A four-day oral treatment regimen for simultaneous micronucleus analyses in the glandular stomach, colon, and bone marrow of rats.

Our aim was to develop a multi-tissue micronucleus (MN) test method for the simultaneous analysis of rat glandular stomach, colon, and bone marrow. We have evaluated the multi-tissue MN test method with a regimen in which rats were administered chemicals orally once per day for four days and the cells of each tissue were collected 24 h after the final dose. The following compounds were studied: N-nitroso-N-methylurea (MNU), 4-nitroquinoline-1-oxide (4NQO), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), N-methyl-N-nitrosourethane (NMUT), 1,2-dimethylhydrazine 2HCl (DMH), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine HCl (PhIP), KBrO(3), amaranth (AM), and quercetin (QN). The gastrointestinal tract carcinogens increased the frequencies of micronucleated (MNed) cells in target tissue in a dose-dependent manner: MNU in gastric- and colonic-cells; 4NQO, MNNG, and NMUT in gastric cells; DMH and PhIP in colonic cells. In immature erythrocytes, MNU, 4NQO, DMH, and PhIP increased the frequency of MNed cells but MNNG and NMUT did not. The food additive KBrO(3), which is known to be a renal carcinogen, increased the frequencies of MNed cells in the glandular stomach and bone marrow. The food additive AM and the plant flavonoid QN, which are non-carcinogenic in most studies, did not cause increased MNed cells in any of the three tissues. Our results indicate that this multi-tissue MN test method is useful for the comprehensive evaluation of the genotoxicity of orally administered compounds.

In vivo rat glandular stomach and colon micronucleus tests: Kinetics of micronucleated cells, apoptosis, and cell proliferation in the target tissues after a single oral administration of stomach- or colon-carcinogens.

We have developed in vivo micronucleus (MN) tests by using an epithelial cell suspension isolated from the glandular stomach and colon of rodents. In the present study, our aim was to demonstrate the characteristics of the glandular stomach and colon MN tests by analyzing time-related changes in MN frequencies, apoptosis and cell proliferation in the target tissues of male CD (SD) rats that were orally administered a single dose of a stomach- or colon-targeted carcinogen, i.e., N-nitroso-N-methylurea (MNU) or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) for the stomach and 1,2-dimethylhydrazine dihydrochloride (DMH) for the colon. After treatment, the MN frequencies significantly increased in the respective target tissues, peaking at 48-96h and decreasing afterwards. The time-response pattern could be explained by the epithelial cell turnover confirmed with a labeling experiment using the thymidine analog, 5-ethynyl-2'-deoxyuridine (EdU). In the study with MNU and DMH, we also prepared paraffin sections of the respective target tissues for the immunohistochemical evaluation of apoptosis and cell proliferation. The incidence of apoptosis increased in the early phase (6 and/or 24h) after treatment, and then decreased. Cell proliferation was depressed when a high incidence of apoptosis was observed, and then it recovered until 72h. MN frequencies increased with the recovery of cell proliferation occurring later than the peak apoptosis response. These results indicated that micronuclei were induced in the glandular stomach and colon epithelial cells by administration of the model chemicals. On the other hand, MNU induced significant increases of MNed cells in both the glandular stomach and bone marrow in the same rats, while MNNG did only in the glandular stomach when administered orally up to 1/4 of the LD50. These results suggest that the glandular stomach- and colon-MN tests would be useful for evaluating the genotoxicity of agents in the gastrointestinal tract.

Persistence and accumulation of micronucleated hepatocytes in liver of rats after repeated administration of diethylnitrosamine.

A repeat-dose micronucleus assay in adult rat liver was recently developed [Mutat. Res. 747 (2012) 234-239]. This assay demonstrated a high detectability of hepatocarcinogens at relatively low doses, as indicated by dose-dependent micronucleus induction. Because the adult rat liver is known to have a long life-span, this desirable property of the assay will be an advantage in detecting micronucleated hepatocytes (MNHEPs) that have persisted for long periods in the liver following repeated dosing. However, no data directly supporting the underlying mechanisms have been published to date. In the present study, we verified the mechanisms by means of pulse-labeling of micronucleated hepatocytes with the thymidine analog 5-ethynyl-2'-deoxyuridine (EdU). The rodent hepatocarcinogen diethylnitrosamine (DEN) was repeatedly administered orally to male Crl:CD (SD) rats (6 weeks old) for up to 2 weeks, and EdU was injected intraperitoneally on days 1, 7, or 14. Hepatocytes were isolated by use of a non-perfusion technique at 24h, 1 week, or 2 weeks after EdU injection and analyzed for EdU incorporation and micronucleus formation. The results of our study confirmed that MNHEPs labeled with EdU on the first day of DEN administration persisted until 2 weeks post-administration in the rat livers. However, the frequency of MHNEPs among EdU-labeled hepatocytes decreased over time. In addition, the number of terminal deoxynucleotidyl transferase-mediated nick end-labeling (TUNEL)-positive cells in the liver tissue increased, suggesting selective removal of micronucleated cells. Theoretical calculation of the cumulative MNHEP frequency on each of the days on which DEN was administered, taking into account the rate of loss, came out closer to the actual value observed in the liver micronucleus test. Taken together, these results indicate that although micronucleated cells induced in rat livers by administration of the genotoxic hepatocarcinogen DEN undergo selective removal, they persist for a long time in a certain proportion, and repeated administration results in their accumulation and increased frequency.

