposed population and its appropriate control. Effects may be produced at this dose, but they are not considered to be adverse. Octanol-Water Partition Coefficient (K,)—The equilibrium ratio of the concentrations of a chemical in n-octanol and water, in dilute solution. Permissible Exposure Limit (PEL)—An allowable exposure level in workplace air averaged over an 8-hour shift. CHLOROFORM 293 9. GLOSSARY qy*—The upper-bound estimate of the low-dose slope of the dose-response curve as determined by the multistage procedure. The q;* can be used to calculate an estimate of carcinogenic potency, the incremental excess cancer risk per unit of exposure (usually pg/L for water, mg/kg/day for food, and ng/m> for air). Reference Dose (RfD)—An estimate (with uncertainty spanning perhaps an order of magnitude) of the daily exposure of the human population to a potential hazard that is likely to be without risk of deleterious effects during a lifetime. The RfD is operationally derived from the NOAEL (from animal and human studies) by a consistent application of uncertainty factors that reflect various types of data used to estimate RfDs and an additional modifying factor, which is based on a professional judgment of the entire database on the chemical. The RfDs are not applicable to nonthreshold effects such as cancer. Reportable Quantity (RQ)—The quantity of a hazardous substance that is considered reportable under CERCLA. Reportable quantities are (1) 1 pound or greater or (2) for selected substances, an amount established by regulation either under CERCLA or under Sect. 311 of the Clean Water Act. Quantities are measured over a 24-hour period. Reproductive Toxicity—The occurrence of adverse effects on the reproductive system that may result from exposure to a chemical. The toxicity may be directed to the reproductive organs and/or the related endocrine system. The manifestation of such toxicity may be noted as alterations in sexual behavior, fertility, pregnancy outcomes, or modifications in other functions that are dependent on the integrity of this system. Short-Term Exposure Limit (STEL)—The maximum concentration to which workers can be exposed for up to 15 min continually. No more than four excursions are allowed per day, and there must be at least 60 minutes between exposure periods. The daily TLV-TWA may not be exceeded. Target Organ Toxicity—This term covers a broad range of adverse effects on target organs or physiological systems (e.g., renal, cardiovascular) extending from those arising through a single limited exposure to those assumed over a lifetime of exposure to a chemical. Teratogen—A chemical that causes structural defects that affect the development of an organism. Threshold Limit Value (TLV)—A concentration of a substance to which most workers can be exposed without adverse effect. The TLV may be expressed as a TWA, as a STEL, or as a CL. Time-Weighted Average (TWA)—An allowable exposure concentration averaged over a normal 8- hour workday or 40-hour workweek. Toxic Dose (TDgp)—A calculated dose of a chemical, introduced by a route other than inhalation, which is expected to cause a specific toxic effect in 50% of a defined experimental animal population. Uncertainty Factor (UF)—A factor used in operationally deriving the RfD from experimental data. UFs are intended to account for (1) the variation in sensitivity among the members of the human population, (2) the uncertainty in extrapolating animal data to the case of human, (3) the uncertainty in extrapolating from data obtained in a study that is of less than lifetime exposure, and (4) the uncertainty in using LOAEL data rather than NOAEL data. Usually each of these factors is set equal to 10. CHLOROFORM A-1 APPENDIX A ATSDR MINIMAL RISK LEVEL The Comprehensive Environmental Response, Compensation, and Liability Act (CERCLA) [42 U.S.C. 9601 et seq.], as amended by the Superfund Amendments and Reauthorization Act (SARA) [Pub. L. 99-499], requires that the Agency for Toxic Substances and Disease Registry (ATSDR) develop Jointly with the U.S. Environmental Protection Agency (EPA), in order of priority, a list of hazardous substances most commonly found at facilities on the CERCLA National Priorities List (NPL); prepare toxicological profiles for each substance included on the priority list of hazardous substances; and assure the initiation of a research program to fill identified data needs associated with the substances. The toxicological profiles include an examination, summary, and interpretation of available toxicological information and epidemiologic evaluations of a hazardous substance. During the development of toxicological profiles, Minimal Risk Levels (MRLs) are derived when reliable and sufficient data exist to identify the target organ(s) of effect or the most sensitive health effect(s) for a specific duration for a given route of exposure. An MRL is an estimate of the daily human exposure to a hazardous substance that is likely to be without appreciable risk of adverse noncancer health effects over a specified duration of exposure. MRLs are based on noncancer health effects only and are not based on a consideration of cancer effects. These substance-specific estimates, which are intended to serve as screening levels, are used by ATSDR health assessors to identify contaminants and potential health effects that may be of concern at hazardous waste sites. It is important to note that MRLs are not intended to define clean-up or action levels. MRLs are derived for hazardous substances using the no-observed-adverse-effect level/uncertainty factor approach. They are below levels that might cause adverse health effects in the people most sensitive to such chemical-induced effects. MRLs are derived for acute (1-14 days), intermediate (15-364 days), and chronic (365 days and longer) durations and for the oral and inhalation routes of exposure. Currently, MRLs for the dermal route of exposure are not derived because ATSDR has not yet identified a method suitable for this route of exposure. MRLs are generally based on the most sensitive chemical-induced end point considered to be of relevance to humans. Serious health effects (such as irreparable damage to the liver or kidneys, or birth defects) are not used as a basis for CHLOROFORM A-2 APPENDIX A establishing MRLs. Exposure to a level above the MRL does not mean that adverse health effects will occur. MRLs are intended only to serve as a screening tool to help public health professionals decide where to look more closely. They may also be viewed as a mechanism to identify those hazardous waste sites that are not expected to cause adverse health