Timothy Lawlor

Comparative mutagenicity of aliphatic epoxides in Salmonella.

37 aliphatic epoxides comprising 6 subclasses (unsubstituted aliphatic epoxides, halogenated aliphatic epoxides, glycidyl esters, glycidates, glycidyl ethers and diglycidyl ethers) were tested, under code, for mutagenicity in Salmonella strains TA98, TA100, TA1535 and TA1537 and/or TA97 with and without metabolic activation using a standardized protocol. The 4 halogenated aliphatic epoxides and the 4 diglycidyl ethers were all mutagenic. The 2 glycidates were negative in all strain/activation systems used while all 5 glycidyl esters were mutagenic. 3 of the 8 unsubstituted aliphatic epoxides and 11 of the 12 glycidyl ethers were mutagenic. Glycidol also was mutagenic whereas 9,10-epoxyoctadecanoic acid, 2-ethylhexyl ester was not mutagenic. Of the 28 mutagenic compounds, all but neodecanoic acid, 2,3-epoxypropyl ester and 2-ethylhexyl glycidyl ether were detected in TA100 without activation. The latter two were detected only with activation in TA100 and TA1535. The majority of the other 26 chemicals were also mutagenic in TA1535 without activation. Good intra- and interlaboratory reproducibility was seen in the results of each of the 4 chemicals tested in more than one set of experiments. The current results confirm and extend the observations of other investigators regarding structural effects on the mutagenicity of members of the aliphatic epoxide class of chemicals.

Mutagenic potential of ammonia-related aflatoxin reaction products in a model system

In a joint research effort, the Food and Drug Administration, the National Toxicology Program and the U.S. Department of Agriculture determined the mutagenic potential of aflatoxin reaction products following ammoniation of aflatoxin B 1 in a pressure chamber used to decontaminate aflatoxin-contaminated cottonseed meal. Uniformly ring-labeled (14C)-aflatoxin B1 was added to nonlabeled B1, distributed on an inert carrier and treated with 4% ammonia at 40 psi, 100 C, for 30 min. Aflatoxin-derived decontamination reaction products were separated, and fractions having a high specific activity were tested for mutagenic activity using the Salmonella/mammalian-microsome mutagenicity assay (Ames test). When concentrations ranging from 3.3 to 100 μg per plate were tested, all fractions exhibited a similar mutagenic response. The observed mutagenic activity was 2,000-20,000 times less than that observed with nonammoniated aflatoxin B1.

Mutagenic potential of ammonia-related aflatoxin reaction products in cottonseed meal

In a joint research effort, the Food and Drug Administration, the National Toxicology Program and the US Department of Agriculture studied the mutagenic potential of aflatoxin reaction products following ammoniation of contaminated cottonseed meal under conditions approximating those approved for commercial ammoniation of nonaflatoxin-contaminated meal. Uniformly ring-labeled14C-aflatoxin B1 was added to cottonseed meal that contained ca. 4000 µg naturally incurred aflatoxin B1/kg. Distribution of the radiolabeled compound was used to trace the modification of aflatoxin B1 after treatment with ammonia. The radioactivity-to-weight ratio of the fractions isolated by solvent extractions and chemical and enzymic treatments was used to measure the relative concentration of an aflatoxin decontamination product. All extract fractions having a radioactivity-to-weight ratio ≥1 were tested for mutagenic activity using theSalmonella/microsome mutagenicity test (Ames test). Purified aflatoxin B1 was mutagenic at a concentration of ca. 0.005 µg/plate. The methylene chloride extract of the ammoniated meal after Pronase digestion exhibited a similar response when 180 µg of this fraction was applied to each plate. This fraction represented 0.16% of the original added radioactivity. The other fractions produced no detectable mutagenic response at the concentrations tested (10–1000 µg/plate) onSalmonella tester strain TA100. Ammonia treatment of aflatoxin-contaminated cottonseed meal significantly decreased aflatoxin levels, and the aflatoxin decontamination products formed by the treatment had little or no mutagenic potential.

Integration of Pig-a, micronucleus, chromosome aberration and comet assay endpoints in a 28-day rodent toxicity study with urethane

As part of the international Pig-a validation trials, we examined the induction of Pig-a mutant reticulocytes and red blood cells (RET(CD59-) and RBC(CD59-), respectively) in peripheral blood of male Sprague Dawley(®) rats treated with urethane (25, 100 and 250mg/kg/day) or saline by oral gavage for 29 days. Additional endpoints integrated into this study were: micronucleated reticulocytes (MN-RET) in peripheral blood; chromosome aberrations (CAb) and DNA damage (%tail intensity via the comet assay) in peripheral blood lymphocytes (PBL); micronucleated polychromatic erythrocytes (MN-PCE) in bone marrow; and DNA damage (comet) in various organs at termination (the 29th dose was added for the comet endpoint at sacrifice). Ethyl methanesulfonate (EMS; 200mg/kg/day on Days 3, 4, 13, 14, 15, 27, 28 and 29) was evaluated as the concurrent positive control (PC). All animals survived to termination and none exhibited overt toxicity, but there were significant differences in body weight and body weight gain in the 250-mg/kg/day urethane group, as compared with the saline control animals. Statistically significant, dose-dependent increases were observed for urethane for: RET(CD59-) and RBC(CD59-) (on Days 15 and 29); MN-RET (on Days 4, 15 and 29); and MN-PCE (on Day 29). The comet assay yielded positive results in PBL (Day 15) and liver (Day 29), but negative results for PBL (Days 4 and 29) and brain, kidney and lung (Day 29). No significant increases in PBL CAb were observed at any sample time. Except for PBL CAb (likely due to excessive cytotoxicity), EMS-induced significant increases in all endpoints/tissues. These results compare favorably with earlier in vivo observations and demonstrate the utility and sensitivity of the Pig-a in vivo gene mutation assay, and its ability to be easily integrated, along with other standard genotoxicity endpoints, into 28-day rodent toxicity studies.

