Michael D. Shelby

Predicting Rodent Carcinogenicity from Four in vitro Genetic Toxicity Assays: An Evaluation of 114 Chemicals Studied by the National Toxicology Program

Abstract Four widely used in vitro assays for genetic toxicity were investigated for their ability to predict the carcinogenicity of chemicals evaluated in long-term rodent studies by the National Toxicology Program (NTP). These assays were mutagenesis in Salmonella typhimurium and in mouse lymphoma cells and chromosome aberrations and sister chromatid exchanges in Chinese hamster ovary cells. Our evaluation compared results previously reported for 73 chemicals (Tennant et al. 1987) with results of similar analyses carried out for 41 additional chemicals tested subsequently by the NTP (Zeiger, Haseman, Shelby, Margolin, and Tennant, in press). For the combined data, Salmonella performed best, achieving a 66% (75/114) concordance, an 89% (32/36) positive predictivity, and a 55% (43/78) negative predictivity with regard to rodent carcinogenicity. The predictivity of Salmonella was even higher when attention was restricted to the multisite and/or two-species carcinogens. Chromosome aberrations also showed a ...

Mutation assays in male germ cells from transgenic mice: overview of study and conclusions

Three confirmed mouse germ cell mutagens, ethyl nitrosourea (ENU), isopropyl methanesulphonate (iPMS) and methyl methanesulphonate (MMS), have been evaluated for their activity as mutagens to the germ cell DNA of two strains of transgenic mice (lac I, Big Blue and LacZ, Muta Mouse). Both testicular DNA and epididymal sperm DNA were evaluated. A range of sampling times was studied, from 3 days post-dosing to 100 days post-dosing. ENU and iPMS were mutagenic to both testicular DNA and epididymal sperm DNA. Mutant frequencies were higher for both chemicals in DNA recovered from testicular tissue than in epididymal sperm DNA. Likewise, mutant frequencies were higher for both DNA samples at the later sampling times. MMS was not mutagenic under any condition of test. A good level of qualitative agreement in test results was seen for the two assays and for the same assays conducted in different laboratories. The level of quantitative agreement was not as high, but was, nonetheless, generally good. Recommendations for the future conduct of transgenic rodent germ cell mutation assays are made. The test data are discussed within the context of the larger question of how such assays should be integrated into the chemical hazard assessment process.

Multilaboratory comparison of in vitro tests for chromosome aberrations in CHO and CHL cells tested under the same protocols

Different test results have been reported for the same chemicals in two in vitro chromosome aberration test systems, CHL cells tested by a Japanese protocol and CHO cells tested by the US National Toxicology Program [Sofuni et al., Mutat Res 241:173–213,1990]. Here, laboratories in Japan, the US and the UK tested 9 such chemicals in CHL and CHO cells using the same protocols and found all 9 positive in both cell types; differences in earlier conclusions with these chemicals were due mainly to test protocol, not to different sensitivities of the cells. The most important protocol difference is sampling time. Chemicals that were negative in the NTP series using a sampling time of 10 to 13 hours often produced positive results when retested here with a 20‐ to 24‐hour sampling time. While positive resultswere obtained in both cell types, CHL cells sometimes had higher aberration levels and survived at higher doses than CHO cells would tolerate. This may reflect some intrinsic difference in sensitivity but may also be affected by factors such as cell cycle length and culture media (e.g., oxygen scavenging capacity). The collaboration reported here also contributed to a better understanding of scoring aberrations, especially “gaps”; there was good agreement on what types of aberrations should be included in the totals when scoring criteria were clearly defined, for example, many changes classified as “gaps” by the Japanese system were classified as “breaks” in the scoring systems used in the United States and the United Kingdom, and were appropriately included in total aberration counts. Environ. Mol. Mutagen 29:189–207, 1997. ® 1997 Wiley‐Liss, Inc.

The induction of micronucleated polychromatic erythrocytes in mice using single and multiple treatments

Studies are reviewed in which the effect of treatment/sample protocol on the induction of micronucleated polychromatic erythrocytes (MN-PCE) in male B6C3F1 mice by 3 carcinogens (benzidine, dimethylbenzanthracene and mitomycin C) were evaluated. 3 different treatment/sampling protocols were used, involving from 1 to 3 consecutive daily treatments and from 3 to 1, respectively, consecutive daily samplings beginning 24 h after the last injection. The results indicate that the 3-day injection/single sample time protocol eliminates the need for multiple sample times, minimizes the number of animals required in a study, decreases the time needed for data collection and simplifies data analysis. A comparison of the frequency of induced MN-PCE in peripheral blood and bone marrow suggests that, following a 3-injection protocol, either tissue can be used with equal efficiency.

Mammalian germ cell mutagenicity of ENU, IPMS and MMS, chemicals selected for a transgenic mouse collaborative study

A collaborative study to systematically assess transgenic mouse mutation assays as screens for germ cell mutagens has been conducted. Three male mouse germ cell mutagens (ENU, iPMS and MMS) were selected for testing. This paper provides a brief review of the effects reported for those 3 chemicals in the most commonly used non-transgenic germ cell mutagenicity assays, namely the dominant lethal, heritable translocation, and specific locus tests. Additionally, information on the DNA reactivity and the molecular nature of mutations induced by these chemicals is summarized.

