Takeshi Morita

Bio: Takeshi Morita is an academic researcher from GlaxoSmithKline. The author has contributed to research in topics: Micronucleus test & Genotoxicity. The author has an hindex of 25, co-authored 49 publications receiving 2960 citations.

Report from the in vitro micronucleus assay working group

Abstract: At the Washington “2nd International Workshop on Genotoxicity Testing” (25–26 March 1999) current methodologies and data for the in vitro micronucleus test were reviewed. As a result, guidelines for the conduct of specific aspects of the protocol were developed. Agreement was achieved on the following topics: choice of cells, slide preparation, analysis of micronuclei, toxicity, use of cytochalasin-B, number of doses, and treatment/harvest times [Environ. Mol. Mutagen. 35 (2000) 167]. Because there were a number of important in vitro micronucleus validation studies in progress, it was not possible to design a definitive, internationally harmonized protocol at that time. These studies have now been completed and the data were reviewed at the Plymouth “3rd International Workshop on Genotoxicity Testing” (28–29 June 2002). Data from studies coordinated by the French Society of Genetic Toxicology, Japanese collaborative studies, European pharmaceutical industry validation studies, along with data from Lilly Research Laboratories were used to prepare conclusions on the main aspects of the in vitro micronucleus protocol. In this paper, the consensus agreements on the protocol for performing the in vitro micronucleus assay are presented. The major recommendations concern: 1. Demonstration of cell proliferation: both cell lines and lymphocytes can be used, but demonstration of cell proliferation in both control and treated cells is compulsory for the acceptance of the test. 2. Assessment of toxicity and dose range finding: assessment of toxicity should be performed by determining cell proliferation, e.g. increased cell counts (CC) or population doubling (PD) without cytochalasin-B, or e.g. cytokinesis-block proliferation index with cytochalasin-B; and by determining other markers for cytotoxicity (confluency, apoptosis, necrosis) which can provide valuable additional information. 3. Treatment schedules for cell lines and lymphocytes. 4. Choice of positive controls: without S9-mix both a clastogen (e.g. mitomycin C or bleomycin) and an aneugen (e.g. colchicine) should be included as positive controls and a clastogen that requires S9 for activity when S9-mix is used (e.g. dimethylnitrosamine, or cyclophosphamide in those cell types that cannot activate this agent directly). 5. Duplicate cultures and number of cells to be scored. 6. Repeat experiments: in lymphocytes, for each experiment blood from 2 different healthy young and non-smoking donors should be compared. In cell lines, the experiments need only to be repeated if the first one is negative. 7. Statistics: statistical significance should not be the sole factor for determining positive results. Biological meaning should serve as a guideline. Examples of statistical analyses are given.

Evaluation of the rodent micronucleus assay in the screening of IARC carcinogens (groups 1, 2A and 2B) the summary report of the 6th collaborative study by CSGMT/JEMS MMS. Collaborative Study of the Micronucleus Group Test. Mammalian Mutagenicity Study Group.

Abstract: To assess the correlation between micronucleus induction and human carcinogenicity, the rodent micronucleus assay was performed on known and potential human carcinogens in the 6th MMS/CSGMT collaborative study Approximately 100 commercially available chemicals and chemical groups on which there was little or no micronucleus assay data were selected from IARC (International Agency for Research on Cancer) Groups 1 (human carcinogen), 2A (probable human carcinogen) and 2B (possible human carcinogen) As minimum requirements for the collaborative study, 5 male mice were treated by intraperitoneal injection or oral gavage once or twice with each chemical at three dose levels, and bone marrow and/or peripheral blood was analyzed Five positives and 2 inconclusives out of 13 Group 1 chemicals, 7 positives and 5 inconclusives of 23 Group 2A chemicals, and 26 positives and 6 inconclusives of 67 Group 2B chemicals were found Such low positive rates were not surprising because of a test chemical selection bias, and we excluded well-known micronucleus inducers The overall evaluation of the rodent micronucleus assay was based on the present data combined with published data on the IARC carcinogens After merging, the positive rates for Groups 1, 2A and 2B were 686, 545 and 456%, respectively Structure-activity relationship analysis suggested that the micronucleus assay is more sensitive to the genetic toxicity of some classes of chemicals Those to which it is sensitive consist of (1) aziridines and bis(2-chloroethyl) compounds; (2) alkyl sulfonate and sulfates; (3) acyl-type N-nitroso compounds; (4) hydrazines; (5) aminobiphenyl and benzidine derivatives; and (6) azo compounds Those to which it is less sensitive consist of (1) dialkyl type N-nitroso compounds; (2) silica and metals and their compounds; (3) aromatic amines without other functional groups; (4) halogenated compounds; and (5) steroids and other hormones After incorporation of structure-activity relationship information, the positive rates of the rodent micronucleus assay became 905, 652 and 600% for IARC Groups 1, 2A and 2B, respectively Noteworthy was the tendency of the test to be more sensitive to those carcinogens with stronger evidence human carcinogenicity

