Development of a repeated-dose liver micronucleus assay using adult rats (II): further investigation of 1,2-dimethylhydrazine and 2,6-diaminotoluene.

Detecting genotoxicity in the liver is considered an effective approach for predicting hepatocarcinogenicity, as many genotoxic chemicals in vivo may act as hepatocarcinogens in rodents. Here, a genotoxic rodent hepatocarcinogen, 1,2-dimethylhydrazine dihydrochloride (1,2-DMH), and a genotoxic (Ames positive) noncarcinogen, 2,6-diaminotolunene (2,6-DAT), were administered orally to rats for up to 28 days, and liver samples were then examined in a repeated-dose liver micronucleus (MN) assay, and additionally tested in the bone marrow (BM) MN assay concurrently. We recently established a simple method to isolate hepatocytes without in situ liver perfusion procedures, and applied this method in the liver MN assay. As a result, 1,2-DMH increased the proportion of micronucleated hepatocytes in both a dose- and duration-dependent manner at relatively low-dose levels that are routinely used in repeated-dose toxicity studies. In contrast to 1,2-DMH, 2,6-DAT did not have a detectable effect. In addition to these two chemicals, two genotoxic rodent hepatocarcinogens, diethylnitrosamine and 2,4-diaminotoluene, which gave positive responses in the liver MN assay in our previous investigation [Narumi et al., Mutat. Res. 747 (2012) 234-239], were subjected to the BM MN assay and histopathological evaluation. All four test chemicals gave negative responses in the BM MN assay. Furthermore, the three hepatocarcinogens displayed hepatotoxicity, including hepatocellular hypertrophy and anisokaryosis, but no abnormal findings were observed in the liver of rats treated with 2,6-DAT. Taken together, the present results indicate that the liver MN assay is effective for predicting hepatocarcinogenicity and may be integrated into repeated-dose toxicity studies without disturbing routine examinations, such as histopathology. Furthermore, with repeat-dose treatment protocols, our findings indicate that the liver MN assay is superior to the BM MN assay for detecting genotoxic or carcinogenic chemicals in rats.

Differential gene expression profiling between genotoxic and non-genotoxic hepatocarcinogens in young rat liver determined by quantitative real-time PCR and principal component analysis.

We recently successfully discriminated mouse genotoxic hepatocarcinogens from non-genotoxic hepatocarcinogens via selected gene expression profiling in the mouse liver based on quantitative real-time PCR (qPCR) and statistical analysis using principal component analysis (PCA). In the present study, we applied these candidate marker genes to rat hepatocarcinogens in the rat liver. qPCR analysis of 33 genes was conducted on liver samples from groups of 4 male 4-week-old F344 rats at 4 and 48 h after a single oral administration of chemicals [2 genotoxic hepatocarcinogens: diethylnitrosamine and 2,6-dinitrotoluene; a non-genotoxic hepatocarcinogen: di(2-ethylhexyl)phthalate; and a non-genotoxic non-hepatocarcinogen: phenacetin]. Thirty-two genes exhibited significant changes in their gene expression ratios (experimental group/control group) according to statistical analysis using the Williams' test and the Dunnett's test. The changes appeared to be greater at 4h than at 48 h. Finally, statistical analysis via PCA successfully differentiated the genotoxic hepatocarcinogens from the non-genotoxic hepatocarcinogen and the non-genotoxic non-hepatocarcinogen at 4h based on 16 genes (Ccnf, Ccng1, Cyp4a10, Ddit4l, Egfr, Gadd45g, Gdf15, Hspb1, Igfbp1, Jun, Myc, Net1, Phlda3, Pml, Rcan1 and Tubb2c) and at 48 h based on 10 genes (Aen, Ccng1, Cdkn1a, Cyp21a1, Cyp4a10, Gdf15, Igfbp1, Mdm2, Phlda3 and Pmm1). Eight major biological processes were extracted from a gene ontology analysis: apoptosis, the cell cycle, cell proliferation, DNA damage, DNA repair, oxidative stress, oncogenes and tumor suppression. The major, biologically relevant gene pathway suggested was the DNA damage response, which signals through a Tp53-mediated pathway and leads to the induction of apoptosis. Immunohistochemical analyses for the expression of Cdkn1a and Hmox1 proteins and the level of apoptosis measured by the TUNEL assay in the liver confirmed the aforementioned results. The present results showed that mouse candidate marker genes are applicable for differentiating genotoxic hepatocarcinogens from non-genotoxic hepatocarcinogens examined in this paper in the rat liver.

