EXPOSURE TESTS FOR ORGANIC COMPOUNDS IN INDUSTRIAL TOXICOLOGY J. S. DEPARTMENT OF HEALTH, EDUCATION, AND WELFARE 3ub|ic Health Service Senter for Disease Control National Institute for Occupational Safety and Health i " „ Air/M7 [[?/( gw ")./"fr If; LEXPOSURE TESTS FOR ORGANIC COMPOUNDS '1} IN INDUSTRIAL TOXICOLOGY Jerzy K. Piotrowski U.S. DEPARTMENT OF HEALTH, EDUCATION, AND WELFARE Public Health Service Center for Disease Control National Institute for Occupational Safety and Health Cincinnati, Ohio September I977 For uh by thc Supel-intendent ol Docmnenu, U.S. Government Printing Office. Washington, D.C. 20402 I v” , 14 .; R A q „„ 1235 ;PG ‘PUBL Mention of company name or product does not constitute endorse- ment by the National Institute for Occupational Safety and Health. DHEW (NIOSH) Publication No. 77-144 PREFACE This monograph was written in the Department of Toxicological Chemistry. Institute of Environmental Research and Bioanalysis. Medical Academy of Lodz. under the auspices of Polish-American Program 05-5l6-l.l6 financed by the National Library of Medicine. Bethesda. Maryland. USA. It was translated from Polish by Julian Liniecki. M.D.. PhD. Dr. Habil. The National Library of Medicine gave the National Institute for Occupational Safety and Health per- mission to publish this monograph in the NlOSH Technical Report Series. ln preparing the manu- script for publication some editorial rewriting was done to make the text conform with standard En- glish sentence construction and word usage. However. special effort was made not to alter the factual material. the intent. and the conclusions of the author. Austin F. Henschel, Ph.D. Editor iii I: :'” .;)lł CONTENTS l. INTRODUCTION 2. ABSORPTION ROUTES Absorption via respiratory tract ............................................. ~ ..... Cutaneous absorption ........................................................... Contact absorption ............................................................. Cutaneous absorption of vapours of organic compounds ............................. Evaluation of cutaneous absorption in conditions of occupational exposure ............. 3. BIOTRANSFORMATIONS lnterspecies differences in the metabolism of organic compounds ...................... Mechanism of biotransformation ................................................. Influence of foreign compounds upon the metabolism of xenobiotics ................... Stimulation of m1crosomal enzymes ............................................... Inhibition of microsomal enzymes ................................................ Influence of physiological factors on the metabolism of foreign compounds ............. Dose-dependent variations in the metabolism of xenobiotics .......................... 4. METABOLIC AND EXCRETION KINETICS Introduction ................................................................... Kinetic models and compartmental analysis ........................................ Real meaning of kinetic parameters ................................................ Simplification of kinetic models .................................................. Rhythms of exposure ........................................................... Continuous exposure ........................................................... Interrupted exposure ............................................................ Cumulation of foreign substances in the body ....................................... Systemic cumulation and exposure tests ........................................... 5. WORKING OUT THE EXPOSURE TESTS ON VOLUNTEERS IN EXPERIMENTAL CONDITIONS Introductory remarks ........................................................... Toxicological chambers ......................................................... Purpose and design of the experiments ............................................ Inhalatory experiment ........................................................... Cutaneous exposure ........................................................... Experimental repeated exposure .................................................. Physiological or ”background” levels of metabolities ................................. Precision of exposure tests ....................................................... Safety problems ................................................................ 6. BENZENE Absorption .................................................................... Metabolism and elimination ..................................................... Determination of benzene and its metabolites ....................................... Determination of benzene in expired air ........................................... Determination of phenol in urine ................................................. Other methods ................................................................. Exposure tests ................................................................. Benzene in expired air ........................................................... Available data on industrial exposure ............................................. V 7. TOULENE Absorption .................................................................... Metabolism and elimination ..................................................... Determination of toulene ........................................................ Determination of benzoic and hippuric acid in urine ................................. Exposure tests ................................................................. Available data on industrial exposure ............................................. 8. XYLENES Absorption .................................................................... Metabolism and elimination ..................................................... Methods for determination of xylene and its metabolites ............................. Exposure tests ................................................................. lnterpretation of the test for simultaneous exposure to xylenes and toluene ............. 9. ETHYLBENZENE Absorption .................................................................... Metabolism and elimination ..................................................... Determination of ethylbenzene and its metabolites .................................. Exposure test .................................................................. Available data on industrial exposure ............................................. 10. STYRENE Absorption .................................................................... Metabolism and elimination ..................................................... Methods for determination of styrene and metabolites ............................... Styrene Mandelic acid in urine Phenylglyoxylic acid in urine Physiological levels ............................................................. Exposure tests ................................................................. Available data on industrial exposure ............................................. ll. PHENOL Absorption ............................................................ « ........ Metabolism and elimination ..................................................... Kinetics of phenol excretion ...................................................... Methods for determination of metabolites .......................................... Other methods ................................................................. Exposure test .................................................................. Available data on industrial exposure ............................................. 12. ANILINE Absorption .................................................................... Metabolism .................................................................... Determination of the metabolites ................................................. Other methods ................................................................. Exposure test .................................................................. Specificity of the exposure test ................................................... Available data on industrial exposure ............................................. l . NlTROBENZEN.E—~~~-~ ”'” /Absorption .................................................................... Metabolism and elimination ..................................................... Metabolic and excretion kinetics .................................................. Methods for the determination of the metabolites ................................... Other methods ................................................................. Exposure test .................................. _ ................................ Specificity of the test ............................ _. ............................... Available data on industrial exposure ............................................. vi 14. BENZIDINE 81 Absorption .................................................................... 81 Metabolism and elimination ..................................................... 81 Determination of benzidine and its metabolites in urine .............................. 81 Identification and determination of individual free diamines using paper chromatography. 82 Approximate evaluation of the total content of aromatic amines in urine ............... 82 Detection and determination of benzidine metabolites in urine ................ - ........ 82 Other methods used in prophylactic examinations ................................... 82 Exposure test .................................................................. 82 Available data on industrial exposure ............................................. 82 15. TRICHLOROETHYLENĘ ,,,,,, 86 Absorption .................................................................... 86 Metabolism .................................................................... 86 Elimination .................................................................... 87 Methods for determination of TR! and its metabolites ............................... 89 Trichloroethylene Trichlo roacetic acid Trichloroethanol Total trichlorocompounds (TTC) Exposure tests ................................................................. 91 TRI in expired air Trlchloroethanol m ur1ne Tr1chloroacet1c ac1d 1n urine Total trichlorocompounds (TTC) ................................................. 94 Available data on industrial exposure ............................................. 94 16. TETRACHLOROETHYLENE 98 T'" ”"”—"Ab 'so'rp't ion .................................................................... 98 Metabolism and elimination ..................................................... 98 Methods for determination of tetrachloroethylene and its metabolites .................. 99 Determination of trichloroacetic acid (TCA) and total trichlorocompounds (TTC) ....... 99 Exposure tests ................................................................. 99 Available data on industrial exposure ............................................. 100 17. CARBON DISULPHIDE 102 Absorption .................... * ................................................ 102 Metabolism and excretion ....................................................... 102 Determination of carbon disulphide and its metabolites .............................. 103 Exposure tests ................................................................. 103 Data on industrial exposure ...................................................... 104 18. PARATHION 107 Absorption .................................................................... 107 Metabolism and elimination ..................................................... 107 Determination of parathion and its metabolites ..................................... 108 lnterpretation of urinary p—nitrophenol determinations ............................... 108 Available data on industrial exposure ............................................. 109 19. FENITROTHION 112 Absorption .................................................................... 112 Metabolism and elimination ..................................................... 112 Methods for determination of p-nitro—m—cresol in urine .............................. 113 Other methods ................................................................. 113 Exposure test .................................................................. 113 Data on industrial exposure ...................................................... 113 20. DDT 115 Absorption .................................................................... 115 Metabolism and elimination ..................................................... 115 vii 21. 22. 23. 24. Determination of DDT and its metabolites ......................................... Determination of DDT and its metabolites in blood and adipose tissue by means of gas chromatography ............................................................. Determination of urinary DDA by means of gas chromatography ..................... Background levels of DDT and its metabolites ...................................... Exposure tests ................................................................. 'DDT in adipose tissue DDT in serum DDA in urine Available data on occupational exposure ........................................... OTHER SUBSTANCES Aliphatic alcohols .............................................................. Formaldehyde .............................................. ' ................... Acetone ....................................................................... Aliphatic alcohol esters .......................................................... Lower chlorinated hydrocarbons .................................................. Aromatic hydrocarbons ......................................................... Aromatic nitro- and aminocompounds ............................................ SELECTED GENERAL BIBLIOGRAPHY AUTHOR INDEX SUBJECT INDEX viii 116 116 117 117 118 ”9 122 I22 122 122 123 123 123 123 127 135 l. INTRODUCTION The strategies for industrial health protection, being developed in numerous countries today, in- clude more efficiently detecting the subtle, initial symptoms of chronic intoxications and dis- eases induced by noxious agents. Practical con- clusions drawn from surveys attempt to set the permissible levels of exposure sufficiently low so as to prevent the manifestation of pathological changes, even after prolonged exposure lasting for several decades. The capital investment in- volved in securing such low levels of exposure is usually enormous; and, therefore, attempts to estimate as precisely as possible the real magni- tude of exposure, without incorporating exces- sively wide margins of safety, is fully justified. Systems for exposure evaluation based on assess- ing concentrations of toxic compounds in the air of the workplace. and safety criteria based solely on the magnitude of these concentrations, are not always satisfactory. First of all, such systems ne- glect absorption of toxic substances by routes other than the respiratory tract. Cutaneous ab- sorption is a matter of concern, and its im— portance becomes all the more appreciated the more thoroughly it is studied. Second, even when substances are considered which are-absorbed chiefly or exclusively via the respiratory tract, bas- ing the estimates of real exposure solely onanal- ysis of air concentrations may be inadequate. Movement from place to place while supervising numerous operations in a manufacturing process, spiked concentrations near or inside installations, and lengthy breaks from exposure which occur when workers are temporarily outside of the premises make reasonable interpretation of mea- sured values very difficult. Additionally, in cases where the toxic substance ShOWS a pronounced ability to accumulate in the body, a body burden attained in preceding periods may be high enough to create a serious danger despite an ac- tual present moderate exposure. The above reasons speak for supplementing air analysis in manufacturing premises with individu- al exposure tests on workers. Knowledge of the real individual exposure could permit flexible ap- plication of various prophylactic measures. These are often less expensive than general reduction of the concentrations in the entire plant, and some- times are much more effective. Theoretically, there are two categories of assays that could be applied as exposure tests: a) deter- mination of a toxic substance or its metabolites in biological media sampled from exposed indi- viduals, and b) following the intensity of the bio- chemical reactions that become manifest under the influence of excessive exposure. In spite of the fact that the latter method would detect an already induced biochemical lesion, review of the literature points to inherent limitations of this technique. For organic compounds two im- portant examples are: inhibition of acetyl- cholinesterase under the influence of phos- phoorganic compounds and carbamates, and methemoglobinemia produced by the metabo- lities of aromatic nitro- and amino compounds. Without depreciating the usefulness and sound- ness of these tests, it appears justified to empha- size that their range of applications is much nar- rower than that of the direct determination of toxic compounds and their metabolites in biolog- ical samples—the concept which is to be further developed in this book. Evaluation of exposure to chemicals is often per- formed by specialists in industrial toxicology. Such specialists may not be competent in medical procedures (e.g., venopuncture). Because of such reasons as relative urine and blood concen- trations, limited availability of frequent blood and tissue samples, etc. the majority of earlier tests were based on analysis of urine, and more recently also on expired air. Thus, exposure tests form a complementary method for the evaluation of exposure in industry, which can be used by those who supervise the air concentration in in- dustrial plants. General knowledge of the situ- ation in a plant makes the interpretation of ex- posure tests easier. This method, which combines the input from exposure tests with general super- vision of occupational conditions, suggests the need for sampling the media to be analysed from workers directly at working place. This approach helps to eliminate instances of misunderstanding that otherwise may be particularly frequent and devastating when interpreting the levels of 'sub- stances that rapidly turn over in the body. The same methods also may be used by medical per- sonnel in ambulatory tests. Knowledge of meta- bolic kinetics permits extrapolations of the levels observed at some point during the day, for in- stance. to that at the end of work shift, and to make both comparable. However, in general, ap- plication of exposure tests in this way has lim- itations; and the interpretation of results in diffi- cult. Exception must be made here, of course, for the substances with pronounced ability to accu- mulate in the body. e.g., DDT. The fate of organic compounds in the animal body became better recognized in the nineteen forties and fifties. The main outlines were de- scribed by R. T. Williams in his Detoxication Mechanisms. During these same years great progress in pharmacokinetics and analytical chemistry was made. At the same time, the idea of utilizing biological analyses for assessment of individual exposure of humans to industrial or- ganic compounds developed in numerous coun- tries. The approach varied from one author to an- other. Thus, whereas Elkins seemed to prefer direct industrial experience, Teisinger and his col- leagues devoted their main efforts to basic in- vestigations on absorption and elimination of or- ganic compounds in volunteers exposed in controlled experimental conditions. The Polish authors have been trying to combine precision of experimental studies with applicability of the tests to industrial conditions. Examples, among others. for benzene, toluene, xylene, phenol, ani- line, nitrobenzene, and carbon disulphide are de- scribed in specific chapters in this book. The exposure tests are not treated at length in textbooks. Some elements of the problem, from various viewpoints, were taken up in the mono- graphs of Teisinger and his coworkers in 1956, and of Elkins in 1959; by the books of Dut- kiewicz et al. in 1964, and by Gadaskina and Filov in l97l. Some elements of the theory are contained in papers by Teisinger, Elkins, Truhaut, and others. some of which have been included in the selected general bibliography. In the present monograph the author has pur- sued the following goals: a) to present actual situations and trends in the development of exposure tests for the more important substances widely elabo- rated in this respect; b) to review the information about the stage of development of the tests for sub- stances less frequently studied, and at the same time to indicate compounds for which the tests could be applied eventually in practice. if further elaborated; c) to present a possibly coherent concept of the exposure tests and the methods for their JERZY K. PIOTROWSKI elaboration and interpretation that would render newly acquired data comparable with each other; and d) to present actual problems in the field calling for. and at the same time permitting, fast experimental solution. The monograph is not a textbook, and therefore detailed analytical procedures have been omitted. For these the reader will have to go to the origi- nal reports. ln the general part of this book only those aspects of absorption, metabolism, and pharmacokinetics are discussed which are rele- vant for the elaboration and application.of ex- posure tests. These chapters were not intended to be exhaustive. either in the treatment of these highly complex problems or in the complete se- lection of a bibliography. Nevertheless, the au- thor hopes they will provide the reader with a key to specialised original articles where more de- tailed and complete information can be found. ln selecting l5 compounds. the information on which is reviewed in detail, the author was mo- tivated by the scope of existing data, but also to some extent by his own experience and personal engagement in the discussed problems. Thus, in spite of the fact thatthe selection was somewhat arbitrary. the author hopes it reflects rather ade- quately the existing body of information. ln spite of the fact that the subject of this mono- graph is rather narrow. the author had to rely on generous assistance of his coworkers. Gratitude for their valuable help in the bibliography search is expressed to Mr. K. Walczak and Mr. A. Podoski; Dr. W. Kaszper, Dr. lwona Balcerska, Mrs. Jadwiga Szymanska, Mrs. Elzbieta Komsta- Szumska and Mirs. Honorata Andrzejczak have taken part in editorial work on some of the chap- ters. The author feels particularly indebted to his secretary. Miss Danuta Poteraj, whose patience. energy. goodwill. and competence have consid- erably facilitated completion of the work. The National lerary of Medicine greatly assis- ted the author in obtaining relevant literature by providing bibliographic surveys through the MED- LARS system involving the MEDLINE and the TOXLINE systems. Assistance in obtaining the information through the MEDLARS system„ given by the project-officer of the program. Mr. R. Worthington. is most sincerely appreciated._ Expressions of gratitude are also forewarded to Dr. Jeanne L. Brand, Chief, International Pro- grams Division. Extramural Programs, who has manifested her enormous goodwill and under- standing for the project throughout the total pe- riod of its realisation. 2. ABSORPTION ROUTES The two essential routes of absorption for indus- trial chemicals are the respiratory tract and the skin. Absorption which takes place through the lungs can be roughly assessed, even in industrial conditions, through air analysis. Results thus ob- tained can be interpreted by comparison with the TLV values. Absorption via the skin, on the oth- er hand, evades this method of measuring ex- posure. This is one of the arguments for strongly advocating the use of exposure tests. ABSORPTION VIA RESPIRATORY TRACT Absorption of organic compounds may take place both in the upper and in the lower parts of the respiratory tract. Two basic processes may be recognized here, depending on the physical prop- erties of the toxic substance. If the toxic sub— stance is present in air in the form of an aerosol absorption will be preceded by deposition of the substance in the upper respiratory passages. When very finely dispersed particles are consid- ered, the deposition will be in the alveolar re- gion. For aerosols of organic compounds these processes have not been sufficiently well studied, either from the viewpoint of particle size distribu- tion for different substances and types of aerosol generation. or from the viewpoint of total per- centage retained in the human respiratory tract. The same refers to the dynamics of the retention in individual parts of the tract. Until more specif- ic data become available, it seems that applica- tion of the general principles of retention dynam- ics for aerosol particles is in order. These have been worked out for radioactive aerosols (see Task group on lung dynamics, 1966). A far greater body of data, however, exists for respiratory absorption of substances that are dis- persed in air as gases and vapours. The basic ab- sorption mechanism is gas diffusion, and the quantity essential for effectiveness of this process is the partition coefficient between air and blood". A more detailed discussion of the prob- lem has been presented elsewhere (Piotrowski, l97l); here only the most essential aspects of the problem will be outlined. Values for the coefficient between air and blood have been estimated for only a few substances; but the coefficients for partition between air and water are available for a far greater number of compounds. These have usually been obtained while working out conditions for absorption of a substance in water with analytical objectives in mind. (The quantity is called the “aeration constant”) The respective values of the air/water partition coefficients for organic compounds may vary by several orders of magnitude: from about 10° for carbon disulphide, through 10”3 for acetone, acrylonitrile and nitrobenzene; to 10'5 for aniline and toluidine. The yield of pulmonary absorption (i.e.. retention of vapours in the lungs) increases with a decreasing partition coefficient for air/ blood (water); however variation of the retention is by far less than the variation of the respective partition coefficients. For example, lung reten- tion of aniline vapour (partition coefficient 10'5) amounts to about 90 per cent; of nitrobenzene (partition coefficient l0‘3) to 80 per cent; of ben- zene (partition coefficient lO") to about 50—75 per cent. and finally of carbon disulphide (par- tition coefficient 10°) to about 40 per cent. ln the course of continuous inhalation exposure to volatile compounds, the concentration of the compound in the blood increases toward an equi- librium between absorption, on the one hand, and metabolism and elimination, on the other. This is accompanied by a decreasing retention. This decrease of retention in the course of con- tinuous inhalation may be observed in practice ' The reciprocal. i..e blood/air partition coefficient Is often used in toxicological literature. In this monograph air/ blood partition coefficient is used in accordance with the authors previous monograph (1971) where it Was incor- porated Into a system ol kinetic description of elimination processes. for compounds whose air/blood (water) partition coefficient is of the order of lO'3 or greater. Among the compounds discussed in this mono- graph, decreasing retention (an example in Fig. 2-I) is characteristic for carbon disulphide, tri- and tetrachloroethylene, benzene, toluene and ni- trobenzene. Nothing comparable has been ob- served for aniline (low value of partition coeffi- cient) and styrene. In the case of styrene, analogous to acrylonitrile (Rogaczewska and Pio- trowski, I968), the justification for lack of time- dependent decrease of retention seems to lie not so much in the magnitude of the physical par- tition coefficient as the reactivity of the vinyl group, which may enter alkylation reactions with blood constituents. Usually, the retention (R) of organic vapour compounds in the respiratory tract has been studied on human volunteers in chamber-type experiments, where it can be determined directly from the ratio of concentrations: R:_i__e (1) C; and Ce denote the concentrations in inhaled and exhaled air, respectively. The technique for the determinations varied widely between au- thors. In older studies some errors, related to ad- sorption of the substance inquestion on tubes providing air for breathing, or to vessels or sacks in which it was collected, could not be excluded. To avoid these complications, when classic meth- ods of air analysis by aspiration are applied in the present author’s laboratory (see chapters on “Toluene” and “PhenoI”). sampling is performed from exhalatory and inhalatory channels directly at the respirator. When gas-chromotographic de- terminations are made utilizing small air samples, the problem of adequate sampling of the exhaled air assumes particular significance. To estimate the retention (balance), the sample should be re- presentative not just of a specific fraction of the breath (e.g. alveolar air), but of the total exhaled air volume (mean concentration). This may be accomplished by using sufficiently large bags, provided the synthetic material of which they are made has no pronounced adsorptive capacity for the compound in question. Examples of an ade- quate selection of the material for a given com- pound may be found in the paper by Smith and Pierce (I970). The most often used one at present is Saran, which is characterized by relatively JERZY K. EIOTROWSKI 0.2' 0.1 30 60 120 180 T. min Fig. 2-]. The retention of carbon disulphide va- pours in the respiratory tract as afunction of time. with continuous exposure (Jakubowski, I 966 ). Taken from: Piotrowski J.: The application Of metabolic and excretion kinetics to problems of industrial taxicologr. US-Governmcnt Printing Office. I97I. Page 77, Fig. 9 weak adsorptivc capacity. Another solution may be application of a respirator with a solid ab- sorber from which the substance to be deter- mined is eluted and analyzed by means of gas chromatography (Sherwood and Carter, I970). The extent to which the technique for estimating retention may affect the results can be appre- ciated from consideration of the wide variation in , data for individual substances given in specific chapters of this book. The total amount of a toxic compound absorbed in the respiratory pathways depends upon its concentration in the air, the duration of ex- posure, and the pulmonary ventilation rate. The last factor, even if most authors have been con- scious of its importance, seems responsible for a lot of misunderstanding in the theory and prac- tice of exposure tests. It is obvious that venti- lation rate, which in a practical field assay can- not be measured, depends upon the physical activity of the individual tested (Zenz and Berg, I970). In chamber-type experiments, in which volunteers are exposed in a sitting position not subject to additional physical effort, the venti- lation rate is usually of the order of 0.3-0.4 and ABSORPTION ROUTES 0.4-0.5 m3/ hr, in females and males, respectively. lt is reasonable to assume that for people engaged in light work, corresponding to a slow walk, the rate is at least doubled. ln calculating the amounts of substances absorbed through the pul- monary tract in industrial conditions, various au- thors assume different average ventilation rates as typical for workers performing light work: from about 0.8 m3/h to about 1.25 m3/h. The latter figure (10 m3 per 8 hour working shift) is often used as the estimate in the U.S.A. Taking the above factors into account, the total amount of a substance absorbed in the respira- tory tract over a period of exposure would be the product of air concentration, duration of ex- posure, ventilation rate, and retention rate. Formulas for calculating the absorbed amounts and air concentrations by extrapolation from chamber exposure studies to field conditions are discussed in the chapter “Elaboration of exposure tests in experimental conditions". CUTANEOUS ABSORPTION The role ofthe skin in the absorption of industrial chemicals is commonly recognized nowadays. For some compounds the skin is the main port of entry under conditions of occupational exposure (cg. benzidine, parathion); for others this route is roughly equivalent to inhalatory absorption (e.g. aniline, nitrobenzene, phenol); even for the bulk of other organic compounds the con- tribution of cutaneous to the total absorption cannot be neglected or excluded. For instance, with a great number of phosphoorganic com- pounds. as studied in animals, intoxication may occur after cutaneous penetration (Kundijev, I967). In the MAC list compiled in recent years (“Threshold limit values...", ACGlH, 1976), cau- tion as to the possibility that cutaneous absorp- tion exists is voiced for about l/4 of the organic compounds, and for a significant number of inor- ganic substances. In cutaneous absorption either transepidermal or transfollicular passage may oc- cur; for organic compounds the transepidermal route seems to be of paramount importance (Djuric, 1966). From the experience gathered so far, it appears that the solubility of a substance both in water and in lipids, reflected in a not too high coefficient of fat/water distribution, is a prerequisite for significant absorption through the skin. This feature may result from structural peculiarities of the skin; a penetrating substance has to pass layers or membranes rich either in protein-water or lipids. From fragmentary experi- mental and numerous practical observations, it follows that the cutaneous absorption rate of or- ganic compounds increases both with increasing temperature and with increasing moisture of the skin and, therefore, becomes facilitated in warm seasons. The absorption of organic compounds may follow surface contamination of the skin (or of clothes); for some compounds it may occur di- rectly from the gaseous phase. CONTACT ABSORPTION Under industrial conditions absorption of a sub- stance from a contaminated skin surface may re- sult in two basic situations: a) When a job requires continuing repeti- tion of dermal contact, absorption of even relatively volatile substances takes place — substances which otherwise would quickly evaporate from the surface of the skin, pre- cluding significant penetration. The follow- ing are examples that may serve here: man- ual applying a glue containing organic solvents, e.g., benzene and its homologues; manual painting with paints containing the same ingredients; binding broken viscose rayon fibres in the coagulating solution, with carbon disulphide as one of the sub- stances present. b) Another situation concerns relatively non—volatile compounds that occasionally contaminate the skin, stay on the skin for a prolonged period, and undergo slow ab- sorption. Typical examples here are ben- zidine' and parathion, and to some degree compounds that may be characterized as liquids of high boiling temperature such as aniline and nitrobenzene. Information on the cutaneous absorbability of these substances after direct contact with the skin comes from various sources which, due to vari- ability of experiments or incidental observations, do not provide a common yardstick for quan- titative comparisons. Available data, applicable directly to man, have been usually derived from following sources: a) Cases of acute intoxication from con- tamination of a large skin surface (exam- ples in the literature may be found for ani- line, nitrobenzene, phenol, parathion, etc.). b) Observations of urinary excretion of pertinent metabolites in workers in whom inhalatory absorption could be excluded (e.g. parathion). However, in both situations (a and b) the data do not permit assessment of the absorption rate. The latter may be estimated from experiments on volunteers. In the laboratory of the author the following method has been applied: a known amount of a substance is applied to the skin in a way excluding evaporation, or enabling cor- rections to be made for losses by evaporation. After a given interval of contact, the amount re- maining at the site of application is determined and, from the differences, the absorption rate is calculated. This type of experiment has been per- formed on animals in the author’s laboratory for aniline (Piotrowski, 1957), for nitrobenzene (Sal- mowa and Piotrowski, I960), for benzene (Hanke et al., 1961), and for acrylonitrile (Rogaczewska and Piotrowski, 1968). The values, representing upper limits of the absorption rate after applica- tion of an infinitely thick layer, were of the order of l mg cm'lhour‘I for aniline and nitrobenzene, and about 3 times lower for benzene and acrylo- nitrile. Experiments performed by Dutkiewicz and Tyras (1968), using a similar technique for toluene, xylene. and styrene, yielded considerably - higher values. The disadvantage of this type of experiment lies in the technical difficulty in secur- ing, conditions for absorption that eliminate los- ses due to evaporation. Thus experimental er- rors usually tend to overestimate the absorption rate. It would be desirable to supplement the re- sults of these experiments with data on the levels of the-absorbed compound in blood, or the ex- cretion of its metabolites in urine. This type of internal control was introduced into the cited ex- periments on aniline and benzene, and proved useful. Technical difficulties arising from the de- termination of metabolites, or from the esti- mation of the absorbed amount of a substance, due to lack of knowledge of the efficiency of in- dividual metabolic pathways, could be overcome by using a labelled compound and estimating the absorbed amount from the total activity of the label (NC) excreted in urine, regardless of its chemical form. This type of experiment is some- times applied in dermatological research (e.g., seez' Feldman and Maibach, 1970). However, the conditions of these experiments differ so much from those required by toxicologists as to render «quantitative comparisons meaningless. This con- clusion is strengthened by the fact that the cited authors classified nitrobenzene as a compound poorly absorbed through the skin. JERZY K. PIOTROWSKI Cutaneous absorption of organic compounds is the most important factor arguing for the devel- opment and application of exposure tests. When determining a given metabolite in urine, one as- sumes that the efficiency with which it is formed after cutaneous absorption is close to that found after inhalation. Further generalization of this as- sumption into an hypothesis on the independence of the metabolism of an organic substance from its route of entry is not required because of the minor role of the gastrointestinal tract as a route of entry. However, a study of Dollery et al. (1970) showed differences in the metabolism of compounds administered via the alimentary tract. With regard to inhalatory and cutaneous absorp- tion, the number of controlled experiments aimed at verifying or discarding the independence of the metabolic pathway from the route of entry hy- pothesis is too limited to serve as a proof. The efficiency of the metabolic pathway of aniline leading to p-aminophenol as the end-product has been studied in man and found independent of route of absorption, whether dermal or in- halatory (Dutkiewicz and Piotrowski, I961). ln addition, no significant difference was found in humans when benzene was considered and phe- nol determined in urine as the metabolite (Hanke et al.. 1961). In rats, no change in excretory ki- netics or ratio of metabolites (p—nitrophenol vs. p-aminophenol) was found when nitrobenzene was administered intravenously or applied to the skin (Piotrowski et al., 1975). On the other hand, fragmentary observations made by Dutkiewicz and Tyras (1969) with regard to some aromatic hydrocarbons pointed to a much lower effi- ciency“ for selected metabolites when skin ab- sorption and inhalatory absorption were com- pared. In the case of parathion, the independence of metabolism from absorption route has been discarded based on observations that after cuta- neous absorption given concentrations of p-nitro- phenol in urine are not accompanied by the ex- pected toxic effects (see “Parathion”). CUTANEOUS ABSORPTION OF VAPOURS OF ORGANIC COMPOUNDS Absorption of some compounds can occur direct- ly through the skin from the gaseous phase. This Efficiency is understood here as a ratio of an amount of metabolite excreted in urine to the absorbed dose. calcu- lated from the amount that could not have been recovered from the application site on the skin. The reservations voiced above with respect to this technique of assessing cutaneous absorption-rate should be born In min . ABSORPTION ROUTES phenomenon was observed and found to play a significant role for aniline, nitrobenzene, and phenol which otherwise are well-absorbed when applied directly. On the other hand, this type of absorption has not been found significant for benzene and toluene. With respect to the former compounds the issue is of practical importance due to the fact that at given air concentrations amounts absorbed via skin are comparable with those which enter the system through the respira- tory tract. An increasing rate was found for cuta- neous absorption of aniline and nitrobenzene with increasing ambient temperature, and for ani- line with increasing humidity. These are the same factors which are known to favour the contact absorption of the same compounds. lt is inter- esting to note that normal work clothes have only a limited effect upon the absorption rate, lowering it by some 20-30% relative to that in nude persons. The mechanism of cutaneous absorption of or- ganic vapours is not well-understood. The rate of absorption is roughly proportional to the air con- centration of the vapours. The process is accom- panied by adsorption of the substances on skin surface, which can be demonstrated by elution. Taking into account the factors affecting both contact and vapour absorption, as well as nega- tive results that have been obtained for highly volatile substances, it cannot be excluded that adsorption on skin surface is a first and indis- pensable factor in the process. If this should prove true, it could be expexted that absorption from the gaseous phase would occur with all compounds of poor volatility that display good adsorption when in direct contact with the skin. With regard to compounds for which cutaneous absorption of vapors is significant, this cutaneous absorption should be considered in elaborating the relationship between absorption and excre- tion (exposure tests). lf in the course of test elab- oration an exposed subject has spent some time in a toxicological chamber, the absorption through the skin is automatically, even if inad- vertently, taken into account. If, on the other hand, as practiced in the author’s laboratory, in- halation and dermal absorption are studied sepa- rately, it is possible to consider and describe both processes independently in a manner presented in the chapter “Experimental elaboration of ex- posure tests“. Information on cutaneous absorption of organic vapours, as discussed above, was obtained from experiments on volunteers; and the evaluation of the absorbed amounts was based on exposure tests. All reservations discussed previously, re- garding possible differences in metabolic rates and efficiencies when organic compounds are ab- sorbed by inhalatory and dermal routes, are also pertinent here. The experiments were made on resting volunteers; for even if strong dependence of cutaneous absorption of vapours on physical activity seems unlikely, there are no experimental data to preclude such a possibility. EVALUATION OF CUTANEOUS ABSORPTION IN CONDITIONS OF OCCUPATIONAL EXPOSURE ln occupational exp0sures quantitative evaluation of the role played by both of the principal ab- sorption routes is possible only by exposure tests. The underlying assumption is that there is no gross difference in metabolism relating to the ab- sorption routes. ln practical conditions two basic procedures have been applied: a) ln individuals working at a controlled rate. the absorption is estimated (exposure test) by eliminating totally one of the ab- sorption routes by means of individual pro- tectors: the inhalatory one by application of respirators, and the cutaneous one by means of an impermeable suit. This meth- od, among others, was used in evaluating parathion absorption (Durham, 1963). The disadvantage of this procedure is a possi- bility of differences in total exposure be- tween the series of investigations; moreover, systemic accumulation of the compound in question from preceding exposure must be taken into account. In addition, protective clothes and gloves made of rubber or syn~ thetics may not completely prevent pene- tration of a substance into the body (Locati et al., 1968). b) The study is made under normal condi- tions of exposure, and total absorption is evaluated from results of exposure tests. The inhalatory absorption is estimated from the concentrations of the test substance in the inspired and expired air over a mea- sured exposure duration. At the same time it should be noted that dermal absorption has two components: vapour and direct (skin) absorption. Total absorbed amount may be expressed by the formula: D(mg) = CT(RV +a) + S (2) where C = air concentrations of a sub- stance, T = duration of exposure, V = pul- monary ventilation rate, R = retention in the respiratory tract, a = coefficient of cu- taneous absorption from the gaseous phase, and S = contact cutaneous absorption. De- veloping further: D(mg) = CTRV + (CTa' + S) (Za) The second term in equation Za represents cutaneous absorption. The disadvantage of this method is the fact that the contribution of skin absorption is estimated indirectly from the difference. This procedure was ap- plied for aniline and nitrobenzne, among others, for which it was possible to detect considerable contact absorption. The potential role of cutaneous absorption can be assessed from data on the extent of con- JERZY K. PIOTROWSKI tamination of skin and working clothes. Exam- ples of such a procedure can be found in the literature for benzidine. aniline, nitrobenzene, and other substances. One of the methods is a pad- test: a textile or paper pad is attached in a stan- dardized way to exposed skin and the pads ana- lyzed for contamination at the end of the work period. This method has found wide application for pesticides, e.g.. DDT and parathion. (Dur- ham and Wolfe, I962). In this way a compara- tive measure of dermal exposure may be obtain- ed in individual workers, or when different technology is being applied. It should be empha- sized. however. that in no case should “dermal exposure“ be identified with the “dermal absorp- tion". As found by Durham (I963) using para- thion as an example. the amounts absorbed via the skin Were two orders of magnitude lower than those found on the skin (dermal exposure). REFERENCES Djuric D.: Biochemija i Biofizyka Industrij ics of Industrial Poisons). Beograd. I966. skich Otrova. (Biochemistry and Biophys- DoIlery C.T.. Davies D.S. and Conolly M.E.: Differences in the metabolism of drugs depending upon their route of administration. Int. Symp. on Clinical Pharmacology, Brussels. St. Catherine Press Ltd.. Bruges. I970, pp. 2I4-2I9. Durham W.F.: An additional note regarding measurement of the exposure of work- ers to pesticides. Bull. Wld. Health Org. 29, 279-28l, l963. Durham W. F. and Wolfe H.R.: Measurement of the exposure of workers to pes- ticides. Bull. Wld. Health. Org. 26, 75-91. I962. Dutkiewicz T. and Piotrowski J.”: Experimental investigations on the quantitative esti- mation of aniline absorption in man. Pure Appl. Chem. 3, 319-323. l96l. Dutkiewicz T. and Tyras H.: Skin abso Brit. .I. lndustr.Med. 25, 243. I968. rption of toluene, styrene and xylene by man. Dutkiewicz T. and Tyras H.: Porównaweze badania nad wchlanianiem toluenu. el_r- Iobenzenu. ksylenu. i styrenu przez skore u ludzi. (Comparative evaluation of dermal absorption of toluene, ethylbenzene, xylene and styrene in man). Med. Pracy 20, 228- 233. I969. Feldmann RJ . and Maibach H.l.: Absorption of some organic compounds through the skin in man. J. Investigative Dermatol. 54, 399-404, I970. Hanke J., Ditkiewicz T..! and Piotrowski J.: Wchłanianie benzenu przez skore u lu- dzi. (The absorption of benzene throughout the skin in men). Med. Pracy 12, 4I3- 426. I96I. K undijev J.I.: Gigienitsheskoje znatshenije problemy wsasywanija fos- foroorganitsheskich insekticidov therez kozshu. (Hygienic importance of dermal ab- sorption of the phosphoroorganic insecticides). DSc thesis, Medicmskij Institut tm. A,A. Bogomolca. Kiev. I967. Locati G.. Cavagna G.. and Bugatti A.: Study on the penetration ofphosdrin through protective gloves. Med. Lavoro 59, 342-345, I968. Piotrowski J.: Quantitative estimation of aniline absorption through the skin in man. J.Hyg.Epidemiol. (Praha) I, l-IO', I957. Piotrowski J.K.. Dutkiewicz B.. and Popinska E.: Wydalanie p-aminofenolu i p-ni- trofenolu w moczu szczurow po podaniu nitrobenzenu droga dozy/na i dermalna. ( The excretion of p-aminophenol and p-nitrophenol in the urine of rats following der- mal and intravenous administration). Bromatol. Chem. Toksykol. 8, 134-135, I975. ABSORPTION ROUTES Rogaczewska T. and .Piotrowski J.: Doswiadczalna ocena drog wchlaniania akrytonitrvlu u ludzi. (Experimental evaluation of the absorption routes of acrylo- nitrile in men.) Med. Pracy 19. 349-354, [968. Salmowa J. and Piotrowski J.: Proba ilosciowej oceny wchłaniania nitrobenzenu w warunkach doswiadczalnych. (Attempt on quantitative estimation of nitrobenzene re- sorption in experimental conditions). Med. Pney ll, l-l4, I960. Sherwood RJ . and Caner F.W.: The measurement of occupational exposure to ben- zene vapour. Ann. Occup. Hyg. 13, 125-146, 1970. Smith 8.5. and Pierce J.O.: The use of plastic bags for industrial air sampling. Am. Ind. Hyg. Assoc. J. 31, 343-348, 1970. Task group on lung dynanTics: Deposition and retention models for internal dosi- metry of the human respiratory tract. Health Physics 12, 173-207, [966. Treshold limit values for chemical substances in workroom air adopted by the Ameri- can gonference of Government Industrial Hygienists for 1973. J. Occup. Med. 16, 39- 44. l 74. Zenz C. and Berg B.A.: The influence of submaximal work on solvent uptake. J. Oc- cup. Med. 12, 367-369, I970. 3. BIOTRANSFORMATIONS The biotransformation of xenobiotics has been a subject of several monographs and textbooks. Among these is the review article by T.E. Gram and l.R. Gillette (1971). Reviews of several more specific subjects may be found in papers by R.T. Williams (1974). D.V. Parke (1972). and J.R. Fouts (1972). Those aspects that are directly rele- vant to exposure tests* will be briefly reviewed. INTERSPECIES DIFFERENCES IN THE METABOLISM OF ORGANIC COMPOUNDS The metabolic pathways of xenobiotics. observed in various animal species. are either identical or very similar. Nevertheless. the rates of the pro- cesses and the efficiency of various pathways. as well as the excretion of individual metabolites. may display pronounced quantitative interspecies differences. ln particular. examples of such dif- ferences may be found for the aromatic compounds. Thus. for instance. aniline is metabolized in some species mainly via hydroxylation in the para-posi- tion (rabbit. rat, man); whereas in others it is metabolized predominantly in the ortho-position (e.g. dog). There are considerable interspecies dif- ferences in the efficiency of biotransformation. particularly in the low dose ranges. ln rabbits the efficiency of p-aminophenol formation decreases with the increasing dose of aniline. while the re- verse is true for man. Nitrobenzene is metabo— lized in rats and rabbits mainly to p-amino- phenol. but in man p~nitrophenol predominates. For the former metabolite the turnover rate in rabbits and rats is high; whereas in man the rate is low. leading to systemic accumulation in re- peated exposures. ln experimental animals. sty- rene yields a constellation of metabolites. among which hippuric acid occupies the main position. In man. on the other hand. styrene is almost completely metabolized to mandelic acid and its oxidation product. phenylglyoxylic acid. "‘ The references omitted from the chapter can be found in chapters dealing with specific subjects. I0 The examples listed above are by no means ex- ceptional. In his review. Williams (1974) gave a series of further examples. pointing to a consid- erable variation in the efficiency with which a given type of transformation proceeds in different species. Such interspecies differences were found for biotransformation reactions of phase 1 (OX- idation. reduction. hydrolysis) for amphetamine. cumarine. biphenyl. quinic acid. and others. For instance. in rats. hydroxylation of amphetamine shows an efficiency of about 80 per cent; whereas in man the same process displays an efficiency of only 5 per cent. Aromatization of quinic acid to ben/oic acid is about 60 per cent efficient in man. and below 5 per cent in commonly used ex- perimental animals (rat. rabbit. dog). Similarly dramatic differences may be found in phase II biotransformations (conjugation). Nine- ty-five per cent of phenol in cat is conjugated with sulphuric acid: whereas in rat the glucuron- idc sulphate ratio is close to unity. On the other hand. in the pig. almost all the phenol is excreted in the form of glucuronide. ln different species conjugation of aromatic acids shows preference to various endogenous components. Thus. in man. arylacetic acids (e.g. phenylaeexienacid) are conjugated almost exclusively glithilglu‘tamine: whereas in non-primates the conjugation occurs mainly with glycine. Close similarities in metabolism of xeonbiotics link man and monkeys. The latter. however. are rarely used for these experiments. There are many exceptions to the idea that monkeys paral- lel man. For quantitative evaluation of human data direct knowledge of the metabolism of xe- nobiotics in man is indispensable. Quantitative interpretation of the data by comparison with available information obtained from animal ex- periments is subject to considerable uncertainty. ' MECHANISM OF BIOTRANSFORMATION Studies performed over the last 30 years have demonstrated that. in mammals, many processes by which xenobiotics are transformed in viva. BIOTRANSFORMATIONS NADP Xreduced flavoprotein Cyt P4503 yin”; NADPHX flavoprotein drug complex Cy? P450 oxidized drlug drug Cyt P450 Cy! P‘”2 V: CO CY' P4503 drug -O 02 drlug CY? 554502? e— I O : 2H+ H20 Fig. 3-1. Electron transport in hepatic microsomes (after Parke, 1972). Taken from: Parke D.V.: The effects of drugs and steroid hormones on the enzymes of the endoplasmic reticulum. In Effects of Drugs on Cellular Control Mechanisms, B.R. Rabin and R.B. Freedman, Editors. Page 70 — scheme. take place in the liver. On the subcellular level, these processes are localized in microsomes. The catalyzing enzymes localized in the smooth mem- branes of the endoplasmic reticulum have been given the name of “microsomal drug metabolizing enzymes". These are not easily identifiable due to their close association with the membranes and the difficulties encountered when solubilization is attempted. On the basis of considerable data it has been accepted that oxidation reactions catalyzed by these enzymes. which lead to incorporation of an oxygen atom into a molecule of a xenobiotic, proceed in the presence of molecular oxygen, and require participation of NADPH; or NADH; (in reduced form). One protein catalyzing a reaction of the type: RH+02+2H— ROH+ H20 (l) is cytochrome P—450. NADPHZ which is the do- nor of hydrogen atoms for reaction (I) does not ' transfer them directly into cytochrome P—450, but indirectly by means of the electron transport chain (Fig. 3—1), of which cytochrome C reductase and a nonidentified mediator (x) are components. An essential component of the discussed electron transport chain is also the second of the cyto- chromes, cytochrome b5; however, its role and closer localization in the chain are not well-un- derstood. This enzymatic complex is responsible, among other "things, for reactions of hydroxyl- ation of aromatic compounds. In this context it 11 appears strange that the same system, and in par- ticular the same enzyme (cytochrome P—450), can catalyze hydroxylation in two different directions (ortho and para) in various animal species. Some light is shed on this problem by observations of spectral changes in cytochrome P-450 that take place in the presence of various xenobiotics. The name cytochrome P—450 derives from the fact that, upon reaction with carbon monoxide, the reduced form of this cytochrome yields a compound with an absorption maximum at 560 nm. a form that is catalytically inactive. In the presence of various xenobiotics, added to a sus- pension of microsomes, two types of spectra orig- inate depending on the compound. Type I spec— trum (absorption minimum at 419-425 nm, the maximum at 385-390 nm) is obtained if pheno- barbital or the inhibitor SKF 525 (B-diethyl- aminoethyl-diphenyI-propylacetate hydrochloride) are added" to the suspension; type II spectrum re— sults from the presence of aniline and of another inhibitor, DPEA (2,4—dichloro—6—phenylphenoxy- ethylamine). Here the minimum and maximum are observed at 390-405 and 426-435 nm, respec- tively. From these observations it has been con- cluded that cytochrome P—450 can react with xenobiotics in two ways: firstly, through a lipoprotein component of the enzyme (type I), and secondly, by forming a complex with the haem-group (type II). The presence of two activ- ity sites in the enzyme could provide an expla- nation for the catalyzing of two different meta- bolic reactions by the 'same compound. Apart from that, it may be legitimately accepted that the protein component of cytochrome P450 is not a single entity. There are good reasons to suggest that at least 2 distinct forms of the en- zyme exist. and this may explain the discrep- ancies between the observed metabolic reactions in which this enzyme participates. For further re- view of this topic, a paper by Gram and Gillette (l97l) should be consulted. To maintain the electron transport chain (pre- sented in Fig 3-l) in action, the regeneration of the oxidized form of NADP by means of reduc- tion is necessary. In contrast to the mito- chondrial respiratory chain that catalyzes normal biological oxidation in the presence of NAD (ox- idized form). the microsomal chain does not con- tain elements that regenerate the oxidized co- enzyme. There must be, therefore, a cooperation between microsomal metabolic processes and other biochemical reactions that are “consuming” the oxidized form of NADP. The process in question here is the pentose cycle of glucose ox- idation, catalyzed by NADP-dependent glucose—6 —phosphate dehyd rogenase. 