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The L5178Y tk +/- mouse lymphoma assay (MLA) has been validated as a sensitive and specific test system for the detection of mutagens/clastogens. There are currently two methodologies for performing the MLA: the original soft agar procedure and the newer microtitre procedure. While both procedures are considered acceptable, a limited amount of comparative information exists for the two methods. In this report the two methods were compared with regard to: (1) spontaneous and induced mutant frequencies; (2) cloning efficiencies; and (3) colony size distributions for mutants. In addition, small and large mutant colonies from microtitre wells were rechallenged for trifluorothymidine (TFT) resistance. In a majority of the cases, cloning efficiency values were higher for the microtitre as were the spontaneous and induced mutation frequency (MF) values. Nevertheless, when responses were compared according to mutation index (fold increase over background MF) the results from the two systems were often similar. More spontaneous small colonies were observed in the microtitre assay. While colony size distribution for induced mutant colonies was compound specific, generally, more small colonies were counted in microtitre. All mutant clones that were rechallenged with TFT demonstrated resistance. Aside from the differences mentioned above, both the microtitre and the soft agar procedures appear equally capable of identifying mutagenic agents.

The L5178Y tk+/- mouse lymphoma assay (MLA) has been in use for more than 15 years as a tool for evaluating the mutagenic potential of various agents. As with other genetic toxicology test systems, one criterion for a positive response has been the requirement of at least a 2-fold increase in mutant frequency (MF) as compared to the respective MF of the solvent controls. More recently, an actual specific increase in MF has been proposed as a criterion for determining a positive response in the MLA; however, this may not be appropriate for laboratories with a low, yet stable, background MF. The twofold rule criterion was evaluated in our laboratory with 66 compounds. The mutagenic status of these compounds was previously determined in other test systems and at one or more laboratories, including Lilly Research Laboratories. The results of this evaluation demonstrate that the twofold rule is an effective method for identifying mutagenic agents in the MLA at LRL where a lower, yet acceptable, background mutation frequency is the norm. A small number of compounds (6) yielded results discordant with the literature; however, these compounds have been previously found to be either difficult to detect in genotoxic assays or to show specific sensitivity in the MLA.

Bacterial test systems have been used extensively to identify the mutagenic potential of new compounds. In particular, the Ames test has gained worldwide acceptance and is required by many regulatory agencies to support product registration. The gradient plate assay (GPA) is a modification of the Ames test. It is used as a high capacity prescreen to detect the mutagenic potential of synthetic intermediates, impurities, and research compounds over a concentration gradient. Since the development of the GPA, over 4000 compounds have been tested in the assay. Selection and use of the GPA in our laboratory is due to many factors: reliability; sensitivity; capacity; timeliness of reporting results; and establishment of safety standard in the laboratory. In this manuscript, results of the GPA method are compared with results from the traditional Ames assay. To date, 113 compounds of identical lots have been evaluated in both tests, and in all but 3 instances the results are the same. Thus, the GPA is an ideal assay for use as a prescreen in determining the ability of a compound to induce bacterial mutation.

The AS52/XPRT mutation assay was examined for sensitivity in the detection of chromosomal mutagens. 6 compounds identified as chromosomal mutagens in the mouse lymphoma assay (MLA) were tested for mutagenicity in AS52 cells using suspension treatment and soft agar cloning. The 6 compounds were benzo[a]pyrene (BAP), 2-acetylaminofluorene (2AAF), methylmethanesulfonate (MMS), methyl acrylate (MCR), benzoin (BZ), and p-aminophenol (AM). BAP, 2AAF and MMS were mutagenic. The mutagenic responses for BAP, 2AAF and MMS included small colony mutants which have been shown to correlate with chromosomal mutation in the MLA. Colonies ranged from approximately 0.2 to 1.5 mm in size. The mutant frequency (MF) for the AS52 cells treated with BAP and 2AAF exceeded that previously reported for the MLA by 2-fold. In contrast, the MF for AS52 cells treated with MMS was one-third that reported in the MLA. The MF obtained in AS52 cells exceeded that reported for the CHO/HGPRT mutation assay for all 3 compounds. MCR, which produces almost entirely small colonies in the MLA, was negative in AS52 cells as were the MLA chromosomal mutagens AM and BZ. However, AM and BZ have only been reported mutagenic in the MLA. Both are considered nongenotoxic and noncarcinogenic. The results with the latter 3 compounds suggest that the AS52 assay is not as sensitive as the MLA for the detection of compounds identified as chromosomal mutagens.