Micronucleus test using formalin-fixed rat glandular stomach and colon.

BACKGROUND: Genotoxicity in tissues other than hematopoietic tissues, such as the liver and gastrointestinal (GI) tract, is an important focus in the risk assessment of chemicals in humans. We previously developed a rat micronucleus test for the GI tract, which is the first contact tissue where chemicals are introduced into the body through oral exposure. Target cells were obtained from fresh tissue samples by ethylenediaminetetraacetic acid disodium salt (EDTA) treatment. As an improvement to this method, we have used formalin-fixed tissues instead of fresh tissues; this approach can be used for tissues that are sampled from other toxicological tests and that are archived for several years. This new method can be used for examining micronucleus induction retrospectively when needed. In the present study, we compared the performance of the EDTA method and the new method with formalin-fixed tissues (formalin-fixation method). RESULTS: Histological examination showed that both the EDTA and formalin-fixation methods could be used for collecting cells located in or above the proliferative zone of the GI tract tissues of rats. In addition, the collected cells were similar in shape. We conducted micronucleus tests with rat GI tract tissues by the two methods using model chemicals, which were used as positive control chemicals (a combination of diethylnitrosamine, 1,2-dimethylhydrazine dihydrochloride, and potassium bromate). The two methods showed similar results. We additionally evaluated the aging effect of tissues stored in formalin fixative. The results showed that 1 year of storage did not affect the frequency of micronucleated cells. CONCLUSION: The equivalence of the EDTA and formalin-fixation methods was confirmed, and micronucleus analysis was possible up to at least 1 year after formalin fixation of the GI tract, indicating that the formalin-fixation method is valuable for the rat GI tract micronucleus test.

In vivo genotoxicity testing strategies: Report from the 8th International Workshop on Genotoxicity Testing (IWGT).

The in vivo working group (WG) considered three topics: acceptable maximum doses for negative erythrocyte micronucleus (MN) tests, validation status of MN assays in non-hematopoietic tissues, and nuisance factors in the comet assay. The WG reached agreement on many issues, including: negative erythrocyte MN studies should be acceptable if dosing is conducted to Organisation for Economic Co-operation and Development (OECD) test guideline (TG) 474 recommendations and if sufficient bone marrow exposure is demonstrated; consensus on the evidence required to demonstrate "sufficient" exposure was not reached. The liver MN test using six-week-old rats is sufficiently validated to develop an OECD TG, but the impact of animal age warrants additional study. Ki-67 is a reliable marker for cellular proliferation in hepatocytes. The gastrointestinal tract MN test is useful for detecting poorly absorbed or rapidly degraded aneugens, and for genotoxic metabolites formed in the colon. Although current validation data are insufficient to support the development of an OECD TG, the methodologies are sufficient to consider as an appendix to OECD TG474. Comparison of comet assay results to laboratory historical control data (HCD) should not be used in data evaluation, unless the HCD distribution is demonstrated to be stable and the predominant source of HCD variation is due to animal, not study, factors. No universally acceptable negative control limit for any tissue was identified. Methodological differences in comet studies can result in variable data interpretations; more data are required before best practice recommendations can be made. Hedgehogs alone are unreliable indicators of cytotoxicity and additional investigations into cytotoxicity markers are required.

Discrimination of genotoxic and non-genotoxic hepatocarcinogens by statistical analysis based on gene expression profiling in the mouse liver as determined by quantitative real-time PCR.