12 From the available data it may be concluded that the oxidation mechanism discussed above is en- gaged in the metabolism of aromatic compounds: oxidation of benzene to phenol; of toluene and xylenes to benzoic acid and toluic acid; of aniline and benzidine to hydroxy derivatives; of styrene to epoxyde; of parathion to paraoxon; etc. Even if this did not follow directly from the mechanism presented in Figure 3-1, the micro- somal enzymes also catalyze a series of other re— actions belonging to phase 1 of biotransforma— tion. Included are reactions of sulphoxidation, de- sulphuration, 0— and N-dealkylation, and some others. The microsomal fraction displays cata- lyzing ability with regard to reactions of reduc- tion, of which some, e.g., reduction of the nitro- group, proceed with the participation of cyto- chrome P—450. In the metabolism of nitro- benzene this system may play a role, as well in the reduction reactions leading to aniline as in hydroxylation of the latter to aminophenols. A reductive dechlorination of DDT, which leads to formation of DDD. also belongs to the reduction reactions taking place in the microsomal fraction. This reaction requires the presence of NADPH2 and can be inhibited by carbon monoxide. which suggests that cytochrome P—450 takes part. The biotransformation mechanisms localized in the liver microsomes are known mostly from animal studies. Evidence exists, however, that similar mechanisms act in the microsomes of the human liver (Ackermann, I972). Biotransformation phase l reactions, taking place in the microsomal metabolic system, are not lim- ited to the liver-cells. The liver, presumably with the highest metabolic activity, has been most in- tensively studied. Other organs have been much less investigated from the viewpoint of ultra— cellular structure or for the possibility of xeno- biotic transformation in various subcellular or- ganellae. Recent data suggest that pulmonary tissue plays a significant role in the bio- transformation of xenobiotics. The lung can be fractionated into subcellular elements that are similar to, but less uniform than, those seen in hepatocytes. The microsomal fraction of pul- monary tissue has the ability to catalyze the me- tabolism of numerous xenobiotics; the mech- anism involved seems to be the same as that in liver cells (Bend et al., 1972). The level of cyto- chromes, both b5 and P—450, in pulmonary mi- crosomes is much lower than in liver, but the ac- tivity of some xenobiotic catabolizing enzymes is similar in both organs; for example, the activity JERZY K. PIOTROWSKI of biphenyl—4—hydroxylase or benzphetamine N— demethylase (Hook et al., l972). The con- tribution of microsomal enzymes of pulmonary tissue to the total metabolism of xenobiotics var- ies from substance to substance. For parathion the contribution is of the order of l/lOO of that of the liver (Neal, I972). The metabolic activity is not localized in alveolar macrophages, known for their role in the defense of the body against bac- teria. viruses. and aerosol particles (Hook et al., l972). Apart from liver and lung reactions of biotransformation, phase I can be observed in microsomal systems of other tissues, e.g., kidneys or intestines for benzpyrene hydroxylase (Chhabra and Fouts, I974) and in other tissues for parathion (Poore and Neal, I972). While the biotransfonnation reactions that take place in the liver microsomes have been most ex- tensively studied, there are also metabolic reac- tions of phase I that are localized in other cellu- lar fractions, for instance: reactions of deamination. oxidation of alcohols and al- dehydes, aromatization of alicyclic compounds. reduction of aldehydes and ketones, and various hydrolytic reactions. It is interesting that in the extramicrosomal fraction (on the basis of another mechanism) a reaction may take place that has been known as a microsomal one. Thus, in the human placenta, aniline is hydroxylated not in microsomes but in postmicrosomal fractions as a reaction catalyzed by Hb and MetHb (Juchau and Symms. l972). Some reactions may also pro— ceed as a result of activity of intestinal bacterial flora, e.g. azoreduction of dyes (Gingell and Walker, l97l). Reaction products of phase l are usually subject to conjugation. Most common reactions of this kind yield glucuronides of phenols, alcohols, a- mines, aromatic acid esters of sulphuric acid (mainly of phenols), or hippuric acids that are formed as products of aromatic acids reacting with gylcine. Usually the reactions of conjugation are multistage processes: a) For the reaction to proceed it is neces- sary that one of the reacting substances be- come activated. Thus glucuronic and sul- phuric acids must be present in the form of UDPGA (uridine diphosphate glucuronic acid) and PAPS (adenosine-3l—phosphate—S’ ‘phosphosulphate), respectively. To enter the reaction with glycine, benzoic acid has , to assume the form of benzoylcoenzyme A. b) Transfer of the respective group into the BIOTRANSFORMATIONS acceptor occurs usually under the influence of the enzyme, a corresponding transferase. Due to this multistep character of a conju- gation reaction. it is difficult to localize the process unequivocally on a subcellular level; e.g.. enzymes synthesizing the active form of glucuronic acid (UDPGA) are present in the soluble fraction of cytoplasm, whereas the transferases (transglucuronylases) are localized in the microsomal fraction. How- ever. this is not a rule: other transferases may be found in the soluble fraction. e.g. sulphokinases that transfer the sulphate group from PAPS. The enzymes which transfer the methyl group from its active form (S—adenosylmethionine) are localed in the soluble subcellular fraction in most tis- sues (adrenals. lungs. liver. kidneys). The enzymes responsible for the transfer of aro- matic acids from their active forms (eg. benzoylcoenzyme A) onto glycine as an ac- ceptor can be found in mitochondria of liv- er and kidney cells. A more detailed review of this problem was published by Gram and Gillette (I97I). An essential feature seems to be that the systems responsible for the reaction of conjugation of xenobiotics are relatively ubiquitous. For instance, activity Of UDP-glucuronyl transferase, an enzyme that transfers the glucuronide onto p-nitro— phenol. has been found in the lungs. intes- tines. and kidneys of rats at levels not lower than in the liver (Chhabra and Fouts, I974). INFLUENCE OF FOREIGN COMPOUNDS UPON THE METABOLISM OF XENOBIOTICS ln general. foreign compounds may accelerate as well as inhibit the reactions involved in the me- tabolism of xenobiotics. The mechanisms re- sponsible are different in either case. Most in- tensively studied has been the stimulating effect of alien compounds on metabolic reactions that take place in liver microsomes. STIMULATION OF MICROSOMAL ENZYMES The elevation of the activity (induction) of micro- somal enzymes under the influence of in vivo 13 exposure of animals to various xenobiotics has been extenisvely studied. The basis for assess- ment of the increased activity is provided usually by measurements of the rate of a model-type re- action of phase I (e.g. hydroxylation of aniline, de- methylation of aminopyrine); among conjugation reactions, the effect of induction may be seen'in relation to the activity of UDP—glucuron- yItransferase (Jansen and Henderson, -l972). The discussed phenomenon occurs only in vivo or in an isolated organ or tissue culture; it could not be reproduced by adding the inducing xenobiotic directly to the suspension of microsomes in i'ilru*. A particularly pronounced effect in vivo has been observed in very young animals (first few weeks of life) in whom the normal activity of liver mi- ' crosomal enzymes is very low. TO a smaller ex- tent the effect can be also reproduced in adults. The increased activity of microsomal enzymes is accompanied by an elevated protein content of the microsomal fraction. and. in general also. by higher concentrations of microsomal components Of the electron transport chain. The stimulatory effect can be abolished by concurrent adminis- tration of inhibitors of protein synthesis (acti- nomycine—D. puromycine). These facts indicate that the stimulatory effect depends upon de novo biosynthesis of microsomal proteins. Induction capability has been demonstrated for numerous xenobiotics that have been divided into two classes on the basis of the underlying mechanisms: a) Compounds with the type of action demonstrated by phenobarbital. including many other barbiturates and drugs. DDT, and probably other chlorinated hydro- carbons used as pesticides, as well as PCB. may be classified into the same category (Klinger et al., I973; Villeneuve et al., l972; Mailman and Hodgson. I972). Inductors of this group stimulate. in a relatively non- specific way. the metabolism of numerous organic compounds: e.g., aniline, parathion. * An exception is formed by so called "biphenyl—Z— hydroxylase,” which may be stimulated by preincubation of microsomes with a number of polycyclic hydrocarbons such as Srmethylcholantrene. 3.4—benzpyrene and others (Bridges et al.. I973). A similar effect was observed with regard to aniline hydroxylation in vitro in the presence of paraoxon (Stevens et al. l972). l4 and other phosphoorganic compounds. The stimulatory effect becomes manifest along with an increase in liver weight. protein and phospholipid content. and the concen- tration of cytochrome P—450, NADPH—cyto- chrome C and NADPH—cytochrome P—450 reductases. b) Compounds of which polycyclic hydro- carbons form the best known representation (3—methyl cholanthrene. 3.4—benz~a-pyrene. fluorene. anthracene. and others). These in- ductors have a much narrower spectrum of activity and display different kinetic fea- tures. The inductory effect is already seen a few hours after administration and is ac- companied by an increased weight of the liver. and of microsomal protein and cyto- chrome P—450 content. No enhancement is seen of the activity of NADPH—cytochrome C and NADPH—cytochrome P—450 re- ductases. For both types of inductors the effect is tran- sient: and few days after discontinuation of their administration. the activity of microsomal en- zymes returns to the normal level. The phenom- ena are discussed in greater detail in the papers by Gram and Gillette (l97l) and Parke (I972). Usually. the induction of microsomal enzymes leads to nonspecific enhancement of activity of the various enzymes. present in the endoplasmic reticulum. that metabolize foreign compounds. However. cases of quite a selective stimulation are known. For example. urethan stimulates se- lectively hydroxylation of aniline without affect- ing the rate of the demethylation processes. which otherwise usually occur. Most likely. this type of stimulation is not based upon the general mechanism of synthesis of microsomal protein and its active components (Schenkman et al.. I974). From the viewpoint of exposure tests. the in- duction of microsomal enzymes is of importance because of three circumstances: a) A worker who is subjected to the test could be under medication. and therefore the metabolic efficiency for a given organic compound may have been altered in com- parison with normal . The following drugs have been shown to act as inductors of mi— crosomal enzymes (of the phenobarbital in- duction type): Phenobarbital. Niketamide. Barbital. Chlorpromazine. Chlorcyclizine. Cyclizine. Glutethimide. Meprobamate. Or- phenadrine. Pentobarbital, Phenylbutazone. Tolbutamide. Ethanamate. Aminopyrine. lmipramine (Gram and Gillette. l97l). This „LX. JERZY K. PIOTROWSKI list is not complete; in practice a majority of organic drugs may be suspected to act in this way. ln relation to many substances the induction-effects have been also observed in animals. Enhanced activity of aniline hy- droxylase is one of the classic tests advo- cated as a proof of the inductory ability of a xenobiotic. The phenobarbital-type effects were seen for the metabolism of styrene (first ‘stage of the pathway. leading to for- mation of the epoxide) (Ohtsuji and lkeda. l97l). Phenobarbital stimulates also the metabolism of trichloroethylene; particu- larly the stage leading from chloral hydrate to trichloroethanol (Leibman and McAlister. 1967). b) ln case of a composite exposure to two or more substances. one of them may pos- sess inductory capability with relation to microsomal enzymes. and. therefore. change the metabolism of a substance to be stud- ied. A typical example is provided by con- current or alternating exposure to phos- phoorganic compounds and chlorinated hydrocarbons. The latter (DDT. Chlordane. Dieldrin. Aldrin. Heptachlor. Hexa- chlorocyclohexane) possess the ability to in- duce the microsomal enzymes and may change. therefore. the metabolism of the phosphoorganic compounds. Activity of mi- crosomal aniline hydroxylase increases un- der the influence of acetone (Clark and Powis. I974). Composite exposure to this compound and to aniline could therefore affect the metabolism of the aniline. To some extent the problem may be important in smokers (induction due to expOsure to polycyclic hydrocarbons) and in individuals who frequently drink alcohol (for dis— cussion. see Parke. I972). c) Substances under study may be in- ductors themselves. and in the course of re- peated exposures may stimulate their own metabolism. This problem. which is essen- tial from the viewpoint of exposure tests. has so far attracted little attention. Gram and Gillette (l97l) maintain that auto- inductory properties are displayed by DDT and benzene among others. The benzene experiments performed on rats in the au- thor‘s laboratory did not confirm this con- cept (see also Cornish et al.. 1970). Similar studies also seem to exclude a significant autoinduction in the case of nitrobenzene. A slight effect of autoinduction was ob- served for aniline (Wisniewska-Knypl et al.. BIOTRANSFORMATIONS l975).'and no effect was demonstrated for trichloroethylene (lkeda and lmmamura. I973). Some effect is probable for carbon disulphide; however final proof seems to be lacking (Sokal. I973). Autoinduction can not be ruled out for some phos- phoorganic insecticides; this problem, however. is still far from being well-docu- mented (McPhillips et al.. l972). Auto- induction could be important especially with regard to the tests worked out experi- mentally on volunteers. Usually from sin— gle-chamber-type experiments the results (correlation between dose and level of re- spective metabolite) are extrapolated to in- dustrial conditions. where workers are ex- posed repeatedly. lf a substance has autoinductive properties. the correlation may be rather different. Due to difficulties in obtaining comparative ex- perimental data for single and repeated. chronic exposure. there has been little direct information available on the degree of real significance this phenomenon might have. Theoretically. one could exptect two kinds of effects: a) As a result of autoinduction. acceler- ation of the metabolism along all parallel pathways could take place. ln a quan- titative sense. an elevated excretion rate of the metabolites would be expected with un- altered general metabolic efficiency (higher urinary levels toward the end of exposure. and more rapid decline of urinary levels af- ter cessation of exposure). b) Due to autoinduction only one metabol- ic pathway could be enhanced. and the oth- er parallel ones left unchanged. The ex- pected overall result would include the changes in the dynamics of the increase and decline of the respective metabolite levels. and the alteration of the metabolic efficiency. It is the present author‘s opinion that the auto- induction phenomenon is probably of subordi- nate importance from the viewpoint of its appli- cability to exposure tests. The supporting argument may be presented as follows: a) Quantitatively. the induction of micro- somal enzymes is of great significance only in very young animals. In adult experi- mental mammals it is less pronounced, and one might expect that also in adult humans metabolism of xenobiotics would not un- dergo dramatic changes due to induction. 15 b) The induction effects have been learned mainly from studies based on isolation of the microsomal fraction of liver cells and in vitro assays of the transformation rate of added substrate. In vivo metabolic trans- formations of xenobiotics do not occur only in liver. and the extent to which mi- crosomal induction takes place in other tis- sues has not been sufficiently studied. lt might be presumed. however. that the changes are significantly less pronounced. This opinion seems to be in line with the direct data of Drew and Fouts (l974). who have studied both the liver and lung microsomes. This might provide an explanation for the fact that in preliminary experiments made in the au- thor’s laboratory on adult rats in vivo, no serious effect of inductors could be demonstrated upon the transformation rate of several organic com- pounds (unpublished data). An example of the difference between in vitro and in vivo situations may be provided by a lack of change in the val-„ ues of CL5„ or DL5„ for benzene in rats under the influence of potent microsomal inductors (pheno- barbital. chlorpromazine), in spite of the fact that distinct stimulation of the metabolism of benzene was observed in vitro in liver microsomes (Drew and Fouts. l974). ' INHIBITION OF MICROSOMAL ENZYMES Inhibition of the metabolism of xenobiotics (lo- calized in liver microsomes) has been observed for various compounds; and it may be presumed that different mechanisms were involved. The compound SKF—525A (2—diethylaminoethyl—2,2- diphenylvalerate) is a potent inhibitor of the me- tabolism of barbiturates. aminopyrine de- methylation. hydrolysis of procaine. and glue- uronide synthesis. but exerts no effect on hydroxylation rate of acetanilide. dealkylation of phenacetin. or reduction of nitro- and azo- groups. Morphine and codeine inhibit some met- abolic reactions in rats. but only in males. ln- hibition of microsomal liver enzymes may follow administration of high doses of nicotinamide. As with numerous other enzymatic processes, the in- hibitory effect may be seen at high concen- trations of heavy metal ions; e.g., repeated ex- posure to mercury causes an inhibition of 75-90 per cent of the normal values (Cornish et al.. l970). Microsomal inhibition has also been dem— onstrated after administration of phosphoor- l6 ganic cholinesterase inhibitors (Rao et al., 1973). Recently, the inhibitory effect of pyridine—and some of its derivatives has been demonstrated us- ing inhibition of p-nitroanisol demethylation and hydroxylation of aniline as the test reactions (Jo- nen et al.. I974). It is postulated that, as demon- strated with relation to other enzymatic pro- cesses, the inhibition may be of a competitive character; two compounds metabolised by the same enzymatic system could mutually decelerate their metabolic transformations. The inhibitory effects could be biphasic; the original inhibition may be followed by induction (Cram and Gil- lette, I97I). These inhibitory effects upon the microsomal en- zyme systems are not well-understood in detail, and the potential importance of these phenomena with regard to interpretation of the exposure tests cannot be assessed at present. INFLUENCE OF PHYSIOLOGICAL FACTORS ON THE METABOLISM OF FOREIGN COMPOUNDS The influence of hormones on the metabolism of xenobiotics in microsomal systems has been doc- umented (Parke. I972). The same, or a similar, microsomal system as that known to be in the liver for the metabolism of xenobiotics has been found in the adrenal cortex, in testicular inter- stitial tissue, and in the placenta. The system is active in the metabolism of cholesterol and ste- roid hormones. Biosynthesis of cholesterol in the liver takes place in microsomes, as well as per- oxidation of the unsaturated fatty acids (Archa- kov et al., I972). Thus, there exists a mutual re- lationship between the level and intensity of the metabolism of cholesterol and steroid hormones on the one hand, and the metabolism of xeno- biotics on the other. For instance, the rate of biosynthesis and degradation of cholesterol is known to be influenced by the inductors and in- hibitors of microsomal enzymes discussed in the preceding section. Phenobarbital induction en— hances the microsomal metabolism of sex-hor- mones (testosterone, androsterone, esterone, pro- gesterone, and others). In rats, the activity of microsomal enzymes, in- volved in the metabolism of xenobiotics, is high- er in males than in females. The difference is re- lated to sex hormones. It is possible to abolish this sex effect by the administration of testos- terone to females and of estrogens to males. In JERZY K. PIOTROWSKI males recovery of the activity of microsomal en- zymes after intensive whole-body or testicular ir- radiation can be attained by administration of testosterone (Knott and Wills, I974). Steroids and xenobiotics form alternative substrates for the same enzymatic systems — thus, the observed metabolic interactions. The existing data for various species are not suf- ficienty consistent to allow unequivocal conclu- sions with regard to sex-related differences in the metabolic rates of various xenobiotics in man. For instance. mice and rats differ in respect to hormonal regulation of the activity of liver mi- crosomal enzymes (Chhabra and Fouts, I974). Nevertheless. the possible existence of these sex- Iinked differences in humans should not be over- looked. In experiments aimed at the elaboration of exposure tests. such a possibility is not always taken into account; and often the experiments are performed on groups including individuals of both sexes. Only a few substances are known to display intersex metabolic differences in human beings; for instance the ratio of two tri- chloroethylene metabolites, trichloroethanol vs. trichloroacetic acid, differs between the sexes (Nomiyama and Nomiyama, I97I). In rats, the most pronounced intersex differences were observed for hepatic metabolism of hexo- barbital and aminopyrine. It is interesting to note that these differences disappear in fasting animals as an effect of depression of activity of micro- somal enzymes in males. Similar effects have been observed after adrenalectomy or administra- tion of thyroid hormones (triiodothyromine, thy- roxine: for further discussion see Cram and Gil- lette. l97l). It should be emphasized that these intersex differences in hepatic microsomal metab- olism were learned mostly from in vitro studies. lt seems also relevant that, at least in the rat, the differences may be absent when metabolic reac- tions catalysed by microsomal enzymes in other organs, e.g., the lungs or kidneys, are concerned. Moreover, in the lungs and kidneys, the sex-re- lated differences may be opposite from those seen for liver microsomal enzymes (Chhabra and Fouts, I974). Among the numerous factors that may exert an influence on the normal or induced activity of mi- crosomal enzymes, a deficiency of ascorbic acid should be mentioned. This deficiency depresses the activity of the enzymes for many of the bio- transformation reactions (for review, see Wag- staff and Street. l97l). In contrast with this find- ing, iron deficiency in the diet enhances the activity of microsomal enzvmes (Becking, I972). BIOTRANSFORMATIONS DOSE-DEPENDENT VARIATIONS IN THE METABOLISM OF XENOBIOTICS For the description of the kinetics of bio- transforrnation and excretion of xenobiotics, it is usually accepted that the processes in question follow the first-order kinetics. With this assump- tion one should not expect any dose-dependent variations in the rate of the metabolic processes, nor in the relative yield of the individual metabo- lites. Although this assumption is useful for sim- plified calculations, in reality it is not always ful- filled. An alternative assumption, that the individual partial processes may differ in the type of kinetics, leads to opposite conclusions. The metabolic processes of xenobiotics are en- zymatic in nature. Thus, they should in general follow the Michaelis—Menten kinetics of en- zymatic reactions where with a high level of the substrate the reaction rate becomes constant, in- dependent of any further increase of the substrate level (saturation of the metabolic pathway). Since the saturation of various metabolic pathways is reached at different levels of the substrate con- centration. the final yield of various metabolites may become variable with changing doses. Let us quote some more typical examples. Variable yield has been found for the excretion of the un- changed substance in animals in the case of ani- line, cyclohexane, 4,6—dinitro-o-cresol, carbon di- sulphide, and fluorobenzene. Also, dose- l7 dependent differences in the excretion yield of in- dividual metabolites were found in animals and/ or humans in the case of ethylene glycol, cy- clohexane, aniline, nitrobenzene, p-nitrophenol, 2 -naphthylamine, and carbon disulphide. The above findings may have a strong bearing both on the fate of individual xenobiotics, and on the use that is made of the metabolic pro- cesses in the exposure tests. For instance, the metabolic pathway of DDT leading through DDD (microsomal reaction) to DDA is acceler- ated in case of higher doses, contributing to rela- tively quick clearance of the xenobiotic when the exposure is high. On the other hand, exposure tests are based on a relationship between the ab- sorbed doses and the excreted amount of a given metabolite. A linear dependence is usually ac~ cepted which facilitates calculations and extrapo- lations. It may be of practical importance to real- ize, therefore, that the metabolic variations in question may be observable even in a relatively narrow range of doses (as in the case of aniline), leading to a curvilinear dependence between the dose absorbed and amount excreted. Such func- tions, if presented for simplicity's sake in the con- ventional linear way, give an extremely high er- ror which in random cases may be used as an argument against the exposure test. On the other hand, exposure tests based on curvilinear func- tions require careful statistical calculations using special methods, especially if the possibility of an extrapolation is considered. REFERENCES Ackermann E.: Dealkylation of ethylmorphine and p-C—hydroxylation of aniline in human and male and female rat liver microsomes. Biochem. Pharmacol 21, 2169- 2180. l972. Archakov A.J., Karnzina J.J., Bokhenko A.J., Aleksandrova T.A., and Panchenko L.F.: Studies on the localization of reaction sites within the NADPH — oxygenase system of rat liver microsomes for the N- and C-oxidation of dimethylaniline and/or the peroxidation of unsaturated fatty acids. Biochem. Pharmacol. 21, 1595—1602, l972. Becking G.C.: Influence of dietary iron levels on hepatic drug metabolism in vivo and in vitro in the rat. Biochem. Pharmacol. 21, 1585-1593, l972. Bend J.R., Hook G.E.R., Easterling R.E., Gram T.E., and Fouts J.R.: A compara- tive study of the hepatic and pulmonary microsomal mixed-function oxidase system in the rabbit. J. Pharmacol. Exp. Therap. 183, 206-2l7, l972. Bridges J.W., McPherson F.J., King L.J., and Parke D.V.: The influence of various chemicals on hepatic drug metabolism systems. ln: Experimental Model Systems in Toxicology and their Significance in Man. Excerpta Med. International Congress, se- ries Nr 311, 1973. PP. 98-l04. Chhabra RS. and Fouts J.R.: Sex differences in the metabolism of xenobiotics by extrahepatic tissue in rats. Drug Metabolism and Disposition 2, 375-379, 1974. 18 JERZY K. PIOTROWSKI Chhabra R.S. and Fouts J.R.: Stimulation of hepatic drug-metabolizing enzymes by DDT. polycyclic hydrocarbons or phenobarbital in adrenalectomized or castrated mice. Toxicol. Appl. Pharmacol. 28, 465-476, 1974. Clark H. and Powis G.: Effect of acetone administered in vivo upon microsomal drug metabolizing activity in the rat. Biochem. Pharmacol. 23, l0I5-l019, 1974. Cornish H.H., Wilson C.E. and Abar E.L.: Efect of foreign compounds on liver mi- crosomal enzymes. Amer. Ind. Hyg. Assoc. J. 1, 605-608, 1970. Drew R.T. and Fouts J .R.: The lack of effect of pretreatment with phenobarbital and chlorpromazine on the acute toxicity of benzene in rats. Toxicol. Appl. Pharmacol. 27, 183-193, 1974. Fouts J .R.: Some studies and comments on hepatic and extrahepatic microsomal tox- ication — detoxication systems. Environ. Health Perspectives (Exper. Issue No. 2- Oct.), 55-66, 1972. Gingell R. and Walker R.: Mechanisms of azoreduction by streptococcus faecalis. II. The role of soluble flavins. Xenobiotica 1, 231-239, 1971. Gram T.E. and Gillette l.R.: Biotransformation of drugs. In: Fundamentals of Rio- chemical Pharmacology. Z.M. Bacq (Executive Editor), R. Capek, R. Paoletti, J. Renson (Editors) Pergamon Press, Oxford, 1971, pp. 571-609. Hook G.E.R., Bend J.R., and Fouts J.R.: Mixed-function oxidases and the alveolar macrophage. Biochem. Pharmacol. 21, 3267-3277, 1972. Hook G.E.R., Bend J.K., Hoel D., Fouts J.R., and Gram T.E.: Preparation of lung microsomes and a comparison of the distribution of enzymes between subcellular fractions of rabbit lung and liver. J. Pharmacol. Exp. Therap. 182, 474-490, 1972. lkeda M. and lmmamura T.: Biological half-life of trichloroethylene and tetra- chloroethylene in human subjects. Int. Arch. Arbeitsmed. 31, 209-224, I973. Jansen B.L.M. and Henderson P.T.H.: Influence of phenobarbital treatment on p—ni— trophenol and bilirubin glucuronidation in Wistar rat, Gunn rat and cat. Biochem. Pharmacol. 21, 2457-2462, 1972. Jonen H.G., Huthwohl B., Kahl R., and Kahl G.F.: Influence ofpyridine and some pyridine derivatives on spectral properties of reduced microsomes and on microsomal drug metabolizing activity. Biochem. Pharmacol. 23, l3l9-l329, I974. Juchau M.R. and Symms K.G.: Aniline hydroxylation in the human placenta; Mech- anistic aspects. Biochem. Pharmacol. 21, 2053-2064, 1972. Knott J.C.A. and Wills E.D.: The role of testosterone in the regulation of oxidative drug metabolism in normal and irradiated animals. Biochem. Pharmacol. 23, 793-800, I974. Klinger W., Gmyrek D., and Grubner 1.: Untersuchung verschiedener Stoffe und Staf- fklassen auf lnduktoreigenschaften. Ill. Chlorierte lnsektizide. Arch. Int. Pharma- codyn. Ther. 202, 270-280, 1973. Leibman K.C. and Mc Allister W..l.: Metabolism of trichloroethylene in liver micro- somes. J. Pharmacol. Exp. Therap. 157, 574-580, 1967. Mailman R.B. and Hodgson E.: The cytochrome P-450 substrate optical difference spectra of pesticides with mouse hepatic microsomes. Bull. Environ. Contam. Tox- icol. 8, 186-192, 1972. Neal R.A.: A comparison of the in vitro metabolism of parathion in the lung and liv- er of the rabbit. Toxicol. Appl. Pharmacol. 23, 123-130, 1972. Mc Phillips J.J., Stevens J.T. and Stitzel R.H.: Effects of anticholinesterase in- secticides on hepatic microsomal metabolism. J. Pharmacol. Exp. Therap. 181, 576- 583, 1972. Nomiyama K. and Nomiyama H.: Metabolism of trichloroethylene in humans. Sex differences in urinary excretion of trichloroacetic acid and trichloroethanol. Int. Arch. Arbeitsmed. 28, 37-48, 1971. Ohtsuji H. and lkeda M.: The metabolism of styrene in the rat and the stimulatory effect of phenobarbital. Toxicol. Appl. Pharmacol. 18, 321-328, 1971. Parke D.V.: The effects of drugs and steroid hormones on the enzymes of the endo- plasmic reticulum. In: Effects of Drugs on Cellular Control Mechanisms. B.R. Rabin and RB. Freedman, Editors, Macmillan Press Ltd. (I972), pp. 69-104. Poore R.E. and Neal R.A.: Evidence for extrahepatic metabolism of parathion. Toxicol. Appl. Pharmacol. 23, 759, I972. Rao H.R. and Anders M.W.: Inhibition of microsomal drug metabolism by anti- cholinesterase insecticides. Bull. Environ. Contam. Toxicol. 9, 4-9, 1973. BIOTRANSFORMATIONS Schenkman J.H., Ritchie A., Cha Y.N., and Sartorelli A.C.: Selective stimulation by urethan of hepatic microsomal aniline hydroxylation. Biochem. Pharmacol. 23, 1148— 1151, 1974. Sokal J.A.: Effect of chronic exposure to carbon disulphide upon some components of the electron transport system in rat liver microsomes. Biochem. Pharmacol. 22, 129-132, 1973. Stevens J.T., Mc Phillips J.J., and Stitzel R.E.z In vitro enhancement of aniline me- tabolism by paraoxon. Toxicol. Appl. Pharmacol. 23, 208-215, 1972. Villeneuve D.C., Grant D.L., and Phillips W.E.J.: Modification of pentrobarbital sleeping times in rats following chronic PCB ingestion. Bull. Environ. Contam. Tox- icol. 7, 264-269. 1972. Wagstaff DJ . and Street J.C.: Ascorbic acid deficiency and induction of hepatic mi- crosomal hydroxylative enzymes by organochlort'ne pesticides. Toxicol. Appl. Phar- macol. 19, 10-19, 1971. Williams R.T.: Inter-species variations in the metabolism of xenobiotics. Bioch. Soc. Trans. 2, 359-377, 1974. Wisniewska-Knypl J.M., Jablonska J.K., and Piotrowski J.K.: Effect of repeated ex- posure to aniline, nitrobenzene and benzene on liver microsomal metabolism in the rat. Brit. J. lndustr. Med. 32, 42-48, 1975. 19 4. METABOLIC AND EXCRETION KINETICS INTRODUCTION In the early period of elaboration of the exposure tests it already became clear that relating levels of a toxic compound, or its metabolite, in biologi- cal media to the magnitude of exposure (dose, concentration in air) was possible only if the rate of metabolism and excretion were considered quantitatively. The first attempts to systematize the concepts and principles of computation for purposes of exposure tests (Soucek, 1952) were made in the period of the development of the ki- netic theory of metabolism and excretion that followed application of radioactive tracers in bio— logical studies. This development has lead to the creation of the method of compartmental anal- ysis (Solomon. 1953). At present numerous au- thors apply the principles of kinetics for pro- cessing the results of experiments aimed at the elaboration of exposure tests, and for the inter- pretation of the data. Attempts to systematize the relevant concepts for purposes of industrial tox- icology, together with the review of possible practical applications, were made by the present author (Piotrowski, 1971). In this chapter the concepts and principles of calculations that seem particularly useful for developing exposure tests will be discussed. KINETIC MODELS AND & COMPARTMENTAL ANALYSIS The grounds for kinetic considerations and re- sulting concepts are provided by observations of the time-course of the blood level or urinary ex- cretion of a substance and its metabolites after intravenous administration. After the single in- troduction of a foreign substance, the level of it or of its metabolite in biological media changes curvilinearly with time in a linear coordinate sys- tem. As with other applications of chemistry and physics, the search for laws governing the process is facilitated by a system of coordinates in which the process may be depicted by a linear function, or an algebraic sum of several linear functions. This condition is fulfilled by the semi-logarithmic 20 system of coordinates, which is very useful in ki— netic studies, in that the time-course of processes in question may be usually depicted by one of the functions as presented in Figure 4-1. The most simple case is represented by curve I. If [S] = the level of a substance in the system (body), t = time of observation after administration of the dose, k = rate constant of disappearance of the substance (in t'I units), the slope of the line a = 0.4343 k, the curve may be ascribed a mathe- matical form: log[S]t = log[S]0 — at (l) 0.1 1 0.05 - 5 I Q „„ 5 001 _ , *3 k 0005‘ c . s ‘6 ~s € @ aoat- @ o 10 20 so 40 so so m 00 90100110 120150140 150 Time [hours] Fig. 4-1. Three basic types of elimination curves following single instantenous exposure, in a semi/ogarithmie system of coordinates ( Domanski and Piotrowski. 1971). Taken from: Piotrowski J.: The application of metabolic and excretion kinetics in industrial toxicologi'. US Government Printing Office, 1971. Page 144. Fig. lii. METABOLIC AND EXCRETION KINETICS OI" [S]I : [SLC—kl (2) where e = the base of natural logarithms. Equa- tion (2) is the most common of kinetic equations. This equation could also be written in the form: [S]. = [sutrat ' (za) However. the original form (2), that makes use of the commonly applied elementary function e‘x. is more communicative and secures easier compara- bility with the results of many studies made so far. Function lll (Fig. 4-l) may be graphically de- picted into two contributing linear functions and may be written in the form: [S]: : (',erlt + Czer:t (3) In formula (3) C, and C2 represent the intercepts of both lines with the ordinate (at t = 0), where- _ A 'L T*: E _ k4 k, !! « A M E _ A k‘ 8 L'! H," E Fig. 4-2. Three basic kinetic models (Domanski and Mikołajczyk, 1971). Taken from: as in Fig. 4-l. Page 132, Table li. 21 as rI and r3 (negative values) provide the re- spective slopes. A similar function can be obtained by graphical analysis of curve ll. ln this case the function may be expressed by a difference between two linear functions of different slopes but equal intercepts (CI = Cl). The function is usually applied for me- tabolite M: [M]t=C2efit—Cler2t (4) The equations (2. 3. 4), as presented above, reflect three different kinetic models, which are pre- sented in Figure 4-2. These models will be re- ferred. to as follows: I -— single compartment model. ll — metabolic model, and lll — two compartment open model. ln all these models it is assumed that partial transfer and excretion processes are of the first order. which means that the rates represented by arrows are directly pro- portional to actual contents (concentrations) of the substance (or metabolite) in a given compart- ment. Thus. change in the systemic content [S] (Fig. 4-2 I) may be described by the most simple differential equaltion: d[S] _ T — k [S] (5) the solution of which (the integral) is provided by equation (2). derived previously from purely em- pirical observations. From the models ll and lll systems of differential equations may be deduced. For instance. the kinetic description of the pro- cesses taking place in model lll, is of the form: 33%;: k] + k2 [SA] +k3[SB] d [S B] d. = kztsn — ka [SB] dLZtE] : kl [SA] The solution (the integral) of this system of dif- ferential equations, in relation to the content of 22 the substances in the first, rapid exchange com- partment A (which is composed of blood plasma, etc.) is given by the general formula of equal tion (3). In the most simple case, when substance S was introduced into the system by means of single in— travenous injection (into the compartment A), the integration constants Cl and C2 appearing in equation (3), equal: _ rl+k3[ ] (|' rl r2 SA0 [SA where the coefficients rl and'r2 may be derived in a_complex way from individual kinetic coeffi- cients ki: (k,+k2+k,)i‘/(k,+k2+k,)2 — 4k,k, (a) 2 (7) r2+k3 C2:rAr O l 2 rig: Similarly. solution of the system of differential equations for the model ll may be obtained in the form: k4 [M]t = W [S]o (e,—ks! _ e-kst> (9) which represents the explicit form of empirical equation (4). The above reasoning points to the possibility of using empirical kinetic curves for reproduction of the model and its coefficients. In most situations, however, when exposure tests are considered, this procedure is not necessary. The interesting infor- mation and calculations may be deduced directly from the general equations (2, 3, 4), which can be obtained by graphical analysis of empirical kinet- ic data. ' The three kinetic models as discussed above re- present only the most simple situations. In real- ity, kinetic models applicable for the purpose in question may be much more complicated, either JERZY K. PIOTROWSKI due to existence of parallel metabolic and excre- tory pathways or because there are, in fact, nu- merous metabolic compartments. An example is the complex metabolic model postulated for tri- chloroethylene (see chapter on “Trichloroethy- lene”) that has not yet been solved. The existence „ of more than 2 compartments presents a serious complication, for the complete solution of such a model is possible only by using a computer. On the other hand, parallel pathways do not intro- duce very serious computational complications. In Figure 4-3, examples are given of models with parallel pathways for which solutions have been developed (Piotrowski, 1971). For practical pur- poses, it is convenient to use a coefficient of loss that denotes a sum of all coefficients of parallel metabolic degradations of a substance and its ex- cretion ( EKEM ). REAL MEANING OF KINETIC PARAMETERS The possibility, mentioned above, of decoding a model with its coefficients from purely empirical kinetic curves implies that some biological mean- ing could perhaps be attributed to kinetic para— meters and concepts. For organic substances, the number of systematic compartments essential from the kinetic view- point may be usually limited to two. In such a case, one of these will be defined as a rapid, and the other as a slow, exchange compartment. The A A ~w M %,1 in in „ A " M A " M17: in i“ f“ " M *:; :, " A ź B l i, ” l, i, "1 Fig. 4-3. Simple kinetic models allowing for parallel processes (Domanski and Piotrowski. 1971). METABOLIC AND EXCRETION KINETICS former (A) always includes blood plasma, usually all extracellular fluids, and perhaps the intra- cellular fluids as well. In the case of organic com- pounds, the slow exchange compartment (B) is usually identified with the adipose tissue; evi- dence in favour of this contention can be found for numerous substances. Agreement between re- ality and kinetic theory would require, however, full applicability of the concepts that follow from the partition of the substance in question be- tween two phases, and, in particular, the consis- tency between coefficients of distribution in ad- ipose tissue plasma and the value which can be deduced from coefficients of the model kz and k3._ However, the data in direct support of this are rather meager. Moreover, an analysis made using carbon disulphide as the compound (Bartonicek, 1959) has shown some problems with the con- cepts, reflected by differences in the desorption rate of the substance deposited in various kinds of adipose tissue. A discussion of this subject, published earlier (Piotrowski, l97l), has pointed to the complexity of the problem and justified re- luctance to attribute precise biological meaning to the kinetic concepts. Similar complications arise when the biological significance of the kinetic coefficients character- izing biotransformation of alien compounds is analyzed. These metabolic reactions are usually enzymatic processes, the course of which in gen- eral should obey the Michaelis-Menten law. The general equation would apply: _Ł£1_ um „_ b+[S] where u = biotransformation rate, and a and b are constants. The above function (10) may be- come transformed into the first order equation postulated by the metabolic and excretion kinet- ics only when concentration of substrate, which is proportional to the content of the substance in the body [S], is very low. Then, for [S] << b the function (10) becomes: u —% [S]= HS] (11) For many situations considered in industrial tox- icology this condition is certainly fulfilled be- cause of low exposure levels, predetermined by the maximum permissible concentrations in the 23 air of industrial premises. Nevertheless, the as- sumption, as it stands, is being accepted unproven in the kinetics of metabolism and excretion and may fail in some cases. SIMPLIFICATIONS OF KINETIC MODELS Kinetic concepts and methods are often dis- regarded even by experienced toxicologists be— cause they are not familiar with the rather com- plex mathematical procedures. On the other hand, the degree of understanding of the real bi- ological meaning of a complex mathematical model as applied to a concrete situation is often limited. ln practical problems related to the ex- posure tests, it seems justified, therefore, to present the models in the most simple fashion. lt is important. however, that the simplification in- troduced has no serious practical bearing on the final calculations. A typical simplification is often applied to or- ganic compounds when their metabolites are be— ing determined in the biological media. Model ll (Fig. 4-2) should find application here, and in the respective equations that follow (e.g. 4). Howev- er, such a procedure is indispensable only if the maximum of the excretion rate of the metabolite is considerably delayed with regard to the end of the exposure interval. If, as often happens, the shift is small, or even undetectable, at the usual time pattern of urine collection (every 2-4 hours), the process may be‘adequately described by means of equation (2), characteristic of the one- compartment model ]. At sufficiently long col- lection intervals. the first term of equation (4) ap- proaches zero, and the whole process may be de- scribed by- the remaining term of lower decay constant “”.r In some cases similar simplification may be applied to the two-compartment open model, on condition that the contribution of the first term of the equation is relatively small. A calculation often used for the characterization of the rate of processes in a single compartment model is the half-time of disappearance (elimi- nation). If the rate constant of disappearance k is known, the half-time may be calculated from equation (2), assuming from the definition that the half-time Tl/g is related to the decay of the substance in the system to half of its initial con- tent. Thus we obtain: k (12) _ow3 ] 2 24 ln tables 4—l and 4-2 the simplified kinetic data for some organic compounds are assembled. RHYTHMS OF EXPOSURE Kinetic equations (2, 3, 4) and their unfolded forms may be applied under the assumption that a foreign substance was introduced into the sys- tem as a result of a single exposure of short du- ration (i.e., directly into the blood—rapid exchange compartment). To obtain the relation between the equations and the absorbed dose, it is sufficient to substitute a magnitude of the single dose Q for [S]0 or [SA]0. For instance, equation (2) will then assume the form: [S], = Qe-kt (l3) However, for industrial toxicology the exposure of interest takes place continuously over several (usually 8) hours, and repeatedly, separated by- periods off work (lasting usually 16 hOurs). The problem of cumulation and the related question of weekends with no exposure, will be discussed separately below. Limiting the problem at first to a description of the process that takes place on one (the first) day. it seems useful to start with continuous ex- posure, which forms the first and principal frag- ment of the daily cycle. CONTINUOUS EXPOSURE To obtain the appropriate description of the pro- cess of continuous exposure, the models pre- sented above (Fig. 4-2) have to be solved under the assumption of continuous absorption of the substance in question into the rapid exchange compartment. Let us assume, that the absorption is characterized by the absorption rate-q, con- stant in time. The scheme on Figure 4-4 applies to model I. The respective differential equation will be: ”%”-m5] (l4) JERZY K. PIOTROWSKI where T = duration of exposure from the onset of absorption. Assuming, that at the beginning, when T = 0, foreign substance is absent from the body [S]0 = 0, the solution of equation l4 will be of the form: [Shug—.(! -e-k,T) (15) l and respectively, the elimination rate “u” will be given by: u:q(._e—k.r) (16) The initial run of the function, presented in Fig- ure 4-5. may serve as an example- For the more complicated kinetic models, the shape of the functions will be similar, but they will be more complex. For instance, for model lll (Fig. 4-2) ___-›s k1 SE Fig. 4-4. Continuous exposure related to one- compartmen! model. METABOLIC AND EXCRETION KINETICS 25 Routes of Medium of Substance Animal loss from determination ŻkEM the organism Methyl chloride ......... rats ....... respiration air, expired air . . . . 2.8 metablism Methyl chloride ......... dogs ............. do ...... blood ........ 2.5 p-nitrophenol .......... man ....... urine, urine ......... 0.7 metabolism Carbon disulfide ....... rats ....... expired air, expired air . . . . 0.5 metabolism Do ................. man ............. do ........... do ..... 0.8 Polyhydroxy-alcohols rabbit ..... urine, blood, urine . . . 0.26-0.37 (1,2-propandiol, metabolism 1,2-butandiol 1,2,4-butantriol) Benzene .............. man ....... expired air, blood, expired 0.23 metabolism air Benzidine ............. rabbit ..... urine, urine ......... 0.035 metabolism Do ................. dog ............. do ........... do ..... 0.12 Benzidine ............. man ....... urine, urine ......... 0.13 metabolism Do ................. . do ...... do ........... do ..... 0.1 2,6-dinitro-o-cresol ...... . do metabolism ...... blood ........ 0.005 Do ................. rat .............. do ........... do ..... 0.04 Do ................. rabbit ........... do ........... do ..... 0.1 Methanol .............. man ....... metabolism, urine ......... 0.1 urine, ex- pired air Fluorobenzene ......... rabbits ..... metabolism expired aid . . . 0.08 expired air Table 4-l. Examples of simplified kinetic data for organic substances. Coefficient of loss calculated from determinations of the parent compound (from: Piotrowski. l97l). Coefficient Substance Animal Investigated of loss metabolite ŻkEM (hr. 1) Carbon disulfide ........ man ......... Metabolite catalyz- 0.5 ing iodine-azide reaction Toluene ................ man ......... Benzoic acid ............ 0.35 Aniline ................. man ......... p-Aminophenol .......... 0.24 Methanol ............... man ......... Formate ................ 0.3 2-Naphthylamine ......... dog .......... 2-Amino-1-naphtol ........ 0.16 2-Naphthylamine ......... rabbit ........ Sum of C‘—" ............. 0.5 Cyanides ............... rat .......... Thiocyanates ............ 0,07 Table 4-2. Examples of simplified kinetic data for organic substances. Coefficient of loss calculated from determinations of the metabolites (from: Piotrowski, I971). 26 the solution will be given by the following formula: where B ,. and B are constants depending upon the coefficient of elimination k, and the complex coefficients r. and r3. The similarity of the func— tions (l7 and 15) is readily apparent. lf continuous absorption takes place over a suf- ficiently long period (T = ~ ), differences between individual models would disappear; and for all of them we would obtain: [51A =:— (18) | and accordingly. the elimination rate would equal the absorption rate: U.... =q (19) Unfortunately. as a rule. in the course of a rela- tively short daily exposure, which is characteristic of industrial situation, thc steady-state will not be attained. and the excretion rate will show a rising trend throughout the whole exposure interval. Nevertheless. a continuous pattern of absorption favours acceptance of the simplified single com- partment model for substances for which the uri- nary metabolites are determined. lt may be shown that the time required to reach maximum excretion rate, measured from termination of continuous exposure, becomes shorter the longer the continuous exposure (Piotrowski, 1971). INTERRUPTED EXPOSURE For a full description of systemic levels and ex- cretion throughout the day, initiated with ex- posure of a few hours‘ duration, the following reasoning may be developed. The time-duration of a continuous exposure may be denoted by T, and the whole time of obser- vation, starting with the onset of continuous ex- posure, by t. The time elapsed since termination of the expOsure will be given by the difference t — T. For the time period of continuous exposure JERZY K. PIOTROWSKI _ P 4 b & lb |'2 1'4 lb la 202224 How: from start of exposure O 2 Fig. 4-5. Excretion rate of phenol as afunt'tion of time of exposure, and after its termination, expressed as afrac'tion of absorption rate. Dotted line — theoretical curve for k = 0.2 hr (Piotrowski./971). Taken from: Piotrowski J. K.: Evaluation o/ ć'X'posulela phenol. Brit. J. lndustr. Med. 28, 172- I78 l97l. Pagel75 Fig. 4. there are valid rules as discussed earlier; and for the one-compartment model, function (16) applies. Beginning with the discontinuation of exposure, the values of function (16) cease to rise any more and start to decline exponentially, in accordance with the magnitude of rate constant k,. Over the entire period of observation, the excretion func— tion will be described by the equation: ut : q (| — e_k' T)e_k' (FT) (20) Figure 4—5 presents the urinary excretion of phe- nol after inhalatory absorption; the curve repres- ents the typical time course of function (20). The figure at the same time provides information about the agreement that can be obtained be- tween the theoretically expected curve and empir- ical data, represented by mean values from vari- ous experiments on volunteers. By analogy to equation (20), respective functions have been calculated for models ll and lll (Fig. METABOLIC AND EXCRETION KINETICS 4-2) (Piotrowski, 1971). However, it seems they have not so far found practical application. CUMULATION OF FOREIGN SUBSTANCES IN THE BODY Cumulation of a substance in the body is under- stood as a process in which the level of the sub- stance in question increases with the time of du- ration of exposure. lt applies both to continuous and to repeated exposure. From the standpoint of exposure tests, the cumulation is taking place when rising levels of a substance occur in the an- alyzed media (urine, blood, expired air). In par- ticular cases, increase of the concentrations in ad- ipose tissue may also be of interest. The cumulation. if it occurs, results from a slow turn-over of the substance in question. Thus it may take place under conditions of every kinetic model. provided the decay-constant is low (long half-time). From theoretical considerations it fol— lows that the highest value of the decay constant (single compartment model) at which cumulation may take place may not exceed 0.1 hour". For substances that are eliminated mainly unaltered in the breath, the cause of slow turn-over may be the low partition coefficient of air/blood; for xx xxxx &Lgłgagk„ .,lSldł,lr'!!In'nnnuunu, kw.! I I I” "*i-Joann Fig. 4-6. The principle of graphic summation assuming regular weekly periods free of exposure (Piotrowski, 1971). Taken from: as in Fig. 4-l, Page l07, Fig. 26. 27 substances excreted by the kidney, a low clear- ance may result from poor glomerular filtration, or intensive tubular reabsorption, or both. In practical situations two further circumstances may be of significance, namely: slow bio- transformation (when excretion takes place in metabolized form) and deposition in adipose tis- sue. These factors may act in combination, as is the case for nitrobenzene or DDT. When exposure tests are considered, the problem of cumulation manifests itself most clearly in the following situation. Classically, the elaboration of a test is based on single exposure of volunteers to the compound of interest. As the result of ab- sorption taking place over several hours, for a given level of exposure, an excretion function is obtained. ln the case of a substance undergoing cumulation in the body, the function obtained af- ter a single exposure will differ from that in peo— ple exposed previously for a longer period. An empirical solution, through a search for a steady- state level of excretion at a given exposure, is rarely feasible. The laboriousness of such a pro- cedure is extreme; and the studies, if performed, are limited in the number of subjects and the range of exposure levels, which in turn limits the credibility of the results obtained. ln principle, in addition to the levels at steady-state situation, the kinetics of attaining these levels may be calcu- lated theoretically, if only the kinetics of the pro- cess are known from single exposure experi- ments. The underlying assumption is that subsequent doses of a given foreign substance do not modify the rates of their own metabolism and excretion. However, it should be mentioned in this place that this assumption may fail when substances are considered that are capable of in- ducing microsomal enzymes, or if one or more metabolic routes become saturated. The mathematical concept of calculation of the time-course of cumulation is based on the prin- ciple of summation of the curves describing the levels of a substance (or its metabolites) after a single exposure over time intervals that are ade- quate for the situation considered. In the field discussed here, the summation of curves is made over intervals of days with weekend inter- missions. The summation itself may be done graphically, as shown in Figure 4-6. When the period considered is long, the graphical pro- cedure is tedious and mathematical summation is preferred. The principle of such a summation is explained below, taking the most simple single compartment model as an example and assuming for sake of simplicity daily instantaneous ex- posure of magnitude Q. 28 The systemic content of a substance after single administration changes as a function of time ac- cording to the formula (13) [S]! = Qe‘" Immediately after administration (t = 0) the body burden will equal Q. One day later it will be Qe ’2‘"; two days later Qe 48' etc. lf at all these intervals repeated doses Q are introduced into the system, the body level at subsequent days will be represented by a sum consisting of “fresh” dose Q plus the amounts that have been retained from the previous doses, and on subsequent days we shall obtain following values: Q; Q + Qe'z‘r; Q + Qe 2” + Qe 48' etc. This series may be ex- pressed by the equation: ~24nr —24(n—l)r - _L:€_ 21 _ Q —24r ( ) l—e [8],, = o"; e where n = number of the consecutive days of ex- posure. At sufficiently long observation (n—mae ) the limit of this function will be given by “m [S]" ": Q TTT—557 (22) "—900 For more complicated models the same principles of calculation apply, and solutions are obtained of a similar shape but only more complex mathe- matically. For each of the models discussed here, the function can be calculated also for daily re- peated exposures of several hours’ duration. ln- terested readers may consult another monograph of the present author (Piotrowski, 1971). The functions describing trends of cumulation in the period of repeated exposure (formula 21) reach asymptotically the limit which denotes equilibrium between daily absorption and elimi- nation. This regular trend is disturbed by the weekend breaks in exposure. Then the body bur- den or metabolite levels in biological media drop. The steeper the slope, the faster is the turnover of a substance; and after the return to exposure the next week, they continue to rise again. JERZY K. PIOTROWSKI The rising trend is most pronounced in the first week of exposure. From theoretical consid- erations, it follows that for substances with a very slow turn-over, after a very long exposure period, the upward trend within the week may be very slight or even inconspicuous due to the usu- al biological scatter of the data. An example of upward trends in the first and in a remote week of exposure for substances of an assumed elimi- nation constant (O.l dayr') is presented in Figure 4-7. The trends displayed by body burden or uri- nary excretion of substances, or their metabo-‘ lites, in weekly cycles may be calculated the- oretically; however, the resulting formulas are quite complex and have not been so far verified experimentally (Piotrowski, 1971). The concept underlying calculation of the time- course of cumulation as presented above, pro- posed in principle by Soucek and Pavelkova (1953), has its adherents, among them Filov (see Goulebev et al., 1973). h should be emphasized, however, that the volume of experimental data that could be found for verification of the meth- od is limited. For substances that are considered in some detail in this monograph, such veri- fication has been obtained for nitrobenzene. For other substances for which the problem of sys- temic cumulation is pertinent (trichloroethylene, tetrachloroethylene, parathion, DDT, etc.), lim- ited kinetic data for repeated expsoures are avail- able. However, they have been confronted with results representative of single exposure, in the manner postulated here. Bearing in mind the limited scope of experimental data, wider appli- cation of the concepts recommended here must 3 W „„,- oo 2 . 1 .. N ”1 1 2 :**—4 5 6 day-— Fig. 4-7. Increasing trends of systemic levels of a substance in the first week (N = l) and in an infinitely distant week (N ~w) of exposure. Calculated for the single-compartment model with coefficient of loss 0.1 day-'. (Piotrowski. 1971). Taken from: as in Fig. 4-1, Page 109, Fig. 27. METABOLIC AND EXCRETION KINETICS be left to the discretion of individual in- vestigators, and their confidence in the kinetic methods of description for the observed regularities. SYSTEMIC CUMULATION AND EXPOSURE TESTS Elaboration of an exposure test, in fact, may be reduced to the assessment of exposure level (E) and the determination of the corresponding level of the foreign substance, or its metabolite, in the biological medium (I). Determination of the lat- , ter must take place at a constant time relative to the end of the daily exposure period. ln the most simple case. the reading of the test (1) is directly proportional to the level of exposure, in accord with the equation: |I =a,E (23) where subscript l denotes first day of the ex- posure cycle (e.g., an experiment on individuals that have not been previously exposed). For sub- stances that do not undergo substantial systemic cumulation, the proportionality coefficient “a” will not vary with consecutive days of exposure (aI : a”. and II = loo, at E = constant). These conditions are fulfilled by such substances as phenol. aniline. carbon disulphide, etc. For sub- stances which undergo cumulation in the body, the relationship (23), obtained from mea- surements in individuals not exposed previously, will not hold under conditions of repeated ex- posure. For further discussion it is convenient to assume that a worker is observed during a week infinitely remote from the onset of exposure, when the relationship would be expressed by the formula: 10‘, = a“, E (24) Thus, for interpretation of the experiments per- formed in toxicological chambers, it is essential to estimate coefficients that would permit de- duction of equation (24) from equation (23). In fact, this is possible when kinetic data are consid- ered which may be obtained additionally from a properly designed chamber experiment. From the latter we have obtained the time-curve of levels 29 of a substance (e.g., urinary excretion rate as a function of time) that, for period T = t — T after discontinuation of exposure, may be expressed by a general formula: #.(7) q („:w—r” +u2cr2' (25) where u |/q is the reading of the test (excretion rate) standardized against absorption rate (or ab— sorbed dose) in a constant exposure period T. Coefficients u, and #2 may be obtained empir- ically as intercepts of the two terms of the excre- tion curve (model ll or lll; in the model I, #2 = 0) for 7 = O. For an infinitely remote week of exposure the following equation holds: T wz Lhc” (26) where Lls and LZs denote borderline values of cumulation coefficients for both terms of equa- tion (30). Exposure is usually measured toward the end of the daily exposure period, at the end of the week. For a chamber experiment this corresponds to the equation in which 7 = 0, and therefore equation (23) will assume the form: ll =( “i + “2)E (27) Assuming further that in industry the mea- surment takes place on the fifth day of an in- finitely remote week, directly after cessation of exposure, the equation corresponding to equa- tions (24) and (26) will be obtained in form: != „L„ (5›+ „21mm (28) where LMS) is an asymptotic value of coefficient Li. calculated for the fifth day of an infinitely re- mote exposure week. Through respective com- parison of equations (27) and (28), and respecting the general form of equations (23) and (24), we obtain: 30 ulLls(5)+ #ZLZSÓ) "I + ”2 (29) aeg—dl Coefficients Li,. in this equation, estimated for T = 0, are given by equation: I Fen—m)ri I—e7ri e(j+m)ri +21 (30) l—eri Lis : I—efi JERZY K. PIOTROWSKI where coefficients L, (L, or L2) correspond to the terms of the equation (excretion) determined by coefficients ri (rI or rz),j = number of a con- secutive day in a given week of exposure, m = number of weekend days (1 or 2, depending on whether there are 5 or 6 working days in a week). Further details concerning formula (30), deduced by Domanski and Mikolajczyk, were given in the previous monograph of the present author (Piotrowski, 1971). This monograph should also be consulted for practical advice re- garding kinetic calculations from experimental data. REFERENCES Bartonicek V.: Distribuce volneho a'kyselou hydrolyzou odstepaneho vazaneho sir- ouhliku v organech bile krysia (The distribution of free and hydrolyzab/e carbon di- sulfide in white rat organs). Prac. Lek. 11, 504-508, 1959. Golubev A.A., Liublina E.l., Tolokoncev N.A., and Filov W.A.: Kolitshestvennaja Toxikologija. (Quantitative Toxicology). Medicina, Leningrad, 1973. Piotrowski J.: The application of metabolic and excretion kinetics to problems of in- dustrial toxicologr. U.S. Government Printing Office, Washington D.C., 1971. Solomon A.K.: 7he kinetics of biological processes. Special problems connected with the use of tracers. Adv. Biol. Med. Physics 3, 65-75, 1953. Soucek B.: Vylucovani latek z organizmu. (Excretion of substances from the or- ganism). Prac. Lek. 4, 188-195, 1952. Soucek B. and Pavelkova E.: Vylucovani kyseliny trichloroctove. (Trichloroacetic acid excretion.) Prac. Lek. 5, 62-64, 1953. 