The mouse lymphoma assay (MLA) and Chinese hamster ovary (CHO) cell assay are sensitive indicators of mutagenicity. The CHO assay has been modified technically to permit treatment in suspension and soft agar cloning comparable to the MLA. This methodology eliminates the risk of metabolic cooperation and the trauma of trypsinization. In addition, a larger population of cells can be treated and cloned for mutant selection. In order to compare the effectiveness of the test systems, 10 chemicals were evaluated for the induction of forward mutations in the CHO and MLA. Several of these chemicals have been reported as clastogenic; therefore, abbreviated colony sizing was performed to gauge the extent of genetic damage to the MLA cells. Both test systems detected benzo[a]pyrene, mitomycin C, acridine orange, and proflavin, and, with the exception of proflavin, more large colonies were present than small colonies. The suspect clastogen, phenytoin, was not mutagenic in the MLA and produced inconclusive results in the CHO. Ethidium bromide, a clastogen and a bacterial mutagen, was not mutagenic in either the MLA or CHO. Four compounds (p-aminophenol, benzoin, methoxychlor, and pyrene) were positive in the MLA, generally inducing a large number of small colonies, while demonstrating no mutagenic activity in the CHO assay. They have also been shown to be generally nongenotoxic in other test systems. Overall, the modified CHO assay did not appear to be better than the MLA for the detection of mutagenic agents. However, the MLA does appear to have lower specificity.

The antihistamine methapyrilene (MP) has been shown to be a potent hepatocarcinogen in rats. However, it has demonstrated little genotoxic activity in a wide variety of short-term tests. In this study, Fischer 344 rats were fed a carcinogenic dose of 0.1% methapyrilene in the diet for 10 weeks prior to sacrifice. S9 was prepared from the livers of the control, MP-treated and Aroclor-induced Fischer 344 rats. Each type of S9 was analyzed for mixed function oxidase activity, cytochrome P-450, and protein content. MP was then evaluated for mutagenicity in 6 strains of S. typhimurium (TA1535, TA1537, TA98, TA100, TA2638 and TA104) and one strain of E. coli (WP2uvrA-) using the standard plate-incorporation assay. MP was not mutagenic in any of the 7 bacterial strains when tested at concentrations < or = 10 mg/ml in the presence of each type of S9. However, in the absence of metabolic activation, an approximate 2-fold increase in revertants was noted with strain TA1535. The data from this study show that MP was not converted to a mutagenic metabolite by any of the three S9 types examined. However, the "weak" positive response with strain 1535 in the absence of metabolic activation indicates that further research is needed to elucidate the mechanism of action of this rat carcinogen.

Iron-supplemented bovine calf serum (ICS) was found to be a viable alternative to fetal bovine serum (FBS) in the growth promotion and cloning efficiency of Chinese hamster ovary (CHO) cells that are used in the HGPRT mutation assay. Suspension cultures of CHO cells had an average generation time of 11.5 h in ICS and 13.6 h for cells maintained in FBS. This slight difference was due to lot variability on the part of FBS and could be eliminated by routine quality control measures. The average cloning efficiencies for CHO cells cloned in either ICS or FBS were 107% and 88%, respectively, and these values were not statistically different. No appreciable difference was noted in the spontaneous mutation rates of cells cloned in either ICS or FBS. Furthermore, the use of ICS in mutagenicity studies with genotoxic agents shows the serum to be at least equal or superior to FBS in the detection of both direct-acting mutagens and promutagens. These data suggest that ICS is an appropriate serum to be used in the CHO/HGPRT test system. Since ICS is more readily available and considerably less costly than FBS, a substantial reduction in the cost of the assay can be realized.

