Assessment of the Pig‐a, micronucleus, and comet assay endpoints in rats treated by acute or repeated dosing protocols with procarbazine hydrochloride and ethyl carbamate

Abstract

The utility and sensitivity of the newly developed flow cytometric Pig‐a gene mutation assay have become a great concern recently. In this study, we have examined the feasibility of integrating the Pig‐a assay as well as micronucleus and Comet endpoints into acute and subchronic general toxicology studies. Male Sprague–Dawley rats were treated for 3 or 28 consecutive days by oral gavage with procarbazine hydrochloride (PCZ) or ethyl carbamate (EC) up to the maximum tolerated dose. The induction of CD59‐negative reticulocytes and erythrocytes, micronucleated reticulocytes in peripheral blood, micronucleated polychromatic erythrocytes in bone marrow, and Comet responses in peripheral blood, liver, kidney, and lung were evaluated at one, two, or more timepoints. Both PCZ and EC produced positive responses at most analyzed timepoints in all tissue types, both with the 3‐day and 28‐day treatment regimens. Furthermore, comparison of the magnitude of the genotoxicity responses indicated that the micronucleus and Comet endpoints generally produced greater responses with the higher dose, short‐term treatments in the 3‐day study, while the Pig‐a assay responded better to the cumulative effects of the lower dose, but repeated subchronic dosing in the 28‐day study. Collectively, these results indicate that integration of several in vivo genotoxicity endpoints into a single routine toxicology study is feasible and that the Pig‐a assay may be particularly suitable for integration into subchronic dose studies based on its ability to accumulate the mutations that result from repeated treatments. This characteristic may be especially important for assaying lower doses of relatively weak genotoxicants. Environ. Mol. Mutagen. 60:56–71, 2019. © 2018 Wiley Periodicals, Inc.

Integrated In Vivo Genotoxicity Assessment of Procarbazine Hydrochloride Demonstrates Induction of Pig‐a and LacZ Mutations, and Micronuclei, in MutaMouse Hematopoietic Cells

“…While Phonethepswath et al () also reported PCH‐induced Pig‐a mutation in mice, it is not productive comparing those results with data reported herein because the previous study utilized an early mouse blood labeling protocol that is now understood to systematically underestimate mutant cell frequency (Labash et al ). Finally, Chen et al () recently reported strong induction of Pig‐a mutations following a 28‐day exposure regimen in rats using daily doses higher than those used here. Thus, in addition to its well‐known clastogenic activity, PCH is also an effective in vivo mutagen when recommended OECD test guideline experimental design considerations are followed.…”

“…PCH appears to be a particularly effective in vivo clastogen inducing both micronuclei in bone marrow (Romagna and Schneider 1990) and DNA breaks as detected by the alkaline elution (Holme et al 1989) and comet (Sasaki et al 1998) assays in multiple tissues including stomach and liver. Furthermore, significant increases in median percent tail DNA were recently reported in bone marrow, liver, kidney, and lungs of rats, 1 day after a 28-day exposure to 30 or 60 mg PCH/kg/day (Chen et al 2019). However, PCH's mutagenic activity is less well-characterized.…”