5. WORKING OUT THE EXPOSURE TESTS ON VOLUNTEERS IN EXPERIMENTAL CONDITIONS INTRODUCTORY REMARKS Experiments made directly on humans in con- trolled conditions, aimed at elaboration of the exposure tests, are usually a final stage of longer preparatory studies that have been performed earlier. Due to difficulties which are generally en- countered in finding a large number of volun- teers, these experiments are designed as a rule to provide answers only to some selected problems, or aspects of a problem, that could not have been solved either by means of animal experi- mentation, or by studies of biological material sampled from individuals exposed in industry. One usually starts working out an exposure test by experiments on humans only when the follow- ing conditions have been fulfilled: a) there is a practical need for the test in question for the evaluation of the workers‘ exposure in industry; b) a general outline of the metabolism of the compound is known from experiments on animals; c) there is a sufficiently sensitive method for determining the compound or its metab- olite in biological material; and d) it. has been proven in industrial condi- tions that the available method yields posi- tive results in exposed people. The experiments on human volunteers are usu- ally conducted in several stages. The first of these may have the objective of obtaining data on the quantitative relations between the metabolites in man and the kinetics of their elimination. It is not exceptional that elucidating the human metab- olism of an industrial poison leads to the selection of a metabolite, for the basis of a test, whose value for such a purpose would not have been recog- nized, if the results of animal experiments alone had been considered. Among the compounds, treated in detail in this monograph, this has hap- pened for ethylbenzene, styrene, and nitro- benzene. Nitrobenzene exemplifies pronounced differences in metabolic kinetics between experi- mental animals and man. The next step consists usually of a basic quantitative experiment in which, on basis of applied inhalatory exposure, a correlation is established between the absorbed amount (air-concentration) and the eliminated amount of the substance or of its selected metab- olite. The third stage may be of a complementary nature and may include, according to the'needs, evaluation of cutaneous absorption and, perhaps, studies of the degree of systemic cumulation un- der conditions of repeated exposure. ln practice it seldom occurs that the stages are followed in the logical sequence as given; this can be worked out and introduced into practical use at an early stage of toxicological reconnaissance when basic toxicity is evaluated, MAC values postulated, or methods worked out for the determination of a substance in air. The tests are, as a rule, developed for substances whose toxicity and potential haz- ards are rather well known, and for which the main practical problem lies in adequate control of the magnitude of exposure. Moreover, taking due account of the fact that elaboration of a test is a lengthy and expensive procedure, and that serious motivation must exist for undertaking any experiments on human beings, working out the tests is undertaken mostly for substances that create a serious health hazard in industry. The degree of health hazard may result either from the seriousness of biological effects (e.g. benzene, benzidine), or from widespread application and size of the exposed population (for example: toluene, styrene, carbon disulphide, DDT). A most obvious motivation for the elaboration of an exposure test seems to exist for those sub- stances for which dermal exposure is of signifi- cance (e.g. benzidine, nitrobenzene, aniline, para- thion). lf several motives exist, an exposure test becomes highly desirable. TOXICOLOGICAL CHAMBERS. The chambers used in experiments on volunteers are relatively simple and can be easily construc- ted in most larger laboratories. The chamber it- 31 32 self consists of a small room (or part of it) of such a volume as to accommodate in a com- fortable standing, sitting, or lying position at least one volunteer. The usual volume would thus be 10-20 m3. The chamber should be in the direct vicinity of a laboratory; the contact should be close enough to render easy service of the cham- per possible (taking the samples and performing analyses). The chamber is usually divided into two spaces: the first one is where preparation of the air to given parameters takes place (i.e., dos- sage of test substance, mixing and conditioning of the air); the second space is occupied by the vol- unteer and provides air with predetermined con- centrations of the test substance. A diagram of a typical toxicological chamber, designed by Du- tkiewicz (I960), which has been used by the present author, is presented in Figure 5-1. The design allows for controlled variation of flow- rate, temperature, and humidity of the air. A ca- pability for maintaining predetermined parame- ters of the microclimate is particularly relevant for experiments on dermal absorption of vapours of organic compounds. The dosage of a test substance needed to attain programmed concentrations can be based on var- ious principles, in the same way as it happens in chambers for animal experiments. In the author's laboratory where liquid non-decomposing com- pounds have been studied (aromatic hydro- carbons and their derivatives, chlorinated ali- phatic hydrocarbons, carbon disulphide) a simple method proved useful: namely, a technique based on evaporation of a liquid from a vessel of a constant surface. The increasing concentrations are easily obtained by raising the temperature of the vessel. and keeping it constant by means of a thermostatically controlled heater. Apart from large chambers, enabling studies both on inhalatory and cutaneous absorption, mini- chambers are used sporadically for inhalation studies. One variant, designed by Senczuk and Orlowski (1974), consists of a helmet covering the head of the subject. In another type this chamber forms an air-tight blouse, attached to a dosing mechanism in such a way that in- vestigation of inhalatory absorption becomes possible. These little chambers seem particularly useful in small laboratories, and, also, for pre- liminary ad hoc estimations of absorption of a given compound in the respiratory tract. ' Concentrations of test substances in the exposure chambers may be measured either in a classical way by aspiration and determination in a solu- tion by one of the available methods, or directly in the gaseous phase using physico-chemical tech- JERZY K. PIOTROWSKI niques. UV-absorption is one of the methods of choice; as an example, a simple Mercury Vapour Concentration Meter (Bardodej, 1964) may be quoted. A system, particularly suitable for anal- ysis of expired air by UV-absorption mea- surements was described by Bocek and Nemecek ([970). lnfra-red absorption measurements are also used, mainly in gas-cuvettes of the multi- reflex type, enabling a light beam pathway of several meters to be obtained. Finally, the most universal method — gas chromatography — is com- monly used at present. The latter may be com- bined with aspiratory sampling of air or with direct injection of the air sample into the col- umn of the instrument. The latter technique has ”X 230cm Fig. 5-1. Example of a chamber designed for experiments on volunteers (Dutkiewicz, 1960). Legend: (I) main chamber space, (2) mixing chamber, (3) vapour-generating device, (4) air flow meter, (5) steam inlet, (6) electric heater, (7) contact thermometers, (8) pendulat ventilator, (9) suction channel, (10) valve, (I I ) and (12) division walls with regulated slits. Maintaining of desired temperature and humidity automatic through (5 ), (6 ). (7 ). Airflow regulated mechanically through ([0). Vapour generation by heating of the liquid substance in a temperature yielding the desired concentration. Taken from: Dutkiewiz T.: Toksykologii-zna komora doswiadczalna. Medycyna Pracy ll, 117- 124. I960. WORKING OUT THE EXPOSURE TESTS ON VOLUNTEERS IN EXPERIMENTAL CONDITIONS 33 been used extensively by R.D. Stewart; the refer- ences to this work are in the chapters on “Sty- rene", “Trichloroethylene”, and s'Tetrachloroethylene”. PURPOSE AND DESIGN OFTHE EXPERIMENTS The experimental design (including the number of persons exposed concurrently), the range of air concentrations. the frequency of urine and breath sampling, etc., depends on the purpose of the ex- periment and the facilities available. Experiments of the first reconnaissance stage aim usually at estimating the respiratory retention, as well as, the amount of excreted metabolities. The experiment, therefore, should incorporate anal— ysis of the inspired and expired air, and mea- surement of its volume. Collection of urine may be performed according to any desired scheme, up to termination of the measurable excretion. For methodological reasons, as a rule, it appears useful to apply a relatively large dose of a sub- stance, and therefore starting the study from a series of self-experiments is desirable. This pre- ‘ liminary experiment permits orientation as to how the kinetics of metabolite excretion look. A series of examples of earlier experiments of this type have been assembled in the monograph by Teisinger et al. (1956). Systematic experiments have to have more pre— cisely defined objectives, namely: a) establishing a relationship between exposure (dose) and urinary level of a metabolite; and b) derivation of kinetic parameters enabling interpretation of the data for repeated exposures. In the author’s laboratory the following experimental design has been accepted as the most useful one: a) The experiment is conducted for air con- centrations in a range below and above the accepted TLV“, incorporating as a rule a series of 3 to 4 concentrations with an in- terval of a factor of 2. For each concen- tration 3 to 5 experiments are made on dif- ferent individuals. The total number of volunteers amounts on the average to 5-10. The duration of exposure has been adapted to the length of a working shift. When for a given substance two daily exposure inter- vals (6 and 8 hr) are met in industry," the ' Threshold limit value. ” ln Poland for highly toxic substances the permissible daily duration of exposure is 6 hr; in the rest of cases 8 hrs; in equivocal situations both lengths of daily exposure are encountered in various branches. experiment is for 8 hours, and the design allows for interpretation of the results with regard to both situations. ln the course of the experiment one or two 1/2 hour breaks are made for meals. b) Urinary samples are collected every 2 hours over the exposure period, and for the rest of the day; the night urine is collected as a single portion. lf collection over the next day appears necessary, it is instituted at liberal intervals. The principle to be ob- served, however, is to collect for analysis complete portions of the urine excreted within an interval, with precise notification of the voiding time; this permits calculation to be made of the excretion rate. ln all samples, concentration of a metabolite and specific gravity or concentration of cre- atinine are measured. These parameters per- mit analysis of the correlation for various variants of an exposure test. INHALATORY EXPERIMENT ln the author‘s laboratory the inhalatory experi- ments are performed in such a way that exposed subjects stay outside the chamber and breathe the air from the chamber through a respirator with two one-way valves. Such a procedure precludes dermal absorption during the experiment. The sampling method of the inhaled air is essential for an appropriate calculation of the absorbed amount of the substance under study. Simple measurements of the concentrations in the cham- ber air cannot be applied in the calculations be- cause of the adhesion of the substance to the tubing; true concentrations in the inhaled air are lower than those in the chamber. For practical purposes, a satisfactory solution would be such that secures sampling of inspired and exhaled air directly from the tubing in the proximity of the subject‘s face. This principle has not been com- monly adopted by all laboratories; in most in- halation experiments the exposed individuals are placed inside a chamber which does not exclude cutaneous absorption. ln these experiments the measure of exposure is the amount of a substance retained in the system in the course of the experiment. To some extent this allows elimination from consideration of one source of variation of the results that would oc- cur if the test were based merely on concentra- tions in the chamber air. These variations depend on individual differences in lung ventilation rate. The necessary step from the doses to concentra- tions is made in computation, assuming a likely 34 ventilation rate in industrial workers, according to the formula: D = CTVR (1) where D = the absorbed dose (in mg), C = con- centration in the inspired air (mg/m3), T = du- ration of the exposure (hr), V = ventilation rate (mJ/hr), R = retention of a substance in the respiratory tract, expressed as a fraction (dimen- sionless). While comparing the doses absorbed by workers with recommended or legally obligatory values of TLV a ventilation rate typical for light Work can be assumed — namely 0.8-1.25 m3/hr. This procedure has not been generally accepted; moreover, experimental designs dominate in which direct concentrations in the air in chamber are taken as the measure of exposure. Examples of both approaches may be found in the chapters devoted to specific compounds. When the experiments have been completed for a series of concentrations, the results may be dia- gramatically presented as in Figure 5-2. In the course of exposure, the concentration of a me- tabolite (excretion rate) rises, then declines there- after with the cessation of absorption. The elimi- nation curves obtained for various doses (air- concentrations) are usually proportional to the doses. For practical purposes values are relevant l I I t (hours) Fig. 5-2. Schematic presentation of the time- course of concentrations in biological media (C), as afunction of time, at various exposure levels ( 5) JERZY K. PIOTROWSKI fa) T fh/ T .l ~J ,l [toz » Fig. 5-3. Schematic presentation of the level of substance (or metabolite) in urine collected to- ward the end of exposure (see Fig. 5-2), as dependent on the exposure level (E) or dose absorbed (D); (a) concentrations uncorrected ( C ); (b) concentrations corrected resp. excretion rate (u). WORKING OUT THE EXPOSURE TESTS ON VOLUNTEERS IN EXPERIMENTAL CONDITIONS 35 [.!/9 _Q5 Fig. 5-4. Excretion rate of a metabolite expressed as afraction of the absorption rate of the parent compound (u/q) observed during exposure and after its cessation. Log ”/9 Fig. 5 -5 . Excretion rate (expressed as afraclion of the absorption rate) as dependent on the time- lapse after cessation of exposure, in a semi- logarithmic system of coordinates. at a constant point in time, usually those ob- served in the last two hours of exposure. The lev- els of metabolite in this interval (shaded area) are correlated with absorbed doses in a way pre- sented in Figure 5-3. Two variants of a test are presented on the same measurements; the graph (b) shows the concentrations in urine as mea- sured directly, and graph (a) presents the same data normalized to standard specific gravity, av- erage creatinine content, or excretion rate. Accu- racy of the correlation and precision of the as- sessment of the absorbed dose may be estimated statistically for each of the variants. As a rule more precise results are obtained if the excretion rate of a metabolite is taken as the variable. This rate is a product of the concentration of the me- tabolites and the excretion rate of urine: u = cd (2) where u = excretion rate of a metabolite (mg/hr); c = concentration of the substance in urine (mg/ ml), and d = excretion rate (mg/hr). Differences in precision of the variants of the test were most pronounced when absorption of phenol has been assessed from urinary excretion of the substance. If direct urinary concentrations were taken as the dependent variable, the absorbed dose could be estimated with precision of about i 80 per cent only; this could be improved to about i 40 per cent when the results are normalized to the stan- dard specific gravity of urine = 1.024, and reach- ed the unusual value of i 6 per cent for the uri- nary excretion rate of phenol. A similar trend, even if less pronounced, was observed when the tests were worked out for aniline, nitrobenzene, and toluene. The experimental design, as outlined above, al- lows for calculation of the kinetics of the process from the experimental data. ln the most simple case, in' all experiments in which the measured values over a given time interval were analytically satisfactory, the data could be presented in the form: u/ q = f(t) (3) where u = excretion rate of a metabolite (mol/ hr), q = absorption rate of a parent substance (mol/hr) (Fig. 5-4). In this arrangement, if meta- bolic efficiency were independent of the dose, the curve would in turn be independent of air con- centrations of the substance. Over the exposure period the value u/q tends to reach an asymp- tote, determined by the metabolic efficiency. In the most favourable case, the value of the latter equals unity (e.g. phenol). The results obtained for the descending slope of the curve, depicted in the semilog coordinates, yield the half-time and excretion constant (Fig. 5-5) that is required for final interpretation of a test (see chapter “Kinet- ics"). For substances whose elimination constants 36 exceed O.! hour ', no cumulation should be ex- pected at repeated industrial exposure; if the va- pours of a substance are not absorbed through the skin, the test is complete. lnterpretation of the test may be then based directly on the graph (Fig. 5-3) or on a formula of the type: ut : uo + aD (4) where Ut = excretion rate of a metabolite (or its concentration) at a constant time relative to the period of exposure, e.g., the last 2 hours of ex- posure; D = the dose absorbed during the experi- ment; 3 = regression coefficient as estimated (usu- ally by means of least squares); Uo = mean back- ground level, assessed in the period preceding the experiment. When the relationship appears curvi- linear, the formula may be calculated by means of one of the nonlinear regression functions. If the elimination constant is less than 0.1 hrl in the final formula that expresses quantitative in- terpretation of the test, a due correction has to be introduced (see chapter “Kinetics"). If the cu- mulative trend is pronounced, the correction can be considerable and its experimental verification in daily repeated exposures may be useful. The above principles, outlined earlier by Pio- trowski (I970) in their basic form, seem also to apply to the tests based on analysis of the breath. Essential difference does not depend here as much on the change of analytical technique, as on the difference in the dynamics of behaviour of volatile substances eliminated in the expired air relative to that inherent for excretion of metabo- lites by the kidneys. As far as the analytical pro- cedure is concerned, it is essential to assure re- producible and well-defined sampling of exhaled air. There seem to exist two possibilities: analysis of alveolar air (an isolated sample collected to- ward the end of expiration), or analysis of the to- tal exhalation. For the latter alternative a useful technique of sampling was described by Sher- wood and Carter (1970). Details of sampling and interpretation of measurements in alveolar air were given by Bocek and Jandrova (I970). How- ever, it seems that detailed principles regarding the time of sampling have not been precisely for- mulated. Difficulties in making a decision about sampling time result from the very fast decline of the concentrations. It appears that sampling in the first few minutes post-exposure should be avoided as desorption of a substance from the upper respiratory passages is still taking place. On the other hand, too long a delay in sampling can be inconvenient due to the considerable decline of the concentrations. Furthermore, the decision be- .IERZY K. PIOTROWSKI comes even more complicated in industrial condi- tions because the time of sampling cannot be scheduled with sufficient precision relative to the moment when exposure was discontinued. It seems, therefore, that optimal sampling time should be selected in such a way as to minimize the error that would result from minor shifts in time. There are also difficulties in the interpretation of the data that derive from different ways in which the concentrations in expired air increase as com- pared with the concentration of metabolites in urine. Usually the latter increases almost linerly with time of exposure and thus the measurements depend not only on the absorption rate but also on the time of exposure; in other words, they re- flect the total absorbed amount (dose). On the other hand. concentrations of volatile substances in the breath increase rapidly at the onset of ex- posure with slow changes afterwards. Therefore, measurements of elimination in the expired air re- flect more likely the actual absorption rate than the accumulated dose. This problem has not been studied in sufficient detail. Of interest is the pro- posal of Sherwood and Carter (1970), who sug- gested with regard to benzene that for assessment of systemic deposits of the substance the expired air should be analyzed as sampled on the next day after exposure, before the start of work. At that time the elimination rate depends more on the systemic deposits than on the recent absorp- tion rate of the previous day. CUTANEOUS EXPOSURE From the point of view of exposure tests, experi- mental evaluations of dermal absorption are par- ticularly essential if the substance in question is absorbed through the skin directly from the gas- eous phase. In such a case it has to be presumed that the TLV for the concentration in air was es- tablished on the assumption that absorption takes place both via the skin and the respiratory tract. This has obvious bearing on the re- lationship between the allowable concentration and the allowable absorbed dose. The only possibility of quantitatively assessing dermal absorption from the gaseous phase rests in the utilization of the previously elaborated ex- posure test, based on the inhalatory penetration of a substance into the system. Determination of a metabolite after dermal exposure permits calcu- lation of the absorbed dose from a previously es- tablished graph or from a proper mathematic re- lationship. There is of course the underlying WORKING OUT THE EXPOSURE TESTS ON VOLUNTEERS IN EXPERIMENTAL CONDITIONS 37 assumption involved that the efficiency of metab- olization, as well as the dynamics of excretion, are not essentially different for both routes of ab- sorption. For further discussion of this issue see the chapter “Absorption routes". Technically the experiment is conducted in such a way that the volunteer is inside the tox- icological chamber but breathes clean air from the outside through a respirator and required tubing. Microclimatic conditions play an essential role in such experiments and should, therefore, be thoroughly controlled. In the subsequent se- ries of experiments the following issues are usu- ally studied: a) influence of the concentration of the substance under study on absorption rate, b) influence of temperature and humidity, and c) ef- fects of clothing. The data permit an assessment of how much the variable conditions in the work- ing environment affect cutaneous absorption from the gaseous phase. Of basic importance for the quantitative inter- pretation of the test is the relationship between the concentration of the substance in air and the dermal absorption rate. In the simplest case it can be linear. and then the absorbed amount would be directly proportional to the concen- tration C and duration of exposure T: D = aCT (5) where a = a proportionality coefficient. If the dose D is expressed in mg, time in hours, air- concentration in mg/m3 then the dimension of the coefficient a would be thr. This provides information about the volume of air “cleared” through dermal absorption of the substance per unit of time. This coefficient is directly compara- ble with the coefficient RV (equation I) for the in- halatory exposure. When both cutaneous and respiratory absorption occurs, the relationship between the absorbed dose and air concentration would be expressed by the formula: D = CT(RV + a) (6) in which the magnitude of both coefficients re- flects the relative role of the two processes in to- tal exposure. Examples have been given in the chapters “Phenol", “Aniline“, and “Nitrobenzene”. For evaluation of the routes of absorption, quan— titative experiments are sometimes made of der- mal absorption from direct contact of a sub- stance with the skin. If it is possible to eliminate the losses by evaporation, or to assess quan- titatively the loss, the amount absorbed may be calculated from the reduction of the substance at the site of application (for examples see the chap- ters: “Aniline”, “Nitrobenzene”, “Benzene”). The technique is rather subtle and subject to numer- ous reservations whose detailed discussion falls outside the scope of present monograph. Basic- ally speaking, however, this is one of the possible ways to test the hypothesis of the independence of the metabolism of a substance from the route of absorption. Another alternative and indirect possibility is provided by a study of the ratio of two different metabolites after administration of a substance by two different routes. In such a case it appears desirable that the metabolites be formed along two independent metabolic path- ways. An example is given in the chapter “Nitrobenzene”. Quantitative data on the cutaneous absorption rate of a liquid or solid substance in direct con- tact with the skin are of comparative significance. The values do not enter the equations upon which the interpretation of exposure tests is based. On the other hand, in industrial condi- tions it is possible to estimate the intensity of dermal absorption. The principle is based on a precise comparison of the effective concentration to which a worker has been exposed during the work shifts with the absorbed dose which is esti- mated by means of an exposure test. The amount exceeding that given by formula (6) is attributed to cutaneous absorption resulting from direct contact of the substance with skin. EXPERIMENTAL REPEATED EXPOSURE Simulation of daily repeated exposure in experi- mental conditions would be highly desirable be- cause of the scarcity of such human data. This is particularly true when the degree of expected cu- mulation in the body is considerable, and there exist reasons to suspect that using the coefficients calculated from kinetic equations may lead to serious errors. Generalizations based on the few existing experi- ments seem premature; the remarks given below should be considered only as suggestions. The basic objective of such experiments is acqui- sition of the data characterizing the steady-state situation. Therefore, the experiments should be continued until the moment when the daily ex- creted amount of a metabolite equals that ex- 38 pected from the metabolic efficiency" which had been determined from a single inhalatory experi~ ment. Observations of the dynamics of the in- creasing urinary levels of a metabolite in sub- sequent days provide basic data to answer the question concerning which day of the week the samples should be taken for evaluation of exposure. Due to easily understandable difficulties in at-, tracting volunteers for experiments of this type, the number of studied individuals and of discrete experiments will be more seriously limited than those of single exposure assays. This means that collecting data for various levels of exposure is particularly difficult, and in practice one is left with direct proportionality extrapolation. There- fore, the data on levels of metabolites at a given magnitude of exposure cannot be presented with very great precision. Moreover, since systemic cu- mulation of organic compounds is determined largely by their deposition in adipose tissue, indi- vidual variation in amounts of body fat may af- fect the magnitude of the cumulation. ln addition any factor that mobilizes a substance from ad- ipose deposits could elevate the urinary levels of the metabolites, to the point that they exceed by far those amounts that could be attributed to the actual absorption rate at a given metabolic efficiency. Therefore, it may be suggested that experiments with repeated exposure should be treated mainly as controls for kinetic extrapolations. If there is a statistically insignificant difference between the two procedures, the choice of appropriate values for interpretation of a test forms a dilemma that must be left to the intuition of the observer. PHYSIOLOGICAL OR “BACK- GROUND” LEVELS OF METABOLITES The relationship given by equation (4) indicates that for the exposure tests the significance of the absolute level of a metabolite after exposure is less than the significance of the increase above the normal “physiological" or “background” lev- el. The increment is attributed to the absorption of the substance in question. The precision of the test decreases sharply at low levels of exposure when physiological levels contribute significantly ‘ This may become difficult in presumably rare situations, when autoinduction or “autoinhibition” could distort the picture; for fuller discussion see the chapter “Biotrans— formations“. JERZY K. PIOTROWSKI to the total level observed. This seriously limits the application of the tests to low levels of ex- ‘posure. Examples are given in specific chapters, mostly those dealing with toluene and carbon disulphide. Direct assessment of the physiological level and of the increment is not possible in the same sam~ ple; evaluation of the physiological or back- ground level always bear the stigma of an extrap- olation. The error of extrapolation is directly related to the magnitude of oscillation of the physiological level. The observer’s influence upon the dispersion of the physiological values is cer- tainly limited; however, applying due corrections (for specific gravity, creatinine content, or di- uresis similar to those discussed above for the very tests themselves) may considerably reduce the variation in some cases. In the author’s labo- ratory the issue of determining a true phys- iological level is usually considered in a separate preliminary experiment in which the urine of non-exposed individuals is sampled in exactly the same way as in the experiment itself. ln addition, each person volunteering for exposure experi- ments collects his or her urine over a complete 24 hour period preceding the experiment, and the mean value of the substance under study is later used as a physiological reference level. ln some cases (toluene, carbon disulphide) phys- iological levels of the metabolites display season- or nutrition-dependent variations. Such a possi- bility should be investigated in preliminary stud- ies for an exposure test. PRECISION OF EXPOSURE TESTS The precision with which absorption of toxic compounds in industry can be assessed by means of an exposure test is debatable. Some light on the subject has been shed by the data from in- halation experiments on human volunteers. The precision of a test may be assessed from the results of an inhalatory experiment only if crite- ria of the magnitude of exposure have been set adequately. Dispersion of results for various indi- viduals may be more apparent than real if they are related directly to mean concentrations in the chamber, disregarding individual variations in re- tention and ventilation rate. Thus, an earlier opinion of Teisinger and his group (Teisinger et al.; 1956), based upon consideration of extreme variations, appears now too pessimistic. From the present author’s own experience, if the ab- sorbed doses are taken as the independent vari- able, a well-controlled test can, as a rule, be WORKING OUT THE EXPOSURE TESTS ON VOLUNTEERS IN EXPERIMENTAL CONDITIONS 39 characterized by a precision of: 20 per cent. Ex- ceptional cases were found in the exposure test for phenol which, due to the simple metabolism and high excretion rate, displayed a precision of belowilO per cent. On the other hand, the pre- cision of the test for nitrobenzene, due to com- plex biotransformation and to cumulation of the substance in the adipose tissue, could not be ob- tained below i 30 per cent. A usually attainable precision of t 20 per cent seems satisfactory as no other method for the evaluation of exposure yields better or even equivalent results. Never- theless, assuming that 20 individuals will have absorbed 100 mg of a substance each, the statis- tics lead to the conclusion that by means of an exposure test, 12 out of 20 will be estimated to lie in the range from 80 to 120 mg, and most of the rest between 60 and 140 mg; 1 individual will fall outside this range. To summarize, in a group of twenty people differences in metabolite levels that vary by a factor of two does not form evi- dence for a real difference in exposure. To reduce the dispersion a so—called “collective" test was proposed (Teisinger). This concept limits dis- cussion to the estimation of the average exposure of a group of individuals engaged in the same type of work. Too rigorous adherence to this concept. however, would preclude identification of individual cases of excessive exposure. Anoth- er variant of the same concept was suggested by the present author, who proposed to assess the mean daily dose of nitrobenzene absorbed by a given worker on the basis of several mea- surements performed on separate days. The con- cept underlying this proposal accounted for both the unsatisfactory precision of the test (t 30 per cent) and the systemic cumulation of the sub- stance. Due to the latter factor the results of the test provide insight into the absorption over the last few days, rather than to that on any one day. Despite the above reasoning, the question of the precision of exposure tests in industrial situations remains open. ln chamber type experiments the rhythm of exposure is constant, and this factor certainly reduces the dispersion of the results. In- troduction into the body of the same dose, but at various time intervals (for instance mainly in the first or in the last hour of exposure), may en- hance considerably the variation of results, and make the precision much worse than that seen in controlled experiments. However, quantitative data with regard to this issue do not seem to be available. SAFETY PROBLEMS The exposure tests for toxic substances are devel- oped for low doses predetermined by the accept- ed TLV’s. Assuming that the TLV’s are intended to be safe for persons exposed daily for the working lifetime, and that they incorporate sig- nificant safety factors, the chance of endangering the health of volunteers by a single exposure seems remote. In spite of this, taking into ac- count the possibilities of hypersensitive volun- teers, unidentified diseases, and finally possible, even if highly unlikely, experimental errors, the experiments on volunteers must observe all possi- ble principles of prudence. In the author’s labora- tory the following principles are routinely observed: I) All individuals who may participate in the experiments are medically examined in a clinic specializing in occupational diseases and intoxications. The examining physician is informed about the nature and mag— nitude of exposure. Selection of the volun- teers is based on the same principles that are applied for admission to work with ex- posures to the substance to be studied. 2) In the course of the experiments there is ready access to a physician, who is notified in advance about the nature of the hazard. 3) In a preparatory period of the in- vestigation, concentrations of the substance to be studied in the chamber are deter- mined by two independent methods in par- allel. When the experiment is in progress, the concentrations are determined at least once every hour. 4) All preparatdry experiments, as well as changes in exposure parameters (concen- tration, duration of exposure, temperature, humidity, absorption route), are preceded by auto-experiments. The investigators who have not been given medical consent for auto-experiments for health reasons are not permitted to participate in experiments on volunteers. 5) The volunteers are thoroughly informed about the nature and magnitude of ex- posure, and about symptoms of poisoning with the substance in question. 6) ln the course of the experiments the chief investigator or his assistant are always present, and continuously observe the be- haviour and reactions of the volunteers. 7) The experiment must be discontinued at 40 JERZY K. PIOTROWSKI once when a volunteer so wishes, regardless of the reason. The strict application of the above principles, over twenty years of research on exposure tests, has resulted in not having a single case of sus- picion of poisoning in a volunteering individual. REFERENCES Bardodej Z.: Metabolismus styrenu. (The metabolism of styrene). Ceskosl. Hyg. 9, 223-239, l964. Bocek K. and Nemecek R.: Mechanismus vstrebovani toxickych par p/icemi. I. Po~ kusna aparatura pro sledovani prubehu koncentrace organic/(ych par ve \{l‘dechovanem vzduchu. (Mechanism of toxic vapour uptake by lungs !. Experi- mental apparatus recording organic vapour concentration in expired air). Prac. Lek. 22, 20l-206. I970. Bocek K. and Jandrova R.: Mechanismus vstrebovani toxickych par plicemi. II. Vliv tle/ky zatlrze dechu a doby expozice na koncentraci trichloroethrlenu v posledni casti wdechu. (Mechanism of toxic vapour uptake by lung. II. Trit'hloraethylene concen- tration in the last portion of expiration — effect of breath-holding period and length of exposure). Prac. Lek. 22, 233-243, 1970. Dutkiewicz T.: Toksykologiczna komora doswiadczalna. (Experimental toxicological chamber.) Med. Pracy ll, 117-124, 1960. Piotroswki J.: Ekspozicjonnyie testy i sistema predelno dopustimych koncentracji to- ksitsheskich veshtshestv w wozdushnoj srede promyshlennych predpijatij. (Exposure tests and the system of TLV in the air of industrial premises). In Principy i metody apriedelenija predelno dopustimych koncentracji v promyshlennosti. Proceedings of a Symposium, Medgiz, Moskwa, 1970. Senczuk W. and Orlowski J.: Opis i charakterystyka inhalactj/nej komory dos- wiadczalnej. (Description and characterization of an inhalation chamber). Bromatol. Chem. Toksykol. 7, 83-85, I974. Sherwood RJ. and Carter F.W.G.: The measurement of occupational expsoure to benzene vapour. Ann. Occup. Hyg. 13, 125-146. 1970. 6. BENZENE ABSORPTION Under industrial conditions, absorption of ben— zene occurs mainly via the respiratory tract. Penetration through the skin may also be of some importance in cases of direct contamination with the liquid. Retention of benzene vapours in the respiratory tract decreases With the duration ot the exposure. At the onset and toward end of an exposure of several hours‘ duration, the values found amounted to 70-80 and 50 per cent (Srbova et al., I949). or 75 and 62 per cent (Dutkiewicz, 1971). respectively. Absorption of the vapours through the skin is negligible. Contact of the liq- uid with the skin leads to significant absorption; the maximal rate of absorption was estimated at about 0.4 mg cm 2hour ' (Hanke et al., I961). METABOLISM AND ELIMINATION The metabolism of benzene has been studied in experimental animals and in man. ln rabbits some 43 per cent of the administered dose of benzene was eliminated unaltered in the expired air. Sulphates and glucuronides of phenol (24%), hydroquinone (5%), pyrocatechol (2%), and hy- droxyhydroquinone (0.3%) were found in urine. A small percentage is excreted after cleavage of the aromatic ring (muconic acid, about 3%). Less than I per cent is excreted in the urine in the form of phenylmercapturic acid. Carbon dioxide from degradation of benzene (experiments with ”C—labelled substance) appears in small amounts (l.5%) in the expired air (Porteous and Williams, 1949; Parke and Williams, I953). The metabolism of benzene in animals is rather fast, and almost.all of the phenol produced is ex- creted within the first 24 hours after adminis- tration. Excretion of hydroquinone and pyro- catechol continues somewhat longer; hydroxyhydroquinone appears even longer in the urine (Porteous and Williams, 1949). Hydroxy- lation, the basic process in the metabolic trans- formation of benzene. is an enzymatic process; and arylhydroxylase, localized in the microsomes of the hepatocytes, is the responsible enzyme. 4] The metabolism of benzene, however, is not lim- ited to the liver (Parke, l968). The rate of the process may be modified both by inhibitors and by inductors of microsomal transformations (Gut. 1971; Lustinec et al., 1969). However, chronic exposure of rats to benzene does not seriously alter the original rate of benzene hy- droxylation, as measured in rat liver homoge- nates in yilro (Wisniewska-Knypl et al., 1975). Humans. exposed experimentally to benzene by inhalation. eliminate part of it in an unaltered form in the expired air (12-16 per cent) and traces of it in the urine (01-02 per cent) during the desaturation period. The metabolites found in the urine were: phenol, 30 per cent; pyrocatechol, 2.9 per cent: and hydroquinone. 1.1 per cent of the absorbed amount (Teisinger et al., 1952; Te- isinger et al.. 0956). In other laboratories a some- what higher fraction of the chief metabolite (phe- nol) was found: about 40 per cent (Dutkiewicz, 1971) or 50—87 per cent (Hunter and Blair, 1972). From experiments on volunteers about 50—90 per cent of the retained amount may be accounted for; the fate of the rest remains obscure. The rate of the metabolic transformation of ben- zene in man has been estimated on the basis of the kinetics of its disappearance from the blood and expired air, and from a similar disap- pearance rate of phenol in the urine. Srbova et al. (1949) found that the disappearance of ben- zene from the blood and exhaled air occurred with the decay coefficient of 0.23 hour". Sher- wood and Carter (1970), on basis of preliminary observations, favour the opinion that in the ex- halation of benzene two phases may be dis- tinguished, with half-times of ] hour and 1 day. Hanke et al. (1961) and Sherwood (1971) estab- lished the rate of disappearance of urinary phe- nol following exposure to benzene at approxi- mately 0.l hr". More complete equations are at least biphasic, as given by Dutkiewicz (l97l) where the respective coefficients of disappearance were 0.35 and 0.077 hr". From these data, a two compartment open model could be postulated (Piotrowski, 1971) in which the rate of the irre- versible loss (expiration and metabolic trans- formation) is represented by a coefficient of 0.18 42 hr-'. The transfer of unaltered benzene from the rapid exchange compartment into the adipose tis- sue, and its return, would occur with respective rate coefficients of 0.08 and 0.19 hr". A more so- phisticated, tricompartment model was proposed recently and solved Using an analogue computer / (Fiserova-Bergerova and Cettl, 1972). From the existing data it follows that, under con- ditions of industrial exposure to benzene, one should not expect a significant rise of phenol in the urine in consecutive days of the work week. However. there is very little systematically col- lected data to support this contention. Thus, Sherwood (1972) has found somewhat elevated phenol levels in the morning urine, even after the weekend break, in one worker exposed to high concentrations of benzene vapours. On the other hand, the possibility of some benzene cumulation in adipose tissue and in the bone marrow may not be excluded. Elimination from these tissues might cause a rising trend of benzene concen- trations in the expired air which is collected be- fore the onset of work on consecutive days (Sher- wood and Carter, 1970). DETERMINATION OF BENZENE AND ITS METABOLITES At present. from the point of view of exposure tests, only the determinations of benzene in ex— pired air and of total urinary phenol appear of importance. DETERMINATION OF BENZENE IN EXPIRED AIR In earlier studies, the determinations of benzene in the air in experimental chambers, in the work environment, and in the exhaled air were usually made by absorbing the compound in a nitrating mixture. Subsequently, the resulting m-dinitro- benzene could be determined by means of polar- ography (Teisinger et al., 1956) or col- orimetrically with butanone, or acetone, and alkali (Schrenk, 1935; Piotrowski, 1954). 1n the more recent reports, gas chromatography is recommended for benzene determination in the expired air; and two different techniques of sam- pling are used: ' (a) A sample is collected into a small glass vessel, coated with a layer of synthetic poly- mer that reduces the adhesion of the ben- zene to the walls. The expired air is passed through the vessel during an entire ex- .IERZY K. PIOTROWSKI piration phase and the final fraction, cor- responding to the alveolar air, is retained in the vessel for analysis. (b) The expired air is collected using a respirator in which an absorbent (silica gel) is mounted beyond the outlet valve. In this case the benzene eliminated in a given time interval (e.g. 10 min.) is collected. By means of this technique, the elimination rate of benzene may be determined. For concurrent evaluation of the concentrations, mea- surements of the volume of air would ap- pear necessary; however, an indirect esti- mate is possible due to the fact that the humidity of the exhaled air is constant. The weight increment of the absorber (25 mg per liter of air) may substitute for a gas- ometric measurement. The concentration obtained using this technique is lower by a factor of 1.5 when compared with the con- centration in the alveolar air (Sherwood and Carter, 1970). The final gas chromatographic determination is made by introducing directly the gas sample or alcohol eluate of the silica-gel absorbent on the column. The convenience of gas-chromatograph determinations of benzene is supported by several reports (e.g., Graziani et al., 1970; Angerer et al., 1973). DETERMINATION OF PHENOL IN URINE See the chapter “Phenol“. OTH ER METHODS In the search for an appropriate exposure test, determinations of benzene have been performed in blood (Fabre and Fabre, 1948; Angerer et al., 1973) and in urine. There have also been at- tempts to utilize the so called “sulphate ratio" in urine (Yant et. al., 1936). This test, however, due to its low sensitivity and poor precision, is only of historic interest. The methods used in the urine test can be found in the older literature (Teisinger et al., 1956), Gadaskina and Filov, (1971). Determinations of pyrocatechol and hy- droquinone in urine (Bergerova and Skramovsky, 1952; Teisinger et al., 1956) have not been uti- lized as exposure tests. BENZENE EXPOSURE TESTS The test for phenol in urine in its original ver- sion. as developed by Teisinger and Fiserova-Ber- gerova (1955), was based on experiments made on volunteers in a toxicological chamber. The de- terminations of phenol (after distillation from an aCid medium that enabled hydrolysis of conju- gated phenol) were made according to Gibbs, as described in the chapter “Phenol”. Interpretation of the test data presented in graphical form made it possible to assess, from urinary excretion of phenol in the daily urine correspond to an effec- in the air to which a subject had been exposed. However, due to the low precision of the evalu- . ation (—25 to +35%), only a crude evaluation of exposure could be attained. At a urinary phenol level below 40 mg/day, average concentrations of benzene in the air were assumed to be below 100 tug/l, even though on the average 100 mg of phenol in the daily urine corresponded to effec- tive concentration of l00 [Ag/l in the air. As a 24 hour collection of urine was necessary for the test, it has not been used in industrial practice. Dutkiewicz (1963, 1965) introduced two substan- tial modifications: (a) basing the test on col- lection of “spot" urine samples toward the end of the exposure period, and (b) acceptance of the dose of benzene absorbed by the inhalatory route, and not the air concentration, as the mea- sure of effective exposure. The values given in Table 6-l refer to the increment in the phenol level in urine above the physiological values (see “Phenol"), and may be used in relation to any particular MPC value. For instance, at the MPC of 20 mg/m3 that has been accepted recently in the USSR. assuming pulmonary ventilation of 7 m3 per 8 hours’ shift, and retention of benzene vapours at 70 per cent, the amount of the sub- stance absorbed into the system would be about 100 mg, and the increment in phenol concen- tration at the end of 8 hours’ exposure would equal about 45 mg/l. The precision of the test, estimated for the range of doses applied, is of the order i 20—25 per cent. Further studies have not introduced essential changes in this interpretation: benzene concen- trations have been accepted again as the indepen- dent variable, and most of the data have been de- rived from field investigations in which precise assessment of exposure seems particularly diffi- cult; some data have been based on single human subjects. The most common methods of inter- pretation are assembled in Table 6-2, where the data have been normalized in such a way as to reflect exposure to a constant concentration of 43 benzene in air at 88 tlg/1 or 25 ppm. The pro- posal of Sherwood (1971, 1972), not based direct- ly on experimental data, differs from field studies of Bardodej (1960), Walkley et al. (1961), Docter and Zielhuis (1967), as well as from the well-con- trolled experiments of Dutkiewicz (1963). Rain- sford and Davies (1965) proposed a simplified method for phenol determination in urine as a screening test; however, their accuracy was far below that of the other tests discussed above. The present author recommends: (3) Application of the Fiserova-Bergerova method of phenol determination in urine as modified by Hanke et al. (1961) based on the Gibbs reaction, or a gas-chro- matographic method in a version yielding still lower physiological phenol levels and equally high efficiency of hydrolysis of the conjugated phenol; (b) Assessment of the exposure from the excretion rate of phenol in urine, collected at the end of exposure, as related to the ab— sorbed dose of benzene (Tab. 1). This cir- cumvents the difficulties resulting from the variation of phenol concentration de- pending on the intensity of diuresis. as the excretion rate is independent of the latter (Bardodej et al.. 1962). Using the absorbed dose as the yardstick of exposure renders assessment of the latter independent of pul- monary ventilation, which is connected in turn with intensity of the physical effort. Variation of physical activity may cause considerable differences in phenol levels at the same air concentration (Benes et al., 1962). (c) Relating the results to exposure on the day of examination, regardless of which day of the working week it is made. ln spite of the above recommendations the present author is of the opinion that the phenol test should be reinvestigated on volunteers under strictly controlled conditions, in a manner similar to that reported by Dutkiewicz (1963), but for much lower concentrations of benzene in the air. The existing experimental data were obtained at relatively high concentrations above 50 mg/m3. The proposed range of concentrations should reach down at least to the MPC, accepted cur- rently in the USSR, i.e. 5 mg/m3. This should permit an assessment of the limits of applicability of the test. lt has to be mentioned that, in contrast to higher exposure levels, at these extremely low air con- centrations the increment of urinary phenol JERZY K. PIOTROWSKI Variants of the test based on: Formula of benzene is estimated Precision with which the absorbed amount Phenol concentration in urine collected between 6th and 8th hour from onset of absorption (mg perliter) ...................... x 1.96y 24% Urinary excretion rate of phenol between 6th and 8th hour from onset of absorption (mg per hour) .......................... x 27.4y 23% Amount of phenol in daily urine 24 hours collection; mg) .............. x 2.27y 16% x - doze of benzene absorbed into the system mg y - excretion of phenol in dimensions given Table 6-1. Variants of the benzene exposure test. Concentration Method of determination; Type of of phenol in Author(s) the physiological level studies urine mg/l Dutkiewicz, Gibbs, version of Fiserova- Experiment on human 1963 Bergerova (1955) modified by volunteers, assumed 200 Honke et al. (1961); physiol. Ve 0.85 mł/hr conc. 9 mg/l Bardodei, With 4 amino-antipyrine field studies 1960, 1962 results correspond to those 180 by Gibbs’ method Walkley, Theis and Benedict (p-ni- field studies et aI., troaniline; p-cresol in- 210 1961 terfers) physiol. conc. 3O mg/I Table 6-2. Assembled data on interpretation of urinary phenol determinations, normalized to air concentrations of benzene of 88 tlg/l (25 ppm). The data refer to .samples of urine collected toward the end of exposure lasting 8 hours. BENZENE above the physiological level will be relatively low. Thus. individual variations of urinary phe- nol (normal background) will be the factor which determines the lower limit of the test. BENZENE IN EXPIRED AIR Based on earlier experiments, Teisinger et al. (1956) and Gadaskina and Filov (1971) did not attribute too much significance to quantitative in- terpretation of free benzene in blood, urine, and exhaled air. The idea prevailed that these deter- minations are of merely qualitative value, supple- menting the less specific phenol test. A renewed interest in the analysis of expired air is part of a more general phenomenon related to the development of gas chromatography. In the case of benzene. there is more data on experi- mental techniques than on interpretation of the results. Sherwood and Carter (1970) and Sher- wood (1972) believe, on a basis of limited data, that directly after exposure (in a sitting position) o—o time 0 °--0 time thr Excreation rate , rng/hr .0- 5" › :~ .0' .w .O F" .” O (II O CII o u o O O l l I I I I I I | 50 ul l I .30 l I I I l I I I I I 50 I00 I50 200 250 300 350 400 450 600 Absorbed dose,mg Fig. 6-1. Excretion rate of benzene with expired air following inhalation of benzene vapour, as dependent on dose retained (Dutkiewicz, 1971). 45 to benzene vapours at a concentration of 25 ppm (88 ug/l) for 4.5 hours (115 ppm-hours), the concentration of the substance in exhalatory air amounts to about 2 ppm (1.5 ppm when a respi- rator, and 2.8 ppm when an alveolar air col- lector, is used). Sixteen hours later (in the morn- ing of the next day) the concentration drops to about 0.2 ppm. The data cited by Sherwood and Carter (I970) are not consistent with the above information. Results of more systematic experimental studies were published by Dutkiewicz (1971) from his_ early experiments, where classical colorimetric methods were still used for the determination of exhaled benzene. Results, obtained for various time intervals following discontinuation of ex- posure. were expressed in terms of exhalation rate; thus comparability with the data quoted above is difficult. It is of interest that, when re- lating the results to the dose absorbed over a 6 hour inhalation period, the precision of the eval- uation was worse as compared with urine anal- ysis ( : 32 per cent, vs 3: 20 per cent, Fig. 6-!) Sherwood (l972) reported that the exhalation ki- netics of benzene could have been described us- ing two exponentials with half-lives of 2,5 and 22 —32 hours. He drew the conclusion that deter- minations performed early after discontinuation of the exposure reflect mainly the absorption over the last 1-2 hours of inhalation, whereas the measurements made on the next day, before the onset of further exposure, provide information about the accumulated amount of benzene in the body. Thus early morning values of benzene in expired air should display a rising trend over consecutive days of an exposure week, reaching roughly a factor of 2 on Friday or Saturday above Monday. This postulate seems consistent with expectations (see uKinetics”). However, it still awaits experimental verification. At present. the determination of benzene in the exhaled air does not contribute significantly to the value of the evaluation of the exposure by means of the urinary phenol test. This subject, however, warrants further studies because of the existing tendency to lower the MPC for benzene. With decreasing concentrations of benzene in the air, the value of the phenol test diminishes rap- idly, most likely because of the interference of the physiological urinary levels of phenol (Du- tkiewicz et al., 1964). 46 AVAILABLE DATA ON INDUSTRIAL EXPOSURE Due to the serious health consequences of exces- sive exposure to benzene and the common exis- tence of control measures, there must exist a great body of data on industrial exposure, the bulk of which, however, remains unpublished. Relatively abundant data have been published in Czechoslovakia (e.g. Kindrichova, l958; Vlasak, I959; Bardodej. 1960; Jindrichova, 1974), where interest of the authors concentrated mainly on the practical value of the phenol test. In Poland, where the phenol test is being used routinely for control of benzene in the industrial environment, the published data are scarce (Dutkiewicz, 1965; Mikulski et al., 1972). The phenol test has been JERZY K. PIOTROWSKI used also in the USA (Walkley et al., 1961) and in the U.K. (Rainsford and Davies, 1965); in the U.K. it was combined with analysis of the ex- pired air (Sherwood, 1971, 1972). Preliminary data were reported also from the USSR (Kanner, l97l). The reports listed above have come from various branches of industry (shoe-manu- facturing. production of telecommunication equipment. production-plants of aromatic com- pounds. ship painting, crude oil pumping sta- tions. etc.). The over-all correlation of urinary phenol concentrations with benzene concen— trations in the air was relatively good; the mag- nitude of exposure varied, of course, with the technological processes and the time over which the determinations were made. REFERENCES Angerer J.. szadkowski D., Manz A., Pett R., and Lehnert G.: Chronische Lo- sungsmillelbelastung am Arbeitsplatz. I. Gaschromatographische Bestimmung von Benzol uml Toluol in der Luft una’ im Damp/mum von Blutproben. (Occupational chronic exposure to organic solvents. I. Gas chromatographic determination of hen- zene and toluene in air and in the vapour phase of blood samples). Int. Arch. Ar- beitsmed. 31, 1-8, 1973. Bardodej Z.: Hodnola a pouziti expozicnich testu. IX. Fenolovy test. (Value and use of exposure Iesls. IX. Phenol test.) Ceskosl. Hyg. 5, 39-46, 1960. Bardodej Z., Bardodejova E., Benes V., Kukackova V., and Vitora A.: Diureza ofen- olovy test. (Diuresis and the phenol test.) Ceskosl. Hyg. 7, 49-52, 1962. Benes V.. Bardodej Z., Bardodejova E., Kukackova V. and Vitora A.: Fizycka nam— aha a/eno/ovy test. (Physical activity and the phenol test). Ceskosl. Hyg. 7, 46-48. 1962. Bergerova V. and Skramovsky S.: Stanoveni dvojmocnych fenolu v biologickem ma- terialy. (Determination of bivalent phenols in biological materials.) Prac. Lek. 4, 64- 75. 1952. Docter H .J . and Zielhuis R.L.: Phenol excretion as a measure of benzene exposure. Ann. Occup. Hyg. lo, 317-326, 1967. Dutkiewicz T.: Quantitative exposure tes (for benzene. Excerpta Med. IV. Int. Congr. Occup. Health, II International Congress, series Nr. 62, 1963 (pp. 431—433). Dutkiewicz T.: Ocena narazenia na benzen z zastosowaniem testuferio/owego w war- unkach przemyslowych. (Evaluation o benzene exposure using the phenol test in in- dustrial conditions). Med. Pracy 16, 2 -30, 1965. Dutkiewicz T.: Wchlanianie par benzenu w tion of benzene vapors in human respiratory 260. 1971. drogach oddechowych u ludzi. (Absorp- tract). Bromat. Chem. Toksykol. 4, 253- Dutkiewicz T.: Wydalanie benzenu przez pluca u osob narazonych na wchłanianie par benzenu. (Pulmonary elimination of benzene in subjects exposed to benzene va- pors). Bromat. Chem. Toksykol. 4, 443-451, 1971. Fabre R. and Fabre A.: Sur le dosage du benzene et du toluene dans l'air des ateliers e; (glans le sang des ouvrieres manipulant ce solvents. Arch. Mal. Prof. 9, 97-103. 1 4 . Fiserova-Bergerova V. and Cettl L.: Elektricky model pro vstrebovani, metabolismus a \{i'lucovani benzenu u cloveka. (Electric model for the absorption, metabolism and excretion of benzene in man). Prac. Lek. 24, 56-61, I972. Graziani G., Fati S., and Pesaresi C.: Gas chromatography studies on the coefficient of Ihe benzene level in the blood in relation to diverse environmental conditions. Folia Med. 53, 51-61, 1970. Gut J.: Dwufazovy ucinek fenobarbitalu na premenu benzenu a toluenu u marca:. (The two-phase effect of phenobarbital upon metabolism of benzene and toluene in guinea pigs). Prac. Lek. 23, 112-1 13, 1971. BENZENE Hanke J., Dutkiewicz T., and Piotrowski J.:” Wchłanianie benzenu przez skore u lu- dzi. (The absorption of benzene throughout the skin in men). Med. Pruey 12, 413- 426, 1961. Hunter C.G. and Blair D.: Benzene: Pharmacokinetic studies in man. Ann. Oeeup. Hyg. 15, 193-199, 1972. Jindrichova J.: Vztach mnovstvifenołu v moci k hla'dine benzenu v ovzdusi, overeny v terennich podminkach. (The relationship between the phenol content in urine and the begnzene content of the atmosphere, verified by field work). Prue. Lek. 10, 131- 134. l 58. Jindrichova J.: Hodnoceni expozice benzenu na pracovistich vychodoceskeho kraja podle fenoloveho expozicniho testu. (Evaluation of benzene exposure using the phe- nol test). Prue. Lek. 26, 260-263, 1974. Kanner N.L.: Sravnitelnaya cennost opredeleniya fenola. glukuront'dov i efirosernych kislot w motshe pri vozideistvi benzola :' toluola. (Comparative value of determining phenol. glucurontdes and sulphates in urine following the action of benzene and tolu- ene). Gig. Tr. Prof. Zubol. 