730 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS register as positive in a feeding experiment if the right convergence of factors is present under the particular experimental circumstances. If the Ames test is less specific in actuality than some of its proponents now believe, a number of unexpected positives are likely to be identified in the near future. What do these positives mean? Are they more truly positives than bioassay positives, or are they less potent carcinogens for the intact mammal? And how should we extrapolate these results to humans? Some advocates propose that the Ames test should be used as a screening test and that all compounds found positive should then be tested by appropriate exposure to intact mammals in vivo. Others propose that compounds found positive by the Ames test should be withdrawn from use until they have undergone bioassay testing in mammals and have been demonstrated negative in two rodent species. Still others maintain that a compound which is Ames-positive and bioassay-negative merely proves that carcinogenic bioassay is insensitive either because of species-specific metabolism or the fi error problem inherent in using practical sample sizes of 50 male and 50 fe- male rodents per dose and controls. Obviously the term "screening test" can have many connotations, especially in regulatory judgments. What a manufacturer chooses to do after voluntarily testing for mutagenesis and find- ing a positive result is still largely a matter of free choice. He can try to eliminate the positive component from his product, he can shift to an alternative compound, or he can undertake lifetime bioassays which are expensive and whose outcome is un- predictable. But in a strict regulatory approach, the number of false positives must be kept to an acceptable level. Undoubtedly, false positives occur in bioassays that problem, however, was identified only after the carcinogenic bioassay system was ac- cepted by most of the scientific community. Even then, some scientists had reserva- tions about whether the bioassay protocols are a relevant model for human exposures that occur at substantially lower levels than those to which test animals are exposed. In summary, the question of how false positives relate to the specificity of the Ames test can be resolved only by expanding the number of chemicals tested however, it is essential not to select only those chemicals known to have the characteristics of carcinogens. The question of relation of false negatives to the specificity of the Ames test does not appear to be a significant problem. Many other criteria can be used to help determine whether a substance should be tested for carcinogenic activity. 3. QUANTITATIVE MEASUREMENT OF THE PARAMETERS OF CONCERN In one sense, the ability of the Ames test to measure the strength of biological activity of the parameter of concern (reverse mutations) over an exceedingly broad range ap- pears better than the ability of the intact animal assay to measure carcinogenesis. However, each test measures different--although probably related--parameters. The carcinogenesis bioassay measures the capability of a substance in a limited system to statistically affect the incidence of tumors in test animals. For true positives, it measures the ability of the substance to express its carcinogenic activity in the intact mammal. Thus penetration through defense mechanisms is involved, and the critical convergence of intrinsic capability (penetration to the target of opportunity and sur- vival in activatable form) has resulted in a carcinogenic hit. In my judgment, dosage must be considered, as well as many parameters of classical toxicology that show the ability of a chemical to penetrate to the target after having survived various defense

REGULATORY CONSIDERATIONS: MUTAGENESIS 731 mechanisms that tend to operate on classical dose response curves. Others believe that any exposure to a carcinogen, however small, presents a substantial risk to human health. The Ames test is sensitive enough to detect activities that may be involved in the causation of cancer, but it does not measure penetrability of the substance nor se- lective parameters such as biological resistance, which are important to evaluation of toxicity, including carcinogenicity. 4. SCIENTIFIC ACCEPTABILITY OF THE TEST The Ames test is very acceptable at present for limited purposes, namely, decision- making on a voluntary basis with considerable latitude for exercising judgment. However, regulatory decision-making requires stricter criteria for applying judgment, and the answer ultimately must be a simple yes or no. At present there still is considerable controversy over the use of in vitro test systems for assessing risk to human health. Some of the controversy is legitimate. If the Ames test is to be used as a screening test, then what is the definitive test--in vivo carcinogenic bioassay? What happens if the latter test is negative? In how many rodent species and strains? How re- liable is this assay system? How much evidence will be needed to make the chemical acceptable if such should be the true and correct response of society? Should the Ames test then be the definitive test? That would be a major decision that would also invoke consideration of changes in how the Delaney Clause is applied. Many issues are involved that should be settled concurrently to the best of our ability as we continue to evaluate the potential utility of the Ames test for regulatory pur- poses. At present, if results of a carcinogenic bioassay were equivocal but selected mutagenicity tests were positive, I would consider that observation to be of regulatory importance. The Ames type of test has some regulatory use for evaluating which me- tabolites of a drug given to humans or to food-producing animals should receive atten- tion if we are to protect the public against carcinogens. However, a decision-tree approach to regulation based solely on positive versus negative results of any single short-term test may cause confusion. For example, what happens if positive results are obtained with natural constituents of foods, spices and flavors? My major recom- mendation is that we devote more effort to expanding the number of substances tested during the next few years before making a final decision. We need further insight into the specificity of the test system to detect mutacarcinogens. It has been said that most chemical carcinogens have mutagenic activity. With some exceptions, I agree. However, a more relevant question is whether or not most mutagenically positive com- pounds are also carcinogenic in the sense of posing a substantial hazard to public health. What regulatory decision should be made about weak mutagens or weak carcinogens? We could, of course, make these decisions on a day-to-day basis, but it would be better to answer some of the pressing questions before we settle on such tests for regulatory decision-making. Otherwise the distinction between a screening test and a definitive test will rapidly disappear. The Ames test and other similar tests have great utility for voluntary decision-making and may be directly useful for regulatory decision-making in the not too distant future. But a premature rush to regulatory judgment may create unanticipated regulatory difficulties and lead to a controversy with connotations similar to the Delaney Clause.