Development of a repeated-dose liver micronucleus assay using adult rats: an investigation of diethylnitrosamine and 2,4-diaminotoluene.

Various liver micronucleus assay methods, such as those involving partial hepatectomy, treatment with mitogens, and the use of juvenile animals, have been developed. These assays have been proven to be of high sensitivity and specificity to predict hepatocarcinogenicity of compounds that cannot be detected by bone marrow micronucleus assays. On the contrary, the existing assays have only been evaluated for their use in detecting micronucleus induction in the settings of relatively short-term cell proliferation. However, the integration of in vivo genotoxicity endpoints into routine toxicity studies is increasingly desired from the viewpoint of animal welfare to reduce the number of animals used. In the present study, the rodent hepatocarcinogens diethylnitrosamine (DEN) and 2,4-diaminotoluene (2,4-DAT) were repeatedly administered orally to male Crl:CD (SD) rats (6 weeks old at the beginning of administration) for 5, 14, and 28 days, and changes in the frequency of hepatocytes with micronuclei in liver tissues that had undergone no artificial treatment to accelerate cell proliferation were evaluated. At the same time, a new method of hepatocyte isolation involving the treatment of a portion of the liver with collagenase in a centrifuge tube, without the use of in situ perfusion, was established. The induction of micronucleated hepatocytes was achieved after the repeated administration of DEN for 5 days or longer and of 2,4-DAT for 14 days or longer. Micronucleus frequencies were increased depending on the number of administrations, indicating that micronucleated hepatocytes had possibly remained for a long period of time and accumulated additively. It therefore appears that even in adult rat liver with low mitotic activity, a repeated-dose of a chemical substance for 14 days or longer enables the detection of micronucleus induction. In addition, the establishment of a method to isolate hepatocytes without perfusion using only a part of the liver enables the integration of liver micronucleus assays into general toxicity studies.

In vivo genotoxicity assessment of a multiwalled carbon nanotube in a mouse ex vivo culture.

BACKGROUND: Multiwalled carbon nanotubes (MWCNTs) are suspected lung carcinogens because their shape and size are similar to asbestos. Various MWCNT types are manufactured; however, only MWNT-7 is classified into Group 2B by The International Agency for Research on Cancer. MWNT-7's carcinogenicity is strongly related to inflammatory reactions. On the other hand, inconsistent results on MWNT-7 genotoxicity have been reported. We previously observed no significant differences in both Pig-a (blood) and gpt (lung) mutant frequencies between MWNT-7-intratracheally treated and negative control rats. In this study, to investigate in vivo MWNT-7 genotoxicity on various endpoints, we attempted to develop a lung micronucleus assay through ex vivo culture targeting the cellular fraction of Clara cells and alveolar Type II (AT-II) cells, known as the initiating cells of lung cancer. Using this system, we analyzed the in vivo MWNT-7 genotoxicity induced by both whole-body inhalation exposure and intratracheal instillation. We also conducted an erythrocyte micronucleus assay using the samples obtained from animals under intratracheal instillation to investigate the tissue specificity of MWNT-7 induced genotoxicities. RESULTS:  We detected a significant increase in the incidence of micronucleated cells derived from the cellular fraction of Clara cells and AT-II cells in both MWNT-7-treated and positive control groups compared to the negative control group under both whole-body inhalation exposures and intratracheal instillation. Additionally, the erythrocyte micronucleus assay detected a significant increase in the incidence of micronucleated reticulocytes only in the positive control group. CONCLUSIONS: Our findings indicated that MWNT-7 was genotoxic in the lungs directly exposed by both the body inhalation and intratracheal instillation but not in the hematopoietic tissue.

