Structure-activity relationships in the rat hepatocyte DNA-repair test for 300 chemicals

312 chemicals/mixtures were tested for genotoxicity in the rat hepatocyte/DNA-repair test. A variety of structure-activity relationships was evident. Of the 309 pure chemicals, 142 were positive. Of these, 43 were judged by IARC to have sufficient or limited evidence of carcinogenicity and none of the remainder was a proven noncarcinogen. Among the 167 negative chemicals, 44 were carcinogens. Some of these are known to be genotoxic in other systems, but based on several lines of evidence, many are considered to be epigenetic carcinogens that lack the ability to react with DNA and rather lead to neoplasia by nongenotoxic mechanisms.     

An evaluation of the E. coli K-12 uvrB/recA DNA repair host-mediated assay. II. In vivo results for 36 compounds tested in the mouse.

The aim of this study was to further evaluate the E. coli K-12 DNA repair host-mediated assay, as a short-term in vivo genotoxicity test, to be used as a complement to the micronucleus test in the routine testing of chemicals and drugs. The assay involves the administration of the test substance to mice by the route of choice, followed by the intravenous administration of a mixture of DNA repair deficient and proficient derivatives of E. coli K-12. After an incubation period the relative survival of the two strains was determined in blood, liver, lungs, kidneys and testes of the host. A significant preferential reduction of the DNA repair deficient strain in any organ indicates that the test substance possesses genotoxic properties. A total of 36 substances, 26 carcinogens, 4 weak or non-carcinogens and 6 unclassified substances, were tested in this assay. Positive results were obtained for 23 compounds. Of the carcinogens 18 were positive and of the non-carcinogens 3 were negative. The overall concordance between the assay and carcinogenicity was 72%. In general, alkylating agents and direct-acting nitroso compounds showed genotoxic activity in all organs tested, while the other substances were positive in a limited number of organs. With oral administration, which was the most commonly used administration route in the study, the organ showing a positive response most often was the blood. The results from the present study were compared with results from the micronucleus test, which were available for 26 of the substances. Results were in agreement for 15 of the substances, while 8 substances were positive in the present assay and negative in the micronucleus test: 4-aminobiphenyl, 2-anisidine, epichlorohydrin, formaldehyde, 1- and 2-naphthylamine, 2-nitrophenylenediamine and 4-nitroquinoline-N-oxide. The substances negative in the E. coli DNA repair host-mediated assay, but positive in the micronucleus test were: benzene, catechol and cyclophosphamide. It is concluded from this evaluation that the E. coli K-12 DNA repair host-mediated assay detects a number of carcinogens that are negative in the micronucleus test, while detecting most of the compounds that are positive in the latter. The advantages of this test are that differential DNA repair measures a broad spectrum of genetic damage, an in vitro/in vivo comparison is possible with the same test organisms, results can be obtained from various organs and the test is rapid.(ABSTRACT TRUNCATED AT 400 WORDS)     

Genotoxicity of a variety of nitroarenes and other nitro compounds in DNA-repair tests with rat and mouse hepatocytes

Genotoxicity of a variety of nitroarenes and other compounds was examined in DNA-repair tests with rat or mouse hepatocytes. Out of 15 nitroarenes tested, 9 compounds, i.e., 1-nitropyrene, 1,3-dinitropyrene, 1,6-dinitropyrene, 1,8-dinitropyrene, 1-nitro-3-acetoxypyrene, 3-nitrofluoranthene, 2-nitrofluorene, 2,7-di-nitrofluorene and 5-nitroacenaphthene elicited positive response of DNA repair in the tests with rat and mouse hepatocytes. Among the positive chemicals, the DNA-repair level of the 3 dinitropyrene isomers was much higher than other nitroarenes. The results indicate that a number of nitroarenes are metabolically activated in the primary culture of rodent hepatocytes, and suggest potential carcinogenicity of 1-nitropyrene and 1-nitro-3-acetoxypyrene the carcinogenicity of which is either not clear or unknown. Of the other nitro compounds, 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide as well as 4-nitroquinoline 1-oxide were clearly genotoxic in the assays with hepatocytes of both species. However, 5-nitro-2-furaldehyde semicarbazone was negative in both assays with hepatocytes of 2 species.     

