Takumi Awogi

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

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 68.6, 54.5 and 45.6%, 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 90.5, 65.2 and 60.0% 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.

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

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.

The micronucleus assay using peripheral blood reticulocytes from mitomycin C- and cyclophosphamide-treated rats.

It used to be believed that the use of rat peripheral blood for the micronucleus assay would be difficult because micronucleated erythrocytes are captured and destroyed by the spleen quickly. We have applied an acridine orange (AO) supravital staining method to rat peripheral blood using AO-coated glass slides. Normal and splenectomized SD rats were treated once with mitomycin C (i.p.) or cyclophosphamide (p.o.), and 5 microliters of blood was collected at intervals from the tail vein between 0 and 72 h after treatment. For comparison, bone marrow cells were smeared conventionally 30 h after treatment. Although the frequencies of spontaneous and chemically induced micronucleated reticulocytes (MNRETs) from normal rats were lower on average in the highest dose group than those of splenectomized rats, the incidence of micronuclei among type I and II reticulocytes in normal rats at 48 h was almost identical to the incidence of RNA-containing erythrocytes with micronucleus in bone marrow. Thus, we suggest that rat peripheral reticulocytes can be used as target cells for the micronucleus assay.

Mouse Lymphoma thymidine kinase locus gene mutation assay : International Workshop on Genotoxicity Test Procedures Workgroup Report

The Mouse Lymphoma Assay (MLA) Workgroup addressed and reached consensus on a number of issues. Discussion focused on five areas: (1) acceptable assay versions; (2) cytotoxicity measure; (3) 24‐hr treatment; (4) microwell colony counting and sizing; and (5) data acceptability/statistical analysis. Although the International Conference on Harmonisation (ICH) indicated a preference for the microwell over the soft agar method, all of the workgroup members agreed that both versions of the MLA are equally acceptable. The workgroup agreed that it is desirable for both assay versions to use the same measure of cytotoxicity to define the acceptable and required concentration range. Currently, laboratories using the microwell version use the relative survival (RS) determined by cloning immediately after the treatment. Laboratories using the soft agar method do not obtain an RS but use the relative total growth (RTG), a combination of the relative suspension growth (RSG) during the expression period and the relative cloning efficiency determined at the time of mutant selection. The workgroup agreed to investigate the RSG, the RS, and the RTG and to develop further guidance. In the interim, the workgroup reached consensus that the RTG be used as the standard measure of cytotoxicity. The ICH recommended a 24‐hr treatment in the absence of S9 when negative results are obtained with short (3–4 hr) treatments. The workgroup agreed to retain this requirement but acknowledged that more data are needed prior to making final recommendations concerning the need for and the specific protocol for the 24‐hr treatment. Environ. Mol. Mutagen. 35:185–190, 2000 Published 2000 Wiley‐Liss, Inc.

Suitable top concentration for tests with mammalian cells: mouse lymphoma assay workgroup.

The Mouse Lymphoma Expert Workgroup of the International Workshop for Genotoxicity Tests (IWGT) met in Basel, Switzerland in August of 2009. The Workgroup (WG) was tasked with discussing the appropriate top concentration for non-pharmaceuticals that would be required for the conduct of the mouse lymphoma assay (MLA) when sufficient cytotoxicity [to between 10 and 20% relative total growth (RTG)] has not been attained. The WG approached this task by (1) enumerating the various regulatory decisions/use for MLA data, (2) discussing the appropriate assays to which MLA data and assay performance should be compared and (3) discussing all the proposals put forth concerning the top concentration for non-pharmaceuticals. In addition, one of the members presented a summary of a re-evaluation of the National Toxicology Program MLA data using the IWGT harmonized guidance that was underway as a separate (non IWGT) activity, being conducted by two members of the Expert WG. The WG was asked to vote on each of the various proposals for top concentration for when cytotoxicity is not concentration limiting. While there was general agreement that the top concentration for non-pharmaceuticals should be re-evaluated and likely lowered from the current recommended levels, there was no agreement on a specific new recommendation.