The effect of aging on the repeated-dose liver micronucleus assay.

BACKGROUND: The liver micronucleus (MN) assay is an effective and important in vivo test for detecting genotoxic compounds. In particular, the repeated-dose liver MN (RDLMN) assay which greatly facilitates incorporation of the liver MN assay into the general toxicity study has been developed. Usefulness of the RDLMN assay was appraised highly in the 7th International Workshops on Genotoxicity Testing (2017 in Tokyo) in that sufficient numbers and types of chemicals were studied and easy integration into the general toxicity study is preferred from the 3R's point of view. However, it was pointed out that it is necessary to evaluate the effect of age at the start of 4-week repeated administration, since there are limited data, where only those of rats of 6 week of age at the start of administration are available. In this study, we conducted the 4-week RDLMN assay using rats of 6 and 8 weeks of age (at the start of administration) to investigate the effect of age on the liver MN inducibility. Clofibrate, a weak inducer of liver MN, was used in this study to detect the slight difference in the liver MN induction. RESULTS: The liver MN induced by clofibrate was detected in both rats of 6 and 8 weeks of age at the start of administration. However, the liver MN induction was lower in rats of 8 weeks of age compared to rats of 6 weeks of age at the start of administration. CONCLUSION: These results suggest that the liver MN inducibility decreases with age. Therefore, we recommend the use of rats of 6 weeks of age at start of administration to reliably detect the liver MN induction in the RDLMN assay.

Evaluation of a liver micronucleus assay in young rats (III): a study using nine hepatotoxicants by the Collaborative Study Group for the Micronucleus Test (CSGMT)/Japanese Environmental Mutagen Society (JEMS)-Mammalian Mutagenicity Study Group (MMS).

We have been investigating a liver micronucleus assay to detect genotoxic chemicals using young rats for several years, and had established its advantages with respect to using autonomous proliferation of young rat hepatocytes. Nine chemicals known to induce hepatotoxic effects such as necrosis (2,6-dinitrotolune, bromobenzene, isoniazid, phenacetin, allyl alcohol and thioacetamide), cholestasis (chlorpromazine hydrochloride and alpha-naphthyl isothiocyanate) and oxidative stress (clofibrate) were selected for this study. A liver micronucleus assay was conducted in 4-week-old male F344 rats using two or three dose levels of test chemicals given orally by gavage to evaluate the compound's ability to induce micronucleated hepatocytes. Several of these test chemicals were additionally examined in a peripheral blood micronucleus assay conducted concurrently and in the same animals. The genotoxic rodent hepatocarcinogen, 2,6-dinitrotoluene showed a positive result in the liver micronucleus assay, but the nongenotoxic hepatocarcinogens, clofibrate and thioacetamide gave negative responses. Bromobenzene, known to produce DNA adducts but is noncarcinogenic in rodent liver, was judged equivocal in this assay. alpha-Naphthyl isothiocyanate is noncarcinogenic and showed negative response in the liver. The other four chemicals, known to be either noncarcinogenic or carcinogenic in other non-liver target organs, showed negative results in the liver micronucleus assay. Based on the results in the present study and previous report described above, it was concluded that this technique is able to effectively predict genotoxic rodent hepatocarcinogenicity, and does not give false positives due to hepatotoxicity.

Evaluation of a liver micronucleus assay in young rats (IV): a study using a double-dosing/single-sampling method by the Collaborative Study Group for the Micronucleus Test (CSGMT)/Japanese Environmental Mutagen Society (JEMS)-Mammalian Mutagenicity Study Group (MMS).