effects. Most MRLs contain a degree of uncertainty because of the lack of precise toxicological information on the people who might be most sensitive (e.g., infants, elderly, nutritionally or immunologically compromised) to the effects of hazardous substances. ATSDR uses a conservative (i.e., protective) approach to address this uncertainty consistent with the public health principle of prevention. Although human data are preferred, MRLs often must be based on animal studies because relevant human studies are lacking. In the absence of evidence to the contrary, ATSDR assumes that humans are more sensitive to the effects of hazardous substance than animals and that certain persons may be particularly sensitive. Thus, the resulting MRL may be as much as a hundredfold below levels that have been shown to be nontoxic in laboratory animals. Proposed MRLs undergo a rigorous review process: Health Effects/MRL Workgroup reviews within the Division of Toxicology, expert panel peer reviews, and agencywide MRL Workgroup reviews, with participation from other federal agencies and comments from the public. They are subject to change as new information becomes available concomitant with updating the toxicological profiles. Thus, MRLs in the most recent toxicological profiles supersede previously published levels. For additional information regarding MRLs, please contact the Division of Toxicology, Agency for Toxic Substances and Disease Registry, 1600 Clifton Road, Mailstop E-29, Atlanta, Georgia 30333. CHLOROFORM A-3 APPENDIX A MINIMAL RISK LEVEL (MRL) WORKSHEETS Chemical name(s): Chloroform CAS number(s): 000067-66-3 Date: March 19, 1997 Profile status: Final Route: [X] Inhalation [ ] Oral Duration: [X] Acute [ ]Intermediate [ ] Chronic Key to figure: 22 Species: Mouse MRL: 0.1 [ ] mg/kg/day [X] ppm [ ] mg/m> Reference: Larson JL, Wolf DC, Morgan KT, et al. 1994c. The toxicity of 1-week exposures to inhaled chloroform in female B6C3F; mice and male F-344 rats. Fund Appl Toxicol 22:431-446. Experimental design: The authors investigated the ability of chloroform vapors to produce toxicity and regenerative cell proliferation in female B6C3F mice and male Fischer 344 rats. Groups of 5 animals were exposed to 0, 1, 3, 10, 30, 100, or 300 ppm chloroform via inhalation for 6 hours a day for 7 consecutive days. Actual exposure concentrations measured for mice were 0, 1.2, 3.0, 10.0, 29.5, 101, and 288 ppm and for rats were 0, 1.5, 3.1, 10.4, 29.3, 100, and 271 ppm. Necropsies were performed on day 8. Animals were administered bromodeoxyuridine (BrdU) via implanted osmotic pump for the last 3.5 days. Cell proliferation was quantitated as the percentage of cells in S-phase (labeling index = LI) measured by the immunohistochemical detection of BrdU-labeled nuclei. Effects noted in study and corresponding doses: Female Mice: 300 ppm: Respiratory NOAEL; proximal tubules of kidney lined by regenerating epithelium (less serious LOAEL). 100 ppm: Renal NOAEL; centrilobular hepatocyte necrosis and severe diffuse vacuolar degeneration of midzonal and periportal hepatocytes (serious LOAEL); weight loss (less serious LOAEL). 30 ppm: Body weight NOAEL 10 ppm: Mild-to-moderate vacuolar changes in centrilobular hepatocytes (less serious LOAEL). 3 ppm: Hepatic effects NOAEL Male Rats: 300 ppm: Swelling and mild centrilobular vacuolation of hepatocytes (less serious LOAEL). 100 ppm: Hepatic effects NOAEL. 30 ppm: Increased number of S-phase nuclei for tubule cells in the renal cortex (less serious LOAEL). 10 ppm: Renal effect NOAEL; decreased body weight gain (less serious LOAEL); epithelial goblet cell hyperplasia and degeneration of Bowman’s glands in olfactory mucosa (less serious LOAEL). 3 ppm: Body weight gain and respiratory NOAEL. CHLOROFORM A-4 APPENDIX A Dose and end point used for MRL derivation: [X] NOAEL [ ] LOAEL: 3 ppm for hepatic effects in mice Uncertainty factors used in MRL derivation: 30 [11 [X]3 []10 (for extrapolation from animals to humans) [11 []3 [X] 10 (for human variability) Was a conversion factor used from ppm in food or water to a mg/body weight dose? If so, explain: No. If an inhalation study in animals, list conversion factors used in determining human equivalent dose: For dosimetry adjustment, the human equivalent concentration (HEC) is calculated based on a NOAEL of 3 ppm using Equation 4-10 (EPA 1990b). This equation was used due to the observation that chloroform achieves “periodicity” within 10% of the exposure duration (see Table A-1 below). Using Equation 4-10, the calculation is: NOAEL yg) = NOAEL x [(blood:air coeff) | se/(blood:air coeff, man] given that the ratio of the blood:air partition coefficients are <1. In the case of chloroform, using the blood:air partition coefficients for the mouse is 21.3 and for the human is 7.34 (Corley et al. 1990), the ratio of mouse:human partition coefficients (21.3/7.34) is >1, therefore a default value of 1 is used to derive the NOAEL jc: NOAELygc = 3 ppm x | NOAELygc = 3 ppm where: NOAEL hg) = Human Equivalent Concentration of the NOAEL (no-observed-adverse effect level) The MRL calculation is as follows: MRL = NOAEL yg) / UF MRL = 3 ppm / 30 MRL = 0.1 ppm Was a conversion used from intermittent to continuous exposure? If so, explain: No. Other additional studies or pertinent information that lend support to this MRL: The Larson et al. (1994¢) study is accompanied by a companion study performed by Mery et al. (1994), which examined the nasal lesions much more closely than this Larson study. The purpose of the Mery et al. study was to determine nasal cavity site-specific lesions and any cell induction/ proliferation associated with varying concentrations of chloroform (0, 1, 3, 10, 30, 100, 300 ppm) inhaled by both rats and mice 6 hours a day for 7 days. Female B6C3F! mice and male Fischer 344 rats were used. Tissue abnormalities seen grossly, histopathologically, enzymatically (cytochrome P-450 levels), and in cell proliferation (BrdU labeling of cells in the S-phase) were reported for both rats and mice. The respiratory epithelium of the nasopharyngeal meatus exhibited an increase in the size of goblet cells at 100 and 300 ppm chloroform, in addition to an increase in both neutral and CHLOROFORM A-5 APPENDIX A acidic mucopolysaccharides. Affected epithelium was up to twice the normal thickness. New bone formation within the nasal region was prominently seen at 10 ppm and above and followed a concentration response curve. At | ppm, only 1 animal showed mild bone enlargement of the first endoturbinate, with no changes seen at 3 ppm. At 10 ppm, minor enlargement was present in all animals. At 30 and 100 ppm, new