15 (10), 60-62, 1971. L'ustinec K., Sedivec V., and Teisinger J.: Vliv SKF na metabolismus benzenu in vi- tro. (The effect of SKF upon benzene metabolism in vitro). Prue. Lek. 21, 299-300, 1969. Mikulski P.J., Wiglusz R., Bublewska A. and Uselis J.: Investigation of exposure of ship painters to organic solvents. Brit. J. lndustr. Med. 29, 450-453, 1972. Parke D.V. and Williams R.T.: The metabolism of benzene containing (C") benzene. Biochem. J. 54, 231-236, 1953. Piotrowski J.: Modyfikacja acetonowej metody oznaczania benzenu i jego nitro- pochodnych w powietrzu. (Modification of acetone method of determining benzene and its nitroderivatives in the air). Med. Pruey 5, 329-335, 1954. Porteous J.W. and Williams R.T.: Studies in detoxication: 20. The metabolism of benzene. Biochem. J. 44, 56-60, 1949. Rainsford 5.0. and Davies T.A.L.: Urinary excretion of phenol by men exposed to vapour benzene: A screening test. Brit. J. Industr. Med. 22, 21-25, 1965. Schrenk H.M., Pearce S.J., and Yant W.P.: A microcolorimetric method for the de- termination of benzene. U.S. Bureuu of Mines, Rep. lnvestig. No. 2287, 1955. Sherwood R.J.: The monitoring of benzene exposure by air sampling. Am. lndustr. Hyg. Assoc. J. 32, 840-846, 1971. Sherwood R.J.: Benzene: the interpretation of monitoring results. Am. Occup. Hyg. 15, 409-421, 1972. Sherwood R.J.: Evaluation of exposure to benzene vapour during the loading of pet- rol. Brit. J. lndustr. Med. 29, 65-69, 1972. Sherwood R.J. and Caner F.W.: The measurement of occupational exposure to ben- zene vapour. Ann. Oeeup. Hyg. 13, 125-146, 1970. Srbova J., Teisinger J., and Skramovsky S.: Vstrebovani a vylucovani benzenu u cloveka. (Absorption and elimination of inhaled benzene in man). Prue. Lek. 1, 1-9, 1949; Arch.lnd.Hyg.Occup.Med. 2, 1-8, 1950. Teisinger J. and Fiserova-Bergerova V.: Vztah siranoveho a phenolickeho testu v moci k koncentraci benzenu ve vzduhu. (Correlation between sulphate and phenolic test in urine and the concentration of benzene in air). Prue. Lek. 7, 1-7, 1955. Teisinger J., Fiserova-Bergerova V., and Kudra J.: Metabolismus benzenu u cloveka. (The metabolism of benzene in man). Prue. Lek. 4, 175-188, 1952. Teisinger J., Skramovsky S., and Srbova J.: Chemicke Methody k Vysetrovani Bio- logickeho Materialu v Przemyslove Toxikologie. Praha, 1956, (Cmng Shramovsky S. and Teisinger J.: Cus. Lek. Ces. 82, 621-625, 1943.) Vlasak R.: Vhodnost fenoloveho testu pro stanoveni stupne expozice zamestnancu pracujicich s benzenem. (The suitability of the phenol test for determining the degrees of exposure of persons working with benzene). Prue. Lek. 11, 469-472, 1959. Walkley J.E., Pagnotto L.D., and Elkins H.E.: The measurement of phenol in urine as an index of benzene exposure. Amer. Ind. Hyg. Assoc. J. 22, 362-367, 1961. Wisniewska-Knypl J.M., Jablonska J.K., and Piotrowski J.K.: Effect of repeated ex- posure to aniline, nitrobenzene and benzene on liver microsomal metabolism in the rat. Brit. J. lndustr. Med. 32, 42-48, 1975. Yam W.P., Schrenk H.H., and Patty F.A.: A plant study of urine sulfate deter- rlrggtgtions as a measure of benzene exposure. Journ. Ind. Hyg. Toxicol. 18, 349-360, Yam W.P., Schrenk H.H., Sayers R.R., Horvath A.A., and Reinhart H.: Urine sul- fate determinations as a measure of benzene exposure. Journ. Ind. Hyg. Toxicol. 18, 69-85, I936. 47 7. TOLUENE ABSORPTION Under industrial conditions, toluene enters the body mainly via the respiratory tract. According to Srbova and Teisinger (1952), the retention of the vapours amounts on the average to 53 per cent (41—63.5%); Piotrowski (1967) found 72 per cent at the onset of inhalation, and 57 per cent at the steady-state (average about 60%). Vapours of toluene do not penetrate the skin (Piotrowski, 1967); however, the liquid applied directly does (Dutkiewicz and Tyras, 1968). METABOLISM AND ELIMINATION In man, of the amount of toluene retained in the body, about 16 per cent is exhaled unaltered and a trace (0.06%) is forind in urine. The chief me- tabolite is benzoic acid, accounting for 72 per cent of the retained toluene (Srbova and Te- isinger, 1953). Another report estimated this frac- tion at 62 per cent (Piotrowski, 1967). Of the uri- nary benzoic acid, 10-20 per cent is conjugated with glucuronic acid and the rest is eliminated in the form of hippurate (Srbova and Teisinger, 1953). ln rabbits about 75 per cent of the admin- istered toluene is excreted in the hippurate form (El Masri et al., 1956). lt may be presumed that the fraction of benzoic acid conjugated with gly- cine should decline somewhat with an increasing dose of toluene due to the limited pool of easily accessible glycine in the body (Arnstein and Neu- berger, I951; Bray et al., 1952). Nevertheless, Ogata et al. (1970) found that hippuric acid ac- counted for 68 per cent (on a molar basis) of the absorbed toluene, with linear proportionality be- tween the hippurate excreted over 24 hours and exposure up to 1500 ppm x hours (i.e. up to 200 ppm = 0.8 mg/ 1). From animal experiments it is known that the yield of urinary hippuric acid may be depressed by simultaneous exposure to trichloroethylene (Ikeda, 1974). No human data are available with respect to the influence of oth- er xenobiotics on the process. After inhalation of toluene vapours the excretion of conjugated benzoic acid is rapid; the excretion half-time is 2-3 hours (Piotrowski, 1967; Ogata et 48 al., 1971). Therefore, 24 hours after exposure, levels of the metabolite decline to control values; and breaks in the Continuous inhalation (alterna- ting 1 hour exposure and 1 hour off) do manifest themselves in the excretion rate of hippuric acid in urine (Ogata et al., 1971). In the case of an 8 hour inhalation exposure, 55-60 per cent of ben- zoic (hippuric) acid is excreted within the ex- posure interval (Ogata et al., 1970). DETERMINATION OF TOLUENE Toluene has been routinely determined (after aer- ation) in the blood and urine, as well as in the breath. The Czech authors (Srbova, 1952) used a method based upon nitration to dinitrotoluene with subsequent polarographic measurement; Fabre et al. (1955) measured the same product colorimetrically after reaction with alkali and butanone, and Piotrowski (1967) after nitration to trinitrotoluene and reaction with alkali and alcohol. At present, in the analysis of blood, air, and par- ticularly of breath, gas chromatography with flame ionization detection is used (Ogata et al., 1970; Mikulski et al., 1972; Angerer et al., 1973; Astrand et al.. 1972). DETERMINATION OF BENZOIC AND HIPPURIC ACID IN URINE In the studies of Srbova and Teisinger (1952, I953), Teisinger and Srbova (1954) and Pio- trowski (1967) a titrimetric method (Kingsbury and Swanson, 1921) was used in which urine was hydrolyzed in an alkaline medium, and the chlo- roform extract was titrated with sodium alcohol— ate. In earlier monographs this method was rec- ommended for exposure tests (Teisinger et al., 1956; Dutkiewicz et al., 1964). Average phys- iological levels of benzoic acid in the urine, as determined by this method, were as follows: 750 TOULENE mg/24 h (Teisinger et al., 1956), 600 (830) mg/l in winter and 750 (970) mg/l in summer (the numbers in parentheses correspond to concen— trations normalized to the specific gravity of urine = l.024). The excretion rate was about 39 mg/h independently of the season (Piotrowski, 1967). Consumption of plums increased substan- tially the excretion of benzoic acid. Flek and Sedivec (1971) proposed that total ben- zoic acid (after alkaline hydrolysis) be determined by means of gas chromatography in the form of methyl ester, obtained from a reaction with di- azomethane. Preliminary data suggest that the physiological levels, obtained in this way, may be lower (20-25 mg/h). The original proposal was related to hippuric, and not to benzoic, acid (Elkins, 1954); the former was titrated after precipitation by the method of Quick (1931). This version had low sensitivity and was useful for analysis of urine only after heavy exposure. At present, a return to the determination of hippuric acid may be noted, and two principal groups of methods are used: a) Colorimetric procedures by which the total hippuric acid (together with methyl- hippurates, if present — see *'Xylene”) is determined in a small sample of urine (l ml or less). These methods stem from studies of El Masri et al. (1956) and Umberger and Fiorese (1963), and are applied in several versons. Ogata et al. (1969) used, with some modifications, a method based on the ex- traction of the metabolite (ether-ethanol or ethyl acetate), and subsequent evaporation and colour reaction with p-dimeth- ylaminobenzaldehyde in the presence of acetic anhydride (plus perhaps FeCIJ). The product, azlactone, shows maximal absorb- ance at 460-470 nm. ln addition, Ogata et al. (1969) described an alternative method with benzcnosulphonyl chloride (absorp- tion maximum - 380 nm). Both methods are characterized by good recovery (94-l00 per cent) and similar sensitivity. Burkiewicz and Zielinska (I972) reported a procedure in which hippuric acid is extracted with chloroform, and color is developed in the presence of acetic anhydride and pyridine. The obtained product, oxazolone, has an absorption maximum at 460 nm. All these methods are sensitive with good precision, and those of Mikulski et al., and of Bur- kiewicz and Zielinska are, also, very simple. The physiological level of hippuric acid var— ies from report to report: 790 1 430 mg/l or › 554 mg/l (Mikulski et al., 1972) when cor- 49 rected for specific gravity of urine and non- corrected, respectively. Burkiewicz and Zi- elinska (1972) reported an excretion rate of 23 i 6.2 mg/ h. These values are similar to the existing data for benzoic acid. Sensi- tivity of the discussed methods is similar for hippuric and methylhippuric acid, and thus they may be used for approximate evalu- ation of simultaneous exposure to toluene and xylene (Mikulski et al., 1972). b) Chromatographic methods (paper and thin-layer chromatography) permit selective determination of hippuric and methyl-hip- puric acid when both are present in the urine. Application of these methods is par- ticularly indicated in case of exposure to xylene; accordingly, they are discussed in the chapter “Xylenes”. Ogata et al. (1969, ' 1970), using these methods, found phys- iological levels to be reasonably low about 300 1r. 100 mg/I. The reasons for this dis— crepancy with the data discussed above is not clear. Apart from the better specifity of the method, differences in nutritional habits of the subjects studied may have con- tributed to the low levels. Other methods for the determination of benzoic and hippuric acids have also found application in the evaluation of toluene exposure. Pagnotto and Lieberman (1967) described a method for hip- puric acid measurement based on ultraviolet spectrophotometry (230 nm) of an urine extract in an i-propanol—ether mixture; the method has been criticised for lack of specificity in the pres- ence of uric acid and other substances (Ogata et al., 1969). Nevertheless, the physiological level, as determined by this method, seems consistent with other data, and amounts, on the average, to about 800 mg/l (Pagnotto and Lieberman, 1967; Kaucka et al., 1973). Recently, Buchet and Lauwerys (1973) proposed the determination of both the hippuric and m- methylhippuric acids using gas chromatography after transformation to methyl esters with diazomethane. EXPOSURE TESTS Relatively exhaustive information is available re- garding determination oftotal benzoic acid for the purpose in question. Teisinger and Srbova (1954) have investigated, in experimental condi- tions on volunteers, the relationship between toluene concentration in the air, in a range from IOO to 1000 mg/m3, and daily excretion of ben- 50 zoic acid. Due to the high level and great scatter of the physiological excretion rate (0.5-1.5 g/ day), confidence limits of the correlation were very wide. The authors concluded that the test may be used only at relatively high toluene con- centrations in the air (of the order of 200 ppm = 800 mg/m3). and then only as a “collective test" (see chapter 5, “Working out exposure tests”). ln this version the test has not found wide practical application. Piotrowski (1967), also in a human experiment (chamber exposure), has based the test on the analysis of a urine fraction, collected toward the end of 8 hours” inhalation exposure, and recommended the evaluation of the absorbed dose of toluene (D. mg) from the excretion rate according to the formula V=39+0.09D (l) where V = excretion rate of benzoic acid over the last 24 hours of the exposure (mg/hour), 39 = mean physiological level of benzoic acid (mg I ano— £ En no E _ .U Mr B o _9 m- 8 it may a; . Jay,/. .700 12100 1500 aim Toluene,mg Fig. 7-1. Excretion rate of benzoic acid (mg/h) in urine collected toward the end of 8-hrs exposure, as dependent on the absorbed dose of toluene (mg/h) (Piotrowski. 1967). Taken from: Piotrowski J.: Ilosciowa ocena wchłaniania toluenu u ludzi, Medycyna Pracy 18, 2l3-223, I967. Page 221, Fig. 8. JERZY K. PIOTROWSKI „% Total excretion of hippuric God or m-(p-) methylhippufic acid (mą) o o 560 nooo Isoo Exposure to rolni: or m- or p-xylene (ppm it how exposed) Fig. 7-2. Relationship between total exposure (ppm X hr) and total excretion of urinary hippuric acid in volunteers (Ogata et al., I 970). hour), 0.09 = coefficient of regression (hour'l); (see Fig. 7-l). ln l8 separate experiments on 6 in- dividuals each. at air concentrations of 100-800 mg/m’. a precision of evaluated absorbed dose of : l80 mg was attained. This means that the test is burdened with an admissible error of i 20 per cent, at doses of 900 mg toluene and more; this corresponds to air concentrations above 200 mg/ m3 (40 ppm). With regard to determinations of urinary hip- puric acid, the first data provided an example of the amounts excreted in urine, collected over the total interval of exposure (9 hours) to various concentrations, from about 0.4 to 3 mg/l. The excreted amounts of hippuric acid were in the range from 700 to 5000 mg. Recently. Ogata et al. (1970) reported data from experiments on volunteers exposed to concen- trations of l00 and 200 ppm (400 and 800 mg/ m3). The data enable a relatively precise cor- relation to be made (Fig. 7-2) of the exposure with 24 hours' excretion of hippuric acid. For practical purposes, however, of most significance is the relationship obtained by these authors be- tween air concentrations and the excretion in urine collected over the last '4 hours of a days’ exposure, with a one hour break in the middle of the interval (Fig. 7-3). These data may be com- TOLUENE pared with those reported by Piotrowski (1967) on the basis of equation (l). For this purpose a pulmonary retention of 0.6 and lung ventilation rate of 0.8 m3/hour may be assumed, and a cor- rection coefficient of 0.7 introduced to account for differences in the molecular weights of benzo- ic and hippuric acids". The data of Ogata et al. when recomputed yielded a formula: V = 12 + 0.08D (2) This equation differs from equation (l) by: a) the lower physiological excretion rate (12 mg/ hour) and b) the regression coefficient of 0.08 in- ' Excretion rate was recalculated from mg per min. to mg per hour. B.: wwa! l 13' xk 8.- E Ę gt,-g & u : = u O c :: u 8 % a v '- 0-4- 0 83 U : a 'z' s ; : |2'+ 4 + | oL o Toluene concentation Fig. 7-3. The level of hippuric acid in urine collected between 4-8 hrs of exposure, as dependent on the concentration of toluene in air (Ogata et al., 1970). Figures 2 and 3 taken from: Ogata M., Tomokuni K., Takatsuka Y.: Urinary excretion of hippuric acid and m- or p-methylhippuric acid in the urine of persons exposed to vapours of toluene and m- or p-xylene as a test of exposure. Brit. J. lndustr. Med. 27, 43-50, 1970. Page 46, Fig. 4 and Page 47, Fig. S-B'. 51 stead of 0.09. The ratio of the latter two values may reflect the efficiency of conjugation of ben- zoic acid with glycine (90 per cent). From the above discussion it follows that the test of Pio- trowski (I967), based on titrimetric determination of benzoic acid, and that of Ogata et al. (1970), based on colorimetric determination of hippuric acid, yield similiar results. For evaluation of exposure to a lesser degree, the determination of unaltered toluene in blood and expired air has been used. Gerarde (1960) pre-- sented a graph relating the blood concentration of toluene following 8 hours exposure vs. the concentration in the air at‘the working place. The graph embraces hi h levels, from 750 to 3000 mg/m3 of air; the córresponding blood con- centrations vary from 5 to 20 ug/ml. Teisinger et al. (1956) presented graphs depicting the kinet- ics of the disappearance of toluene from the blood and expired air after cessation of the ex- * posure. When the fastest elimination in the breath was completed, the decay of concen- trations both in the breath and in the blood was characterized by a half-time in the order of 2 hours. Recently, the use of toluene determinations in the expired air and blood has gained new adherents (Angerer et al., 1973; Szadkowski et al., 1973; Astrand et al., 1972). The background level of toluene in blood, when determined by gas chro- matography, in nonexposed people, has not ex- ceeded 0.l5 ppm (Szadkowski et al., 1972). ln short-time exposure it has been shown that, both in alveolar air and in arterial blood, a steady- state is rapidly reached, usually within 30 minutes. At the air concentration of 100 ppm, the concentration of toluene in alveolar air and in arterial blood was about 18 ppm and 1 ppm, respectively. At 200 ppm in air, values twice as high were obtained. These values, characteristic of the end of a thirty minute exposure in subjects at rest, showed further rise if, in the following period, exposed volunteers were also subjected to exercise (300-900 kpm/ min). There was a close linear correlation between the toluene concen- tration in alveolar air and in arterial blood, irre- spective of the type and magnitude of exposure (Astrand et al., I972). However, the above data seem still inadequate for routine toluene deter- mination in expired air or blood, when evalu- ation of industrial exposure is the objective. The present author proposes to use, for purposes of current control of the'exposure, either the test of Piotrowski (1967) or that of Ogata et al. (1970). On the other hand a low physiological 52, level of hippuric acid, as seen by Ogata et al., would all for clarification as to whether it was due to the particular specificity of the method, or to the dietary habits of the studied individuals. 1f the former is true, the method of Ogata et al. (1969) should have priority over all other meth- ods used so far for determination of benzoic and hippuric acid in urine. Even at the low physiological levels of hippuric acid, reported by Ogata et al., the applicability of the test is limited to moderate and high toluene concentrations in the air. At concentrations equal to or lower than 50 mg/m3 (12 ppm), none of the published tests will provide acceptable quan- titative data for individual measurements. Studies seem warranted to investigate further the use- fulness of blood and breath analysis for toluene by means of gas chromatography. AVAILABLE DATA ON INDUSTRIAL EXPOSURE Although exposure tests based on hippuric and benzoic acid assays are routinely applied in some countries for exposure control, published data on this topic are scarce. Recently published data on occupational exposure may be found in the fol- lowing reports. Pagnotto and Lieberman (1967) studied the exposure of workers employed in leather finishing and rubber coating plants, where average tOluene concentrations in the air ranged from 53 to 112 ppm. Using spot samples of urine collected at the end of working shifts, these au— thors found a high correlation between the air concentration of toluene and urinary level of hip- puric acid. Exposure to 100 ppm was reflected by high hippuric acid concentration of about 4 mg/ ml. Cappellini and Alessio (1971) found in indus- trial conditions somewhat lower values at com- parable exposure levels. Ogata et al. (1971) stud- JERZY K. PIOTROWSKI ied the correlation between urinary concentrations of hippuric acid and air concen- trations of toluene in industrial conditions with rather poor results (r = 0.67). Mikulski et al. (1972) assessed the exposure of workers engaged in ship painting (benzene, toluene, xylene) and found high levels of hippuric acids (methyl- hippuric inclusive), on the average from 1800 to 5500 mg/l depending on working conditions. The correlation between the sum of toluene and xylene air concentrations in ppm and concentrations of hippuric acids was high (r = 0.81). Kaucka et al. (1973) used the test (hippuric acid, method of Pagnotto and Lieberman) for evalu- ation of worker exposures at concentrations of toluene of the order of 100-500 mg/m3; and found high urinary concentrations, up to 5000 mg/l. Szadkowski et al. (I973), studying the exposure of printing workers, found no correlation be- tween the concentrations of toluene in the air and of hippuric acid in the urine; however, a cor- relation between the former and the toluene con- centration in blood did exist. These observations are, however, of limited value regarding the evaluation of exposure tests, since the basis for comparisons (effective exposure) cannot be properly estimated in industrial condi- tions, especially if skin absorption cannot be ex— cluded. Moreover, poor correlations are to be ex- pected because of the differences in lung ventilation in various individuals. Additionally, taking the urinary concentration (instead of the excretion rate) as the measure of the metabolite level reduces significantly the accuracy of the evaluation (see “Working out of exposure tests”). From personal information collected by the present author it folloWs that, in Poland, prac- tical application of the test meets difficulties re- sulting from the low level of exposure found in industry. REFERENCES Angerer J., Szadkowski D., Manz A., Pett R., and Lehnert G.: Chronische Lo- sungsmittelbelastung am Arbeitsplatz. I. Gaschromatographische Bestimmung von Benzol und Tolual in der Luft und im Dampfraum von Blutproben. (Occupational chronic exposure to organic solvents. 1. Gas chromatographic determination of ben- zene and toluene in air and in the vapour phase of blood samples). Int. Arch. Ar- beitsmed. 31, l-8, 1973. TOLUENE Amstein H.R.V. and Neuberger A.: Hippuric acid synthesis in the rat. Biochem. J. 50, 154-162, 1951. Astrand l., Ehrner—Samuel H., Kilbom A., and Ovrum P.: Toluene exposure. I. Con- centration in alveolar air and blood at rest and during exercise. Work, Environ., Health 9, 119-130, 1972. Bray. H.C., Clowes R.C., Thorpe W.V., White K., and Wood P.B.: The ate of cer- tain organic acids and amides in the rabbits. Biochem. J. 50, 583-592, 195 . Buchet J.P. and Lauwerys R.R.: Measurement of urinary hippuric and m-methyl- hippuric acids by gas chromatography. Brit. J. lndustr. Med. 30, 125-128, 1973. Burkiewicz C. and Zielinska H.: Metoda oznaczania kwasu hipurowego u ludzi nar- azonych na toluen. (A method for hippuric acid determination in persons exposed to toluene). Med. Pracy 23, 487-496, 1972. Capellini A. and Alessio L.: Urinary excretion of hippuric acid in workers exposed to toluene. Med. Lav. 62, 196-201, 1971. Dutkiewicz T. and Tyras H.: Skin absorption of toluene, styrene and xylene by man. Brit. J. lndustr. Med. 25, 243, 1968. Elkins H.B.: Analyses of biochemical materials as indices of exposure to organic sol- vents. Arch. Ind. Hyg. Occup. Med. 9, 212-217, 1954. El Masri A.M., Smith J.N., and Williams R.T.: Studies in detoxication, 69. The me- tabolistn of alkylbenzenes: n-propylbenzene and n-butylbenzene with further obser- vations on ethyl benzene. Biochem. J. 64, 50-56, 1956. Fabre R.. Truhaut R., and Laham M.: Recherches toxicologiques sur le solvents de replacement du benzene. II. Etude du~toluene. Arch. Mal. Prof. 16, 167-170, 1955. Pick J. and Sedivec V.: Kyselina benzoova v moci osob exponovanych parami tolu- enu: Stanoveni plynovou chromatografii. (Benzoic acid in urine of persons exposed (137tloluene vapours: Determination by gas chromatography). Prac. Lek. 23, 309-312, Gerarde H.W.: Toxicology and Biochemistry of Aromatic Hydrocarbons. Elsevier Publishing Co.. New York. 1960. Ikeda M.: Reciprocal metabolic inhibition of toluene and trichloroethylene in vivo and in vitro. lnt. Arch. Arbeitsmed. 23, 125-130, 1974. Kaucka J.. Brazdova D., Pospisilova E., and Minarikova J.: Kore/ace expozicnich testu (kyseliny hipurove) v moci a hodnot toluenu v ovzdusi ve dvou vybranych por- vozech. (Correlation of exposure tests (of hippuric acid) in urine and toluene values in the atmosphere in two selected enterprises). Prac. Lek. 25, 327-330, 1973. Kingsbury F.B. and Swanson W.W.: A rapid method for the determination ofhip- puric acid in urine. J. Biol. Chem. 48, 13-20, 1921. Mikulski P. and Wiglusz R.: A simple micromethod of determination of hippuric acid in the urine. Bull. Inst. Mar. Med., Gdansk 21, 129-138, 1970. Mikulski P., Wiglusz R., Bublewska A., and Uselis J.: Investigation of exposure of ship painters to organic solvents. Brit. J. Industr. Med. 29, 450-453, 1972. Ogata M., Takatsuka Y.. and Tomokuni K.: Excretion of hippuric acid and m- or -- methylhtppuric acid in the urine of persons exposed to vapours of toluene and m- or p-xylene in an exposure chamber and in workshops, with specific reference to repeat- ed exposures. Brit. J. lndustr. Med. 28, 382-385. 1971. Ogata M., Tomokuni T., and Takatsuka Y.: uantitative determination in urine of hiplpuric acid and m- or p-methylhippuric aci , metabolites of toluene and m- or p- xy ene. Brit. J. lndustr. Med. 26, 330-334, 1969. Ogata et al.: lbid. (Citing Von Oettingen W.F., Neal P.A. and Donahue D.D.: The toxicity and potential dangers of toluene. J. Amer. Med. Ass. 118, 579-584, 1942.) Ogata M., Tomokuni K., and Takatsuka Y.: Urinary excretion of hippuric acid and m- or p-methylhippuric acid in the urine of persons exposed to vapours of toluene and m— or p-xylene as a test of exposure. Brit. J. lndustr. Med. 27, 43-50, 1970. Pagnotto L.D. and Lieberman L.M.: Urinary hippuric acid excretion as an index of toluene exposure. Amer. lndustr. Hyg. Assoc. J. 28, 129-134, 1967. Piotrowski J.: Ilosciowa ocena wchłaniania toluenu u ludzi. (Quantitative evaluation of exposure to toluene in men). Med. Pracy 18, 213-223, 1967. Quick A.J.: The conjugation of benzoic acid in man. J. Biol. Chem. 92, 65-85, 1931. Srbova J. and Teisin er J.: Vstrebovani a vylucovani toluenu u cloveka. (Absorption and elimination of to uene in man). Prac. Lek. 4, 41-47, 1952. 53 JERZY K. PIOTROWSKI Srbova J. and Teisingcr J.: 0 metabolismu toluenu. (On the metabolism of toluene). Pnc. lck. 5, 259-263. 1953. Srbova J.: Polarograflcke stanoveni toluenu ve vzduchu a v biologickem materialu. (Polarographic determination of toluene in the air and in biological material). Prac. Lek. 4, 47-51, L952. Szadkowski D., Pett R., An erer J., Manz A., and Lehnert G.: Chronische Lo- sungsmittelbelastung am Ar eitsplatz. ll. Schadstojfspiegel im Blut und Metabo- litenelimination im Ham in ihrer Bedeutung als Uberwachungskriterien bei tolu- oIexponierten Tiefdruckem. (Occupational exposure to organic solvents. ll. Toluene concentrations in blood and excretion rates of metabolites in urine in the supervision of printing workers). Int. Arch. Arbeitsmed. 31, 265-276, 1973. . Teisinger J. and Srbova J.: Vztah vylucovani kyseliny benzoove v moci k maximalne pripustne davce toluenu ve vzduchu. (Correlation between excreting of benzoic acid in urine and the maximal permitted doses of toluene in the air). Prac. Lek. 6, 332- 335, I954. Umberger CJ . and Fiorese F.F.: Colorimetric method for hippuric acid. Clin. Chem. 9, 91-96. I963. Von Oettingen W.F., Neal F.A., Donahue D.D., et al.: The toxicity and potential dangers of toluene with special re erence to its maximal permissible concentration. U.S. Publ. Health Serv. Bull. No. 79, 1942. 8. XYLENES Xylene, when used as an industrial solvent, is usually composed of three isomers with m-xylene accounting for 75-85 per cent of the total. ABSORPTION lt appears that absorption of xylenes in humans has not yet been subjected to systematic studies. By analogy with toluene, it is believed that under conditions of industrial exposure the respiratory tract forms the main route of entry (Gadaskina and Filov, l97l). Experimental data of Ogata et al.. (1970) indicate that retention of m-xylene va- pours in the respiratory system reaches about 87 percent and is greater than that oftquene‘The in- vestigation of Orlowski (1972) showed a retention of 70-87 per cent at the beginning of exposure, with a slight downward trend after several hours of inhalation (65 to 85 per cent). With direct ap- plication to the skin, xylene is absorbed through this route also (Dutkiewicz and Tyras, I968). METABOLISM AND ELIMINATION ln the body, xylene undergoes oxidation leading to the formation of toluic acids, and, to a smaller degree. of xylenols. The m- and p-toluic acids are coupled to glycine, forming m- and p-methyl- hippuric acids. whereas o-toluic acid undergoes conjugation with glucuronic acid. The oxidation of m- and p-xylenes to toluic acids is highly effi- cient. in the order of 90 per cent with all the toluic acid bound to glycine (Bray et al., 1949, 1950; Fabre et al., 1960). The excreted m-methyl- hippuric acid has been found to account for 72 per cent of the absorbed m—xylene when studied on volunteers; and the metabolic efficiency for m- and p-xylene remained unchanged, up to the exposure product of 1500 ppm x hours. Data re- ported by Orlowski (I972), based also on the ob- servation of human beings, point to an efficiency of m-xylene transformation into m-methylhip- puric acid of about 90 per cent. The excretion rate was similar to that of toluene, followed con- currently, indicating a half-life in the range of 2-3 hours (Ogata et al., 1970). Senczuk and Orlowski 55 (in press) showed that the dynamics of m-methyl- hippuric acid excretion in humans, during ex- posure to m-xylene and after its discontinuation, may be satisfactorily described using a single rate coefficient of k = 0.5 hour"; this corresponds to a half-life of about 1.5 hours. METHODS FOR DETERMINATION OF XYLENE AND ITS METABOLITES The methods applied originally for the deter- mination of xylene were similar to those used for toluene. Fabre et al. (1960) determined xylene in biological materials by means of nitration and color reaction with butanone and alkali. Methods used at present for analysis in expired air, both for xylene and toluene, are based on gas chro- matography with flame ionization detection (Ogata et al., I970; Senczuk and Orlowski, 1974). These determination methods have not been used so far in exposure tests. Of the metabolites of m- and p-xylenes, the re- spective methylhippuric acids are determined in urine. They may be determined directly in urine extracts, similarly to hippuric acid; however, this procedure does not seem satisfactory due to the interference of high and variable levels of hip- puric acid in the urine of non-exposed people (see Toluene). Therefore, the exposure tests for m- and p-xylenes have become more attractive since the chromatographic separation of their metabolites have been worked out. In an original method of Gaffney et al. (1954), paper chromatography is applied with the colour product of hippuric acid being obtained directly on the paper. Ogata et al. (1969) found this pro- cedure poorly reproducible, and proposed a mod- ification of the thin layer chromatography tech- nique using Silica Gel G: the ethylacetate extract of urine applied to the slab as the solvent is de- veloped using toluene. acetic acid, and water in the proportion of 100, 50, 2.3. The colour reac- tion is produced by spraying the chromatogram with p-dimethylaminobenzaldehyde; the spots are eluted with ethanol; and absorption is measured 56 at 460-470 nm. The molar extinctions of azolactones, obtained from the hippuric, as well as m- and p-methylhippuric acids, are similar and amount to 1.5 x 104 and L4 x 104, respectively. The paper chromatography technique has been refined, and after selection of optimal parameters related to drying and colour development gives re- producible. quantitative results (Orlowski, 1974). EXPOSURE TESTS Ogata et al. (I970) performed chamber experi- ments on volunteers exposed to vapours of m-xy- lene (100 and 200 ppm) and p-xylene (100 ppm) for 3 and 8 hours. Air determinations were made using gas chromatography; the methylhippuric acids in the urine were determined by means of paper chromatography. The total excretion of methylhippuric acids (in 24 hrs. urine) was found to be directly proportional to the product of air concentration and duration of exposure (see also “Toluene"). For practical purposes, relevant results can be obtained from exposures of 8 hours” du- ration and from fractions of the urine collected over the last 4 hours of the exposure. Figure 8-1 shows that for both isomeres the results were rel- atively well comparable for concentrations nor- malized to a urine specific gravity of 1.024, and for the excretion rates. For m-xylene the propor- tionality" between these values and the air concen- tration is clearly apparent. Senczuk and Orlowski (in press), on the basis of human experiments, proposed to assess the ab- sorbed dose from urinary excretion of the metab- olites over the last 2 hours of exposure. The ex- cretion rate (u. mg/hour) may be related to the absorbed amount of m-xylene (D; mg) by a formula D = 4.9u INTERPRETATION OF THE TEST FOR SIMULTANEOUS EXPOSURE TO XYLENES AND TOLUENE The chromatographic method, as proposed by Ogata et al. (1969) renders separate deter- mination possible for hippuric and methyl- hippuric acids. The efficiency with which these metabolites are formed from toluene and xylenes is similar (see “Toluene”), as are the values obtain- ed from analysis of urine collected toward the end of daily exposure to both compounds at equal concentrations. If the maximum allowable JERZY K. PIOTROWSKI concentrations of toluene and xylenes are equal (e.g. in USSR, see “Sanitary Standards”), the measured total concentration of a mixture in the air may be compared with that calculated from the urinary excretion of the metabolites. ln such a case. a common method for determination of total hippuric acids without separation may be applied (Mikulski et al., 1972). On the other hand, if the TLV’s for the com- pounds are different. the air concentrations of toluene and xylenes have to be calculated from the levels of hippuric and methylhippuric acids in the urine. For the interpretation of the results, the principle of summation should be applied as used for mixtures in the air (Ogata et al.. 1970). mmm ~MM- at odd _ E : Ę 2 Ś 1:4" 8 ” O % 1-3 : 2 3 8;- 25 : ą ‘é 8 a. 32- a. a + 4 I o- o Fig. 8-1. The concentration of m-methylhippuric acid in urine as dependent on the concentration of tit-xylene in air (Ogata et al., 1970). Taken from: Ogata M., Tomoluni K. and Tak- atsuka Y.: Urinary excretion of hippuric acid and m- or p-methylhippuric acid in the urine of persons exposed to vapours of toluene and m- or p-xy/ene as a test of exposure. Brit. J. lndustr. Med. 27, 43-50, 1970. Page 47, Fig. 5-B—2 (including table with numerical values). XYLENES REFERENCES Bray H.C.. Humphris B.C., and Thorpe W.V.: Metabolism of derivatives of toluene. 3. 0-. m- and p-xvlenes. Biochem. J. 45, 241 -244, 1949. Bray H. G., Humphris B. G. and Thorpe W V.: Metabolism of derivatives of toluene. 5. The fate of the xvlenols in the rabbit, with further observations on the metabolism of the x_i/.enes Biochem. J. 47, 395- 398, 1950. Dutkiewicz T. and Tyras H.: Skin absorption of toluene. styrene and xylene by man. Brit. J. lndustr. Med. 25, 243-245, I968. Fabre R.. Truhant R., and Laham S.: Recherches toxicologiques sur les solvants de remplacement de benzene. IV. Etude des xylenes. Arch. Mal. Prof. 21, 301-303, 1960. Gaffney G.W., Schreier K., DiFerrante N., and Altman K.J.: The quantitative deter- mination of hippuric acid. J. Biol. Chem. 206, 695-698, 1954. Mikulski P.1., Wilgusz R., Bublewska A., and Uselis J.: Investigation of exposure of ship painters to organic solvents. Brit. J. Industr. Med. 29, 450—453, 1972. Ogata M., Takatsuda Y., and Tomokuni K.: Excretion of hippuric acid and m- or p- methylhippuric acid in the urine of persons exposed to vapours of toluene and m- or p-xylene in an exposure chamber and in workshops, with specific reference to repeat- ed exposures. Brit. J. lndustr. Med. 28, 382-385, 1971. Ogata M. Tomokuni K. and Takatsuka Y.. Quantitative determination in urine of hippuric acid and m- or p-methvlhippuric acid, metabolites of toluene and m- or p- x_i/ene. Brit. J. lndustr. Med. 26, 330- 334, 1969. Ogata M.. Tomokuni K., and Takatsuka Y.: Urinary excretion of hippuric acid and m- or p-melhylhippuric acid in the urine of persons exposed to vapours of toluene and m- or p-xylene as a test of exposure. Brit. J. lndustr. Med. 27, 43-50, 1970. Orlowksi J.: Ocena narazenia na m-ksylen w warunkach przemyslowych w oparciu 0 test ekspozycyjny. (Evaluation of industrial exposure to m-xylene based on the ex- posure test). PhD thesns, Medical Academy m Poznan, 1972. Orlowski J.: KolorI'metrvczna metoda oznaczania kwasu m-metylohipurowego w mo- czu oraz przebieg wydalania. (Colorimetric method of determination of m-methyl- hippuric acid in the urine). Bromat. Chem. Toksykol. 7, 87-91, 1974. Sanitarnyje normy projektirovanija promishlennych predpijatij SN 245-71. (Sanitary standards for the designing of industrial establishments). Gosudarstwiennyj Komitet Soveta Ministrov, USSR, 1971. Senczuk W. and Orlowksi J.: Opis i charakteristyka inhalacIjnej komorv dos- wiadczalnej. (Description and characterization of an inhalation chamber). Brom. Chem. Toksykol. 7,83-85. 1974. Senczuk W. and Orlowski J.: Absorption of m-xylene vapours through the respira- tory tract and excretion of m-methylhippuric acid in urine. Brit. J. lndustr. Med. (in press). 57 9. ETHYLBENZENE ABSORPTION Under conditions of industrial exposure, respira- tory absorption fonns the main route of entry for ethylbenzene. Retention of the vapours in the respiratory tract of volunteers, in controlled ex- periments at concentrations in the range of 100 to 8l0 mg/m3, amounts on the average to 60-64 per cent (Bardodej and Bardodejova, 1966a, b). ln an earlier study by the same authors (1961) the retention had been estimated at 45-50 per cent. The authors express the view that absorp- tion of ethylbenzene vapours through the skin is not significant, and consider appreciable cutane- ous absorption of the liquid substance as unlike- ly. However. Dutkiewicz and Tyras (1967) were able to demonstrate that liquid ethylbenzene is absorbed to a significant degree through the skin, if applied directly. METABOLISM AND ELIMINATION The data at hand allow the conclusion that al- most all the ethylbenzene absorbed into the sys- tem is metabolised. Only a minor amount is elim- inated unaltered in the urine and exhaled air (Bardodej and Bardodejoba, 1966a). Sum- marizing the earlier animal studies, the metabolic transformation of ethylbenzene occurs via two parallel metabolic pathways that start with ox— idation of the carbon atom in position 1 or 2 of the side-chain. The metabolites of the first path- way are phenylmethylcarbinol glucuronide and hippuric acid (30-35 per cent each), with a minor amount of mandelic acid (1-2 per cent) as well. The other pathway eventually reaches phen- aceturic acid (IS-20 per cent) through phenyl ace- tate. Hydroxylation of the aromatic ring, yielding p-ethylphenol, only accounts for traces of the metabolite pool (0.3 per cent; Bakke‘and Sche- line, 1970). The metabolism in man is somewhat _ different. Thus Bardodej and Bardodejova (1966a) suggest that the second pathway, which starts with oxidation of carbon atom 2, is of no significance. The first pathway, on the other hand, starts with phenylmethylcarbinol (excreted as glucuronide in small amounts of the order of 5 58 per cent), and ends up with phenylglyoxylic and mandelic acid, both probably in mutual redox equilibrium. The latter acids form the main me- tabolites ofethylbenzene in man, and account for some 64 and 25 per cent of the amount absorbed into.the system, respectively. No hippuric acid (one of the main metabolites in animals) has been detected in man and the same may be said of mercapturic acid and phenylacetylglutamine. The balance, as presented by the authors, leaves no free space for appreciable amounts of other metabolites. The metabolism of ethylbenzene in man is relatively rapid, the half-time of the excre— tion rate of mandelic acid being close to 5 hours (Bardodej and Bardodejova, 1966a). The metabo- lism in man is schematically presented in Figure 9-1. The same authors discussed all the aspects summarized above in a review article in 1970 (Bardodej and Bardodejova, 1970). DETERMINATION OF ETHYL- BENZENE AND ITS METABOLITES ' Determination of ethylbenzene in air and in breath has been performed directly by UV ab- sorption, applying the “Mercury Vapour Concen- tration Meter" (Hendrey Relays), or spec- trophotometrically in ethanol or heptan solutions at the wavelength of 262 nm (Bardodej and Bard- odejoba, l966a). For determination of the metabolites (mandelic , and phenylglyoxylic acids) see “Styrene”. Pha/wywijanym acid —> R ' CO ' C O O H 1‘ I \N ' if R'CHOH'CHZOH —> R‘CHOH 'COOH Mandel/”c acid R—co—CH3 R 'CHz—CH3_R'CHOH'CH3 L other bio transformat/ons R=©>— Fig. 9-1. Metabo/ic pathways of ethylbenzene in humans (Bardodej and Bardodejova, 1966). ETHYIBENZENE 1000 500 — Monde/ic acid in urine ( mg/l) 0.2 0.4 fray/benzene in air (mg/l) Fig. 9-2. The concentration of mandelic acid in urine as dependent on the concentration of ethylbenzene in the air (Bardodej and Bardodeiova. I961). EX POSURE TEST The basis for the interpretation of mandelic acid in urine was worked out by Bardodej and Bard- odejova (l96l). They relied upon data from hu- man experiments when the subjects were exposed in a toxicological chamber for 8 hours to in- halation of ethylbenzene vapours at concen- trations of l00-400 mg/m‘. With an average ven- 59 tilation rate of 7.6 l/min, the numerical values are presented in graphic form in Figure 9-2. The assessment of exposure is based on measurements of the concentration of mandelic acid in samples of urine collected in the two last hours of the ex- posure lasting for 8 hours. In the earlier paper (l96l). these authors suggested a straight-line re- lationship in which the urinary‘concentration of 900 mg/l corresponded to an air concentration of 400 mg/m-‘. These data as shown suggest a trend to reduced efficiency of mandelic acid formation with increasing concentrations of ethylbenzene in air. Due to higher ventilation rates in working sub- jects. the authors propose, for interpretation of field determinations (in industry), a relationship in which the amounts of mandelic acid cor- responding to ethylbenzene air-concentrations are higher than those displayed in Figure 9-2. The 200 mg/m3 (46 ppm; ethylbenzene) should be re- presented by about l000 mg of mandelic acid per liter of urine, when the urine is normalized to a specific gravity of l.024; or, if expressed as a ra- tio to creatinine concentration, 0.7 mg per mg creatinine. AVAILABLE DATA ON INDUSTRI- AL EXPOSURE In studies of urinary excretion of mandelic acid in workers exposed in chemical plants, it was found that the concentrations were below 1500 mg/l, or 1.2 mg per mg creatinine. REFERENCES Bakke OM. and Scheline R.R.: Hydroxylation of aromatic hydrocarbons in the rat. Toxicol. Appl. Pharmacol. 16, 691-700, 1970. Bardodej Z. and Bardodejova E.: Hodnota a pouziti expozicnich testu. X. Expozicni test pro etylbenzen. (Value and application of exposure tests. X. Exposure test for ethylbenzene). Ceskosl. Hyg. 6, 537-545, 1961. Bardodej Z. and Bardodejova E.: Metabolismus etylbenzenu. (Metabolism of ethyl- benzene). Ceskosl. Hyg. 11, 226-235, I966a. Bardodej Z. and Bardodejova E.: The metabolism of ethylbenzene, styrene and alpha- melhylstvrene. Proc. XV Int. Congr. Occup. Health, Vienna, vol. “-1, 1966b, pp. 456-460. Bardodej Z. and Bardodejova E.: Biotransformation of ethyl benzene, styrene and a!- pha-methylstyrene. Am. Ind. Hyg. Assoc. J. 31, 206-209, 1970. Dutkiewicz T. and Tyras H.: A study of the skin absorption of ethylbenzene in man. Brit. J. lndustr. Med. 24, 330-332, 1967. 10. STYRENE ABSOR PTION Respiratory absorption is probably the main route of entry for styrene into the body. Data on the retention of styrene vapours are divergent. According to Bardodej et al. (1961), the retention reaches 45—50 per cent; however, Bardodej (1964), as well as Fiserova-Bergerova and Te- isinger (1965), have reported a figure of 60 per cent. Stewart et al. (1968) obtained data for re- tention in the range of 70-75 per cent. Fiserova- Bergerova and Teisinger observed that, in con- trast with other volatile organic compounds (e.g. benzene, toluene, trichloroethylene), the retention of styrene in the respiratory tract remained con- stant throughout the whole exposure time. After direct contact of fluid styrene with the skin, sys- temic absorption was observed by Dutkiewicz and Tyras (I968). It seems that cutaneous absorption of the vapours has not been studied. METABOLISM AND ELIMINATION Almost all of the absorbed styrene is metabo- lized. In man, the proportion of unchanged sub- stance eliminated in the expired air is very small. Fiserova-Bergerova and Teisinger (1965) were un- able to find any exhaled styrene after cessation of exposure; Bardodej and Bardodejova (1966) found only traces; and Stewart et al. (1968), ap- plying gas chromatography, estimated the share of its elimination by this route at somewhat more than 1 per cent of the retained dose. This would conformwith earlier information on sty- rene metabolism in animals in whom elimination of the unaltered substance in exhaled air had been estimated at 2~3 per cent (Danishefsky and Willhite, I954; El Masri et al., 1958). Practically, metabolic changes of styrene affect only the side chain: the amount of detected phenol (4—viny1- phenol) accounted for only 0.1 per cent of the administered dose (Bakke and Scheline, 1970). The scheme of the metabolic pathway of styrene is still controversial. In Figure 10-1 the metabolic scheme is presented as proposed by Ohtsuji and lkeda (1971); it should be of assistance while fur- ther discussing the subject. 1n experimental animals hippuric acid is the main metabolite; this was stressed particularly in ear- lier reports (Spencer et al., 1942; Carpenter et al., 1944; El Masri et al., 1958). Bardodej and co- workers (1961) demonstrated that mandelic acid is the principal, and perhaps the only, metabolite of ' styrene in man. ln later studies mandelic acid has 60 been found in significant amounts also in experi- mental animals. Thus Vrba et al. (1964) found that in rats the excreted mandelic acid could ac- count for 2l-28 per cent of the styrene dose; and in the same animals Ohtsuji and lkeda (1971) found that the amount of this metabolite, ex- creted over the first 20 hours of observation, cor- responded to 15 per cent of the dose. ln rabbits 20 to 30 per cent of the administered styrene was excreted in the form of mandelic acid (Rubin- skaja, 1965). When ether extracts of rats” urine were analyzed (Vrba et al., 1967) by means of gas chromatography, apart from mandelic acid, phenylglyoxylic acid and four other metabolites were found. The latter were not identical with styrene styrene styrene mandelic phenylglyoxylic oxide glycol acid acid cu, enzo 914on coon coon CH CH’ CHOH CHOH C=O © — © ~ © — © — © cnzon coon courier-wean banzyl benlaic hippuric alcohol acid acid Fig. 10-1. Metabolic pathways of styrene (Ohtsuji and Ikeda. 1971). Taken from: Ohtsuji H. and lkeda M.: The metabolism of styrene in the rat and the stim- ulatory effect of phenobarbital. Toxicol. Appl. Pharmacology l8, 321-328, 1971. Page 326, Fig. 1. STYRENE those known already from the earlier studies. The results of recent investigations by Bardodej et al. (1971) demonstrated that in rats both phenyl- glyoxylic and mandelic acid may be taken as the principal metabolites of styrene. ln addition to mandelic acid, phenylglyoxylic acid was also demonstrated in man (Bardodej, 1964; Bardodej and Bardodejova, 1966; Huzl et al., 1967). These observations were subsequently confirmed by Ohtsuji and lkeda (1970). No appreciable el- evation of benzoic or hippuric acid levels has been found in humans, either in earlier or in more recent studies (Bardodej et al., 1960; Stew- art et al., 1968; Ohtsuji and lkeda, 1970). Ohtsuji and lkeda (1971) presume, therefore, that the metabolic pathway leading to mandelic acid is common for man and for most experimental ani- mals. ln the former, the efficiency of mandelic acid decarboxylation, responsible for further me- tabolization to hippuric acid (Fig. 10-1), is low. Therefore, mandelic and phenylglyoxylic acids probably form the only metabolic end-products of styrene in man. Ohtsuji and lkeda (1971) also demonstrated that phenobarbital stimulates styrene metabolism in rats in a manner typical for microsomal in- duction. They further found that it is the first stage of the metabolism which is thereby stimu- lated, leading to the formation of a more toxic epoxide (styrene oxide). lt seems that the kinetics ~of styrene metabolite excretion in man have not been systematically studied. Bardodej (1964), as well as Gadaskina and Filov (1971), reported that after styrene inhalation, the half-time of excre- tion of mandelic acid was 7 hours; whereas after administration of the mandelic acid itself, it amounted only to 2 hours. METHODS FOR DETERMINATION OF STYRENE AND METABOLITES Styrene: For the determination of styrene in the air, breath, and blood a spectrophotometric method has been applied, with the final mea- surement being made in pentane at 245 nm (Fil- ov and Rusin, 1960; Bardodej et al., 1961; Bard- odej, I964). The measured concentrations in the final solution were within the range from 0.5 to l0 lug/ml. For automatic analysis of air in an experimental chamber, Bardodej (1964) applied, among others, the “Mercury Vapor Concen- tration Meter" (Hendrey Relays, Ltd.); whereas Stewart et al. (1968) applied, for the same pur- pose, continuous measurements with an infra-red spectrophotometer at 11.0 um wavelength di- 6| rectly in the gas medium in a ten meter path length gas cell. The latter authors applied gas chromatography (hydrogen flame ionization de- tector) for the determinations of styrene in breath and blood, after extracting the compound with carbon disulphide. Mandelie acid in urine: Bardodej and Bard- odejova (1961) determined mandelic acid follow- ing oxidation to benzaldehyde and distillation. For analysis of the distillate three methods were porposed: a) titrimetric—the excess of iodine will be titrated using 0.01 or 0.1 n thiosulphate de- pending on the concentration of benzaldehyde, b) polarographic (described later in detail by Bard- odej et al., 1964)—the range to be determined 0— 1000 or 0—5000 mg mandelic acid, c) spec- trophotometric—measurements are made in a wa- ter solution at 260 nm. Bardodej (1964) proposed a simpler method, consisting of ether extraction, oxidation with FeCl3, and light absorption mea- surement at 405 nm. Ohtsuji and lkeda (I970) proposed a spec- trophotometric determination of mandelic acid at 450 nm following ether extraction of urine, evap- oration of the extract, and reaction with for- maldehyde in sulphuric acid (El Masri et al., 1958), The range of values to be determined var- ies from 40—400 ug in the sample. Slob (1973) described a method based on a gas- chromatographic assay of a derivative which may be obtained from reaction of mandelic acid with N,O—bis(thiomethylsilyl)acetamide; the intro- ductory step of the analysis consists of extracting the acidified urine with ethyl acetate and sepa- ration of the fraction by means of paper chromatography. Phenylglyoxylic acid in urine: Bardodej (1964) proposed a spectrophotometric determination at 380 nm of a product of the colour-forming reac— tion with dinitrophenyl hydrazine, following ex- traction with ethylbenzene and reextraction. Due to the interference of pyruvic acid (which enters the same reaction) at low exposure values, a pre- liminary separation of phenylglyoxylic acid by means of paper chromatography was proposed. Bardodej and Bardodejova (1966) applied polar- ography for the determination of phenylglyoxylic acid. Ohtsuji and lkeda (1970) made use of the same procedure as that applied for mandelic acid, having only shifted the wavelength to 350 nm. Whereas phenylglyoxylic acid does not inter- fere with the determination of mandelic acid, the reverse is not true, and due corrections are re- quired. The authors recommend concurrent de— termination of both metabolites by means of nor- 62 mal spectrophotometric analysis of a mixture of two substances, applying a system of two alge— braic equations. ~ PHYSIOLOGICAL LEVELS Bardodej et al. (1961) reported that the concen- tration of mandelic acid in the urine of nonex- posed individuals was relatively constant and did not exceed 5 mg per liter; in 1964 Bardodej re- ported concentrations of about 20 mg/l; whereas Gadaskina and Filov (1971) present the opinion that readings in the urine of nonexposed people, obtained via benzaldehyde, are due to the inter- ference of other compounds, which correspond to concentrations of mandelic acid on the average from 14 to 18 mg/I. Applying Bardodej’s (1964) method, Hinkova (1972) found a mean phys- iological level of mandelic acid of 31.5 mg/l; Bolanowska and Sapota (in press) reported a mean value of 25 mg/l (: 35%). Using their own method Ohtsuji and lkeda found a mean level of 92 (47-178) mg/l in nonexposed persons; these values seem to exclude their method from assays when evaluation of low-level exposure is the objective. Applying gas chromatography for the purpose in question, Slob (I973) has observed physiological levels of0.6-7.2 mg/l, or 0.3—0.8 mg per 8 hours. For phenylglyoxylic acid, with the polarographic method. Bardodej and Bardodejova (1966) re- ported physiological levels below 50 mg/l; ap- plying the spectrophotometric method of Bard- odej (1964), Bolanowska and Sapota obtained a mean concentration of 18 mg/l (Ł 81%); spec- trophotometric determinations made by Ohtsuji and lkeda (their own method) yielded similar val- ues with a mean of 19 mg/l (range 11-34). EXPOSURE TESTS Bardodej et al. (1961) proposed a test for styrene using human volunteers, under controlled condi- tions. and based on the determination of man- delic acid in urine. The subjects were exposed to relatively high concentrations of styrene, 550 and 1000 mg/m3 for 8 hours; as a dependent variable the concentration of mandelic acid in the fraction of urine collected toward endxof the exposure in- terval was chosen. At the lower and higher ex- posure levels mean concentrations in the urine of 6 subjects amounted to 1340 (1015—1750) and 2760 (2330-3180) mg/l, respectively (Fig. 10-2). It must be born in mind, however, that the data, as JERZY K. PIOTROWSKI reported by the authors, lead to the conclusion that 100 per cent of the systemically absorbed styrene was metabolised to mandelic acid, leaving no room for phenylglyoxylate. An attempt to use the test for lower concen- trations of styrene in air was made by Hinkova (I972). The study was performed similarly to that of Bardodej, but at concentrations of 5 and 50 mg/m’; the exposure was also shorter, lasting 6 hours. The mean levels found in urine were 75 and 145 mg/l (corrected for specific gravity), re- spectively. These values should be viewed in re- lation to the rather high mean physiological con- centration of3l.5 mg/l. As a measure of the internal consistency of the results reported in these two papers, the mean ra- tios of concentrations in urine (U = mg/l) and in the air (A = mg/m3) may be applied. The U/A ratio of the means for the higher and lower con- centrations were respectively, 2.3 and 8.6 (Hin- Ko % % I000 Mandel/c acid [n urine , rng/l § 42 0.4 0.6 0,8 1,0 Styrene zh air, rng/[ Fig. 10-2. The concentration of mandelic acid in urine collected toward the end of exposure, as dependent on the styrene concentration in the air (Bardodej and Bardodejova, 1961). Taken from: Bardodej Z. and Bardodejova E.: Hodnota a pouziti expozicnich testu. Exposicni test pro ethylbenzen. Cesk. Hygiene 6, 537-545, 1961. Page 544, Fig. 8. STYR ENE kova, I972). From this comparison it follows that the test, at least in its general form, may be applied to air concentrations of styrene down to abbut 50 mg/m3; for lower concentrations the test is still not applicable. The present author is of the opinion that the cited experimental data may form only a tentative basis for the inter- pretation of urinary mandelic acid concen- trations. Thus more complete elaboration of the test. under experimental conditions and for a sat- isfactorily wide range of the concentrations, is still needed. . In view of the lack of feasibility of using the in- crement of hippuric acid“ in the urine as a mea- sure of exposure to styrene, Stewart (1968) pro- posed to base the test upon styrene determination in the breath, even if the fraction eliminated this way is rather minute. In 6 volunteers, who were exposed to air concentrations of styrene of about 100 ppm (425 mg/m3) for 7 hours, he found a concentration in the breath of 2.5 ppm immedi- ately after discontinuation of inhalation, and it decreased rapidly thereafter. After an initial rapid decay (over 1 hr), the concentration fell to about 0.7 ppm, diminishing over the next 5 hours to about 0.3 ppm. From experiments in which other styrene concentrations were applied for 1 or 2 hours, it seems to follow that the concentration in the breath directly after cessation of the in- halation reflects the final, preceding absorption rate rather than total absorbed dose of styrene. The reviewed data do not permit as yet a conclu- sion as to whether, and under which conditions, the method could be applied as an exposure test. AVAILABLE DATA ON INDUSTRIAL EXPOSURE Bardodej et al. (1961) presented the results of ex- posure assessment in several factories where lami- nates were manufactured. When two plants with negligible exposure were eliminated from consid- eration, mean urinary concentrations of mandelic acid fell within the range of 130 to 1650 mg/l. Mean U/A** ratios ranged from 2.3 to 4.7, with a general mean of about 3.0. Simko et al. (1966) found mean urinary concen- trations of mandelic acid from 685 to 1330 mg/l ' The author did not cite the work of Bardodej, who intro- duced the test based upon mandelic acid determination; earlier attempts of Bardodej et al. (1960) to base the test uporli excretion of benzoic acid had also yielded negative resu ts. "" Calculations were made by the present author for 5 fac- tories, where more than 6 air determinations were made. 63 in 4 groups of workers (6-23 people each) ex- posed to styrene while manufacturing laminates. These data, representing a cross-section for the years l96l-65, are not adequate for a correlation analysis with air concentrations. However, ap— proximate U/A ratios were within the limits of 1.4—3.4. The same authors provided information on the dynamics of the increase in the urinary concentrations in co'urse of a work shift. Mean values in the 27 persons studied at the beginning, in the middle, and at the end of the shift were about 360, 600, and 1300 mg/l, respectively. Sim- ilar observations were made recently by Bur- kiewicz et al. (1974). The starting level, approxi- mately 1/4 of the final value, indicates a possibility of a significant systemic cumulation of styrene. However, no increasing trend was found within the week’s exposure period (Burkiewicz et al., 1974). Huzl et al. (1967) have evaluated the exposure accompanying the production of laminates and observed urinary concentrations of mandelic acid of the order of 200-400 mg/l, the U/A ratio be- ing about 1.0. Goetełl et al. (1972) determined the mandelic and phenylglyoxylic acid in the urine of workers ex— posed to styrene concentrations ranging up to 300 ppm (1300 mg/m’). In the group of lower exposure (up to 150 ppm), mandelic acid concen- trations were below the limits of 3000 mg/l (mean AU/A ratio 4.3) and the phenylglyoxylic up to about 500 mg/I ( AU/A 0.6). In the groups exposed to lower concentrations an increase of both metabolites was observed that was propor- tional to the concentration of styrene in the air. In those, however, who were exposed to concen- trations above 200 ppm, an unexpected drop of metabolite concentration was found with an in- crease of styrene concentrations in the air. Hinkova (1972) determined the mandelic acid in the urine of people working in contact with poly- ester paints used for furniture finishing. ln winter and summer, the mean air concentration of sty- rene was 4.8 and 47 mg/m3, respectively. The corresponding mean urinary concentrations of mandelic acid were 38 and 55 mg/l, at the phys- iological reference level of 21 mg/l. Ohtsuji and Ikeda (1970) performed a deter- mination of mandelic and phenylglyoxylic acids in the urine of workers employed in the manu- facture of plastic tanks, at moderate air concen- trations of styrene. ln groups exposed to styrene in the range of concentrations of 10-30 ppm (40— 120 mg/mJ), 7-20 ppm (30—80 mg/m3) and 1-20 ppm (4—80 mg/m3), the corresponding mean uri- 64 nary levels of mandelic and phenylglyoxylic acids were: 875, 473 and 310 mg/l (approximate U/A ratios 8-4), and 381, 287 and 201 mg/l, respectively. Bolanowska and Sapota studied the urinary ex- cretion of mandelic and phenylglyoxylic acids in the workers of several industrial plants where av- erage concentrations of styrene vapours were in the range of ll to 130 mg/mJ. Mean mandelic and phenylglyoxylic acid concentrations were from 65-650 mg/l (mean U/A ratios 3.5-9) and from 60 to 200 mg/l, respectively. The ratio mandelic acid : phenylglyoxylic acid varied from l.l—l.3 in those with low-level exposure to 3.2 in those exposed to the highest concentrations. JERZY K. PIOTROWSKI From the above review, and mainly from the variable W A ratios reported, it may be conclud- ed that the results are still internally inconsistent and warrant further studies, mainly in the range of low-level exposure. An attempt to make use of the determination of styrene in the breath has been described by Goe- tell et al. (1972). ln subjects exposed to styrene concentrations in ranges of 17—32 ppm, 88—139 ppm. and 235-239 ppm, the styrene concen- trations in the breath measured immediately after / discontinuation of exposure were 0.7, 4.5, and 9.0 ppm, respectively. ln 5 hours they dropped to about 0.2. 