Report from working group on in vitro tests for chromosomal aberrations.

Abstract: The following summary represents a consensus of the working group except where noted. The items discussed are listed in the order in which they appear in the OECD guideline (473) for easy reference. Metabolic activation. S9 from animals induced either with Aroclor 1254 or with the combination of phenobarbital with beta-naphthoflavone is acceptable, and other systems could be used with suitable justification. Exposure concentrations. The upper limit of testing should be 10 mM (or 5 mg/ml where molecular weight is not known or mixtures are being tested), whichever is lower. Where this limit is inappropriate the investigator should give detailed justification of the choice of top concentration. Cytotoxicity should be measured not only in range-finding tests but also concurrently with the assay for chromosomal aberrations. Cytotoxicity should be assessed by measurements of cell growth such as cell counts or confluence estimation. Mitotic index data alone are not a sufficient measure of cytotoxicity, except in the case of blood cultures for which other methods are impractical. Cytotoxicity at the top dose should be greater than 50% of concurrent negative/solvent controls, if this can be achieved without exceeding a concentration limit of 10 mM or 5 mg/ml. There should be at least three concentrations scored for aberrations (each with and without S9), covering a toxicity range down to a concentration giving little or no cytotoxicity. This will usually mean that the concentrations scored will be quite closely spaced. It was not possible to reach a consensus on the issue of solubility limits. The group did not agree on whether (a) solubility rather than cytotoxicity should be the limiting factor, such that only one top dose with evident precipitate should be scored even if toxicity is not observed, or (b) several concentrations with evident precipitate should be scored for aberrations if this were necessary to obtain cytotoxicity. It was agreed that evidence of precipitation should be determined in the final culture medium. Controls. Concurrent positive controls are required but the working group thought it inappropriate to specify the control chemicals or the degree of response that should be obtained, leaving it up to the test laboratory to demonstrate that the system was working adequately based on historical data within the laboratory. It is not necessary to include both negative and solvent controls concurrently with the aberration test; solvent controls alone are acceptable provided that the laboratory has data to demonstrate that there is no effect of the solvent on baseline values. Preparation of cultures.(ABSTRACT TRUNCATED AT 400 WORDS)

Evaluation of the rat micronucleus test with bone marrow and peripheral blood: Summary of the 9th collaborative study by CSGMT/JEMS. MMS

Abstract: The mouse has traditionally been used for the micronucleus test, with bone marrow the usual target organ. The aim of the 9th collaborative study by CSGMT was to evaluate the suitability of the rat for the micronucleus test, with bone marrow and peripheral blood as the target organ. Since the rat spleen eliminates circulating micronucleated erythrocytes, a rat peripheral blood micronucleus assay might not be feasible. Thirty-four Japanese laboratories and six overseas laboratories participated in this collaboration, and 40 chemicals were studied. As a rule, rat bone marrow and peripheral blood were analyzed using acridine orange staining. Among 36 mouse micronucleus-positive rat carcinogens, 34 of which had been evaluated by CSGMT, we observed 33 positive and three negative results with rat bone marrow and 30 positive, three equivocal, and three negative responses with rat peripheral blood. Of the two mouse micronucleus-negative rat carcinogens, acrylonitrile was positive in rat bone marrow and 4,4'-methylene bis(2-chloroaniline) was negative in both rat bone marrow and peripheral blood. Two chemicals reported to be mouse micronucleus-negative and rat-positive, azobenzene and Solvent Yellow 14, and one chemical reported to be mouse-positive and rat-negative, 1,2-dimethylhydrazine, gave positive responses in rat bone marrow and peripheral blood. The concordance between bone marrow and peripheral blood with rats was 92%. The concordance between rat and mouse erythrocytes was 88%. We concluded that the rat micronucleus assay, using either bone marrow or peripheral blood, can be used as an alternative to the mouse micronucleus assay.

Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing.

Abstract: Atthe International Workshop on Genotoxicity Test Procedures (IWGTP) held in Washington, DC, March 25-26, 1999, an expert panel met to develop guidelines for the use of the single-cell gel (SCG)/Comet assay in genetic toxicology. The expert panel reached a consensus that the optimal version of the Comet assay for identifying agents with genotoxic activity was the alkaline (pH > 13) version of the assay developed by Singh et al. [1988]. The pH > 13 version is capable of detecting DNA single-strand breaks (SSB), alkali-labile sites (ALS), DNA-DNA/DNA-protein cross-linking, and SSB associated with incomplete excision repair sites. Relative to other genotoxicity tests, the advantages of the SCG assay include its demonstrated sensitivity for detecting low levels of DNA damage, the requirement for small numbers of cells per sample, its flexibility, its low costs, its ease of application, and the short time needed to complete a study. The expert panel decided that no single version of the alkaline (pH > 13) Comet assay was clearly superior. However, critical technical steps within the assay were discussed and guidelines developed for preparing slides with agarose gels, lysing cells to liberate DNA, exposing the liberated DNA to alkali to produce single-stranded DNA and to express ALS as SSB, electrophoresing the DNA using pH > 13 alkaline conditions, alkali neutralization, DNA staining, comet visualization, and data collection. Based on the current state of knowledge, the expert panel developed guidelines for conducting in vitro or in vivo Comet assays. The goal of the expert panel was to identify minimal standards for obtaining reproducible and reliable Comet data deemed suitable for regulatory submission. The expert panel used the current Organization for Economic Co-operation and Development (OECD) guidelines for in vitro and in vivo genetic toxicological studies as guides during the development of the corresponding in vitro and in vivo SCG assay guidelines. Guideline topics considered included initial considerations, principles of the test method, description of the test method, procedure, results, data analysis and reporting. Special consideration was given by the expert panel to the potential adverse effect of DNA degradation associated with cytotoxicity on the interpretation of Comet assay results. The expert panel also discussed related SCG methodologies that might be useful in the interpretation of positive Comet data. The related methodologies discussed included: (1) the use of different pH conditions during electrophoreses to discriminate between DNA strand breaks and ALS; (2) the use of repair enzymes or antibodies to detect specific classes of DNA damage; (3) the use of a neutral diffusion assay to identify apoptotic/necrotic cells; and (4) the use of the acellular SCG assay to evaluate the ability of a test substance to interact directly with DNA. The alkaline (pH > 13) Comet assay guidelines developed by the expert panel represent a work in progress. Additional information is needed before the assay can be critically evaluated for its utility in genetic toxicology. The information needed includes comprehensive data on the different sources of variability (e.g., cell to cell, gel to gel, run to run, culture to culture, animal to animal, experiment to experiment) intrinsic to the alkaline (pH > 3) SCG assay, the generation of a large database based on in vitro and in vivo testing using these guidelines, and the results of appropriately designed multilaboratory international validation studies.

Occurrence, genotoxicity, and carcinogenicity of regulated and emerging disinfection by-products in drinking water: a review and roadmap for research.