osseous spicules was present at the beginning of the first endoturbinate, while at 300 ppm, the width of the new bone was almost doubled compared to controls receiving no chloroform, with lesions extending to involve up to 75% of the turbinate in all of the sites studied. Enzymatically, staining for P-450-2E1 was most prominent in the control animals in the cytoplasm of olfactory epithelial sustentacular cells and in the acinar cells of Bowman’s glands, and more intense in the superficial cells than in the deep cells. In general, starting at about 3 ppm, increasing the chloroform concentration tended to decrease the amount of P-450 staining. Exposure to chloroform resulted in a dramatic increase in the number of S-phase nuclei. A clear proportional concentration-related effect was observed, with the proliferative response confined to activated periosteal cells, including both osteogenic (round) and preosteogenic (spindle) cells. The proximal and central regions of the first endoturbinate had the highest increase of cell proliferation, while the distal part had only a moderate response, with this response being statistically significant from controls at concentrations of greater than 10 ppm. Decreased body weight was observed at 300 ppm only (data not provided). In mice, decreased body weight was observed at 100 and 300 ppm (data not provided). The only treatment-related histologic change observed in female mice was a slight indication of new bone growth in the proximal part of the first endoturbinate in one mouse exposed to 300 ppm chloroform. The S-phase response was observed at chloroform concentration of 10 ppm and higher. Using the Corley PBPK model for chloroform (Corley et al. 1990) in the Scop version (courtesy of Dr. Nancy Chiu, USEPA) to simulate the mouse exposure of chloroform by inhalation. This data is presented below: Table A-1. Corley PBPK Model for Chloroform to Simulate Mouse Exposure by Inhalation Time (hrs): Blood Concentration (CA) (mg/L): 0.00 0.014 0.25 0.040 0.50 0.041 0.75 0.041 1.25 0.042 1.50 0.042 1.75 0.042 2.00 0.042 2.25 0.042 2.50 0.042 3.375 0.042 4.5 0.042 5.625 0.042 6.75 0.0006 ource: Corley et al. cop version (courtesy o ancy Chiu, CHLOROFORM A-6 APPENDIX A The data furnished by this model demonstrates that the arterial blood concentration (CA) of chloroform in the mouse exposed to 3 ppm of chloroform for 6 hours reached “periodicity” within 15 minutes following exposure. This data allowed the use of EPA (1990b) Equation 4-10 to derive the acute-duration inhalation MRL for chloroform exposure. Agency Contact (Chemical Manager): Selene Chou CHLOROFORM A-7 APPENDIX A MINIMAL RISK LEVEL WORKSHEET Chemical name(s): Chloroform CAS number(s): 000067-66-3 Date: March 19, 1997 Profile status: Final Route: [X] Inhalation [ ] Oral Duration: [ ] Acute [X] Intermediate [ ] Chronic Key to figure: 39 Species: Human MRL: 0.05 [ ] mg/kg/day [X] ppm [ ] mg/m> Reference: Phoon WH, Goh KT, Lee LT, et al. 1983. Toxic jaundice from occupational exposure to chloroform. Med J Malaysia 38:31-34. Experimental design: The study describes outbreaks of toxic hepatitis in workers occupationally exposed to chloroform in two different factories. Mostly women were employed in both places. Effects noted in study and corresponding doses: The workers in the first outbreak were exposed to concentrations up to 400 ppm chloroform in the workplace. No other chemical was involved. Blood chloroform levels in exposed workers ranged from 0.10 to 0.29 mg/100 mL. Workplace concentration levels of chloroform ranged from 14 to 50 ppm in the second outbreak. Vomiting and toxic hepatitis were noted to occur at an inhaled concentration of 14 ppm (less serious LOAEL). All affected workers were exposed to chloroform for less than six months. The patients exhibited anorexia, nausea, vomiting, and jaundice without fever. The subjects had originally been diagnosed with viral hepatitis, however the diagnosis of toxic hepatitis due to chloroform exposure was based upon epidemiological considerations. Dose and end point used for MRL derivation: [ ] NOAEL [X] LOAEL : 14 ppm for hepatic effects Uncertainty factors (UF) used in MRL derivation: 100 [11 [13 [X] 10 (for use of a LOAEL) [11 [13 [X] 10 (for human variability) Modifying factor (MF) used in MRL derivation: []1 [X]3 []10 (for insufficient diagnostic data to determine the seriousness of hepatotoxic effects) Was a conversion factor used from ppm in food or water to a mg/body weight dose? If so, explain: No. If an inhalation study in animals, list conversion factors used in determining human equivalent dose: No factors were used to convert to a human equivalent dose, since the data obtained from this study was obtained from human exposures to chloroform. CHLOROFORM A-8 APPENDIX A The MRL calculation is as follows: MRL = LOAEL / (UF x MF) MRL = 14 ppm / (100 x 3) MRL = 0.05 ppm Was a conversion used from intermittent to continuous exposure? If so, explain: No. Other additional studies or pertinent information that lend support to this MRL: The study by Bomski et al. (1967) noted similar finding in a group of 68 workers occupationally exposed to chloroform for 1-4 years in a pharmaceutical plant. Inhaled chloroform concentrations ranged from 0.01 to 1 mg/L. Other solvents were reported in the air in trace amounts. Hepatomegaly was found in 25% of chloroform-exposed workers. Toxic hepatitis was found in 5.6% of the liver enlargement cases. The workers were diagnosed as having hepatosplenomegaly, enhanced serum glutamic pyruvic transaminase [SGPT] and serum glutamic oxaloacetic transaminase [SGOT] activities, and hyper-gammaglobulinemia. Hepatosteatosis (fatty liver) was detected in 20.6% of liver-enlargement cases. Chloroform-exposed workers had a higher frequency of jaundice over the years than the control group. Agency Contact (Chemical Manager): Selene Chou CHLOROFORM A-9 APPENDIX A MINIMAL RISK LEVEL WORKSHEET Chemical name(s): Chloroform CAS number(s): 000067-66-3 Date: March 19, 1997 Profile status: Final Route: [X] Inhalation [ ] Oral Duration: [ ] Acute [ ]Intermediate [X] Chronic Key to figure: 51 Species: Human MRL: 0.02 [ ] mgkg/day [X] ppm [] mg/m’ Reference: Bomski H, Sobolewska A, Strakowski A. 1967. [Toxic damage of the liver by chloroform in chemical industry workers.] Int Arch F Gewerbepathologie u. Gewerbehygiene 24:127-134 (German). Experimental design: A group of 68 workers occupationally exposed to chloroform for 1-4 years in a pharmaceutical plant were examined. Doses of inhaled chloroform ranged from 2 to 205 ppm over a 1-4-year-period. Air concentrations of chloroform ranged from 0.01 mg/L to 1 