0.3, and 0.9 ppm, respectively. REFERENCES Bakke O.M. and Scheline R.R.: Hydroxylation of aromatic hydrocarbons in the rat. Toxicol. Appl. Pharmacol. 16, 691-700, 1970. Bardodej Z.: Metabolismus styrenu. (The metabolism of styrene). Ceskosl. Hyg. 9, 223-239, 1964. Bardodej Z. and Bardodejova E.: The metabolism of ethylbenzene, styrene and alpha- methylstyrene. Proc. XV lnt. Congr. Occup. Health, Vienna, vol. 11-1, 1966, pp. 456- 460. Bardodej Z. and Bardodejova E.: Hodnota a pouziti expozicnich testu. X. Expozicni test pro ethylbenzenu. (Value and application of exposure tests. X. Exposure testfor ethylbenzene). Ceskosl. Hyg. 6, 537-545, 1961. Bardodej Z. and Bardodejova E.: Metabolismus etylbenzenu. (Metabolism of ethyl- benzene). Ceskosl. Hyg. 11, 226-235, 1966. Bardodej Z.. Bardodejova E., and Malek B.: Hodnota a pouziti expozicnich testu. X I. Expozicni test pro styren. (Value and application of exposure tests. X l. Exposure testfor styrene). Ceskosl. Hyg. 6, 546-552, 1961. Bardodej Z., Bardodejova E., and Gut J.: Metabolismus styrenu u krys. (The metab- olism of styrene in the rat). Ceskosl. Hyg. 16, 243-245, 1971. ' Bardodej Z., Fiserova-Bergerova V., and Ledrer E.: Polarograficke stanoveni kyseliny mandlove v mcoi. (Polarographic determination of mandelic acid in urine). Prac. Lek. 16, 414-415, I964. Bardodej Z., Malek Z., and Volfova B.: Riziko styrenu pri vyrobe skelnych laminata. (The hazard of styrene in the production of glass laminates). Ceskosl..Hyg. 5, 541- 546, 1960. Bolanowska W. and Sapota A.: Proba zastosowania testu Bardodeja dla oceny ekspozyq‘i przemysłowej na styren. (Attempt ta apply the Bardodej test for evaluation of industrial styrene exposure). Med. Pracy (in press). Burkiewicz C., Rybkowska J., and Zielinska H.: Ocena ekspozycji na styren u ludzi w warunkach przemyslowych. (Exposure to styrene in industrial workers). Med. Pracy 25, 305-310, !974. Carpenter C.F., Schaffer G.B., Weil C.S., and Smith A.F.: Studies on the inhalation of 1.3—butadiene, with a comparison of the narcotic effect with benzol, toluol and styrene. and a note on the elimination of styrene in human. J. 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Hinkova L.: Studies of styrene exposure test at maximum allowable concentration 5 rng/m3. Works of the United Research institute of Hygiene and Industrial Safety, vol. 23, pp. 77-79, 1972. (Ed.: Medicina i Fizkultura, Sofia). Huzl F., Sykora J., Meinerova F., Jankova J., Srutek J., Junger V., and Lahn V.: Olazka rizika pri praci se styrenem. (To the question of health hazard during the work with styrene). Prac. Lek. 19, 121-125, 1967. Ohtsuji H. and lkeda M.: A rapid colorimetric method for the determination of phenng/yoxylic and mandelic acids: Its application to the urinalysis of workers ex- posed to styrene vapour. Brit. .l. lndustr. Med. 27, 150-154, 1970. Ohtsuji H. and lkeda M.: The metabolism of styrene in the rat and the, stimulatory effect of phenobarbital. Toxicol. Appl. Pharmacol. 18, 321-328, 1971. Rubinskaja S.E.: 0 prewrastshenijach stirola w organizmie experimentalnych zhivotnych. (On the metabolism of styrene in experimental animals). Gig. Tr. Prof. _Zebol. Nr. 11, 29-33, 1965. Simko A., Jindrichova J., and Pultarova H.: Vliv styrenu na zdravomi stav prac- ujicich pri vyrobe laminatu. (The effect of styrene on the health state of workers em- ployed in laminate production). Prac. Lek. 18, 348-352, 1966. Slob A.: A new method for determination of mandelic acid excretion at low level sty- rene exposure. Brit. .1. lndustr. Med. 30, 390-393, 1973. Spencer H.C., lrish D.D.. Adams E.M., and Rowe V.K.: The response oflaboratory animals to monomeric styrene. J. Ind. Hyg. Toxicol. 24, 295, 1942. (Cited by Gad- askina l.D. and Filov W.A. 1971). Stewart R.D., Dodd H.C., Baretta E.D., and Schaffer A.W.: Human exposure to sty- rene vapor. Arch. Environ. Health 16, 656-662, 1968. Vrba J., Madlo Z., and Ledrer E.: K premene styrenu. etylbenzenu a kyseliny man- dlove u krys. (Styrene. ethylbenzene, and mandelic acid metabolism in rats). Ceskosl. Hyg. 9, 436-442, I963. Vrba J.. Madlo Z., Kovar V.: Studium metabolismu styrenu u krys pomoci plynove chrotnato rafie. (A study of styrene metabolism in the rat by means of gas chro- matograp y). Ceskosl. Hyg. 12, 477-478, 1967. 65 ll. PHENOL ABSORPTION In industrial conditions phenol is absorbed main- ly via the respiratory tract and through the skin. Retention in the respiratory tract is highly effi- . cient; about 70 per cent on the average, starting with 80 percent at the beginning and declining to about 68 per cent toward the end of an 8 hours‘ inhalation period. The phenol vapours are also absorbed directly through the skin. For persons in working denims the amount absorbed into the system over 6 hours ranged from 5 to 12 and from 30 to 65 mg, at average concentrations of 5 and 25 mg/m3, re- spectively. On the average the cutaneous absorp- tion rate in exposed persons equalled that of an air concentration of 0.35 m3. The working clothes did not reduce the cutaneous absorption rate of the vapours as judged from comparisons with a group of naked persons (Piotrowski, l97l). The possibility of direct cutaneous phenol ab- sorption was pointed out by numerous in- vestigators who studied incidental poisonings in human beings after contact of the substance sole- ly with skin (Watorski, 1950; Wroniewicz, 1953; Duverneuil and Ravier, 1962). METABOLISM AND ELIMINATION The absorbed phenol is distributed in a relatively uniform fashion in the blood and other tissues (Oettingen, 1949; cited by Gadaskina and Filov, 1971). The metabolism of phenol is quite simple: in rabbi-ts, for instance, a small fraction under- goes hydroxylation to hydroquinone and pyro- catechol, 10 and 1 per cent, respectively. The phenol and diphenols are also conjugated with sulphuric and glucuronic acid; at low doses cou- pling mainly with the former, and at higher doses with the latter acid. Phenol combined with these acids is excreted in the urine with an efficiency exceeding 90 per cent of the absorbed dose (Parke and Williams, 1953). Low doses of phenol are excreted totally in a combined form; at toxic levels a substantial part of the free compound is excreted. In human volunteers, at doses of the order of 20-60 mg absorbed by the inhalatory 66 route in well-controlled experimental conditions, the efficiency of total phenol urinary excretion was close to 100 per cent (Piotrowski, 1971). High efficiency of absorption and elimination of phenols is also supported by approximate esti- mates of Ohtsuji and lkeda (l972) for field con- ditions. Administering MC-phenol (0.01 mg/kg) orally to volunteers, Capel et al. (1972) found that the urinary metabolites were present mostly in form of the phenylsulphate (77%) and phenyl glucuronide (16%). KINETICS OF PHENOL EXCRETION ln animals, small doses of phenol are excreted completely within 24 hours; at higher levels, 2—5 per cent may be still detected in the body after this interval (Porteous and Williams, 1949). In man after inhalation and systemic absorption, the kinetics of phenol excretion can be adequately described using a simple one compartment model with an elimination constant of 0.2 hour". It may be concluded, therefore, that in man, signifi- cant cumulation of phenol should not occur un- der conditions of daily repeated industrial ex- posure to this compound (Piotrowski, 1971). This conclusion is also consistent with the data of Ohtsuji and lkeda (I972), who were unable to demonstrate unequivocally an elevation of morn- ing, preexposure phenol concentrations in urine collected at the end of a working week compared to those seen on the first post-weekend morning. METHODS FOR DETERMINATION OF METABOLITES The exposure test is based on the determination of total urinary phenol (Piotrowski, l97l). To as- sess the exposure to phenol, the procedure usu- ally recommended for its determination in urine is based on distillation from the acid medium with subsequent colorimetric measurement of the product of this reaction with Gibb‘s reagent— 2,6—dibromoquinone-chlorimide. A practical ver- sion of the method is the one derived from a pro- cedure developed for animal urine by Porteous PHENOL and Williams (1949), and modified later for lower phenol concentrations in human urine by Fis- crova-Bergerova (I954). This version combines acid hydrolysis of the coupled phenol with its distillation in one short procedure. The method is recommended by Teisinger and coworkers (I956). With slight modifications consisting of increased amounts of the reagent (Hanke et al., 1961; For- ster and Malinakova, 1961), the method has been recommended by Dutkiewicz et al. (1964), by Gadaskina and Filov (I97l), as well as by the au- thor of the present review. Other methods of phenol determination in urine, reported in the literature, differ widely in all pos- sible parameters and procedures, namely those of: a) hydrolysis, b) separation, and c) final de- termination of the phenol. The procedure of hydrolysis, as proposed origi- nally by Porteous and Williams, forms a separate part of the analysis and is quite drastic; its condi- tions could be justified by difficulties encountered while hydrolyzing the coupled compounds of phenol. lkeda (I964), who had been hydrolyzing the material in sealed glass ampoules, has shown that the use of IO n acid does not lead to higher readings than does the use of 0.25 n. Buchet and- coworkers (I972), who have applied hydrochloric acid for the purpose in question, were unable to demonstrate any significant influence of the HCl concentration, or duration of the procedure, upon efficiency of the hydrolysis. Controlled analyses performed in the laboratory of the present author show that the combination of hy- drolysis with steam distillation, as proposed by Fiserova-Bergerova (1954), assures complete hy- drolysis of the coupled phenol. Van Haaften and Sie (1965) reported that heating urine with phos- phoric acid (lzl) directly in the heated section upstream of the chromatographic column of GLC apparatus provides a method for quan- titative hydrolysis. lkeda (I964) and Ohtsuji and lkeda (I972) have substituted ether-extraction for steam distillation of phenol from an acid medium. lt should be mentioned, however, that the high physiological levels of phenol in'urine reported by these au- thors are likely to be due to the presence of other unidentified phenolic compounds extracted concurrently. Final determination of phenol is sometimes per- formed also by the method of Theis and Benedict (coupling with diazo-p-nitroaniline), or using the Emerson colour reaction with 4—aminoantipyrme (Emerson, I943; Kukachova and Naus, 1956; Bardodej, 1960; Bardodej and Krivucova, I961). 67 In the presence of p-cresol which is found in physiological urine, the former reaction is non- specific. This refers to both reactions relative to - o- and m—cresol, if present. Gas chromatography is most specific due to the nature of the process utilized by the method. However, following some procedures a preliminary preparation of a urine high “background" may be seen, resulting from interference of substances which had not been separated from urinary phenol at the preliminary stage of the procedures. (Van Haftcn and Sie, 1965; Buchet et al., 1972). The method recommended above for use in the exposure test is characterized by the lowest phys- iological level for phenol: below 20 mg/day (Teisinger and coworkers, I956), or 16 mg/l mean: 7 i 3 mg/l — Dutkiewicz et al., (1964); more systematic investigations of Piotrowski (1971) yielded the following physiological values: 8.7 t 2 mg/day or 9.5 t 3.6 mg/I of urine stan- dardized to specific gravity of 1.024. OTHER METHODS Determination of phenylsulphuric and phenyl- glucuronic acids may not serve as a basis for evaluation of exposure of a low or moderate magnitude (Ohtsuji and lkeda, I972). EXPOSURE TEST The exposure test, as proposed by Piotrowski (I97I). is based on total phenol determination in urine voided over the last two hours of exposure on any working day. The results are expressed in excretion rate. mg/h, from which the absorbed dose. through all routes, is calculated according to the formula: - y=0.44+0.108x (I) where y = the excretion rate of phenol in mg/ hour, x = the absorbed dose in mg, and the con- stant 0.44 denotes the average excretion rate of phenol in physiological urine. The precision of this test has been found to be extremely good (i 2.2 mg). Another version of the test is based on the concentration of the ex- creted phenol in urine; if results are normalized to the standard specific gravity of the latter (1.024) the regression equation assumes the form: y=0.5+ I.6x (2) 68 where y = concentration of phenol in urine mg/l, standardized to specific gravity of l.024, x = ab- sorbed dose of phenol mg 0.5 — respective phys- iological concentration. The precision of this ver— sion of the test. however. is much worse (L 15 mg). Calculation of the absorbed dose according to equations (l) and (2) may apply equally well to 6 and 8 hours‘ exposures (Mogilnicka and Pio-. trowski. I974). Evaluation of absorbed amounts of phenol from the standpoint of maximum al- lowable concentrations in the air (C) can be based on the following relationship: D = CT(RV + a) (3) in which D = the absorbed dose as determined by means of the test. T = duration of daily exposure (6 or 8 hours. depending on the circumstances). R = fraction of the substance retained in the respiratory tract. V = lung ventillation (for high work it may be assumed as 0.8 m3 per hour). a = skin absorption coefficient expressed in volume of air cleared of phenol per unit time (0.35 m3 hour). For the MAC of5 mg/m‘ (USSR) and I9 mg,‘m~‘ (USA) the value of D amounts to 35 and I35 mg. respectively. Expressed in the form of urinary excretion rate (U). the corresponding val- ues would be 4.4 and 15.3 mg per hour. AVAILABLE DATA ON INDUSTRIAL EXPOSURE ln bakelite plants. at phenol concentrations in the air reaching l2.5 mg/m-‘. Ohtsuji and lkeda (I972) found urinary concentrations of total phe- nol in the range of 200 - 350 mg,/1. Mogilnicka and Piotrowski (I974) have studied the exposure of workers in plants manufacturing phenol from chlorobenzene and cumene. and also in factories in which caprolactam and phenolic resins were produced using phenol as a starting material. At an air concentration of phenol well below 5 mg) JERZY K. PIOTROWSKI Excretion rate (mq lhr ) o to is so 40 so bo Dose (mg) Fig. 11-]. EXcretion rate of phenol in urine collected during the last 2 hr of exposure as a junction of absorbed dose of phenol (Piotrowski, 1971). Taken from: Piotrowski J.: Evaluation of exposure to phenol. Brit. J. Industr. Med. 28, 177- 178, 1971. Page 176, Fig.5. m3. the absorbed doses did not usually exceed 35 mg. with sporadic cases reaching a level of 70 mg per working day. These authors found reasonable agreement between average amounts of absorbed phenol. evaluated either on a basis of the excre- tion rate or the urinary concentration. when the urinary concentration was normalized to urine of specific gravity = 1.024. This latter method. how- ever. yielded sporadic results differing widely from those obtained using the excretion rate as the basic parameter, of the test. From estimates presented by Mogilnicka and Piotrowski (I974). it may be concluded that. under prevailing indus- trial conditions studied. the contamination of skin and clothes does not seem to have a signifi- cant contributing role in the total exposure (absorption). REFERENCES Bardodej Z.: Hodnola a pouziti expozicnich testu. IX. Fenolovy test. (Value and use ofexposure tests. IX. Phenol test). Ceskosl. Hyg. 5, 39-46. I960. Bardodej Z. and Krivucova M.: Metabolismus fenolu u morcat. (Phenol metabo/sm in guinea pigs). Ceskosl. Hyg. 6, 553-554. l96l. Buchet J.P.. Lauwerys R., and Cambier M.: An improved gas chromatographic meth- PHENOL 051741» the determination of phenol in urine. J. Europeen dc Toxicologie 5, 27-31, 1 . Capcl 1.D., French M.R., Millbum P., Smith R.L., and Williams R.T.: Fate of l4-C phenol in various species. Xenoblotlcl 2, 25-34, 1972. Duverneuil G. and Ravicr E.: Toxicite suraique du phenol par voie transcutanee. Arch. Mal. Prof. 23, 830-833, 1962. Fgmgrson E.: A new color test for the phenolic compounds. J. Org. Chem. 8, 417-437, 4 . Fiscrova-Bcrgerova V.: Stanoveni fenolu v moci. (Determination of phenol in urine). Pracowni Lekarstvi 6, 352-354, 1954. Forster V. and Malinakova H.: Pripominky k metodice stanoveni fenolu v moci di- bromchinonchlormidem. (Comments on the method of estimating phenol in the urine by means of dibromoquinone chlorimide). Pracowni Lekarstvi 13, 82-83, 1961. Hanke J., Dutkiewicz T., and Piotrowski J.: Wchłanianie benzenu przes skore u lu- d;:5 (lghelt absorption of benzene throughout the skin in men). Med. Pracy 12, 413— 4 . 6 . Ikeda M.: Enzymatic studies on benzene intoxication. The Journal of Biochemistry 55, 231-243, 1964. Kukackova V. and Naus A.: Prispevek k stanoveni fenolu. (A contribution to the de- termination quhenol). Ceskosl. Hyg. 1, 282-283, 1956. Mogilnicka E. and Piotrowski J.K.: Test ekspozycji na fenol w swietle badan tere- migacz; (97;he exposure test for phenol in light of the field study). Med. Pracy 25, I -l . I 4. Ohtsuki H. and Ikcda M.: Quantitative relationship between atmospheric phenol va- gour an? phenol in the urine of workers in bakelite factories. Brit. J. lndustr. Med. 9, 70-7 , 1972. Parke D.V. and Williams R.T.: The metabolism of benzene. (a) The formation of phenyloglucuronide and phenylosulphuric acid "C benzene, (b) The metabolism of "C phenol. Biochem. J. 55, 337-343, 1953. Piotrowski J.K.: Evaluation of exposure to phenol: Absorption of phenol vapour in the lungs and through the skin and excretion of phenol in urine. Brit. J. lndustr. Med. 28, 172-178, 1971. ' Proteous J.W. and Williams R.T.: Studies in detoxication. 19. The metabolism of benzene: (a) The dtermination of phenol in urine with 2.6—dichlo- roquinonechloroimide. Biochem. J. 44, 46-55, 1949. Ilhcis7R.9Cź and Benedict S.R.: Determination of phenols in the blood. J. Biol. Chem. , 6 . 1 4. Van Haaften A.B. and Sic S.T.: The measurement of phenol in urine by gas chro- tltgatgmraphy as a check of benzene exposure. Amer. Ind. Hyg. Assoc. J. 26, 52-65, 6 . Watorski K.: Zatruciafenolem przez powierzchnie skory. ( Phenol poisoning imbibed through the skin). Med. Pracy 1, 119-131, 1950. Wroniewica Z.: Zatrucia przemyslowe wywolane działaniem fenolu na skore. (Indus- trt'5a3l poisonings caused by the phenol's action on the skin). Med. Pracy 4, 39-46, 19 . 69 12. ANILINE ABSORPTION ln industrial conditions absorption of aniline takes place in the respiratory tract and through the skin. The retention of aniline vapours in the respiratory tract is high (about 90 per cent), and does not vary with duration of the exposure. The vapours also penetrate the skin. The rate of sys- temic absorption via the skin at a given concen- tration of aniline in air is comparable with the rate of absorption in the respiratory tract; at low concentrations the skin absorption is even greater (Fig. l2-l). Elevated temperature of the environ- ment enhances somewhat the cutaneous absorp- tion rate (with temperature rise from 25 C to 30 C by about 20 per cent). The influence of in- creased air humidity manifests itśelf in a similar direction (increase of the humidity from 35 to 75 per cent leads to a rise of the absorption by 30 per cent). The above data relate to individuals ll absorption rate , ing/hr 20 l 0 —› .50 l 30 4 air concentration, mg/m3 |O Fig. 12-1. Absorption rate of aniline vapours through the respiratory tract (dotted line) and skin (solid line) as dependent on the air concentration (Dutkiewicz, 1961). 70 exposed naked under experimental conditions. Normal Working clothes reduce the cutaneous ab- sorption rate by about 40 per cent (Dutkiewicz, I961). ln experimental conditions it was demonstrated that contact of the skin with liquid aniline (satur- ated layer) yields absorption rates of the order 0.2-0.7 mg cm”2 hour". The rate does not seem to change with duration of the contact, but it shows a rising trend with increasing skin tem- perature and becomes considerably enhanced when the skin is moist (Piotrowski, 1957). In in— dustry this absorption process may play a sub- stantial role because, in some occupations (e.g., dye-stuff industry), a significant contamination of the skin (up to 2 mg/cmz) and clothes (up to 25 mg/cml) has been found (Trojanowska, 1959). From estimates of the total systemic absorption by means of exposure tests and by taking the an- iline air concentrations into account, Dutkiewicz (l96l) demonstrated that in industrial conditions the skin formed the main route of absorption; the dominant role in the process was played by direct cutaneous contamination with aniline. Similar conclusions were reached in earlier stud- ies in which aniline concentrations in the air in industry were compared with the f_requency of subacute aniline poisoning in various seasons (Jamrog et al., 1954). METABOLISM The metabolism of aniline has been learned mainly from animal experiments; the human data are sketchy. Oxidation of aniline leads mainly to the formation of 0- and p-aminophenols; the ra- tio of the two varies considerably from species to species (Parke and Williams, 1956). After the ad- ministering the '4C—labelled compound to rabbits, it was possible to prove that m-aminophenol is formed, but with low efficiency. The excreted un- changed aniline accounted usually for less than l per cent of administered dose; however the per- centage increased at high doses (Parke, 1960; Pi- otrowski, l96l). The aminophenols are excreted ANILINE in the urine, conjugated with glucuronic and sul- phuric acids (Smith and Williams, 1949). Biological hydroxylation of aniline is localized in the liver as a typical process that depends on the microsomal electron transport chain with NA- DPHgas hydrogen donor and atmospheric oxy— gen (Parke, 1968). From this it would be ex- pected that the rate of aniline transformation in the body will increase under the influence of typi- cal inducers of microsomal enzymes, such as phe- nobarbital and others. ln rats, dietary iron defi- ciency may play the role of an inducing factor (Becking, 1972). Ability to induce slightly the mi- crosomal enzymes in rats was shown by aniline itself if given repeatedly (Wisniewska-Knypl et al., 1975). The metabolism of aniline has been less in- vestigated in human tissues than in those of ani- mal species. In humans the process also takes place in the microsomes of hepatocytes, but at a rate slower than in rats and not correlated with cytochrome P—450 level (Ackermann, 1972). How- ever, it appears that the process discussed is not the only one possible. For instance, no metabolic activity of the microsomal fraction has been dem- onstrated in human placenta. Nonetheless, me- tabolism of aniline was localised in the cytoplasmic fraction (104,000 g supernatant), and proceeded in the presence of NADH or NADPH as the hydrogen donors, with molecular oxygen and hemoglobin or methemoglobin as catalyzers (Juchau and Symms, 1972). lt was demonstrated that, in man, p-aminophenol forms the principal metabolite of aniline. The metabolite is excreted in urine, conjugated with glucuronic and sulphuric acid. The efficiency with which the metabolite is formed seems to rise, within the range from 15-60 per cent, with increasing doses of aniline. The metabolism—in- cluding oxidation, conjugation, and excretion—is relatively rapid. With some approximation, out- lined in the chapter “Kinetics", the process may be given a first order kinetic description with rate constant in the range of 0.1-0.3 hour". Under conditions of repeated industrial exposure, there- fore, there should be no cumulation of aniline in the body (Piotrowski, 1957; Dutkiewicz, 1961). DETERMINATION OF THE METABOLITES After hydrolysis, the total p-aminophenol may be assayed colorimetrically making use of the in- dophenol reaction. Complexity of the method de- 71 pends on the range of concentrations to be deter- mined and the required or assumed accuracy. Robinson et al. (1951) has made the reaction di- rectly in a diluted hydrolyzate of animal urine, coupling phenol to p-aminophenol in sodium car- bonate solution in the presence of potassium fer- ricyanide as the accelerating oxidant. Piotrowski (1954, I957) determined urinary p-aminophenol in humans in a similar manner, but with different dilutions, and using phenol in ammonia without an oxidant. The method is not very accurate and the limit of detection is about 10 ug/ml urine. Because of its simplicity, the method has been recommended for practical use in the exposure test (Teisinger et al., 1956; Dutkiewicz et al., 1964). Gadaskina and Filov (1971) recommended the somewhat more accurate method of Dut- kiewicz (I960). Developed for human urine, the indophenol reaction is carried out with l—naph- thol in diluted urine—hydrolyzate from which col- ouring substances were removed previously by extraction with n-butanol in acid medium. The reaction itself is made in ammonia solution, the indophenol for colorimetry is extracted sub- sequently with n-butanol. Dutkiewicz and Pio- trowski (1961) have used both the methods in ex- periments on humans. and obtained consistent results. The difficulties encountered while determining low concentrations of p-aminophenol in the urine are related, on the one hand, to the presence of substances inhibiting the indophenol reaction (as- corbic acid, uric acid. creatinine, colouring sub- stances of urine), and, on the other hand, to the low stability of the compound to be measured in the neutral medium which is optimal for quantita- tive extraction. Greenberg and Lester, cited by Dutkiewicz (l960), proposed a radical procedure to circumvent the difficulties, namely: to extract p-aminophenol from a small volume of urine with dichloroethylene, followed by reextraction with hydrochloric acid. The physiological concen- trations obtained using this method (mean = 3.2 ug/ml) were similar to those reported by Du- tkiewicz (I960). who had used his own method (mean = 3.7 ug/ml). OTHER METHODS Aniline itself can be determined in blood after deproteinization with trichloroacetic adic. The di- azo reaction is carried out and the product con- jugated with l—naphthol; the resulting coloured compound may be determined colorimetrically after extraction with n-butanol. P-aminophenol 72 can also be determined in blood. The blood is deproteinized using zinc- and barium sulphates; and the free, as well asthe conjugated, p-amino— phenol (after hydrolysis) is determined by means of indophenol reaction, using for this purpose l— naphthol in an alkaline medium. These methods were used, so far, only in animal experiments (Salm. 1958). For approximate evaluation of exposure to ani- line and other aromatic amines, Marhold (1953), Hill (1953) and Dieteren (1965) have proposed determination of the total urine content of sub— stances yielding a diazo product. The methods are non-specific. and there does not seem to exist a satisfactory basis for interpretation of the results. EXPOSURE TEST Piotrowski (1957) originally worked out the ex- posure test by exposing volunteers to aniline, ap- plied directly to skin in a manner enabling assess- ment of the absorbed dose, followed by determination of urinary p-aminophenol. Later. subjects were studied by Dutkiewicz (l96l), who exposed volunteers by the inhalatory route, while 0 Skin absorption l x Lung absorption mg/h as 1 w b 01 I I l Excretion rate N | . \ )(. I ..,! ;;;.O 1i l 1 l l l l l l l l O 20 40 _ | _ I so eo 100 120' Aniline mg Fig. 12-2. Excretion rate of p-aminophenol in urine collected toward the end of exposure as dependent on the absorbed dose of aniline (Dutkiewicz and Piotrowski, 1961). Taken from: Dutkiewicz T. and Piotrowski J.:” Pure Appl. Chem. 3, 319-323, 1961. Page 320, Fig. 3. JERZY K. PIOTROWSKI preventing at the same time contact of the va- pours with the skin. Piotrowski demonstrated that the amount of absorbed aniline (in the range from 10 to 100 mg) may be assessed with reason- able accuracy. accepting as a measure the excre- tion rate of p-aminophenol in the last portion of urine voided in the exposure interval. Estimates based upon concentrations of p-amino- phenol, even after normalization of the urine to constant specific gravity (1.024), are much less accurate. The relationship between the absorbed doseand p-aminophenol excretion rate is curvi- linear (concave); this results from the increasing efficiency of the metabolic pathway with rising doses of aniline. Relations reported by both the authors are consistent, as shown in Figure 12-2. For the range of doses beyond those studied ex- perimentally, Piotrowski proposed a formula per- mitting the extrapolation: 0.l2D2 U =— 66 + D (1) where U : urinary excretion rate of p-amino- phenol measured between 4 and 6 hours of ex- posure (mg/hours), D = absorbed dose of aniline my. For practical use of the test, the" following pro— cedure is proposed: from the excretion rate of p- aminophenol, the absorbed dose is read from the curve in Figure l2-2 or calculated from formula (I). To relate the obtained result to MAC of ani- line vapours, a general formula may be applied: D=CnRv+a) a) where D = absorbed dose (mg), T = duration of daily exposure (hour), R = retention of the va- pours in the respiratory tract = 0.9, V = lung ven- tilation rate (for light work 0.8 m3/hour may be substituted), and a = proportionality coefficient for skin absorption of the vapours. From Figure l2-l it can be read that the value of a varies with aniline concentrations, decreasing from about 0.6 thour for low concentrations (5 mg/m3) to about 0.2 m3/hour at 50 mg/m3. The author of this review proposed to use, for practical purposes. the values of a cor- responding to MAC values in a given country (e.g. 0.6 and 0.3 mJ/hour for 5 mg/m3 (Poland) and 19 mg/m3 (USA), respectively. The value of C, obtained from transformation of equation (2), may be directly compared with the ANILINE respective MAC values adopted in any given coun- try. For the values given above for Poland and the USA, the derived permissible daily doses, at assumed 6 and 8 hours of daily exposure, amount. respectively, to 35 and 150 mg. The cor- responding excretion rates would be 1.5 and 13 mg/hour. The relationship between the absorbed dose of aniline and the concentration of p-aminophenol in urine sampled toward the end of exposure is burdened with much larger errors. Figure I2-3 may be used as a basis of calculations of data en- countered in the literature. The sensitivity of the exposure test allows the estimation of daily doses of aniline above a threshold of 20-30 mg. It is useless, however, at exposure levels as low as the recent MAC level in the USSR (0.1 mg/m3 of air) (Cahiers de Notes Documentaires, I974). The latter value has been proposed based on obser- vations performed on humans exposed in indus- try to aniline concentrations not exceeding on the average 3 mg/m3, where subtle changes in the blood picture as well as in the nervous system were observed (Vasilenko et al., I972 a). Other data indicate, in humans, a slight elevation of the methemoglobin level after oral doses exceeding 25 mg (corresponding to air concentrations of 2-3 mg/m-‘) (Jenkins et al., I972). It seems, therefore, likely that the MAC level will be considerably re- duced in the future, and the postulated low ex- posure level will not be measurable anymore us- ing the described exposure test. SPECIFICITY OF THE EXPOSURE TEST The test, based upon the determination of uri- nary p-aminophenol, is nonspecific in situations where an individual has absorbed other aromatic compounds metabolised to the same end-product, i.e. p-aminophenol. From the practical stand- point, the most important role is played by the analgesic phenacetin, an ingredient of anti-head- ache pills. A few hours after intake of phenacetin the concentrations of p-aminophenol may reach values in the order of 200 mg/I and one day later oscillate still around 30 mg/l (Simko and Has- sman, 1960). It is therefore necessary to ascertain whether a person whose urine is to be analyzed did take headache pills or other drugs over the preceding two days. As a control it is recommen— ded that urine voided before onset of the ex- posure be analyzed; the result should be negative. Nitrobenzene, absorbed in small amounts (up to 70 mg daily) does not interfere with the p-amino- 73 E l PAP, mg/l 3 o | 50 IN Dose , mg Fig. 12-3. Maximum urinary concentrations of p- aminophenol. as dependent on the dose of ani/ine absorbed through the skin (Piotrowski, 1960). phenol exposure test for aniline (Salmowa et al., I963). In workers exposed to dimethylaniline, a slight elevation of urinary p-aminophenol was observed (Vasilenko et al., 1972 b). AVAILABLE DATA ON INDUSTRIAL EXPOSURE The bulk of the available data refer to earlier years, and should not be taken as reflecting the present hazard of exposure to aniline in industry. The data reported by Dutkiewicz (I96I) and by Kodura and Sniady (1963) indicated that the dai- ly absorption did not exceed 45 mg. Piotrowski (I968), who has studied more recently the ex- posure of workers engaged in the manufacture of aniline, found daily absorption between 30 and 80 mg. Exposure of the same order of magnitude has been reported from the Hungarian industry (Pacseri, I959). Most of the recent data from the USSR point to a very low exposure of workers manufacturing diphenylamine and other organic amino com- pounds. The average concentrations of p-amino- phenol in urine did not exceed 20 ug/ml (Vasi- lenko et al., I972 b). A slight rising trend in urinary p-aminophenol concentrations in sub- sequent days of the working week was reported. 74 JERZY K. PIOTROWSKI REFERENCES Ackerman E.: Enta/kylierung von Athylmorphin und p-c-Hydroxylierung von Ani/in in Lebertnikrosomen von Menschen und von mannlichem und weiblichem Rutten. Biochem. Pharmacol. 21, 2169-2180, 1972. Becking G.C .: Influence of dietary iron levels on hepatic drug metabolism in vivo and in vitro in the rat. Biochem. Pharmacol. 21, 1585-1593, 1972. Dieteren A.M.L.: Urinary amine output estimation. Arch. Environ. Health 10, 816- 820, 1965. Dutkiewicz T.: Wchłanianie par aniliny u ludzi. (Aniline vapours absorption in men). Med. Pracy 12, 1-15, 1961. Dutkiewicż T.: Metoda oznaczania fizjologicznego poziomu p-aminofenolu w moczu. (The method of determination and the physiological level of p-aminophenol in the urine). Med. Pracy 11, 167-182, 1960. Dutkiewicz T. and Piotrowski J.: Experimental investigations on the quantitative esti- mation of aniline absorption in man. Pure Appl. Chemistry 3, 319-323, 1961. Hill D.L.: Excretion of diazotizable metabolites in man after aniline exposure. Arch. lnd. Hyg. Occup. Med. 9, 347-349, 1953. Jamrog D.. Kesy J., Piotrowski J. and Zaremba Z.: Wyniki badan toksykologicznych wfabryce barwnikow organiczynch. (Toxicological investigations in the dye industry). Med. Pracy 5, 287-298, 1954. ' Jenkins F.P.. Robinson J.A., Gellatly J.B.M. and Salmond G.W.A.: The no-ef/ect dose of aniline in human subjects and a comparison of aniline toxicity in man and the rat. Food. Cosmet. Toxicol. 10, 671-679, 1972. Juchau M.R. and symms K.G.: Aniline hydroxylation in the human placenta; Mech~ anistic aspects. Biochem. Pharmacol. 21, 2053-2064, 1972. Kodura J. and Sniady H.: Wartosc oznaczania p-aminofenolu i p-nitrofenolu w mo- czu jako wskaznik stopnia narazenia na aniline i nitrobenzene. (Value of p-amino- phenol and p-nitrophenol determination in urine as indication of degree of exposure to aniline and nitrobenzene). Med. Pracy 14, 15—21, 1963. Marhold J.: Orientacni methoda k stanoveni diazotovatelnych latek v moci. (Orien- tational method of estimation of diazotisable substances in the urine). Prac. Lek. 5, 348-349, 1953. Parke D.V. and Williams R.T.: Species differences in the 0- and p-hydroxylation of aniline. Biochem. J. 63, 1956. Parke D.V.: Studies in detoxication. 84. The metabolism of ("C) aniline in the rabbit and other animals. Biochem. J. 77, 493-503, 1960. Pacseri I.: p-Aminophenol excretion as an index of aniline exposure. Prac. Lek. 11, 165-166. 1959. Piotrowski J.: Proba zastosowania biochemicznych wskaznikow wchlaniania aniliny. nitrobenzene i benzenu u pracownikow przemyslu barwnikarskiego. (Attempt to ap- ply the biochemical indicators to define aniline, nitrobenzene and benzene absorption among the dye industry workers). Med. Pracy 5, 299-307, 1954. Piotrowski J.: Quantitative estimation of aniline absorption through the skin in man. J. Hyg. Epid. Microbiol. and Immunol. l, 1-23, 1957. Piotrowski J.: Metabolizm niskich dawek aniliny u krolikow. (Metabolism of small doses ,ofaniline in rabbits). Med. Pracy 12, 309-317, 1961. Piotrowski J.: Chemiczne zagadnienia przemysłowej toksykologii nitrobenzeneu. (Chemical aspects of industrial toxicology of nitrobenzene). Dsc thesis, Medical Acad- emy in Gdansk. I968. Piotrowski J.: Niektore zagadnienia metabolizmu aniliny. (Selected problems of ani— line metabolism) PhD thesis, University of Lodz, 1960. Robinson D., Smith W.J., and Williams R.T.: The metabolism ofnitrobenzene in the rabbit: o, m- and p-nitrophenols, m- and p-aminophenols and 4—nitrocatech0l as me- tabolites ofnitrobenzene. Biochem. J. 50, 228-234, 1951. Salm J.: Oznaczanie aniliny i p-aminofenolu we krwi. (The determination of aniline and p-aminophenol in blood). Med. Pracy 9, 455-462, 1958. Salmowa J.. Piotrowski J., and Neuhorn U.: Evaluation of exposure to nitrobenzene. Absorption of nitrobenzene vapour through lungs and excretion of p-nitrophenol in urine. Brit. J. lndustr. Med. 30, 41—46, 1963. ANILINE Simko A. and Hassman P.: Hodnoty p-aminofenolu u osob pracujicich s anilinem. (p-Aminophenol values in people exposed to the hazard of aniline). Prac. Lek. 12, 249-25I. I960. Smith J.N. and Williams R.T.: The fate of aniline in the rabbit. Biochem. J. 44, 242- 250. 1959. Trojanowska B.: Zanieczyszczenia nitro- i amino-zwiazkami skory i odziezy och- ronnej u pracownikow przemyslu barwnikarskiego. (The pollution with nitro- and amino-compounds of the work clothes and of the skin, of the dye industry workers). Med. Pracy 6, 387-392, I959. Vasilenko N.M., Volodchenko V.A., Khizhnyakova L.N., Zvezdai V.l., Manfanovskii V.V.. Anatovskaya V.S., Krylova E.V., Voskoboinikov N.A., Gnezdilova A.I., and Sonki LS.: Materialy po obosnovaniu snishenia predelno dopustimoi koncentrat-ii an- ilina v vozduche rabotshei zony. (Information to substantiate a decrease of the max- imum permissible concentration of aniline in the air of working zones). Gig. Sanit. 375, 3l-34, I972a. Vasilenko N.M., Guezdilova A.I., and Shevchenko N.F.: Sodershanie para- aminofenola v motshe kak kriterii kontakta rabotayushtshich s anilinem i yego deri- vatami. (Paraaminophenol content in the urine as a criteria of exposure of workers to aniline and its derivatives). Gig. Tr. Prof. Zabol. 16(10), 54-56, l972b. Wisniewska-Knypl J.M., Jablonska J.K., and Piotrowski J.K.: The effect of repeated exposure to aniline. nitrobenzene and benzene on liver microsomal metabolism in the rat. Brit. .]. Indusu-. Med. 32, 42-48, 1975. Valeurs Iimites de concentration des substances toxiques dans I'air. Cahiers de Notes Documentaires, No. 74, 99-123, 1974. Institut National de Recherche et de Securite, Paris. 75 13. NITROBENZENE ABSORPTION ln industry, nitrobenzene may be absorbed into the body by all possible routes, but mainly via the respiratory tract and through the skin. Reten- tion of nitrobenzene vapours in the respiratory tract is high; on the average 80 per cent, from 87 per cent at the beginning down to 73 per cent af- ter 6 hours of inhalation (Salmowa et al., 1963). The vapours pass directly through the skin. The rate of absorption is roughly proportional to air concentration. from I mg,/hr to about 9 mg/hr at S and 20 mg/mJ. respectively. The temperature of the air in the range from 25 C to 30 C did not influence the absorption rate; increasing the rela- tive humidity from 35 to 67% increased the rate of absorption by about 40 per cent. Normal working denims reduced the cutaneous absorp- tion rate of the vapours, relative to the values in persons exposed naked, by about 20 per cent (Pi- otrowski, I967). The cutaneous absorption of nitrobenzene also takes place after direct application to the skin (contamination). The maximal absorption rate resulting from creation of a saturated layer at the skin surface reaches values from 0.2 to 3 mg/ cmlh", rising with skin temperature. The absorp- tion rate. measured as a decrement of nitro- benzene from the contacting layer, decreased with duration of contact (Salmowa and Pio- trowski, I960). Absorption of nitrobenzene as a result of direct contamination of skin (and clothes) may be of practical importance in indus— try. As demonstrated by Trojanowska (l959), the contamination of the skin and working clothes of workers in the dye manufacturing industry may reach values of 2 and 25 mg/cm3, respectively. Also, in a field study performed in a nitro- benzene manufacturing plant, Piotrowski (I966) has shown that the absorbed doses of the com- pound. estimated by means of exposure tests, ex- ceeded considerably the amounts that could be attributed to absorption of the vapours. METABOLISM AND ELIMINATION Nitrobenzene is quite soluble in lipids; the coeffi- cient of oil—water distribution is about 800 (Du- 76 tkiewicz et al„ l964). In rats, one hour after in- travenous administration of a small dose of the substance, the ratio of concentrations of un- altered nitrobenzene, adipose tissue vs. blood, in internal organs and muscles was approximately l0 : l (Piotrowski. I968). Information on the metabolism of nitrobenzene is based mainly on animal experiments; the hu- man data are fragmentary. The main pathways are directed in two ways: a) reduction to aniline with subsequent hydroxylation to aminophenols, and b) direct hydroxylation of nitrobenzene with formation of nitrophenols. Partial further reduc- tion of nitrophenols to aminophenols may take place. The reduction of nitrobenzene to aniline proceeds through the unstable intermediate products, ni- trosobenzene and B-phenyl hydroxylamine, which are highly toxic and possess a pronounced methemoglobinemic activity (Uehleke, l963). The process takes place mainly in the liver under the influence of “mammal nitro-reductase”, localized partly in the microsomal fraction and partly in the cytoplasm. The process depends upon NA- DPH2 and requires the presence of flavoprotein; oxygen exerts an inhibitory influence. The mech- anism of further hydroxylation of aniline to ami- nophenols is probably the same as discussed in the chapter on “Aniline". The hydroxylation of nitrobenzene to nitro- phenols is not a microsomal process and is diffi- cult to reproduce in animal tissues “in vitro”. The process proceeds under the influence of per- oxidase in the presence of oxygen (Buhler and Mason. l96l). ln rabbits, p-aminophenol is the main (3l per cent) metabolic product of biotransformation; other metabolites are p- and m—nitrophenols (9 per cent each), and o- and m-aminophenols (each 3-4 per cent). Only a minor fraction of nitro- benzene is excreted unaltered with the expired air; aromatic ring cleavage with resulting MC02 expiration after administration of the labelled compound accounts only for about I per cent of the administered dose. The nitro— and amino- phenols are excreted in urine conjugated with sulphuric and glucuronic acid (Robinson, et al., I951). NITROBENZENE Quantitative relations between metabolites vary with animal species and dose of nitrobenzene. In rabbits the share of p—aminophenol rises from 2- 6 to 15-27 per cent with increase of the dose from 10 to 50-100 mg/kg, respectively; whereas the efficiency of the formed p—nitrophenol seems constant, of the order of 5-15 per cent. In con- trast, in rats the efficiency of p—nitrophenol for- mation decreases with an increasing dose of ni- trobenzene, the percentage of p—aminophenol formed remaining constant (Salmowa and Pio- trowski, 1960; Salmowa, 1961). Excretion of two metabolites in man—p-amino- phenol and p—nitrophenol—have been studied. ln experimental conditions of inhalatory exposure to nitrobenzene, the efficiency of p-nitrophenol for- mation was of the order of 6-21 per cent (in one individual up to 37 per cent); mean 13-16 per cent (Salmowa et al., 1963; PiotroWski, 1967). The respective efficiency for p-aminophenol may be estimated only indirectly from observation of acute poisoning cases, where the molar ratio of excreted p-nitrophenol to p-aminophenol was about 2:1 (lkeda and Kita, I964; Myslak et al., 1971). Combined excretion of these two metabo- lites in man accounts for not more than 20-30 per cent of the nitrobenzene dose. The fate of the rest remains obscure. METABOLIC AND EXCRETION KINETICS Experimental investigations of nitrobenzene me- tabolism in animals, in which large doses of com- pound were administered, pointed to a prolonged excretion of the metabolites. At moderate and low doses, however, the excretion of p-nitro- phenol and p—aminophenol obeyed a first order kinetics with a rate constant of about 0.09 hour '. At a daily dosage of 10—20 mg/kg for a week, the cumulative effect was slight; and in- crease in the excretion rate did not exceed 40 per cent relative to the first day. In rats the excretion was somewhat slower, and the rate constants, ob- tained from following the urinary levels of p-ni- trophenol and p-aminophenol, were about 0.08 and 0.06 hour ', respectively (Salmowa and Pio- trowski, 1960; Salmowa, 1961). From a study of nitrobenzene concentrations in the blood of an acutely poisoned individual, Te- isinger et al. (I956) concluded that the compound persists in the human body for a prolonged time. Similar observations were made with respect to urinary excretion of the metabolites, p-nitro- phenol and p-aminophenol, by patients treated 77 for acute or subacute poisoning. The excretion coefficient for p-nitrophenol, followed in urine for 3 weeks, amounted to about 0.008 hour ' (lkdea and Kita, 1964; Myslak et al., 1971). lt may be concluded, therefore, that the metabolic transformation and excretion of nitrobenzene in man is slower by an order of magnitude than in rabbits or rats. A study of the metabolic kinetics of nitrobenzene in man, by means of the urinary excretion rate of p-nitrophenol after experimental exposure to low doses of nitrobenzene, demon- strated that it is a biphasic process that may be expressed by an equation: Ut/UO : 0.66 e—0J4t + 0,34 e410115: (1) where t = time in hours. This curve characterizes the, so-called, two com- partment open mode] in which the role of the slowly turning over compartment can be attri- buted to adipose tissue. The prolonged systemic retention of nitrobenzene in man, and slow ex-_ cretion of its metabolites, is predetermined by the low rate of the metabolic transformation of ni- trobenzene itself, namely of reduction and hy- droxylation; whereas the coupling and excretion of the metabolites (p-nitrophenol) is a rapid pro- cess. In accord with theoretical expectation in man, nitrobenzene cumulates in the course of re- peated daily exposures, reaching an equilibrium after 34 days, at which time p-nitrophenol level in urine is 2—3 fold higher than on the first day of‘exposure (Piotrowski, 1967). This allows cal- culation of cumulative trends on the basis of the kinetic data derived from investigation of single exposures (Piotrowski, I971). METHODS FOR THE DETER- MINATION OF THE METABOLITES The exposure test is based upon determination in the urine of p—nitrophenol after hydrolysis. Basic methods for respective determination have been developed for assessment of exposure to para- thion. All the methods include removal of inter- fering colour substances, hydrolysis, extraction of p-nitrophenol, re-extraction into the aqueous me- dium, reduction to p-aminophenol, and in- dophenol reaction yielding a blue product. The detection limit is similar for all methods (about 5 [Lg in a sample); and final detectability results 78 from the volume of urine taken for analysis (0.1 ;: g/mI at a 50 ml, 0.5 ug/ml at a 10 ml sam- ple). Various authors have recommended differ- ent methods or their modifications. The method of Salmowa has been applied in elaboration of the exposure test (Salmowa et al., 1963; Pio- trowski, 1967). The method includes acid hydro- lysis, oxidation of urine dyes with perhydrol in an alkaline medium, extraction of p-nitrophenol with a mixture of solvent naphtha and ethyl- ether, re-extraction to the alkaline aqueous me- dium, reduction with zinc in an acid medium, and development of the colour with phenol in presence of ammonia. The limit of detection (for a 10 ml sample of urine) amounts to 0.5 11 /ml; the precision of the method is about t 6 per cent. The methods published recently for p-nitrophenol in urine make use of gas chromatography (see “Parathion"). OTHER METHODS Determination of p-aminophenoI—see: “Aniline”. For assessment of exposure to nitrobenzene and other aromatic mono-nitrocompounds, Ajtai and Csayi (I956) proposed polarographic deter- mination of unaltered nitrocompou‘nds after des- tillation of urine. No quantitative interpretation of this assay has been proposed. EXPOSURE TEST Originally, a test based upon determination of p- aminophenol in urine was recommended (Von Oettingen, I94I; Piotrowski, 1954; Teisinger et al., 1956). Later studies by Salmowa and PiotrOWSki (I960) and by Salmowa (1961) showed that the test could not be applied for the evaluation of low-level exposure due to poor sensitivity and lack of specificity of the method in the low con- centration range. Salmowa (1961) proposed to base the test upon the determination of p-nitrophenol; and experi- ments involving single (Salmowa et al., 1963) and repeated exposure (Piotrowski, 1967) demon— strated that the test may be applied for the as- sessment in man of an absorbed nitrobenzene dose (or average doses) above 10 mg. However, the test, as discussed here, should be treated as semiquantitative because the precision in the rec- ommended version was not better than i 10 mg nitrobenzene. In the test, urine samples are collected in the last 2-3 hours of a working shift. Since in the first 3 JERZY K. PIOTROWSKI days of the working week there is a systemic cu- mulation of nitrobenzene, it was suggested that the test be performed on the 4th, 5th, and possi- bly 6th day of the week and that the estimate of absorption be based on the mean values for indi- vidual workers. The test is based on the measurement of the ex- cretion rate of p-nitrophenol (ug/hour); for ac- cording to Salmowa et al. (1963), this parameter leads to better accuracy than the measurement of the concentration of the metabolite itself. ln the case of single exposures, interpretation of the test is provided by equation: I = 3.6 E (2) where: 1 = excretion rate measured toward the end of daily exposure (# g/hour); E = absorbed dose in mg. In the case of daily repeated exposures, if the measurement is made at the end of week, the re- lationship should undergo a change, the expected magnitude of which may be derived from param- eters of equation (I).- The result would be: | : 10.8 E (3) indicating a threefold increase of excretion rela- tive to the first day of exposure. The experi- mental relationship obtained by Piotrowski (I967) was: I = 7.9 E (3a) Equations 3 and 3a denote the range of uncer- tainty in the interpretation of the test in condi- tions of repeated exposure. Figure 13-1 presents the dynamics of increase of p-nitrophenol in urine. found experimentally, after repeated daily exposure of a constant magnitude. The final interpretation of the test is derived by relating the absorbed dose to the permissible dose Dm; resulting from exposure to the max- . imal pemiissible concentration in air Cm. The re— lation between these two parameters is as follows: Dm : CmT(VR + a) (4) Mean values of the parameters may be chosen as follows: lung ventilation V = 0.8 m3/hour (light work), lung retention R = 0.8; coefficient of va- NITROBENZENE pour skin absorption a = 0.25 m3/hour. The permissible dose is determined, therefore, by two factors (daily exposure time T and maximum permissible concentration in air Cm), depending on the regulations, specific for a given country. In the USSR, at 6 hours daily work with aro— matic nitro- and aminocompounds, and a MAC in the air of 3 mg/m3 (Sanitarnyje Normy, SN 245—7l). the permissible dose Dm will be 15 mg; in the USA, at 8 hours of daily exposure and Cm = 5 mg/mJ, Dm = 35 mg. According to equations 3 and 3a these values corresponded to the per- missible urinary excretion rate of p-nitrophenol of 130-180 and 280—390 [tg/hour, respectively. SPECIFICITY OF THE TEST Among the aromatic nitrocompounds tested thus far in rats, a positive reaction for p-nitrophenol in urine was given by o-chloronitrobenzene and 2.5—dichloronitrobenzene, for which relative to equimolar doses of nitrobenzene the readings were 34 and 10 per cent—respectively. Other aro- matic nitrocompounds, such as m-nitrobenzene, 0- and p-nitrotoluene, 2,4—dinitrotoluene, 2,4,6- trinitrotoluene, 0- and p-ethylnitrobenzene, p- chloronitrobenzene, m- and p-nitroaniline, and ! ~nitronaphthalene— have yielded either negative or equivocal results (Piotrowski, 1967). On the other hand, Kodura and Sniady (1963) reported from a field study positive results with the p-ni- trophenol test in workers exposed to p—chloro- ' nitrobenzene. However, due to the character of production in the factory, concurrent exposure of the studied individuals to o-chloronit'robenzene could not be excluded. 79 | | l I l 1 [25'4567891011121 days Fig. 13-1. Increasing trend of p—nitrophenol in urine. following daily exposure to nitrobenzene. Ratio of daily excreted amounts on the sub- sequent n-th day to the first day of exposure. Solid line — theoretical trend, dotted line —— mean experimental data (Piotrowski, 1966). AVAILABLE DATA ON INDUSTRIAL EXPOSURE The early published data related to Polish indus- try. Serious systemic absorption (doses of the or— der of several hundred mg per day( had been described by Piotrowski in workers in a nitrobenzene producing plant as early as 1954. When the working conditions were improved, at similar production levels 10 years later, the ab- sorbed daily doses were estimated at 20—65 mg, with sporadic values up to 130 mg. Exposure to nitrobenzene in course of aniline production was not appreciable. absorbed doses being about 7—9 mg (Piotrowski, 1966). A moderate exposure to nitrobenzene, apart from nitrobenzene manu- facture, was found earlier in other studies, mostly in the Polish dye-stuff industry (Salmowa, 1961; Kodura and Sniady, 1963). REFERENCES Ajtai J. and Csanyi G.: Arbeitsmethoden zur Polarographischen Bestimmung einiger Aromatischer Nitroverbindungen itn Dienste der arbeitshygienischen Untersuchungen. Acta Chimica 9, 463-470, 1956. Buhler DR. and Mason H.S.: Hydroxylation catalyzed by peroxidase. Arch. Bio- chem. Biophys. 92, 424-437, 1961. lkeda M. and Kita A.: Excretion of p-nitrophenol and p-aminophenol in the urine of a patient exposed to nitrobenzene. Brit. J. lndustr. Med. 21, 210-213, 1964. Kodura J. and Sniady H.: Wartosc oznaczania p-aminofenolu i p-nitrofenolu w mo- czu jako wskaznik stopnia narazenia na anline i nitrobenzen. (Value of p-amino- 80 JERZY K. PIOTROWSKI phenol and p-nitrophenol determination in urine as indication of degree of exposure to aniline and nitrobenzene). Med. Pracy 14, 15-21, 1963. Lawford DJ. and Harvey D.G.: Determination of p-nitrophenoł in urine and in blood by the indophenol reaction. Analyst 78, 63-65, 1953. Mountain J .T., Zlotolow H. and O'Conor C.T.: Determination of paranitrophenoł in urine in parathion poisoning cases. Industrial Health Monthly 11, 88, 1951. Mysiak Z., Piotrowski J.K., and Musialowicz E.: Acute nitrobenzene poisoning. Arch. Toxikol. 28, 208-213, 1971. Piotrowski J.: Proba zastosowania biochemicznych wskaznikow wohlaniania aniliny, nitrobenzenu i benzenu u pracownikow przemyslu barwnikarskiego. (Attempts to ap- ply the biochemical indicators to define aniline, nitrobenzene and benzene absorption among the dye industry workers). Med. Pracy 5, 299-307, 1954. Piotrowski J.: Chemiczne zagadnienia przemysłowej toksykologii nitrobenzenu. (Chemical problems of the industrial toxicology of nitrobenzene). Med. Pracy 17, 519-534. 1966. Piotrowski J.: Further investigations on the evaluation of exposure to nitrobenzene. Brit. J. lndustr. Med. 24, 60-65, 1967. Piotrowski J.: Chemiczne zagadnienia przemysłowej toksykologii nitrobenzenu. (Chemical problems on the industrial toxicilogy of nitrobenzene). Dsc thesis, Medical Academy. Gdansk I 968. Robinson D., Smith J.N., and Williams R.T.: Nitro compounds. The metabolism of 0-. m- and p-nitrophenols in the rabbit. Biochem. J. 50, 221-227, 1951. Salmowa J.: !losciowa ocena wchłaniania itrobenzenu. ll. p-Nitrofenoł jako metabolit nitrobenzenu w niskim zakresie dawek. (Quantitative evaluation of nitrobenzene ab- sorption. II. p-Nitrophenol as metabolits of nitrobenzene in the low range of ab- sorbed doses). Med. Pracy 12, 145-155, 1961. Salmowa J. and Piotrowski J.: Proba ilosciowej oceny wchłaniania nitrobenzenu w warunkach doswiadczalnych. (Attempt on the quantitative estimation of the nitro- benzene resportion in experimental conditions). Med. Pracy ll, 1-14, 1960. Salmowa J., Piotrowski J., and Neuhorn U.: Evaluation of exposure to nitrobenzene. Absorption of nitrobenzene vapour through lungs and excretion of p-nitrophenol in urine. Brit. J. lndustr. Med. 20, 41-46, 1963. Trojanowska B.: Zanieczyszczenia nitro- o aminozwiazkami skory i odziezy och- ronnej u pracowrtikow przemyslu barwnikarskiego. (The pollution with nitro and aminocompounds of the work clothes and the skin of the dye industry workers). Med. Pracy 10, 387-392, 1959. Uehleke H.: Nitrobenzene and phenylhydraxylamine as intermediates in the biologi- cal reduction of nitrobenzene. Naturwissenschaften 50, 335-336, 1963. Vlachova D.: Stanoveni p-nitrofenołu v moci exponowanych osob. (Determination p- nitrophenol in the urine of exposed persons). Prac. Lek. 8, 283-288, 1956. Von Oettingen W.F.: The aromatic amino and nitro compounds. U.S. Publ. Health Bull No. 271, Washington, 1941. Sanitamjje Normy Projektirowanija Promishlennych Predpijatij, SN-245-7l (Sanitary standards for designing of industrial premises). Gosurdarstwiennyj Komitet Sowieta Mmistrow. USSR pro Delam Stroitelstwa, 1971. 14. BENZIDINE ABSORPTION Under industrial conditions, benzidine may pass into the system via all routes: the respiratory, gastrointestinal, and the skin (Dutkiewicz et al., I964). Absorption through the skin seems to be dominant under normal conditions; this is borne out by experiments on volunteers (Meigs et al., 1951), as well as by observations made in indus- try. It has been demonstrated repeatedly that a positive correlation exists between benzidine con- centrations on the skin, underwear, and clothes of workers and its concentration in the urine. Furthermore, when a daily change of underwear and working denims and an afterwork bath were introduced, the urinary level of benzidine was re- duced by a factor of 2 to 3 (Meigs et al., I95]; Meigs et al., I954; Ader et al., I958). METABOLISM AND ELIMINATION In the body, benzidine undergoes conversion mainly into 3—hydroxybenzidine. In mice, this metabolite and its N-acetyl derivative is excreted predominantly estrified with sulphuric and gluc- uronic acids; these esters account for about 2/3 of all the metabolites. Of the others, mono- and diacetyl benzidine, as well as N-sulphuric and N- glucuronic derivatives, were identified in urine (Seiarini and Meigs, 1961). In this and an earlier ' paper (1958), these authors were unable to con— firm the data on biotransformation of benzidine to the dihydroxy-derivative. Part of the absorbed benzidine is excreted un- altered in the urine; the percentage varies among the species. In the first experiments on humans, Meigs et al. (1951) recovered 10 per cent of the compound unchanged in urine. According to Sci- arini and Meigs (I961), who investigated urinary excretion in three workers exposed to consid- erable amounts of benzidine in industry, the free compound and mono- and diacetylbenzidine ac- counted for 3.6-5.6 and 6.7—15.9 per cent, re- spectively, of the total excretion; the rest was present in the form of 3—hydroxybenzidine (esterified). 81 The dynamics of benzidine metabolism may be evaluated on the basis of its rate of disap- pearance from urine. Ader (I957) estimated an elimination half-time at about 20, 5.8 and 5.4 hours in the rabbit, dog, and man, respectively. From these values it follows that after repeated industrial exposure, systemic cumulation of ben- zidine in man is unlikely. DETERMINATION OF BENZIDINE AND ITS METABOLITES IN URINE Glassman and Meigs (I951) developed a method for the determination of free benzidine in urine based on ether extraction from the alkaline me- dium, evaporation of the solvent, and reaction of the compound with chloramine T which yields a coloured product (merichinoid, with an absorp- tion maximum at 440 nm). When fifty milliliters of urine were used, the limit of detection was at about 20 ug/l; however, quantitative deter- mination was possible only above 100 ug/l, Modification of this method (Ader and Chrzaszczewska, I957) simplified somewhat the analytical procedure. leaving however the basic parameters unchanged. Sciarini and Mahew (I955) developed a rapid method recommended also by Gadaskina and Filov (1971), in which a smaller volume of urine (IS ml) is extracted with ethyl acetate; the colorimetric determination, however. is based on the same principle as in the previously described procedures. Again, the quantitative range for this method lies above 150 ug/l. A method based on the same principle suitable for lower concentrations was described recently by Piotrowski and coworkers (1971); 150 —250 ml of urine is continuously extracted with ether, benzidine is reextracted from the latter into a small volume of hydrochloric acid, and the colour reaction is performed with chloramine T. The resulting dye is extracted again with a small amount of chloroform and read at.445 nm. The limit of detection is about 2 ug/l with quan- titative determination possible above 6 ug ben- zidine per liter urine (precision t 9%). 