j. Soc. Cosmet. Chem., 31,145-151 (May/June 1980) The cost and utility of several microbial genetic toxicology tests JOHN R. ROHEIM and JOHN E. HEINZE, Armour and Company, Armour Research Center, Scottsdale, AZ 85260 Received May 7, 1979. Presented at Annual Scientific Seminar, Society of Cosmetic Chemists, May 1979, Dallas, Texas. Synopsis In order to gain experience with genetic toxicology testing and to be able to comment on any proposed governmental regulations, we have instituted a program to evaluate several genetic toxicology assays. The complete program consists of a computerized literature search and an evaluation process for short-term in vitro assays. Using this program, we have determined the ACCURACY, REPRODUCIBILITY, COST, and TIME requirements of SEVERAL MICROBIAL GENETIC TOXICOLOGY TESTS. INTRODUCTION Genetic toxicology testing has been the subject of recent discussions by the Society of Cosmetic Chemists (1,2,3). These tests are gaining wider acceptance and may at some future date be required for safety evaluation (4,5). Therefore, a program has been initiated to evaluate a variety of genetic toxicology tests. This report describes the process of evaluation used and the investigation of three different types of microbial tests. Programs similar in nature are in progress in other laboratories (6,7). GENETIC TOXICOLOGY EVALUATION PROGRAM The genetic toxicology program consists of a two-part evaluation process. The first part makes use of computerized literature search techniques (8) to efficiently obtain current scientific literature on test methods and controls to be used in the evaluation. The second part consists of a comparative evaluation of several tests using reference compounds. Based on the results of the literature search, three types of microbial tests were chosen for further consideration: bacterial mutation assays, bacterial DNA repair tests, and tests using eukaryotic microorganisms, i.e., yeast. BACTERIAL MUTATION ASSAYS A number of tests using bacteria have been developed to measure the mutagenic potential of a chemical, i.e., the ability of the chemical to produce forward or reverse 145

146 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS mutations. However, consideration of the published information on the various bacterial mutation assays reveals that the Ames (9) test has been studied much more intensively than any of the other tests. For this reason, the Ames test was selected for further study. The Ames test uses five histidine requiring strains of Salmonella typhimurium which were specifically constructed to be sensitive to the effects of a wide spectrum of mutagens. These strains have lost their ability to synthesize histidine and consequently are unable to grow in the absence of histidine. However, each strain reverts to histidine independence at a low but reproducible rate. Since revertants arise through a mutational mechanism, the presence of a mutagen can be expected to increase the reversion frequency. Thus, the essence of the Ames test is a comparison of the histidine reversion frequency of each strain in the presence of a chemical substance to that frequency in the absence of the chemical. A positive test result, indicative of mutagenic activity, occurs if the number of chemically induced revertants in any strain is significantly higher than the number of spontaneous revertants. An important point is that a number of mutagens are not directly active but require metabolic conversion into reactive forms. In mammals, this function is usually performed by liver enzymes of the microsomal P-450 type. Since microorganisms generally lack this type of enzyme system, the Ames test includes provisions for supplying this activity through the use of a rat liver microsomal preparation called an "S-9 mix." The use of an S-9 mix allows the Ames test to detect mutagens which would otherwise be missed. The ability of the Ames test to detect carcinogens has been demonstrated with a larger number of chemicals than any other genetic toxicology test. The results of five independent studies (10-15) have indicated that the Ames test detects 65 to 90% of the several hundred known carcinogens examined. Moreover, 75 to 90% of the several hundred presumed noncarcinogens examined are negative in the Ames test. Thus, the Ames test seems to be accurate although it does poorly in certain classes of carcinogens such as chlorinated pesticides, metals, estrogens, and nitrosamines. For this reason, other types of microbial tests were also considered. BACTERIAL DNA REPAIR TESTS Bacterial DNA repair tests measure the potential of a chemical to damage DNA. Such tests may detect a broader spectrum of chemicals which produce genetic damage than mutagenicity tests. Based on the literature search, three bacterial DNA repair tests were selected for evaluation: the Escherichia coli PolA test (16), the Bacillus subtilis Rec-assay (17), and the E. coli RecBC test (18). The essence of the bacterial DNA repair tests is a comparison of the size of the zone of inhibition produced by a chemical against a DNA repair deficient strain and an otherwise identical DNA repair normal strain. A positive test is shown by larger zones of inhibition against the repair deficient strain. Positive results indicate that the chemical causes damage to bacterial DNA which the DNA repair deficient strain cannot repair as effectively as the normal strain. Alternatively, a negative test is reflected by equal size zones of inhibition against both strains, indicating that the chemical does not damage DNA. The test is also considered negative if no zones of inhibition are produced by the chemical. Analysis of the published information on the