mg/L. Other solvents were reported in the air in trace amounts. Effects noted in study and corresponding doses: A systemic LOAEL (hepatomegaly) of 2 ppm was determined from the data presented in this study. Hepatomegaly was found in 25% of chloroform exposed workers. The results were compared with a group of unexposed controls, and a group of persons who had infectious hepatitis during the last 1-4 years. Toxic hepatitis was found in 5.6% of the liver enlargement cases (the workers were diagnosed as having hepatosplenomegaly, enhanced SGPT and SGOT activities, and hyper-gammaglobulinemia). Hepatosteatosis (fatty liver) was detected in 20.6% of liver-enlargement cases. Functional tests were negative in most of the subjects; a biopsy was not performed in any case. Chloroform-exposed workers had a higher frequency of jaundice over the years than the control group. The authors speculated that a viral infection might have been promoted in the chloroform damaged liver. Dose end point used for MRL derivation: [ ] NOAEL [X] LOAEL: 2 ppm for hepatic effects Uncertainty factors used in MRL derivation: 100 [11 []3 [X] 10 (for use of a LOAEL) [11 [13 [X] 10 (for human variability) Was a conversion factor used from ppm in food or water to a mg/body weight dose? If so, explain: No. If an inhalation study in animals, list conversion factors used in determining human equivalent dose: No factors were used to convert to a human equivalent dose, since the data obtained from this study was obtained from human exposures to chloroform. CHLOROFORM APPENDIX A The MRL calculation is as follows: MRL = LOAEL / (UF) MRL = 2 ppm / (100) MRL = 0.02 ppm Was a conversion used from intermittent to continuous exposure? If so, explain: No. Other additional studies or pertinent information that lend support to this MRL: Agency Contact (Chemical Manager): Selene Chou CHLOROFORM A-11 APPENDIX A MINIMAL RISK LEVEL WORKSHEET Chemical name(s): Chloroform CAS number(s): 000067-66-3 Date: March 19, 1997 Profile status: Final Route: [ ] Inhalation [X] Oral Duration: [X] Acute [ ]Intermediate [ ] Chronic Key to figure: 28 Species: Mouse MRL: 03 [X] mg/kg/day []ppm [] mg/m’ Reference: Larson JL, Wolf DC, Butterworth BE. 1994b. Induced cytotoxicity and cell proliferation in the hepatocarcinogenicity of chloroform in female B6C3F | mice: comparison of administration by gavage in corn oil vs ad libitum in drinking water. Fund Appl Toxicol 22:90-102. Experimental design: This study was designed to identify a relationship between the magnitude and duration of chloroform- induced histopathologic and proliferative responses for female mice dosed with chloroform in the drinking water vs those dosed in corn oil via gavage. Authors placed 0, 60, 200, 400, 900, or 1,800 ppm of chloroform in drinking water; however, due to decreased water intake, the authors’ calculation of consumed chloroform was 0, 16, 26, 53, 81, or 105 mg/kg/day. Effects noted in study and corresponding doses: In the 400, 900, and 1,800 ppm treatment groups, the livers had tinctorial changes characterized by pale cytoplasmic eosinophilic staining of centrilobular hepatocytes compared to the periportal hepatocytes and controls. Livers from mice treated with 200 ppm (26 mg/kg/day actual intake) chloroform or less failed to showed significant histologic changes when compared to controls. Thus the dose of 26 mg/kg/day was considered the NOAEL for hepatic effects in these mice. Chloroform exposure did cause a slight dose dependent decrease in number of cells in S-phase in the kidneys, mainly in the cortex, while there was an increase in these type of cells in the outer medullary region. Decreased body weight was observed at the two highest doses. After 4 days treatment, serum clinical chemistry analyses were not different from controls in either liver alanine aminotransferase (ALT) or sorbitol dehydrogenase (SDH) at any dose. Dose end point used for MRL derivation: [X] NOAEL [ ] LOAEL: 26 mg/kg/day for hepatic effects in mice CHLOROFORM A-12 APPENDIX A Uncertainty factors used in MRL derivation: 100 ]1 [13 []10 (for use of a LOAEL) [11 [13 [X] 10 (for extrapolation from animals to humans) 11 [13 [X] 10 (for human variability) Was a conversion factor used from ppm in food or water to a mg/body weight dose? If so, explain: Conversion of the ppm concentration of chloroform in the drinking water to a mg/kg/day dose was provided by the authors of the paper. The MRL calculation is as follows: MRL = NOAEL / UF MRL = 26 mg/kg/day / 100 MRL = 0.3 mg/kg/day If an inhalation study in animals, list conversion factors used in determining human equivalent dose: Was a conversion used from intermittent to continuous exposure? If so, explain: Other additional studies or pertinent information that lend support to this MRL: Larson et al. (1995) also studied the dose response relationships for the induction of cytolethality and regenerative cell proliferation in male Fischer 344 rats given chloroform in corn oil by gavage or in the drinking water. Groups of 12 rats were administered oral doses of 0, 3, 10, 34, 90, and 180 mg/kg/day chloroform in corn oil by gavage for 4 or for 5 days a week for 3 weeks. BrdU was administered via an implanted osmotic pump to label cells in S-phase. Statistically significant decreases in body weight gains were observed in the 180 mg/kg/day dose group at 4 days and in the 90 and 180 mg/kg/day dose groups at 3 weeks. At 34 mg/kg/day, slight-to-mild centrilobular sinusoidal leukostasis was observed after 4 days of exposure. The livers of rats given 90 mg/kg/day for 4 days had a slight increase in centrilobular pallor and necrosis of hepatocytes surrounding the central vein; the remaining central and some mid-zonal hepatocytes were swollen and displayed a cytoplasmic granularity. After 3 weeks of exposure, livers of rats in the 34 or 90 mg/kg/day dose groups did not differ from controls. In the 180 mg/kg/day dose group, the livers of rats after 4 days had scattered individual cell necrosis throughout the central and midzonal regions. The cytoplasm of the centrilobular hepatocytes was pale eosinophilic and mildly vacuolated. In the 180 mg/kg/day dose group, after 3 weeks effects were similar to those seen at 4 days after exposure. Dose-dependent increases in both ALT and SDH were observed at 4 days in the 90 and 180 mg/kg/day dose groups and at 3 weeks in the 180 mg/kg/day dose group only. A dose-dependent increase in LI was seen in rat liver after 4 days of treatment with 90 and 180 mg/kg/day by gavage, but the LI remained elevated after 3 weeks of treatment only at the 180 mg/kg/day dose. At doses of 0, 60, 200, 400, 900, and 1,800 ppm