82 The methods listed above are nonspecific in the sense that, like benzidine, the derivatives—cg. di— chlorobenzidine, dianisidine, tolidine—~will be de- termined if they are present. The limits of de- tection for all these compounds are similar. IDENTIFICATION AND DETER- MINATION OF INDIVIDUAL FREE DIAMINES USING PAPER CHROMATOGRAPHY Meigs et al. (1954) and Ader and Chrzaszczewska (1957) separated benzidine from its derivatives (o-tolidine, o-dichlorobenzidine, o-dianisidine) by means of paper chromatography. Partly evapo- -rated ether extract of urine was applied to the paper, developed with petroleum—ether, and the resulting patches stained using chloramine T. Ghetti et al. (1968) developed the chromatogram with a mixture of iso-butanol, acetic acid and water (3:l:l). Half of the strip was used for the determination of positions of the compounds by staining with p-(dimethylamino)benza1dehyde. The remaining half was cut in pieces, extracted, and the substances determined by diazotization and coupling with N—l—naphthylethylenediamine (absorption measurement of 575 nm). According to the authors, the limit of detection lies at about 5 II g ofdiamine per liter of urine. APPROXIMATE EVALUATION OF THE TOTAL CONTENT OF ARO- MATIC AMINES IN URINE Marhold (1953) described a method*, based on diazotization of amines and coupling with phenyl-7-acid directly in a sample of urine (50 ml). The resulting dye is absorbed on a piece of cotton fabric and, after drying, the colour is compared with the standards; the sample may be preserved as a lasting evidence of the level of the exposure. The method, as described, is nonspe- cific and of low sensitivity. The sensitivity was considerably increased by Ghetti et al. (1968) who performed the reaction in a small volume of the extract, obtained from the original volume of urine = 1.5 l. According to these authors, the sen- sitivity of this version of the method is to 6 ug/ ]. ' FIAT. 1313. vol. 4. page 400. JERZY K. PIOTROWSKI DETECTION AND DETER- MINATION OF BENZIDINE METAB- OLITES IN URINE For detection of individual benzidine metabolites in urine, Laham et al. (1970) applied paper chro- matography with subsequent staining with Ehrlich or Gibbs reagents. The method, as devel- oped, enables the detection in urine of benzidine, mono- and diacetylbenzidine, as well as of 4,4— diaminodipheny1—3-sulphate and its monoacetyl derivative. The acetyl derivatives of benzidine and 4,4—diaminodiphenyl—3—sulphate yield col- oured products after acid hydrolysis. Sciarini and Meigs (1958, 1961) proposed meth- ods for the quantitative determination of these substances, based upon two reactions: a) diazo- tization and coupling with N—l—naphthylethyl- enediamine (Bratton and Marshall, 1939) (the re- acting substances are: benzidine, 3— hydroxybenzidine, and its sulphuric ester), and b) reaction with nitrous acid (Burkhardt and Wood, 1929) (yellow colour) for the determination of 3— hydroxybenzidine. Both reactions are made di- rectly in a small volume (1—4 ml) of urine; the sensitivity seems too low for evaluation of indus- trial exposure to benzidine. The same authors proposed (1961) analyzing the urine of people ex- posed to significant amounts of benzidine by the above methods—however, after extraction of the metabolites from urine with ethyl acetate. ln this way the sensitivity was considerably improved, rendering determination of 3—hydroxybenzidine possible at concentrations of the order of 4—20 mg per liter of urine. OTHER METHODS USED IN PRO- PHYLACTIC EXAMINATIONS The basic hazard of benzidine is related to the in- creased incidence of urinary bladder cancer (see Case et al., 1954; Chwat, 1957). Thus, addi- tionally, or irrespective of the chemical evalu- ation of the magnitude of the exposure, biochem- ical and morphological tests are being applied for early detection of precancerous stages. Beta-gluc- uronidase activity levels in the urine were el- evated in the exposed people, particularly in those in whom urological changes were already found by cystoscopy (Popler et al., 1964; Kleinbauer et al., 1969). Urine may also be screened for the presence of cancerous cells, a sufficienty sensitive technique has been developed by Rofe (1957). BENZIDINE EXPOSURE TEST As yet, for the evaluation of exposure to ben- zidine, only detection of the free compound in the urine has been developed. Apart from the preliminary observations of Meigs et al. (1951), no experiments have been performed on volun- teers; and thus the interpretation of the benzidine concentrations in urine is mainly comparative. Attempts to assess the absolute amounts ab- sorbed are based on more or less speculative as- sumptions. The latter may be summarized as follows: (i) The maximum urinary excretion rate oc- curs about 2-3 hours after termination of the exposure, declining thereafter with a half time of 5 to 6 hours (Ader, 1957). This precludes significant systemic'cumulation of benzidine, and the levels found in urine may be legitimately ascribed to the absorp- tion on a given day. (ii) The concentration of benzidine in urine, collected directly after termination of the exposure (C), is related in a simple way to the daily amount excreted in 24 hrs urine (m) by a formula (Ader, 1957): m = 2C. (iii) From data of Meigs et al. (1951), as well as of Sciarini and Meigs (1961), it fol- lows that in man 4—10 per cent of the ab- sorbed benzidine is excreted unaltered in urine; by analogy to the canine excretory pattern, the Polish authors (Ader et al., 1958; Bolanowska et al., 1972) assumed an average value of 7 per cent. Quantitative assessment of the absorbed amounts has found application for the evaluation of the relative importance of the role of the various ab- sorption routes under industrial conditions. Com- parison of the evaluated absorbed amount in benzidine, based on urinary excretion, with that calculated from concentrations of the compound in air, provides relevant information on the role of dermal absorption. The maximum permissible absorption rate of benzidine and its maximum permissible concen- tration in the urine have not yet been defined. From the older literature it follows that in peri- ods, when urinary concentrations of the order of 100 [lg/1 and above were common, the incidence of the cancer of urinary bladder was appreciable (see Ader et al., 1958; Chwat, 1957). A drastic re- duction of exposure to benzidine in Poland has been accomplished only recently; and the follow- up period is too short to see the effect of this 83 new exposure, lower by an order of magnitude, upon the incidence of this occupational cancer. Tentative values of the maximum permissible uri— nary concentrations of benzidine resulted mainly from the sensitivity of the methods applied for its determination. ln Poland the earlier and contem- porary values applied were 20 and 2 ug/l, respectively. AVAILABLE DATA ON INDUSTRIAL EXPOSURE First reports on the magnitude of occupational exposure of workers to benzidine in manu- facturing plants were published in the fifties in the USA (Meigs et al., 1951, 1954) and in Poland (Ader et al., 1958); later data come from the same centers (Sciarini and Meigs, 1961; Bolanowska et al., 1972). The earlier data published in the fifties had been collected under conditions of relatively primitive production procedures with hygienic precautions not carefully observed. Urinary concentrations of benzidine (and its derivatives such as dichlo- robenzidine, tolidine, dianisidine, that had been determined jointly at a similar sensitivity of method as then applied) were high, of the order of 100 ug/l or more. Current interpretation would indicate a daily absorption rate in the or- der of several milligrams. At the same time, air concentrations of benzidine in the industrial premises reached the level of 0.09 mg/mJ air (Meigs et al.. 1951). If a more efficient method of benzidine collection from the air were applied, the concentrations appeared even higher, and in a Polish plant, as investigated by Ader et al. (1958), the mean concentrations clustered in the range form 0.15 to 0.4 mg/mJ. Calculations made by all the authors pointed to the dominant role of dermal absorption. Ader et al. (1958) esti- mated the dermal route contribution to the total absorption at 80 to 90 per cent. The urinary lev- els were higher in summer; moreover, the levels found in consecutive days of the week displayed a steep rising trend. The latter resulted from in- creasing contamination of clothes and of the skin (Fig. 14-1). Massive exposure of workers to ben- zidine continued in the USA later. Thus Sciarini and Meigs (1961) reported mean urinary concen- trations of 230 i 158 and 674 t 534 ug/l in the cool and warm season, respectively. The most re- cent data indicate an extremely high incidence of 400' 300' ”% F 1200 i O": :; too Non. Toes Nedn. Thurs. Fri Sat Fig. I4-l. The increase of benzidine concentrations in urine (II) compared with the ris- ing contamination of the skin (I) in subsequent days of the working week (Ader et al., (1958). Taken from: Ader D..,Piotrowski J., Zaremba Z.: Chemiczna ocena narazenia na benzydyne w przemysle. Medycyna Pracy 9, 207-217, 1958. Page 21 ], Fig. 2. JERZY K. PIOTROWSKI bladder cancer in workers manufacturing ben- zidine; on the other hand, concentrations of ben- zidine in urine (below 160 ug/l) may indicate some drop in actual exposure as compared with earlier data (Zavon et al., 1973). The necessary practical steps resulting from the picture presented above were taken in Poland in the late sixties. Improvements in manufacturing technology and the introduction of strictly con— trolled hygienic procedures led to a reduction in exposures estimated from all the indices by at least one order of magnitude: mean concen- trations in the air were down to 7-11 tig/m3, amounts in the working clothes to about 200 mg/mz, on the skin (chest) to 5-8 mg/mz, and urinary levels to 9 u g/l (Bolanowska et al., l972). lt should be also noted that at present in Poland people below the age of 40 are not allow- ed occupational contact with benzidine. For those above that age, the permissible period of exposure is 3 years. Considerable reduction of exposure to benzidine in a newly constructed manufacturing plant is born out in the reports of Czechoslovakian au- thors (Popler et al., 1964). These data, however, could not be easily compared with previously re- viewed reports due to the lack of data on concen- trations of benzidine in the urine of workers. In the laboratory of these authors (see also Kleinbauer et al., 1969) evaluation of exposure to benzidine was based upon concentrations of the compound in the air, on clothes and skin (these parameters are used by others mainly as supple- mentary data), and upon activity of beta-gluc- uronidase in urine. REFERENCES Ader D.: Chemiczna ocena narazenia na benzydyne w przemysle. Il. Wydalanie wol- nej benzydyny w moczu. (Chemical evaluation of industrial exposure to benzidine. Il. Free benzidine excretion in urine). Med. Pracy 8, 329-334, 1957. Ader D. and Chrzaszczewska A.: Oznaczanie benzydyny i jej pochodnych w moczu. (Determination of benzidine and its derivatives in urine). Med. Pracy 8, 15-14, 1957. Ader D., Piotrowski J ., and Zaremba Z.: Chemiczna ocena narazenia na benzydyne w przemysle. ll. Proba oceny wchłaniania benzydyny w warunkach przemyslowych. (Chemical evaluation of industrial exposure to benzidine. ll. Attempt to evaluate the absorption of benzidine in industrial conditions). Med. Pracy 9, 207-217, l958. Bolanowska W., Sa ma A., and Mogilnicka E.: Ocena aktualnej ekspozycji na ben- zydyne u robotni ow zatrudnionych w produkcji benzydyny. (Evaluation of the present exposure to benzidine). Med. Pracy 23, 129-138, 1972. Bratton A.C. and Marshall E.K.: New coupling component for sulfanilamide deter- mination. .l. Biol. Chem. 128, 537-550, 1939. BEN ZIDINE Burkhardt G.N. and Wood H.: Nitroarylsulfuric acids and their reduction products. J. Chem. Soc. 131, 141-152, 1929. Case R.A.M., Hosker M.E., Mc Donald D.B., and Pearson J.T.: Tumours of the uri- nary bladder in workmen engaged in the manufacture and use of certain dyestuff in- termediates in the British chemical industry. I. The role of aniline, benzidine. alpha- napthylamine and beta-naphthylamine. Brit. J. lndustr. Med. 11, 75-104, 1954. Chwat S.: Patogeneza i zwalczanie zawodowego raka pecherza moczowego. (Patho- genesis and prevention of occuprational vesical tumours). Med. Pracy 8, 159-180, 1957. Ghetti G., Bartalini E., Armeli G., and Prozzoli L.: Se aration and determination of aromatic amines (benzidine, o-tolidine, dianisidine, «lichlorobenzidine, alpha-naph- thyloamine, and beta-naphtyloamine) in various substrates. Calibration of new anal- ytical method for use in industrial hygiene laboratories. Lav. Um. 20, 389—400, 1968; (cited in Chem. Abstr. 72, 24552y, 1971. Glassman J.M. and Meigs J.W.: Benzidine (4,4—diaminobiophenyl) and substituted benzidines. A microchemical screening technique for estimation levels of industrial exposurefrom urine and air samples. Arch. Ind. Hyg. Occup. Med. 4, 519-532, 1951. Kleinbauer V., Kunor V., Popler A., and Vlasak R.: Sledovani expozice zamestnancu pri vyrobe benzidinu. (Observation of personnel exposure in benzidine production). Ceskosl. Hyg. 14, 150-159, 1969. Laham S.. Farant J., and Potvin M.: Biochemical determination of urinary bladder carcinogens in human urine. Occup. Health Review 21, 14—20, 1970. Marhold J.: Orientacni methoda k stanoveni diazovatelnych latek v moci. (A screen- ing methodfor the diazotizable amines in urine). Prac. Lek. 5, 348-350, 1953. Meigs J.M., Brown R.M., and Sciarini L.J.: A study of exposure to benzidine and sugstituted benzidines in a chemical plant. Arch. Ind. Hyg. Occup. Med. 4, 533-540, 19 I. Meigs J.M., Sciarini L.J., and Van Sandt W.A.: Skin penetration by diamines of the benzidine ggroup. Arch. Ind. Hyg. Occup. Med. 9, 122-132, 1954. Piotrowski J.K., Bolanowska W., and Sapota A.: Oznaczanie niskich stezen ben- zydyny w moczu. (The determination of low benzidine concentrations in urine). Med. Pracy 22, 307-131, 1971. Popler A.. Selucky M., and Vlasak R.: Sledovani exposice zamestnancu ve vyrobe benzidinu. (Follow up of exposure in people working the production of benzidine). Prac. Lek. 16, 147-152, 1964. Rofe P.: A routine method for the preparation of the cells in urine. Brit. J. lndustr. Med. 14, 164-167, 1957. Sciarini L.J. and Mahew J.A.: A rapid technique for estimating benzidines in indus- trial exposure. AMA Arch. Ind. Health 11, 420—424, 1955. Sciarini LJ. and Meigs J.W.: The biotransformation of benzidine (4,4—di- tsttznlirgggtphgesng'l) an industrial carcinogen, in the dog (I). AMA Arch. Ind. Health 18, ~ , I . Sciarini L.H. and Meigs J.W.: The biotransformation of benzidine. Il. Studies in mouse and man. Arch. Environm. Health 2, 423-430, 1961. Zavon M.R., Hoegg U., and Bingham E.: Benzidine exposure as a cause of bladder cancer. Arch. Environ. Health 27, 1-7, 1973. 85 15. TRICHLOROETHYLENE ABSORPTION Under industrial conditions the respiratory tract forms the main route for trichloroethylene (TRI) absorption. The retention of the TRI vapours measured under similar circumstances amounted on the average to about 70 per cent (Grandjean et al., 1955). Studying the retention in controlled experiments. the Czech authors found the re- tained percentage varied between 58 and 70 (mean of 64, Soucek and Vlachova, 1960) or 51 to 64 (mean 58, Bartonicek, 1962). At variance with the above data are results obtained by the Japanese investigators (Nomiyama and Nomi- yama, 1971) who found a rather low retention of about 36 per cent. Skin absorption of tri- chloroethylene is probably of minor importance (Frant and Westendorp, 1950; Smith, 1966), al- though it may take place when direct con- tamination of the skin occurs (Stewart and Dodd, I964). It appears that the absorption of cucn : ccn2 CH Cl _ COOH Ck 2 _ . cą- cnc: Monochlovoocehcocnd * cc13- cno CCI3— CH(OH)2 cc13— cuz -OH CCIS- coon 1 TCA ech-cng— o -C6H906 nc:3 Urochloric acid Chlo'otorm Fig. I5-I . Metabo/ic pathways of tri- chloroelhylene (Williams, 1959). 86 the vapours through the skin has not been stud- ied in controlled conditions. METABOLISM The absorbed trichloroethylene is rather uni- formly distributed in various tissues. However, the earlier data reviewed by Gadaskina and Filov (1971) indicated that the lowest concentrations are encountered in muscle and the highest ones in adipose tissue. According to Soucek and Vlachova (1960) and Bartonicek (1962) some 70-80 per cent of system- ic trichloroethylene is metabolised in man. The principal metabolites are: trichloroacetic acid (TCA) and trichloroethanol (TCE). TCE is ex- creted in urine as glucuronide-urochloralic acid. Smaller amounts of monochloroacetic acid (MCA) (Soucek and Vlachova, 1954) and chloro- form (the same authors, 1955) are also found. Based upon earlier studies, Gadaskina and Filov (1971) proposed a scheme of metabolic pathways for trichloroethylene as presented in Figure 15-1. The metabolites of the main pathway (tri- chloroethylene epoxide, trichloroacetic aldehyde, and chloral hydrate) are partially hypothetic. lt was only in recent studies that Kimmerle and Eben (l973a) were able to detect chloral hydrate in the blood of rats exposed to trichloroethylene; this observation, however, could not be con- firmed in a human experiment (Kimmerle and Eben, I973). The presumption that two principal metabolites (TCA, TCE) are direct products of a common precursor (chloral hydrate) lacks direct experimental support. In man the most abundant metabolic product is TCE (about 50%), then TCA (about 19%), and MCA (about 4%) (Soucek and Vlachova, 1960). The ratio of the two principal metabolites, TCE/ TCA, disputed at length in the literature, varied in individual reports from 2.6 (Soucek and Vlachova, 1960) through 2.1 (Bardodej and Krivucova, I958) to 1.4 (Barbonicek, I962). It seems that the principal source of discrepancy lies in the fact that frequently the ratio has been based on the concentrations of the metabolites TRICHLOROETHYLENE found in spot urine samples. This procedure seems to be inadequate because of the pro- nounced differences in the kinetics of excretion of TCE and TCA, leading to considerable vari- ations of the ratio with time after cessation of the exposure. The ratio seems to be higher in males than in females (Nomiyama and Nomiyama, 1971). Relatively copious data have been accumulated on the systemic behaviour of trichloroacetic acid. In guinea pigs, tissue distribution of this metabo— lite is relatively uniform with highest concen- trations in the adrenals, spleen, and testes. Blood has been most extensively studied; however, the concentrations of TCA are not high relative to other tissues (Fabre and Truhant, 1952). Most of the blood TCA is found in the plasma (Soucek, 1955). ln human beings the concentration ratio of plasma/red cells amounted to 4.8 (Bartonicek, I962). It is a commonly accepted view that in plasma the compound is protein bound. Whether administered directly into the blood stream in the form of a sodium salt or originating metabolic- ally from TRI, TCA shows the same systemic ef- fect (Smith, I966). The biochemistry of TRI metabolism has not been fully clarified. Smith, (1966), relying on ear- lier studies of Butler (I949), expressed the view that the formation of TCA is not limited to one organ only. Fabre and Truhant (1952) found in their study on rats that the highest metabolic ac- tivity is displayed by the lungs and spleen. In dogs and rats, the formation of TCA is consid- erably diminished by the administration of tet- raethyl thiuram disulphide (TETD) (Forssman et al., I955). In humans, TETD inhibits the'for- mation of both TCA and TCE, with TCE be- ing least affected (Bartonicek and Teisinger, I963). lntraveous administration of glucose and insulin enhances the formation of all TRI metab- olites (Soucek and Vlachova, 1960). These facts point to a similarity of TRI metabolism with that of ethanol. It was found that alcohol dehy- drogenase catalyzes one of the postulated meta- bolic steps, the transformation of chloral hydrate into TCE (Friedman and Cooper, 1960). The transformation of TRI into the former and of chloral hydrate to TCE (in presence of NADH) or to TCA (in presence of NAD), could be ac- complished in post mitochondrial supernatants of hamster and rat liver. The Michaelis constants Km are: IO'ZM, 7 x IO'3M; and 6 x 10'4M for the transformations of TRI + chloral hydrate, chlo- ral hydrate —> TCA; and chloral hydrate —> TCE, respectively (lkeda and lmmamura, 1973). It was also reported that the transformation rate of TRI 87 can be enhanced by administration of pheno- barbital, a well known inductor of microsomal enzymes (Leibman and Mac Allister, 1967). The values of Km did not change; only the maximal rate of reaction, as measured in vitro, was in- creased. Repeated exposure of animals to low TRI concentrations did not stimulate the metab- olism; however, the stimulatory effect was seen after massive exposure (lkeda and lmmamura, 1973). The biotransformation of trichloroethylene is partly inhibited by the presence of toluene (lkeda, I974). ELIMINATION Reviewing the early studies by Czech authors, Teisinger and coworkers (1956) concluded that, in human beings, after cessation of a single ex- posure to TRI vapours, its metabolites are ex- creted in the following fashion: unaltered TRI mainly in the expired air (19%) and only traces in urine (up to 0.6%); TCA, TCE and MCA leave the body in the urine in amounts of about 16, 35 and 3-6 per cent of the retained amount, respectively. With considerable delay chloroform appears in the exhaled air, in varying amounts, reaching in some cases 15 per cent of the TRI on a molar basis. This efficiency of elimination dis- plays wide variation, and thus the numerical val- ues reported from various laboratories differed considerably. Due to prolonged excretion of TRI metabolites and the varying time of observation, the reported values are not always directly com- parable. Bartonicek (1962) concluded that other routes of elimination of TRI metabolites (e.g. faeces, sweat, saliva) are of secondary importance. The kinetics of elimination of TRI on one hand, and of its metabolites on the other, show basic differences. Unchanged TRI is eliminated directly in exhaled air after discontinuation of exposure, and its concentrations drop fast with time. From the data of Stewart et al. (1962, 1970), it follows that the disappearance rate of TRI from the ex- haled air varies with the duration of exposure, the rate being inversely correlated with the du- ration. The elimination curves are multi- exponential, and therefore the half-time, as deter— mined, depends upon the interval when the measurements are made: over the first two hours after cessation of an‘exposure of 1 to 4 hours, the half-time is of the order of 0.5 hour; after longer exposures and determined later, it in- creases up to 15 to 20 hours. Up to 2.5 to 5 hours after discontinuation of exposure, other authors reported curves with two exponential terms. The half-time at the beginning of obser- vation was of the order of 0.5 hour, increasing to 1 to 2 hours toward the end of the period (Klyin et al., 1967; Nomiyama and Nomiyama, 1971; Kimmerle and Eben, 1973 b). TRI metabolites excreted in the urine reach their maxima at different times after discontinuation of the exposure. The kinetics of their disap- pearance vary considerably with the metabolite. The concentration of monochloroacetic acid reaches a maximum directly after cessation of TRl inhalation and decays relatively fast; the half-time amounts to about 15 hours. The max- imal concentrations of trichloroethanol are ob- served several hours later and their decay is bi- phasic: at first a half-time of 24 hours predominates, later a half-time of 40 hours is ob- served. Trichloroacetic acid reaches a maximum only after I to 2 days, and the biphasic decay fol- lows with half-times of 50 and 70 hours (Soucek and Vlachova, 1960). Similarly lkeda et al. (1971) observed kinetics of elimination of the metabo— lites which may be characterized with the follow- ing two half-times of urinary concentrations: TCE = 6 and 40 hours; TCA = 25 and 85 hours. The data reported by other authors seem basic- ally consistent with the above picture (Bar- tonicek. 1962; Stewart et al. I970). The kinetics of elimination of TRI and its metabolites is sche- matically presented in Figure 15-2; a multi-com- partment model has been also presented that lilzII--IEI+ anni—I 0 .. O L c .9 .. O L u x m ___ -._ \‘\_ ~._____ ~ . "'." 4 5 Fig. [5-2. Schematic presentation of the kinetics of excretion of trichloroethylene and its metabolites. following single exposure. Lower diagram: A — trichloroethylene in expired air, B — MCA, C— TCE, D — TCA, allin urine. Up- per scheme — the corresponding model, leading to excretion of. trichloroethylene (I), MCA (V), TCE (VI). TCA (VII) and chloroform (VIII). JERZY K. PIOTROWSKI could form a basis for a mathematical descrip- tion of the metabolic kinetics of TRI. As a mat- ter of fact, the complexity of the model may be advocated to explain why the elimination pro- cesses have never been described as a whole, and individual authors have limited their description only to the presentation of the half-times. Re- gardless of its basic relation to material truth (ex- istence of two intermediate metabolic precursors of TCE and TCA is assumed), the model could be solved only with the use of a computer. Approximate analysis of the kinetics of elimi- nation of TRl metabolites, as postulated by the model, makes it possible to predict some phe- nomena. For instance, after a single exposure the ratio of excretion rates of the two principal me- tabolites, TCE/TCA, should be a steeply de- creasing function of time. In fact, Nomiyama and Nomiyama (1971) found that the ratio declined from about 30. directly after exposure, to a value of 0.2 several days later. Applying a simple principle of graphic sum- mation (as proposed by Soucek and Pavelkova, I953). it may be predicted that, after daily re- peated exposures, the level of TRI metabolites in biological materials will be the greater 1) the longer the time required, after a single exposure, for the maximum concentration to appear in the material; and 2) the slower is the rate of decay of that concentration. Experimental data indicate that in man, trichloroethylene itself is not subject to significant cumulation (Stewart et al., 1970; Kimmerle and Eben, 1973). The theoretical con- siderations of lkeda and lmmamura (1973) pos- tulated a 2 weeks' period of repetitive exposures for reaching a steady-state; this seems exagger- ated due to assumed arbitrary values taken for the computation. A significant cumulation is seen for TCE and TCA. Of Course, this must hold whether the predictions are based on urinary or blood concentrations. As might have been pre- dicted, the concentration ratio TCE/TCA de- creases gradually with time. The experimentally determined growth of TCE urinary concentration over 5 consecutive ex- posure days was not appreciable: about 60 per cent relative to day l, and a steady-state was reached after 3 days. TCA concentrations in the urine rose almost linearly with the duration of exposure, reaching a level 7—12 times higher than those on the first days; no steady-state was reach- ed after 5 days. In the same period the TCE/ TCA concentration ratio decreased from about ID to 2 (Ertle et al., 1972; Fig. 15-3). A similar picture was seen by Muller and cow0rkers (1972), TRICHLOROETHYLENE who measured the concentrations in blood (Fig. 15-4). The difference in excretory kinetics of the two metabolites, TCE/TCA ratio may be used to evaluate the character of exposure and the assess- ment of the time elapsed between cessation of the exposure and sampling of the urine. Under con- ditions of repetitive exposure the steady-state is represented by a TCE/TCA ratio of l.S—3.0. Val- ues substantially higher represent early periods after single exposures; values of the ratio below 1 are obtained several days after discontinuation of the exposure. Apart from this potential use- fulness, the differences in the elimination kinetics of TCE and TCA complicate the picture signifi- cantly. However, it is interesting to note that the problem may be considerably simplified if only a sum of all trichlorocompounds in the urine is de- termined. From the data of Nomiyania (1971, Fig. 15—5), it follows that after a single exposure the excretion rate of trichloroderivatives in the urine declines monoexponentially with a half- time of about 1 day. On the other hand, lkeda et al. (1971) reported that, after a single inhalatory exposure. the maximum excretion rate of total "'9 m Soupmlshjdays ”9 ”Tri 250/5ppmrsh5aoys 200 ?. E :1“) E . < 0 ›- . e f |— "9 . Em Tn I00ppm/6h,5days ‘o' I- 200 100 O ZL 1-8 72 96 120 l“ 168 hrs Fig. [5-3. Urinary excretion of triehloroethanol and trichloroacetic acid in daily intervals (mg/24 hr)following repeated exposure on 5 consecutive days (Ertle et al., 0972). Taken from: Ertle T., Henschler D., Muller G., and Spassovski M.: Metabolism of tri- chloroethylene in man. Arch. Toxikol. 29, 171- 188, 1972. Page. I79, Fig. 2. Fig. [5-4. The increasing trends of TCA in plasma (upper graph) and TC E in whole blood (lower graph) in volunteers exposed to trichloroethylene for 5 consecutive days (Muller et al., 1972). Taken from: Muller G., Spassovski M., and Henschler D.: Trichloroelhylene exposure and tri- t-hloroerhylene metabolites in urine and blood. Arch. Toxikol. 29, 335-340, 1972, Page 337, Fig. l. trichlorocompounds appeared 2 to 4 hours after discontinuation of inhalation; over the first 24 hours the concentration declined with a half-time of about 8 hours. The same authors, however, observed a patient hospitalized several days after intoxication; the decline of excretion of total tri- chlorocompounds could be characterized by a half-time of 3 to 4 days. Ikeda and lmmamura (1973), summarizing the data of various authors, concluded that the'half- time of total trichlorocompounds in urine, when studied on volunteers under controlled condi- tions, was bracketted by the values 3| to 50 hours. The sameparameter, when studied on workers under industrial conditions, fell in the range of 26 to 50 hours. The mean for both groups was close to 40 hours. METHODS FOR DETERMINATION OF TRI AND ITS METABOLITES Trichloroethylene. ln earlier studies TRl was de- termined in expired air and in biological materi- mg! 12 hrs ... 8 8 ... O .- V‘ I . Urinary ucretion ol Total Trichloro-Compounds . › . - . . 0 l 2 3 4 5 o Days after Trichloroethylene Exposure Fig. 15-5. Urinary excretion of total trichloro- compoundsjollowing single short exposure to Iri- ('hloroelhylene (Nomiyama, I 971). Taken from: Nomiyama K.: Estimation of tri- chloroelhylene exposure by biological materials. Int. Arch. Arbetismed. 27, 281-292, 1971, Page 285, Fig. 2. als (blood, urine) after distillation or aeration, followed by absorption in pyridine, with sub- sequent color reaction of Fujiwara. The earlier methods have been reviewed by Smith (1966). Teisinger et al. (1956) recommended the method as presented by Soucek and Franklova (1952). Ehmer-Samuel (1963) proposed a‘determination in blood by extraction with isooctane, with sub- sequent gas chromatographic measurement (ni- trogen, electron capture detector). Gas chro- matography in different modifications has been used in more recent reports (Kylin et al., 1967; Ertle et al., 1972; Stewart et al., 1962, 1964, 1970). Stewart et al. also used infrared spec- trophotometry ( A = 11.78 # in gas-cuvettes of 10 m light-path length). Trichloroacetic acid. The usually applied col- orimetric method makes use of Fujiwara’s colour reaction, performed by heating the alkaline pyri- dine extract. Teisinger et al. (1956) recommended the method in the version of Soucek and Frank- lova (1952). The same, or a very similar mod- JERZY K. PIOTROWSKI ification, has been used by Bardodej and Vyskocil (1956), Soucek and Vlachova (1960), Bartonicek (1962), Kundig and Hogger (1970), and Laham ( 1970). ln the most recent studies the same method was used in the modification of Tanaka and 1keda (1968) (1keda et al., 1971; Er- tle et al., 1972; Kimmerle and Eben, 1973). Ehmer-Samuel et al. (1973) recently proposed a gas—chromatogrphic method for TCA deter- mination in urine. A toluene extract of urine containing TCA is methylated using boron- trifluoride-methanol reagent, and TCA methyl es- ter is then determined with a Varian gas chro- matograph electron capture detector. The method is suitable for serial determinations, and its level of detection has been evaluated at about 3 # g/ m1. Trichloroethanol. ln most studies a modified method has been used: TCE present in urine in the conjugated form with glucuronic acid is hy- drolyzed by prolonged heating with a strong acid in a sealed glass ampoule. Trichloroacetic acid is then removed from the solution on an ionic ex- changer and TCE is oxidized to TCA by means of dichromate and then determined. A version of this procedure as described by Vlachova (1957) has been recommended by Teisinger et al. (1956), Dutkiewicz et al. (1964), and by Gadaskina and Filov (1971). Bardodej (1962) proposed a method according to which TCE is steam-distilled and determined col- orimetrically (240 nm.), without oxidation, after reaction with pyridine and alkali. For tissue as- says Cabana and Gessner (1967), and for urine Ogata et al. (1970), conducted the reaction with alkali and pyridine without separation of the me- tabolites. TCE and'TCA concentrations are cal- culated after measurements at two wave-lengths (440 nm and 530 nm). Vlachova (1956), Seto and Schultz (1956), and Tanaka and 1keda (1968) conducted two parallel determinations: TCA di- rectly and a sum of trichlorocompounds after hy- drolysis and oxidation. TCE was calculated from the difference. Sedives and Flek (1969) proposed TCE deter- mination in urine by means of gas chro- matography with flame ionization detection. Eben developed a method which was applied lat- er by Ertle et al. (1972) in which TCE is deter- mined using gas chromatography with an electron capture detector. For acid hydrolysis of uro— chloralic acid several authors (Ogata et al., 1970; Ertle et al., 1972; Kimmerle and Eben, 1973) sub- stituted enzymatic hydrolysis using - glucuronidase. TRICHLOROETHYLENE Total trichlorocompounds (TTC). The principle of this method for TTC consists of hydrolyzing the sample, with subsequent oxidation and utili- zation of the Fujiwara reaction as described above for trichloroethanol. Recently, lmamura and lkeda (1973) proposed a modification of the method for rapid determination: a sample of urine is heated with CrO3 in HNOJ, alkalised af- ter cooling, and pyridine added. Short heating in a boiling water bath then follows, with extinction measurement at 530 nm. EXPOSURE TESTS Introductory remarks. It seems that a practical importance should be ascribed to the following determinations: l) TRI in expired air, and also 2) TCE, 3) TCA and 4) TTC in urine. The concen— tration of TRl itself in exhaled air may provide information on the magnitude of exposure on the day of sampling; urinary TCE should give an evaluation of the mean exposure over the last 2-3 days; and finally, TCA and TTC in urine will provide information about the averaged exposure in the period of preceeding week or more. Al- though the data regarding the kinetics of TRI elimination in the exhaled air are not fully con- sistent, it seems that those obtained in controlled human experiments (single exposure) may serve as a basis for interpretation. When urinary TCE is considered, the data obtained by those authors who had carried out experimental inhalations studies on human volunteers, over several con- secutive days until the steady-state was reached, may be utilized. For TCA and TTC in urine, it should be noted that the exposures have not been continued for a sufficiently long period in any of the experiments. In this case the urinary levels re- flecting steady-state may be obtained from field studies (bearing in mind the lack of precision in assessing the real concentrations to which work- ers have been exposed over preceeding period of some 10-20 days), or by means of calculation from kinetic extrapolation. A basis for the latter was created for TCA in urine by Soucek and Pavelkova (1953). Finally for TTC, due to relatively simple kinetics of its excretion, the sum of TCA + TCE data for the steady-state may be computed from kinetic equations, assuming an excretion half-time of about 40 hours. TRI in expired air. For evaluation of exposure, determination of TRI in expired air sampled af- ter inhalation has been recommended by Boettner and Muranko (1969), Stewart et al. 91 (1970), Nomiyama (1971), and by Pfaffli and Backman (1972). All these authors have applied gas chromatography for analysis of the air sam- pled into Saran bags. The proposal regarding in- terpretation of the results put forward by Nomi- yama (1971) and its rationale, as understood by the present author, may be presented as follows. In its simplest form, the concentration of TRI in expired air, as a function of time after discon- tinuation of exposure, may be presented by a curve as in Figure 15-6. Immediately after cessa- tion of TRl inhalation, the concentration forms a given fraction of that in the inspired air during exposure and decays relatively fast. After a short time this rapid elimination ceases and then fol- lows a period of slower elimination with concen- trations following an exponential function. If the intercept of this second exponential at t = 0 is denoted as “‘b', and the concentration of the inhaled TRI as “a", and assuming b:a = constant, we obtain: b _ Cet TC. e k‘ (I) The purpose of the assay is to evaluate the con- centration .Ci from Cet- After transformation of equation (I) one obtains: i %Cet ekt (2) IogC E xposun Fig. [5-6. The principle under/dying evaluation of exposure to Iriehloroethylene from its level in expired air, in the procedure of Nomiyama (I970). 92 According to recalculated data of Nomiyama (I971), a/b = 12.5 and k = 0.3 h-l. ' The infomation assembled by Nomiyama on the comparison of the calculated values of Ci (equa- I0,000: Ą—A POWELL , |945 EA—A soucex,1952 . o—o BARTONICEKJQGZ 4’000- o—o STEWART, r970 3°00, O_O KYLIN, 1967 I o—e NOMIYAMA, 1971 2,000- I,000 , 2‘ 500_ I” % soo ," ‘—’ 200 U U l00 300 600 ION 2000 99m Fig. 15-7. Evaluation of the Nomiyarna's method of exposure evaluation from expired air analysis by comparison of respective data of various authors. . 500_ o---o sruov sms „, o—o nas? DAY sxposune _- — z ' '- . :5 , . ' \ 400' , ' O ' E 0’ . 3 _ ,' w 00 ,' e ,' . I :~ 200- „X O : I O .: 1’ : |ool- I I I A I I 1 V 50 100 I50 200 TRI in air , ppm Fig. [5-8. Urinary excretion of trichloroethanol following one day and 3-5 days exposure, as dependent on the concentration of tri- chloroelhylene in the air. Data of various authors standardized for 7—hrs exposure duration. JERZY K. PIOTROWSKI tion 2) with those directly determined are pre- sented in logarithmic coordinates in Figure 15-7. From the graph it follows that: i) existing data cover only the range of high concentrations above l00 ppm; ii) the method ‘yields calculated values of Ci with an uncertainly factor of 4 (Le. the concentration as determined may actually lie in the range from 100 to 400 ppm). To improve the interpretation of this exposure test will require more experimental studies. It would be desirable to refrain from too much so- phistication in designing th experiments, because exhalation of TRl must be a composite function of many variables. For practical purposes experi- ments will suffice in which a wide range of low TRI concentrations in the air are used, with con- stant duration of exposure (e.g., 7 hours cor- responding to the effective duration of an indus- trial shift) and constant sampling time, sufficiently early (for instance 1 hour after the end of shift) to render the test feasible without retaining a worker for too long in the factory af- ter work. Final interpretation could be worked out directly from empirical data. Trichloroethanol in urine. Several authors have recently conducted experiments on TCE excre- tion in urine in subjects exposed daily until a steady-state in the excretion rate was reached (Stewart et al., 1970; Ertle et al., 1972; Muller et al., I972; Kimmerle and Eben, 1973). TCE has been determined in 24-hours samples of urine, but this is not practical under industrial condi- tions. In the experiments reviewed here, the du- ration of daily exposure varied from 4 to 7 hours. The data were normalized for 7 hours of exposure assuming this is the effective duration of exposure during a normal 8 hours working shift. All the data reported in the papers cited above consistently indicate that TCE rises through the 5th day of exposure, tapering off thereafter. To calculate the steady-state values, the means of the excretion rate were determined on days 3 through 5. The results are presented in Figure l5-8. It appears that the relation between the magnitude of exposure and the daily urinary excretion of TCE shows a satisfactory precision. The mean of all individual relations may be rec- ommended for practical purposes. It should be noted that the relationship (exposure — excre- tion) seems to be curvilinear; this may point to the fact that the proportion of TCE in all TRI metabolites diminishes with increasing absorbed dose. The data of Muller et al. (1972) on the TCE lev- els in the blood (Fig. 15—4) suggest a rather fast TRICHLOROETHYLENE increase and decay of TCE in blood, related to the rhythm of exposure. lt should be expected, therefore, that similar fluctuations might be seen in the urine. A given daily urinary excretion of TCE might be accompanied by considerable vari- ation of the concentrations in individual fractions .of urine, the maximum occuring directly after discontinuation of the exposure and the mini- mum in the morning of the following day. Ogata et al. (1971) found that at the end of exposure to concentrations of 100 ppm of TRI in air, the ex- cretion concentration of TCE in urine was 530 mg/l. Trichloroacetic acid in urine. Attempts to use TCA in urine as a measure of exposure to TRI were made in the late 1940's utilizing empirical correlations between TR1 concentration in air on the one hand, and urinary TCA on the other. These early studies Were reviewed by Bardodej and Vyskocil (1956), Teisinger et al. (1956), and Dutkiewicz et al. (1964). It is accepted that both variables are linked by an approximately linear correlation, represented by a line on a graph (Fig. 15-9) as proposed by Bardodej and Krivucova (1955). Assuming linear propor- tionality, a concentration of TRI in the air of 0.4 mg/l should be followed by mean concentrations of TCA in urine of: 140 mg/l (Bardodej and Krivucova, 1955), 150 mg/l (Frant and West- endorp, 1950), and 220 mg/l (Friberg et al., 1953; Grandjean et al., 1955). Interpretation of the test, as accepted in practice, is usually based upon the data of Bardodej and Krivucova, (1955). The above data, obtained from field studies, have not been verified in controlled experimental stud- ies. Stewart et al. (1970), Ertle et al. (1972) and Kirnmerle and Eben (1973) have studied TCA ex- cretion in the urine of volunteers exposed for 5 consecutive days. The results (Fig. 15-3) indicated a linear progression of the concentrations with duration of the studies. It should be expected, therefore, that steady-state values must be appre- ciably higher. Nonetheless, by the 5th day of dai- ly exposure, the authors found high urinary lev- els of TCA. Normalizing the data in such a way that they corresponded to a 7 hour exposure to TR1 at 0.4 mg/ l, and assuming a mean daily vol- ume of urine of 1.2 1., the resulting TCA concen- trations would be directly comparable with those obtained from the field studies. The data were: according to Stewart et al. 130 mg/l; according to Ertle et al., in a different series, from 160 to 190 mg/l. ln general, the TCA concentrations ex- ceeded those reported by Bardodej and Krivucova (1955) even if the steady-state had not been reached. The problem certainly calls for fur— 93 160 CC13 COOH in urine,mg/I 201- tl 1 1 l 0.05 0.2 0.4 CClZICHCI in oir, mg/l Fig. I5-9. TCA concentration in. urine as dependent on the trichloroethylene concentration in air ( Bardodej and Krivucova, 1955). ther experimental investigations. At the same time it should be mentioned that urinary concen- trations, as seen in field studies, may be either higher than in a laboratory experiment due to greater lung ventilation, or IOWer as a result of shorter effective real exposure as compared with the product of official working time and average concentration at a given work site. The com- parison as presented above might be interpreted as suggesting that these differences in actual ex- posure could have led to the discrepancy. On the other hand, too little is still known about possi- ble 'changes in the metabolism of TRI in the course of prolonged exposure to exclude the pos- sibility of lowered efficiency with which TCA is produced in the body, as was suggested by Bard- odej and Krivucova in 1955 and supported recen- tly by lkeda et al. (1972). It should also be recollected that Grandjean et al. (1955) pointed to a phenomenon that the ratio U/A (U = TCA concentration in urine, mg/l; A = TRI in the air, ppm) is related to age, varying from the value of 6 in young subjects to only 2 in elderly people. A sex effect on TRI metabolization to TCA also appears to exist, as the ratio TCE/TCA, was higher by a factor of 2 in males (Nomiyama and Nomiyama, 1971). Assuming that reasons for the discussed discrepancy should be sought in vari- ation of TRl metabolism in the body (variation of proportions of TCE and TCA in the total 94 pool of metabolites), a more universal approach to the problem would consist of a shift to deter- mination of the sum of trichlorocompounds, as suggested by Nomiyama and Nomiyama (1971). TOTAL TRICHLOROCOMPOUNDS (TTC) The sum represents the total of TCA and TCE, and thus the bulk of TRI metabolites in urine. The metabolic yield of TTC does not depend on sex, and the kinetics of excretion may be expressed by a single exponential equation (Nomiyama and Nomiyama, 1971). Utilizing TTC for the evalu- ation of exposure to TRI has been suggested by numerous authors, namely: Bardodej and Krivucova (1958), Medek (1958), Tanaka and lkeda (1968), and Nomiyama (1971). Inter- pretation of the test is based on the U/ A ratio being equal to 6 (U = TTC in urine; mg/l; A = TRI in the air, ppm). Due to the simple charac- teristics of the excretion, the results may be easily interpreted even if the determinations are made several days after cessation of the exposure, as~ suming an average half-time of TTC excretion of 40 hours (see “Elimination”). AVAILABLE DATA ON INDUSTRIAL EXPOSURE There are voluminous data on industrial ex- posure, obtained utilizing the exposure tests, mainly of TCA excretion. Apart from the papers cited above there should be included the reports of: Ahlmark and Forssman (1961), Waelschova (1954), Teisinger et al. (l95_5),_Hickish et al. JERZY K. PIOTROWSKI (1956), Medek (1958), Sukhotina (1969), Kundig and Hogger (1970), Ikeda et al. (1972), Rosowski et al. (1974). The infomation relates to the ex- posure in the course of TRl production, and var- ious applications of the substance, mainly in dry cleaning and degreasing of metals. Concen-- trations or daily excreted amounts of TCA in urine have encompassed a wide range of values, from those close to zero up to 300—400 mg/ l; and in earlier studies even up to 700-1000 mg/l. One" of the problems very often discussed in ear- lier studies was the evaluation of TCA deter- minations in urine for the diagnosis of chronic TRI intoxications. From the onset opinions were divided. Frant and Westendorp (1950) were un- able to find a significant correlation between TCA level and intensity of the intoxication symp- toms. However, it has been claimed that in most workers exposed for a prolonged period to TRI and displaying urinary concentrations of TCA above 150 mg/l, symptoms of chronic poisoning were present. Frequency of the symptoms were significantly increased at TCA concentration above 20 mg/l, a value which has been recom- mended as safe. It is interesting to note that this value corresponds to the air concentration of TRI = 0.05 mg/l, which is accepted as the max- imum acceptable one in the Soviet Union. Although at present the use of urinary TCA as an individual diagnostic testis not being dis- cussed, the accumulated data proved that TRI exposure, reflected by urinary TCA excretion at concentrations of more than 100 mg/l, may lead after prolonged exposure to symptoms accepted at present as signs of deleterious action of TRI on the human organism (Bardodej and Byskocil; 1956; Grandjean et al., 1955; Waelschowa, 1954; Ertle et al., 1972). REFERENCES Ahlmark A. and Forssman S.: Evaluating triehloroethylene exposures by urine- analysis/br lrichloroacelic acid. Arch. Ind. Hyg. Occup. Med. 3, 386-398. 1951. Bardodej Z.: Hodnota a pouziti expozit-m'eh testu. VIII. Test tekaveho organic/who ('h/oridu v mot-i. (Value and use of exposure test. VIII. Volatile chloride test in the urine). Cs. Hyg. 7, 234-239, I962. 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Hawai H., and Kuniyoski M.: Excretion kinetics of urinary tznetabglites in a patient addicted to trichloroethylene. Brit. J. lndustr. Med. 28, 203- 06. 1 I. Immamura T. and lkeda M.: A time-saving procedure for the determination of total igg'hloro—compounds in human urine samples. lnt. Arch. Arbeitsmed. 3|, 333-338, 3. Kimmerle G. and Eben A.: (a) Metabolism, excretion and toxicology of tri- chloroethylene after inhalation and toxicology o trichloroethylene after inhalation. l. Experitnental exposure on rats. Arch. Toxikol. , 115-126, 1973. Kimmerle G. and Eben A.:, (b) Metabolism, excretion and toxicology of tri- tl'gśoitścśthylerśe after inhalation. ll. Experimental human exposure. Arch. Toxikol. 30, - . 197 . JERZY K. PIOTROWSKI Kundig S. and Ho er D.: The importance of the determination o tri- and per- chlorethylene metabo ites in the urine. Int. Arch. Arheltsrned. 26, 306- 15, I970. Kylin B., Axell K., Samule H.E., and Lindborg A.: Effect of inhaled tri- chloroethylene on the CNS. Arch. Environ. Health 15, 48-52, 1967. 197%", S.: Studies in placental transfer: Trichloroethylene. Industr. Med. 39, 46-49, Leibman K.C. and Me Allister W.J.: Metabolism of trichloroethylene in liver micro- somes. Ill. Induction of the enzymic activity and its effect on excretion of metabo- lites. J. mamami. Exp. Ther. 157, 574-580, 1967. Medek V.: Vztah trichlorethanolu a kyseliny trichloroctove v maci k trichlorethylenu v ovzdust‘. ziskany merenim w terennych podminkach. (The relationship between tri- chloroethanol and trichloroacetic acid in the urine and trichloroethylene in the atmo- sphere. as ascertained by field work). Prue. Lek. 10, 135-138, 1958. Muller G., Spassovski M., and Henschler D.: Trichloroethylene exposure and tri- chloroethylene metabolites in urine and blood. Arch. Toxikol. 29, 335-340, 1972. Nomiyama K.: Estimation o trichloroethylene exposure by biological materials. th. Arch. Arbcituned. 7, 281-29 , 1,971. Nomiyama K. and Nomiyama H.: Metabolism of trichloroethylene in human: Sex difference in urinary excretion of trichloroacetic acid and trichloroethanol. Int. Arch. Arbeit-neu. 28, 37-48, 1971. - Ogata M.. Takatsuka Y. and Tomokuni T.: A simple method for the quantitative analysis of urinary trichloroethanol and trichloroacetic acid as an index of tri- chloroethylene exposure. Brit. J. Industr. Med. 27, 378-381, 1970. Ogata M.. Takatsuka Y., and Tomokuni K.: Excretion of organic chlorine com- pounds in the urine of persons exposed to vapours of trichloroethylene and tet- rachloroethylene. Brit. .I. Industr. Med. 28, 386-391, 1971. Pfaffli P. and Backman A.L.: Trichloroethylene concentrations in blood and expired air as indicators 0 occupational exposure. A preliminary report. Work Environ. Health 9, I40-I44. I 72. _ Rosowski W.. Gozdzik H., Menzel-Lipinska M., Potoniec-Malinowska E., Salamon K., Lipinski E.. and Gruszka A.: Przewlekle narazenie na trojchloroetylene w swietle niektorych badan toksykologicznych. kliniczrtvch i biochemicznych. (Chronic ex- posure to trichloroethylene on the basis of some toxicological, clinical and biochem- ical studies). Med. Pracy 25, 179-185. 1974. Sedivec V. and Flek J.: Stanoveni toxickych latek a jeich metabolitu v biologickych tekutinach metodom plynove chromatografia !. Trichloretanol v moci. (The deter- mination of toxic compounds and their metabolites in biological media by gas chro- matography. I. Trichloroethanol in urine). Prac. Lek. 21, 301—305, 1969. Seto T.A. and Schultze M.O.: Determination of trichloroethylene. trichloroacetic acid and trichloroethanol in urine. Analyt. Chem. 28, I625-1629, 1956. Smith G.F.: Trichloroethylene: A review. Brit. J. Industr. Med. 23, 249-262. I966. Soucek B.: Rozdelovaci koe/icientt' kyseliny, trichloroctove. (Distribution coefficients of trichloroacetic acid). Prac. Lek. 7, 89-90, 1955. Soucek B. and Franklova E.: Stanoveni malych mnozstvi trichloroethylenu a kyseliny trichloroctove. (Estimation of small quantities of trichloroethylene and trichloroacetic acid). Prue. Lek. 4. 264-273, 1952. Soucek B. and Pavelkova E.: Bylucovani k_rseliny trichloroctove. ( Trichloroacetic acid excretion). Prac. Lek. 5, 62-64, 1953. Soucek B. and Vlachova D.: Dalsi metabolity trichlorethylenu u cloveka. (Further metabolites of trichloroethylene in man). Prac. Lek. 6, 330-332, 1954. Soucek B. and Vlachova D.: Chloroform jako metabolit trichlorethylenu. (Chloro- form as a melabolite of-trichloroethvlene). Prac. Lek. 7, 143—146, 1955. Soucek 8. and Vlachova D.: Excretion of trichloroethylene metabolites in human urine. Brit. J. Industr. Med. 17, 60-64. 1960. Stewart R.D.. Gay H.H.. Erley D.S., Hake C.L., and Peterson J.E.: Observations on the concentrations of trichloroethylene in blood and expired air following exposure of humans. Amer. Industr. Hyg. ASsoc. J. 23, 167-170, 1962. Stewart R.D. and Dodd H.C.: Absorption of carbon tetrachloride. trichloroethylene. tetrachloroethylene, methylene chloride and l,l,l—trichloroethane through the human skin. Am. Industr. Hyg. Assoc. J. 25, 439—446, 1964. Stewart R.D.. Dodd H.C., Gay H.H., and Erley D.S.: Experimental human exposure to trichloroethylene. Arch. Environ. Health 20, 64-71, 1970. TRICHLOROETHYLENE Sukhotina K.I.: Materialy isledowaniia metabolitow trichloroeliclena u rabomich proizwodstw trichloroett'lena i monochloruksusnok kisloty. (Data on a study o/me- taholites of trichloroelhylene in- workers engaged in the production of tri- cltloroetltrlene and monochloracetie acid). Gig. Tr. Prof. Zabol. 13, 35-38, I969. Tanaka S. and Ikeda M.: A method for determination oftrichloroethano/ and tri- chloroacetic acid in urine. Brit. J. lndustr. Med. 25, 2I4-2I9, I968. Teisinger J.. Styblova V.. and Vlachova D.: Vyznam stanoveni trichlorethanolu v moci u pracuiicich s trichlorethylenem. (The importance of determination of tri- ch/oroethanol in the urine of workers with trichloroethylene). Prac. Lek. 7. 258-260. I955. Vlachova D.: Jednoducha metoda na stanoveni trichloroethano/u r moci. (A simple method/or the determination o_l'trichloroethanol in the urine). Prac. Lek. 8. 433—435. I956. VIachovu D.: Determination of trichloroethanol in the urine alter exposure to tri- (liloroctltrlene. .I. Hyg. Expidem. (Praha) 1, 225-229. I957. Waclschova A.: Pracovni risiko mameslnancu v chemickych cistrinach tri- chloethylenem. (The working risk in chemical cleaning establishments using tri- chloroetlt_rlene). Prac. Lek. 6, 265-168. I954. 97 16. TETRACHLOROETHYLENE' ABSORPTION Pulmonary absorption constitutes the main route of entry for tetrachloroethylene into the body. Under experimental conditions in man, it was found that at the beginning the retention of tetra- chloroethylene in the respiratory tract amounted to about 74 per cent; this decreased slightly in the course of the exposure and after 2 hours sta- bilized at 62 per cent (Bolanowska and Golacka, I972). These values are close to those observed in mice given the I**C—labelled compound by in- halation (70 per cent, Yllner, 1961). Tet- “rachlorethylene undergoes cutaneous absorption if applied as a liquid to the skin (Stewart and Dodd. 1964). lt seems that systemic absorption of the vapours through the skin has not been studied. METABOLISM AND ELIMINATION From animal experiments it appears that tet— rachloroethylene is metabolized only to a very limited degree, being mostly eliminated from the body unaltered in the breath. Daniel (1963) ad- ministered 36Cl-tetrachloroethylene orally to rats and recovered 98 per cent of the radioactivity in the exhaled air. The data reported by Dmitriewa (1967) and Yllner (1961) indicated that elimi- nation via the respiratory tract accounted for only about 70 per cent of the total. Among the metabolites observed in animals were: tri- chloroacetic acid and ionic chlorine (Daniel, I963), and ethylene glycol and oxalic acid (Dmitriewa, 1967). Both authors maintained that chloral hydrate should not be listed among the intermediate metabolic products (see: “Trichloroethylene“). The metabolism of tetrachloroethylene in man is poorly understood. Judging from the 60 per cent steady-state respiratory retention, Bolanowska and Golacka (1972) presumed that this is the per- centage that undergoes biotransformation in the body. On the other hand, excreted trichloroacetic acid, which has attracted most of the attention, accounted only for few per cent of the systemic retention. Two per cent of the inhaled tet- 98 rachlorethylene was excreted as TCA in the urine over a 67 hours collection period. There are no human data on other metabolites. Elimination of tetrachloroethylene in the breath may be related to the blood level. The coefficient of distribution blood/air, studied in vitro, assumed a value of about 9 (Morgan et al., 1970). The disappearance rate of tet- rachloroethylene from the blood is quite high; 30 minutes after discontinuation of inhalation the compound could not be detected. On the other hand, due to the sensitivity of the determination, elimination of the unchanged compound in the expired air could be followed for prolonged peri- ods (Stewart et al., 1961). Stewart et al. (1970) followed the elimination of tetrachloroethylene for about ›100 hours in human subjects after a single exposure, and for about 330 hours after re- peated daily exposures for 5 days (about 100 ppm, 7 hours a day). From the latter data it follows that, over the first few days. the compound disappears from the breath with a half-time of about 70 hours. Bolanowska and Golacka (1972), who have ana- lyzed the elimination curve more closely for 40 hours after cessation of inhalation, found 4 con- tributing exponential terms in the equation for expressing disappearance in the expired air, of which the fastest one could have been neglected due to its quantitatively minor role. The elimination equation, related to the intercept at time t = 0 (the first very fast term neglected), assumes the form: Et/Eo : 0.61 c—l-Żt + 0.25 e-OJ“t + 0.14 e-0-02t (l) where E0 = concentration of tetrachloroethylene in breath at discontinuation of inhalation, E. the same concentration at time = to (hours). The corresponding half-times are 0.6, 4.8, and 34 hours. After repeated exposure, some increase of tetrachloroethylene concentration in the breath is observed (Fig. [6-1, Stewart et al., 1970). Bolanowska and Golacka '(1972), who evaluated TETRACHLOROETHYLENE the phenomenon from a kinetic viewpoint, con- cluded that the maximal increase above the con- centration on day l should be in the order of 20 per cent. However, the metabolites of tetrachloroethylene, excreted in the urine, should most likely display a considerable cumulation in the course of re- peated exposures. lkeda and lmmamura (1973) observed that the half-time of urinary excretion of total trichlorocompounds in workers exposed to tetrachloroethylene is twice as long as that af- ter inhalation of trichloroethylene, and amounts to I44 hours. Tada (I969), on the other hand, found maximum urinary levels of trichloroacetic acid between days 3 and 5 in human subjects ex- posed, under controlled experimental conditions, to tetrachloroethylene at concentrations of 50-160 ppm for 4-6 days. This picture did not differ from similar observations for trichloroethylene exposure. From the work reviewed, however, it seems rather unlikely that TCA levels in the urine can cumulate so as to reach the order of magnitude characteristic for exposure to trichloroethylene. METHODS FOR DETERMINATION OF TETRACHLOROETHYLENE AND ITS METABOLITES The methods based on the Fujiwara reaction are not suitable for tetrachloroethylene deter- mination, because the sensitivity of the reaction is 20 times lower than for trichloroethylene (Lug- ge, 1966). Recently, for the analysis of air in toxicological chambers and in human breath, either direct in- frared absorptiometry in gasphase in [0 m long cuvettes (Stewart et al., 1961; Stewart et al., 1970) or, more often, gas chromatography has been applied. ln the latter case, the selection of a flame ionization detector has been most usual (Stewart and Dodd, 1964; Stewart et al., 1970; Ogata et al., 1971); less frequently electron cap- ture (Stewart and Dodd, 1964); and sometimes even argon ionization detectors (Golacka and Bolanowska, 1972) were in use. For breath sam- pling glass pipettes, Saran sacks (Stewart et al., 1970), or sacks made of aluminum foil coated with polyethylene (Golacka and Bolanowska, 1972) were employed. DETERMINATION OF TRICHLOROACETIC ACID (TCA) AND OF TOTAL TRICHLORO- COMPOUNDS (TTC) See section on “Trichloroethylene”. 99 ś 3 S n 'ItALHLOHOETHYLENE (( ›N( !NWATION pn .. 7 HOUR VAPOR EXPOSURES O I 2 3 4 5 6 7 E O IO 11 12 l3 14 TIME IN DAYS Fig. Ió-I . Breath concentrations of tet- rachloroethylene in the post-exposure period on successive days of repeated exposure (Stewart et al.. l970). Taken from: Stewart R.D., Baretta E.D., Dodd H.C. and Torkelson T.R.: Experimental human exposure to tetrachloroethylene. Arch. Environ. Health 20, 224—229. 