Micronucleus test with benzo[a]pyrene using a single peroral administration and intraperitoneal injection in males of the MS/Ae and CD-1 mouse strains

The effect of route of administration on the outcome of the micronucleus test was examined by administering benzo[a]pyrene (B[a]P) perorally (p.o.) and intraperitoneally (i.p.) to males of the MS/Ae and CD-1 mouse strains. This study consisted of 3 parts. First, an acute toxicity study lasting 3 days was done to estimate LD50s. The LD50 was larger than 1600 mg/kg for both routes in the 2 strains. Second, pilot micronucleus tests were carried out, on the basis of which an appropriate sampling time (48 h) and dose levels (62.5, 125, 250, and 500 mg/kg) were chosen for both routes and both strains. Third, full-scale micronucleus tests were done, which indicated that (1) B[a]P induced micronuclei dose-dependently by each administration route in each strain, (2) the i.p. route induced frequencies of micronuclei almost equal to or slightly higher than did the p.o. route, and (3) the MS/Ae strain was the higher responder.

Induction of micronucleated reticulocytes by potassium bromate and potassium chromate in CD-1 male mice

Micronucleus tests of potassium bromate (KBrO3) and potassium chromate (K2CrO4) were conducted with peripheral blood reticulocytes (PB-RETs) of CD-1 male mice dose intraperitoneally. Peripheral blood cells collected from the tail were stained supravitally with acridine orange (AO) using AO-coated glass slides. Both KBrO3 and K2CrO4 induced micronuclei in PB-RETs in the same manner as in polychromatic erythrocytes of bone marrow.

Inter‐laboratory validation of the modified murine local lymph node assay based on 5‐bromo‐2′‐deoxyuridine incorporation

The murine local lymph node assay (LLNA) is a well‐established alternative to the guinea pig maximization test (GPMT) or Buehler test (BT) for the assessment of the skin sensitizing ability of a drug, cosmetic material, pesticide or industrial chemical. Instead of radioisotope using in this method, Takeyoshi M. et al. ( 2001 ) has developed a modified LLNA based on the 5‐bromo‐2′‐deoxyuridine (BrdU) incorporation (LLNA:BrdU‐ELISA). The LLNA:BrdU‐ELISA is practically identical to the LLNA methodology excluding the use of BrdU, for which a single intraperitoneal injection of BrdU is made on day 4, and colorimetric detection of cell turnover. We conducted the validation study to evaluate the reliability and relevance of LLNA:BrdU‐ELISA.

Nuclear localization of alkaline phosphatase in cultured human cancer cells

 The nucleolar localization of alkaline phosphatase (ALPase) was observed by electron microscopic cytochemistry. The culture cells used in this study were normal human cells (fibroblast, WI-38) and human cancer cells (hepatocellular carcinoma, Hep-G2; malignant melanoma, A-375; pancreatic carcinoma, BxPC-3). Cultured cells in almost all strains contained high ALPase activity in the nucleolus, and the localization of ALPase changed during the cell cycle stages. The pattern of ALPase localization during the interphase was divided into three groups: cytoplasmic type, nucleus type, and both types. Moreover, at the mitotic phase, the reaction products were observed on the chromosome. In the cultured malignant melanoma cells, the appearance ratio of ALPase reaction products on the nucleolus (33.9%) showed a higher ratio compared with normal cultured fibroblasts (6.3%). This phenomenon suggests that the high level of the ALPase reaction product may be related to the high level of proliferation of cancer cells.

The strains recommended for use in the bacterial reverse mutation test (OECD guideline 471) can be certified as non-genetically modified organisms

The bacterial reverse mutation test, commonly called Ames test, is used worldwide. In Japan, the genetically modified organisms (GMOs) are regulated under the Cartagena Domestic Law, and organisms obtained by self-cloning and/or natural occurrence would be exempted from the law case by case. The strains of Salmonella typhimurium and Escherichia coli recommended for use in the bacterial reverse mutation test (OECD guideline 471), have been considered as non-GMOs because they can be constructed by self-cloning or naturally occurring bacterial strains, or do not disturb the biological diversity. The present article explains the reasons why these tester strains should be classified as non-GMOs.