The hair-dye ingredients, HC Blue No. 1 (HCB1) and HC Blue No. 2 (HCB2), were tested for the induction of bacterial mutation using Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100; and Escherichia coli strains WP2uvrA-. In addition, both dyes were evaluated in the mouse lymphoma L5178Y TK+/- assay (MLA) for the potential to induce forward mutation. A liver homogenate (S9) prepared from Aroclor 1254-induced male Fischer 344 rats was used to provide a means for metabolic activation. HCB1 was not mutagenic in the Ames assay, but was weakly mutagenic in the MLA only in the presence of metabolic activation. In contrast, HCB2 was a strong mutagen in the Ames assay in tester strain TA98 both in the presence and absence of metabolic activation. A positive response was also noted with HCB2 in the MLA, both in the presence and absence of metabolic activation. Negative findings from the Ames assay of this study agree with other published results where an identical lot of HCB1 was used. Using the same lot, a weak positive result was observed in the MLA, however, the activation requirements and magnitude of the response were different from that of a lot evaluated by the NTP. In contrast, HCB2 appears to be both a bacterial and mammalian cell mutagen independent of lot variability.

The Chinese hamster ovary cell assay (CHO), which measures forward mutation of the HGPRT locus, is used in several laboratories for the detection of mutagens. A procedure involving treatment of CHO cells in suspension culture and mutant selection in soft agar cloning has been developed (Oberly TJ, Bewsey BJ, Probst GS (1987): Mutat Res 182:99-111). In order to evaluate the effectiveness of these modifications, 33 chemicals representing six chemical classes were tested, and the results were compared to findings obtained in other tests for genotoxicity at Lilly Research Laboratories (LRL). A positive response was obtained with 21 chemicals, all of which are recognized mutagens. Of the 12 compounds that produced negative results, 4 were considered to be mutagens and/or carcinogens. Twelve of the compounds mentioned in this report have been previously tested in the CHO/HGPRT assay by other laboratories, and the results showed strong agreement between laboratories. These findings support the conclusion that the use of suspension cultures and soft agar cloning in the CHO assay provides a sensitive test for the identification of mutagens and is a viable alternative to the traditional monolayer procedure of O'Neill et al. (O'Neill JP, Couch DB, Machanoff R, San Sebastian JR, Brimer PA, Hsie AW (1977): Mutat Res 45:103-109).

Eleven metals were examined for their potential to induce forward mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells. The materials tested included AlCl3, CdSO4, HgCl2, K2CrO4, K2Cr2O7, MgCl2, MnCl2, NaAsO2, Na2HAsO4, NaCl, and Pb(NO3)2. Strong positive responses at survivals greater than 10% were observed with CdSO4, K2CrO4, K2Cr2O7, and MnCl2. Weak positive responses, yielding 2- to 3-fold increases in mutation frequency above the solvent control at greater than 10% survival, were seen with HgCl2, NaAsO2, Na2HAsO4, and Pb(NO3)2. Negative responses were obtained with MgCl2, NaCl, and AlCl3.

L5178Y/TK+/- cells were treated with 3-methylcholanthrene (3MC) in order to obtain thymidine-kinase-deficient mutants (TK-/-) which were resistant to trifluorothymidine (TFTr). Clones of TK-/- cells were harvested from soft agar and adapted to growth in suspension culture. The phenotype of the TK-/- and TK+/- clones was confirmed by measuring thymidine kinase activity. These studies were undertaken with cells from 16 3MC-induced large colony clones (lambda TK-/-), 21 3MC-induced small colony clones (sigma TK-/-), and 51 spontaneous sigma TK-/- clones. Thymidine kinase activity was absent in all of the lambda TK-/- and sigma TK-/- 3MC-induced clones and also in 49 of 51 sigma TK-/- spontaneous clones. After at least 50 generations in suspension culture, TFTr was retained by 80% of the 3MC-induced lambda TK-/- cells, by 75% of the 3MC-induced sigma TK-/- cells, and by 89% of the spontaneous sigma TK-/- cells. The collective results showed that 86 of the 88 TFTr colonies examined lacked thymidine kinase activity and indicate that at least 98% of all TFTr colonies seen in the L5178Y assay are true TK-/- mutants.

