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Unpredictability: An essay in toxicology
Fd Chem. Toxic. Vol. 24, No. 4, pp. 343-349, 1986 Printed in Great Britain
0278-6915/86 $3.00+0.00 Pergamon Journals Ltd
Review Section UNPREDICTABILITY: AN ESSAY IN TOXICOLOGY* I. F. H. PURCHASE Imperial Chemical Industries pie, Central Toxicology Laboratory, Alderley Park, Macclesfield, Cheshire SK10 4TJ, England (Received 4 January 1986)
"Extrapolation in toxicology is more than using carbon paper" A. F. Rahde
Toxicology is one of the few scientific disciplines that can have an immediate and enormous effect on public policy. It is difficult to think of any other scientific discipline that, from the simplest result, can create major policy impacts. A recent example is that of nitrophenyipentadiene (NPPD--otherwise known as spy dust) which is alleged by the US State Department to be used as part of Soviet surveillance of US diplomats in Moscow. The fact that NPPD is mutagenic in the Salmonella mutation assay (Ames test) turned the allegation from a minor incident into a cancer scare with international repercussions. Toxicology plays a key role in preventative medicine by providing information on the human hazard of natural and synthetic chemicals that may be widely distributed in the environment and used for a wide variety of purposes in modem society. The importance of the role of toxicology and the consequences of interpretations of toxic hazard place a great premium on accurate interpretation and assessment of toxicological data. It is a privilege to be able to use the opportunity of the Annual BIBRA Scientific Lecture to address a distinguished audience on a subject of such importance. There is a long history of general public interest in preventing disease. This can be traced back to the laws of Moses, which prohibited the consumption of pork, presumably because of the health hazard presented by the cysticercosis transmitted through incompletely cooked pork. I am intrigued by a statement by Pliny the Elder in the first century AD who said "So many poisons are employed to force wine to suit our taste and we are surprised it is not wholesome". His comment seems relevant today as the Austrian Government struggles with the consequences of the deliberate addition of diethylene glycol to wine to enhance its flavour. It is interesting to reflect on how Pliny was able to identify poisons and it must be presumed that this was based on the observation of illness in people after injection of these chemicals. It was not until 19 centuries later that
techniques other than observations on man were used to assess the toxicity of chemicals. In the early 20th century, Dr Harvey Wiley, the Director of the Bureau of Chemistry which subsequently became the Food and Drug Administration in the USA, was faced with the problem of assessing the safety of a variety of chemicals used in food (Hutt, 1985). His response was to set up a "poison squad" of 12 young male employees. He then carried out feeding experiments using five chemicals that were widely used in foodstuffs at the time. This probably represents one of the earliest attempts at the scientific investigation of the safety of chemicals. During the next 20 years the ethical concerns associated with the use of human volunteers in experiments of this type led to the use of animals in safety testing. The development of strains of laboratory animals, and of methods of maintaining them in laboratories under conditions of good health, made it possible to carry out reproducible experiments on animals and led to the beginnings of experimental toxicology. Ever since the time that toxicologists came to rely on the use of animals as surrogates for man, accurate predictability has been a major issue in assessing human hazard. For this lecture, I have chosen three examples where there is sufficient knowledge to suggest that straightforward prediction from simple results would be misleading; it is for that reason that I have included the word "unpredictability" in the title. In the majority of situations in toxicology, reasonably straightforward predictions based on animal data have proved to be sufficiently accurate for preventative purposes. By studying the exceptions to this general rule, there are some valuable lessons to be learnt about the techniques used in human hazard assessment. Short-term tests for carcinogens: an unpredictable paradox The introduction of the Salmonella microsome assay by Ames in 1973 filled a long-felt need for a cheap and quick method for predicting chemical carcinogenicity. The utility of this and other techniques was initially demonstrated by a variety of validation studies (Table 1). The early studies suggested that it was possible to achieve sensitivity and specificity values of around 90%, giving an overall accuracy of prediction of around 90% for both
*Address to the 1986 BIBRA Annual Scientific Meeting, held on 14 October at the Royal College of Physicians, London. 343
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I. F. H. PURCHASE Table 1. Performance characteristics of the Ames test No. of compounds Sensitivity* Speciflcityt Reference 300 90 87 McCann, Choi, Yamasaki & Ames (1975) 120 91 93 Purchase et al. (1978) 146 93 77 Sugimura, Sato, Nagao et al. (1976) 86 50 78 Poirier & de Serres (1979) 42 42-68 59-82 de Serres & Ashby (1981) *The percentage of positive results from the carcinogens tested. i'Tbe percentage of negative results from the non-carcinogens tested. Table 2. Number of positive results to be expected if 1000 chemicals with a known prevalenceof carcinogens are tested in a test of specified accuracy* No. of positive resultsl" Test accuracy Prevalenceof (%) carcinogens (%)... 1 5 99 20 (50%) 59 (I 6%) 90 I08 (92%) 140 (68%) *It is assumed that the sensitivityand specificityvalues are identical and equal to the accuracy. ?The figures in brackets are the percentage of the positive results that would be "false' (i.e. positive results for non-carcinogens).
carcinogens and non-carcinogens. Some more recent studies have shown lower predictivity values and it is worth examining the reasons for the differences in results from the various validation studies. It is fairly obvious that test performance and test reproducibility are primary variables influencing the predictivity results obtained from validation studies. We pointed out in the report of the large validation study carried out in ICI's Central Toxicology Laboratory (Purchase, Longstaff, Ashby e t al. 1978) that at least two other factors influenced the outcome. The first of these was the selection of the chemicals used in the validation study. It has become even clearer now that it is possible to select a group of carcinogens and non-carcinogens in such a way as to produce excellent or p o o r results from a validation study. This has an influence on the way in which studies are designed and interpreted and also on the interpretation of the results of testing a single compound. In the latter situation the use of 'chemical class control pairs' is a considerable aid to the judgement of the relevance of the results (Ashby & Purchase, 1977). The second feature that needs to be considered in evaluating validation studies is the influence of prevalence on sensitivity and specificity. The issue here is that the fewer the carcinogens in the sample of chemicals tested, the greater the number of falsepositive results obtained (Table 2 and Fig. 1) and v i c e v e r s a . In the limiting case in which a group of
chemicals, all of which are non-carcinogenic, are tested in the Ames test, all positive results will be wrong. There arc thus three major variables that influence the outcome of a validation study of this type, namely, testperformance (including reproducibility), chemical type and prevalence. One can assume that by suitable technical control, the test performance and reproducibilitycan be optimized. However, when testing a single chemical, one has no idea about the prevalence of carcinogens unless a good deal of judgement is used about the type of chemical structure being tested.
One of the consequences of using tests that have a finite false-negative rate has been the suggestion that batteries of tests will increase the probability of detecting carcinogenic chemicals. While this may well be true, it is not generally recognized that a very severe penalty is paid for the improvement in sensitivity provided by a battery of short-term tests; the penalty is that the specificity o f testing decreases appreciably with each additional test that is added. This first became obvious in the analysis of the results of the "International Collaborative Programme on Short-term Tests for Carcinogens" (de Serres & Ashby, 1981). In this international study, 42 chemicals (25 carcinogens, 13 non-carcinogens and four
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Fig. 1. The effect of prevalence (% carcinogens in the sample) on sensitivity (or probability that a compound is a carcinogen after an Ames-positive result). Sensitivity values are calculated from data in Purchase et al. (1978). In the limiting case, where there are no carcinogens in the sample, all positive results are false. Similarly, where all chemicals are carcinogens, there are no false positive results.
