T.J. Oberly

Guide for performing the mouse lymphoma assay for mammalian cell mutagenicity

J Wellcome Research Laboratories, 3030 Cornwallis Road, Research Triangle Park, NC 27709 (U.S.A.), 2 National Toxicology Program, NIEHS, P.O. Box 12233, Research Triangle Park, NC 2 7709 (U.S.A.), 3 Sitek Research Laboratories, 12111 Parklawn Drioe, Rockoille, M D 20852 (U.S.A.), 4 Environmental Protection Agency, MD 68, Research Triangle Park, NC 27711 (U.S.A.), 5 The Upjohn Company, Kalamazoo, M1 49001 (U.S.A.) and 6 Eli Lilly and Co., Bldg. 240, Rm. 228, Greenfield, I N 46140 (U.S.A.)

Evaluation of ochratoxin A for mutagenicity in a battery of bacterial and mammalian cell assays

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.

Genotoxicity studies on the preemergence herbicide trifluralin

This paper reports the results of studies conducted within the Lily Research Laboratories and discusses additional studies with trifluralin that have been reported in the literature

A comparison of the CHO/HGPRT+ and the L5178Y/TK+/- mutation assays using suspension treatment and soft agar cloning : results for 10 chemicals

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.

An evaluation of 6 chromosomal mutagens in the AS52/XPRT mutation assay utilizing suspension culture and soft agar cloning

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.

An evaluation of the twofold rule for assessing a positive response in the L5178Y TK+/− mouse lymphoma assay

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.

The gradient plate assay: a modified Ames assay used as a prescreen for the identification of bacterial mutagens

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.

IDENTIFICATION OF GENOTOXINS: A CORRELATION OF BACTERIAL MUTATION WITH HEPATOCYTE DNA REPAIR

Since the mid-1970s a variety of short-term tests have been developed for the identification of mutagens and carcinogens.’ In addition to the Salmonella/mammalian microsome test of Ames,” which has found widespread application, other tests that measure direct DNA dama e and r e ~ a i r , ~ ~ mutation in cultured mammalian cells,’”’2 chromosomal effects,”-”and in virro cell tran~forrnation’~-’~ have also been proposed as predictive for carcinogenic chemicals. A common feature of these systems is that the end point measured reflects the interaction of a chemical with DNA and, therefore, only carcinogens that express their activity through alterations to the genetic material are detected. Accordingly, this new research area has been named genetic toxicology and agents that elicit a positive response in these tests are operationally defined as genotoxins.*’ Within this framework, substances for which there is evidence of carcinogenicity and that are believed to exert a carcinogenic effect through pathways not involving DNA, such as hormonal imbalance, immune suppression, chronic tissue injury, or solid-state effects, are operationally classified as epigenetic carcinogens.” Presently, no reliable test for epigenetic carcinogens has been developed; however, recent progress with assays for tumor may contribute to our understanding of epigenetic carcinogenesis. Considerable progress has been made in the development of tests for genotoxic chemicals, and an important outcome of the efforts from many laboratories has been the recognition that no single system is a definitive predictor of genotoxicity. Consequently, many industrial laboratories have adopted a testing strategy incorporating a battery of assays, which provides a biological hierarchy of testing using systems having diverse genetic end points and dissimilar mechanisms for metabolic activation. For the past several years this laboratory has been committed to the development of a genetic toxicology test battery that now serves to interface with a research and development program to predict the genotoxicity of candidate pharmaceuticals and agriculturals. The test battery is conducted in tier fashion using systems of increasing biological complexity and relevance, and includes tests for bacterial mutation, DNA repair, mammalian cell mutation, and chromosomal effects in vivo. The largest number of compounds have been tested in the bacterial mutation and hepatocyte DNA-repair assays, and results in these systems provide the basis for the following discussion. The relatively large data base developed in these assays has allowed the selection of more interesting compounds for evaluation in the latter two tests. Pertinent findings will also be discussed below. The experience with this battery has reinforced the belief that no single test can adequately predict mutagenic or carcinogenic potential and that the collective findings of a test battery are essential for the comprehensive assessment of genotoxicity. 334

Mutagenicity evaluation of HC Blue No. 1 and HC Blue No. 2. III. Effects in the Salmonella typhimurium/Escherichia coli reversion assay and the mouse lymphoma L5178Y TK+/− forward mutation assay

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.

Iron-supplemented bovine serum as an alternative to fetal bovine serum in the CHO/HGPRT mutation assay

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.