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.

Biological materials can release amino acids during the course of bacterial mutation testing. Low levels of released amino acids from soluble materials can cause moderate increases in the number of revertant colonies on the plate, whereas higher levels lead to overgrowth of the background lawn, making counting of revertant colonies impossible. For poorly soluble material, the released amino acids can be present at high levels in localized spots on the plate, leading to the growth of 'pseudorevertant' colonies. The 'treat and wash' modified preincubation method employed here is an adaptation of the treat and plate method (used for evaluation of antibiotics) and involves washing the bacteria free of test compound after a 90 min exposure prior to plating out on minimal plates. The MC overlay method is a modified version of the standard plate incorporation assay, in which a top overlay containing 4% high viscosity methylcellulose is used in place of agar to stabilize the test compound in solution, preventing precipitation and subsequent localized amino acid release. Both modified methods produce the expected results for negative and positive controls. Peptides [synthetic curtailed analogs of human parathyroid hormone, PTH(1-34) and Ostabolin-C] that produced false positive results or could not be evaluated owing to overgrowth of the background lawn using standard methods, showed no artifacts and no evidence of genotoxicity using the modified methods. It is concluded that the treat and wash and MC overlay methods are valid versions of the bacterial mutation test for avoiding complications associated with released amino acids.

There is widespread dietary exposure to capsaicin in the form of chili peppers, while capsaicin's analgesic qualities have led to increased use of a topical herbal remedy in various impure forms. Most recently, injection of pure capsaicin has been proposed as a means of relieving a variety of debilitating diseases, in which case tissues would receive relatively high and direct exposure. The purpose of the present study, where a series of standard assays were performed in accordance with the Organisation for Economic Cooperation and Development guidance, was to clarify earlier conflicting reports concerning potential genotoxicity of capsaicin prior to administering it to patients in an injectable form. The results confirm the absence of genotoxic activity of high-purity capsaicin in the bacterial mutation and chromosome aberration tests. In addition, no evidence of cytotoxicity or genotoxicity was seen in the rat bone marrow micronucleus test, where systemic exposure to pure capsaicin was achieved using the subcutaneous route and a rising dose toleration protocol. It is concluded that pure capsaicin is not active in the standard battery of genotoxicity assays recommended by the International Conference on Harmonisation for evaluation of new medicines; earlier reported in vitro genotoxic activity is probably associated with mutagenic impurities in commercial grades of the material.

"Ames" bacterial mutation tests are widely performed for evaluation and registration of new materials including industrial chemicals, agrochemicals, medical devices, pharmaceuticals, pharmaceutical impurities and other materials. Tests are used to predict their potential long-term adverse health effects (including carcinogenicity). Given their importance, pre-screening 'miniaturized' versions have been developed which allow higher throughput and use less test material, including the widely-employed 24-well micro-Ames (µAmes) test which uses 20 times less material. However, little quantitative information has been published on the methodology or sensitivity of this system. We describe methods and results used in direct comparisons of the sensitivity of micro and standard systems using the same cultures, formulations, etc. Initial testing utilized the plate incorporation method and, later, the pre-incubation method. In a subsequent phase of testing, a four-way direct comparison was made between the pre-incubation and plate incorporation methods in both systems using some direct-acting mutagens. Tests used only those strain/S9/chemical combinations where a response was expected. Historical control results accumulated during testing are also presented. Spontaneous and induced revertant colony counts for the µAmes system were consistently proportionate and approximately 1/20th those for the standard Ames test. Sensitivities of the two systems were found to be nearly identical in almost all cases for a wide variety of weak and strong inorganic and organic mutagens. Standardized procedures and increased reliability of the estimate of the background revertant frequency in the µAmes system means that the two systems give equivalent results and are expected to be highly predictive of one another. Environ. Mol. Mutagen. 57:687-705, 2016. © 2016 Wiley Periodicals, Inc.

A continuous hydrogenation device was evaluated for the detoxification of selected tri-, tetra-, or pentacyclic polyaromatic hydrocarbon (PAH) compounds {anthracene, phenanthrene, chrysene, and benzo[a]pyrene (B[a]P)} by hydrogenation. A substrate stream in hexane, 0.05-1.0% (w/v), was mixed with hydrogen-carbon dioxide (H2-CO2, 5-30% v/v) and delivered to a heated reactor column (25 cm x 1 cm) containing palladium supported on gamma alumina (Pd0/gamma-Al2O3) that was terminated with a capillary restrictor. The flow rate from the reactor, approximately 800 mL min(-1) decompressed gas, corresponded to 4 mL min(-1) fluid under the operating conditions of the trials. Reaction products were recovered by passing the reactor effluent through hexane. At 90 degrees C, the anthracene or phenanthrene substrate was hydrogenated only partially to octahydro and dodecahydro species and contained only a minor quantity of totally hydrogenated products. For substrates with increasing numbers of fused aromatic rings, the hydrogenation efficiency was decreased further. However, at an increasing temperature (90-150 degrees C) and increasing mobile phase flow rate (20.68 MPa corresponding to 2100 mL min(-1) decompressed gas), B[a]P and chrysene were hydrogenated, virtuallytotally, to their corresponding perhydro analogues (eicosahydrobenzo[a]pyrenes and octadecahydrochrysenes), respectively. That this approach might be useful for decontaminating soil extracts was supported by companion in vitro trials in which the substrate and products were assayed for mutagenic activity with five bacterial strains that are auxotrophic for histidine (Salmonella typhimurium TA98, TA100, TA1535, and TA1537) or tryptophan (Escherichia coliWP2 uvrA), using the bacterial reverse mutation assay (modified Ames test). Generally, substantial increases in revertant colony counts were not observed with any of the strains following exposure to the hydrogenation products in the absence or presence of the 10 or 30% S9 mix, which is consistent with the loss of mutagenic activity from these hydrogenation products.