345
Unpredictability: an essay in toxicology Table 3. The number of positive outcomes to be expected from testing a group of 100 non-carcinogens with between one and four in vitro short-term predictive tests of 90% accuracy No. of positive outcomes
No. of tests
0
1
2
3
4
At least 1
I 2 3 4
90 81 72.9 65.6
l0 18 24.3 29.2
-1 2.7 4.9
--0.1 0.3
---<0.1
10 19 27.1 34.4
compounds of unknown carcinogenicity) were supplied to a variety of laboratories which used a total of 35 short-term tests in a very carefully controlled comparative study. It was possible to examine the consequences of using batteries of tests on the overall ability to predict carcinogenicity and non-carcinogenicity by selection of results from this database (Purchase, 1980). Eight tests were selected on the basis of the criteria laid down in the EPA guidelines for testing pesticides under the Federal Insecticide, Fungicide and Rodenticide Act. These guidelines require that new pesticides should be tested in three point mutation assays, three chromosomal level assays, and two assays capable of assessing damage to DNA. Eight tests were selected from the International Study and all the tests selected had sensitivity and specificity values between 60 and 85%. When the carcinogens were subjected to eight tests, all 25 had between one and eight positive results. At the same time, however, ten of the 13 non-carcinogens also had at least one positive result. It is possible to calculate the likely outcome of testing chemicals with short-term tests of varying accuracy. In Table 3 the likely outcome of using four tests of 90% accuracy for the testing of a group of non-carcinogens is presented. This illustrates the point that the more tests are used, the lower the specificity of the overall prediction will be. These calculations can be taken a stage further by considering the likely outcome of using eight tests of, say, 90% accuracy to test a group of I00 chemicals, half of which are carcinogens and half non-carcinogens. In Table 4 the distribution of results derived from such a calculation is presented. This can be compared with the results obtained from the International Collaborative Study presented in the same table. The practical results from the international study are clearly incapable of classifying the chemicals clearly
into groups of carcinogens and non-carcinogens solely on the basis of the results of eight tests. The most likely explanation for the discrepancy between the results from the international study and the theoretical results is that the average sensitivity and specificity values of the eight tests were in the region of 70%. This can be confirmed by calculation (Table 4). This example is based on a number of assumptions such as the statistical independence of the tests used, and the identification of a chemical as carcinogenic on the basis of a single positive result in the battery of eight tests. These assumptions may not be realistic but they are used here to illustrate the importance of using judgement in the interpretation of the results of short-term tests. The paradox of obtaining a rfiajority of incorrect predictions when using a battery of useful tests should serve to reinforce this point. In the next two examples I shall consider the human hazard from two chemicals (trichloroethylene and hexachlorobutadiene) which have the common feature of being non-mutagenic in the Ames test but carcinogenic to animals. Trichloroethylene-a carcinogen?
Trichloroethylene increases the incidence of hepatocellular carcinoma in mice when administered by gavage at daily doses of about 1 and 2 g/kg. Similar doses in rats produce no carcinogenic response (National Cancer Institute, 1976; National Toxicology Program, 1983). Trichloroethylene is negative in the majority of in vitro and in vivo mutagenicity tests and does not produce a mutagenic response in the Ames test (Bartsch, Malaveille, Barbin & Planche, 1979; Greim, Bonse, Radwin et al. 1975; Simmon, Kaubarter & Tardiff, 1977; Slacik-Erben, Roll, Franke & 'Uehleke, 1980; Waskell, 1978). This pattern of results raises the question of how to assess the human health hazard from exposure to trichloroethylene. Trichloroethylene is metabolized predominantly to trichloroacetic acid, which appears to be the active metabolite in both rats and mice. The first difference in response between the mouse and the rat can be discerned by estimating the amount of trichloroethylene metabolized at various doses (Fig. 2). In the case of the mouse, there is a linear relationship between the administered dose and the amount metabolized over the range of doses tested. However, in the case of the rat, doses above about 500 mg/kg did not produce a marked increase in the amount metab-
Table 4. Theoretical or actual percentages of compounds giving positive results when tested with different batteries of eight independent tests Percentage of compounds giving positive results on 0 ~ tests
No. of positive results...
0
1
2
3
4
5
6
7
8
Tests of 90% accuracy* Tests of 70% accuracyt Tests from Purchase (1980)2
22 3 7
17 10 10
8 15 19
2 15 19
0 14 14
2 15 5
8 15 17
17 10 2
22 3 7
Test battery (8 tests)
*Results calculated assuming eight independent tests of 90% accuracy and test chemicals that were 50% carcinogens and 50% non-carcinogens. tResults calculated assuming eight independent tests of 70% accuracy and test chemicals that were 50% carcinogens and 50% non-carcinogens. :[:Eight tests were selected from the International Collaborative Programme on Short-term Tests for Carcinogens (de Serres & Ashby, 1981) and the percentages of positive results were recorded (Purchase, 1980). O f the 42 compounds tested, 25 were carcinogens.
346
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1985).
et al.
olized, suggesting that the primary metabolic pathway is saturated at high dose levels (Prout, Provan & Green, 1985). The administration of trichloroethylene to mice results in a substantial increase in the number of peroxisomes present in the hepatocytes. There is once again a more or less linear relationship between the dose administered and the level of peroxisome proliferation (Fig. 3). In the rat, however, there is little evidence of an increase in peroxisomes even at 2000mg/kg (Elcombe, 1985). The reason for this difference in response appears to be the different rate at which trichloroacetic acid is produced in the two
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species. Trichloroacetic acid itself is an effective peroxisome proliferator in both rats and mice (Elcombe, 1985); the absence of peroxisome proliferation in the rat after administration of trichloroethylene must be attributed to the relatively low levels of trichloroacetic acid produced even at high doses of trichloroethylene. There are a number of chemicals that induce liver cancer in rodents after inducing peroxisome proliferation. It has been suggested that the induction of peroxisomes is a necessary step for the induction of hepatocellular carcinoma after administration of these chemicals (Reddy & Lalwani, 1983). The precise mechanism by which an increased level of peroxisomes induces cancer is not known, although it is speculated that an increased production of oxygen radicals may be an important step. A direct comparison of metabolism and peroxisome proliferation in mice, rats and man, at the sort of doses that induce cancer in mice, is clearly not ethically acceptable. It is, however, possible to compare the metabolic capacity and the ability to induce peroxisomes in isolated hepatocytes from the three species. The kinetics of metabolism of trichloroethylene in isolated hepatocytes demonstrates that the mouse has a greater relative metabolic capacity than the rat and that bepatocytes from both rodent species have a greater capacity to metabolize trichloroethylene to trichloroacetic acid than have human hepatocytes (Table 5; Elcombe, 1985). These results mimic the results obtained in rats and mice and suggest that man will have a much lower capacity than either rodent species to metabolize trichloroethylene to trichloroacetic acid. In isolated hepatocytes, trichloroacetic acid induces peroxisomes (measured as cyanide-insensitive palmitoyl CoA oxidation) at about the same level in both mouse and rat hepatocytes. Once again, this mimics the result observed in v i v o (Elcombe, 1985).
