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A working group convened at the 2009 5th IWGT to discuss possibilities for improving in vivo genotoxicity assessment by investigating possible links to standard toxicity testing. The working group considered: (1) combination of acute micronucleus (MN) and Comet assays into a single study, (2) integration of MN assays into repeated-dose toxicity (RDT) studies, (3) integration of Comet assays into RDT studies, and (4) requirements for the top dose when integrating genotoxicity measurements into RDT studies. The working group reviewed current requirements for in vivo genotoxicity testing of different chemical product classes and identified opportunities for combination and integration of genotoxicity endpoints for each class. The combination of the acute in vivo MN and Comet assays was considered by the working group to represent a technically feasible and scientifically acceptable alternative to conducting independent assays. Two combination protocols, consisting of either a 3- or a 4-treament protocol, were considered equally acceptable. As the integration of MN assays into RDT studies had already been discussed in detail in previous IWGT meetings, the working group focussed on factors that could affect the results of the integrated MN assay, such as the possible effects of repeated bleeding and the need for early harvests. The working group reached the consensus that repeated bleeding at reasonable volumes is not a critical confounding factor for the MN assay in rats older than 9 weeks of age and that rats bled for toxicokinetic investigations or for other routine toxicological purposes can be used for MN analysis. The working group considered the available data as insufficient to conclude that there is a need for an early sampling point for MN analysis in RDT studies, in addition to the routine determination at terminal sacrifice. Specific scenarios were identified where an additional early sampling can have advantages, e.g., for compounds that exert toxic effects on hematopoiesis, including some aneugens. For the integration of Comet assays into RDT studies, the working group reached the consensus that, based upon the limited amount of data available, integration is scientifically acceptable and that the liver Comet assay can complement the MN assay in blood or bone marrow in detecting in vivo genotoxins. Practical issues need to be considered when conducting an integrated Comet assay study. Freezing of tissue samples for later Comet assay analysis could alleviate logistical problems. However, the working group concluded that freezing of tissue samples can presently not be recommended for routine use, although it was noted that results from some laboratories look promising. Another discussion topic centred around the question as to whether tissue toxicity, which is more likely observed in RDT than in acute toxicity studies, would affect the results of the Comet assay. Based on the available data from in vivo studies, the working group concluded that there are no clear examples where cytotoxicity, by itself, generates increases or decreases in DNA migration. The working group identified the need for a refined guidance on the use and interpretation of cytotoxicity methods used in the Comet assay, as the different methods used generally lead to inconsistent conclusions. Since top doses in RDT studies often are limited by toxicity that occurs only after several doses, the working group discussed whether the sensitivity of integrated genotoxicity studies is reduced under these circumstances. For compounds for which in vitro genotoxicity studies yielded negative results, the working group reached the consensus that integration of in vivo genotoxicity endpoints (typically the MN assay) into RDT studies is generally acceptable. If in vitro genotoxicity results are unavailable or positive, consensus was reached that the maximum tolerated dose (MTD) is acceptable as the top dose in RDT studies in many cases, such as when the RDT study MTD or exposure is close (50% or greater) to an acute study MTD or exposure. Finally, the group agreed that exceptions to this general rule might be acceptable, for example when human exposure is lower than the preclinical exposure by a large margin.

We examined the dose-dependency of gene expression changes for 51 genes in mouse liver treated with two N-nitroso genotoxic hepatocarcinogens, diethylnitrosamine (DEN) and ethylnitrosourea (ENU) by quantitative real-time PCR (qPCR). DEN (3, 9, 27 and 80mg/kg bw) or ENU (6, 17, 50 and 150mg/kg bw) was injected intraperitoneally into groups of five male 9-week-old B6C3F(1) mice and the livers were dissected after 4h and 28 days. Total RNA from pooled livers was reverse-transcribed to cDNA and the amount of each gene was quantified by qPCR. Results were analyzed by hierarchical and k-means clustering and ingenuity pathway analysis (IPA). The most characteristic result was a similar dose-dependency of gene expression changes with DEN and ENU. Twenty-one genes exhibited a distinct dose-dependent increase in expression at 4h for both carcinogens [Bax, Btg2, Ccng1, Cdkn1a, Cyp4a10, Cyp21a1, Fos, Gadd45b, Gdf15, Hmox1, Hspb1, Isg20l1, Jun, Mbd1, Mdm2, Myc, Net1, Plk2, Ppp1r3c, Rcan1 and Tubb2c], although the increase in gene expression due to ENU was generally weaker than that due to DEN. Only Gdf15 showed a dose-dependent increase in expression at 28 days for both carcinogens. The differences between DEN and ENU were in the expression of additional genes (7 for DEN and 8 for ENU). IPA extracted five gene networks: Network-1 included genes related to cancer and cell cycle arrest and associated with Bax, Btg2, Ccng1, Cdkn1a, Gadd45b, Gdf15, Hspb1, Mdm2 and Plk2 and Network-2 was related to DNA replication, recombination, repair and cell death and associated with Cyp21a1, Gdf15, Ppp1r3c, Rcan1 and Tubb2c. The present results show a distinct dose-dependency of gene expression changes induced by DEN and ENU. These changes were associated with cancer, cell cycle arrest, DNA replication, recombination, repair and cell death and were seen not only at 4h but also, for some, at 28 days after administration.