Screening for carcinogens by the DNA synthesis inhibition test using human fibroblasts

The Ames test has probably been the most common prescreening test for potential carcinogens. This system, however, occasionally presents false-positive or false-negative results for certain kinds of chemicals. We chose 24 agents, most of which showed a discrepancy in their results with the animal carcinogenicity test and the Ames test, and screened them by the DNA synthesis inhibition test using human fibroblasts. Among 22 agents, 13 (59%) showed consistent results, 6 (27%) showed discrepant results with those obtained by animal tests and 3 (14%) showed ambiguous results. The majority of the DNA synthesis inhibition test results for these chemicals were consistent with the results obtained by whole animal tests, suggesting that the DNA synthesis inhibition test with mammalian cells in culture is a reasonably reliable prescreening test for potential carcinogens.     

Genotoxicity of epoxy resin hardeners in the hepatocyte primary culture/DNA repair test

The genotoxicity of 9 chemicals used as epoxy resin hardeners was examined in the DNA repair test with rat hepatocytes. DNA repair synthesis was elicited by 7 chemicals, i.e., 4-aminodiphenyl ether, 4,4-diaminodiphenyl ether, 3,4,4′-triaminodiphenyl ether, 3,3′-dichloro-4,4′-diaminodiphenyl ether, 1,3-phenylenedi-4-aminophenyl ether, 4,4′-diaminodiphenyl methane and 4,4′-methylene-bis(2-chloroaniline).The positive results obtained with 4 epoxy resin hardeners of unknown carcinogenicity, i.e., 4-aminodiphenyl ether, 3,4,4′-triaminodiphenyl ether, 3,3′-dichloro-4,4′-diaminodiphenyl ether and 1,3-phenylene-di-4-aminophenyl ether suggest that they may be carcinogens. The genotoxicity of 1,4-phenylene-di-4-aminophenyl ether, of unknown carcinogenicity, and 4,4′-diaminodiphenyl sulfone, for which there is no sound proof of carcinogenicity, was not confirmed in the DNA repair test. The result with 4,4′-diaminodiphenyl sulfone was in agreement with its lack of mutagenicity in Salmonella typhimurium.     

Definitive relationships among chemical structure, carcinogenicity and mutagenicity for 301 chemicals tested by the U.S. NTP.

An analysis is presented in which are evaluated correlations among chemical structure, mutagenicity to Salmonella, and carcinogenicity to rats and mice among 301 chemicals tested by the U.S. NTP. Overall, there was a high correlation between structural alerts to DNA reactivity and mutagenicity, but the correlation of either property with carcinogenicity was low. If rodent carcinogenicity is regarded as a singular property of chemicals, then neither structural alerts nor mutagenicity to Salmonella are effective in its prediction. Given this, the database was fragmented and new correlations sought between the derived sub-groups. First, the 301 chemicals were segregated into six broad chemical groupings. Second, the rodent cancer data were partially segregated by target tissue. Using the previously assigned structural alerts to DNA reactivity (electrophilicity), the chemicals were split into 154 alerting chemicals and 147 non-alerting chemicals. The alerting chemicals were split into three chemical groups; aromatic amino/nitro-types, alkylating agents and miscellaneous structurally-alerting groups. The non-alerting chemicals were subjectively split into three broad categories; non-alerting, non-alerting containing a non-reactive halogen group, and non-alerting chemical with minor concerns about a possible structural alert. The tumor data for all 301 chemicals are re-presented according to these six chemical groupings. The most significant findings to emerge from comparisons among these six groups of chemicals were as follows: (a) Most of the rodent carcinogens, including most of the 2-species and/or multiple site carcinogens, were among the structurally alerting chemicals. (b) Most of the structurally alerting chemicals were mutagenic; 84% of the carcinogens and 66% of the non-carcinogens. 100% of the 33 aromatic amino/nitro-type 2-species carcinogens were mutagenic. Thus, for structurally alerting chemicals, the Salmonella assay showed high sensitivity and low specificity (0.84 and 0.33, respectively). (c) Among the 147 non-alerting chemicals less than 5% were mutagenic, whether they were carcinogens or non-carcinogens (sensitivity 0.04).     

Genotoxicity of quinone pigments from pathogenic fungi

The genotoxicity and mutagenicity of several kinds of quinone pigments from pathogenic fungi were examined by means of the hepatocyte primary culture (HPC)/DNA repair test and of Ames test with TA98 and TA100. Clear genotoxicity of the two quinone chemicals, xanthomegnin and luteosporin were observed in the HPC/DNA repair test, though definite mutagenicity was not detected in the Salmonella microsome test. These two pigments are thus suspected to be genotoxic carcinogens.     