A collaborative study was conducted to evaluate whether a liver micronucleus assay using four-week-old male F344 rats can be used to detect genotoxic rat hepatocarcinogens using double-dosing with a single-sampling 4 days after the second dose. The assay methods were thoroughly validated by the seven laboratories involved in the study. Seven chemicals, 2,4-diaminotoluene, diethyl nitrosamine, p-dimethylaminoazobenzene, 1,2-dimethylhydrazine dihydrochloride, 2,4-dinitrotolunene, 2,6-dinitrotoluene and mitomycin C, known to produce positive responses in the single-dosing/triple-sampling method were selected for use in the present study, and each chemical was examined in two laboratories with the exception of 2,4-dinitrotolunene. Although several of the compounds were examined at lower doses for reasons of toxicity than in the single-dosing/triple-sampling method, all chemicals tested in the present study induced micronuclei in liver cells indicating a positive result. These findings suggest that the liver micronucleus assay can be used in young rats to detect genotoxic rat hepatocarcinogens using a double-dosing/single-sampling procedure. Further, the number of animals used in the liver micronucleus assay can be reduced by one-third to a half by using the double-dosing/single-sampling method. This reduction in animal numbers also has significant savings in time and resource for liver perfusion and hepatocyte isolation.

Evaluation of a liver micronucleus assay with 12 chemicals using young rats (II): a study by the Collaborative Study Group for the Micronucleus Test/Japanese Environmental Mutagen Society-Mammalian Mutagenicity Study Group.

The partial hepatectomy method, co-treatment method with mitogens and an in vivo/in vitro assay method have been reported as in vivo liver micronucleus (MN) assays. These methods have disadvantages with respect to widespread use as an in vivo assay, i.e. they are time consuming, labour intensive and there is the possibility of interaction with the mitogens used. Therefore, we have attempted to develop a new method to overcome these disadvantages. The assay as described herein utilises the autonomous proliferation of hepatocytes of young rats. Nine chemicals have been evaluated using this method thus far. We have also assessed the sensitivity and detectability according to the following methods. A liver MN assay was performed in two strains of young rats using one or two doses of 12 chemicals to investigate the inducibility of micronucleated hepatocytes. For some of the chemicals, a peripheral blood MN assay was performed concurrently in the same animals. The following chemicals were used: diethylnitrosamine (DEN), 2-acetylaminofluorene (2AAF), 2,4-diaminotoluene (2,4-DAT), quinoline, p-dimethylaminoazobenzene (DAB), dimethylnitrosamine (DMN), ethylmethanesulphonate, 5-fluorouracil, mitomycin C (MMC), 1,2-dimethylhydrazine.2HCl, cyclophosphamide and 2,4-dinitrotoluene (2,4-DNT). The rodent hepatocarcinogens, quinoline, DAB and DMN showed positive responses in previous assays. The results of the present assay revealed new positive responses for single doses of 2AAF, 2,4-DAT, MMC, 1,2-dimethylhydrazine.2HCl and 2,4-DNT. These chemicals are known rodent hepatocarcinogens, whose clastogenicity is believed to be related to the formation of reactive metabolites generated through enzymatic activation, or the chemicals act directly. Two doses of 2AAF and DMN appeared to be more effective than a single dose in terms of MN induction. Although there were quantitative differences in the incidences of MNs, both strains of rat (F344 and SD) responded positively after treatment with DEN, DMN, 2,4-DAT, DAB, quinoline and 2AAF, suggesting that both strains are appropriate for the assay. Based on these results, it is concluded that this technique could be effective for detecting chemical clastogenicity in hepatocytes in vivo.

Optimization of specimen preparation from formalin-fixed liver tissues for liver micronucleus assays: Hepatocyte staining with fluorescent dyes.

The liver micronucleus (MN) assay is an effective and important in vivo test for detecting genotoxic compounds, particularly those that require metabolic activation. For this assay, hepatocytes (HEPs) can be isolated by collagenase treatment but without requirement for in situ liver perfusion. Consequently, the liver MN assay can be integrated into a general repeated-dose (RD) toxicity study. The method is also applicable to liver MN assays involving partial hepatectomy or the use of juvenile rats. Here, we propose an improved method for staining HEPs prepared from formalin-fixed liver tissues for MN assays, without collagenase treatment. HEP suspensions are prepared by treating the tissues with concentrated KOH and a fluorescent dye, SYBR(®) Gold (SYGO), is used for staining. Visualization of the MN in SYGO-stained HEPs is clearer than with Wright-Giemsa staining. We compared the induction of MN as measured with our new method versus the conventional method using collagenase dispersion. Our method not only enables the integration of the liver MN assay into a general RD toxicity study but also allows it to be conducted retrospectively.