1970. Page 228, Fig. 2. EXPOSURE TESTS Attempts to use TCA or TTC in urine for the evaluation .of systemic tetrachloroethylene ab- sorption were made by several authors —— howev- er. with negative results (Friborska, 1969; Tada, 1969; Kundig and Hogger, 1970; Ogata et al., 1971; Bolanowska and Golacka, 1972). Ikeda and Ohtsuji (1972) demonstrated that relative to tri— chloroethylene, when calculated per total amount of excreted trichlorocompounds (TTC), tet- rachloroethylene yields only 8 per cent of the amount of Fujiwara reaction positive compounds. Therefore, at similar conditions of experimental exposure (200 ppm) to both the compounds, the TTC urinary concentration for tet- rachloroethylene is lower by an order of mag- nitude. Moreover, at concentrations of the latter in air above 100 ppm, the TTC urinary level be- comes stabilized at relatively low values of about 50 mg, l. Under these conditions, growing importance should be attributed to the attempts to base the exposure test upon tetrachloroethylene deter- mination in the breath. From the data of Stewart et al. (I96l), it may be concluded that, at a given time after discontinuation of inhalation, the con- centration of tetrachloroethylene in the breath may be related both to the concentration in the inhaled air and to the duration of the exposure. Directly after cessation of a TCE inhalation of 7 hours’ duration at the concentration of 100 ppm, the concentration in the breath is of the order of 20 ppm; slightly less after the first, and mod- 100 erately above this value after exposure on sub- sequent days (Stewart et al., 1970). However, as may be judged from Figure 16-1, in time inter- vals that are suitable for performance of ex- posure tests, the magnitude of exposure could not be deciphered with reasonable precision from breath concentrations of tetrachloroethylene. From the data of Bolanowska and Golacka (1972), it could be calculated that 15 minutes af- ter discontinuation of 6 hours’ exposure to the vapours at a concentration of 380 mg/m3, the ex- halation rate is of the order of 15 mg/hour. The data referred to above provide general an- swers to the basic questions and give guidance to JERZY K. PIOTROWSKI further studies aimed at development of a test. However, they do not form as yet a basis for quantitative evaluation of exposure. AVAILABLE DATA ON INDUSTRIAL EXPOSURE Due to poorly advanced exposure tests for tet- rachloroethylene, there are no data available from field studies that would contribute signifi- cant information to the subject. REFERENCES Bolanowska W. and Golacka J.: Wchlanianie i wydalanie czterochloroetylenu u ludzi w warunkach ekspoerrmenta/nych. (Inhalation and excretion of tetrachloroethy/ene in man in experimental conditions). Med. Pracy 23. 109-119, 1972. Daniel J.W.: The metabolism of 3ÓC I -l abe/led trichloroethylene and tet— rachloroethy/ene in the rat. Biochem. Pharmacol. 12, 795-802, l963. Dmitriewa N.W.: K metabolismu letrachloretilena. (On the metabolism of tet- rach/oroethy/ene). Gig. Truda Prof. Zabol. ll, Nr. 1, 54-58, I967. Friborska A.: The phosphatases of peripheral white blood cells in workers exposed to Irichloroethi'lene and perchloroethylene. Brit. J. Industr. Med. 26, 159-161, I969. Golacka J. and Bolanowska W.: Oznaczanie par czterochloroetylenu w komorze dos- wiadczalnej i powietrzu wydechowym za matographic determination of tetrachlor pomoca chromatografii gazowej. (Gas chro- oethylene vapours in the air of experimental chamber and in the expired air). Chem. Anal. 17, 1375-1379, 1972. lkeda M. and lmmamura T.: Biological half-life of trichloroethylene and tet- rachloroethylene in human subjects. lnt. Arch. Arbeitsmed. 21, 209-224, 1973. lkeda M. and Ohtsuji H.: A comparative study of the excretion of Fujiwara reaction- positive substances in urine of humans and rodents given trichloro- or tet- rachloroderivatives of ethane and ethylene. Brit. J. lndustr. Med. 29, 99-!04, 1972. lkeda M.. Ohtsuji H., lmmamura T., and Komosike Y.: Urinary excretion of total trich/oro-compounds, tetrichloroethanol and TCA as a measure of exposure to TRI and tetrachloroethylene. Brit. J. lndustr. Med. 29, 328-333, 1972. Kundig S. and Hogger D.: Die Bedeutung der Tri— and Perchlorathylen metaboliten in Urin. (The importance of the determination of tri- and perchlorethylene metabo- lites in the urine offactort' workers). lnt. Arch. Arbetismed. 25, 306-315, 1970. Lugge G.A.: Fujiwara reaction and determination of carbon tetrachloride. chloro- form. tetrachloroethane and trichloroethylene in air. Anal. Chem. 38, 1532-1533, 1966. Morgan A., Black A., and Belcher D.R.: The excretion in breath of some aliphatic halogenated hydrocarbons following administration by inhalation. Am. Occup. Hyg. 13, 2l9-233, I970. Ogata M., Takatsuka. Y., and Tomokuni K.: Excretion of organic chlorine com- pounds in the urine of persons exposed to vapours of trichloroethylene and tet- rachloroethylene. Brit. J. lndustr. Med. 28, 386-39], 1971. Stewart R.D., Arbor A., Gay H.H., Erley D.S., Hake CL. and Schaffer A.W.: Human exposure to tetrachloroethylene vapor. Arch. Environ. Health 2, 516-522. 1961. Stewart R.D., Baretta E.D., Dodd H.C., and Torkelson T.R.: Experimental human exposure to tetrachloethylene. Arch. Environ. Health 20, 224-229, 1970. TETRACHLOROETHYLENE 101 Stewart RD. and Dodd H.C.: Absorption of carbon tetrachloride, trichloroethylene, tetrat-hloroethylene. methylene chloride and 1.1,1-trichloroethane through human skin. Amer. Ind. Hyg. Assoc. J. 25, 438-443, 1964. Tada 0.: On the methods of evaluating the exposure to some chlorinated hydro- carbons. J. Sci. of Labour 45, 757-760, 1969. Yllner S.: Urinary metabolites of ”C tetrachloroethylene in mice. Nature I91, 820- 821. I961. 17. CARBON DISULPHIDE ABSORPTION Carbon disulphide is absorbed into the system by all routes; however. under conditions of industri- aI exposure the inhalatory route is of primary importance. Due to rapid saturation of the blood. the retention of carbon disulphide vapours in the respiratory tract decreases rapidly with the duration of inhalation. reaching a steady-state level after about 3 hours. According to the data of Teisinger. Soucek, and coworkers (Teisinger et al., I956). the initial and steady-state values of retention amount to about 80 and 15-40 per cent, respectively. Later investigations of Demus (1964) and Jakubowski (1966) yielded the corresponding values of 30—60 and 35-50 per cent. .The com— pound in fluid form, as well as its water solu- tions, are absorbed through the skin. This may be of practical importance in viscose rayon spin- ning mills (Baranowska, 1965). In the rabbit, der- mal absorption of carbon disulphide vapours was demonstrated by Cohen et al. (1958) and Petrun (1967); in man this route seems of little, if any, significance (Baranowska, 1965). METABOLISM AND EXCRETION A large proportion of absorbed carbon disul- phide is eliminated unchanged in the expired air. The data from animal experiments vary widely, depending on the species studied, the dose, and the route of administration. The extremes range from almost 100 per cent (rats, intragastric ad- ministration in doses of 80 and 200 mg per kg; Soucek, 1959) to 13-25 per cent (mice, studies us- ing JsS-labelled compound; Strittmatter et al. 1950). Data obtained from human inhalation ex- periments point to a considerable recovery of CS: in expired air in the period of desaturation. Quantitative assessment of the magnitude of this fraction is subject to discussion because of the difficulty in defining the absorbed dose in in- halation experiments which last for several hours, when the low “retention” may be partly account- ed for by the parallel elimination of the CS: via the same route. Thus, in early Czechoslovakian publications, the amount of CS: exhaled, relative 102 to net retention in the body, varied from 5 to 23 per cent of the dose (Teisinger and Soucek, 1949; Soucek and Pavelkova, 1953; Soucek, 1959). A less disputable assessment was obtained from studies with short inhalation periods (0.5-2 hours), in which the aVerage exhalation was about 23 per cent (Harashima and Masuda, 1962). The process in itself is quite rapid; the half-time of disappearance from rat tissues is 0.5- l.25 hours (Freundt and Schnapp, 1970), that of exhalation in rats is about 1.4 hour (Soucek, l959). and in humans less than I hour (Demus, 1964). These data relate to the main phase of elimination and disregard the very fast decline in concentration of CS: in expired air seen directly after discontinuation of the exposure. A later. very slow phase of elimination can be demon- strated which most likely should be linked with liberation of CS: from adipose tissue and from the so-called “bound carbon disulphide”. ln urine. carbon disulphide is found in minor quantities (less than l% of absorbed dose) in the free and bound form (subject to aeration from acidified urine) (Bartonicek, 1958); the unbound fraction amounts to about 30 per cent of the to- tal urinary amount (Demus. 1964). From these data it follows that about 25 per cent of the absorbed CS: is eliminated unaltered, and about 50 percent is metabolized. The inter- mediate products of the biotransformation con- sist most probably of compounds resulting from CS: reactions with proteins, peptides, and amino acids. The latter have been studied in vitro and in vivo, and were identified as dith- iocarbaminocarbonic acids (Soucek and Madlo. I956). From reactions with peptides, following cyclization. compounds of the thiazolidone type are produced. (Cohen eta1., l959). The globulins display little reactivity with CS:, whereas albumins form relatively stable com- pounds possessing free-SH groups, which in con- trast to dithiocarbaminocarbonates do not liber- ate CS: in acid media (Bobsien. 1954; Madlo and Soucek, 1956). The terminal metabolic products of CS: are sul- phates. mainly the inorganic ones. which are ex- CARBON DISULI’HIDE creted in urine in amounts of about 30 per cent of C3582 dose, as a well as small quantity (1—2% of the dose) as sulphur compounds, whose char- acteristic feature is a capacity to catalyze the io- dine-azide reaction (Yoshida, 1955; Jakubowski, 197l). Jakubowski (1968) has demonstrated that the latter compounds are not identical with those found in normal urine. Pergal et al. (1972 a, b) identified two CS; metabolites in human urine: thiourea, and 2—mercapto—Z—thiazolin—S—on, the former in greater abundance than the latter. The urinary excretion of iodine-azide catalyzing compounds is a rapid process with a half-time in humans of about 1.4 hr. (Jakubowksi, 1966). However, Djuric et al. (1965) and Graovac-Lep- osavic et al. (1966) have observed that in some workers the compound catalyzing iodine-azide re- action can be found in high concentration in urine in the morning before the start of con- secutive daily exposures. These authors proposed to use this phenomenon as an early diagnostic test of carbon disulphide intoxication. DETERMINATION OF CARBON DI- SULPHIDE AND ITS METABOLITES Free and bound CS; in blood and urine: A re- view of the older methods was published by Djuric (1966). The method recommended by Te— isinger and coworkers (1956) consists of aerating CS;, using nitrogen, transferring the latter to an absorbing solution containing diethylamine and copper acetate, and colorimetric or polarographic determination of the formed Cu-diethyl- dithiocarbamate. A similar procedure may be used for the determination of the bound urinary carbon disulphide after acid hydrolysis of the sample (Bartonicek, 1958). The compounds catalyzing the iodine-azide reac- tion: The original method developed by Vasak (Vasak, 1963; Vasak et al., 1963) is based upon the measurement of the time necessary for the oxidation of sodium-azide by a given amount of iodine. The rate of this reaction depends on the type and concentration of the catalyzing com- pounds present in the sample of urine. The reading of the test is expressed as a coefficient of exposure E, calculated from the formula: E = c log t—where t = reaction time in seconds, c = concentration of creatinine in mg/ml. The reaction is also seen in the urine of nonexposed people; but the time for the reaction is then long, and the values of E fall usually above 6.5. The correlation between the concentration of C82 metabolites and the values of exposure coefficient E is a negative one. 103 The chronometric method has proved useful for evaluating relatively high exposures to CS; (Jakubowski, 1965; Locati and Sassi, 1966; Sal- vadeo et al., 1967; Djuric et al., 1968). When the eXposure is to low concentrations of CS„ the sensitivity of the method is not satisfactory; moreover, difficulties are also encountered be- cause of the long reaction time‘ in samples of urine with a low concentration of creatinine (Tiller — see Djuric, 1966; Jakubowski and Pio- trowski, 1966). Jakubowski and Piotrowski (1966) have elabo- rated a modification of the method based on ti- trimetric determination of the iodine consumed in the reaction. The reaction is performed under conditions which discriminate in part the influ- ence of the catalyzing compounds present in physiological urine. The results are expressed in mg iodine per 1 ml urine, corrected for a stan— dard concentration of creatinine of 1.5 mg/ml. Physiological values are usually in the range from 8 to 12 mgl;/ 1.5 mg creatinine. The read— ings are directly proportional to the concentration of the catalyzing compounds. The results obtain- ed by this method correlate with those from the Vasak test, and approximate mutual translation of the data is possible. The method of Jakubowski and Piotrowski (1966) can be used at lower CS; exposure levels than the original Vas- ka-test, and it is the former upon which the ex- posure test of Jakubowksi (1966) was based. The amount of iodine used in the iodine-azide re- action can also be determined colorimetrically, as described by Magos (1972), for the urine of rats. The latter method has not been applied so far for assessment of industrial exposure to CS;. For this purpose, at low levels of the exposure, the elaboration of a method for the deter- mination of CS; metabolites catalyzing the io- dine-azide reaction seems desirable. This method would eliminate the influence of compounds with similar properties encountered in normal urine. EXPOSURE TESTS A correlation between CS; concentration. in blood and/or urine and the magnitude of ex- posure (CS; concentration in the air) has been sought for the past 20 years. The relevant studies have been reviewed by Djuric (1966). Bobsien (1954) and Weist (1959) have found a positive correlation between CS; in the blood and in the air. Demus (1964) investigated a similar urine/ air relationship. - 104 The data reported by Demus (1964) relate to a high leVel of exposure, in the range of 100-450 mg/m’. The concentration ratio urine/air (both expressed as ug/l) was about 4. Teisinger and coworkers (1956) concluded that analysis of blood, urine, and exhaled air for CS; may lead only to very approximate evaluation of the exposure. ' The interest in the CS; exposure test was revived after the study by Vasak et al. (1963) of the test based on analysis of urine for its content of com- pounds catalyzing the iodine-azide reaction. The chronometric method, referred to above, was de- rived by the authors from experiments on hu- mans exposed to CS; under controlled condi- tions; however, the range of exposures applied was limited to rather high values. The proposed interpretation of the test was approximate, based Ama. . l. 20 15 -. . l I .I 10 .' . ' I 5 ' _ l 0 so 100 150 zoo 250 300 356 400 ascii-scs. Fig. I7-I. Exposure test for carbon disulphide. Increase of iodine consumption (mg) per 1.5 mg of ereatinine. against the absorbed dose of C S; (Jakubowski and Piotrowski, I 966). Taken from: Jakubowski M. and Piotrowski J.: Practical evaluation of the iodine-azide test for the estimation of exposure to carbon disulphide. ln: Excerpta Medica Monograph “Toxicology of carbon disulphide”, Proceedings of a Symposium, Prague. I966. Page 70. Fig. 1. JERZY K. PIOTROWSKI on classification of exposure coefficient (E) val- ues into rather broad categories. A modified version of the test was developed by Jakubowski (I966). The differences, relative to Vasak‘s test, consist of: a) application of io- dometric titration, and b) reference of the results to the absorbed dose of carbon disulphide. The interpretation is based upon the increment of the amount of iodine used, relative to physiological urine. Due to considerable seasonal fluctuation of physiological values (6—11 and 8—18 mg 12/ 1.5 mg creatinine in winter and spring (May), re- spectively). the author proposed to base the eval- uation of the exposure on the difference between pre- and postwork readings for a given subject. The coefficient of correlation between the amount of CS3 absorbed into the system over 6 or 8 hours of exposure and the reading of the test was r = 0.88, with a precision of dose esti- mation of t 25 per cent. The relationship is pre- sented in Figure l7-l. DATA ONINDUSTRIAL EXPOSURE A large proportion of the information reviewed above was obtained from direct observations un- der conditions of industrial exposure, and repeti- tion is not warranted. ln the fifties and early six- ties the concentrations of carbon disulphide in the air in numerous viscose rayon plants was very high. Jindrichova still reported in I957 aver- age concentrations in the order of 250 mg/m3. At such levels of exposure, positive results could have been obtained when exposure tests based on CS2 determinations in. urine or blood were used. Jindrichova found blood and urinary concen- trations of 10-70 ug/lOO mg and 50-600 lig/l, respectively. The ratio of the two concentrations (blood/urine) was close to unity. The data on CS: concentrations in air and urine are in basic ac- cord with the relation found by Demus (1964). From investigations of both the authors it fol- lows that urinary elimination is a rather slow process: the concentrations in the morning urine of the exposed workers amount to some 25 to 33 per cent of the maximal workday values. In the early nineteen-sixties, in most viscose ray- on plants, carbon disulphide concentrations in the air were lowered to a range of 50-150 mg/m’, with higher values seen only exceptionally. Under these conditions the Vasak-test in its original form was used with success (Roubal et al., 1963; Jakubowski. 1965; Djuric et al., 1965; Kaszper and Rogaczewsak, 1965). New technological im- provements have lead to further reduction of air CARBON DISULPHIDE CS; concentrations in many of the viscose rayon plants. Poland may serve here as an example — by 1964/65 average concentrations clustered around 20-30 mg/m3, with only a few de- partments in variousvplants having higher con- centrations (Jakubowski and Piotrowski, 1966). 105 At these levels of exposure Jakubowski's iodine- azide reaction may be applied — however, most- ly as a collective test. None of the tests developed so far permit evaluation of exposures below the level of 20 mg/m3. REFERENCES Baranowska B.: Einschatzung der Haut als Resorptionsweg fur Schwefelkohlenstoffl (Evaluation of skin absorption of carbon disulphide). lnt. Archiv. Gewerbepath. Gewebehyg. 21, 362-368, 1965. Bartonicek V.: Distribuce volneho a kyselou hydrolysou odstepneho vazaneho sir- ouhliku v organech bile krysy. (The distribution of free and bound carbon disulphide liberated by acid hydrolysis, in the organs of white rats). Prac. Lek. 10, 334-338, 1958. , Bobsien K.: Untersuchung uber die Aufnahme und Bindung von C 52 an Ko- rpereigene Stofle mit neuer Bestimmungsmethode. (On the absorption and binding of C S) on the systemic compounds. as judged using a new method of determinations). Arch. Gewerbepath. Gewerbehyg. 13, 193-203, 1954. Cohen A.E., Pulus H.J., Keenan R.G., and Scheel L.D.: Skin absorption of carbon disulphide vapor in rabbits. Arch. lnd. Health 17, 164-169, 1958. Cohen A.H., Scheel L.D., Kopp J.F., Stockell F.R., Keenan R.G., Mountain J.T.. and Pulus H.J.: Biochemical mechanisms in chronic carbon disulphide poisoning. Am. Ind. Hyg. Assoc. J. 20, 303-323, 1959. Demus H.: Uber die Aufnahme, chemische Umsetzung und Auscheidung des Schwefelkoh/enstoffes durch den menschlichen korper. (On the absorption, chemical transformation and elimination of carbon disulphide in the human body). lnt. Arch. Gewerbepath. Gewerbehyg. 20, 507-536, 1964. Djuric D.: Determination of carbon disulphide and its metabolites in biological mate- rial. Toxicology ol' Carbon Disulphide, Proc. Sysn. P., Prague, 52-60, 1966. Djuric D., Graovac-Laposavic L., and Rezman l.: Use of the iodine-azide test in oc- cupational health. Arch. Hig. Rada Toksikol. 19, 245-250, 1968. Djuric D., Surducki N., and Berkes 1.: Iodine-azide test in urine of persons exposed to carbon disulphide. Brit. J. Industr. Med. 22, 321-323, 1965. Freundt KJ . and Schnapp E.:l Distribution and elimination of carbon disulphide in rats following inhalation. Neunyn Schmiedebergs Arch. Pharmakol. 266, 324-325, 1970. Graovac-Laposavic L., Djuric D., Pavlovic A., and Jovicic: The use of iodine-azide test for early diagnosis of C S, poisoning. Toxicology of Carbon Disulphide, Excerpta Medica Monograph, Prague, 1966. Harashima S. and Masuda Y.: Quantitative determination of absorption and elimi- nation of carbon disulphide through different channels in human body. Int. Arch. Gewerbepeth. Gewerbehyg. 19, 263—269, 1962. Jakubowski M.: Babania nad ocena stopnia ekspozycji na dwusiarczek wegla. (Evalu- ation of the degree of exposure to carbon disulphide). Med. Pracy 16, 1-8, 1965. Jakubowski M.: Badania nad ocena stopnia ekspozycji na dwusiarczek wegla. III. Ilosciowa ocena wchłaniania par dwusiarczku wegla u ludzi w warunkach dos- wiadczalnych na podstawie testu jodo-azydkowego. (Evaluation of exposure to car- bon disulphide. [II. Experimental studies on the absorption of carbon disulphide va- pours and its relation to the iodine-azide test). Med. Pracy 17, 388-400, 1966. Jakubowski M.: Badania nad przemiana dwusiarczku wegla do zwiazkow ka- talizujacych reakcje jodo-azydkowa: l. Badania chromatograflczne. (Studies on the conversion of carbon disulphide to compounds catalysing the iodine-azide reactions. I. Chromatographic studies). Med. Pracy 19, 244-251, 1968. 106 JERZY K. PIOTROWSKI Jakubowski M.: Badania and Przemiana dwusiarczku wegla znakowanego siarka S- 35. (Studies on the carbon disulphide metabolism to compounds catalysing the io- dine-azide reaction. II. Studies with use of carbon disulphide labelled with sulphur S- 35). Med. Pracy 22, 195-206, 1971. Jakubowski M. and Piotrowski J.: Practical evaluation of the iodine-azide test for the estimation of exposure to carbon disulphide. Toxicology of carbon disulphide, Excerpta Medica Monography, Prague, 1966, p. 70-75. Jakubowski M., and Piotrowski J.: Badania nad ocena ekspozycji na dwusiarczek wegla. II. Dobor warunkow przeprowadzania reakcji jodo-azydkowej w moczu. (Evaluation of exposure to carbon disulphide. II. A modified procedure for the io- dine-azide test in urine). Med. Pracy 16, 86-95, 1965. Jindrichova l.: Zdravotni stav pracujt'cich pri vyrobe kordovych vlaken. (The health of workers engaged in the production of cordfibres). Prac. Lek. 9, 10-17, 1957. Kaszper W. and Rogaczewska T.: Higieniczna ocena nowokonstruowanych maszyn przedzalniczvch do produkcji wiskozowych wlokien cietych. (Sanitary evaluation of the newly constructed spinning machines for the production of cut viscose fibres). Med. Pracy 16, 9-23, 1965. Locati G. and Sassi C.: The iodine-azide test as an index of exposure to carbon disul- phide of workers in viscose plants. Med. Lav. 58, 431-437, 1966. Madlo Z. and Soucek B.: Reaktion des Schwefelkohlenstoffs mit Proteinen. (There- action of carbon disulphide with proteins). Arch. Gewerbepath. Gewerbehyg. 14, 554- 557, 1956. Magos L.: Relevancy of bivalent sulphur excretion to carbon disulphide exposure in different metabolic conditions. Brit. J. lndustr. Med. 29, 90-94, 1972. Pergal M., Vukojevic N., Cirin-Popov N., Djuric D., and Bojovic T.: Carbon disul- phide metabolites excreted in the urine of exposed workers. I. Isolation and identi- fication of2—mercapto—2—thiazolin—5—one. Arch. Environ. Health. 25, 38-4], 1972(a). Pergal M., Vukojevic N., and Djuric D.: Carbon disulphide metabolites excreted in urine of exposed workers. Arch. Environ. Health 25, 42-44, 1972(b). Petrun N.M.: V lianie serougleroda na niekotorve biochimitsheskie pokazateli so- stoyania organisma pri kozshnom puti postuplenia. (Effect of CS on some biochem- ical indicatices of the state of the organism following dermal absorption). Gig. Tr. Prof. Zabol. 11 (No. 7), 50-53, I967. Roubal J., Vasak V., and Kimmelova B.: Hygienicke problematika vyroby bis- kozovych kordu. (Hygienic problems associated with the production of viscose cards): Ceskosl. Hyg. 8, 265-272, 1963. Salvadeo A., Catenacci G., and Maugeri U.: Value and limits of the iodine-azide test for the evaluation of exposure to carbon disulphide. Med. Lavoro 58, 245-249, 1967. Soucek B.: Absorpce a eliminace sirouhliku. (Absorption and excretion of carbon di- sulphide). Prac. Lek. 11, 403-407, 1959. Soucek B. and Madlo Z.: Dithiocarbamincarbonsauren als Abbauprodukte des Schwe/elkohlenstofls). Arch. Gewerbepath. Gewebehyg. 14, 511-521, 1956. Soucek B. and Pavelkova E.: Vstrebovani, metabolismus a pusobeni sirouhliku v or- ganismu. (A bsorption. metabolism and action of carbon disulphide in the body). Prac. Lek. 5,181-191, 1953. Strittmatter C.F., Peters T., and Mc Kee R.: Metabolism of labeled carbon disul- phide in guinea pigs and mice. Arch. Ind. Hyg. Occup. Med. 1, 54-64, 1950. Teisinger J. and Soucek B.: Absorption and elimination of carbon disulphide in man. .I. Ind. Hyg. Toxicol. 31, 67, 1949. Vasak V.: Hodnoceni expozice zamestnancu v prostredi znecistenem parami sir- ouhliku. I. Uvodni sdeleni. (Assessment of exposure of workers to carbon disulphide vapours. I. Preliminary report). Prac. Lek. 15, l43-l44, 1963. Vasak V., Vanecek M., and Kimmelova B.: Hodenoceni exposice zamestnancu v prostredi znecistenem parami sirpuhliku. II. Pouziti jodo-azidove reakce k dukazu a stanoveni sirnych metabolitu v moci. (Assessment of exposure of workers to carbon disulphide vapours. II. Application of the iodine-azide reaction for the detection and estimation of carbon disulphide metabolites in urine). Prac. Lek. 15, l45-l49, 1963. Weist H.: Schwefelkohlenstoffbestimmungen im Blut ohne und bei Schwefelkohlenstoff-Exposition. (The determinations of carbon disulphide in blood in exgygosed and unexposed persons). Arch. Gewerbepath. Gewerbehyg. 17, 430-441, 9 . Yoshida K.: 0n the transformation of carbon disul tde in body, Report 2: on the syn- thesis of thi—keton compound. J. Sci. Labour. 3], 09, I955 (cit. by Vasek V., 1963). 18. PARATHION . (0,0-diethyl O-p-nitrophenyl phosphorothioate) ABSORPTION Parathion may be absorbed through all routes. The essential question of the relative importance of parathion absorption via the respiratory tract and through the skin prompted many controlled laboratory experiments and field studies. lt seems beyond reasonable doubt now that both routes are important in conditions of occupational ex- posure; differences in opinions relate only to the biological consequences that may be attributed to absorption via each route. Thus Hartwell et al. (1965) and Hartwell et al. (1964), on the basis of their controlled studies on volunteers and field type observations, emphasize that cholinergic ef- fects are particularly pronounced when in- halatory absorption is considered. These authors maintain that for prevention of cholinesterase in- hibition, respiratory protection is of particular importance; cutaneous absorption, quantitatively comparable, is not followed by the expected bio- logical effects (Hayes et al., 1964). The latter opinion however, should be received with some caution: in inhalatory experiments in which Hart- well et al, (1964) applied a thermal generation technique for attaining the desired concen- trations, a more toxic paraoxan was formed in the gaseous phase. Moreover, other observations point to the occurrence of serious parathion in- toxications in humans due to cutaneous absorp- tion (Prinz, 1969). A high toxicity of parathion applied to the skin in experimental animals was also observed (Gainess, 1969). The available data do not support the contention that parathion un- dergoes hydrolysis (inactivation) in the skin (Fre- driksson et al., 1961). Therefore, some authors maintain that the transformation rate of para— thion is independent of the route by which the compound reaches the systemic circulation (Nabb et al., I966). These authors applied the deter- mination of cholinesterase activity in the serum for the assessment of parathion absorption rate through the skin in rabbits, and estimated the rate at about 3.5 mg/cm2.hour. The technique, however, as pointed out by Arterberry et al. (1961), could withstand criticism only if there were a correlation between parathion absorption, p-nitrophenol excretion, and cholinesterase inhibition. For cutaneous absorption of parathion to occur, it is not necessary to contaminate the skin direct- ly with the compound; the gaseous phase pene- trates the skin leading, nevertheless, to analy- tically measurable amounts on the skin surface (Hayes et al., 1964). The available data demon- strate unequivocally that under the conditions in which parathion is applied in agriculture, the po- tential hazard related to dermal absorption by far outweighs the risk of inhalatory intoxication (Batchelor and Walker, 1954; Durham et al., 1972). METABOLISM AND ELIMINATION The metabolism of parathion has been intensively studied due mainly to its toxic action, particu- larly the inhibition of acetylcholine esterase. The basic relevant reports have been reviewed in ear- lier monographs, e.g. O'Brien (1960) and Heath (1961). The present views on parathion metabo- Iism "in mammals, corresponding also to infor- mations on the human metabolism, are presented in Figure 18-1. According to this scheme the compound is metabolised mainly via two parallel pathways: the first leads to hydrolysis with the formation of nontoxic diethyl phos- phorothionate; the other one leads through acti- vation to highly toxic paraoxon that in turn un- dergoes hydrolysis to diethylorthophosphate. The hydrolysis of parathion, as well as that of para- oxon, liberates p-nitrophenol which is excreted in the urine. From the metabolic quantitative point of view, the relevant underlying mechanism is that related to the mixed function oxidase which is localised in microsomes; the liver is the chief but not the only metabolising organ (Norman et al., 1973; Poore and Neal, 1972). Organs which accumulate parathion in significant amounts (e.g. kidneys, lungs, intestinal walls —— Fredriksson and Bigalow, 1961), display metabolic activity in- dependent of that present in liver. Hydrolysis of paraoxon proceeds to a substantial degree in the 107 108 S u (CgH50)z PO EGHA N02 _. Par-athlon (ZV \4, .? % (cargo); P o CSHANOZ (causa)z p on « H0 can, N02 P D erb t -N h na! araoxon pbospgorothtmc P ierP e Acrd ł a3? 0 II (CZH50)zP-0H ' H0 EGHA-NOŻ methyl phuphate minor unknown me la aan tes p - Mtraphenol Fig. I8-I. The melabolism of parathion by mammalian hepatic microsomes (after Poore and Neal, 1972). Taken from: Poore R.E. and Neal R.A.: Evidence for extrahepatt‘t' metabolism of parathion. Toxicol. Appl. Pharmacol. 23, 759-768, 1972, Page 760. Fig. I. blood under the influence of phosphatases, as well as a relatively specific hydrolase, called para- oxonase (Geldmacher et al., 1973). Dependence of parathion toxicity upon the route of adminis- tration, as discussed above, could result from the varying activity of the two main metabolic path- ways (Fig. 18-l) in different tissues (organs), par- ticularly those of the liver and lungs. The avail- able data however, do not permit an unequivocal assessment of the extent the above explanation is likely to be true (for discussion see Neal, 1972). In any case, it appears certain that the final me- tabolite in both cases is p-nitrophenol, regardless of the share of the total taken by either of the metabolic pathways. Thus, at least theoretically, the determination of the total excreted p-nitro- phenol may be a measure of the quantity of parathion absorbed into the system. The metabolic rates vary considerably with ani- mal species. For instance, one step of the trans- formation, hydrolysis of paraoxon under the in- fluence of blood phosphatases, is 50 times faster in rabbit than in man (O‘Brien, I960). Urinary excretion of p-nitrophenol in the cynomolgus monkey lasts for a month after administration of parathion; for more than the first ten days, the decline of the excretion rate is marginal (Lieben, et al., 1952; Waldman et al., 1954). The same au- thors have observed prolonged excretion of p-ni- trophenol in humans exposed to parathion (Lieb- en et al., 1953); however, the data reported by JERZY K. PIOTROWSKI others indicate that the process is in fact much faster. From the data of Vlachova (1956), the maximal excretion rate of the metabolite occurs within the first day after exposure; thereafter the decline is so fast that within a day the rate de- clines to about 1/ l0 of the maximum values. The data of Hayes et al. (1964), obtained from repeat- ed cutaneous exposures, indicate a fast decline of excretory levels between consecutive daily ex- posures also. Therefore, the excretion of p-nitro- phenol becomes stabilised already after the sec- ond day of exposure, and does not indicate any further cumulation of parathion in the body. DETERMINATION OF PARATHION AND ITS METABOLITES The methods relevant for purposes of exposure evaluation are those for nitrophenol deter- mination in urine, discussed in the section “Ni- trobenzene“. ln recent years wide application of the sensitive method of Elliot et al. (1960) has oc- curred. At present the prevailing tendency is to introduce gas chromatography with electron cap- ture detector by which a sensitivity in the order of 50 ppb from a small amount of urine may be reached. An increase in the volatility of p-nitro- phenol silanization has been proposed by means of hexamethyldisilizane (HMDS) (Cranmer 1970) or transformation into ethyl ether using dialo— ethane (Bradway and Shafik, 1973). Until recently determination of the unaltered parathion itself has not been considered for ex— posure evaluation. The issue may be of intrinsic interest due to possibility of gas-chromatographic determination of the compound in the serum. It has been suggested that this procedure may form a basis for an exposure test of still greater sensi- tivity than determination of p-nitrophenol in urine (Roan et al., 1969; Watanabe, 1972). INTERPRETATION OF URINARY P- NITROPHENOL DETERMINATIONS It appears that experimental evidence is lacking which would allow a direct estimate of parathion absorption from the urinary excretion of p-nitro- phenol in man. Approximate assessment of the absorbed amounts seems possible if the following assumptions are accepted: a) At repeated exposure the excretion of p- nitrophenol becomes stabilized by the sec- ond day on a level related to the magnitude of exposure (absorbed dose). PARATHION b) Maximum excretion rate is usually reached several hours after discontinuation of the exposure, and therefore repres- entative samples of urine cannot be obtain- ed by sampling directly after the end of a work shift. Bearing this in mind it appears reasonable to base quantitative assessment on the determination of p—nitrophenol in 24 hours urine. c) The assumption seems rather well found- ed that in man virtually all p-nitrophenol is, eliminated in the urine. Therefore a daily urinary amount of p-nitrophenol should di- rectly correspond, on a molar basis, to the amount of parathion absorbed on the day of assay. This assumption is not inconsis- tent with the data of Vlachova ( 1958, 1962) who found in rats a urinary excretion of p- nitrophenol corresponding to 40-60 per cent of the administered parathion (depending on the sex); the same percentage is excreted by these animals after administration of p- nitrophenol itself. The parathion exposure test used most common- ly is based on the degree of blood cholinesterase inhibition. Total agreement as to the existence of a correlation between both tests is lacking, and it is beyond doubt that determination of p-nitro- phenol is a more sensitive assay. Nonetheless, in animal experiments (rats) a relationship was found between the dose of parathion and the de- gree of cholinesterase inhibition (Fiserova-Ber- gerova, 1962). Arterberry et al. (1961) also found a correlation between the two discussed indices. A similar conclusion seems to follow from the study of Hartwell et al. (1964), with the reserva- tion that in this case inhalatory exposure was in- volved. On the other hand, lack of the re- lationship was found by Roan et al. (1969). 1n discussing the mutual relationship of both tests the following phenomena, which make un- equivocal and simple conclusions less likely, should be borne in mind: a) At a constant daily exposure to para- thion the excretion of p-nitrophenol reaches a plateau already on the second day; where- as the inhibition of cholinesterase proceeds gradually for 2-3 weeks, and becomes sta- bilized only afterwards at a level reflecting the magnitude of the daily exposure (Glome and Swensson, 1958). 109 b) In experiments on human volunteers, low doses of parathion do not lead to a de- pression but to an elevation of the cholinesterase activity in both erythrocytes and serum; inhibitory effects may be ex- pected only after daily doses via the ali- mentary tract in excess of 0.05 mg per kg body weight (Williams et al., 1958). c) There seems no definitive answer to the question whether blood cholinesterase in- hibition is, for a given dose of the com- pound, independent of the route of, absorption From a practical viewpoint, information is need— ed on the concentrations (amounts) of p-nitro- phenol in urine at which no inhibition of blood cholinesterase occurs. ln human beings these con- centrations seem to be in the order of 70-150 ug/I (Lieben et al., 1953) or 200 ug/l in 24 hour urine (Vlachova, 1956). A similar order of values follows from the investigation of Hartwell et al. (1964), and from computation of the doses that had been applied by Williams et al. (1958) at which no decline of cholinesterase activity was found. The concentrations of p-nitrophenol in 24 hour urine, corresponding to maximum permissible concentrations in the work environment (0.05 mg/m3 —- USSR; 0.1 mg/m3 —— USA), may be calculated assuming full efficiency of absorption in the respiratory tract, ventilation of 0.8 m3/h for 8 hours, and similar cutaneous absorption from the gaseous phase. At 100 per cent trans- formation into p-nitrophenol the concentrations of the latter m daily urine should be of the order of 250-500 ug/l AVAILABLE DATA ONINDUSTRIAL EXPOSURE The data on the magnitude of exposure to para- thion in the course of its manufacture are not vo- luminous. The reviewed literature relates mainly to the issues resulting from application of in- secticides in agriculture. In addition to the papers cited above, others include: Wolfe, 1972; Wa- tanabe, 1972; and Guthrie et al., 1972. 110 › mm K. PIOTROWSKI REFERENCES Arterber J.D., Durham W.F., Elliot J.W., and Wolfe H.R.: Exposure to parathion: Measure by blood cholinesterase level and urinary p-nitrophenol excretion. Arch. Environ. Health 3, 476-485, 1961. . Batchelor C.S. and Walker K.C.: Health hazards involved in use of parathion in fruit %%%? of north central Washington. Arch. Indusu. Hyg. Occup. Med. 10, 522- Bradway D.F. and Shafik T.M.: Parathion exposure studies. A gas chromatographic method for the determination of low levels of p-nitrophenol in human and animal urine. Bull. Environ. Contam. Toxicol. 9, 134—139, 1973. Cranmer M.F.: Determination of p-nitrophenol in human urine. Bull. Environ. Con- tam. Toxicol. 5, 329-332, 1970. Durham W.F., Wolfe H.R., and Elliot J.W.: Absorption and excretion of paralhion by spraymen. Arch. Environ. Health 24, 381-387, 1972. Elliot J.W., Walker K.C., Penick A.E., and Durham W.F.: Insecticide exposure: A sensitive procedure for urinary p-nitrophenol determination as a measure of exposure to parathion. J. Agr. Food Chem. 8, 111-113, 1960. Fiserova-Bergerova V.: Aktivita cholinesterazy po opakovane aplikaci malych dawek parathionu krysam. ( C holinoesterase activity after repeated application of small doses of parathion of rats). Prac. Lek. I4, 87-92, I962. Fredriksson T.. Farrior W.L. Jr., and Witter R.F.: Studies on the percutaneous ab- sorption of parathion and paraoxon. Acta Dermatovener. 4], 335-343, 1961. Fredriksson T. and Bigelow J .K.: Tissue distribution of ”P—labeled parathion: Auto- radiographic technique. Arch. Environ. Health 2, 663-667, I96I. Gainess T.B.: Acute toxicity of pesticides. Toxicol. Appl. Pharmacol. 14, SIS-533. I969. Geldmacher-Von Malinckrodt M.. Petenyi M.. Fluegel M., Burgis H.. Dietzel B.. Metzner H.. Nirschl H., and Renner O.: Specifity of human serum paraoxonase. Hoppe-Seylers Z. Physiol. Chem. 354, 337-340, I973. Glome J. and Swensson A.: Studies on the risks associated with the use of parathion impregnated gauze strips in fly control. Brit. J. Industr. Med. 15, 62—66, 1958. Guthrie F.E., Tappan W.B., Jackson M.D., Smith F.D., Krieger H.C., and Chasson A.L.: C holinesterase levels of cigar-wrapper tobacco workers exposed to parathion. Arch. Environ. Health 25, 32-37, 1972. Hartwell W.V., Hayes G.R.Jr., and Funckes A.J.: Respiratory exposure of volunteers to parathion. Arch. Environ. Health 8, 820-825, 1964. Hartwell W.V. and Hayes J.R.: Respiratory exposure to organic phosphorus in- secticides. Arch. Environ. Health 11, 564-568, 1963. Hayes G.R., Funckes A.J., and Hartwell W.V.: Dermal exposure of human volun- teers to parathion. Arch. Environ. Health 8, 829-833, 1964. Heath D.F.: Organophosphorus Poisons. Anticholinesterases and related compounds. Pergamon Press, New York, 1961. Lieben J.. Waldman R.K., and Krauso L.: Urinary excretion of paranitrophenolfol— lowing parathion exposure. Arch. Ind. Hyg. Occup. Med. 6, 49l-495, I952. Lieben J.. Waldman R.K., and Krause L.: Urinary excretion of paranitrophenol fol- lowing exposure to parathion. Arch. Ind. Hyg. Occup. Med. 7, 93-98, 1953. Nabb D.P., Stein W.J., and Hayes W.J.Jr.: Rate ofskin absorption ofparathion and paraoxon. Arch. Environ. Health 12, 501-555, 1966. Neal R.A.: Studies of the enzymic mechanism of the metabolism of diethyl 4—nitro- phenyl phosphorothionate (parathion) by rat liver microsomes. Biochem. J. 105, 289- 297. 1967. Neal R.A.: A comparison of the in vitro metabolism of parathion in the lung and liv- er of the rabbit. Toxicol. Appl. Pharmacol. 23, 123-130, 1972. Norman J.B., Baughn W.K„Aand Neal R.A.: Studies of the mechanisms of metabo- lism of diethyl p-nitrophenyl phosphorotionate (parathion) by rat liver microsomes. Biochem. Pharmacol. 22, I09I-I IOI, 1973. O'Brien R.D.: Toxic Phosphorus Esters: Chemistry. Metabolism and Biological Ef- , fects. Academic Press, New York, 1960. Piotrowski J.: Further investigations on the evaluation of exposure to nitrobenzen'e. Brit. J. lndustr. Med. 24, 60-65, I967. PARATHION Poore R.E. and Neal R.A.: Evidence for extrahepatic metabolism of parathion. Toxicol. Appl. Pharmacol. 23, 759-768, 1972. Prinz H.J.: Severe percutoneous poisoning with parathion. Arch. Toxikol. 25, 318— 328. 1969. Roan C.C.. Morgan D.P., Cook N., and Paschal E.H.:Blood cholinesterases, serum parathion concentrations and urine p-nitrophenol concentrations in exposed individu- als. Bull. Environ. Contam. Toxicol. 4, 362-369, 1969. Vlachova D.: Stanoveni p-nitrofenolu v moci exponovanych osob. (Determination of p-nitrophenol in the urine of exposed persons). Prac. Lek. 8, 283-288, 1956. Vlachova D.: Intoxikace parathionem u krysich samcu a samic. (Parothion intoxi- cation in male andfemale rats). Prac. Lek. 10, 331-334, 1958. Vlachova D.: Vylucovani p-nitrofenolu a chovani cholinesterazy po opakovanem podani parathionu u krys. (Excretion of p-nitrophenol and behaviour of cholitéesterasze following repeated administration of parathion in rats). Prac. Lek. 14, 323-3 5. 196 . Waldman R.K., Lieben J.. and Krause L.: Physiological responses to parathion ex- posure: Correlation between serum cholinesterase activity and urinary para- nitrophenol excretion. Arch. Ind. Hyg. Occup. Med. 9, 37-44, 1954. Watanabe S.: Detection of organophosphate pesticides in blood serum from the pa- tients suspected of acute and chronic pesticide poisonings and its clinical significance. Tohoku J. Exp. Med. 107, 30l-302, I972 (Abstract supplied by MEDLARS). Watanabe S.: Poisoning by parathion used in apple orchards. Baioteku 3, 268-274. I972 (Jap.) (Abstract supplied by MEDLARS). Williams M.W.. Cook J.W., Blake J.R., Jorgenscn P.S., and Frawley J.P.: The effect oś parathitsm ggshuman red blood cell and plasma cholinesterase. Arch. Ind. Health I ,441-44 , l . Wolfe H.R.: Protection of individuals who mix or apply pesticides in the field. ln: Proceedings of the National Conference on Protective Clothing and Safety Equip- mean]; for Pesticide Workers, Washington, 1972, pp. 35-39 (Abstract supplied by M LARS). lll 19. FENITROTHION (Metathion, Sumithion) Fenitrothion, 0,0—dimethyl—O—(3—methyl—4—ni- trophenyl) phosphorothioate, is an analogue of parathion, of pronounced toxicity for insects and relatively low toxicity for mammals. ABSORPTION The compound can be absorbed into the system by all routes: pulmonary, cutaneous, and through the alimentary tract (Matyushina, 1966). A negli- gible difference in the efficiency and dynamics of excretion of the main metabolite (p-nitro-m-cre- sol) in rats after intraperitoneal and intragastric administration of fenitrothion points to a rapid and efficient absorption from the gastro-intestinal tract (Hladka and Nosal, 1967). Absorption through the skin is also easy. DL50 after dermal application is higher by a factor of two than after cn. (› cum Ę ' cu,o\. >P-0 NO. , P-o no, no no Fig. 19-1. Initial metabolic pathways of fenitrothion (Miyamoto et al., 1963). Taken from: Miyamoto J., Sato Y., Kadota T., Fuunami A., Endo M.: Agricultural and llłiokłlgical Chemistry 27, 381-389, 1963. Page 389, ig. . alimentary administration (Matyushina, 1966). Observations exist that suggest initial retention of the compound in the skin with somewhat delayed resorption into the bloodstream (Chruscielska, l968). METABOLISM AND ELIMINATION Miyamoto et al. (1963) studied the fate of the compound, labelled with 32P, in rats and guinea- pigs, and proposed a scheme of metabolic path- ways as presented in Figure 19-1. According to these authors, fenitrothion and also its de- sulphuration product, fenitrooxon, are subject primarily to hydrolysis yielding either methanol or p-nitro—m-cresol. The latter is the chief metabolite in rats (Hladka and Nosal, 1967) and in man (Nosal and Hladka, 1968), accounting respectively ‘for 62-66 and 48- 59 per cent of the administered dose‘(in man af- ter oral administration). At increasing doses in man the efficiency of this metabolic pathway declines. A small percentage of the dose (3%) was found in rats‘ urine in the form of a reduction product, p-amino-m-cresol '(Hladka, 1969). The bio— transformation mechanism of fenitrothion is probably analogous to that found for parathion. Chronic exposure to organohalogen insecticides (e.g. heptachlor) increases the toxicity of fen- itrothion by elevating the activation rate to fen- itrooxon, with a concomitant increased excretion of p-nitro-m-cresol; these facts point to induction of microsomal enzymes (Mestitzova et al., 1970). The metabolism of fenitrothion is relatively rap- id. ln rats, excretion of p—nitro-m—cresol is bi- phasic, and over the first 24 hours after intra- peritoneal administration about 80 per cent of total originating p-nitro-m-cresol is eliminated (Hladka and Nosal, 1967). lts ‘metabolism and elimination in man appear to be still faster. Al- most all the p-nitro-m-cresol is eliminated within 24 hours after absorption, and maximal excretion rate follows directly the oral administration of 112 FENITROTHION fenitrothion (Melichar and Franz, I966; Nosal and Hladka, I968). These observations clearly show that the cumulative effects of fenitrothion found by Matyushina (I966) could not have re- sulted from material cumulation of the com- pound in the course of repeated exposure. METHODS FOR DETERMINATION OF P-NITRO-M-CRESOL IN URINE The methods proposed for the evaluation of ex- posure in humans are based upon colorimetric determination of yellow nitro—cresolate in an al- kaline medium. The introductory stage of the procedure includes hydrolysis and extraction with acetonitrile. To partly eliminate the influence of urinary pigments, Melichar and Franz (1966) in- troduced (H202) oxidation. To improve sepa- ration, Hladka and Hladky (1966) proposed thin- layer chromatography. In the latter version the method includes acid hydrolysis, extraction with acetonitrile from the alkaline medium, repeated extraction with a mixture of solvent naphtha and ethyl ether from the acidified medium, and thin- layer chromatographic separation on silica-gel. The colour is developed with ammonia vapour, and spectrophotometric determination is carried out at 402 nm in pyridine eluate. The method en- ables detection of 2 ug of p—nitro-m-cresol in 20 ml of urine (Hladka, I969). As with p-nitrophenol (see: “Nitrobenzene”, and “Parathion”), it seems possible to determine p-ni- tro-m-cresol in urine by means of gas chro- matography, as proposed for analysis of other media (Bowman and Beroza, 1969). OTHER METHODS For diagnosis of intoxication with phos- phoroorganic compounds, including parathion and fenitrothion, determination of unlayered compounds in blood-serum by means of gas chromatography has been proposed (Watanabe, 1972 a, b). EXPOSURE TEST With respect to man, preliminary experimental data by Melichar and Franz (1966) and results of more systematic studies by Nosal and Hladka (I968), in which p-nitro-m-cresol has been deter- mined, have been reported by Hladka (1969). In the latter studies fenitrothion was administered orally to volunteers in gelatin capsules in doses 113 mg NC/ZAHRS N w 1‘ 01 O7 xl I l I 1 L I O 2,5 5 IO 15 mg 20 mg Fenitrothion Fig. [9-2. Dependence of the quantity of nilroeresol dishcarged in 25 hours' urine of persons tested in relation to the dose of fenitrothion (Nosal and HIadka, 1968 . Taken from: Nosal M. and Hladka A.: Int. Arch. geerr-beruth. Gewerbehyg. 25, 28-38, 1968. Page , 1g. . from 2.5 to 20 mg. The relationship between the dose and daily urinary excretion of p-nitro-m- cresol is preented in Figure l9-2. This re- lationship should also be valid for repeated ex- posures, because the authors experimentally dem- onstrated in human subjects that no essential cumula—tion of the metabolite occurred. From the point of view of the route of adminis- tration of fenitrothion, reservations are pertinent that relate to the efficiency of absorption from the gastrointestinal tract and the degradation of the compound in the intestinal content (with pos- sible participation of bacterial flora, etc.). The in- terpretation of the test with regard to dermal ex- posure does not seem possible. The same applies to the interpretation of results obtained from analysis of spot urine samples. The above test may not be replaced by an assay of cholinesterase activity in blood, because, as shown by Nosal and Hladka (1968), changes in the latter (as well as in erythrocytes in the plas- ma) are not detectable after one- or four-days’ exposure at the levels above the sensitivity limits of the test. DATA ON INDUSTRIAL EXPOSURE No data have been published so far on applica- tion of the test in industrial conditions. 114 JERZYK.HOTROWSKI REFERENCES Bowman M.C. and Beroza M.: Determination ofaccothion (feniotrothion) its oxygen analogue and its cresol in maize. grass and milk gas chromatography. J. Agr. Food Chem. 17. 272-276. I969. Chruscielska K.: Szybkosc Wchłanianiafenitrotionu znakowanego P32 poprzez skore szczura. (The absorption rate offenitrothion labelled ”P through the skin in the rat). I Symposium on Toxicology, Wroclaw, 1968: Abstracts, pp. 98-99. Hladka A: Stanovenie p-amino- -m—krezolu v moci bielIch potkanov po aplikacii I'en- itrotionu. ( The determination of p-amino-m- -cresol in the urine of rats following ad- ministration o_l'jenitrothion). Prac. Lek. 21, 60-62, 1969. Hladka A.: Stanoveni p-nitro- -m-krezolu ako degradacneho produktu fenitrotionu |' ludskom moci. (Determination of p-nitro-m-cresol as a degradation product o/'_/en- itrolhion in human urine). Prac. Lek. 21, 405—408, 1969. Hladka A. and Hladky Z. Separation of p- nitrophenol and p- nitro- m- -cresol from urine pigIIIents hI thin lą'_.|erthromatograph|' J. Chromatogr. 22,457-459, 1966. Hladka A. and Nosal M.: The determination of the exposition to Metathion (fen- ilrothion) on the basis of excreting its metabolite p-nitro-m-cresol through urine in rats. lnt. Areh. Gewerbepath. Gewerbehyg. 23, 209- 214,1967. Malyushina V.l.: K ohosnovaniiu predelno dopustimoi kom entraI II met|lnitrophosa |' rozduche rahotshey zon_'.| (Substantiation of the maximum permissible c'onIen- Iration oj' met/|_|'lnitrophos in the air of the working zone). Gig. Sanit. 31, 12- 17. 1966. Melichar B. and Franz J.. Stanoveni p- -nitro- m- -kresolu |' moc i po expozici metalionu (0. 0— („methyl-0 (3— methy—II 4— nitroI'enII)—thiofosfatu). (Determination of p- nitro- m- crew] in urine al'ler exposure to Metathion).Prac.Lek.18,112-115. 1966. Mestitzova M.. Kovac J.. Durcek K. and Hladka A.: Toxicita organoI'osI'atu (I'en- itrotiott) u potkanoI' chronic/(y otravenI'ch ch/oroorganickI'm insekticidem (hep- Iachlor). ( The toxicity o‘I'I'enitrothion in rats chronically intoxicated with the chloro— organic inIeI tit ide heptach.lor) Prac. Lek. 22, 361-365. I970. Miyamoto .I. Sato J. Kadota T. Fujinami A. and Endo M.. Studies on the mode of m lion o/ organu-phosphorus compounds. l. Metabo/it Iate ol' P3’ labeled SuIIIithion (Ian/3. Met/III parathion in guinea pig and white rat. Agr. Biol. Chem. 27, 381 -389, l 6 Nosal M. and Hladka A: Determination of the exposure to fenttrothton (0,0-dimeth- |'—I 0 (3IIIeIIIrl— 4— —nitrophenII)—Iiophosphate) on the basis of the excretion oI p- nitto- m-cresol by the urine 0/ the persons tested. lnt. Arch. Gewerbepath. Gewerbehyg. 25, 28—38.1968. Watanabe S.: Detection of organophosphorus insecticides from sera of patients stricken with acute poisoning with organa-phosphorus insecticides and its significance (Jap.) Nippon Noson. lgakkai Zasshi (J. Jap. Ass.) 21, 9-17, 1972(a). Abstract suppli- ed by NLM/MEDLINE. Watanabe S.: Detection o_/' organophosphate pesticides in blood serum from the pa- tients suspected of acute and chronic pesticides poisonings and its clinical signifi- cance. Tohoku J. Exp. Med. 107, 301-302. !972(b). Abstract supplied by NLM MEDLIN E. 20. DDT (pp-dichlorodiphenyl-trichloroethane) ABSORPTION The absorption routes of DDT— under conditions of occupational exposure are not well» under- stood. lt is obvious that absorption of DDT aerosols and vapours via the respiratory tract is possible, but there are no specified-ata as to the efficiency of the process. A part of the aerosol is most probably deposited in the upper respiratory airways and is subsequently swallowed. This part would be absorbed as if the compound were giv- en orally. Most model experiments on the metab- olism and systemic cumulation of DDT have been performed with oral administration of the compound. Generalization from these experi- ments involves the implication that intestinal ab- sorption is highly efficient. Some doubt is cast on the validity of this presumption by the experi- mental data of Jensen et al. (1957), who demon- strated that considerable amounts of DDT were present in feces representing the unabsorbed frac- tion of the compound. lt appears that cutaneous absorption of DDT has not been thoroughly studied. METABOLISM AND ELIMINATION Gadaskina and Filov (1971) claimed that after al- imentary administration of DDT the compound appears rapidly in the blood, and the concen- trations reach maximum after 6-8 hours, de- clining relatively rapidly thereafter. Organ distri- bution of DDT is rather uniform with the exception of adipose tissue, where the concen— trations are much higher than the average. The same refers to organs rich in lipids, such as bone marrow, adrenal glands, etc. In the course of re- peated exposures, it is possible to accumulate in animal adipose tissue total amounts of DDT much in excess of the lethal dose. DDT, as well as its metabolites DDD and DDE are present in liver, mainly in the soluble fraction (cytoplasm) and partly in the mitochondria. The nuclei contain relatively small amounts (Ku- zminskaja and Girenko, 1973). 1t is far from clear whether DDT in blood and tissues is 115 present in a free form, or as biocomplexes (Hat- anaka et al., 1967; Dale et al., 1967; Schoor, 1973). In the body DDT is subject to complex metabol- ic transformations that follow two directions: the first pathway goes through DDD, DDM U, and a chain of intermediate metabolites to DDA, which is excreted as the final metabolic product. The alternative pathway leads to DDE. This compound displays little reactivity; and, if metabolized at all (to DDMU), the process must be slow. Therefore, because DDE is lipophilic (as in DDT itself), in course of prolonged DDT exposure the metabolite undergoes substantial cumulation in adipose tissue. On the diagram presented in Figure 20-1 the met- abolic pathways are given according to Peterson and Robinson (1964). The dotted arrows depict the somewhat different results of later studies by Datta (1970). Datta postulated further trans- formation of DDE with a common metabolic pathway starting with DDMU. The data of Roan et al. (l97l), however, argue against further met- abolic transformation of DDE to DDA in man. The mechanisms of biotransformation of DDT to DDD on one hand, and to DDE on the other, are probably different. The data of various au- thors, reviewed by Alary et al. (1971), indicate „an VII xxx )(ch CC)2 cng—mrn-—›DcR2—)~(ER-—»HCR2 (DDD) II | I HCR _ co), 2 ((co) MEDIATES›H2C0H~--MEDIATES-> coon (DDT)\HCCIZ (DDuu) (DDOH) (DDA) (DDD) R-©Cl Fig. 20-1. Metabolic pathways of DDTaccording to: Peterson and Robinson (1964); Dana (1970). 