Comparative evaluation of different pairs of DNA repair-deficient and DNA repair-proficient bacterial tester strains for rapid detection of chemical mutagens and carcinogens

The aim of the present study was to evaluate the usefulness of different pairs of DNA repair-deficient and DNA repair-proficient bacterial tester strains in a mutagenicity/carcinogenicity screen, possibly as complements to the Ames test. 70 carcinogenic and non-carcinogenic compounds, representing a variety of chemical structures, were tested for their DNA-damaging effects, using 6 different DNA-repair-deficient bacterial strains. 2 Bacillus subtilis systems, H17/M45 and HLL3g/HJ-15, were used. The susceptibility of Escherichia coli AB1157 was compared with the susceptibility of 4 recombination-deficient mutants, JC5547, JC2921, JC2926 and JC5519. The test compounds were applied onto paper disks (spot test, ST), or incorporated into a top agar layer (agar-incorporation test, AT). The 2 B. subtilis systems were generally found to be more sensitive and reliable than the assays using E coli. The incorporation of the test compounds in the agar increased the sensitivity of the test for polycyclic aromatic hydrocarbons and other poorly water-soluble compounds. Hydrazines and several other highly polar chemicals could be tested more efficiently when applied onto paper disks. About 30% of the test compounds did not induce any growth inhibition and so could not be tested properly. In order to evaluate the ability of these DNA-repair tests to complement the Ames Salmonella mutagenicity test in a genetic toxicology screening program, results from this study were compared with published data both on mutagenicity in the Ames test and on carcinogenicity. 8 carcinogens generally found to be non-mutagenic for Salmonella were tested: 2 showed DNA-damaging properties (mitomycin C, 1,2-dimethylhydrazine), 5 failed to do so (actinomycin D, griseofulvin, thioacetamide, diethylstilbestrol, safrole), and one (thiourea) was not toxic, so that no classification was possible. 2 non-carcinogenic bacterial mutagens were examined; one, sodium azide, was equitoxic for repair-proficient and -deficient strains, while the other, nitrofurantoin, primarily inhibited repair-deficient strains. The DNA-repair tests failed to indicate the mutagenic and carcinogenic properties of acridine orange. Nalidixic acid, a non-mutagenic DNA synthesis inhibitor, damaged bacterial DNA. Apart from the differences summarized above, carcinogenicity was indicated correctly by the Salmonella S9 assay and most sets of DNA-repair-deficient and DNA-repair-proficient tester strains evaluated in this study. Thus, several more carcinogens could be detected by performing the Ames test and the bacterial DNA-repair tests in tandem than by using either test alone. Nevertheless, the use of both bacterial in vitro systems in a battery of short-term tests for mutagenicity/carcinogenicity evaluation is not considered to be ideal, since the Ames test and the pairs of DNA-repair-deficient and DNA-repair-proficient tester strains used had several shortcomings in common under the conditions of this study.     

Classification according to chemical structure, mutagenicity to Salmonella and level of carcinogenicity of a further 42 chemicals tested for carcinogenicity by the U.S. National Toxicology Program

This paper is an extension and update of an earlier review published in this journal (Ashby and Tennant, 1988). A summary of the rodent carcinogenicity bioassay data on a further 42 chemicals tested by the U.S. National Toxicology Program (NTP) is presented. An evaluation of each chemical for structural alerts to DNA-reactivity is also provided, together with a summary of its mutagenicity to Salmonella. The 42 chemicals were numbered and evaluated as an extension of the earlier analysis of 222 NTP chemicals. The activity patterns and conclusions derived from the earlier study remain unchanged for the larger group of 264 chemicals. Based on the extended database of 264 NTP chemicals, the sensitivity of the Salmonella assay for rodent carcinogens is 58% and the specificity for the non-carcinogens is 73%. A total of 32 chemicals were defined as equivocal for carcinogenicity and, of these, 11 (34%) are mutagenic to Salmonella. An evaluation is made of instances where predictions of carcinogenicity, based on structural alerts, disagree with the Salmonella mutagenicity result (12% of the database). The majority of the disagreements are for structural alerts on non-mutagens, and that places these alerts as a sensitive primary screen with a specificity lower than that of the Salmonella assay. That analysis indicates some need for assays complementary to the Salmonella test when screening for potential genotoxic carcinogens. It also reveals that the correlation between structural alerts and mutagenicity to Salmonella is probably greater than 90%. Chemicals predicted to show Michael-type alkylating activity (i.e., CH2 = CHX; where X = an electron-withdrawing group, e.g. acrylamide) have been confirmed as a structural alert, and the halomethanes (624 are possible) have been classified as structurally-alerting. To this end an extended carcinogen-alert model structure is presented. Among the 138 NTP carcinogens now reviewed, 45 (33%) are non-mutagenic to Salmonella and possess a chemical structure that does not alert to DNA-reactivity. These carcinogens therefore either illustrate the need for complementary genetic screening tests to the Salmonella assay, or they represent the group of non-genotoxic carcinogens referred to most specifically by Weisburger and Williams (1981); the latter concept is favoured.     