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Fig. 3. Changes in peroxisome levels (as measured by cyanide-insensitive palmitoyl CoA oxidation) in the livers of rats ( I ) and mice (O) given trichloroethylene in a range of doses (after Elcombe, 1985). Asterisked values differ significantly from the control.
Unpredictability: an essay in toxicology Table 5. Kinetics of trichloroethylene metabolism to trichloroacetic acid (TCA) in isolated hepatocytes Formation of TCA
Species Mouse Rat Man
Enzyme affinity (Km)
Rate of conversion (Vmax)
Relative metabolizing capacity
28 151 165
107 19 6
120 4 1
After Elcombe (1985).
Human hepatocytes do not produce any increase in peroxisomes after administration of trichloroacetic acid, in spite of the fact that other indices of cell survival indicate that the hepatocytes are fully competent. The overall picture that emerges from this work is that the mouse is susceptible to the induction of hepatocellular carcinoma after the administration of trichloroethylene because of the high levels of trichloroacetic acid produced consequent on the linear kinetics of metabolism. Trichloroacetic acid is the proximate toxin inducing peroxisome proliferation and it is presumed that this leads to the induction of liver cancer. In the rat, the amount of trichloroethylene metabolized to trichloroacetic acid is limited by saturation of the metabolic pathway, with the consequence that there is no peroxisome proliferation and no increase in the incidence of hepatocellular carcinoma after the administration of high doses of trichloroethylene. In man, it is likely that the amount of trichloroacetic acid produced will be substantially lower than that seen in the mouse or the rat. A further difference is that human hepatocytes are not responsive to the effects of trichloroacetic acid. This evidence on the mechanism by which trichloroethylene produces cancer in mice and the fact that the mechanisms do not operate in human cells suggest that it is unlikely that trichloroethylene will be carcinogenic in man. This general conclusion is supported by the limited epiderniological evidence that is available. In spite of this information, trichloroethylene is now controlled as a carcinogen in the USA using risk assessment techniques that assume a non-threshold dose response and human sensitivity equal to that of the mouse. Hexachlorobutadiene--a carcinogen! Hexachlorobutadiene (HCBD) is negative in the standard Ames test (de Meester, Mercier & Poncelet, 1981; Stott, Quast & Watanabe, 1981) but increases the incidence of cancer of the kidney after administration to rats (Kociba, Schwetz, Keyes et al. 1977). Acute administration of HCBD produces damage to the proximal convoluted tubule in both mouse and rat kidney (Lock & Ishmael, 19~/9; Ishmael, Pratt & Lock, 1984). Extensive studies have been carried out on the metabolism and mode of action of HCBD at ICI's Central Toxicology Laboratory. The first step in the metabolism of HCBD is conjugation with glutathione and excretion in the bile (Wolf, Berry, Nash et al. 1984; Nash, King, Lock & Green, 1984). After cannulation of the bile duct, HCBD does not produce nephrotoxicity and confirmation of this mechanism is obtained by the
347
administration of HCBD-glutathione conjugate, which does produce nephrotoxicity. The second step in the metabolism is the reabsorption of the HCBD-glutathione conjugate, possibly after cleavage of the glutathione in the intestine. The remaining cysteine conjugate is then accumulated by the kidney (Lock & Ishmael, 1985) where the enzyme fl-lyase further metabolizes the cysteine conjugate. Confirmation of this route of metabolism to the ultimate toxin was obtained by the administration of the N-acetylcysteine conjugate (Nacetylpentachlorobutadienylcysteine) which produced an effect on kidney function similar to that of the parent compound, HCBD (Nash et al. 1984). Rat kidney fl-lyase converts the cysteine conjugate to pyruvate, ammonia and a reactive thiol moiety, as would be predicted by a consideration of the structure (Green & Odum, 1985). The various steps in the metabolism of hexachlorobutadiene to the active thiol compound are presented in Fig. 4. The final piece of information that explains the metabolic activation of HCBD is that the H C B D cysteine conjugate produces mutagenic effects in the Ames test when incubated with rat kidney S-9 (Green & Odum, 1985). This result explains why HCBD itself is not mutagenic when tested in a standard Ames test using rat liver S-9. The complex metabolic pathway results in the appearance of the HCBD-cysteine conjugate in the kidney after administration of HCBD, and further metabolism of this conjugate produces the proximate toxin and carcinogen. Knowledge of metabolic processes in man suggests that the same metabolic pathway will occur in man and therefore it is reasonable to assume that HCBD represents a carcinogenic hazard to man. Both trichloroethylene and HCBD are negative in the standard Ames test but carcinogenic when tested in certain rodent carcinogenicity studies. A simplistic conclusion would be that for trichloroethylene the Ames result provides an accurate prediction, but the mouse liver careinogenicity is a false positive; the converse is true for HCBD, where the mechanism suggests that the Ames result is a false negative and that HCBD represents a carcinogenic hazard to man. Improving hazard assessment--an urgent challenge Modern toxicology is an important branch of preventative medicine and contributes specifically to the assessment of human hazard from exposure to chemicals. Its techniques are laboratory studies and epidemiology and the challenge lies in predicting what effects will occur in man when the only information that is available is that from animals. Over the last 10 years or so there have been major international initiatives aimed at harmonizing methods of testing for new chemicals. This is typified by the OECD guidelines which set out in general terms the protocols that need to be followed in order to assess particular aspects of the toxicology of new chemicals. The EEC and various national authorities also have guidelines that set out the requirements for toxicity testing and, by and large, these are now reasonably consistent with one another. The advantages of having guidelines of this type are that international trade, which requires a common assessment of the hazards of chemicals, is greatly facili-
348
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tated. The main disadvantage is that the assessment of chemical toxicity typically follows the guidelines rigidly and there is little incentive to carry out work that might improve the hazard assessment. For new chemicals, this approach seems to be relatively sarisfactory and it allows a reasonably efficient assessment of hazard without too much disagreement between different national authorities. The situation with older established-products is substantially different. For many of them, evidence from many years of use can provide information about their safety under conditions of use. The application of rigid testing guidelines, which work relatively well for new chemicals, can be less than satisfactory when applied too rigidly to the problem of assessing human hazard from established chemicals. It appears that over the last few years, particularly in the USA and particularly for carcinogenicity, the regulatory assessment has become so rigid that it is losing credibility not only among toxicologists but also among the general public. An example of this is the way in which the application of the Delaney amendment in the case of saccharin was overturned by the United States Congress. The results of toxicological studies have an important bearing on public health and are therefore subject to much political pressure. There is, however, a common duty among all those involved in toxicological assessment, whether they are from industry, from governments or from academic institutions, to improve the process of human hazard assessment and to improve the methods used in toxicology. Industrial toxicologists, who have a particular inter-
est in making accurate hazard assessments, have a major role to play in this field. The industrial contribution is through its own in-house laboratories and also through collaborative efforts such as those carfled out at BIBRA. My fear for the future is that these efforts, which rely heavily on industrial funding, can easily be smothered if they are not encouraged. If the interpretation of government regulations follows the rigid and simplistic acceptance of animal toxicology data as indicative of human hazard, there will be little incentive to improve methods of assessing human hazard. This type of toxicology, aptly referred to by Professor Rahde as carbon paper extrapolation, ignores knowledge of mechanisms of action and therefore provides less than satisfactory assessment of human hazard. Toxicology has developed dramatically in the last few decades to the stage where we can now see how the assessment of human hazard can be improved. This is particularly true for environmental chemicals and chemicals that have a long history of use. Those involved in making regulatory decisions about chemicals for which there are good data have it in their power to encourage the development of improved methods of hazard assessment. By incorporating data on mechanisms of action and interspecies differences in toxicity into hazard assessments, it is possible to provide an incentive to all parties to improve methods of hazard assessment. By doing this, and using the currently available and somewhat fragmentary information about the toxicity of chemicals, it will be possible to encourage industry to harness its resources, apply its skills and develop new methods so that hazards that
Unpredictability: an essay in toxicology currently look unpredictable may become more easily predictable. Acknowledgements--I wish to thank my colleagues, particularly Drs J. Ashby, C. R. Elcombe, T. Green and E. A. Lock and Mr Godley for their help in the preparation of this manuscript.