Vehicle and positive control values from the in vivo rodent comet assay and biomonitoring studies using human lymphocytes: historical database and influence of technical aspects.

There is increased interest in the in vivo comet assay in rodents as a follow‐up approach for determining the biological relevance of chemicals that are genotoxic in in vitro assays. This is partly because, unlike other assays, DNA damage can be assessed in this assay in virtually any tissue. Since background levels of DNA damage can vary with the species, tissue, and cell processing method, a robust historical control database covering multiple tissues is essential. We describe extensive vehicle and positive control data for multiple tissues from rats and mice. In addition, we report historical data from control and genotoxin‐treated human blood. Technical issues impacting comet results are described, including the method of cell preparation and freezing. Cell preparation by scraping (stomach and other GI tract organs) resulted in higher % tail DNA than mincing (liver, spleen, kidney etc) or direct collection (blood or bone marrow). Treatment with the positive control genotoxicant, ethyl methanesulfonate (EMS) in rats and methyl methanesulfonate in mice, resulted in statistically significant increases in % tail DNA. Background DNA damage was not markedly increased when cell suspensions were stored frozen prior to preparing slides, and the outcome of the assay was unchanged (EMS was always positive). In conclusion, historical data from our laboratory for the in vivo comet assay for multiple tissues from rats and mice, as well as human blood show very good reproducibility. These data and recommendations provided are aimed at contributing to the design and proper interpretation of results from comet assays. Environ. Mol. Mutagen. 55:633–642, 2014.

Genotoxicity of 6 oxime compounds in the Salmonella/mammalian-microsome assay and mouse lymphoma TK+/− assay

To aid in the selection of chemical candidates for in vivo tests, the mutagenicity of 6 oxime compounds was evaluated in the Salmonella plate incorporation assay and mouse lymphoma L5178Y TK +/- assay. All of the oximes were mutagenic in the mouse lymphoma assay in the absence of exogenous metabolic activation. Acetaldehyde oxime was also mutagenic in the presence of S9 activation. In contrast to these results, a positive response was noted only for 2-(hydroxyimino)-N-phenyl-acetamide oxime in strain TA1535 in the absence of activation in the Salmonella/microsome test.

Summary of in vitro genetic toxicology assay results: Expected and unexpected effects of recent study design modifications

Key modifications to in vitro genetic toxicology testing have been made in the last 5 years including the use of optimization approaches such as structure–activity relationships and screening assays to identify and eliminate potentially genotoxic chemicals from further consideration, better guidance on cytotoxicity assessment and dose selection, and greater use of p53‐competent human cells. To determine the effect of these changes on testing outcomes, the pattern of positive results across assays conducted by BioReliance from 2005 to 2010 was examined. Data were tabulated for good laboratory practice (GLP)‐compliant Ames, mouse lymphoma (MLA), chromosome aberration in Chinese hamster ovary (CHO) cells, and in human peripheral blood lymphocytes (HPBL) assays along with non‐GLP screening Ames assays. A decrease in percentage of positive results in MLA and CHO chromosome aberration assays was observed, whereas the percentage of positive Ames assays remained consistent. This was not unexpected because MLA and CHO cytogenetic assays have undergone the most substantive changes (e.g., the establishment of the Global Evaluation Factor for the MLA and the use of the relative increase in cell counts in CHO chromosome aberration assays). Over the last 5 years, there has been an increase in the percentage of positive results observed in the chromosome aberration assay in HPBL. It is speculated that this may have led to an increase in HPBL‐positive results if the chemicals routed to HPBL had previous positive genotoxicity results. Another factor may be the lack of a reliable cytotoxicity measurement in the HPBL assay. Environ. Mol. Mutagen., 2012.

Combination comet/micronucleus assay validation performed by BioReliance under the JaCVAM initiative

In the international validation study of the in vivo rat alkaline comet assay (comet assay), the Japanese Center for the Validation of Alternative Methods (JaCVAM) provided three coded chemicals to BioReliance, 1,3-dichloropropene, ethionamide and busulfan, to be tested in a combined in vivo comet/micronucleus assay. Induction of DNA damage (comet) in liver, stomach and jejunum (1,3-dichloropropene only) cells, and induction of MNPCEs in bone marrow, were examined in male Sprague-Dawley (Hsd:SD) rats following oral administration of the test chemical for three consecutive days. A dose range finding (DRF) test was performed with each chemical to determine the maximum tolerated dose (MTD). Based on the results of the DRF test; 1,3-dichloropropene was tested at 50, 100 and 200 mg/kg/day; ethionamide was tested at 125, 250 and 500 mg/kg/day, and busulfan was tested at 10, 20 and 40 mg/kg/day. The results indicated that 1,3-dichloropropene induced DNA damage only in liver cells at all three test article doses, while no effects were observed in the stomach and jejunum cells. Additionally, it did not increase MNPCEs in the bone marrow. 1,3-Dichloropropene was concluded to be negative in the MN assay but positive in the comet assay. Ethionamide did not induce DNA damage in liver. However, in stomach, statistically significant decreases (although still within historical range) in % tail DNA at all test article doses compared to the vehicle control were observed. There was no increase in MNPCEs in the bone marrow. Thus, ethionamide was concluded to be negative in the comet/MN combined assay. Busulfan did not induce DNA damage in any of the organs tested (liver and stomach) but it did induce a significant increase in MNPCEs in the bone marrow. Busulfan was concluded to be negative in the comet assay but positive in the MN assay.