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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., (© 2023 Environmental Mutagenesis and Genomics Society. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.)

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., (© 2023. The Author(s).)

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., (© 2022. The Author(s).)

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., (© 2021. The Author(s).)

We report on a 16-year-old Japanese boy in whom an esophageal squamous cell carcinoma (ESCC) developed 12 years after allogeneic hematopoietic stem cell transplantation was performed for aplastic anemia. A high frequency of microsatellite instability was detected in samples of ESCC. Moreover, the detection of pathogenic variants, including single nucleotide substitution of TP53 (c.346C>T) and BRCA2 (c.6952C>T) and splicing of KDM6A (c.1194+2T>G), suggest that the development of ESCC in the patient was triggered by impairment of checkpoint and repair for DNA damage and epigenetic modification through accumulation of gene mutations induced by chronic graft-versus-host disease and prolonged administration of tacrolimus.

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., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Segmentation is a fundamental process in biomedical image analysis that enables various types of analysis. Segmenting organs in histological microscopy images is problematic because the boundaries between regions are ambiguous, the images have various appearances, and the amount of training data is limited. To address these difficulties, supervised learning methods (e.g., convolutional neural networking (CNN)) are insufficient to predict regions accurately because they usually require a large amount of training data to learn the various appearances. In this paper, we propose a semi-automatic segmentation method that effectively uses scribble annotations for metric learning. Deep discriminative metric learning re-trains the representation of the feature space so that the distances between the samples with the same class labels are reduced, while those between ones with different class labels are enlarged. It makes pixel classification easy. Evaluation of the proposed method in a heart region segmentation task demonstrated that it performed better than three other methods.

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., Competing Interests: The animal experiments were approved by the Institutional Animal Care and Use Committee of each testing facility prior to conducting the experiments.Not applicableThe authors declare that they have no competing interests.Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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., (Copyright © 2018. Published by Elsevier B.V.)

Background: Parents of children with cancer are susceptible to psychological distress; however, many parents also report posttraumatic growth (PTG). The objective of this study was to explore the variables associated with PTG in parents of children with cancer who were either on treatment or off treatment., Methods: One hundred and nineteen parents (71 mothers and 48 fathers) of children with cancer completed self-report questionnaires, including the PTG Inventory, Center for Epidemiologic Studies Depression Scale, State-Trait Anxiety Inventory, and Impact of Event Scale-Revised. Demographic data and children's medical information were also collected. Multivariate linear regression analyses were conducted to investigate the variables associated with PTG., Results: The mean age of participants was 41.4 years (SD = 6). Higher PTG Inventory scores were associated with parents' lower trait anxiety (P = .028), parents' sex (female; P = .004), treatment status (within 12 months from treatment end compared with on-treatment; P = .048), surgery (P = .007), and late effects (P = .01)., Conclusions: Parents' PTG was associated with children's clinical characteristics, parents' sex, and parents' anxiety levels. When dealing with PTG, the parents' psychological characteristics and children's clinical characteristics should be considered. Particularly for parents with high trait anxiety, it is important to reduce anxiety first before addressing PTG., (Copyright © 2016 John Wiley & Sons, Ltd.)

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., (Copyright © 2016 Elsevier B.V. All rights reserved.)

We report an 11-month-old breast-fed boy with feeding difficulties, lethargy, and developmental delay. Blood examination showed pancytopenia and decreased serum levels of vitamin B12. Anisocytosis and poikilocytes were detected in his peripheral blood, and increased megaloblastosis without leukemic cells was detected in his bone marrow. After the diagnosis of megaloblastic anemia due to vitamin B12 deficiency, symptoms were improved by vitamin B12 administration. Further investigation of the mother identified Crohn's disease and suggested that the supply of vitamin B12 from the mother to the infant, via the placenta during pregnancy and via breast milk after birth, was decreased due to impaired absorption of vitamin B12 in the mother's small intestine. Magnetic resonance imaging of the boy's brain on admission showed cerebral cortex atrophy which had improved by the age of 1 year and 10 months after vitamin B12 treatment, though developmental delay was still evident at the age of 3 years. Infantile vitamin B12 deficiency often presents with nonspecific manifestations, such as developmental delay and failure to thrive, in addition to anemia and is thus not easily diagnosed. To prevent severe neurological sequelae, this condition must be rapidly diagnosed, because a prolonged duration increases the risk of permanent disabilities.

Maffucci syndrome is a nonhereditary congenital disorder characterized by multiple enchondromas and with soft-tissue hemangiomas. Somatic mutations of the isocitrate dehydrogenase (IDH) gene have been detected in enchondroma and hemangioma tissue from patients with Maffucci syndrome. The rate of malignant transformation in Maffucci syndrome is high, with enchondromas transforming into chondrosarcomas and the development of secondary neoplasms, including pancreatic and hepatic adenocarcinoma, mesenchymal ovarian tumors, and brain tumors such as glioma. However, hematopoietic malignancies arising in Maffucci syndrome are rare. We report a 7-year-old girl with Maffucci syndrome in whom acute myeloid leukemia (AML) with cup-like nuclear invagination developed. Both leukemic cells and hemangioma had the same gene mutations: an insertion frameshift c.863_864insTCTG (p.W288 fs) in the nucleophosmin (NPM1) gene and a missense mutation c.392_395GTCG > CTCT (p.G131_R132 > AL) in the IDH1 gene. However, buccal mucosa cells and peripheral blood mononuclear cells harvested after two cycles of chemotherapy showed wild-type genotypes. These results suggest that the multiple somatic mutations of the IDH1 and NPM1 genes in hemangioblasts are related to the development of cup-like AML associated with Maffucci syndrome. However, further studies are needed to identify additional molecular events in AML but not in hemangioma.

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., (Copyright © 2014 Elsevier B.V. All rights reserved.)

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., (Copyright © 2015 Elsevier B.V. All rights reserved.)

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., (Copyright © 2014. Published by Elsevier B.V.)

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., (Copyright © 2014 Elsevier B.V. All rights reserved.)

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., (Copyright © 2015 Elsevier B.V. All rights reserved.)

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., (Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.)

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., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Repeated-dose liver, bone marrow, and gastrointestinal tract micronucleus assays that use young adult rats were evaluated in a collaborative study that was organized by the Japanese Environmental Mutagen Society-Mammalian Mutagenicity Study Group. A genotoxic hepatocarcinogen quinoline was orally administered to independent groups of five Crl:CD (SD) male rats at doses of 30, 60 and 120mg/kg for 14 days and at doses of 15, 30 and 60mg/kg for 28 days. After treatment, the livers were harvested and hepatocytes were isolated by collagenase treatment. The frequency of micronucleated hepatocytes (MNHEPs) increased significantly in both the 14- and 28-day repeated dose studies. However, the frequency of micronucleated cells did not increase in the bone marrow, stomach or colon cells, which were not quinoline-induced carcinogenic target organs in the rats. These results indicate that a repeated-dose liver micronucleus (RDLMN) assay using young adult rats is capable of detecting the genotoxicity of quinoline at the target organ of carcinogenicity. The protocol may also permit the integration of the genotoxic endpoint into general repeated-dose toxicity studies. Furthermore, we elucidated that conducting the micronucleus assay in multiple organs could potentially assess organ specificity., (Copyright © 2015 Elsevier B.V. All rights reserved.)