Abstract: Disinfection by-products (DBPs) are formed when disinfectants (chlorine, ozone, chlorine dioxide, or chloramines) react with naturally occurring organic matter, anthropogenic contaminants, bromide, and iodide during the production of drinking water. Here we review 30 years of research on the occurrence, genotoxicity, and carcinogenicity of 85 DBPs, 11 of which are currently regulated by the U.S., and 74 of which are considered emerging DBPs due to their moderate occurrence levels and/or toxicological properties. These 74 include halonitromethanes, iodo-acids and other unregulated halo-acids, iodo-trihalomethanes (THMs), and other unregulated halomethanes, halofuranones (MX [3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone] and brominated MX DBPs), haloamides, haloacetonitriles, tribromopyrrole, aldehydes, and N-nitrosodimethylamine (NDMA) and other nitrosamines. Alternative disinfection practices result in drinking water from which extracted organic material is less mutagenic than extracts of chlorinated water. However, the levels of many emerging DBPs are increased by alternative disinfectants (primarily ozone or chloramines) compared to chlorination, and many emerging DBPs are more genotoxic than some of the regulated DBPs. Our analysis identified three categories of DBPs of particular interest. Category 1 contains eight DBPs with some or all of the toxicologic characteristics of human carcinogens: four regulated (bromodichloromethane, dichloroacetic acid, dibromoacetic acid, and bromate) and four unregulated DBPs (formaldehyde, acetaldehyde, MX, and NDMA). Categories 2 and 3 contain 43 emerging DBPs that are present at moderate levels (sub- to low-mug/L): category 2 contains 29 of these that are genotoxic (including chloral hydrate and chloroacetaldehyde, which are also a rodent carcinogens); category 3 contains the remaining 14 for which little or no toxicological data are available. In general, the brominated DBPs are both more genotoxic and carcinogenic than are chlorinated compounds, and iodinated DBPs were the most genotoxic of all but have not been tested for carcinogenicity. There were toxicological data gaps for even some of the 11 regulated DBPs, as well as for most of the 74 emerging DBPs. A systematic assessment of DBPs for genotoxicity has been performed for approximately 60 DBPs for DNA damage in mammalian cells and 16 for mutagenicity in Salmonella. A recent epidemiologic study found that much of the risk for bladder cancer associated with drinking water was associated with three factors: THM levels, showering/bathing/swimming (i.e., dermal/inhalation exposure), and genotype (having the GSTT1-1 gene). This finding, along with mechanistic studies, highlights the emerging importance of dermal/inhalation exposure to the THMs, or possibly other DBPs, and the role of genotype for risk for drinking-water-associated bladder cancer. More than 50% of the total organic halogen (TOX) formed by chlorination and more than 50% of the assimilable organic carbon (AOC) formed by ozonation has not been identified chemically. The potential interactions among the 600 identified DBPs in the complex mixture of drinking water to which we are exposed by various routes is not reflected in any of the toxicology studies of individual DBPs. The categories of DBPs described here, the identified data gaps, and the emerging role of dermal/inhalation exposure provide guidance for drinking water and public health research.

Cytokinesis-block micronucleus cytome assay

Abstract: The cytokinesis-block micronucleus cytome assay is a comprehensive system for measuring DNA damage, cytostasis and cytotoxicity. DNA damage events are scored specifically in once-divided binucleated (BN) cells and include (a) micronuclei (MNi), a biomarker of chromosome breakage and/or whole chromosome loss, (b) nucleoplasmic bridges (NPBs), a biomarker of DNA misrepair and/or telomere end-fusions, and (c) nuclear buds (NBUDs), a biomarker of elimination of amplified DNA and/or DNA repair complexes. Cytostatic effects are measured via the proportion of mono-, bi- and multinucleated cells and cytotoxicity via necrotic and/or apoptotic cell ratios. Further information regarding mechanisms leading to MNi, NPBs and NBUDs formation is obtained using centromere and/or telomere probes. The assay is being applied successfully for biomonitoring of in vivo genotoxin exposure, in vitro genotoxicity testing and in diverse research fields such as nutrigenomics and pharmacogenomics as well as a predictor of normal tissue and tumor radiation sensitivity and cancer risk. The procedure can take up to 5 days to complete.

Oecd guideline for testing of chemicals

Abstract: 1. The in vivo alkaline comet (single cell gel electrophoresis) assay (hereafter called simply the comet assay) is used for the detection of DNA strand breaks in cells or nuclei isolated from multiple tissues of animals, usually rodents, that have been exposed to potentially genotoxic material(s). The comet assay has been reviewed and recommendations have been published by various expert groups (1) (2) (3) (4) (5) (6) (7) (8) (9) (10). This Test Guideline is part of a series of Test Guidelines on genetic toxicology. A document presented as an Introduction to the Test Guidelines on genotoxicity (11) can also be referred to and provides succinct and useful guidance to users of these Test Guidelines.

International conference on harmonisation of technical requirements for registration of pharmaceuticals for human use

Abstract: A new tripartite, harmonised guideline is proposed in the Quality topic area on the “Comparability of Biotechnological / Biological Products Subject to Changes in their Drug Substance and /or Drug Product Manufacturing Process”. For establishing these comparisons, it is necessary to address both product and process aspects which, while in some instances region-specific, are uniform in their principles and are necessary to support changes in manufacturing processes yielding products defined within the scope of Q6B. When the Quality aspects have been addressed, experts from the Safety and Efficacy groups will be invited to consider issues in the preclinical and in the clinical areas, as appropriate.