The general aim of the present study is to discriminate between mouse genotoxic and non-genotoxic hepatocarcinogens via selected gene expression patterns in the liver as analyzed by quantitative real-time PCR (qPCR) and statistical analysis. qPCR was conducted on liver samples from groups of 5 male, 9-week-old B6C3F(1) mice, at 4 and 48h following a single intraperitoneal administration of chemicals. We quantified 35 genes selected from our previous DNA microarray studies using 12 different chemicals: 8 genotoxic hepatocarcinogens (2-acetylaminofluorene, 2,4-diaminotoluene, diisopropanolnitrosamine, 4-dimethylaminoazobenzene, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, N-nitrosomorpholine, quinoline and urethane) and 4 non-genotoxic hepatocarcinogens (1,4-dichlorobenzene, dichlorodiphenyltrichloroethane, di(2-ethylhexyl)phthalate and furan). A considerable number of genes exhibited significant changes in their gene expression ratios (experimental group/control group) analyzed statistically by the Dunnett's test and Welch's t-test. Finally, we distinguished between the genotoxic and non-genotoxic hepatocarcinogens by statistical analysis using principal component analysis (PCA) of the gene expression profiles for 7 genes (Btg2, Ccnf, Ccng1, Lpr1, Mbd1, Phlda3 and Tubb2c) at 4h and for 12 genes (Aen, Bax, Btg2, Ccnf, Ccng1, Cdkn1a, Gdf15, Lrp1, Mbd1, Phlda3, Plk2 and Tubb2c) at 48h. Seven major biological processes were extracted from the gene ontology analysis: apoptosis, the cell cycle, cell proliferation, DNA damage, DNA repair, oncogenes and tumor suppression. The major, biologically relevant gene pathway suggested was the DNA damage response pathway, resulting from signal transduction by a p53-class mediator leading to the induction of apoptosis. Eight genes (Aen, Bax, Btg2, Ccng1, Cdkn1a, Gdf15, Phlda3 and Plk2) that are directly associated with Trp53 contributed to the PCA. The current findings demonstrate a successful discrimination between genotoxic and non-genotoxic hepatocarcinogens, using qPCR and PCA, on 12 genes associated with a Trp53-mediated signaling pathway for DNA damage response at 4 and 48 h after a single administration of chemicals.

Evaluation of a 4-day repeated-dose micronucleus test in rat glandular stomach and colon using aneugens and non-genotoxic non-carcinogens.

BACKGROUND: We previously developed a rodent gastrointestinal (GI) tract micronucleus (MN) test using the glandular stomach and/or colon, and evaluated this test method using several genotoxic carcinogens (clastogens) and genotoxic non-carcinogens; we demonstrated that this test method could detect genotoxic stomach and/or colon carcinogens with target organ specificity. In the present study, we further evaluated the sensitivity and specificity of the MN test for the rat glandular stomach and colon using three aneugens (colchicine, vinblastine sulfate, and docetaxel hydrate) and two non-genotoxic non-carcinogens (sodium chloride and sucrose). RESULTS: Male Crl:CD (SD) rats were administered test compounds through clinical administration route (orally or intravenously) for four consecutive days and then examined for the micronucleated cell frequencies in the glandular stomach and colon. We observed that all three aneugens significantly and dose-dependently increased the micronucleated cell frequencies in the stomach and colon. In contrast, neither of the two non-genotoxic non-carcinogens increased the micronucleated cell frequency in these tissues. Notably, an increase in cell proliferation was observed in the glandular stomach of rats administered a stomach toxicant, sodium chloride, but this increase did not affect the induction of micronuclei in the gastric cells. CONCLUSIONS: In the present study, it was demonstrated that the glandular stomach and colon MN tests could detect aneugens as positive and could adequately evaluate non-genotoxic non-carcinogens as negative, including a chemical that enhances cell proliferation. These results provide important evidence supporting good performance of the rat glandular stomach and colon MN tests with a 4-day treatment regimen.

Detection of hepatocarcinogens by combination of liver micronucleus assay and histopathological examination in 2-week or 4-week repeated dose studies.