DNA repair assays as tests for environmental mutagens: A report of the U.S. EPA gene-tox program

A literature review was undertaken to determine the usefulness of DNA repair assays, other than unscheduled DNA synthesis, as screening techniques for mutagenic carcinogens. 92 reports were found to contain useful data for 49 chemicals using 6 techniques, namely, (1) cesium chloride equilibrium density gradients to study repair replication, (2) benzoylated naphthoylated diethylaminoethyl cellulose columns to study repair replication, (3) 313-nm irradiation of DNA containing bromodeoxyuridine to study repair replication, (4) alkaline elution to study repair of single-strand breaks and crosslinks, (5) alkaline sucrose gradients to study repair of single-strand breaks, and (6) direct assays for removal of adducts from DNA. Almost all of the 49 chemicals studied were known mutagens or carcinogens and/or known inducers of DNA repair. 9 compounds failed to elicit DNA repair by at least 1 assay technique, and at least 3 of these were not tested by the most appropriate and sensitive method. Nevertheless, although valid for studying repair phenomena in eukaryotic cells, these assays are not considered useful for screening. They are time-consuming, expensive, and/or require highly specialized skills. Despite the high frequency of positive reports, it is obvious from the literature that repair assays will fail to detect, or will detect with low efficiency, those agents whose main action is either intercalation or induction of strand breaks. For these and other reasons, DNA repair as a basis for screening for mutagenic carcinogens is not considered to be a useful concept.     

Evaluation of the alkaline elution/rat hepatocyte assay as a predictor of carcinogenic/mutagenic potential

We have recently developed an alkaline elution/rat hepatocyte assay to sensitively measure DNA single-strand breaks induced by xenobiotics in non-radiolabeled rat hepatocytes. Here we have evaluated this assay as a predictor of carcinogenic/mutagenic activity by testing 91 compounds (64 carcinogens and 27 non-carcinogens) from more than 25 diverse chemical classes. Hepatocytes were isolated from uninduced rats by collagenase perfusion, exposed to chemicals for 3 h, harvested, and analyzed for DNA single-strand breaks by alkaline elution. DNA determinations were done fluorimetrically. Cytotoxicity was estimated by glutamate-oxaloacetate transaminase release or by trypan blue dye exclusion. The assay correctly predicted the reported carcinogenic/non-carcinogenic potential of 92% of the carcinogens tested and 85% of non-carcinogens tested. The assay detected a number of compounds, including inorganics, certain pesticides, and steroids, which give false-negative results in other short-term tests. Only 2 rat liver carcinogens were incorrectly identified; the other carcinogens incorrectly identified are weakly or questionably carcinogenic (i.e., they cause tumors only in one species, after lifetime exposure, or at high doses). Some chemicals cause DNA damage only at cytotoxic concentrations; of 16 such compounds in this study, 12 are weak carcinogens suggesting a link between DNA damage caused by cytotoxicity and carcinogenesis. Our data indicate that this assay rapidly, reproducibly, sensitively, and accurately detects DNA single-strand breaks in rat hepatocytes and that the production of these breaks correlates well with carcinogenic and mutagenic activity.     

