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A novel galacto-oligosaccharide (GOS) manufactured by a two-step enzyme reaction of lactose was examined in a comet assay for its potential to induce DNA damage in vivo by estimating the DNA fragmentation level in the cellular nuclei of the glandular stomach, colon, and peripheral blood. GOS was orally administered at doses of 0 (vehicle alone), 500, 1000, and 2000 mg/kg/day to five male Crl: CD(Sprague Dawley) rats per group three times (48, 24, and 3 h before the animals were terminated). The specimens were prepared in accordance with the standard protocol (version 14.2) of the “International Validation of the In Vivo Rodent Alkaline Comet Assay for the Detection of Genotoxic Carcinogens” organized by the Japanese Center for the Validation of Alternative Methods. No significant differences in the percentage of DNA in the tail were observed between the GOS-treated groups and vehicle controls in any of the organs evaluated. Additionally, no GOS-related clinical signs or effects on body weight were seen. Based on these results, the comet assay of GOS in the glandular stomach, colon, and peripheral blood using rats was judged negative. Therefore, it is concluded that GOS did not induce DNA damage in vivo under the conditions employed in this study. [ABSTRACT FROM AUTHOR]

Abstract Background The PedsQL 3.0 Cancer Module is a widely used instrument to measure pediatric cancer specific health-related quality of life (HRQOL) for children aged 2 to 18 years. We developed the Japanese version of the PedsQL Cancer Module and investigated its reliability and validity among Japanese children and their parents. Methods Participants were 212 children with cancer and 253 of their parents. Reliability was determined by internal consistency using Cronbach's coefficient alpha and test-retest reliability using intra-class correlation coefficient (ICC). Validity was assessed through factor validity, convergent and discriminant validity, concurrent validity, and clinical validity. Factor validity was examined by exploratory factor analysis. Convergent and discriminant validity were examined by multitrait scaling analysis. Concurrent validity was assessed using Spearman's correlation coefficients between the Cancer Module and Generic Core Scales, and the comparison of the scores of child self-reports with those of other self-rating depression scales for children. Clinical validity was assessed by comparing the on- and off- treatment scores using Kruskal-Wallis and Mann-Whitney U tests. Results Cronbach's coefficient alpha was over 0.70 for the total scale and over 0.60 for each subscale by age except for the 'pain and hurt' subscale for children aged 5 to 7 years. For test-retest reliability, the ICC exceeded 0.70 for the total scale for each age. Exploratory factor analysis demonstrated sufficient factorial validity. Multitrait scaling analysis showed high success rates. Strong correlations were found between the reports by children and their parents, and the scores of the Cancer Module and the Generic Core Scales except for 'treatment anxiety' subscales for child reports. The Depression Self-Rating Scale for Children (DSRS-C) scores were significantly correlated with emotional domains and the total score of the cancer module. Children who had been off treatment over 12 months demonstrated significantly higher scores than those on treatment. Conclusions The results demonstrate the reliability and validity of the Japanese version of the PedsQL Cancer Module among Japanese children.

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).)