Results of rat Pig-a/PIGRET assay with a single dose regimen of 1,3-propane sultone and 2-acetyl aminofluorene.

The Pig-a assay is a useful in vivo mutation detecting test and is easier to perform than the in vivo transgenic mutation assay. This assay is now recognized to be able to detect a number of mutagenic chemicals administered to rats in sub-acute or sub-chronic dose studies. The present investigation was conducted to evaluate the usefulness of peripheral blood Pig-a assays with total red blood cells (RBC Pig-a assay) and with reticulocytes (PIGRET assay) using two genotoxic rodent carcinogens, 1,3-propane sultone (1,3-PS) and 2-acetylaminofluorene (2-AAF). Male rats were orally administered a single dose of each test compound, and both the RBC Pig-a and PIGRET assays were performed using flow cytometry to measure the Pig-a mutant frequency (MF) before and after dosing on Days 8, 15 and 29. In the experiment with 1,3-PS, significant increases in Pig-a MF were observed from Day 15 and Day 8 in the RBC Pig-a and PIGRET assays, respectively. The results of both assays demonstrated that the increases in Pig-a MF were detectable after a single treatment with 1,3-PS. Furthermore, the difference in the kinetics of the increase in Pig-a MF between the RBC Pig-a and PIGRET assays with 1,3-PS suggests that the PIGRET assay has an advantage in detecting the mutant erythrocytes earlier than the RBC Pig-a assay. In contrast, no significant increases were observed in the Pig-a assays using either RBC or reticulocytes with 2-AAF. The negative results in both assays with 2-AAF may indicate the limitation of the single dose method; however, further investigation at higher doses is necessary to determine limitation of the single dose method.

Evaluation of the sensitivity and specificity of in vivo erythrocyte micronucleus and transgenic rodent gene mutation tests to detect rodent carcinogens.

Sensitivity and/or specificity of the in vivo erythrocyte micronucleus (MN) and transgenic rodent mutation (TGR) tests to detect rodent carcinogens and non-carcinogens were investigated. The Carcinogenicity and Genotoxicity eXperience (CGX) dataset created by Kirkland et al. was used for the carcinogenicity and in vitro genotoxicity data, i.e., Ames and chromosome aberration (CA) tests. Broad literature surveys were conducted to gather in vivo MN or TGR test data to add to the CGX dataset. Genotoxicity data in vitro were also updated slightly. Data on 379 chemicals (293 carcinogens and 86 non-carcinogens) were available for the in vivo MN test; sensitivity, specificity or concordances were calculated as 41.0%, 60.5% or 45.4%, respectively. For the TGR test, data on 80 chemicals (76 carcinogens and 4 non-carcinogens) were available; sensitivity was calculated as 72.4%. Based on the recent guidance on genotoxicity testing strategies, performance (sensitivity/specificity) of the following combinations was calculated; Ames+in vivo MN (68.7%/45.3%), Ames+TGR (83.8%/not calculated (nc)), Ames+in vitro CA+in vivo MN (80.8%/21.3%), Ames+in vitro CA+TGR (89.1%/nc), Ames+in vivo MN+TGR (87.5%/nc), Ames+in vitro CA+in vivo MN+TGR (89.3%/nc). Relatively good balance in performance was shown by the Ames+in vivo MN in comparison with Ames+in vitro CA (74.3%/37.5%). Ames+TGR and Ames+in vivo MN+TGR gave even higher sensitivity, but the specificity could not be calculated (too few TGR data on non-carcinogens). This indicates that in vivo MN and TGR tests are both useful as in vivo tests to detect rodent carcinogens.