Structural basis of carcinogenicity in rodents of genotoxicants and non-genotoxicants

A set of 189 chemicals tested in the National Toxicology Program Cancer Bioassay was subjected to analysis by CASE, the Computer-Automated Structure Evaluation system. In the data set, 63% of the chemicals were carcinogens, approx. 40% of the carcinogens were non-genotoxic, i.e., they possessed neither "structural alerts" for DNA reactivity as defined by Ashby and Tennant, 1988, nor were they mutagenic for Salmonella. The data base can be characterized as a "combined rodent" compilation as chemicals were characterized as "carcinogenic" if they were carcinogenic in either rats or mice or both. CASE identified 23 fragments which accounted for the carcinogenicity, or lack thereof, of most of the chemicals. The sensitivity and specificity were unexpectedly high: 1.00 and 0.86, respectively. Based upon the identified biophores and biophobes, CASE performed exceedingly well in predicting the activity of chemicals not included among the 189 in the original set. CASE predicted correctly the carcinogenicity of non-genotoxic carcinogens thereby suggesting a structural commonality in the action of this group of carcinogens. As a matter of fact biophores restricted to non-genotoxic carcinogens were identified as were "non-electrophilic" biophores shared by genotoxic and non-genotoxic carcinogens. The findings suggest that the CASE program may help in the elucidation of the basis of the action of non-genotoxic carcinogens.     

The genotoxicity of a variety of aniline derivatives in a DNA repair test with primary cultured rat hepatocytes

The genotoxicity of a variety of aniline derivatives was examined by a DNA repair test with rat hepatocytes. Out of 37 aniline derivatives, 6 chemicals, i.e., 2,4,6-trimethylaniline (mesidine), 2,4-xylidine, 3,5-diaminobenzoic acid, 3,4-diaminochlorobenzene, 2-chloro-4-methylaniline and 4-chloro-N-methylaniline, elicited positive DNA repair responses. The results are in agreement with the bacterial mutagenicities with or without norharman of these compounds. Positive compounds of unknown carcinogenicity in the present assay, i.e., 3,5-diaminobenzoic acid, 2-chloro-4-methylaniline and 4-chloro-N-methylaniline are suspected of being potentially carcinogenic.     

Genotoxicity of pyrene oxide and 1-nitropyrene oxides in hepatocyte primary culture/DNA repair test

The genotoxicity of a pyrene oxide, 1-nitropyrene (NP) oxides and other related compounds was examined in the hepatocyte primary culture (HPC)/DNA repair test. Pyrene 4,5-oxide and both 1-NP-4,5-oxide and 1-NP-9,10-oxide elicited clearly positive responses of DNA repair. In this assay, 1-NP itself was weakly positive. However, other related chemicals such as pyrene, 1-nitro-3-hydroxypyrene, 1-nitro-6-hydroxypyrene, and 1-nitro-8-hydroxypyrene did not generate positive responses.     

Comparison of the continuous rat hepatoma cell line 2sFou with primary rat hepatocyte cultures for the induction of DNA repair synthesis by nitrosamines, benzo[a]pyrene and hydroxyurea

We have examined the suitability of the continuous rat hepatoma cell line 2sFou for testing the genotoxicity of chemicals in comparison with that of primary rat hepatocyte cultures (HPC). The capacity of the cells for metabolic activation was assessed by measuring induction of DNA-repair synthesis and inhibition of replicative DNA synthesis by the test compounds dimethylnitrosamine (DMN), diethylnitrosamine (DEN), hydroxyurea (HU) and benzo[a]pyrene (BaP), which are substrates for major hepatic and extrahepatic forms of cytochrome P-450 dependent monooxygenases. The cellular capacity for DNA-repair synthesis was assessed using UV-light as a DNA-damaging agent. Repair-specific incorporation of [3H]deoxycytidine (3H-dCyd) caused by UV-light was higher in 2sFou cells than in HPC. In contrast, background repair incorporation of 3H-dCyd in 2sFou cells was only 1/3 that found in HPC. All the test agents induced DNA repair and inhibited DNA synthesis in both 2sFou cells and HPC. The two nitrosamines were more effective in HPC than in 2sFou cells. HU and BaP affected DNA repair and DNA synthesis in the two cell systems at a similar range of concentrations. In general, DNA repair in the 2sFou cells increased near linearly with the concentrations of the test compounds. The data indicate that 2sFou cells are capable of activating hepatotropic pro-mutagens/carcinogens such as dialkylnitrosamines, and are sensitive indicators of DNA damage. In contrast, BaP, a non-hepatotoxic compound, caused only little DNA repair in these cells. Thus, continuously growing cells, such as 2sFou, show a qualitatively similar response to genotoxic chemicals as HPC and offer a potential alternative to HPC for genotoxicity testing.     