BACKGROUND: Currently, revisions to the ICH S1 guidance on rodent carcinogenicity testing are being proposed. Application of this approach would reduce the use of animals in accordance with the 3Rs principles (reduce/refine/replace). The method would also shift resources to focus on more scientific mechanism-based carcinogenicity assessments and promote safe and ethical development of new small molecule pharmaceuticals. In the revised draft, findings such as cellular hypertrophy, diffuse and/or focal cellular hyperplasia, persistent tissue injury and/or chronic inflammation, preneoplastic changes, and tumors are listed as histopathology findings of particular interest for identifying carcinogenic potential. In order to predict hepatocarcinogenicity of test chemicals based on the results from 2- or 4-week repeated dose studies, we retrospectively reanalyzed the results of a previous collaborative study on the liver micronucleus assay. We focused on liver micronucleus induction in combination with histopathological changes including hypertrophy, proliferation of oval cells or bile duct epithelial cells, tissue injuries, regenerative changes, and inflammatory changes as the early responses of hepatocarcinogenesis. For these early responses, A total of 20 carcinogens, including 14 genotoxic hepatocarcinogens (Group A) and 6 non-liver-targeted genotoxic carcinogens (Group B) were evaluated. RESULTS: In the Group A chemicals, 5 chemicals (NPYR, MDA, NDPA, 2,6-DNT, and NMOR) showed all of the 6 early responses in hepatocarcinogenesis. Five chemicals (DMN, 2,4-DNT, QUN, 2-AAF, and TAA) showed 4 responses, and 4 chemicals (DAB, 2-NP, MCT, and Sudan I) showed 3 responses. All chemicals exhibited at least 3 early responses. Contrarily, in the Group B chemicals (6 chemicals), 3 of the 6 early responses were observed in 1 chemical (MNNG). No more than two responses were observed in 3 chemicals (MMC, MMS, and KA), and no responses were observed in 2 chemicals (CP and KBrO3). CONCLUSION: Evaluation of liver micronucleus induction in combination with histopathological examination is useful for detecting hepatocarcinogens. This assay takes much less time than routine long-term carcinogenicity studies.

Markedly enhanced micronucleus induction by 1,2-dimethylhydrazine dihydrochloride in colonic cells of rats with bacterial colonization in the intestine.

To investigate how intestinal bacteria affect host cytogenetic alterations in the early initiation step of colon carcinogenesis, we conducted a comet assay and micronucleus (MN) test using germ-free (GF) and conventionalized (Cvd) rats after a single subcutaneous injection of the carcinogen, 1,2-dimethylhydrazine dihydrochloride (DMH). DNA damage was also determined in the liver in comet assays, as DMH is metabolized and activated in this organ. The time-response patterns of DNA damage in the liver and colon were similar in both rats, and maximum values were observed at 3 h after the treatment. In contrast, the maximum frequency of micronucleated (MNed) colonic cells was markedly higher in the Cvd rats than in the GF rats and was observed after 72 h and 120 h, respectively. The frequency of MNed cells in non-treated animals was similar in the GF and Cvd rats. In addition, we determined time-responses in the incidence of apoptosis and cell proliferation indices, i.e., the numbers of BrdU-labeled cells, mitotic cells in the crypts, and crypt column heights, using histological sections of the colons in these rats. Maximal incidence of apoptosis was observed at 6 and 24 h in the Cvd and GF rats, respectively. The value in the Cvd rats tended to be higher than that in the GF rats. Cell proliferation was suppressed until 24 and 48 h in the Cvd and GF rats, respectively, and increased subsequently. The rebound response of cell proliferation was more pronounced and occurred earlier in the Cvd rats than that in the GF rats. We demonstrated that cytogenetic alterations other than DNA damage, particularly the MNed colonic cell induction by DMH, were markedly enhanced in rats with bacterial colonization in the intestine compared to those in GF rats.

Evaluation of a 28-day repeated-dose micronucleus test in rat glandular stomach, colon, and liver using gastrointestinal tract-targeted genotoxic-carcinogens and non-carcinogens.

The usefulness of the rat repeated-dose liver and gastrointestinal (GI) tract micronucleus (MN) tests to detect site-specific carcinogens has been shown previously using 22 chemicals in a study conducted by the Mammalian Mutagenicity Study group in the Japanese Environmental Mutagen Society. However, in the 6th International Workshop on Genotoxicity Testing, the need for further data to identify the sensitivity and specificity of the GI tract MN test and the specificity of the liver MN test, for the purpose of regulatory use, was mentioned. In the present study, we conducted additional studies to validate the performance of the 28-day repeated-dose GI tract and liver MN tests using genotoxic stomach carcinogens, N-nitroso-N-methylurea (MNU) and N-methyl-N-nitrosourethane (NMUT); genotoxic colon carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine hydrochloride (PhIP), and non-carcinogens, sodium chloride, sucrose, and amaranth. Male Crl:CD (SD) rats were administered with each chemical by oral gavage for 28 days and the micronucleated cell frequencies in the glandular stomach, colon, and liver were monitored. MNU and NMUT showed positive results in the glandular stomach, and PhIP did so in the colon. These carcinogens showed negative results in the liver, which is not a target organ for these chemicals. Negative results were obtained for all three non-carcinogens in the glandular stomach, colon, and liver. Therefore, it was shown that the glandular stomach and colon MN tests with 28-day repeated-dose regimen have a good sensitivity for detecting genotoxic GI tract carcinogens as positive and have a good specificity to determined non-carcinogens as negative. The negative results with these six chemicals in the liver provide additional evidence supporting the good specificity of the 28-day repeated-dose liver MN test.