REFERENCES Ashby J. & Purchase I. F. H. (1977). The selection of appropriate chemical class controls for use with short term tests for potential carcinogenicity. Ann. occup. Hyg. 20, 297. Bartsch H., Malaveille C., Barbin A. & Planche G. (1979). Mutagenic and alkylating metabolites of haloethylenes, chlorobutadienes and dichlorobutenes produced by rodent or human liver tissues. Archs Toxicol. 41, 249. de Meester C., Mercier M. & Poncelet F. (1981). Mutagenic activity of butadiene, hexachlorobutadiene and isoprene. In Industrial and Environmental Xenobiotics. Edited by I. Gut, M. Cikrt & G. L. Plaa. p. 195. Springer Verlag, New York. de Scrres F. J. & Ashby J. (Editors) (1981). Evaluation of Short-term Tests for Carcinogens: Report of the International Collaborative Programme. Elsevier, Amsterdam. Elcombe C. R. (1985). Species differences in carcinogenicity and peroxisome proliferation due to trichloroethylene: a biochemical human hazard assessment. Archs Toxicol. Suppl 8, 6. Green T. & Odum J. (1985). Structure/activity studies of the nephrotoxic and mutagenic action of cysteine conjugates ofchloro and fluoralkenes. Chemico-Biol. Interactions 54, 15. Gmim H., Bonse G., Radwin Z. Reichert D. & Henschler D. (1975). Mutagenicity /n vitro and potential carcinogenicity of chlorinated ethylenes as a function of metabolic oxirane formation. Biochem. Pharmac. 24, 2013. Hutt P. (1985). Use of quantitative risk assessment in regulatory decision making under Federal health and safety statutes. In Risk Quantitation and Regulatory Policy. Edited by D. G. Hoel, R. A. Merrill & F. P. Perera. Banbury Report 19. p. 15. Cold Spring Harbor Laboratory, New York. Ishmael J., Pratt I. S. & Lock E. A. (1984). Hexachloro-l,3butadiene-induced renal tubular necrosis in the mouse. J. Path. 142, 195. Kociba R. J., Schwetz B. A., Keyes D. G., Jersey G. C., Ballard J. J., Dittenber D. A., Quast J. F., Wade C. E. & Humiston C. G. (1977). Chronic toxicity of reproduction studies of hexachlorobutadiene in rats. Envir. Hlth Perspect. 21, 49. Lock E. A. & Ishmael J. (1979). The acute toxic effects of hexachlorobutadiene on the rat kidney. Archs Toxicol. 43, 47. Lock E. A. & Ishmael J. (1985). Effect of the organic acid transport inhibitor probenecid on renal cortical uptake and proximal tubular toxicity of hexachloro-l,3-
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butadiene and its conjugates. Toxic. appl. Pharmac. 81, 32. McCann J., Choi E., Yamasaki E. & Ames B. N. (1975). Detection of carcinogens as mutagens in the Salmonella/microsome test. Part 1. Assay of 300 chemicals. Proc. natn. Acad. Sci. U.S.A. 72, 5135. Nash J. A., King L. J., Lock E. A. & Green T. (1984). The metabolism and disposition of hexachloro-l,3-butadiene in the rat and its relevance to nephrotoxicity. Toxic. appl. Pharmac. 73, 124. National Cancer Institute (1976). Carcinogenesis Bioassay of Trichloroethylene. CAS No. 79-01-6, DHEW Publ. No (NIH) 76. National Toxicology Program (1983). National Toxicology Program draft report abstracts on nine chemical carcinogenesis bioassays. Chem. Regul. Rep. 6, 767. Poirier L. & de Serres F. J. (1979). Initial National Cancer Institute Studies on mutagenesis as a prescreen for chemical carcinogens: an appraisal. J. hath. Cancer Inst. 62,_ 919. Prout M. S., Provan W. M. & Green T. (1985). Species differences in response to trichloroethylene. I. Pharmacokinetics in rats and mice. Toxic. appl. Pharmac. 79, 389. Purchase I. F. H. (1980). Appraisal of the merits and shortcomings of tests of mutagenic potential. In Mechanisms of Toxicity and Hazard Evaluation: Proceedings o f the Second International Congress o f Toxicology. Edited by B. Holmstedt, R. Lawerys, M. Mercier & H. Roberfroid, p. 105. Elsevier, Amsterdam. Purchase I. F. H., Longstaff E., Ashby J., Styles J. A., Anderson D., Lefevre P. A. & Westwood F. R. (1978). An evaluation of 6 short term tests for detecting organic chemical carcinogens. Br. J. Cancer 37, 873. Reddy J. K. & Lalwani N. D. (1983). Carcinogenesis by hepatic peroxisome proliferators: evaluation of the risks of hypolipidemic drugs and industrial plasticizers to humans. CRC Crit. Rev. Toxicol. 13, 1. Simmon V. F., Kaubaner K. & Tardiff R. G. (1977). Mutagenic activity of chemicals identified in drinking water. In Progress in Genetic Toxicology. Edited by B. A. Bridges & F. H. Sobels. p. 249. Elsevier, Amsterdam. Slacik-Erben R., Roll R., Franke G. & Uehleke H. (1980). Trichloroethylene vapours do not produce dominant lethal mutations in male mice. Archs Toxicol. 45, 37. Stott W. T., Quast J. F. & Watanabe P. G. (1981). Differentiation of the mechanisms of oncogenicity of 1,4-dioxane and 1,3-hexachlorobutadiene in the rat. Toxic. appl. Pharmac. 60, 287. Sugimura T., Sato S., Nagao M,, Yahagi T., Marsushima T., Semo Y., Takeuchi M. & Kawachi T. (1976). Overlapping of carcinogens and mutagens. In Fundamentals in Cancer Prevention. Edited by P. N. Magee et aLp. 191. University of Tokyo Press. Waskell L. (1978). A study of the mutagenicity of anaesthetics and their metabolites. Mutation Res. 57, 141. Wolf C. R., Berry P. N., Nash J. A., Green T. & Lock E. A. (1984). Role of microsomal and cytosolic glutathione S-transferases in the conjugation of hexachloro1,3-butadiene and its possible relevance to toxicity. J. Pharmac. exp. Ther. 228, 202.