Mutation tests in Neurospora crassa. A report of the U.S. Environmental Protection Agency Gene-Tox Program

Many mutation tests have been developed in Neurospora crassa during the almost 40 years of its use in mutation research. These tests detect two major classes of mutation: gene mutation and meiotic nondisjunction. Within the first class, forward- and reverse-mutation tests have been used. The forward-mutation tests include those that detect mutations at many loci and at specific loci. Both kinds of forward-mutation tests have been done in homokaryons (n) and heterokaryons (n + n'). From the publications that were not rejected by our pre-established criteria, data were extracted for 166 chemicals that had been tested for mutagenicity. Only 6 of the 166 chemicals have been tested in one or more gene mutation test and the meiotic nondisjunction test; these 6 chemicals were positive in the first and negative in the second. Of the 102 chemicals tested in one or more gene mutation tests, 94 were positive and 8 were negative. Of the 70 chemicals tested in the meiotic nondisjunction test, 7 were positive and 63 were negative. Two tests, the ad-3 forward-mutation test and the meiotic nondisjunction test, have been used most frequently. These two tests are especially important for hazard evaluation, because each detects a class of mutations that is likely to be deleterious or lethal in the F1 - disomics by the meiotic nondisjunction test and multilocus deletions by the ad-3 forward-mutation test in heterokaryons. Generally, direct-acting chemicals are mutagenic in the gene mutation tests, but few chemicals that required metabolic activation have been tested. Only 31 of the 166 chemicals tested in N. crassa have been tested for carcinogenicity. Among these chemicals, there is a good association between mutagenicity in gene mutation tests and carcinogenicity but a poorer association between meiotic nondisjunction and carcinogenicity; however, only a small number of chemicals has been tested in the meiotic nondisjunction test. Further use and development of certain mutation tests in N. crassa are desirable.     

Genetic toxicology data in the evaluation of potential human environmental carcinogens.

In 1969, the International Agency for Research on Cancer (IARC) initiated the Monographs Programme to evaluate the carcinogenic risk of chemicals to humans. Results from short-term mutagenicity tests were first included in the IARC Monographs in the mid-1970s based on the observation that most carcinogens are also mutagens, although not all mutagens are carcinogens. Experimental evidence at that time showed a strong correlation between mutagenicity and carcinogenicity and indicated that short-term mutagenicity tests are useful for predicting carcinogenicity. Although the strength of these correlations has diminished over the past 20 years with the identification of putative nongenotoxic carcinogens, such tests provide vital information for identifying potential human carcinogens and understanding mechanisms of carcinogenesis. The short-term test results for agents compiled in the EPA/IARC Genetic Activity Profile (GAP) database over nearly 15 years are summarized and reviewed here with regard to their IARC carcinogenicity classifications. The evidence of mutagenicity or nonmutagenicity based on a 'defining set' of test results from three genetic endpoints (gene mutation, chromosomal aberrations, and aneuploidy) is examined. Recommendations are made for assessing chemicals based on the strength of evidence from short-term tests, and the implications of this approach in identifying mutational mechanisms of carcinogenesis are discussed. The role of short-term test data in influencing the overall classification of specific compounds in recent Monograph volumes is discussed, particularly with reference to studies in human populations. Ethylene oxide is cited as an example.     

Unscheduled DNA synthesis tests. A report of the U.S. Environmental Protection Agency Gene-Tox Program

The utility of unscheduled DNA synthesis (UDS) testing for screening potentially hazardous chemicals was evaluated using the published papers and technical reports available to the UDS Work Group. A total of 244 documents were reviewed. Based on criteria defined in advance for evaluation of the results, 169 were rejected. From the 75 documents accepted, results were reviewed for 136 chemicals tested using autoradiographic approaches and for 147 chemicals tested using liquid scintillation counting (LSC) procedures; 38 chemicals were tested by both approaches to measure UDS. Since there were no documents available that provided detailed recommendations of UDS screening protocols or criteria for evaluating the results, the UDS Work Group presents suggested protocols and evaluation criteria suitable for measuring and evaluating UDS by autoradiography in primary rat hepatocytes and diploid human fibroblasts and by the LSC approach in diploid human fibroblasts. UDS detection is an appropriate system for inclusion in carcinogenicity and mutagenicity testing programs, because it measures the repair of DNA damage induced by many classes of chemicals over the entire mammalian genome. However, for this system to be utilized effectively, appropriate metabolic activation systems for autoradiographic measurements of UDS in human diploid fibroblasts must be developed, the nature of hepatocyte-to-hepatocyte variability in UDS responses must be determined, and the three suggested protocols must be thoroughly evaluated by using them to test a large number of coded chemicals of known in vivo mutagenicity and carcinogenicity.     