Evaluation of the novel liver micronucleus assay using formalin-fixed tissues.

BACKGROUND: The repeated-dose liver micronucleus (RDLMN) assay is an effective and important in vivo test for detecting genotoxic compounds, particularly for those that require metabolic activation to show genotoxicity. In a collaborative study by the Collaborative Study Group for the Micronucleus Test (CSGMT)/The Japanese Environmental Mutagen Society (JEMS) - Mammalian Mutagenicity Study Group (MMS), micronucleus induction of 22 chemicals with the RDLMN assay employing the collagenase digestion method was examined and reported on. Recently, we have developed a method which enables retrospective evaluation of micronucleus induction in formalin-fixed liver tissues (the formalin-fixed method) obtained in general toxicity studies completed in the past. Using this method, we were able to easily evaluate clastogenic potential of chemicals from the formalin-fixed tissues obtained in the general toxicity studies.In this study, to evaluate the usefulness of the formalin-fixed method, we have conducted a liver micronucleus assay using the formalin-fixed liver samples obtained from the above collaborative study (18 of 22 test chemicals) and carried out a comparison with the results obtained by the collagenase digestion method. RESULTS: Comparison of the collagenase digestion and formalin-fixed methods was conducted using the results of the micronucleus assays with a total of 18 test chemicals which included 12 genotoxic hepatocarcinogens (Group A), 4 genotoxic carcinogens but not liver targeted (Group B), and 2 nongenotoxic hepatocarcinogens (Group C). The formalin-fixed method obtained the similar results as the collagenase digestion method in 10 out of the 12 chemicals of Group A, and all chemicals of Group B and Group C. Although the results were statistically contradictive due to different levels of concurrent negative control, the 2 other chemicals of Group A showed comparable responses between the two methods. CONCLUSION: The present study shows that the formalin-fixed method is capable of detecting liver carcinogens with sensitivity equal to or higher than that of the collagenase digestion method. We recommend use of the formalin-fixed method because of its capability of enabling retrospective evaluation of micronucleus induction in the formalin-fixed liver tissues obtained in general toxicity studies completed in the past.

In vivo genotoxicity testing strategies: Report from the 7th International workshop on genotoxicity testing (IWGT).

The working group reached complete or majority agreement on many issues. Results from TGR and in vivo comet assays for 91 chemicals showed they have similar ability to detect in vivo genotoxicity per se with bacterial mutagens and Ames-positive carcinogens. TGR and comet assay results were not significantly different when compared with IARC Group 1, 2 A, and unclassified carcinogens. There were significantly more comet assay positive responses for Group 2B chemicals, and for IARC classified and unclassified carcinogens combined, which may be expected since mutation is a sub-set of genotoxicity. A liver comet assay combined with the bone marrow/blood micronucleus (MNviv) test would detect in vivo genotoxins that do not exhibit tissue-specific or site-of-contact effects, and is appropriate for routine in vivo genotoxicity testing. Generally for orally administered substances, a comet assay at only one site-of-contact GI tract tissue (stomach or duodenum/jejunum) is required. In MNviv tests, evidence of target tissue exposure can be obtained in a number of different ways, as recommended by ICH S2(R1) and EFSA (Hardy et al., 2017). Except for special cases the i.p. route is inappropriate for in vivo testing; for risk evaluations more weight should be given to data from a physiologically relevant administration route. The liver MN test is sufficiently validated for the development of an OECD guideline. However, the impact of dosing animals >6 weeks of age needs to be evaluated. The GI tract MN test shows promise but needs more validation for an OECD guideline. The Pig-a assay detects systemically available mutagens and is a valuable follow-up to in vitro positive results. A new freeze-thaw protocol provides more flexibility. Mutant reticulocyte and erythrocyte frequencies should both be determined. Preliminary data are available for the Pig-a assay in male rat germ cells which require validation including germ cell DNA mutation origin.

Detection of micronucleated cells and gene expression changes in glandular stomach of mice treated with stomach-targeted carcinogens.