0278-6915/86 $3.00+0.00 Pergamon Journals Ltd
Review Section UNPREDICTABILITY: AN ESSAY IN TOXICOLOGY* I. F. H. PURCHASE Imperial Chemical Industries pie, Central Toxicology Laboratory, Alderley Park, Macclesfield, Cheshire SK10 4TJ, England (Received 4 January 1986)
"Extrapolation in toxicology is more than using carbon paper" A. F. Rahde
Toxicology is one of the few scientific disciplines that can have an immediate and enormous effect on public policy. It is difficult to think of any other scientific discipline that, from the simplest result, can create major policy impacts. A recent example is that of nitrophenyipentadiene (NPPD--otherwise known as spy dust) which is alleged by the US State Department to be used as part of Soviet surveillance of US diplomats in Moscow. The fact that NPPD is mutagenic in the Salmonella mutation assay (Ames test) turned the allegation from a minor incident into a cancer scare with international repercussions. Toxicology plays a key role in preventative medicine by providing information on the human hazard of natural and synthetic chemicals that may be widely distributed in the environment and used for a wide variety of purposes in modem society. The importance of the role of toxicology and the consequences of interpretations of toxic hazard place a great premium on accurate interpretation and assessment of toxicological data. It is a privilege to be able to use the opportunity of the Annual BIBRA Scientific Lecture to address a distinguished audience on a subject of such importance. There is a long history of general public interest in preventing disease. This can be traced back to the laws of Moses, which prohibited the consumption of pork, presumably because of the health hazard presented by the cysticercosis transmitted through incompletely cooked pork. I am intrigued by a statement by Pliny the Elder in the first century AD who said "So many poisons are employed to force wine to suit our taste and we are surprised it is not wholesome". His comment seems relevant today as the Austrian Government struggles with the consequences of the deliberate addition of diethylene glycol to wine to enhance its flavour. It is interesting to reflect on how Pliny was able to identify poisons and it must be presumed that this was based on the observation of illness in people after injection of these chemicals. It was not until 19 centuries later that
techniques other than observations on man were used to assess the toxicity of chemicals. In the early 20th century, Dr Harvey Wiley, the Director of the Bureau of Chemistry which subsequently became the Food and Drug Administration in the USA, was faced with the problem of assessing the safety of a variety of chemicals used in food (Hutt, 1985). His response was to set up a "poison squad" of 12 young male employees. He then carried out feeding experiments using five chemicals that were widely used in foodstuffs at the time. This probably represents one of the earliest attempts at the scientific investigation of the safety of chemicals. During the next 20 years the ethical concerns associated with the use of human volunteers in experiments of this type led to the use of animals in safety testing. The development of strains of laboratory animals, and of methods of maintaining them in laboratories under conditions of good health, made it possible to carry out reproducible experiments on animals and led to the beginnings of experimental toxicology. Ever since the time that toxicologists came to rely on the use of animals as surrogates for man, accurate predictability has been a major issue in assessing human hazard. For this lecture, I have chosen three examples where there is sufficient knowledge to suggest that straightforward prediction from simple results would be misleading; it is for that reason that I have included the word "unpredictability" in the title. In the majority of situations in toxicology, reasonably straightforward predictions based on animal data have proved to be sufficiently accurate for preventative purposes. By studying the exceptions to this general rule, there are some valuable lessons to be learnt about the techniques used in human hazard assessment. Short-term tests for carcinogens: an unpredictable paradox The introduction of the Salmonella microsome assay by Ames in 1973 filled a long-felt need for a cheap and quick method for predicting chemical carcinogenicity. The utility of this and other techniques was initially demonstrated by a variety of validation studies (Table 1). The early studies suggested that it was possible to achieve sensitivity and specificity values of around 90%, giving an overall accuracy of prediction of around 90% for both
*Address to the 1986 BIBRA Annual Scientific Meeting, held on 14 October at the Royal College of Physicians, London. 343
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I. F. H. PURCHASE Table 1. Performance characteristics of the Ames test No. of compounds Sensitivity* Speciflcityt Reference 300 90 87 McCann, Choi, Yamasaki & Ames (1975) 120 91 93 Purchase et al. (1978) 146 93 77 Sugimura, Sato, Nagao et al. (1976) 86 50 78 Poirier & de Serres (1979) 42 42-68 59-82 de Serres & Ashby (1981) *The percentage of positive results from the carcinogens tested. i'Tbe percentage of negative results from the non-carcinogens tested. Table 2. Number of positive results to be expected if 1000 chemicals with a known prevalenceof carcinogens are tested in a test of specified accuracy* No. of positive resultsl" Test accuracy Prevalenceof (%) carcinogens (%)... 1 5 99 20 (50%) 59 (I 6%) 90 I08 (92%) 140 (68%) *It is assumed that the sensitivityand specificityvalues are identical and equal to the accuracy. ?The figures in brackets are the percentage of the positive results that would be "false' (i.e. positive results for non-carcinogens).
carcinogens and non-carcinogens. Some more recent studies have shown lower predictivity values and it is worth examining the reasons for the differences in results from the various validation studies. It is fairly obvious that test performance and test reproducibility are primary variables influencing the predictivity results obtained from validation studies. We pointed out in the report of the large validation study carried out in ICI's Central Toxicology Laboratory (Purchase, Longstaff, Ashby e t al. 1978) that at least two other factors influenced the outcome. The first of these was the selection of the chemicals used in the validation study. It has become even clearer now that it is possible to select a group of carcinogens and non-carcinogens in such a way as to produce excellent or p o o r results from a validation study. This has an influence on the way in which studies are designed and interpreted and also on the interpretation of the results of testing a single compound. In the latter situation the use of 'chemical class control pairs' is a considerable aid to the judgement of the relevance of the results (Ashby & Purchase, 1977). The second feature that needs to be considered in evaluating validation studies is the influence of prevalence on sensitivity and specificity. The issue here is that the fewer the carcinogens in the sample of chemicals tested, the greater the number of falsepositive results obtained (Table 2 and Fig. 1) and v i c e v e r s a . In the limiting case in which a group of
chemicals, all of which are non-carcinogenic, are tested in the Ames test, all positive results will be wrong. There arc thus three major variables that influence the outcome of a validation study of this type, namely, testperformance (including reproducibility), chemical type and prevalence. One can assume that by suitable technical control, the test performance and reproducibilitycan be optimized. However, when testing a single chemical, one has no idea about the prevalence of carcinogens unless a good deal of judgement is used about the type of chemical structure being tested.
One of the consequences of using tests that have a finite false-negative rate has been the suggestion that batteries of tests will increase the probability of detecting carcinogenic chemicals. While this may well be true, it is not generally recognized that a very severe penalty is paid for the improvement in sensitivity provided by a battery of short-term tests; the penalty is that the specificity o f testing decreases appreciably with each additional test that is added. This first became obvious in the analysis of the results of the "International Collaborative Programme on Short-term Tests for Carcinogens" (de Serres & Ashby, 1981). In this international study, 42 chemicals (25 carcinogens, 13 non-carcinogens and four
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Fig. 1. The effect of prevalence (% carcinogens in the sample) on sensitivity (or probability that a compound is a carcinogen after an Ames-positive result). Sensitivity values are calculated from data in Purchase et al. (1978). In the limiting case, where there are no carcinogens in the sample, all positive results are false. Similarly, where all chemicals are carcinogens, there are no false positive results.
