Use of a standardized JaCVAM in vivo rat comet assay protocol to assess the genotoxicity of three coded test compounds; ampicillin trihydrate, 1,2-dimethylhydrazine dihydrochloride, and N-nitrosodimethylamine

Abstract

As part of the Japanese Center for the Validation of Alternative Methods (JaCVAM)-initiative international validation study of the in vivo rat alkaline comet assay (comet assay), our laboratory examined ampicillin trihydrate (AMP), 1,2-dimethylhydrazine dihydrochloride (DMH), and N-nitrosodimethylamine (NDA) using a standard comet assay validation protocol (v14.2) developed by the JaCVAM validation management team (VMT). Coded samples were received by our laboratory along with basic MSDS information. Solubility analysis and range-finding experiments of the coded test compounds were conducted for dose selection. Animal dosing schedules, the comet assay processing and analysis, and statistical analysis were conducted in accordance with the standard protocol. Based upon our blinded evaluation, AMP was not found to exhibit evidence of genotoxicity in either the rat liver or stomach. However, both NDA and DMH were observed to cause a significant increase in % tail DNA in the rat liver at all dose levels tested. While acute hepatoxicity was observed for these compounds in the high dose group, in the investigators opinion there were a sufficient number of consistently damaged/measurable cells at the medium and low dose groups to judge these compounds as genotoxic. There was no evidence of genotoxicity from either NDA or DMH in the rat stomach. In conclusion, our laboratory observed increased DNA damage from two blinded test compounds in rat liver (later identified as genotoxic carcinogens), while no evidence of genotoxicity was observed for the third blinded test compound (later identified as a non-genotoxic, non-carcinogen). This data supports the use of a standardized protocol of the in vivo comet assay as a cost-effective alternative genotoxicity assay for regulatory testing purposes.

Oxidative stress, cholinesterase activity, and DNA damage in the liver, whole blood, and plasma of Wistar rats following a 28-day exposure to glyphosate

“…The frequency of such comets ("hedgehogs" or "clouds") was determined based on visual scoring among 100 nucleoids per sample. According to literature data, they may represent DNA damage resulting from cytotoxicity (59). We did not measure the abnormalsized tail parameters, as we find them inappropriate for this study.…”

Re-analysis results using medians of the data from the JaCVAM-organized international validation study of the in vivo rat alkaline comet assay

“…Data re-analysis was conducted by two types of investigations, i.e., one used each laboratory analysis report [4][5][6][7][8][9][10][11][12][13][14][15][16] and the other was our unified analysis. For both analyses, based on TG 489 [3], the median % tail DNA for each slide was determined, the mean of the 2 median values was calculated for each animal, and then the mean of the individual animal means was determined to give a group mean.…”

“…In laboratory analysis, statistical significance between a negative control group and chemical treatment groups except for the positive control group was analyzed with a one-way analysis of variance (ANOVA) using log-transformed values (3 labs; Lab B: one-sided Simple meanapproach Significant 18 3 3 24 Significant in either statistic 0 5 3 8 Non-significant 2 4 47 53 Number of data 20 12 53 85 Significant: statistically significant in Dunnett's and linear trend test, non-significant: no statistically significant in both statistical analysis methods, significant in either statistic: statistically significant in either but not both of the two statistical analysis methods. and p < 0.03 [4], Lab D: p < 0.05 [6], Lab G: p < 0.001 [9]) followed by a multiple-comparison such as Dunnett's test (3 labs; Lab C: p < 0.05 [5], Lab L: two-sided and p < 0.05 [14], Lab M: two-sided and p < 0.05 [15]), a multiple-comparison with Dunnett's test for nonlog-transformation values (5 labs; Lab E: two-sided and p < 0.05 [7], Lab F: two-sided and p<0.05 [8], Lab I: two-sided and p < 0.05 [11], Lab J: p < 0.05 [12], Lab N: two-sided and p < 0.05 [16]), or a post-hoc right one-sided pairwise comparison using log-transformed values (1 lab; Lab K: one-sided and p < 0.05 [9]). One laboratory (Lab H) did not use statistics because the values in all treatment groups were lower than the respective control values [10].…”

“…One laboratory (Lab H) did not use statistics because the values in all treatment groups were lower than the respective control values [10]. Dose-dependency was also examined in 8 of 12 laboratories with a statistical analysis method, such as a linear trend test [4,7,8,[11][12][13][14]16]. …”

“…Here, we refer to the former method of analysis as the simple mean approach and the latter as the median based approach. Due to TG 489, the individual laboratory reports of the main validation study (Phase 4-2) from most of the participating laboratories were revised and described using their own statistical approach based on using medians (and means) [4][5][6][7][8][9][10][11][12][13][14][15][16]. Also, we separately re-analyzed the Phase 4-2 data based on the median based approach in the same manner as the original statistical analysis of the JaCVAM-organized validation study as reported by Uno et al [2] in order to compare the sensitivity/specificity using the simple mean approach with the median based approach.…”