To assess the genotoxicity of chemicals on the stomach, we developed in vivo assays that can detect micronucleus induction and gene expression changes in epithelial cells of the glandular stomach in mice. Male BALB/c mice were orally given a single dose (100 mg/kg) of N-nitroso-N-methylurea (MNU) or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) as stomach-targeted carcinogens. The glandular stomach was excised at 4h, 3 and 4 days after administration, and a single cell suspension of epithelial cells was prepared from the everted glandular stomach by EDTA treatment. For determination of micronucleus induction, gastric epithelial cells on days 3 and 4 after administration were fixed with 10% neutral-buffered formalin, stained with a combination of AO-DAPI, and analyzed under fluorescence microscopy. We also examined the induction of micronuclei in peripheral blood of these mice on days 2 and 3 after administration. Moreover, total RNA was extracted from gastric epithelial cells at 4h after administration, and p21 and plk2 expression was analyzed using a quantitative RT-PCR technique. 1) A significant increase of micronucleated cells was observed in the glandular stomach in mice treated with N-nitroso-N-methylurea (MNU) or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) compared to mice treated with vehicle. 2) In peripheral blood, induction of micronuclei was observed in mice treated with MNU but not with MNNG. 3) p21 and plk2, which related to cell cycle arrest, were up-regulated in the glandular stomach in mice treated with MNU or MNNG compared to mice treated with vehicle. The present study showed that these assays using glandular stomach may help to evaluate the genotoxicity of chemicals after oral administration.

In vivo erythrocyte micronucleus assay III. Validation and regulatory acceptance of automated scoring and the use of rat peripheral blood reticulocytes, with discussion of non-hematopoietic target cells and a single dose-level limit test.

The in vivo micronucleus assay working group of the International Workshop on Genotoxicity Testing (IWGT) discussed new aspects in the in vivo micronucleus (MN) test, including the regulatory acceptance of data derived from automated scoring, especially with regard to the use of flow cytometry, the suitability of rat peripheral blood reticulocytes to serve as the principal cell population for analysis, the establishment of in vivo MN assays in tissues other than bone marrow and blood (for example liver, skin, colon, germ cells), and the biological relevance of the single-dose-level test. Our group members agreed that flow cytometric systems to detect induction of micronucleated immature erythrocytes have advantages based on the presented data, e.g., they give good reproducibility compared to manual scoring, are rapid, and require only small quantities of peripheral blood. Flow cytometric analysis of peripheral blood reticulocytes has the potential to allow monitoring of chromosome damage in rodents and also other species as part of routine toxicology studies. It appears that it will be applicable to humans as well, although in this case the possible confounding effects of splenic activity will need to be considered closely. Also, the consensus of the group was that any system that meets the validation criteria recommended by the IWGT (2000) should be acceptable. A number of different flow cytometric-based micronucleus assays have been developed, but at the present time the validation data are most extensive for the flow cytometric method using anti-CD71 fluorescent staining especially in terms of inter-laboratory collaborative data. Whichever method is chosen, it is desirable that each laboratory should determine the minimum sample size required to ensure that scoring error is maintained below the level of animal-to-animal variation. In the second IWGT, the potential to use rat peripheral blood reticulocytes as target cells for the micronucleus assay was discussed, but a consensus regarding acceptability for regulatory purposes could not be reached at that time. Subsequent validation efforts, combined with accumulated published data, demonstrate that blood-derived reticulocytes from rats as well as mice are acceptable when young reticulocytes are analyzed under proper assay protocol and sample size. The working group reviewed the results of micronucleus assays using target cells/tissues other than hematopoietic cells. We also discussed the relevance of the liver micronucleus assay using young rats, and the importance of understanding the maturation of enzyme systems involved in the processes of metabolic activation in the liver of young rats. Although the consensus of the group was that the more information with regard to the metabolic capabilities of young rats would be useful, the published literature shows that young rats have sufficient metabolic capacity for the purposes of this assay. The use of young rats as a model for detecting MN induction in the liver offers a good alternative methodology to the use of partial hepatectomy or mitogenic stimulation. Additional data obtained from colon and skin MN models have been integrated into the data bases, enhancing confidence in the utility of these models. A fourth topic discussed by the working group was the regulatory acceptance of the single-dose-level assay. There was no consensus regarding the acceptability of a single dose level protocol when dose-limiting toxicity occurs. The use of a single dose level can lead to problems in data interpretation or to the loss of animals due to unexpected toxicity, making it necessary to repeat the study with additional doses. A limit test at a single dose level is currently accepted when toxicity is not dose-limiting.