for 4 days, no microscopic alterations were seen in the kidneys after 4 days of treatment. As a general observation, rats treated for 3 weeks with 200 ppm chloroform and greater had slightly increased numbers of focal areas of regenerating renal proximal tubular epithelium and cell proliferation than were CHLOROFORM A-13 APPENDIX A noted in controls, but no clear dose response relationship was evident. However, the overall renal LI was not increased at any dose or time point. Similarly, only mild hepatocyte vacuolation was observed in rats given 900 or 1,800 ppm in water for 4 days and in rats given 1,800 ppm in water for 3 weeks. No increase in the hepatic LI was observed at any time point. When chloroform was administered in the drinking water at doses of 0, 60, 200, 400, 900, and 1,800 ppm for 3 weeks, no microscopic alterations were seen in the kidneys after 4 days of treatment. As a general observation, rats treated for 3 weeks with 200 ppm chloroform and greater had slightly increased numbers of focal areas of regenerating renal proximal tubular epithelium and cell proliferation than were noted in controls, but no clear dose response relationship was evident. However, the overall renal LI was not increased at any dose or time point. Similarly, only mild hepatocyte vacuolation was observed in rats given 900 or 1,800 ppm in water for 4 days and in rats given 1,800 ppm in water for 3 weeks. No increase in the hepatic LI was observed at any time point. The authors noted that these data indicated more severe hepatic and renal toxicity when chloroform is administered by gavage than in the drinking water. Agency Contact (Chemical Manager): Selene Chou CHLOROFORM A-14 APPENDIX A MINIMAL RISK LEVEL WORKSHEET Chemical name(s): Chloroform CAS number(s): 000067-66-3 Date: March 19, 1997 Profile status: Final Route: [ ] Inhalation [X] Oral Duration: [ ] Acute [X] Intermediate [ ] Chronic Key to figure: 68 Species: Dog MRL: 0.1 [X] mg/kg/day [] ppm [] mg/m’ Reference: Heywood R, Sortwell RJ, Noel PRB, et al. 1979. Safety evaluation of toothpaste containing chloroform. III. Long-term study in beagle dogs. J Environ Pathol Toxicol 2:835-851. Experimental design: An intermediate oral exposure MRL of 0.1 mg/kg/day was derived using the study by Heywood et al. (1979). The study was 7.5 years in duration in which 8 male and 8 female Beagle dogs were exposed to chloroform in toothpaste capsules, with doses of 0, 15, and 30 mg/kg/day, 6 days a week for 6 weeks. Clinical chemistry parameters were monitored at 6 and 13 weeks of exposure and thereafter at intervals of 8-32 weeks. Effects noted in study and corresponding doses: Serum glutamic pyruvic transaminase (SGPT) activity was significantly increased (p<0.05) in the 30 mg/kg/day group beginning at 6 weeks and at every interval thereafter. SGPT activity was not increased in the 15 mg/kg/day group until week 130. Thus, 15 mg/kg/day is the NOAEL for intermediate duration exposure. Dose end point used for MRL derivation: [X] NOAEL [ ] LOAEL : 15 mg/kg/day for hepatic effects Uncertainty factors used in MRL derivation: 100 [11 [13 [X] 10 (for extrapolation from animals to humans) [11 [13 [X] 10 (for human variability) Was a conversion factor used from ppm in food or water to a mg/body weight dose? If so, explain: No. If an inhalation study in animals, list conversion factors used in determining human equivalent dose: Was a conversion used from intermittent to continuous exposure? If so, explain: (15 mg/kg/day) x 6/7 days = 12.9 mg/kg/day CHLOROFORM A-15 APPENDIX A The MRL calculation is as follows: MRL = 12.9 mg/kg/day/ 100 MRL = 0.1 mg/kg/day Other additional studies or pertinent information that lend support to this MRL: Liver effects in animals have been reported in numerous oral studies of intermediate duration. Fatty changes, necrosis, increased liver weight, and hyperplasia have been observed in rats exposed to >150 mg/kg/day chloroform in drinking water for 90 days (Palmer et al. 1979). An increased incidence of sporadic, mild, reversible liver changes occurred in mice exposed to chloroform in drinking water at doses of 0.3-114 mg/kg/day for 90 days, but the incidences were not significantly higher than the incidences in controls (Chu et al. 1982a). Fatty and hydropic changes, necrosis, and cirrhosis were observed in mice treated by gavage with >50 mg/kg/day chloroform in oil for 90 days (Bull et al. 1986; Munson et al. 1982) or at 86 mg/kg/day in drinking water for 1 year (Klaunig et al. 1986). In contrast, centrilobular fatty changes observed in mice at 64 mg/kg/day chloroform in drinking water for 90 days appeared to be reversible (Jorgenson and Rushbrook 1980), and no liver effects were found in mice treated with >50 mg/kg/day in aqueous vehicles (Bull et al. 1986). In addition, hepatocellular degeneration was induced in F| females in a 2-generation study in which mice were treated by gavage with 41 mg/kg/day chloroform in oil (Gulati et al. 1988). Agency Contact (Chemical Manager): Selene Chou CHLOROFORM A-16 APPENDIX A MINIMAL RISK LEVEL WORKSHEET Chemical name(s): Chloroform CAS number(s): 000067-66-3 Date: March 19, 1997 Profile status: Final Route: [ ] Inhalation [X] Oral Duration: [ ] Acute [ JIntermediate [X] Chronic Key to figure: 89 Species: Dog MRL: 0.01 [X] mg/kg/day []ppm [] mgm’ Reference: Heywood R, Sortwell RJ, Noel PRB, et al. 1979. Safety evaluation of toothpaste containing chloroform. III. Long-term study in beagle dogs. J Environ Pathol Toxicol 2:835-851. Experimental design: Eight male and 8 female Beagle dogs were exposed to chloroform in toothpaste capsules. Doses used were 0, 15, and 30 mg/kg/day, 6 days a week for 7.5 years. Clinical chemistry parameters were monitored at 6 and 13 weeks of exposure and thereafter at intervals of 8-32 weeks for 7.5 years. Effects noted in study and corresponding doses: SGPT activity was significantly increased (p<0.05) in the 30 mg/kg/day group beginning at 6 weeks and at every interval thereafter. SGPT activity was not increased in the 15 mg/kg/day group until week 130. No treatment-related body weight changes were observed in chloroform exposed dogs. No hematological changes were found. Increased SGPT levels, and less distinct elevation of SGOT and SAP seemed to be dose-related. However, the SGPT levels tended to return to normal during the recovery period. Bromsulphalein retention test was performed during the sixth year of the study; no treatment-related abnormality was found. No organ weight changes were found in the exposed groups. No remarkable histopathological differences were observed in dogs regarding the cardiovascular system. Fatty cysts