Measuring reproducibility of dose response data for the Pig-a assay using covariate benchmark dose analysis.

The reproducibility of the in vivo Pig-a gene mutation test system was assessed across 13 different Japanese laboratories. In each laboratory rats were exposed to the same dosing regimen of N-nitroso-N-ethylurea (ENU), and red blood cells (RBCs) and reticulocytes (RETs) were collected for mutant phenotypic analysis using flow cytometry. Mutant frequency dose response data were analysed using the PROAST benchmark dose (BMD) statistical package. Laboratory was used as a covariate during the analysis to allow all dose responses to be analysed at the same time, with conserved shape parameters. This approach has recently been shown to increase the precision of the BMD analysis, as well as providing a measure of equipotency. This measure of equipotency was used here to demonstrate a reasonable level of interlaboratory reproducibility. Increased reproducibility could have been achieved by increasing the number of cells scored, as this would reduce the number of zero values within the mutant frequency data. Overall, the interlaboratory trial was successful, and these findings support the transferability of the in vivo Pig-a gene mutation assay.

The PIGRET assay, a method for measuring Pig-a gene mutation in reticulocytes, is reliable as a short-term in vivo genotoxicity test: Summary of the MMS/JEMS-collaborative study across 16 laboratories using 24 chemicals.

The in vivo mutation assay using the X-linked phosphatidylinositol glycan class A gene (Pig-a in rodents, PIG-A in humans) is a promising tool for evaluating the mutagenicity of chemicals. Approaches for measuring Pig-a mutant cells have focused on peripheral red blood cells (RBCs) and reticulocytes (RETs) from rodents. The recently developed PIGRET assay is capable of screening >1×106 RETs for Pig-a mutants by concentrating RETs in whole blood prior to flow cytometric analysis. Additionally, due to the characteristics of erythropoiesis, the PIGRET assay can potentially detect increases in Pig-a mutant frequency (MF) sooner after exposure compared with a Pig-a assay targeting total RBCs (RBC Pig-a assay). In order to test the merits and limitations of the PIGRET assay as a short-term genotoxicity test, an interlaboratory trial involving 16 laboratories was organized by the Mammalian Mutagenicity Study Group of the Japanese Environmental Mutagenicity Society (MMS/JEMS). First, the technical proficiency of the laboratories and transferability of the assay were confirmed by performing both the PIGRET and RBC Pig-a assays on rats treated with single doses of N-nitroso-N-ethylurea. Next, the collaborating laboratories used the PIGRET and RBC Pig-a assays to assess the mutagenicity of a total of 24 chemicals in rats, using a single treatment design and mutant analysis at 1, 2, and 4 weeks after the treatment. Thirteen chemicals produced positive responses in the PIGRET assay; three of these chemicals were not detected in the RBC Pig-a assay. Twelve chemicals induced an increase in RET Pig-a MF beginning 1 week after dosing, while only 3 chemicals positive for RBC Pig-a MF produced positive responses 1 week after dosing. Based on these results, we conclude that the PIGRET assay is useful as a short-term test for in vivo mutation using a single-dose protocol.

Results of the International Validation of the in vivo rodent alkaline comet assay for the detection of genotoxic carcinogens: Individual data for 1,2-dibromoethane, p-anisidine, and o-anthranilic acid in the 2nd step of the 4th phase Validation Study under the JaCVAM initiative.