Detection of Pig-a gene mutants in rat peripheral blood following a single urethane treatment: A comparison of the RBC Pig-a and PIGRET assays.

The rat red blood cell (RBC) Pig-a assay has been recommended by an expert working group of the International Workshop on Genotoxicity Testing as a potential new method to evaluate in vivo gene mutations in regulatory genotoxicity risk assessments. In a collaborative study in Japan, an improved Pig-a assay using reticulocytes (PIGRET assay) with magnetic enrichment of CD71-positive cells was evaluated, and it was revealed that this assay could detect the mutagenicity of chemicals earlier than the RBC Pig-a assay could. To verify further the suitability of the PIGRET assay for an in vivo short-term genotoxicity screening test, a joint research study was conducted by the Japanese Environmental Mutagen Society, and 24 compounds were evaluated. One of the compounds evaluated in this study was urethane, a multi-organ rodent carcinogen. Urethane (250, 500, and 1000mg/kg body weight) was orally administered once to 8-week-old male Crl:CD (SD) rats. Blood samples were collected at 1, 2, and 4 weeks after the administration and processed for the RBC Pig-a and PIGRET assays. In the PIGRET assay, the Pig-a mutant frequency (MF) significantly increased at both 2 and 4 weeks after the treatment of 1000mg/kg of urethane. However, in the RBC Pig-a assay, a significant increase in the Pig-a MF was observed only at 1 week after the treatment with 500mg/kg, but the MF value was within our historical control range; therefore, it was judged to be negative. These results suggest that the PIGRET assay might be useful for evaluating the in vivo mutagenicity more clearly than the RBC Pig-a assay after a single treatment of test compounds.

Repeated-dose liver and gastrointestinal tract micronucleus assays with CI Solvent Yellow 14 (Sudan I) using young adult rats.

The in vivo genotoxicity of CI Solvent Yellow 14 (Sudan I) was examined using repeated-dose liver and gastrointestinal tract micronucleus (MN) assays in young adult rats. Sudan I is a mono-azo dye based on aniline and 1-amino-2-hydroxynaphthalene. This dye was demonstrated as a rat liver carcinogen in a National Toxicology Program (NTP) bioassay, and genotoxicity was noted in a rat bone marrow micronucleus (BMMN) assay. In the present study, Sudan I was administered orally to rats for 14-days, and the MN frequency in the liver, stomach, colon, and bone marrow were analyzed. The frequency of micronucleated hepatocytes (MNHEPs) was not significantly increased by the administration of the Sudan I. Gastrointestinal tract MNs were also not induced. However, in the BMMN assay, a significant increase in micronucleated immature erythrocytes (MNIMEs) was observed in a dose-dependent manner. While Sudan I has been reported to lack hepatic genotoxicity, it has also exhibited tumor-promoting activities. These results are consistent with the lack of induction of MN in the hepatocytes. The lack of MN induction in cells of the gastrointestinal tract was also logical because azo-compounds are reported to be unlikely to induce DNA damage in the rat gut. The repeated-dose rat liver and gastrointestinal tract MN assays have the potential to be used in the evaluation of the genotoxicity of a chemical in each organ in accordance with its mode of action.

Repeated dose liver and gastrointestinal tract micronucleus assays using N-methyl-N'-nitro-N-nitrosoguanidine in young adult rats.

N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG) is a direct-acting mutagen that induces tumors in the glandular stomach, but not in the liver or colon, of rats after oral administration. To evaluate the performance of repeated dose liver and gastrointestinal tract micronucleus (MN) assays in young adult rats, MNNG was administered by oral gavage to male CD (SD) rats aged 6 weeks at doses of 0 (vehicle; 2.5% DMSO aqueous solution), 3.125, 6.25, 12.5, and 25mg/kg/day once daily for 14 and 28 days, and the MN frequencies were examined in the hepatocytes, glandular stomach cells, and colonic cells. The MN induction in immature erythrocytes in the bone marrow of these animals was also simultaneously evaluated. The frequencies of micronucleated (MNed) glandular stomach cells were significantly increased in all MNNG treatment groups in a dose-dependent manner in both repeated dose studies. In contrast, the frequencies of MNed hepatocytes and colonic cells were not significantly increased compared to the vehicle control. In the bone marrow, a small but significant increase in the frequency of MNed immature erythrocytes was observed only at the highest dose in the 28-day study. Since a clear positive result in the glandular stomach agrees with the tissue specificity of tumor induction by this chemical, the MN assay with the glandular stomach, which is a direct contact site with high concentrations of test substances administered by oral gavage, may be useful for detecting genotoxic compounds that are short-lived in vivo, such as MNNG.