were observed in the liver in all groups; however, in females the incidence seemed to be dose-related (3 of 12, 5 of 8, 7 of 8). Fat deposition in renal glomeruli was reportedly higher in the 30 mg/kg/day chloroform group. Dose end point used for MRL derivation: [ 1 NOAEL [X] LOAEL: 15 mg/kg/day for hepatic effects Uncertainty factors used in MRL derivation: 1000 [11 [13 [X] 10 (for use of a LOAEL) [11 [13 [X] 10 (for extrapolation from animals to humans) [11 [13 [X] 10 (for human variability) Was a conversion factor used from ppm in food or water to a mg/body weight dose? If so, explain: No. If an inhalation study in animals, list conversion factors used in determining human equivalent dose: CHLOROFORM A-17 APPENDIX A Was a conversion used from intermittent to continuous exposure? If so, explain: (15 mg/kg/day) x 6/7 days = 12.9 mg/kg/day. The MRL calculation is as follows: MRL = LOAEL spy / UF MRL = 12.9 mg/kg/day / 1000 MRL = 0.01 mg/kg/day Other additional studies or pertinent information that lend support to this MRL: Numerous chronic-duration oral studies examined hepatic and renal end points as well as neurological and cancer effects. Serious effects occurred at higher doses; 15 mg/kg/day was the lowest dose used in available animals studies. A NOAEL of 2.46 mg/kg/day for liver and kidney effects (SGPT, SGOT, BUN and SAP) was found in humans who used a dentifrice containing 0.34% or a mouthwash containing 0.43% chloroform for 1-5 years (DeSalva et al. 1974). Agency Contact (Chemical Manager): Selene Chou CHLOROFORM B-1 APPENDIX B USER’S GUIDE Chapter 1 Public Health Statement This chapter of the profile is a health effects summary written in non-technical language. Its intended audience is the general public especially people living in the vicinity of a hazardous waste site or chemical release. If the Public Health Statement were removed from the rest of the document, it would still communicate to the lay public essential information about the chemical. The major headings in the Public Health Statement are useful to find specific topics of concern. The topics are written in a question and answer format. The answer to each question includes a sentence that will direct the reader to chapters in the profile that will provide more information on the given topic. Chapter 2 Tables and Figures for Levels of Significant Exposure (LSE) Tables (2-1, 2-2, and 2-3) and figures (2-1 and 2-2) are used to summarize health effects and illustrate graphically levels of exposure associated with those effects. These levels cover health effects observed at increasing dose concentrations and durations, differences in response by species, minimal risk levels (MRLs) to humans for noncancer endpoints, and EPA’s estimated range associated with an upper- bound individual lifetime cancer risk of 1 in 10,000 to 1 in 10,000,000. Use the LSE tables and figures for a quick review of the health effects and to locate data for a specific exposure scenario. The LSE tables and figures should always be used in conjunction with the text. All entries in these tables and figures represent studies that provide reliable, quantitative estimates of No-Observed-Adverse- Effect Levels (NOAELSs), Lowest-Observed- Adverse-Effect Levels (LOAELS), or Cancer Effect Levels (CELs). The legends presented below demonstrate the application of these tables and figures. Representative examples of LSE Table 2-1 and Figure 2-1 are shown. The numbers in the left column of the legends correspond to the numbers in the example table and figure. LEGEND See LSE Table 2-1 (1) Route of Exposure One of the first considerations when reviewing the toxicity of a substance using these tables and figures should be the relevant and appropriate route of exposure. When sufficient data exists, three LSE tables and two LSE figures are presented in the document. The three LSE tables present data on the three principal routes of exposure, i.e., inhalation, oral, and dermal (LSE Table 2-1, 2-2, and 2-3, respectively). LSE figures are limited to the inhalation (LSE Figure 2-1) and oral (LSE Figure 2-2) routes. Not all substances will have data on each route of exposure and will not therefore have all five of the tables and figures. (2) Exposure Period Three exposure periods - acute (less than 15 days), intermediate (15-364 days), and chronic (365 days or more) are presented within each relevant route of exposure. In this example, an inhalation study of intermediate exposure duration is reported. For quick reference to health effects CHLOROFORM : B-2 3 @ 6) 6 (7 ® © (10) (In APPENDIX B occurring from a known length of exposure, locate the applicable exposure period within the LSE table and figure. Health Effect The major categories of health effects included in LSE tables and figures are death, systemic, immunological, neurological, developmental, reproductive, and cancer. NOAELSs and LOAEL:s can be reported in the tables and figures for all effects but cancer. Systemic effects are further defined in the "System" column of the LSE table (see key number 18). Key to Figure Each key number in the LSE table links study information to one or more data points using the same key number in the corresponding LSE figure. In this example, the study represented by key number 18 has been used to derive a NOAEL and a Less Serious LOAEL (also see the 2 "18r" data points in Figure 2-1). Species The test species, whether animal or human, are identified in this column. Section 2.4, "Relevance to Public Health," covers the relevance of animal data to human toxicity and Section 2.3, "Toxicokinetics," contains any available information on comparative toxicokinetics. Although NOAELs and LOAELSs are species specific, the levels are extrapolated to equivalent human doses to derive an MRL. Exposure Frequency/Duration The duration of the study and the weekly and daily exposure regimen are provided in this column. This permits comparison of NOAELs and LOAELSs from different studies. In this case (key number 18), rats were exposed to toxaphene via inhalation for 6 hours per day, 5 days per week, for 3 weeks. For a more complete review of the dosing regimen refer to the appropriate sections of the text or the original reference paper, i.e., Nitschke et al. 1981. System This column further defines the systemic effects. These systems include: respiratory, cardiovascular, gastrointestinal, hematological, musculoskeletal, hepatic, renal, and dermal/ocular. "Other" refers to any systemic effect (e.g., a decrease in body weight) not covered in these systems. In the example of key number 18, 1 systemic effect (respiratory) was investigated. NOAEL A No-Observed-Adverse-Effect