Evaluation of the rodent micronucleus assay by a 28‐day treatment protocol: Summary of the 13th Collaborative Study by the Collaborative Study Group for the Micronucleus Test (CSGMT)/Environmental Mutagen Society of Japan (JEMS)–Mammalian Mutagenicity Study Group (MMS)

Abstract

To examine whether micronucleus tests can be incorporated into general toxicology assays, we performed micronucleus tests applying the treatment protocols typically used in such assays. In this 13th Collaborative Study of the CSGMT, both rats and mice were tested, although rats were used in the majority of the studies. Fifteen mutagens were tested in rats, mainly by oral (p.o.) administration. Micronucleus induction was evaluated 2, 3, and 4 days, and 1, 2, 3, and 28 days after the beginning of the treatment in the peripheral blood, and at 28 days in the bone marrow. Of the 15 chemicals that induced micronuclei in rats in short-term assays, two chemicals (1,2-dimethylhydrazine.2HCl and mitomycin C) were negative in all our experiments, possibly because of insufficient dose levels. The remaining 13 were positive within the estimated dose range of a general toxicology assay, suggesting the possibility of integrating the micronucleus assay into general toxicology assays. Three patterns were observed in micronucleus induction during the period of repeated treatment: (1) gradual increases in micronucleus frequency with sequential doses, (2) a peak at 3-5 days followed by gradual decreases in micronucleus frequency with sequential doses, and (3) a rapid increase in micronucleus frequency followed by a plateau. We evaluated factors that might have been involved in those patterns, such as the spleen function, target organ exposure, extramedullary hematopoiesis, hypothermia, and hypoxia. Another factor we considered was dosage. Because the dosages employed in a general toxicity assay are usually lower than those used in short-term micronucleus assays, this discrepancy was considered the greatest potential problem for integrating the micronucleus assay into general toxicology assays. Our results indicate that the integration of the micronucleus assay into a 28-day toxicological assay is feasible. To serve this purpose, blood samples collected 4 days after the beginning of treatment and blood and bone marrow samples collected at autopsy should be examined. Furthermore, although it is recognized that mice may be suitable for performing independent micronucleus assays, we propose that rats can provide biologically important and relevant information regarding potential chemical mutagens that can be evaluated under conditions used in the conduct of general toxicology studies.

Genotoxic Effects of Chlorpyrifos in Freshwater Fish Cirrhinus mrigala Using Micronucleus Assay

“…Additional blood sampling has been considered useful on days 2-4 after exposure to toxicant [23]. Keeping these points in consideration, specimens were exposed for 4-5 weeks with initial sampling on days 2, 4, and 8 and thereafter at weekly interval.…”

Effective use of the <i>Pig-a</i> gene mutation assay for mutagenicity screening: measuring CD59-deficient red blood cells in rats treated with genotoxic chemicals

“…Previous Pig-a mutant manifestation studies indicate that ENU-induced Pig-a mutant RBCs in rats persist for at least 6 months after the first treatment with ENU, and that Pig-a mutant RBCs accumulate after repeat dosing in a near-additive fashion Phonethepswath et al, 2010;Miura et al, 2009). Micronucleus induction is usually short-lived after single doses, but pseudo-persistent responses can appear to occur after daily repeated doses (Hamada et al, 2001a). Thus, it would be possible to evaluate both chromosomal aberration and gene mutation if the Pig-a assay and the micronucleus assay were integrated into 28-day toxicology studies.…”

Evaluation of a general toxicity study incorporating phototoxicity assessments in Sprague-Dawley rats

“…Additionally, the Guidance for Industry Photosafety Testing (2003) published by the Food and Drug Administration (FDA) specifies, "assessments of photoirritation may be incorporated into ongoing general toxicity studies in some circumstances." Toxicology studies incorporating genotoxicity (Hamada et al, 2001) and pharmacological safety (Luft and Bode, 2002) assessments have already been reported. These reports indicate that combining multiple studies shortens the research and development period and reduces the number of experiments and animals required.…”

“…These reports indicate that combining multiple studies shortens the research and development period and reduces the number of experiments and animals required. In addition, Hamada's report (Hamada et al, 2001) suggests that information concerning toxicokinetics and other diverse toxicity information can be obtained simultaneously from the same animals, which is beneficial for the comprehensive safety evaluation of chemicals. Thus, the ability to incorporate phototoxicity assessments into general toxicity studies could be of huge benefit for accelerating the testing of new drugs and reducing the number of animal experiments required during drug testing.…”