345
Unpredictability: an essay in toxicology Table 3. The number of positive outcomes to be expected from testing a group of 100 non-carcinogens with between one and four in vitro short-term predictive tests of 90% accuracy No. of positive outcomes
No. of tests
0
1
2
3
4
At least 1
I 2 3 4
90 81 72.9 65.6
l0 18 24.3 29.2
-1 2.7 4.9
--0.1 0.3
---<0.1
10 19 27.1 34.4
compounds of unknown carcinogenicity) were supplied to a variety of laboratories which used a total of 35 short-term tests in a very carefully controlled comparative study. It was possible to examine the consequences of using batteries of tests on the overall ability to predict carcinogenicity and non-carcinogenicity by selection of results from this database (Purchase, 1980). Eight tests were selected on the basis of the criteria laid down in the EPA guidelines for testing pesticides under the Federal Insecticide, Fungicide and Rodenticide Act. These guidelines require that new pesticides should be tested in three point mutation assays, three chromosomal level assays, and two assays capable of assessing damage to DNA. Eight tests were selected from the International Study and all the tests selected had sensitivity and specificity values between 60 and 85%. When the carcinogens were subjected to eight tests, all 25 had between one and eight positive results. At the same time, however, ten of the 13 non-carcinogens also had at least one positive result. It is possible to calculate the likely outcome of testing chemicals with short-term tests of varying accuracy. In Table 3 the likely outcome of using four tests of 90% accuracy for the testing of a group of non-carcinogens is presented. This illustrates the point that the more tests are used, the lower the specificity of the overall prediction will be. These calculations can be taken a stage further by considering the likely outcome of using eight tests of, say, 90% accuracy to test a group of I00 chemicals, half of which are carcinogens and half non-carcinogens. In Table 4 the distribution of results derived from such a calculation is presented. This can be compared with the results obtained from the International Collaborative Study presented in the same table. The practical results from the international study are clearly incapable of classifying the chemicals clearly
into groups of carcinogens and non-carcinogens solely on the basis of the results of eight tests. The most likely explanation for the discrepancy between the results from the international study and the theoretical results is that the average sensitivity and specificity values of the eight tests were in the region of 70%. This can be confirmed by calculation (Table 4). This example is based on a number of assumptions such as the statistical independence of the tests used, and the identification of a chemical as carcinogenic on the basis of a single positive result in the battery of eight tests. These assumptions may not be realistic but they are used here to illustrate the importance of using judgement in the interpretation of the results of short-term tests. The paradox of obtaining a rfiajority of incorrect predictions when using a battery of useful tests should serve to reinforce this point. In the next two examples I shall consider the human hazard from two chemicals (trichloroethylene and hexachlorobutadiene) which have the common feature of being non-mutagenic in the Ames test but carcinogenic to animals. Trichloroethylene-a carcinogen?
Trichloroethylene increases the incidence of hepatocellular carcinoma in mice when administered by gavage at daily doses of about 1 and 2 g/kg. Similar doses in rats produce no carcinogenic response (National Cancer Institute, 1976; National Toxicology Program, 1983). Trichloroethylene is negative in the majority of in vitro and in vivo mutagenicity tests and does not produce a mutagenic response in the Ames test (Bartsch, Malaveille, Barbin & Planche, 1979; Greim, Bonse, Radwin et al. 1975; Simmon, Kaubarter & Tardiff, 1977; Slacik-Erben, Roll, Franke & 'Uehleke, 1980; Waskell, 1978). This pattern of results raises the question of how to assess the human health hazard from exposure to trichloroethylene. Trichloroethylene is metabolized predominantly to trichloroacetic acid, which appears to be the active metabolite in both rats and mice. The first difference in response between the mouse and the rat can be discerned by estimating the amount of trichloroethylene metabolized at various doses (Fig. 2). In the case of the mouse, there is a linear relationship between the administered dose and the amount metabolized over the range of doses tested. However, in the case of the rat, doses above about 500 mg/kg did not produce a marked increase in the amount metab-
Table 4. Theoretical or actual percentages of compounds giving positive results when tested with different batteries of eight independent tests Percentage of compounds giving positive results on 0 ~ tests
No. of positive results...
0
1
2
3
4
5
6
7
8
Tests of 90% accuracy* Tests of 70% accuracyt Tests from Purchase (1980)2
22 3 7
17 10 10
8 15 19
2 15 19
0 14 14
2 15 5
8 15 17
17 10 2
22 3 7
Test battery (8 tests)
*Results calculated assuming eight independent tests of 90% accuracy and test chemicals that were 50% carcinogens and 50% non-carcinogens. tResults calculated assuming eight independent tests of 70% accuracy and test chemicals that were 50% carcinogens and 50% non-carcinogens. :[:Eight tests were selected from the International Collaborative Programme on Short-term Tests for Carcinogens (de Serres & Ashby, 1981) and the percentages of positive results were recorded (Purchase, 1980). O f the 42 compounds tested, 25 were carcinogens.
346
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1985).
et al.
olized, suggesting that the primary metabolic pathway is saturated at high dose levels (Prout, Provan & Green, 1985). The administration of trichloroethylene to mice results in a substantial increase in the number of peroxisomes present in the hepatocytes. There is once again a more or less linear relationship between the dose administered and the level of peroxisome proliferation (Fig. 3). In the rat, however, there is little evidence of an increase in peroxisomes even at 2000mg/kg (Elcombe, 1985). The reason for this difference in response appears to be the different rate at which trichloroacetic acid is produced in the two
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species. Trichloroacetic acid itself is an effective peroxisome proliferator in both rats and mice (Elcombe, 1985); the absence of peroxisome proliferation in the rat after administration of trichloroethylene must be attributed to the relatively low levels of trichloroacetic acid produced even at high doses of trichloroethylene. There are a number of chemicals that induce liver cancer in rodents after inducing peroxisome proliferation. It has been suggested that the induction of peroxisomes is a necessary step for the induction of hepatocellular carcinoma after administration of these chemicals (Reddy & Lalwani, 1983). The precise mechanism by which an increased level of peroxisomes induces cancer is not known, although it is speculated that an increased production of oxygen radicals may be an important step. A direct comparison of metabolism and peroxisome proliferation in mice, rats and man, at the sort of doses that induce cancer in mice, is clearly not ethically acceptable. It is, however, possible to compare the metabolic capacity and the ability to induce peroxisomes in isolated hepatocytes from the three species. The kinetics of metabolism of trichloroethylene in isolated hepatocytes demonstrates that the mouse has a greater relative metabolic capacity than the rat and that bepatocytes from both rodent species have a greater capacity to metabolize trichloroethylene to trichloroacetic acid than have human hepatocytes (Table 5; Elcombe, 1985). These results mimic the results obtained in rats and mice and suggest that man will have a much lower capacity than either rodent species to metabolize trichloroethylene to trichloroacetic acid. In isolated hepatocytes, trichloroacetic acid induces peroxisomes (measured as cyanide-insensitive palmitoyl CoA oxidation) at about the same level in both mouse and rat hepatocytes. Once again, this mimics the result observed in v i v o (Elcombe, 1985).