116 that the transformation of DDT to DDD occurs predominantly in the liver, and is catalysed by microsomal enzymes that take part in the metab- olism of foreign compounds. To some extent the transformation can be accelerated by induction of these enzymes with typical inductors, e.g. phe- nobarbital. The transformation of DDT to DDE may be, at least partly, a non-enzymatic process catalysed by iron porphyrins. DDT itself, and to smaller degree its metabolites, have inductive properties with regard to the ac- tivity of microsomal enzymes (Street et al., 1966; Gillett et al., 1966). lt may be presumed, there- fore, that autoinduction occurs — however, only after high doses of DDT (Gillett, 1968). Elimination from the body takes place via urine and faeces; the main eliminated product in both cases is DDA. The DDA reaches the intestinal content mainly with bile where complexes of DDA are formed, most likely with cholic acids. Considerable amounts of DDT or DDE in bile have not been found. Pronounced elimination of DDT with faeces represents most likely the un- absorbed fraction of the compound (Jensen et al., 1957). The so called “neutral metabolites" of DDT are also absent, at least in considerable amounts in the urine of humans exposed oc- cupationally to DDT (Cueto and Biros, 1967). DETERMINATION OF DDT AND ITS METABOLITES For a long time DDT and its metabolites had been determined by use of a colorimetric method, known as Schechter-Haller method. In this pro- cedure DDT and its metabolites are converted into tetranitro derivatives that yield colour prod- ucts upon reaction with potassium ethylate (Schechter et al., 1945). lnitially DDT had been isolated from the adipose tissue by extraction with carbon tetrachloride or chloroform preceded by sulfonation of the fat. Later Mattson et al. (1953) developed a method for separation of DDE from DDT, using a modified Davidov’s column and applying alumina as the sorbent and acetone as the eluent. The Schechter-Haller method is still in use in numerous laboratories; attempts are being made to adjust the procedure for the determination of the individual metabo- lites by introducing separation of coloured prod- ucts by means of thin-layer chromatography (Krechniak and Dubrawski, 1971). However, a contemporary approach to the determination of DDT and its metabolites is based totally on ap- plication of gas chromatography. JERZY K. PIOTROWSKI From the viewpoint of exposure evaluation, use may be made of DDT (and possibly DDE) deter- mination in the blood, in the adipose tissue (ob- tained by means of biopsy), and of DDA deter- mination in urine. DETERMINATION OF DDT AND ITS METABOLITES IN BLOOD AND ADIPOSE TISSUE BY MEANS OF GAS CHROMATOGRAPHY ln principle, the gas-chromatographic methods ap- plied here consist of extraction of the sample with hexane, introduction of an adequate amount of the dessicated extract into a chromatographic column, and measurement at adequately selected conditions (type of column and detector; tem- perature of separation technique). Among the most widely known procedures are methods de- veloped by Hayes et al. (1965) for adipose tissue and by Dale et al. (1966) for blood. In the latter, a first attempt was made to leave out intro- ductory clean-up of the extract before the chro- matographic step; this tendency has been domi- nant in further methodologic studies. As stressed by Radomski and Fiserova (1965, 1967), clean- ing-up of the extracts may be omitted while ana- lyzing fat-tissue provided that a top class gas- chromatograph is used, equipped with a good electron-capture detector and able to work in the range from 2 to 50 picograms. It should be mentioned here, that when compared with the tritium detector the 63NI EC detector is of an ad- vantage because the latter is less susceptible to contamination and may be used at higher tem- peratures than the former. The simplicity of this procedure may be essential for determination in biopsy-samples because a small amount of the ma- terial (200 mg) is sufficient for the analysis. Sen- sitive and precise methods of tissue analysis, in which a complete purification of the extract is applied before chromatography, require in gener- al a substantially greater amount of the material to start with (for example, see Saschenbrecher and Ecobichon, 1967). However, there are meth- ods available, developed more recently in which clean-up is performed on a column filled with ac- tivated magnesium silicate (fluorisil) that enable the analysis to be completed on a small amount of the adipose tissue, between 50 and 200 mg (Morgan and Roan, l97l). Thus, procedures in- cluding the clean-up step are still being developed (McLeod and Wales, 1972). When analyzing blood for its content of DDT and metabolites, doubt has been expressed DDT whether simple extraction with hexane is satis- factorily efficient due to a possibility that DDT may be present in complexes which are not ex- tracted. An alternative technique, proposed by Dale et al. (I967), is based on the principle of volatilizations. A small amount of plasma is put in a tube stoppered with siliconized glasswool for capture of sublimed pesticides. Hexane extraction is performed of both the residual content of the tube as well as of the glass wool. Using this technique higher results have been obtained in blood analysis relative to simple hexane extrac- tion, but the results for standards were poor. Therefore the method has not been put into com- mon use. Another, more recent attempt to im- prove the method is based on the modification of the extraction conditions. The extraction is made in the presence of anhydrous sodium phosphate and formic acid; the extract, after washing with potassium carbonate solution, is applied directly to the column. The role of formic acid is to im— prove considerably the separation (Palmer and Kolmodin-Hedman, 1972). Apart from the meth- od of Dale et al. (I966) for analysis of blood, other chromatographic techniques are sporad- ically used. Morgan and Roan (1971) have used preliminary extraction with acetone and iso- octane. purification on a column with n-pentane extraction. followed by evaporation and analysis of the extract. DETERMINATION OF URINARY DDA BY MEANS OF GAS CHROMATOGRAPHY As demonstrated by Cueto and Biros (I967), oc- cupational exposure to DDT is not followed by an increased elimination of DDT and, so-called, neutral metabolites (mainly DDE) in urine. Ear— lier, the determination of urinary DDA was per- formed colorimetrically after separation of the metabolite from urine by means of ion-exchange chromatography (Cueto et al., 1956). Recently, gas—chromatographic methods are widely used. Laws et al. (1967) extract DDA from urine with chloroform after hydrolysis. After evaporation, methylation is performed by heating with anhy- drous methanol in the presence of sulphuric acid; n-hexane extract is applied to the gas chro- matographic column. Cranmer et al. (1969) used hexane extraction from an acidified medium fol- lowed (after evaporation) by esterification with borontrifluoride in methanol, purification on the fluorosil micro-column, and gas-chromatographic determination. To suppress the influence of 117 “background", particularly that caused by minute amounts of DDT and its neutral metabolites against which the electron-capture detector is very sensitive, the authors proposed to use in- stead a Coulson conductivity detector. Roan et al. (I97l) apply preliminary heating of urine with sulphuric acid followed by ether extraction of DDA. Esterification is then made with dimeth- yIsquhate and the extract is analysed using a gas chromatograph with a microcolorimetric de- tector. The sensitivity of the method in this ver- sion reached I ng/ ml. BACKGROUND LEVELS OF DDT AND ITS METABOLITES Background levels of DDT and its metabolites in human biological material depend on the scale at which DDT has been used. Thus the levels vary with time and geography. In general the sup- pression of the use of DDT has lead in recent years to a reduction of levels of DDT and its me- tabolites in human tissues. In adipose tissue, obtained usually at autopsy and less frequently by biopsy, DDT or DDE, or a sum of both. have been determined. In Great Britain the average concentrations (sum of both substances) amounted to 2.2 ppm (Hunter et al., I963) whereas at the same time in the USA the level reached 10-12 ppm (with individual values up to 22 ppm; Hofman et al., I964; Quinby et al., 1965). Hunter (1968) reported that the lowest concentrations (l.8 ppm) were found in Australia and the highest (about 19 ppm) in Israel. In Po- land, in one agricultrual area, the maximal levels noted in I968 reached 30 ppm and were still high in I970 — I6 ppm (Ochynski and Bronisz, I972). Of the above values obtained for people not ex- posed occupationally to DDT, DDT comprised 28 to 34 per cent, the rest was due to DDE. The approximate ratio DDE/DDT in the adipose tis— sue is thereforc close to 2. At steady-state, the relation of concentration of DDT and its metabolites in blood to those in ad- ipose tissue is relatively constant: at absolute concentrations in blood of about 20 ppb the con- centration ratio fat/blood equalled about I40 (Brown I972). Thus, similar to the DDT content in fat, the total blood DDT in occupationally nonexposed individuals varied from report to re- port. Perron and Barrentine (1970) reported val- ues in the range of 5-25 ppb, of which 80-100 per cent was DDE. Apple et al. (1970) estimated the relation of DDT to DDE in serum; DDE con- 118 centrations of 5-25 ppb were accompanied by DDT at the levels of 2 to 5 ppb. Relatively less information is available with re- gard to background levels of DDA in urine. In the experiments of Hayes et al. (1956), the levels in control groups were in the range of 0-90 ppb; Ortelee (I958) reported, for a group of individu- als with a lowest daily intake of DDT of about 0.2 mg, urinary DDA concentrations in the range 40-150 ppb. Durham et al. (1965) found, in a general population, levels from below 20 up to 350 ppb. Hayes et al. (1971) reported the most frequent values in the range of 3-lSO.ag/hr, which would correspond to 60-3000 ppb; the au— thors, however, were unable to exclude erroneous sporadic inclusion into the control group of indi— viduals exposed experimentally to DDT. Cranmer et al. (I969) reported values in the range from 8 to 19 ppb for occupationally non- exposed, whereas Edmundsen et al, ( 1970) have found concentrations on the order of 7-22 ppb in people exposed to a mild degree. The last two sets of data. obtained by gas-chromatographic methods inspire the most confidence. EXPOSURE TESTS Studies on volunteers given known amounts of DDT orally will be reviewed and discussed. This is the only type of controlled experiments on hu- man volunteers that involved significantly in- s 4000 : ~ & 5 /‘ J Ż I :S .? too _*a „_ : „ . w ›- @» -o 10 _ O .S +— Q . ... .... . ...i . .. . _ Q 0.01 04 1 to 100 DDT dose (mg/man/dog) Fig. 20-2. Storage of DDT in the body fat (ppm) as dependent on the daily dosage (mg per man per day) in humans. Taken from: Durham W.F., Armstrong J.F., nguóiśiby G.E.: Arch. Environ. Health 11, 641-647, JERZY K. PIOTROWSKI creased absorption of DDT. All reservations that were raised previously are pertinent here because of: a) non-complete absorption of the substance from GI. tract, and b) a possibility of bio- transformation of DDT in intestines under, for instance. the influence of bacterial flora. DDT in adipose tissue. Hayes et al. (1956) con- ducted basic experiments on prisoner-volunteers and obtained the data from adipose tissue biop- sy. They found that after a year of continuous administration of a technical preparation of DDT its cumulation in the tissue was completed and the steady-state values attained vere: 25-40 and 210 ppm for the daily intakes of 3.5 mg and 35 mg, respectively (the values for pure p,p’- DDT were higher). Recently the repeated system- atic studies by Hayes et al. (1971) have pointed to a longer period necessary for attaining equi- librium (2 years) and steady-state values some- what higher than the earlier ones; at the daily in- takes of 3.5 mg and 35 mg, these were 30-50 and 200-300 ppm, respectively. Morgan and Roan (1971), in a shorter study (l/2 year) when the ris- ing trends were still apparent, obtained toward the end of exposure adipose tissue levels ofw50 and 120 ppm for respective intake rates of 7.7 and 15.4 mg per day. From these studies it seems to follow that: a) the tissue levels approach equi- librium late, after 1-2 years; and b) the concen- trations of DDT in the fat are approximately proportional to the daily dose. Durham et al. (I965). on the basis of the studies by Hayes et al. (1956) and their own investigation in which a low controlled dietary intake of DDT was used, have assumed for interpretation a simple propor- tionality in a log-log coordinate system (Fig. 20- 2). This could be modified only slightly in view of later studies. For assessment of DDT absorption, the levels of DDE in adipose tissue seem to be of little use. Hayes et al. (1956) and Morgan and Roan (1971) have seen some increase of DDE concentrations after DDT, given in doses of 7-35 mg/day; how- ever. the increase was much smaller than that of DDT itself. and displayed a much larger scatter of individual values. DDT in serum. Apple et al. (1970) performed ex- periments on volunteers to whom DDT was ad- ministered orally for about 1/2 year in daily dos- es of 10 and 20 mg. The serum levels rose over the whole interval of exposure, reaching the re- spective values of about 150 and 300 ppb. In a later experiment of the same group, Morgan and Roan (1971) confirmed the rising trend of serum DDT over the whole l/2 year period of ex- DDT posure, and obtained final concentrations of about 200 and 400—600 ppb for the daily intakes of 7. 7 and I5. 4 mg DDT respectively. From these data one could conclude that, similar to DDT in the fat, serum DDT may be used as a measure of chronic absorption of the substance. The data of all the cited authors, however, in particular of Apple et al. (1970), argue against the possibility of assessing the absorption rate from DDE levels in serum. DDA in urine. Hayes et al. (I956) maintain that about 20 per cent of a DDT dose is excreted as urinary DDA. For daily oral DDT doses of 3.5 and 35 mg they found urinary concentrations of DDA (toward the end of exposure period) of 50- 300 and 900-6000 ppb, respectively. Ortelee (I958) presented a relationship between daily DDT dose and DDA concentrations in urine in the form of a curve in log-log coordinates (Fig. 20-3). Roan et al. (1971) found in volunteers who were given I5 mg of DDT daily that the DDA levels in urine rose over the whole period of ex- periment lasting for [/2 year, and reached at the end a daily excretion rate of 2 mg/24 hours, i.e. about 2000 ppb. This concentration was higher than predicted by Ortelle’s curve. Finally, un— expectedly high DDA urinary levels were obtain- ed by Hayes et al. (1971) in individuals adminis- tered a daily DDT dose of 35 mg. The excretion rate reached 2-3 mg/hour, and this value exceed- ed by an order of magnitude those expected from the data reported by others. Discrepancies in the reviewed data warrant fur— ther well-controlled human studies. AVAILABLE DATA ON OCCUPA- TIONAL EXPOSURE Hayes et al, (I958) claimed that people exposed occupationally to DDT have greater deposits of the substance in their adipose tissue than nonexposed individuals. Also the contribution of DDE to the so-called “total DDT" should be lower in the former. Ortelee (I958) studied work- ers engaged in DDT manufacture and evaluated their exposure via analysis of urine for DDA content. The author concluded that the workers in the plants studied were absorbing daily 10-50 mg of DDT. Durham et al. (I965) found that at massive occupational exposure the urinary DDA exceeded by far (120-7500 ppb) the values ob- served in the control group, and reached values close to those observed after daily ingestion of 35 mg (volunteers). Laws et al. (1967) assessed DDT 119 .- O I N - DDA IN URINE (mm) a % .? Si...? „ę 8 api .. . . div—ti‘fatuiasofi 05m M7 m (n./mlm" Fig 20-3. C omemralion of DDA in the urine of men Hi!/1 known dai/„t oral intake of DDT (Ortelee I958). Taken from: Ortelee, M. F.. Arch. Ind. Health 18, 433-440,1958 Page 435 absorption in workers by two independent meth- ods: a) determining DDT in the adipose tissue and interpreting the data according to Durham et al. (1965); b) measuring DDA urinary concen- trations and applying Ortelee’s diagram (I958). The" mean values obtained for three groups of workers by either way were consistent with each other: I8, 6.3, and 3.6 against 17.5, 8.4, and 6.3 mg DDT/day, respectively. Edmundson et al. (I969a) studied changes in blood DDT and DDE, and urinary DDA, in workers after a sin- gle massive exposure to DDT; the steepest rise was found for urinary DDA. The same authors (I969b) recommended DDE determination in blood for the purpose of exposure evaluation. In a still later report. the same group (Edmundson et al., 1970) expressed the opinion that in oc- cupationally exposed individuals there is an in- crease in the role of the detoxication mechanism that leads to the formation of DDA. These authors were unable to find a direct re- lation between actual exposure, on one hand, and the DDT and DDE levels in blood and DDA in urine, on the other. This conclusion is strengthened by existing data on the kinetics of DDT turnover in the body. Perron and Barrentine (I970) found only minor differences in serum DDT concentrations between controls and oc- 120 cupationally exposed individuals; on the other hand a pronounced seasonal variation has been observed in both groups. The authors suggested that the variation resulted from seasonal applica- JERZY K. PIOTROWSKI tion of DDT in the region in which the study rectly in the manufacture of DDT. had been performed; the rise in all studied REFERENCES Alary J.G.. Guay P. and Brodeur J.: Elfen ofphenobarbital pretreattnent on the me- tabolism of DDT in the rat and the bovine. Toxicol. Appl. Pharmacol. 18, 457-468. 1971. Apple G_.. Morgan DP. and Roan C.C.: Determinants of serum DDT and DDE centrations. Bull. Environ. Cont. Toxicol. 5, 16-23, 1970. Brown J.R.: Concentration of chlorinated hydrocarbon pesticides in human blood and their relation to the concentration in depot fat. Toxicol. Appl. Pharmacol. 22, 327-328. I972. Cranmer M.F.. Carrol 1.1., and Copeland M.F.: Determination of DDTand metabo- lites including DDA in human urine by gas chromatography. Bull. Environ. Contam. Toxicol. 4, 214-223. 1969. Cueto C.. Barnes A.C.. and Mattson A.M.: Determination of DDA un urine using an ion exchange resin. J. Agric. Food Chem. 4,943-945, 1956. C ueto C. and Biros F.J.: Chlorinated insecticides and related materials in human urine. Toxicol. Appl. Pharmacol. 10, 261-269, I967. Dale W.E., Curley A.. and Cueto H.: Hexane extractable chlorinated insecticides in human blood. Life Sci. 5, 47-54, 1966. Dale W.E.. Curley A., and Hayes W.J.: Determination of chlorinated insecticides in human blood. lnd. Med. Surg. 36, 275-280, !967. Datta P.R.: In vivo detoxication of p,p- DDT via p,p— DDE Io p,p- DDA in rats. lndustr. Med. Surg. 39, 190-194, I970. Durham W.F., Amstrong J.F., and Quinby G.E.: DDA excretion levels. Arch. Envi- ron. Health 11, 76-79. 1965. Edmundson W.F., Davies „LE., Cranmer M., and Nachman G.A.: Levels of DDT and DDE in blood and DDA in urine of pesticide formulators following a single in- tensive exposure. Ind. Med. Surg. #8, 145-150, 1969(a). Edmundson W.F., Davies J.E., Nachman G.A., and Roeth R.L.: P,p- DDTand p,p- 5DDE ig blood samples of'occupationallv exposed workers. Pub. Health Rep. 84, 53- 8. 196 (b). Edmundson W.F., Davies J.E., and Cranmer M.: DDT and DDE in blood and DDA un urine of men exposed to 3 percent DDT aerosol. Public Health Rep. 85, 457-463. I970. Gillett J.W.: No effect level on DDTin induction o/‘microsomal epoxidation. J. Agr. Food. Chem. 16, 295-297. 1968. Gillett J.W.. Chan T.M., and Terriere L.C.: Interactions between DDT analogs and mircosomal epoxidase systems. J. Agr. Food Chem. 14, 540-545, 1966. Hatanaka A.. Hilton B.D.. and O'Brien R.D.: The apparent binding of DDTto tis- sue components. J. Agr. Food Chem. 15, 854-857, 1967. Hayes W.J.Jr.. Dale W.E.. and Burse V.W.: Chlorinated hydrocarbon pesticides in the/at ofpeople in New Orleans. Life Sci. 4, 1611-1615. 1965. Hayes W.J.Jr.. Dale W.E.. and Pirkle C.l.: Evidence o/"sq/ety of long term high oral doses ofDD T_/or man. Arch. Environ. Health 22, 119-135, 1971. Hayes W.J.Jr., Durham W.F.. and Cueto C.Jr.: The effect of known repeated oral doses ol'clzlorophenothane (DDT) in man. J. Am. Med. Assoc. 162, 890-897. 1956. Hayes W.J.Jr., Quinby G.E., Walker K.C., Elliott J.W., and Upholt W.M.: Storage of DDT and DDE in people with different degrees of exposure to DDT. Arch. Ind. Health 18. 398-406, 1958. groups seemed to have been induced by environ- mental exposure. Mean DDT and DDE levels of the order of 650 ppb (DDT) and 600 ppb (DDE) were found. in the serum of workers engaged di- DDT Hofman W.S.. Fishbein W.J.. and Andelman M.B.: The pesticide content of human _lat tissue. Arch. Environ. Health 9, 387-393, 1964. Hunter C.G.: Allowable human body concentrations of organo-chlorine pesticides. Med. Lavoro 59. 577-583, 1968. Hunter C.G.. Robinson J.. and Richardson A.: Chlorinated insecticide content ofhu- man body/at in Southern England. Brit. Med. J. 26, 22l-224, I963. Jensen J.A.. Cueto C., Dale W.E., Rothe C.F., Pearce O.W.. and Mattson A.M.: DDTmetabo/ites in feces and bile of rats. J. Agr. Food Chem. 5, 9I9-925, I957. Krechniak J. and Dubrawski R.: Chromatografia cienkowarstwowa czteronitropochodnych DDT i jego metabolitow. (Thin-layer chromatography of tet- ranitro-derivatives of DDT and its metabolites). Bromat. Chem. Toksykol. 4, 211- 213. 1971. Kuzminskaja K.A., and Girenko D.B.: Raspriedieleniie i metabolism DDT w sub- kletotshnich fraki-[jack pietsheni experimentalnych zshivotnych i tsheloveka. (Distri- bution and metabolism of DDT in the subcellular fractions of the liver in experi- mental anitttals and man). Gig. Sanit. Nr 12, 59-62, 1973. Laws E.R.. Curley A., and Biros F.J.: Men with intensive occupational exposure to DDT. Arch. Environ. Health 15, 766-775, 1967. Mattson A.M.. Spillane J.T., Baker C., and Pearce O.W.: Determination of DDT and related substances in human fat. Anal. Chem. 25, 1065—4070, 1953. McLeod H.A. and Wales P.J.: A low temperature cleanup procedure for pesticides and their metabolites in biological samples. J. Agr. Food Chem. 20, 624-627. 1972. Morgan D.P. and Roan C.C.: Absorption, storage and metabolic conversion of in- gested DDTand DDTmetabo/ites in man. Arch. Environ. Health 22, 301-308. 1971. Ochynski J. and Bronisz H.: Zawartosc DDTi DDE w tkance tlus2czowei ludnosci woj. lubelskiego w latach 1969-1970. (DDT and DDE content in adipose tissue of Lublin province population in the period of 1969-1970). Bromat. Chem. Toksykol. 5, 345-349. 1972. Ortelee M.F.: Study ofmen with prolonged intensive occupational exposure to DDT. Arch. lnd. Health I8, 433-440, 1958. Palmer L. and Kolmodin-Hedman B.: Improved quantitative gas-chromatographic method for the analysis of small amounts of chlorinated hydrocarbon pesticides in human plasma. J. Chromatography 74, 21-30, 1972. Perron R.C. and Barrentine B.F.: Human serum DDT concentrations related to en- vironmental DDTexposure. Arch. Environ. Health 20, 368-376, 1970. Peterson J.E. and Robinson W.J.: Metabolic products o/'p,p- DDT in the rat. Toxicol. Appl. Pharmacol. 6 321- 327 1964. Quinby G. E.; Hayes W. J. Armstrong J. F. and Durham W. F. DDT storage in the US population. J. Am. Med. Assoc. 191, I75-I79, 1965. Radomski J.L. and Fiserova— —Bergerova V.: The determination of pesticides in tissues with the electron capture detector without prior clean-up. lnd. Med. Surg. 34, 934- 939. 1965. Radomski J.L. and Fiserova V.: Determination of chlorinated hydrocarbon pesticides in, human and animal tissues. lnd. Med. Surg. 36, 281-285, 1967. Roan C.. Morgan D., and Paschal E.H.: Urinary excretion of DDA following inges- tion ofDDTand DDTmetabolites in man. Arch. Environ. Health 22, 309-315. 1971. Saschenbrecker R.W. and Ecobichon D.J.: Extraction and gas chromatographic anal- ysis of chlorinated insecticides from animal tissues. J. Agr. Food Chem. 15, I68-l70, I967. Schechter M.S.. Soloway S.B.. Hayes R.A.. and Haller H.L.: Colorimetric deter- t7nination of DDT: Color tests for related compounds. Ind. Eng. Chem., Anal. Ed. 17, 04-709. I945. Schoor W.P.: In vivo binding ofp.p- DDE to human serum proteins. Bull. Environ. Contam. Toxicol. 9, 70-74. I973. Street J.C.. Chadwick R.W.. Wang M., and Phillips R.L.: Insecticide interactions af- fecting residue storage in animal tissues. J. Arg. Food. Chem. 14, 545-549, 1966. 121 21. OTHER SUBSTANCES The author did not make a systematic literature survey with regard to substances that have not been discussed in detail in this book. Some infor- mation was obtained, however, in a preliminary literature search. For the period of 1969-1971 the literature was searched more systematically using MEDLINE as the information source. Earlier re- ports were included in older monographs on the subject. Thus, even if the information for the substances mentioned below may not be regarded as exhaustive, it should permit basic orientation as to the scope and soundness of the existing data. ALIPHATIC ALCOHOLS For ethyl alcohol a voluminous literature exists, mainly on methods of determination in blood. Essential information is contained in older mono- graphs (Teisinger et al., 0956; Dutkiewicz et al., I964; Gadaskina and Filov, 1971). The gas—chro- matographic methods, not reviewed there, recen- tly attracted the attention of two authors: Jain (1971) and Laplace and his coworkers (I971). The blood level may be also estimated indirectly by means of breath analysis (Yamamoto and Ueda, I972). Interpretation of the results, howev- er, may be regarded more as a practical task in forensic medicine, than as an exposure test in the sense of this book (Dutkiewicz et al., 1964). A contrasting view was however, expressed by Spassovski and Benchev (I969), who suggested that already at concentrations of ethanol in air of the order of I000 mg/m3 an elevated level in blood occurs both in volunteers and in workers exposed chronically in. occupational conditions. Methanol was extensively covered earlier in the older monographs referred to in this book. Both the methods of determination and the inter- pretation of the results relate mainly to evalu- ation of acute intoxications. Nevertheless, an ap- proximate interpretation of urinary levels of methanol has been proposed for purposes of ex- posure evaluation in industrial conditions. The basic experimental data on humans were report- ed by Leaf and Zatman (1952), who found that a dose of 100 mg/kg was accompanied by a uri- 122 nary concentration of about 190 mg/l. The re- sults of field studies, made by Dutkiewicz and Blochowicz (I967), when recalculated, yield a similar relationship. More recent data, obtained * from experiments on volunteers and from field studies in industrial conditions (Spassovski and Benchev, 1969) suggest that, numerically, the ef- fective air concentrations of methanol (mg/m3) to which a worker had been exposed may be ob- tained by multiplying urinary concentrations (mg/ I) by a factor of 3. For higher aliphatic alcohols, it seems, even pre- liminary human data are absent. There are, how- ever, limited phannacokinetic data for some of them. e.g. 2—propanol, obtained in animal studies (Abshagen and Rietbrock, 1969). FORMALDEHYDE An evaluation of exposure based on the deter- mination of the substance itself in urine has been proposed (Spassovski, 1965, 1966), and later blood assays were suggested (Volkova and Sidorova, 1971). The latter authors, who studied the prob- lem in field conditions, reported that at the air concentration of 7 mg/m3 the blood levels were in the range from 0.06 to 0.4 mg%. This infor- mation still should be regarded as preliminary. ACETONE ' Studies on volunteers were conducted by Spas- sovski et al. (I967) and Di Vincenzo et al (1973). The first author recommended urinary deter- minations of the substance for exposure evalu- ation; at air concentrations of 200 and 2400 mg/ m3, 40 and 100 mg/l had been found in urine, re- spectively. Di Vincenzo et al. (1973), applying gas-chromatographic methods, were also unable to find a direct proportionality between concen- trations in air and in urine; however, direct pro- portionality was found between concentrations in the air and those in the blood and the breath. The half-life of acetone in blood in humans is close to 3 hours, and this seems to preclude any possibility of cumulation. It seems these two OTHER SUBSTANCES studies exhaust the available human data with re- gard to acetone. ALIPHATIC ALCOHOL ESTERS lt appears that no attempts have been made so far to undertake elaboration of exposure tests for esters of aliphatic alcohols. An exception seems to exist here with regard to glycol dinitrate and diethylene glycol dinitrate. Both compounds have been studied in rats. Moreover, for the latter of two compounds some preliminary data for hu- man subjects exposed in industry have been ob- tained. From the results, it seems to follow that evaluation of the exposure could be based, per- haps, upon urinary determination of nitrates that form the final metabolites of both compounds. LOWER CHLORINATED HYDROCARBONS There exist data for some chloroderivatives of methane, ethane, ethylene and acetylene. Of this group, tri- and tetrachloroethylene have been dis- cussed in separate chapters in this book. At- tempts to evaluate exposure to other compounds of this group have been based almost exclusively upon breath analysis. The general problems of this methodology have been discussed by Boettner and Muranko (1969) and by Backman and Pfaffli (1972). A general picture of the fate of methane chloroderivatives such as methyl- chloride, chloroform and carbon tetrachloride had been obtained earlier from animal experi— ments (Sperling et al., 1950; Soucek, 1962; Sou- cek, l961a, b). Recently, elimination of methy- lene—chloride in the expired air has been studied in volunteers (Riley et al., 1966), as well as blood and urinary concentrations of the compound (Di Vincenzo et al., 1971); the results seem to be encouraging. Metabolism of l,l,l—trichloroethane has been studied in rats (Hake et al., 1960) and the domi- nant role of elimination of the unaltered com- pound with the expired air was demonstrated. Stewart (1968) recommended breath analysis for the evaluation of exposure in man, and both in this work and in the report of Gazzaniga et al. (1969) there exists a basis for approximate inter- pretation of the results. Similar analysis was made for vinyl-chloride (Baretta et al., 1969). An exception in this group is represented by l,l,l,2— tetrachloroethane, which like trichloroethylene undergoes metabolisation predominantly to tri- chloroethanol and trichloroacetic acid (Phu-Lich et al., 1971). 123 AROMATIC HYDROCARBONS Apart from benzene, toluene, xylene, ethyl- benzene, and styrene, on which the available in- formation has been reviewed extensively in other chapters of this monograph, there are isolated pieces of information on mesitylene, i-pro- pylbenzene, and alpha-methylstyrene. For the first of these compounds methodical data are available on urinary determination of the main metabolite, mesitylenic acid (3,5—dimethylbenzoic acid) in exposed individuals. The interpretation is, however, of a preliminary character (Laham and Matutina, 1973). For i-propylbenzene there are available relatively exhaustive data obtained in a study on volunteers. The results, yet un- published, suggest that the exposure evaluation might be based upon determination of dimethyl- phenylcarbinol in urine (Litewka, 1974). Alpha- methylstyrene was shown to be metabolised in the body similarly to styrene; the main metabo- lite in man is atrolactic acid (Bardodej and Bard- odejova. 1966). AROMATIC NITRO- AND AMINOCOMPOUNDS Apart from nitrobenzene discussed extensively early in this book. others of importance are the following nitroderivatives of benzene: tri— nitrotoluene, dinitrophenol, and dinitro-o-cresol. Among aromatic amines, besides aniline and ben- zidine, of significance are their derivatives, as well as, the carcinogenic beta-naphthylamine. It seems that recently exposure tests for this group of compounds have not been attracting much in- terest; and the basic amount of information known from earlier reviews, and in particular from the monograph by Gadaskina and Filov, has not been expanded significantly. Of other aromatic compounds, some interest has centered on pentachlorophenol. The metabolic data available are those obtained from animal experiments only (Pleskova and Bencze, 1959). With the evaluation of human exposure in mind, methods have been developed and refined only for the determination of the compound in urine (Bencze and Pleskova, I959; Cranmer and Freal, 1970). Similar attention has been devoted to p-di- chlorobenzene (McKinney et al., 1970): a gas- chromatographic method was developed for the determination after hydrolysis of its metabolite, 2,5—dichlorophenol in urine. Among heterocyclic compounds, preliminary data are available for furfural, which finds appli- cation in the refining of mineral oils. As shown 124 in volunteers, and confirmed in industrial condi- tions, the exposure evaluation can be based on the determination in urine of the metabolite, fur- oylglycine (Kemka and Klucik, 1962). Among other compounds not discussed in detail in this book, relatively exhaustive information exists for an organohalogen pesticide: aldrin and its epoxide, dieldrine; dieldrine is also a metabo- lite of aldrin. The situation in general is very sim- ilar to that for DDT (see chapter: DDT). Both aldrin and dieldrin are lipophilic and, therefore, their levels in adipose tissue and in blood in- crease with the duration of the exposure. Both are also found in individuals from the general population, and this circumstance has a bearing on the evaluation of occupational exposure (Robinson and Roberts, 1969). Methods for the JERZY K. PIOTROWSKI determination of dieldrin in the blood have been discussed by Robinson et al. (1967) and Rich- ardson et al. ( I967). Based on this determination, a “diagnostic” version of the test was developed (Brown et al., 1964); however, the determination of the metabolites in urine has also aroused some interest (Cueto and Hayes, 1962). As the half-life of dieldrin in the human body is about 3 months, the problem of steady-state levels in tissue as a function of dose and duration of the exposure was studied in individuals exposed for a long pe- riod via the alimentary route, similar to that de- scribed for DDT (Hunter and Robinson, 1967). There are some data available on blood levels in people exposed occupationally, and on the bio- logical effects (Mick et al., 1972; Avar and Czegledi—Janko, 1970; Versteeg and Jager, 1973). REFERENCES Abshagen U. and Rietbroch N.: Kinetik a'er Elimination von 2—Propanol and seines Metabo/Hen Aceton bein Hund und Ratte. (Elimination of 2—propano/ in dogs and rats). Naunyn-Shcmiedebergs Arch. Pharmak. 264, 110-118, 1969. Avar P. and Czegledi-Janko G.: Occupational exposure to aldrin: Clinical and labo- ratorr/imlings. Brit. J. lndustr. Med. 27, 279-282, 1970. Backman A.L. and Pfaffli P.: Concentrations of halogenated hydrocarbons in the ex- pired air as indicators ofexposure. Work Environ. Health 9, 140-144, 1972. Bardodej Z. and Bardodejova E.: Kyselina atrolaktova metabolitem al/ametylstyrenu. (A trolactic acid as a metabolite of alpha-inethylstyrene). Ceskosl. Hyg. ll, 302-304. 1966. Baretta E.D.. Stewart R.D., and Mutchler J.E.: Monitoring exposures to vinyl chlo- ride vapour: Breath analysis and continuous air sampling. Amer. Ind. Hyg. Assoc. J. 30, 537—544. 1969. Bencze K. and Pleskova A.: Stanovenie pentachlorofenolu so 4-aminoantypirynom v biologic/(om materiali. (The estimation of pentachlorophenol with 4—aminoantipyrine in biological material). Prac. Lek. 11, 354-358, 1959. ' Boettner E.A. and Muranko H.J.: Animal breath data/or estimating the exposure of humans to chlorinated hydrocarbons. Amer. lnd. Hyg. Assoc. J. 30 , 437-442. 1969. Brown V.K.H., Hunter C.G., and Richardson A.: A blood test diagnostic ofexposure lo aldrin and dieldrin. Brit. J. lndustr. Med. 21, 283-286. 1964. Cranmer M. and Freal J.: Gas chromatographic analysis of pentachlorophenol in hu- man urine by'formation o_l'alkyl ethers. Life Sci. 9, lZl-l28, 1970. C ueto C .Jr. and Hayes W.J.Jr.: The detection of dieldrin metabolites in human urine. J. Agr. Food Chem. 10, 366-369, 1962. Di Vincenzo GD.. Yanno F.J., and Astill B.D.: The gas chromatographic analysis of methylene chloride in breath blood and urine. Am. lnd. Hyg. Assoc. J. 32, 387-39l, 1971. Di Vincenzo C.D.. Yanno F.J., and Astill B.D.: Exposure of man and 'dog to low concentrations ofacetone vapor. Am. lnd. Hyg. Assoc. J. 34, 329-336, 1973. Dutkiewicz T. and Blochowicz A.: Ocena narazenia na metanol na tle badan tere- nowych. (Evaluation of exposure to methanol in view of field investigations). Med. Pracy 18, 132-141. 1967. Gazzaniga G.. Binaschi S., Sportelli A., and Riva M.: L’eliminazione nell aria alveo- lare dell uomo dell l.l,l—trichlor0ethano dopo esposizione a 600 ppm per 3 ore. OTHER SUBSTANCES 125 (Elimination in human alveolar air of 1.1.1 —trichloroethane after exposure to concen- trations 0/600 ppm for 3 hrs). Bol. Soc. ltal. Biol. Sper. 45, 97-99, I969. Hake C.L., Waggoner T.B., Robertson D.N. and Rowe V.K.: The metabolism of l.l.l—trichloroethane by the rat. Arch. Environ. Health 1, 101-105. 1960. Hunter C.G. and Robinson J.: Pharmacodynamics of dieldrin (HEOD). I. Ingestion by human subjects/or l8 months. Arch. Environ. Health 15, 614—626, 1967. Jain N.C .: Direct blood-injection method for gas-chromatographic determination of alcohol: and other volatile compounds. Clin. Chem. 17, 82-85, 1971. Kemka R. and Klucik l.: Navrh expozicneho testu prefurfural na zaklade stanovenia _litroylglrcinu ako metabolitufurfuralu v moci. (A proposed exposure testforfurfura/ based on the estimation of furoylglycine as afurfural metabolite in the urine). Prac. Lek. 14. 331-337. 1962. Laham S. and Matutina E.O.: Microdetermination of mesitylenic acid in human urine. Arch. Toxikol. 30, l99-205, 1973. Laplace M.. Boncour R.. Lebbe J. et al.: Application of gas chromatography to the examination of blood volatile components. I]. Determination of blood alcohol. Arch. Mal. Prof. 32, 265-270. 1971. Leaf G. and Zatman L.J.: A study of the conditions under which methanol may exert a toxic hazard in industry. Brit. J. lndustr. Med. 9, 19-25, 1952. Litewka B.: Przebieg wchlaniania kumenu u ludzi droga oddechowa i wydalanie du'umetrlo/enylokarbinolu z moczem. (lnhalatory absorption of cumene in humans and urinary excretion ofdimethy'lphenylcarbinol). PhD thesis. Medical Academy in Poznan. 1974. Mc Kinney J.D.. Fishbein L.. Fletcher C.E. and Barthel W.F.: The electron capture gas chromatography of paradichlorobenzene metabolites as a measure of exposure. Bull. Environ. Cont. Toxicol. 5. 354-361. 1970. Mick D.L.. Long K.R.. and Bonderman D.P.: Aldrin and dieldrin in the blood of pesticideformulators. Amer. Ind. Hyd. Assoc. J. 33, 94-99, I972. Phu-Lich N.. Cluet J.L.. and Dutertre-Catella H.: Metabo/istne du tetrachloro l.l,l,2 —ethane. (Metabolism oftetrach/oro l,l,l,2—ethane). C.R. Acad. Sc. Paris. 272. 1173- 1176. 1971. Pleskova A. and Bencze K.: Toxicke vlastnosti pentachlorfenolu. (Toxic properties of pentachlorophenol). Prac. Lek. ll. 348-354, 1959. Richardson A.. Robinson J.. Bush B., and Davies J.M.' Determination of dieldrin (HEOD) in blood. Arch. Environ. Health, 14, 703-708, 1967. Riely E.C.. Fassett D.W., and Sutton W.L.: Methylene chloride vapor in expired air o/‘human subjects. Am. lnd. Hyg. Assoc. .l. 27, 341-348, 1966. Robinson J.. Richardson A. and Davies J.M.: Comparison of analytical methods/or determination o_l'diela'rin (HEOD) in blood. Arch. Environ. Health 15, 67-69. 1967. Robinson J. and Roberts M.: Estimation of exposure of the general population to di- eldrin (HEOD). Food Cosmet. Toxicol., 50l-514, 1969. Soucek B.: Osud choroformu v organismu krysy. ( The fate of chloroform in the or- ganism of the rat). Prac. Lek. 13, 228-232, 1961. Soucek B.: Zadrzovani. vylucovani a rozlozeni chloridu uhliciteho v organismu krysy. (Retention. excretion and distribution of carbon tetrachloride in the rat organism). Prac. Lek. 13, 287-290, 1961. Soucek B.: Zadrzovani a cylucovani methylchloridu u krys. (Retention and elimi- nation o/‘methylchloride in rats). Prac. Lek. 14, 117-119. 1962. Spassovski M.: Bestimmung des Formaldehyds im Ham der Werktatigen als Indi- kation des sanitaren Zustandes in Industriebetrieben. (Determination o/formaldehyde in the urine of workers as a measure of the sanitary conditions of the factors). Z. Ges. Hyg. 11, 199-202. 1965. Spassovski M.: Expositionstest des Formaidehids. Proc. XV lnt. Congr. Occup. Health, A-ll-180, 1966, 811-815. . Spassovski M. and Benchev I.: Expozicionen test za metilow alkohol. (Exposure test for methylalcohol). Higiena i Zraveopasvane 12, 333-339. 1969. Spassovski M. and Benchev l.: Further investigations on the exposure test of ethyl al- cohol. Institute of Occupational Health, Sofia, Bulgaria, 1969 (reprint). Spassovski M.. Benchev 1., and Kaloyanova F.: Expozicyonen test za acetona. (Ex- posure testfor acetone). Gigiena i Zdraveopazvane 10, 503-508. 1967. 126 JERZY K. nomowsxi Sperling F.. Macri F.J.. and von Gettingen W.F.: Distribution and excretion of intra- vgnously administered methyl chloride. Arch. Ind. Hyg. Occup. Med. 1, 215-224. l 50. Stewart R.D.: The toxicology of 0,1,1—triehloroethane. Ann. Occup. Med. 11, 71-79, l968. Vasak V.: Stanoveni dusicanu v moci jako expozicni test pri pracy s dinitro- dig/_rkolem. (Determination of nitrates in the urine as exposure test in work with di- nitrotliglrcol). Prac. Lek. 17, 47-50, I965. Versteeg I.P.J. and Jager K.W.: Long-term occupational exposure to the insecticides aldrin. dieldrt'n. eldrin and telodrt'n. Brit. J. lndustr. Med. 30, 201-202, 1973. Volkova Z.A. and Sidorova EA.: Sodershant'eformaldegida v krovi rabotayushtshich w kontakte s motshevinoę/ormaldegidm'mi smolami. (The level of formaldehyde in blood of workers being in contact with urea-formaldehyde polymers). Gig. Truda Prof. Zlbol. No. 5, 44-46, I971. Yamamoto K. and Ueda M.: Studies on breath alcohol analysis/or the estimation of blood alcohol levels. Forens. Sci. l, 207-224, I972. 22. SELECTED GENERAL BIBLIOGRAPHY Djuric D.: Biochemija i biofizyka industrijskich otrova. (Biochemistry and Biophysics of Industri- al Poisons). Beograd, 1966. Dutkiewicz T.: Poznamky k metodice oceneni ex- pozice prumyslovymi jea'y. (Notes on the meth- ods of assessing exposure to industrial poisons). Prac. Lek. 16, 56-59, 1964. Dutkiewicz T.: Chemia toksykologiczna. (Toxi- cological Chemistry). PZWL, Warszawa, 1974. Dutkiewicz T., Piotrowksi J., and Kesy-Dab- rowska l.: Chemiczne badania materialu biologi- cznego w toksykologii przemysłowej. (Chemical assays of biological media in industrial tox- icology). PZWL, Warszawa, 1964. Elkins H.B.: Analyses of biological materials as indices of exposure to organic solvents. Ind. Hyg. Occup. Med., 212-222, 1954. Elkins H.B.: The Chemistry of Industrial Toxi- cology. 2nd Ed. J. Wiley Inc., New York, 1959. Elkins H.B.: Excretory and biologic threshold limits. Cummings Memorial Lecture — 1967, Amer. Ind. Hyg. Hyg. Assoc. J., 305-314, 1967. Gadaskina 1.D. and Filov W.A.: Prevrashtsheniya i opredelenie promishlennych yadov w organisme. (The biotransformation and determinations of industrial poisons in the body). Medicina, Leningrad, 1971. Golubev A.A., Liublina E.1., Tolokoncev N.A., and Filov W.A.: Kolitshestvennaya toxikologiya. (Quantitative toxicology). Medicina, Leningrad, 1973. ' Gram T.E. and Gillette J.R.: _Biotransformation of drugs. In: Fundamentals of Biochemical Phar- macology, Z.M. Bacq (Ed.), Pergamon Press, Oxford, 1971. Parke D.V.: The Biochemistry of Foreign Com- pounds.›Pergamon Press, Oxford, 1968. Piotrowski J.: Expozicyonnye testy i sistema pri- edelno dopustimych koncentracii toxitsheskich veshtshestv. (Exposure tests and the system of TVL values for toxic compounts) In: Principy i metody ustanovleniya predielno dopustimych koncentracii vrednych veshtshestv v vozduche proizvodstvennych pomeshtshenii. Medicina, Moskva. 1970. Piotrowski J.: The application of metabolic and excretion kinetics to problems of industrial tox- icology. U.S. Government Printing Office, Wash- ington D.C., 1971. Piotrowski J.K.: Certain problems of exposure tests for aromatic compounds. Prac. Lek. 24, 94- 97, 1972. Stewart C.P. and Stolman A.: Toxicology: Mechanisms and analytical methods. Academia Press, New York, 1960 (vol. I). Stolman A. and Stewart C.P.: The absorption, distribution and excretion of poisons and their metabolites. Progress in Chemical Toxicology (A. Stolman, Ed.) Acad.Press, New York, 1965. Teisinger J.: Tests biologiques d'exposition. (Bio- logical exposure tests). Prac. Lek. ll, 153-161, 1959. Teisinger J.: Cinnost odboru prumyslove tox- ikologie. (The activity of laboratory for industrial toxicology). Prac. Lek. 15, 15-18, 1963. Teisinger J.: Biologicke expozicni- testy. (Biologi- cal exposure tests). Prac. Lek. 21, 387-395, 1969. Teisinger J., Skramovsky S., and Srbova J.: C hemicke methody k vysetrovani biologickeho materialu v prumyslove toxikologii. (Chemical methods for the investigation of biological media in industrial toxicology). SZN, Praha, 1956. Williams R.T.: Detoxication mechanisms. Chapman and Hall Ltd., London, 1959. 127 23. AUTHOR INDEX (A list of authors whose works are cited or referenced in the text.) Abar EL. 18 Abshagen U. 122, 124 Ackerman E. 12, 17, 71, 74 Adams E.M. 65 Ader D. 81, 82, 83, 84 Ahlmark A. 94 Ajtai 1. 78, 79 Alary LG. 115, 120 Aleksandrova T.A. 17 Alessio L. 52. 53 Altman K.J. 57 Anatovskaja V.S. 74 Andelman M.B. 121 Anders M.W. 18 Angerer 1. 42, 46, 48, 51, 52 Apple C.117,118,119,120 Arbor A. 100 Archakov A1 16, 17 Armeli C. 85 Armstrong 1.F. 120, 121 Arnstein M.R.V. 48, 53 Arterberry LD. 107, 109, 110 Astrand 1. 48, 51, 53 Astill BD. 124 Avar P. 124 Axell K. 95 Axelson O. 65 Backman A.L. 91, 96, 123, 124 Bakke O.M. 58, 59, 60, 64 Balmer D.K. 96 Baranowska B. 102, 105 Bardodej Z. 32, 40, 43, 46, 58, 59, 60, 61, 62, 63, 64, 67, 68, 85, 90, 93, 94, 95, 123, 124 Bardodejova E. 46, 58, 59, 60, 61, 62, 64, 123, 124 Baretta E.D. 45, 65,100, 123, 124 Barnes A.C. 120 Barrentine B.F. 117, 119, 121 Barret H.N. 86 Bartalini E. 85 Barttel W.V. 125 Bartonicek V. 23, 30, 86, 87, 88, 95, 102, 103, 105 Batchelor C.S. 107, 110 128 Becking C.C. 16, 17, 71, 74 Bedford 1. 95 Beker C. 121 Belcher DR. 100 Benchev I. 122, 125 Bencze K. 123, 124, 125 Bend 1.R. 12, 17, 18 Benedict R.C. 67, 69 Benes V. 43, 46 Berg B.A. 4, 9 Bergerova V. 42, 46 Berkes 1. 105 Beroza M. 113,114 Bigelow J.K. 107, 110 Binaschi S. 124 › Bingham E. 85 Biros F.J.116,117,120,121 Black A. 100 Blair D. 41. 43, 45, 47 Blake J.R. 111 Blochowicz A. 122. 124 Bobsien K. 102, 105 Bocek K. 32, 36, 40 Boettner E.A. 91. 96. 123, 124 Bojovic T. 106 Bokhenko A.J. 17 Bolanowska W. 62, 64, 83, 84, 85, 98, 99, 100 Boncour R. 125 Bonderman D.P. 125 Bowman M.C. 113.114 Bradway D.F. 108, 110 Bratton A.C. 82 Bray H.C. 48, 53, 55, 57 Brazdova D. 53 Bridges J.W. 13,17 ' Bradeur J. 120 Bronisz H. 117, 121 Brown J.R. 117.121 Brown R.M. 85 Brown V.K.H. 124 Btblewska A. 47, 53, 57 Buchet J.P. 49, 53, 64, 68 Bugatti A. 8 Buhler DR. 76, 79 Burgis H. 110 Burkhardt G.N. 82 Burkiewicz C. 49. 53. 63. 64 Burse V.W. 120 Butler T.C. 86. 87. 90 Cabana B.E. 90. 97 Cambier M. 69 Capellini A. 52. 53 Carpenter GP. 60. 64 Carrol 1.1. 120 Carter F.W. 4. 9. 36. 40. 41. 42. 45 Case R.A.M. 82.85 Catenacci G. 106 Cavagna G. 8 Cettl L. 42. 46.47 Cha Y.N. 19 Chadwick R.W. 121 Chan T.M. 120 Chasson A.L. 110 Chhabra R.S.12.13.16.17 Chruscielska K. 112. 114 Chrzaszczewska A. 81. 82. 85 Chwast S. 82. 83. 85 Cirin-Popow N. 106 Clark H. 14. 18 Clowes RC. 53 Cluet J.L. 125 Cohen A.F. 102. 105 Conolly NE. 8 Cook N. 111 Copeland M.F. 120 Cornish H.H.l4.15.18 Cooper J.R. 87. 95 Cranmer M.F.108.110.117.118.120 Cranmer N. 123. 124 Csanyi Gy. 78. 79 Cueto C. 116.117.120.121 Cueto C. Jr. 124 Curley A. 120. 121 Czegledi-Janko C. 124 Dale W.E.115.116.117.120. 121 Daniel J.W. 98. 100 Danishevsky 1. 60. 64 Datta P.R. 115.120 Davies D.S. 8 Davies J.E. 120 Davies J.M. 125 Davies T.A.L. 43. 46. 47 Demus H. 102. 104. 105 129 Dieteren A.M.L. 72. 74 Dietzel B. 110 Die Ferrante N. 57 Di Vincenzo G.D. 122. 123. 124 Djuric D. 5. 8. 103. 104. 105. 106 Dmitriewa N.W. 98. 100 Docter H.J. 43. 46 Dodd H.C. 65. 86.96.98. 99. 101 Dollery CT. 6. 8 Domanski T. 20. 30 Mc Donald D.B. 85 Donahue D.D. 53. 54 Drew R.T. 15. 18 Dubrawski R. 116. 121 Durcek K. 114 Durham W.F. 7. 8. 107. 110. 117. 118. 119. 120. 121 Dutertre-Catella H. 125 Dutkiewicz T. 2. 6. 8. 32. 40. 41. 43. 45.46. 47. 48. 53. 55. 57. 58. 59. 60. 65. 67. 68. 69. 70. 71. 72. 73. 74. 76. 78. 81. 90. 93. 122. 124. 127 Duvernenil G. 66. 69 Easterling R.E. 17 Eben A. 86. 87. 88. 90. 92. 93. 95 Ecobichon D.J. 116. 121 Edmundson W.F. 118. 119. 120 Ehrner-Samuel H. 53. 90.95 Elkins H.B. 2. 3. 29. 47. 49. 53. 78. 93. 122. 127 Elliot J.W. 108.110.120 El Masri A.M. 48. 49. 53. 60. 61. 65 Emerson E. 67. 69 Endo M. 114 Erley D.S. 96. 100 Ertle T. 88. 89.90. 92. 93. 94. 95 Fabre A. 42. 46 Fabre R. 42. 46. 48. 53. 55. 57. 87. 95 Farant J. 85 Farrior W.L. 110 Fassett D.W. 125 Fati S. 46 Feldmann RJ. 6. 8 Filov W.A. 2. 30. 42. 45. 55.61. 62.64. 65.66. 67. 69. 71.78. 81.86. 90.115.122.123.127 Fiorese F.F. 49. 54 Fiserova-Bergerova V. 2. 42. 43. 46. 47.60. 64. 65. 67.69.109.110.116.121 Fishbein L. 125 Fishbein W.J. 121 Flek J. 49. 53. 90. 96 Fletcher C.E. 125 130 Flueoel M. 110 Fouts J.R.10,l2,13,15,16,17,18 Forssman S. 87. 94. 95 Forster V. 67, 69 Franklova E. 90. 96 Frant R. 86.93. 94. 95 Franz J. 113.114 Frawley J.P.'111 Freal J. 123. 124 Fredriksson T. 107, 110 Freundt K.J. 102. 105 Friberg L. 93. 95 Friborska A. 99. 100 Friedman P.J. 87. 95 Fujinami A. 114 Funckes A.J. 110 Gadaskina J.D. 2, 42, 45, 55, 61, 64, 66. 67. 69. 71. 78. 81, 86. 90, 115, 122, 123, 127 Gaffney G.W. 55. 57 Gainess T.G. 107, 110 Gay H.H. 96. 100 Gassaniga G. 123, 124 Geldmacher M. 108. 110 Gellatly J.B.M. 74 Gerarde H.W. 51. 53 Gessner P.K. 90 Ghetti G. 82. 85 Gillette J.R. 10.11.13,14,16, 18, 127 Gillet! J.W. 116. 120 Gingell R. 12. 18 Girenko D.B. 115.121 Glassman J.M. 81, 85 Glome J. 109, 110 Gmyrek D. 18 Gnezdilova A.J. 75 Golubev A.A. 28. 30. 127 Golacka J. 98. 99. 100 Gozdzik H. 96 Goetell P. 63, 65 Gram T.E. 10.11.13.14.16. 18, 127 Grandjean E. 86. 93. 94. 95 Grant D.L. 19 Graovac-Laposavic L. 103, 105 Graziani G. 42. 47 Grubner l. 18 Gruszka A. 96 Guay P. 120 Gut J. 41, 46, 64 Guthrie F.E. 109, 110 Hass P.A. 95 Hake CL. 96, 100, 123, 125 Haller H.L. 121 Hanke J. 6, 8, 41.43. 47, 67, 69 Harashima S. 102, 105 Hartwell W.V. 107, 108, 109, 110 Harvey D.G. 78, 80 Hassman P. 73. 75 Hatanaka A. 115, 120 Hayes CR. 107, 108, 110 Hayes G.R.Jr. 110 Hayes R.A. 121 Hayes WJ. Jr. 110. 116. 117, 118, 120, 124 Heath D.F. 107. 110 Henderson P.T.H. 13, 18 Henschler D. 96 Hickish D.E. 94. 95 Hill D.L. 72. 74 Hilton BD. 120 Hinkova L. 62. 65 Hladka A. 112.113.114 Hladky Z. 113.114 Hodgson E. 13, 18 Hoegg'U. 85 Hoel D. 18 Hofman W.S. 117.121 Hogger D. 90. 94, 96, 99, 100 Hook G.E.R. 12.17.18 Horvath AA. 47 Hosker M.E. 85 Humphris 3.0. 57 Hunter C.G. 41.47. 117, 121, 124, 125 Huthwohl B. 18 Huzl F. 61. 63. 65 lkeda M. 14. 15. 18, 48. 53.- 60. 61, 62, 65, 66, 67, 68. 69. 77. 79. 87. 88. 89. 90, 91, 94, 95, 99, 100 lmamura T. 15, 18.87.88. 91. 95. 99. 100 Irish D.D. 65 Jablonska J.K. 19, 47. 75 Jackson M.D. 110 Jager K.W. 124, 126 Jain N.C. 122. 125 Jamrog D. 70. 74 Jandrova R. 36. 40 Jankova J. 65 Jakubowski M. 102, 103, 104, 105 Jansen B.L.M. 13, 18 Jenkins RP. 73, 74 ‘ Jensen U.A.115,116,121 Jindrichova J. 46, 47, 65, 104, 106 Johnston J.M. 86 Jonen H.G. 16, 18 Jorgensen P.S. 107 Juchau M.R. 12,18, 71, 74 Junger V. 65 Kadota T. 114 Kahl R. 18 Kahl G.F. 18 Kanner N.L. 46, 47 Karnzina J.J. 117 Kaszper W. 104, 106 Kaucka J. 49. 52, 53 Kawai H. 95 Keenan R.G. 105 Kemka R. 124, 125 Kesy-Dabrowska 1. 74, 127 Khizhnyakova LN. 75 Kilbom A. 53 Kimmelova B. 106 Kimmerle G. 86, 88, 90, 92, 93, 95 King L.J. 17 Kingsbury F.B. 48, 53 Kita A. 79 Kleinbauer A. 83, 84, 85 Lkinger W. 13, 18 Klucik 1. 124, 125 Knoepfel H.K. 95 Knott J.C.A. 16, 18 Kodura J. 73, 74, 78 Kolmdodin-Hedman B. 117, 121 Komosike Y. 100 Komike J. 95 Kopp J.F. 105 Kovac J. 114 Kovar J. 65 Krause L. 78, 110, 111 Krechniak J. 116, 121 Krieger H.G. 110 Krivucova M. 67, 68, 86, 93, 94 Krylova E.V. 74 Kudra J. 47 Kukackova V. 47, 67, 69 Kundig S. 90, 94, 96, 99, 100 Kundijev J.1. 6, 8 Kuniyoski M. 95 Kunor V. 85 Kuzminskaja K.A. 115, 121 Kylin B. 88, 90, 95, 96 131 Laham M. 53 Laham S. 57, 82, 85, 90, 96, 123, 125 Lahn V. 65 Laplace H. 122, 125 Lauwerys R. 69 Lawford DJ. 78, 80 Laws E.R. 117,119,120,121 Leaf G. 122, 125 Lebbe J. 125 Ledrer E. 64, 65 Lehnert G. 47, 52, 54 Leibman K.C. 14, 18, 87, 86 Lieben J. 108,109, 110, 111 Lieberman L.M. 49, 52, 53 Lindberg A. 96 Lindelof B. 65 Lipinski E. 96 Litewka B. 123, 125 Liublina E.1. 30. 127 Locati G. 7, 8, 103, 106 Long K.R. 125 Lugge 0.5. 99, 100 Lustinec K. 41, 47 Macri F.J. 126 Madlo Z. 65, 102, 105, 106 Magos L. 103, 106 Mahew J.A. 81, 85 Maibach H.1. 6, 8 Mailman R.B.'13. 18 Malek B. 64 Malinakova H. 67, 69 Manfanovski V.V. 74 Mangeri U. 106 Manz A. 46, 54 Marhold J. 72, 74, 82, 85 Marshall E.K. 82,84 Mason HS. 76, 79 Masuda Y. 102, 105 Mattson A.M. 116,119,121 Matutina E.O. 123, 125 Matyuskina V.1. 112, 113, 114 Mc Allister WJ. 14, 18, 87, 96 Mc Kee R. 106 Mc Kinney J.D. 123, 125 McLeod H.A. 116,121 Mc Pherson F.J. 17 Mc Phillips J.J. 18, 19 Medek V. 94, 96 Meigs J.N. 81, 82,83, 85 Melichar B. 113, 114 132 Menzel-Lipinska M. 96 Mestitzova M. 112, 114'- Metzner H. 110' Mick D.L. 124, 125 Mikołajczyk L. 30 Mikulski P.]. 46. 47, 48, 49, 52, 53, 57 Minarikova J. 53 Miyamoto J. 114 Mogilnicka E. 68, 69, 84 Morgan A. 98, 100 Morgan D.P. 111,116, 117, 118,120, 121 _Mountain J.T. 78, 80, 105 Muranko H.J. 91, 95, 123, 124 Musialowicz E. 80 Mutchler J.E. 124 Muller D. 95 Muller G. 88, 92, 96 Munchinger R. 95 Myslak Z. 77, 80 Nabb DR 107, 110 Nachman G.A. 120 Naus A. 67, 69 Neal R.A. 12, 18, 53, 54, 108, 110 Nemecek R. 32, 40 Neuberger A. 48, 53 Neuhom U. 80 Nirschl H. 110 Niyamoto J. 112. 114 Nomiyama H. 16, 18, 86,87, 88, 91,94, 96 Nomiyama K. 16, 18, 86, 87, 88, 91, 92, 94, 96 Norman J.B. 107, 110 Nostrom A. 95 Nosal M. 112, 113, 114 O‘Brien RD. 107, 108, 110, 120 OchynskiJ. 118,121 O'Connor G.T. 80 Ogata M. 48, 49,50, 51, 52, 53, 55, 56, 57, 90, 93, 96, 98, 99, 100, 126 Ohtsuji H. 14, 18, 60, 61, 62, 63, 65, 66, 67, 68, 69, 95, 96, 100 Orlowski J. 32, 40, 55, 56, 57 Ortelee M.F. 117,119,121 Owe—Larsson A. 95 Ovrum P. 53 Pacseri 1. 73, 74 Pagnotto L.D. 47, 49, 52, 53 Palmer L. 117, 121 Paluch E. 78 Panchenko LF. 17 Paschal E.H. 111, 121 Parke D.V. 10, 14, 16, 17, 18, 41, 47, 66, 69, 71, 74, 77, 127 Patty F.A. 47 Pavelkova E. 28, 30, 88, 91, 96, 102, 106 Pavlovic A. 105 Pearce O.W. 121 Pearce S.J. 47 Pearson J.T. 85 Penick A.E. 110 Pergal M. 103. 106 Perron R.C.117,119,121 Pesaresi C. 47 Peters T. 106 Peterson J.E. 115, 121 Petrun N.M. 102, 106 Pett R. 46. 52 Pfaffli P. 91,96, 123, 124 Phillips R.L. 121 Phillips W.E.J. 19 Phi-Lich N. 123, 125 Pierce JŻO. 4, 9 Piotrowski J.K. 3, 4, 6, 8, 9, 19, 20, 22, 23, 26, 27, 28, 30, 36, 40, 41, 42, 47, 48, 50, 51, 53, 66,67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 82, 84, 85,103,105,106,110,127 Pleskova A. 123, 124, 125 Poore R.E. 12, 18, 107, 111 Pirkle C.1. 120 Popinska E. 8 Popler A. 83, 84, 85 Porteous J.W. 41, 47, 66, 67, 69 Pospisilova E. 53 Potoniec-Malinowska E. 96 Powell J.F. 86 Powis G. 14,18 Petenyi M. 110 Pozzoli L. 85 Potvin M. 85 Prinz H.J. 107, 111 Pultarova H. 65 Pulus H.J. 105 Quick A.J. 49, 53 Quinby G.E. 117, 120 Radomski J.L. 116, 121 Rainsford 5.0. 43, 46, 47 Rao H.R. 16,18 Ravier E. 66, 69 Reinhart H. 47 Renner O. 110 Rezman l. 105 Richardson A. 121, 124, 125 Rietbrock N. 122. 124 Riley E.C. 123. 125 Ritchie A. 19 Riva M. 124 Roan C.C. 108, 109,111,115,116,117, 118,119, 120,121 Roberts M. 124, 125 Robertson D.N. 124 Robinson D. 71. 74, 76, 80 Robinson J. 74, 121. 124, 125 Robinson W.H. 115, 121 Roeth R.L. 120 Rofe P. 85 Rogaczewska T. 4, 6, 9, 104, 106 Rosowski W. 94, 96 Rothe C.F. 121 Roubal J. 104. 106 Rowe V.K. 65. 124 Rubinskaya S.E. 60. 65 Rusin W.J. 61, 65 Rybkowska J. 65 Salmowa J. 6. 9. 74. 76. 77. 78. 79. 80 Salm J. 72. 74 Salvadeo A. 103, 106 Samuel H.E. 96 Sapota A. 62. 64. 65. 84. 85 Sartorelli A.C. 19 Saschenbrecher R.W. 116. 121 Sassi C. 103.106 Sato J. 114 Sayers R.R. 47 Schaffer A.W. 65. 100 Schaffer GB. 64 Schechter M.S. 116. 121 ScheelLD. 105 Scheline R.R. 58. 59. 60, 64 Schenkman J.B. 14. 19 Schnapp E. 102, 105 Schoor W.P. 115. 121 Schreier K. 57 Schrenk H.N. 42. 47 Schultze MO. 90. 96 Sciarini L.J. 81. 82. 83. 85 Sedivec V. 47. 49. 53. 90. 96 133 Selucky N. 85 Senczuk W. 32, 40. 55, 56. 57 Serebrnaya S.C. 115 Seto T.A. 90. 96 Shafik T.M. 108, 110 Sherwood R.J. 4. 9. 36. 40. 41. 42. 43. 45. 46. 47 Shevchenko N.F. 74 Sidorova E.A. 122. 126 Simko A. 63. 65. 73. 75 Skog E. 95 SkramOVSky St. 42. 46.47. 127 Slob A. 61.62. 65 Smith A.F. 64 Smith B.S. 4. 9 Smith G.F. 86.87.90. 96 Smith 1-‘.D. 110 Smith .1.H. 95 Smith .I.N. 53. 65. 71. 75. 80 Smith W.J. 75 Sniady H. 73. 75. 79. 80 Sokal .1.A. 15. 19 Solomon A.K. 20. 30 Soloway SB. 121 Sonkin J.S. 75 Soucek B. 2. 20. 28. 30. 86. 87. 88. 89. 90. 96. 102. 106. 123. 125 ' Spassovski M. 95. 122. 123. 125 Spencer H.C. 60. 65 Sperling F. 123. 126 Spillane .1.T. 121 Sportelli A. 124 Srhova .1. 41. 47.48.49. 54. 127 Srutek .l. 65 Stein W.J. 110 Stewart CR 127 Stewart RD. 33. 60. 61.63.65. 86. 87. 88. 89. 90. 92. 96. 98. 99. 100.101. 123. 124. 126 Stevens .1.T. 13. 18. 19 Stitzel R.E. 18. 19 Stockell F.R. 105 Stolman A. 66. 127 Street J.C. 16. 19. 116.121 Strittmatter C.F. 102. 106 Styblova V. 97 Sukhotina K.1. 94. 97 Surducki M. 105 Sutton W.Z. 125 Swanson W.W. 48. 53 Swensson A. 109. 110 Sykora J. 65 Symms K.G. 12.18.71. 75 Szadkowski D. 47. 51. 52. 54 134 Tada 0.99. 101 Tanaka S. 90.194. 97 Takatsuka Y. 53. 57. 96. 100 Tappan W.B. 110 Teisinger J. 2. 33. 38. 39. 41. 42, 43, 45. 47, 48, 50. 51. 54.60. 65. 67. 69, 71. 77. 78. 87'. 88, 89. 90. 93. 95.97. 102. 103. 104. 106. 122. 127 Terriere L.C. 120 Theis R.C. 67. 69 Theorsell W. 95 Thorpe W.V. 53. 57 Tomokuni K. 53. 57. 96. 100 Tolokoncev NA. 30. I27 Torkelson T.R. I00 Trojanowska B. 70. 75. 76. 80 Truhaut R. 2. 53. 57. 87. 95 Tyras H. 6. 8. 48. 53. 55. 57. 58. 59.60.65 Ueda N. 122. 126 Uehleke H. 76. 80 Umberger C.J. 49. 54 Upholt W.M. 121 Uselis J. 47. 53. 57 Vanacek M. 106 Vaughn W.K. 110 .Van Haaften A.H. 67. 69 Van Sandt WA. 85 Vasak V. 2. 103. 106. 123. 126 Vasilenko N.M. 73. 75 Versteeg |.P.J. 124. 126 Villeneuve D.C. 13. 19 Vitora A. 46 Vlachova D. 2. 78. 80. 86. 87. 88. 90. 97. 108. 109. 1 1 1 Vlasak R. 46.47. 85 Volfova B. 64 Volkova Z.A. 122. 126 Volodchenko V.A. 75 Von'Oettingen W.F. 49. 50. 54. 78. 801 Voskoboinikov N.A. 75 Vrba J. 60. 65 Vukojevic N. 106 Vyskocil J. 90. 93. 94.95 Waelschova A. 94. 97 Waggoner T.B. 125 Wagstaff B.J. 16. 19 Waldmann R.K.78.108.110. 111 Wales P..l. 116. 121 Walker K.C. 107. 110. 120 Walker R. 12. 18 Walkley J.E. 43. 46. 47 Watanabe S. 108.109. 111.113. 114 Wang M. 121 Watrorski K. 66. 69 Weil C.S. 64 Weist H. 103. 106 Westendorp J. 86. 93. 94. 95 White K. 53 Wiglusz R. 47. 53 WiIlhite M. 60. 64 Williams M.W. 108. 109. 111 Williams R.T. 2. 10. 19. 41.47. 58. 65. 66. 67. 69. 71. 73. 75. 76. 80. 81. 86. 127 Wills B.D. l6. 18 Wilson G.E. 18 Witter R.F. 110 WisnieWska-Knypl J.M. 14. 19.41.47. 71. 75 Wolfe H.R.8.109. 110.111 Wood P.B. 53 Wroniewicz Z. 66. 69 Yamamoto K. 122. 126 Yanno F.J. 125 Yam W.P. 42. 47 Yllner S. 98. IOI Yoshida J. 103. 106 Zatman L.J. 122. 125 Zaremba Z. 74. 84 Zenon M.R. 84. 85 ch7 C. 4. 9 Zielinska H. 49. 53 Zielhuis R.L. 43.46 Zlotolov H. 80 Zvezdai U. 74 24. SUBJECT INDEX Absorption routes 3 lungs 3 skin 4 contact absorption 5 absorption of vapours 6 in occupational exposure 7 (see also” individual substances) Acetone 122 Aldrin 124 Aliphatic alcohols 122 p-Amino-m—cresol 112 o-Aminophenol 70, 76 m-A'minophenol 70, 76 p-Aminophenol 71, 72, 73, 76 as metabolite of aniline 70 determination of physiological concentrations 72 as exposure test to aniline 72 as metabolite of nitrobenzene 76 Aniline 70, 76 absorption 70 metabolism 10. 70 determination 71 determination of metabolites 71 exposure test 72 specifity of 63 industrial exposure 73 MAC values 72, 73 Aromatic amines determination in urine 82 Atrolactic acid 123 Autoinduction of biotransformations 15 Azlactone 49 Benzene 4| absorption 41 biotransformation 41 elimination 41 determination 42 determination of metabolites 42 exposure tests 43, 46 industrial exposure 46 Benzidine 81 absorption 8| biotransformation 81 elimination 81 determination of 81 determination of metabolites 82 135 medical control 82 exposure test 83 industrial exposure 83 bladder cancer 83 Benzoic acid 48 determination of 48 physiological levels 48. 50 as exposure test for toluene 49 as styrene metabolite 6| Biotransformation 10 interspecies differences 10 mechanisms 10 stimulation of 13 inhibition of 15 physiological factors 16 (see also individual substances) Carbon disulphide 102 absorption 102 metabolism 102 elimination 102 determination 103 determination of metabolites 103 exposure test 103 industrial exposure 104 Carbon tetrachloride 123 Chloral hydrate 86, 98 Chlorinated hydrocarbons 123 Chloroform 86. 123 Cholinesterase activity 107, 109 Collective exposure tests 39 Conjugation 12 Cumulation 27 (see also individual substances) Cytochrome b5 ll Cytochrome P-450 11 DDA -— 115 determination 117 background levels 117 as exposure test for DDT 118 in subjects exposed professionally 119 DDD115 DDE115 DDMU115 DDT 115 absorption 115 136 metabolism 115 elimination 116 determination 116 determination of metabolites 117 background levels 117 exposure tests 118 occupational exposure 119 Dianisidine 82 Dichlorobenzidine 82 p-Dichlorobenzene 123 2,5—Dichlorophenol 123 Dieldrin 124 Diethyleneglycol dinitrate 123 Dithiocarbaminocarbonates 102 Ethyl alcohol 122 Ethylbenzene 58 absorption 58 metabolism 58 elimination 58 determination 58 determination of metabolites 58 exposure test 59 industrial exposure 59 Ethylene glycol 98 p-Ethylphenol 58 p-Ethylphenol 58 Expired air analysis 31 Exposure chambers 32 Exposure tests elaboration of 31 on volunteers 31 toxicological chambers 32 design of eXperiments 33 inhalatory experiments 33 cutaneous eXposure 36 repeated exposure 37 precision of 38 safety problems 39 Extrahepatic metabolism 12 Extramicrosomal biotransformations 12 Fenitrothion 112 absorption 112 metabolims 112 elimination 112 determination in blood 113 exposure test 113 Formaldehyde 122 Furfural 123 Furoilglycine 123 Glycol dinitrate 123 Hippuric acid as metabolite of styrene 61 as metabolite of ethylbenzene 58 as metabolite of toluene 48 determination of 48 physiological levels 49 as exposure test for toluene 50 Hydroquinone 41, 42, 66 3—Hydroxybenzidine 81 Hydroxyhydroquinone 41 Hydroxylation 11 Induction of microsomal enzymes 13 see also individual substances lnhalatory absorption 33 Inhibition of metabolism 15 Iodine-azide reaction 103 Kinetics 20 kinetic models 20 half-time 23 rhythms of exposure 24 continuous exposure 24 interrupted exposure 26 cumulation 27 principle of summation 27 weekly rhythm 28 in exposure tests 29 Mandelic acid as ethylbenzene metabolite 58 as styrene metabolite 60 determination 61 physiological levels 62 as exposure test for styrene 62 as exposure tests for ethylbenzene 59 2—Mercapto—Z—thiazolin—S—on 103 Merichinoid 81 Mesithylene 123 Mesithylenic acid 123 Methanol 122 Methemoglobin 76 Methyl chloride 123 Methylene chloride 123 alpha-Methylstyrene 123 Methylhippuric acids 49, 55 as xylenes metabolites 55 determination 55 as exposure tests for xylenes 55 Metabolism see biotransformation Microsomal metabolism 11 Monochloroacetic acid 86 Muconic acid 41 Nitrobenzene 73. 76 absorption 76 metabolism 10, 76 metabolic kinetics 77 determination of metabolites 77 exposure test 78 specifity of 79 industrial exposure 79 m-Nitrophenol 76 p-Nitrophenol as metabolite of nitrobenzene 76 determination in urine 77, 108 as exposure test for parathion 108 as exposure test for nitrobenzene 78 p-Nitro-m-cresol 112 determination in urine 112 Nitrocompounds aromatic in urine determination of 78 Nitrosobenzene 76 Oxalic acid 98 Oxazolone 49 Paraoxonase 107 Parathion 107 absorption 107 metabolism 107 excretion 107 determination 108 exposure test 108 industrial exposure 109 Pentachlorophenol l23, 124 Phenol 11 absorption 66 metabolism 66 elimination 66 determination 66 exposure test 67 industrial exposure 68 as benzene metabolite 41 physiological urinary levels 67 Phenol test for phenol 67 for benzene 43 Phenylglyoxylic acid as styrene metabolite 60 determination of 61 physiological levels 62 as ethylbenzene metabolite 58 beta-Phenylhydroxylamine 76 Phenaceturic acid 59 Phenyl acetate 58 Phenylmercapturic acid 41 Phenyl-methylcarbinol 58 Phenacetin 73 2—Propanol 122 i-Propylbenzene 123 Phosphatases 108 137 Pyrocatechol 41, 42, 66 Retention in lungs 3 see also ind iv1dual substances Respiratory absorption 3 see also indivrdual substances Skin absorption 4 see also individual substances Stimulation of metabolism 13 Styrene 60 absorption 60 metabolism 10, 60 determination 61 determination of metabolites 61, 62 exposure tests 62, 63 individual exposure 63, 64 Styrene oxide 61 l.l.l.2-Tetrachloroethane 123 Tetrachloroethylene 98 Tetraethylthiuram disulphide 87 Thiazolidone 102 Thiourea 102 Tolidine 82 Toluene 48 absorption 48 metabolism 48 excretion 48 determination 48 exposure tests 49, 50, 51 industrial exposure 52 kinetics 51 Toluic acids 55 Total trichlorocompounds (TTC) as metabolites of tetrachloroethylene 99 as metabolites of trichloroethylene 88, 89 kinetics of excretion 88 determination 91 Trichloroacetic acid (TCA) as tetrachloroethylene metabolite 98, 99 as trichloroethylene metabolite 86 tissue distribution 87 kinetics of excretion 88 determination 90 Trichloroethanol (TCE) as trichloroethylene metabolite 86 kinetics of excretion 88 determination 90 Trichloroethylene epoxide 86 l,l,l—Trichloroethane 123 Trichloroethylene 86 absorption 86 tissue distribution 86 metabolism 86, 87 elimination 87 138 elimination kinetics 87, 88, 89 cumulation of metabolites 88 determination 89 determination of metabolites 89, 90 exposure tests 91, 92, 93, 94 industrial exposure 94 Urochloralic acid 86 see also trichloroethanol Vinyl chloride I23 4—Vinyl phenol 60 Viscose rayon plants 104 Xylenes 49, 55 absorption 55 metabolism 55 determination 55 . determination of metabolites 55 exposure tests m-Xylene 55 p-Xylene 55 o-Xylene 55 Xylenols 55 71k 0. 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