A procedure involving treatment of cells in suspension culture and soft-agar cloning was developed for measuring mutation of Chinese hamster ovary (CHO) cells to 6-thioguanine (6TG) resistance. The use of suspension cultures precluded the need for trypsinization and also permitted a 5-fold increase in cell population for compound exposure and mutant selection as compared to former monolayer techniques. Soft-agar cloning reduced the opportunity for metabolic cooperation and permitted the use of non-dialyzed fetal calf serum which resulted in spontaneous mutant frequencies of 6.6 +/- 3.2 X 10(-6) and cloning efficiencies of 91 +/- 18%. Relative total growth values were calculated based on suspension growth and cloning efficiencies such that an assessment of toxicity could be estimated from treatment through cloning. Dose-dependent mutagenic responses were observed in CHO cells following treatment with ethyl methanesulfonate, methyl methanesulfonate, 4-nitroquinoline-1-oxide, methylnitrosourea and N-methyl-N'-nitro-N-nitrosoguanidine. Clones of 6TG-resistant cells harvested from soft agar maintained 6TG resistance and methotrexate sensitivity and did not incorporate [3H]hypoxanthine into DNA. These preliminary findings indicate that the use of suspension cultures and soft-agar cloning has improved the efficiency and cost effectiveness of the CHO/HGPRT mutation assay.

Ochratoxin A (OA), a nephrotoxic mycotoxin, was evaluated for genotoxic potential in a battery of in vitro and in vivo assays. OA was not mutagenic to Salmonella typhimurium, either with or without metabolic activation, in the plate incorporation (Ames) test at concentrations of 50-600 micrograms OA/plate or in the gradient plate assay at concentrations of 0.1-1000 micrograms OA/ml. No induction of unscheduled DNA synthesis was evident in primary cultures of rat hepatocytes exposed to concentrations of OA ranging from 0.000025 to 500 micrograms/ml. In the mouse lymphoma forward mutation assay, exposure of L5178Y TK+/- mouse lymphoma cells to OA did not increase the numbers of L5178Y TK-/- mutants. There was no significant difference between the numbers of sister-chromatid exchanges in cells from OA-treated Chinese hamsters and those in cells from the negative-control animals.

The L5178YTK+/- mouse lymphoma assay (MLA) has been utilized in several laboratories as a short-term test for chemical-induced forward mutation in cultured mammalian cells. In order to evaluate several technical modifications to the MLA, 42 chemicals representing 9 chemical classes were tested and the results were compared with those published elsewhere as well as with findings in a genetic toxicology test battery currently used in this laboratory. A positive response for the induction of TK-/- mutants was obtained for 26 chemicals. With the exception of p-aminophenol, all of these compounds were recognized mutagens or carcinogens and were representative of direct-acting and activation-dependent genotoxins. 16 compounds did not induce TK-/- mutants and among these were 5 compounds that were considered to be mutagens or carcinogens. A comparison of the results of this study with those published elsewhere revealed a strong agreement among findings for this test irrespective of minor technical variations. It was concluded that the MLA is a useful system for identifying chemical mutagens in mammalian cells and can serve as a valuable component in a genetic toxicology test battery.

The mutagenic potential of glycidol and 7 alkyl glycidyl ethers having straight alkyl chains of 2, 4, 6, 8, 10, 12, and 14 carbon atoms were examined in a battery of in vitro assays. The battery consisted of the Salmonella/mammalian microsome assay, the L5178Y mouse lymphoma assay, and unscheduled DNA synthesis using W138 cells. The mutagenic potential of the compounds ranged from strongly mutagenic to non-mutagenic; glycidol exhibited the greatest activity. All the ethers through C-4 showed a definite response whole the C-8 or higher ethers showed very weak or no responses. Dose-response curves were obtained by all 3 assays for those compounds that exhibited mutagenic activity. The sensitivity of each assay is discussed, as are the effects of the liver microsome systems used for metabolic activation.