As part of the Japanese Center for the Validation of Alternative Methods (JaCVAM)-initiative International Validation Study of an in vivo rat alkaline comet assay, we examined 1,2-dibromoethane (DBE), p-anisidine (ASD), and o-anthranilic acid (ANT) to investigate the effectiveness of the comet assay in detecting genotoxic carcinogens. Each of the three test chemicals was administered to 5 male Sprague-Dawley rats per group by oral gavage at 48, 24, and 3h before specimen preparation. Single cells were collected from the liver and glandular stomach at 3h after the final dosing, and the specimens prepared from these two organs were subjected to electrophoresis under alkaline conditions (pH>13). The percentage of DNA intensity in the comet tail was then assessed using an image analysis system. A micronucleus (MN) assay was also conducted using these three test chemicals with the bone marrow (BM) cells collected from the same animals simultaneously used in the comet assay, i.e., combination study of the comet assay and BM MN assay. A genotoxic (Ames positive) rodent carcinogen, DBE gave a positive result in the comet assay in the present study, while a genotoxic (Ames positive) non-carcinogen, ASD and a non-genotoxic (Ames negative) non-carcinogen, ANT showed negative results in the comet assay. All three chemicals produced negative results in the BM MN assay. While the comet assay findings in the present study were consistent with those obtained from the rodent carcinogenicity studies for the three test chemicals, we consider the positive result in the comet assay for DBE to be particularly meaningful, given that this chemical produced a negative result in the BM MN assay. Therefore, the combination study of the comet assay and BM MN assay is a useful method to detect genotoxic carcinogens that are undetectable with the BM MN assay alone.

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.

Micronucleus induction in rat liver and bone marrow by acute vs. repeat doses of the genotoxic hepatocarcinogen monocrotaline.

The liver micronucleus (MN) assay is useful for predicting genotoxic rodent hepatocarcinogenicity. We have recently established the repeated-dose liver MN (RDLMN) assay in rats for integration into general toxicity studies. To investigate the effectiveness of the RDLMN assay, the genotoxic rodent hepatocarcinogen, monocrotaline (MCT), was administered by oral gavage to 6-week old male rats once daily for 14 days at 0.5 and 1.5mg/kg/day, and for 28 days at 0.15, 0.5, 1.5, 3.75, 7.5 and 15mg/kg/day. Then, MN induction was measured in the liver and bone marrow (BM), and histopathological hepatotoxicity was examined. Additionally, in order to evaluate the effects of repeated dosing periods on MN inducibility, a double-dose examination of MCT at doses of 15, 30 and 60mg/kg/day in juvenile (26-days old) and young adult (7-weeks old) rats was also conducted, as an acute dose MN assay. The peripheral blood (PB) and liver were sampled at 48h and 4 days after the second dosing, respectively. In the repeated-dose MN assay, MCT produced a positive result in the liver at a non-hepatotoxic lower dose level, but not in the BM at any dose level. In contrast, in the double-dose MN assay, MCT showed a negative result in the young adult rat livers, although it gave positive responses in the livers of juvenile rats and in the PB with both age groups. The maximum dose used in the repeated-dose assay was considerably lower than that used in the acute dose assay. These results suggest that a repeated dosing regimen is more suitable for the liver MN assay using young adult rats than an acute dose regimen, and the RDLMN assay might be capable of detecting genotoxic rodent hepatocarcinogens at dose levels that are typically undetectable in BM MN assays.

Evaluation of a repeated dose liver micronucleus assay in rats treated with two genotoxic hepatocarcinogens, dimethylnitrosamine and 2-acetylaminofluorene: the possibility of integrating micronucleus tests with multiple tissues into a repeated dose general toxicity study.

As part of a collaborative study by the Collaborative Study Group for Micronucleus Test (CSGMT) of the Mammalian Mutagenicity Study Group (MMS) in the Japanese Environmental Mutagen Society (JEMS), the present study evaluated the effectiveness of the repeated dose liver micronucleus (RDLMN) assay. Two genotoxic hepatocarcinogens, dimethylnitrosamine (DMN) and 2-acetylaminofluorene (2-AAF), were administered orally to male rats (6 weeks old at the initial dosing) once daily for 14 and 28 days to evaluate the micronucleus (MN) inducibility in the liver. In addition, these chemicals were evaluated for MN inducibility in the bone marrow (BM) and gastrointestinal (GI) tract, i.e. glandular stomach and colon of the same animals used in the RDLMN assay. As a result, both chemicals produced positive results in the liver, although a weak positive response was given by 2-AAF. DMN gave negative results in the tissues other than the liver. 2-AAF produced positive responses in the BM and glandular stomach, and a prominent response was particularly observed in the glandular stomach, which is directly exposed to the test chemicals by gavage. The present results suggest that the RDLMN assay is a useful method for detecting genotoxic hepatocarcinogens, and that it is especially effective for evaluating test chemicals, such as DMN, undetectable by the BM and GI tract MN assay. Moreover, the results in this investigation indicate that the use of multiple tissues in the study integrating the MN tests is more effective than using a single tissue, for detection of the MN induction produced by chemical exposure to rats, and helps to determine the characteristics of the test chemicals.