We studied five carcinogens for (a) organ-specific mutagenicity and expression time in the transgenic (TG) mouse mutation assay and (b) clastogenicity in the peripheral blood micronucleus assay in the same mice. Groups of mice were injected intraperitoneally (ip) with N-nitroso-di-n-propylamine (NDPA), propylnitrosourea (PNU), 7, 12-dimethylbenz[a]anthracene (DMBA), 4-nitroquinoline-1-oxide (4NQO), or procarbazine (PCZ); 4NQO was also administered orally. LacZ mutant frequencies (MF) of various organs, sampled 7, 14 and 28 days after treatment, were analyzed by galE positive selection. At least 5 organs were analyzed in each experiment. Bone marrow, liver, and testis were always analyzed, as were each chemical's target organs. All chemicals, except NDPA, induced micronuclei. All chemicals increased lacZ MF in all of their target organs for carcinogenesis and, to a lesser extent, in some non-target organs. That suggests that an organ that has a positive response to a chemical in the TG mouse mutation assay is likely to develop tumors on exposure to that chemical, but it does not always happen. The time-course of MF increases (7-28 days) differed among tissues. In general, time-dependent increase in MF occurred in organs with a low cell proliferation rate whereas no increase, or even a decrease, occurred in organs with a high proliferation rate. Our results demonstrated that the TG mouse mutation assay is effective for the detection of chemical mutagenesis in the target organs for carcinogenesis, and organ and time-course variations in chemical mutagenesis are important issues for the establishment of an optimal protocol for the assay.

As part of a collaborative study, the Mammalian Mutagenesis Study Group (MMS), a sub-organization of the Environmental Mutagen Society of Japan (JEMS) conducted mutagenicity tests in MutaMouse. Using a positive selection method, we studied the organ-specificity and time dependence of mutation induction by 4-nitroquinoline 1-oxide (4NQO). A single dose of 4NQO was administered intraperitoneally (7.5 or 15 mg/kg) or orally (200 mg/kg) to groups of male mice. On days 7, 14 and 28 after treatment, we isolated the liver, kidney, lung, spleen, bone marrow, testis and stomach in the intraperitoneal administration experiment and the liver, lung, bone marrow, testis and stomach in the oral administration experiment. In addition, we performed the peripheral blood micronucleus test to evaluate clastogenicity. In the mice treated intraperitoneally at 7.5 mg/kg, we found increased mutant frequency (MF) only in the lung, where the MF did not vary with expression time. In the mice treated at 15 mg/kg, we found increased MF in the liver, bone marrow and lung. In orally treated mice, the MF was high in the lung and liver and very high in the bone marrow and stomach while the increase in the testis was negligible. As the expression time was prolonged, the MF tended to increase in the liver, decrease in the bone marrow, and remain stable in the lung, testis and stomach. The incidence of micronucleus induction in peripheral blood cells was significantly increased (p0.01) in the 4NQO groups when compared with the vehicle control group by intraperitoneal treatment. Thus, these assay systems appeared to be of use in detecting not only genetic mutation but also chromosomal aberration.

Crocidolite, a carcinogenic asbestos in humans, specifically induces mesothelioma. We investigated the cytogenotoxic effects of crocidolite in a human mesothelioma cell line, MSTO211H, and a human promyelocytic leukemia cell line, HL60. Using confocal laser scanning microscopy, we found that the MSTO211H cells had phagocytotic activity, whereas the HL60 cells did not. In the MSTO211H cells, crocidolite decreased the cell population and increased the numbers of polynucleated cells (PN) and tetraploid cells, and increased the coefficients of variation (CV) of DNA contents in G0/G1 cells and the formation of 8-hydroxydeoxyguanosine. In contrast, crocidolite showed none of these cytogenotoxic effects in HL60 cells. To investigate the importance of phagocytosis in the cytogenotoxicity of crocidolite, we sorted the crocidolite-phagocytosed cells from less-phagocytosed cells by fluorescence-activated cell sorting, and studied the differences in cytogenotoxicity between these two cell groups. We found significant increases in the numbers of PN and tetraploid cells and the CV in the crocidolite-phagocytosed cells compared to the less-phagocytosed cells. These findings indicate that MSTO211H cells are susceptible to the cytogenotoxic effects of asbestos due to their phagocytotic activity, and that the MSTO211H cell line is suitable for the detection of such effects on human cells by asbestos and other materials which need to be phagocytosed to exert their toxicity.

The effect of intraperitoneal injection (i.p.) versus oral gavage administration (p.o.) of potassium bromate was examined using the micronucleus test in 2 strains of male mice (MS/Ae and CD-1). First, a small acute toxicity test and a pilot micronucleus experiment were performed to determine the appropriate dose range and sampling time for the full-scale micronucleus test. The full-scale test was carried out using doses of 18.8, 37.5, 75, and 150 mg/kg in the i.p. test and of 37.5, 75, 150, and 300 mg/kg in the p.o. test. The sampling time was 24 h for both mouse strains. Potassium bromate induced micronucleated polychromatic erythrocytes (MNPCEs) dose-dependently by both routes of administration in both mouse strains. No distinct difference in route of administration was observed in the test with MS/Ae mice. In CD-1 mice more MNPCEs were induced by the i.p. route than by the p.o. route.