Summary report of the working group on mammalian germ cell tests

The two tests considered by the Working Group were the mammalian germ cell cytogenetic assay and the rodent dominant lethal test. It was agreed that both tests were mainly used for identification of germ cell hazards, however, that the commonly applied protocol of the dominant lethal assay often supplied information for hazard characterization such as sensitivity of particular developmental stages of male germ cells. No particular species or strains were indicated. Concurrent solvent controls were regarded as indispensable for both tests. In the discussion of the mammalian germ cell cytogenetic assay, harmonization was obtained to a large extent with the cytogenetic bone marrow assay regarding the number of animals (5), the number of cells analyzed per animal (200), the highest exposure dose (MTD) and sampling times (twice within 24 and 48 h after dosing). However, it was pointed out that only the single acute exposure was adequate for the mammalian germ cell cytogenetic assay. Furthermore, it was stated that only structural chromosome aberrations could be analyzed and that it was not informative to score polyploidies or aneuploidies. In the discussion of the rodent dominant lethal test, it was stated that the assay was generally performed with treated males, however, increasing concern about female specific effects required that a protocol for female dominant lethal testing should be developed and validated. Acute and subacute treatment schedules were considered equally acceptable. It was regarded as highly important that the entire male germ cell development from meiosis to mature sperm was covered in the test protocol either by the appropriate mating schedules after single dosing or by subchronic dosing during the respective period. Postimplantation loss, preimplantation loss and fertility rate were the main parameters to be assessed in the rodent dominant lethal tests. It was agreed that the size of the experiment depended on the spontaneous frequency of dead implants, the mating scheme and the statistical design of the experiment.

Induction of micronucleated erythrocytes in rodents by diisopropylcarbodiimide and dicyclohexylcarbodiimide: dependence on exposure protocol.

The induction of micronucleated erythrocytes by diisopropylcarbodiimide (DIC) and dicyclohexylcarbodiimide (DCC) was investigated as part of a U.S. National Toxicology Program (NTP) evaluation of the subchronic toxicity of these chemicals. Analysis of peripheral blood smears from male and female B6C3F1 mice exposed to 17.5–140.0 mg DIC/kg/day by skin painting for 13 weeks revealed dose‐related increases in the frequency of micronucleated normochromatic erythrocytes (MN‐NCE) in both sexes. Results of a similar 13‐week peripheral blood micronucleus (MN) test with DCC (1.5–12.0 mg/kg/day) were also positive, although the increases in MN‐NCE were not as great as those observed with DIC. In contrast to the positive results of the subchronic skin‐painting studies in mice, acute bone marrow MN studies with DIC and DCC in male F344 rats, using intraperitoneal (i.p.) injection, yielded negative results. Both the acute and the subchronic exposures included doses that produced clinical signs of toxicity. Acute mouse bone marrow MN tests with DIC administered in single or triple i.p. injection protocols were subsequently conducted to determine if the differing responses between mice and rats were due to species or protocol differences. The results of these acute tests were negative or equivocal. Because the subchronic studies produced positive results, it was hypothesized that these carbodiimides required multiple treatments over an extended period of time to produce an increase in MN‐erythrocytes. To confirm the original response, a second dermal subchronic study was conducted with DIC; the protocol was modified to include sequential blood samplings to permit monitoring MN frequencies over time. The data demonstrated a small but consistent induction of micronucleated erythrocytes in mice treated with DIC by skin painting. Environ. Mol. Mutagen. 33:65–74, 1999

ENU mutagenesis in the mouse electrophoretic specific-locus test. 2. Mutational studies of mature oocytes.

Experiments were conducted using the biochemical specific-locus test to assess the mutagenicity of N-ethyl-N-nitrosourea (ENU) in mature oocytes of mice. C57Bl/6J females were treated with 100 mg/kg ENU by intraperitoneal injection and mated to untreated DBA males for 1 week following treatment. 1447 progeny were screened for evidence of mutations affecting the electrophoretic mobility of 32 different proteins; two mutants were detected by electrophoretic analyses. These results provide evidence that ENU is a germ-cell mutagen in mouse mature oocytes, although the frequency of mutants is somewhat lower than that obtained from spermatogonia treated with the same dose.

Ethylene dibromide : negative results with the mouse dominant lethal assay and the electrophoretic specific-locus test

Ethylene dibromide (1,2-dibromoethane; EDB) was tested for the induction of dominant lethal and electrophoretically-detectable specific-locus mutations in the germ cells of DBA/2J male mice. Males were treated with a single intraperitoneal injection of 100 mg/kg EDB and mated to two C57BL/6J females. In the dominant lethal assay, matings were carried out to measure the effect of EDB on meiotic and postmeiotic stages; germ cells representing spermatogonial stem cells were analyzed in the electrophoretic specific-locus test. Neither of these germ cell tests produced any evidence that EDB is a germ cell mutagen. It appears from these data and those reported in the literature that EDB, a genotoxic carcinogen that affects male fertility in some mammalian species, is not mutagenic in the germ cells of the male mouse.

Mutagenicity of 4-chloromethylbiphenyl in the salmonella/microsome assay

The mutagenic activity of 4-chloromethylbiphenyl was determined in Salmonella typhimurium strains TA1535, TA1537, TA98, and TA100 using the plate-incorporation assay with and without rat-liver S9. The compound was positive in strains TA1537, TA98, and TA100 both with and without S9 activation. A weakly positive response was seen with strain TA1535 tested without activation. This study was conducted as part of the U.K. Environmental Mutagen Society, Genetic Toxicology Trial.