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Fig. 3. Changes in peroxisome levels (as measured by cyanide-insensitive palmitoyl CoA oxidation) in the livers of rats ( I ) and mice (O) given trichloroethylene in a range of doses (after Elcombe, 1985). Asterisked values differ significantly from the control.
Unpredictability: an essay in toxicology Table 5. Kinetics of trichloroethylene metabolism to trichloroacetic acid (TCA) in isolated hepatocytes Formation of TCA
Species Mouse Rat Man
Enzyme affinity (Km)
Rate of conversion (Vmax)
Relative metabolizing capacity
28 151 165
107 19 6
120 4 1
After Elcombe (1985).
Human hepatocytes do not produce any increase in peroxisomes after administration of trichloroacetic acid, in spite of the fact that other indices of cell survival indicate that the hepatocytes are fully competent. The overall picture that emerges from this work is that the mouse is susceptible to the induction of hepatocellular carcinoma after the administration of trichloroethylene because of the high levels of trichloroacetic acid produced consequent on the linear kinetics of metabolism. Trichloroacetic acid is the proximate toxin inducing peroxisome proliferation and it is presumed that this leads to the induction of liver cancer. In the rat, the amount of trichloroethylene metabolized to trichloroacetic acid is limited by saturation of the metabolic pathway, with the consequence that there is no peroxisome proliferation and no increase in the incidence of hepatocellular carcinoma after the administration of high doses of trichloroethylene. In man, it is likely that the amount of trichloroacetic acid produced will be substantially lower than that seen in the mouse or the rat. A further difference is that human hepatocytes are not responsive to the effects of trichloroacetic acid. This evidence on the mechanism by which trichloroethylene produces cancer in mice and the fact that the mechanisms do not operate in human cells suggest that it is unlikely that trichloroethylene will be carcinogenic in man. This general conclusion is supported by the limited epiderniological evidence that is available. In spite of this information, trichloroethylene is now controlled as a carcinogen in the USA using risk assessment techniques that assume a non-threshold dose response and human sensitivity equal to that of the mouse. Hexachlorobutadiene--a carcinogen! Hexachlorobutadiene (HCBD) is negative in the standard Ames test (de Meester, Mercier & Poncelet, 1981; Stott, Quast & Watanabe, 1981) but increases the incidence of cancer of the kidney after administration to rats (Kociba, Schwetz, Keyes et al. 1977). Acute administration of HCBD produces damage to the proximal convoluted tubule in both mouse and rat kidney (Lock & Ishmael, 19~/9; Ishmael, Pratt & Lock, 1984). Extensive studies have been carried out on the metabolism and mode of action of HCBD at ICI's Central Toxicology Laboratory. The first step in the metabolism of HCBD is conjugation with glutathione and excretion in the bile (Wolf, Berry, Nash et al. 1984; Nash, King, Lock & Green, 1984). After cannulation of the bile duct, HCBD does not produce nephrotoxicity and confirmation of this mechanism is obtained by the
347
administration of HCBD-glutathione conjugate, which does produce nephrotoxicity. The second step in the metabolism is the reabsorption of the HCBD-glutathione conjugate, possibly after cleavage of the glutathione in the intestine. The remaining cysteine conjugate is then accumulated by the kidney (Lock & Ishmael, 1985) where the enzyme fl-lyase further metabolizes the cysteine conjugate. Confirmation of this route of metabolism to the ultimate toxin was obtained by the administration of the N-acetylcysteine conjugate (Nacetylpentachlorobutadienylcysteine) which produced an effect on kidney function similar to that of the parent compound, HCBD (Nash et al. 1984). Rat kidney fl-lyase converts the cysteine conjugate to pyruvate, ammonia and a reactive thiol moiety, as would be predicted by a consideration of the structure (Green & Odum, 1985). The various steps in the metabolism of hexachlorobutadiene to the active thiol compound are presented in Fig. 4. The final piece of information that explains the metabolic activation of HCBD is that the H C B D cysteine conjugate produces mutagenic effects in the Ames test when incubated with rat kidney S-9 (Green & Odum, 1985). This result explains why HCBD itself is not mutagenic when tested in a standard Ames test using rat liver S-9. The complex metabolic pathway results in the appearance of the HCBD-cysteine conjugate in the kidney after administration of HCBD, and further metabolism of this conjugate produces the proximate toxin and carcinogen. Knowledge of metabolic processes in man suggests that the same metabolic pathway will occur in man and therefore it is reasonable to assume that HCBD represents a carcinogenic hazard to man. Both trichloroethylene and HCBD are negative in the standard Ames test but carcinogenic when tested in certain rodent carcinogenicity studies. A simplistic conclusion would be that for trichloroethylene the Ames result provides an accurate prediction, but the mouse liver careinogenicity is a false positive; the converse is true for HCBD, where the mechanism suggests that the Ames result is a false negative and that HCBD represents a carcinogenic hazard to man. Improving hazard assessment--an urgent challenge Modern toxicology is an important branch of preventative medicine and contributes specifically to the assessment of human hazard from exposure to chemicals. Its techniques are laboratory studies and epidemiology and the challenge lies in predicting what effects will occur in man when the only information that is available is that from animals. Over the last 10 years or so there have been major international initiatives aimed at harmonizing methods of testing for new chemicals. This is typified by the OECD guidelines which set out in general terms the protocols that need to be followed in order to assess particular aspects of the toxicology of new chemicals. The EEC and various national authorities also have guidelines that set out the requirements for toxicity testing and, by and large, these are now reasonably consistent with one another. The advantages of having guidelines of this type are that international trade, which requires a common assessment of the hazards of chemicals, is greatly facili-
348
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Fig. 4. Metabolic activation of hexachloro-l,3-butadiene (HCBD) in male rats, a proposed mechanism for HCBD-mediated nephrotoxicity (after Nash et al. 1984).
tated. The main disadvantage is that the assessment of chemical toxicity typically follows the guidelines rigidly and there is little incentive to carry out work that might improve the hazard assessment. For new chemicals, this approach seems to be relatively sarisfactory and it allows a reasonably efficient assessment of hazard without too much disagreement between different national authorities. The situation with older established-products is substantially different. For many of them, evidence from many years of use can provide information about their safety under conditions of use. The application of rigid testing guidelines, which work relatively well for new chemicals, can be less than satisfactory when applied too rigidly to the problem of assessing human hazard from established chemicals. It appears that over the last few years, particularly in the USA and particularly for carcinogenicity, the regulatory assessment has become so rigid that it is losing credibility not only among toxicologists but also among the general public. An example of this is the way in which the application of the Delaney amendment in the case of saccharin was overturned by the United States Congress. The results of toxicological studies have an important bearing on public health and are therefore subject to much political pressure. There is, however, a common duty among all those involved in toxicological assessment, whether they are from industry, from governments or from academic institutions, to improve the process of human hazard assessment and to improve the methods used in toxicology. Industrial toxicologists, who have a particular inter-
est in making accurate hazard assessments, have a major role to play in this field. The industrial contribution is through its own in-house laboratories and also through collaborative efforts such as those carfled out at BIBRA. My fear for the future is that these efforts, which rely heavily on industrial funding, can easily be smothered if they are not encouraged. If the interpretation of government regulations follows the rigid and simplistic acceptance of animal toxicology data as indicative of human hazard, there will be little incentive to improve methods of assessing human hazard. This type of toxicology, aptly referred to by Professor Rahde as carbon paper extrapolation, ignores knowledge of mechanisms of action and therefore provides less than satisfactory assessment of human hazard. Toxicology has developed dramatically in the last few decades to the stage where we can now see how the assessment of human hazard can be improved. This is particularly true for environmental chemicals and chemicals that have a long history of use. Those involved in making regulatory decisions about chemicals for which there are good data have it in their power to encourage the development of improved methods of hazard assessment. By incorporating data on mechanisms of action and interspecies differences in toxicity into hazard assessments, it is possible to provide an incentive to all parties to improve methods of hazard assessment. By doing this, and using the currently available and somewhat fragmentary information about the toxicity of chemicals, it will be possible to encourage industry to harness its resources, apply its skills and develop new methods so that hazards that
Unpredictability: an essay in toxicology currently look unpredictable may become more easily predictable. Acknowledgements--I wish to thank my colleagues, particularly Drs J. Ashby, C. R. Elcombe, T. Green and E. A. Lock and Mr Godley for their help in the preparation of this manuscript.
