3Rs friendly study designs facilitate rat liver and blood micronucleus assays and Pig‐a gene mutation assessments: Proof‐of‐concept with 13 reference chemicals

Abstract

Regulatory guidance documents stress the value of assessing the most appropriate endpoints in multiple tissues when evaluating the in vivo genotoxic potential of chemicals. However, conducting several independent studies to evaluate multiple endpoints and/or tissue compartments is resource intensive. Furthermore, when dependent on visual detection, conventional approaches for scoring genotoxicity endpoints can be slow, tedious, and less objective than the ideal. To address these issues with current practices we attempted to (1) devise resource sparing treatment and harvest schedules that are compatible with liver and blood micronucleus endpoints, as well as the Pig‐a gene mutation assay, and (2) utilize flow cytometry‐based methods to score each of these genotoxicity biomarkers. Proof‐of‐principle experiments were performed with 4‐week‐old male and female Crl:CD(SD) rats exposed to aristolochic acids I/II, benzo[a]pyrene, cisplatin, cyclophosphamide, diethylnitrosamine, 1,2‐dimethylhydrazine, dimethylnitrosamine, 2,6‐dinitrotoluene, hydroxyurea, melphalan, temozolomide, quinoline, or vinblastine. These 13 chemicals were each tested in two treatment regimens: one 3‐day exposure cycle, and three 3‐day exposure cycles. Each exposure, blood collection, and liver harvest was accomplished during a standard Monday–Friday workweek. Key findings are that even these well‐studied, relatively potent genotoxicants were not active in both tissues and all assays (indeed only cisplatin was clearly positive in all three assays); and whereas the sensitivity of the Pig‐a assay clearly benefitted from three versus one treatment cycle, micronucleus assays yielded qualitatively similar results across both study designs. Collectively, these results suggest it is possible to significantly reduce animal and other resource requirements while improving assessments of in vivo genotoxicity potential by simultaneously evaluating three endpoints and two important tissue compartments using fit‐for‐purpose study designs in conjunction with flow cytometric scoring approaches. Environ. Mol. Mutagen., 60:704–739, 2019. © 2019 Wiley Periodicals, Inc.

Absence of hydroxyurea‐induced mutational effects supports higher utilisation for the treatment of sickle cell anaemia

“…Specifically, these assay results indicate that hydroxyurea is not mutagenic, but rather exhibits clastogenic potential. This important point was convincingly demonstrated in vivo by the discrepant results for the MN‐RET and Pig‐a end‐points 38,39 . Although the assay end‐points consider the same target cell population (bone marrow erythroid cells), only the clastogenic end‐point was positive.…”

“…As discussed in the in vitro genotoxicity section above, this profile of non‐DNA‐reactive effects conveys a much lower risk of carcinogenesis. Finally, as demonstrated by erythroid versus liver micronucleus results indicate, in vivo clastogenic potential is limited to select cell types, even when maximally tolerated dose levels are utilised 39 …”

Study of Combinatorial Drug Synergy of Novel Acridone Derivatives With Temozolomide Using in-silico and in-vitro Methods in the Treatment of Drug-Resistant Glioma

“…Rats in two groups were given acridone derivatives alone at a dose of 300 and 2,000 mg, rats in two groups were given a combination of Temozolomide : acridone derivatives at dose of 10:1 mg/kg and 15:1.5 mg/kg body weight and one group was kept as control. Dose of Temozolomide was taken as per previously published article (50). Compounds alone and combination were suspended in 2.0% of Tween 80 in normal saline and control group was taken for vehicle only.…”

Permitted daily exposure limits for noteworthy N‐nitrosamines

“…Other studies were rejected as inadequate, with too few dose levels (Ashby et al, 1994; Souliotis et al, 1998; Suzuki et al, 1996) or the exposure, expression, or recovery periods were too short (Butterworth et al, 1998; Jiao et al, 1997). Studies of a different mutation target, the Pig‐a gene, using circulating red blood cells (Dertinger et al, 2019) were also considered; however, for these metabolically activated mutagens, mutations in liver cells of the transgenic rodents were considered the most relevant endpoint for derivation of a PDE based on mutagenicity‐data.…”