The alkaline single cell gel electrophoresis assay with mouse multiple organs: results with 30 aromatic amines evaluated by the IARC and U.S. NTP

The genotoxicity of 30 aromatic amines selected from IARC (International Agency for Research on Cancer) groups 1, 2A, 2B and 3 and from the U.S. NTP (National Toxicology Program) carcinogenicity database were evaluated using the alkaline single cell gel electrophoresis (SCG) (Comet) assay in mouse organs. We treated groups of four mice once orally at the maximum tolerated dose (MTD) and sampled stomach, colon, liver, kidney, bladder, lung, brain, and bone marrow 3, 8 and 24 h after treatment. For the 20 aromatic amines that are rodent carcinogens, the assay was positive in at least one organ, suggesting a high predictive ability for the assay. For most of the SCG-positive aromatic amines, the organs exhibiting increased levels of DNA damage were not necessarily the target organs for carcinogenicity. It was rare, in contrast, for the target organs not to show DNA damage. Organ-specific genotoxicity, therefore, is necessary but not sufficient for the prediction of organ-specific carcinogenicity. For the 10 non-carcinogenic aromatic amines (eight were Ames test-positive and two were Ames test-negative), the assay was negative in all organs studied. In the safety evaluation of chemicals, it is important to demonstrate that Ames test-positive agents are not genotoxic in vivo. Chemical carcinogens can be classified as genotoxic (Ames test-positive) and putative non-genotoxic (Ames test-negative) carcinogens. The alkaline SCG assay, which detects DNA lesions, is not suitable for identifying non-genotoxic carcinogens. The present SCG study revealed a high positive response ratio for rodent genotoxic carcinogens and a high negative response ratio for rodent genotoxic non-carcinogens. These results suggest that the alkaline SCG assay can be usefully used to evaluate the in vivo genotoxicity of chemicals in multiple organs, providing for a good assessment of potential carcinogenicity.     

Prevalidation study of the BALB/c 3T3 cell transformation assay for assessment of carcinogenic potential of chemicals

The cell transformation assays (CTAs) have attracted attention within the field of alternative methods due to their potential to reduce the number of animal experiments in the field of carcinogenicity. The CTA using BALB/c 3T3 cells has proved to be able to respond to chemical carcinogens by inducing morphologically transformed foci. Although a considerable amount of data on the performance of the assay has been collected, a formal evaluation focusing particularly on reproducibility, and a standardised protocol were considered important. Therefore the European Centre for the Validation of Alternative Methods (ECVAM) decided to coordinate a prevalidation study of the BALB/c 3T3 CTA. Three different laboratories from Japan and Europe participated. In the study the following modules were assessed stepwise: test definition (Module 1) consisted of the standardisation of the protocol, the selection of the cell lineage, and the preparation of a photo catalogue on the transformed foci. The within-laboratory reproducibility (Module 2) and the transferability (Module 3) were assessed using non-coded and coded 3-methylcholanthrene. Then, five coded chemicals were tested for the assessment of between-laboratory reproducibility (Module 4). All three laboratories obtained positive results with benzo[a]pyrene, phenanthrene and o-toluidine HCl. 2-Acetylaminofluorene was positive in two laboratories and equivocal in one laboratory. Anthracene was negative in all three laboratories. The chemicals except phenanthrene, which is classified by IARC (http://monographs.iarc.fr) as group 3 "not classifiable as to its carcinogenicity to human", were correctly predicted as carcinogens. Further studies on phenanthrene will clarify this discrepancy. Thus, although only a few chemicals were tested, it can be seen that the predictive capacity of the BALB/c 3T3 CTA is satisfactory. On the basis of the outcome of this study, an improved protocol, incorporating some changes related to data interpretation, has been developed. It is recommended that this protocol be used in the future to provide more data that may confirm the robustness of this protocol and the performance of the assay itself. During the study it became clear that selecting the most appropriate concentrations for the transformation assay is crucial.