Level (NOAEL) is the highest exposure level at which no harmful effects were seen in the organ system studied. Key number 18 reports a NOAEL of 3 ppm for the respiratory system which was used to derive an intermediate exposure, inhalation MRL of 0.0005 ppm (see footnote "b"). LOAEL A Lowest-Observed-Adverse-Effect Level (LOAEL) is the lowest dose used in the study that caused a harmful health effect. LOAELSs have been classified into "Less Serious" and "Serious" effects. These distinctions help readers identify the levels of exposure at which adverse health effects first appear and the gradation of effects with increasing dose. A brief description of the specific endpoint used to quantify the adverse effect accompanies the LOAEL. The respiratory effect reported in key number 18 (hyperplasia) is a Less serious LOAEL of 10 ppm. MRLs are not derived from Serious LOAELSs. Reference The complete reference citation is given in chapter 8 of the profile. CEL A Cancer Effect Level (CEL) is the lowest exposure level associated with the onset of carcinogenesis in experimental or epidemiologic studies. CELs are always considered serious effects. The LSE tables and figures do not contain NOAELSs for cancer, but the text may report doses not causing measurable cancer increases. CHLOROFORM B-3 APPENDIX B (12) Footnotes Explanations of abbreviations or reference notes for data in the LSE tables are found in the footnotes. Footnote "b" indicates the NOAEL of 3 ppm in key number 18 was used to derive an MRL of 0.0005 ppm. LEGEND See Figure 2-1 LSE figures graphically illustrate the data presented in the corresponding LSE tables. Figures help the reader quickly compare health effects according to exposure concentrations for particular exposure periods. (13) Exposure Period The same exposure periods appear as in the LSE table. In this example, health effects observed within the intermediate and chronic exposure periods are illustrated. (14) Health Effect These are the categories of health effects for which reliable quantitative data exists. The same health effects appear in the LSE table. (15) Levels of Exposure concentrations or doses for each health effect in the LSE tables are graphically displayed in the LSE figures. Exposure concentration or dose is measured on the log scale "y" axis. Inhalation exposure is reported in mg/m> or ppm and oral exposure is reported in mg/kg/day. (16) NOAEL In this example, 18r NOAEL is the critical endpoint for which an intermediate inhalation exposure MRL is based. As you can see from the LSE figure key, the open-circle symbol indicates to a NOAEL for the test species-rat. The key number 18 corresponds to the entry in the LSE table. The dashed descending arrow indicates the extrapolation from the exposure level of 3 ppm (see entry 18 in the Table) to the MRL of 0.0005 ppm (see footnote "b" in the LSE table). (17) CEL Key number 38r is 1 of 3 studies for which Cancer Effect Levels were derived. The diamond symbol refers to a Cancer Effect Level for the test species-mouse. The number 38 corresponds to the entry in the LSE table. (18) Estimated Upper-Bound Human Cancer Risk Levels This is the range associated with the upper-bound for lifetime cancer risk of 1 in 10,000 to 1 in 10,000,000. These risk levels are derived from the EPA’s Human Health Assessment Group’s upper-bound estimates of the slope of the cancer dose response curve at low dose levels (q;*). (19) Key to LSE Figure The Key explains the abbreviations and symbols used in the figure. WHO40HOTHO TABLE 2-1. Levels of Significant Exposure to [Chemical x] — Inhalation Exposure LOAEL (effect) Key to frequency/ NOAEL - . figure? Species duration ~~ System (ppm) Less serious (ppm) Serious (ppm) Reference INTERMEDIATE EXPOSURE Systemic l l l ! | 18 Rat 13 wk Resp 10 (hyperplasia) Nitschke et al. 5d/wk 1981 6hr/d > 0 m et ——————————— a — —— -——————————————————— — ———— ~~ ———-—— = CHRONIC EXPOSURE g @ Cancer 38 Rat 18 mo (CEL, multiple Wong et al. 1982 5d/wk organs) 7hr/d 39 Rat 89-104 wk 10 (CEL, lung tumors, NTP 1982 5d/wk nasal tumors) 6hr/d 40 Mouse 79-103 wk 10 (CEL, lung tumors, NTP 1982 5d/wk hemangiosarcomas) 6hr/d 2 The number corresponds to entries in Figure 2-1. b Used to derive an intermediate inhalation Minimal Risk Level (MRL) of 5 x 103 ppm; dose adjusted for intermittent exposure and divided by an uncertainty factor of 100 (10 for extrapolation from animal to humans, 10 for human variability). v-8 [13] — > Figure 2-1. Levels of Significant Exposure to [Chemical X] — Inhalation Acute Intermediate (£14 days) (15-364 days) Systemic Systemic N N cr 0 x4 mH of 5 5 xO © NS 3° } © : & & 2° " & 3° a & & —> (ppm) py XR EN 2? NR & o &R 3 Q > NS Q <& NS NS <& oO 10000 — 1000 | 0 . Oo ® 37m 38 100 - ©09® o O @® 30r 3ar % 17r Q 20m» OD 20m 31r d a 35m o 16r 2491 D 22921r O 28m® 29r O 27x 40m : [18] = : uo ” Q 18r 1 We | . 0.1 | 10-5 | Estimated [18] Y Upper Bound 0.01 — 5 | Human Cancer Key 10 Risk Levels 0.001 — r Rat @ LOAEL for serious effects (animals) !' Minimal risk level for effects 1077 ; : |" other than cancer m Mouse (P LOAEL for less serious effects (animals) 1 0.0001 — h Rabbit (OO NOAEL (animals) g Guinea Pig CEL - Cancer Effect Level The number next to each point 0.00001 % Monkey * corresponds to entries in the accompanying table. 0.000001 + * Doses represent the lowest dose tested per study that produced a tumorigenic response and do not imply the existence of a threshold for the cancer end point. 0.0000001 — 9 XIAN3ddVY WHO40OHO THO S-9 CHLOROFORM B-6 APPENDIX B Chapter 2 (Section 2.4) Relevance to Public Health The Relevance to Public Health section provides a health effects summary based on evaluations of existing toxicologic, epidemiologic, and toxicokinetic information. This summary is designed to present interpretive, weight-of-evidence discussions for human health endpoints by addressing the following questions. 1. What effects are known to occur in humans? 2. What effects observed in animals are likely to be of concern to humans? 