JaCVAM-organized international validation study of the in vivo rodent alkaline comet assay for detection of genotoxic carcinogens: II. Summary of definitive validation study results.

The in vivo rodent alkaline comet assay (comet assay) is used internationally to investigate the in vivo genotoxic potential of test chemicals. This assay, however, has not previously been formally validated. The Japanese Center for the Validation of Alternative Methods (JaCVAM), with the cooperation of the U.S. NTP Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM)/the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM), the European Centre for the Validation of Alternative Methods (ECVAM), and the Japanese Environmental Mutagen Society/Mammalian Mutagenesis Study Group (JEMS/MMS), organized an international validation study to evaluate the reliability and relevance of the assay for identifying genotoxic carcinogens, using liver and stomach as target organs. The ultimate goal of this exercise was to establish an Organisation for Economic Co-operation and Development (OECD) test guideline. The study protocol was optimized in the pre-validation studies, and then the definitive (4th phase) validation study was conducted in two steps. In the 1st step, assay reproducibility was confirmed among laboratories using four coded reference chemicals and the positive control ethyl methanesulfonate. In the 2nd step, the predictive capability was investigated using 40 coded chemicals with known genotoxic and carcinogenic activity (i.e., genotoxic carcinogens, genotoxic non-carcinogens, non-genotoxic carcinogens, and non-genotoxic non-carcinogens). Based on the results obtained, the in vivo comet assay is concluded to be highly capable of identifying genotoxic chemicals and therefore can serve as a reliable predictor of rodent carcinogenicity.

Evaluation of the repeated-dose liver and gastrointestinal tract micronucleus assays with 22 chemicals using young adult rats: summary of the collaborative study by the Collaborative Study Group for the Micronucleus Test (CSGMT)/The Japanese Environmental Mutagen Society (JEMS) - Mammalian Mutagenicity Study Group (MMS).

The repeated-dose liver micronucleus (RDLMN) assay using young adult rats has the potential to detect hepatocarcinogens. We conducted a collaborative study to assess the performance of this assay and to evaluate the possibility of integrating it into general toxicological studies. Twenty-four testing laboratories belonging to the Mammalian Mutagenicity Study Group, a subgroup of the Japanese Environmental Mutagen Society, participated in this trial. Twenty-two model chemicals, including some hepatocarcinogens, were tested in 14- and/or 28-day RDLMN assays. As a result, 14 out of the 16 hepatocarcinogens were positive, including 9 genotoxic hepatocarcinogens, which were reported negative in the bone marrow/peripheral blood micronucleus (MN) assay by a single treatment. These outcomes show the high sensitivity of the RDLMN assay to hepatocarcinogens. Regarding the specificity, 4 out of the 6 non-liver targeted genotoxic carcinogens gave negative responses. This shows the high organ specificity of the RDLMN assay. In addition to the RDLMN assay, we simultaneously conducted gastrointestinal tract MN assays using 6 of the above carcinogens as an optional trial of the collaborative study. The MN assay using the glandular stomach, which is the first contact site of the test chemical when administered by oral gavage, was able to detect chromosomal aberrations with 3 test chemicals including a stomach-targeted carcinogen. The treatment regime was the 14- and/or 28-day repeated-dose, and the regime is sufficiently promising to incorporate these methods into repeated-dose toxicological studies. The outcomes of our collaborative study indicated that the new techniques to detect chromosomal aberrations in vivo in several tissues worked successfully.