Recommended protocols for the liver micronucleus test: Report of the IWGT working group.

At the 6th International Workshop on Genotoxicity Testing (IWGT), the liver micronucleus test working group discussed practical aspects of the in vivo rodent liver micronucleus test (LMNT). The group members focused on the three methodologies currently used, i.e., a partial hepatectomy (PH) method, a juvenile/young rat (JR) method, and a repeated-dose (RD) method in adult rodents. Since the liver is the main organ that metabolizes chemicals, the LMNT is expected to detect clastogens, especially those that need metabolic activation in the liver, and aneugens. Based on current data the three methods seem to have a high sensitivity and specificity, but more data, especially on non-genotoxic but toxic substances, would be needed to fully evaluate the test performance. The three methods can be combined with the micronucleus test (MNT) using bone marrow (BM) and/or peripheral blood (PB). The ability of the PH method to detect both clastogens and aneugens has already been established, but the methodology is technically challenging. The JR method is relatively straightforward, but animal metabolism might not be fully comparable to adult animals, and data on aneugens are limited. These two methods also have the advantage of a short testing period. The RD method is also straightforward and can be integrated into repeated-dose (e.g. 2 or 4 weeks) toxicity studies, but again data on aneugens are limited. The working group concluded that the LMNT could be used as a second in vivo test when a relevant positive result in in vitro mammalian cell genotoxicity tests is noted (especially under the condition of metabolic activation), and a negative result is observed in the in vivo BM/PB-MNT. The group members discussed LMNT protocols and reached consensus about many aspects of test procedures. However, data gaps as mentioned above remain, and further data are needed to fully establish the LMNT protocol.

Micronucleus test in rodent tissues other than liver or erythrocytes: Report of the IWGT working group.

At the 6th International Workshop on Genotoxicity Testing, the liver micronucleus test (MNT) working group briefly discussed the MNT using tissues other than liver/erythrocytes. Many tissues other than liver/erythrocytes have been studied, primarily for research purposes. They have included the colon and intestinal epithelium, skin, spleen, lung, stomach, bladder, buccal mucosa, vagina, and fetal/neonatal tissues. These tissues were chosen because they were target sites of carcinogens, and/or relevant to a specific route of exposure. Recently, there has been particular focus on the gastrointestinal (GI) tract as it is a contact site associated with high exposure following oral gavage. Furthermore GI tumors are observed with high frequency in human populations. A collaborative study of the rat glandular stomach and colon MNT was conducted in conjunction with a collaborative study of the repeated-dose liver MNT. Based on limited data currently available, the rodent MNT using the glandular stomach and/or colon seems to detect genotoxic carcinogens with GI tract target-organ specificity. The working group concluded that the GI tract MNT would be a promising method to examine clastogenicity or aneugenicity of test chemicals in the stomach and/or colon. Further data will be needed to fully establish the methods, and to identify the sensitivity and specificity of the GI tract MNT.

Evaluation of repeated dose micronucleus assays of the liver and gastrointestinal tract using potassium bromate: a report of the collaborative study by CSGMT/JEMS.MMS.

The food additive potassium bromate (KBrO3) is known as a renal carcinogen and causes chromosomal aberrations in vitro without metabolic activation and in vivo in hematopoietic and renal cells. As a part of a collaborative study by the Mammalian Mutagenicity Study group, which is a subgroup of the Japanese Environmental Mutagen Society, we administered KBrO3 to rats orally for 4, 14, and 28 days and examined the micronucleated (MNed) cell frequency in the liver, glandular stomach, colon, and bone marrow to confirm whether the genotoxic carcinogen targeting other than liver and gastrointestinal (GI) tract was detected by the repeated dose liver and GI tract micronucleus (MN) assays. In our study, animals treated with KBrO3 showed some signs of toxicity in the kidney and/or stomach. KBrO3 did not increase the frequency of MNed cells in the liver and colon in any of the repeated dose studies. However, KBrO3 increased the frequency of MNed cells in the glandular stomach and bone marrow. Additionally, the MNed cell frequency in the glandular stomach was not significantly affected by the difference in the length of the administration period. These results suggest that performing the MN assay using the glandular stomach, which is the first tissue to contact agents after oral ingestion, is useful for evaluating the genotoxic potential of chemicals and that the glandular stomach MN assay could be integrated into general toxicity studies.