3. What exposure conditions are likely to be of concern to humans, especially around hazardous waste sites? The section covers endpoints in the same order they appear within the Discussion of Health Effects by Route of Exposure section, by route (inhalation, oral, dermal) and within route by effect. Human data are presented first, then animal data. Both are organized by duration (acute, intermediate, chronic). In vitro data and data from parenteral routes (intramuscular, intravenous, subcutaneous, etc.) are also considered in this section. If data are located in the scientific literature, a table of genotoxicity information is included. The carcinogenic potential of the profiled substance is qualitatively evaluated, when appropriate, using existing toxicokinetic, genotoxic, and carcinogenic data. ATSDR does not currently assess cancer potency or perform cancer risk assessments. Minimal risk levels (MRLs) for noncancer endpoints (if derived) and the endpoints from which they were derived are indicated and discussed. Limitations to existing scientific literature that prevent a satisfactory evaluation of the relevance to public health are identified in the Data Needs section. Interpretation of Minimal Risk Levels Where sufficient toxicologic information is available, we have derived minimal risk levels (MRLs) for inhalation and oral routes of entry at each duration of exposure (acute, intermediate, and chronic). These MRLs are not meant to support regulatory action; but to acquaint health professionals with exposure levels at which adverse health effects are not expected to occur in humans. They should help physicians and public health officials determine the safety of a community living near a chemical emission, given the concentration of a contaminant in air or the estimated daily dose in water. MRLs are based largely on toxicological studies in animals and on reports of human occupational exposure. MRL users should be familiar with the toxicologic information on which the number is based. Chapter 2.4, "Relevance to Public Health," contains basic information known about the substance. Other sections such as 2.6, "Interactions with Other Substances," and 2.7, "Populations that are Unusually Susceptible" provide important supplemental information. MRL users should also understand the MRL derivation methodology. MRLs are derived using a modified version of the risk assessment methodology the Environmental Protection Agency (EPA) provides (Barnes and Dourson 1988) to determine reference doses for lifetime exposure (RfDs). CHLOROFORM B-7 APPENDIX B To derive an MRL, ATSDR generally selects the most sensitive endpoint which, in its best judgement, represents the most sensitive human health effect for a given exposure route and duration. ATSDR cannot make this judgement or derive an MRL unless information (quantitative or qualitative) is available for all potential systemic, neurological, and developmental effects. If this information and reliable quantitative data on the chosen endpoint are available, ATSDR derives an MRL using the most sensitive species (when information from multiple species is available) with the highest NOAEL that does not exceed any adverse effect levels. When a NOAEL is not available, a lowest-observed-adverse-effect level (LOAEL) can be used to derive an MRL, and an uncertainty factor (UF) of 10 must be employed. Additional uncertainty factors of 10 must be used both for human variability to protect sensitive subpopulations (people who are most susceptible to the health effects caused by the substance) and for interspecies variability (extrapolation from animals to humans). In deriving an MRL, these individual uncertainty factors are multiplied together. The product is then divided into the inhalation concentration or oral dosage selected from the study. Uncertainty factors used in developing a substance-specific MRL are provided in the footnotes of the LSE Tables. CHLOROFORM ACGIH ADME atm ATSDR BCF BSC C CDC CEL CERCLA CFR CLP cm CNS d DHEW DHHS DOL ECG EEG EPA EKG F Fy FAO FEMA FIFRA fpm ft FR g GC gen HPLC hr IDLH IARC ILO in Kd kg kkg oC ow C-1 APPENDIX C ACRONYMS, ABBREVIATIONS, AND SYMBOLS American Conference of Governmental Industrial Hygienists Absorption, Distribution, Metabolism, and Excretion atmosphere Agency for Toxic Substances and Disease Registry bioconcentration factor Board of Scientific Counselors Centigrade Centers for Disease Control Cancer Effect Level Comprehensive Environmental Response, Compensation, and Liability Act Code of Federal Regulations Contract Laboratory Program centimeter central nervous system day Department of Health, Education, and Welfare Department of Health and Human Services Department of Labor electrocardiogram electroencephalogram Environmental Protection Agency see ECG Fahrenheit first filial generation Food and Agricultural Organization of the United Nations Federal Emergency Management Agency Federal Insecticide, Fungicide, and Rodenticide Act feet per minute foot Federal Register gram gas chromatography generation high-performance liquid chromatography hour Immediately Dangerous to Life and Health International Agency for Research on Cancer International Labor Organization inch adsorption ratio kilogram metric ton organic carbon partition coefficient octanol-water partition coefficient CHLOROFORM L LC LCi, LCs0 LD, LDs, LOAEL LSE m mg min mL mm mm Hg mmol mo mppcf MRL MS NIEHS NIOSH NIOSHTIC ng nm NHANES nmol NOAEL NOES NOHS NPL NRC NTIS NTP OSHA PEL pg pmol PHS PMR ppb ppm ppt REL RfD RTECS sec SCE SIC SMR APPENDIX C liter liquid chromatography lethal concentration, low lethal concentration, 50% kill lethal dose, low lethal dose, 50% kill lowest-observed-adverse-effect level Levels of Significant Exposure meter milligram minute milliliter millimeter millimeters of mercury millimole month millions of particles per cubic foot Minimal Risk Level mass spectrometry National Institute of Environmental Health Sciences National Institute for Occupational Safety and Health NIOSH’s Computerized Information Retrieval System nanogram nanometer National Health and Nutrition Examination Survey nanomole no-observed-adverse-effect level National Occupational Exposure Survey National Occupational Hazard Survey National Priorities List National Research Council National Technical Information Service National Toxicology Program Occupational Safety and Health Administration permissible exposure limit picogram picomole Public Health Service proportionate mortality ratio parts per billion parts per million parts per trillion recommended exposure limit Reference Dose Registry of Toxic Effects of Chemical Substances second sister chromatid exchange Standard Industrial Classification standard mortality ratio C-2 CHLOROFORM STEL STORET TLV TSCA TRI TWA U.S. UF yr wk R WR LIANA NIV YV TE 0 3 APPENDIX C short term exposure limit STORAGE and RETRIEVAL threshold limit value Toxic Substances Control Act Toxics Release Inventory time-weighted average United States uncertainty factor year World Health Organization week greater than greater than or equal to equal to less than less than or equal to percent alpha beta delta gamma micrometer microgram C-3 vr U.S. GOVERNMENT PRINTING OFFICE: 1997-538-161 NTT LES BERKELEY LIBRARIES CD8LAYYELS