REFERENCES Ashby J. & Purchase I. F. H. (1977). The selection of appropriate chemical class controls for use with short term tests for potential carcinogenicity. Ann. occup. Hyg. 20, 297. Bartsch H., Malaveille C., Barbin A. & Planche G. (1979). Mutagenic and alkylating metabolites of haloethylenes, chlorobutadienes and dichlorobutenes produced by rodent or human liver tissues. Archs Toxicol. 41, 249. de Meester C., Mercier M. & Poncelet F. (1981). Mutagenic activity of butadiene, hexachlorobutadiene and isoprene. In Industrial and Environmental Xenobiotics. Edited by I. Gut, M. Cikrt & G. L. Plaa. p. 195. Springer Verlag, New York. de Scrres F. J. & Ashby J. (Editors) (1981). Evaluation of Short-term Tests for Carcinogens: Report of the International Collaborative Programme. Elsevier, Amsterdam. Elcombe C. R. (1985). Species differences in carcinogenicity and peroxisome proliferation due to trichloroethylene: a biochemical human hazard assessment. Archs Toxicol. Suppl 8, 6. Green T. & Odum J. (1985). Structure/activity studies of the nephrotoxic and mutagenic action of cysteine conjugates ofchloro and fluoralkenes. Chemico-Biol. Interactions 54, 15. Gmim H., Bonse G., Radwin Z. Reichert D. & Henschler D. (1975). Mutagenicity /n vitro and potential carcinogenicity of chlorinated ethylenes as a function of metabolic oxirane formation. Biochem. Pharmac. 24, 2013. Hutt P. (1985). Use of quantitative risk assessment in regulatory decision making under Federal health and safety statutes. In Risk Quantitation and Regulatory Policy. Edited by D. G. Hoel, R. A. Merrill & F. P. Perera. Banbury Report 19. p. 15. Cold Spring Harbor Laboratory, New York. Ishmael J., Pratt I. S. & Lock E. A. (1984). Hexachloro-l,3butadiene-induced renal tubular necrosis in the mouse. J. Path. 142, 195. Kociba R. J., Schwetz B. A., Keyes D. G., Jersey G. C., Ballard J. J., Dittenber D. A., Quast J. F., Wade C. E. & Humiston C. G. (1977). Chronic toxicity of reproduction studies of hexachlorobutadiene in rats. Envir. Hlth Perspect. 21, 49. Lock E. A. & Ishmael J. (1979). The acute toxic effects of hexachlorobutadiene on the rat kidney. Archs Toxicol. 43, 47. Lock E. A. & Ishmael J. (1985). Effect of the organic acid transport inhibitor probenecid on renal cortical uptake and proximal tubular toxicity of hexachloro-l,3-
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butadiene and its conjugates. Toxic. appl. Pharmac. 81, 32. McCann J., Choi E., Yamasaki E. & Ames B. N. (1975). Detection of carcinogens as mutagens in the Salmonella/microsome test. Part 1. Assay of 300 chemicals. Proc. natn. Acad. Sci. U.S.A. 72, 5135. Nash J. A., King L. J., Lock E. A. & Green T. (1984). The metabolism and disposition of hexachloro-l,3-butadiene in the rat and its relevance to nephrotoxicity. Toxic. appl. Pharmac. 73, 124. National Cancer Institute (1976). Carcinogenesis Bioassay of Trichloroethylene. CAS No. 79-01-6, DHEW Publ. No (NIH) 76. National Toxicology Program (1983). National Toxicology Program draft report abstracts on nine chemical carcinogenesis bioassays. Chem. Regul. Rep. 6, 767. Poirier L. & de Serres F. J. (1979). Initial National Cancer Institute Studies on mutagenesis as a prescreen for chemical carcinogens: an appraisal. J. hath. Cancer Inst. 62,_ 919. Prout M. S., Provan W. M. & Green T. (1985). Species differences in response to trichloroethylene. I. Pharmacokinetics in rats and mice. Toxic. appl. Pharmac. 79, 389. Purchase I. F. H. (1980). Appraisal of the merits and shortcomings of tests of mutagenic potential. In Mechanisms of Toxicity and Hazard Evaluation: Proceedings o f the Second International Congress o f Toxicology. Edited by B. Holmstedt, R. Lawerys, M. Mercier & H. Roberfroid, p. 105. Elsevier, Amsterdam. Purchase I. F. H., Longstaff E., Ashby J., Styles J. A., Anderson D., Lefevre P. A. & Westwood F. R. (1978). An evaluation of 6 short term tests for detecting organic chemical carcinogens. Br. J. Cancer 37, 873. Reddy J. K. & Lalwani N. D. (1983). Carcinogenesis by hepatic peroxisome proliferators: evaluation of the risks of hypolipidemic drugs and industrial plasticizers to humans. CRC Crit. Rev. Toxicol. 13, 1. Simmon V. F., Kaubaner K. & Tardiff R. G. (1977). Mutagenic activity of chemicals identified in drinking water. In Progress in Genetic Toxicology. Edited by B. A. Bridges & F. H. Sobels. p. 249. Elsevier, Amsterdam. Slacik-Erben R., Roll R., Franke G. & Uehleke H. (1980). Trichloroethylene vapours do not produce dominant lethal mutations in male mice. Archs Toxicol. 45, 37. Stott W. T., Quast J. F. & Watanabe P. G. (1981). Differentiation of the mechanisms of oncogenicity of 1,4-dioxane and 1,3-hexachlorobutadiene in the rat. Toxic. appl. Pharmac. 60, 287. Sugimura T., Sato S., Nagao M,, Yahagi T., Marsushima T., Semo Y., Takeuchi M. & Kawachi T. (1976). Overlapping of carcinogens and mutagens. In Fundamentals in Cancer Prevention. Edited by P. N. Magee et aLp. 191. University of Tokyo Press. Waskell L. (1978). A study of the mutagenicity of anaesthetics and their metabolites. Mutation Res. 57, 141. Wolf C. R., Berry P. N., Nash J. A., Green T. & Lock E. A. (1984). Role of microsomal and cytosolic glutathione S-transferases in the conjugation of hexachloro1,3-butadiene and its possible relevance to toxicity. J. Pharmac. exp. Ther. 228, 202.