Confidence with which data of animal safety toxicity
Confidence with which data of animal safety/toxicity studies can be translated to human depends upon the knowledge that whether humans would be exposed to similar chemical entities (parent and its metabolites) as the animals exposed during toxicity studies. In this regard, in vitro qualitative evaluation of interspecies difference in metabolite profile is important for selection of most appropriate animal species for toxicity studies which is close to human in terms of metabolite pattern. Thus, as majority of the toxicity studies are conducted in rats, in vitro metabolite profiles for BNZ were generated in human and rat for interspecies comparison (Whalley et al., 2017). Investigating the biotransformation pathways, reduction and fluvastatin were the major metabolic routes observed. Four oxidative metabolites and four reduced metabolites were formed in human liver microsomes. Similar metabolites were formed in rat liver microsomes as well, except that only two of the four reduced metabolites were observed and a single oxidative plus reduced metabolite was also observed in rat. These qualitatively different metabolites warrant further investigation to explore the metabolites formed in vivo upon systemic administration. In a qualitative study carried out by Das et al., seven metabolites of BNZ were identified when incubated with rat liver microsomes (Das et al., 1989). This commensurates with the findings of our present study. The inhibitory effect of BNZ on the activity of CYP isoforms was measured to evaluate the probability of CYP mediated metabolic interactions. BNZ was observed to have moderate inhibitory effect only on CYP1A2 with IC50 value of 5.82 μM. Such high systemic levels are very unlikely to be reached upon environmental exposure, thus reducing the possibility of metabolic interactions upon co-exposure with other xenobiotics. Similar findings have also been reported in a study conducted by Das et al. where BNZ was found to be an inhibitor of hepatic aminopyrine N-demethylase and ethoxyresorufin-O-deethylase activities, both in vitro and in vivo, in rat. However, these inhibitory effects were observed at relatively higher concentrations (Das et al., 1991). The weak inhibitory effect of BNZ on CYP isoforms further reduces the uncertainty in assessing the risk of BNZ exposure in humans.
It has been shown that given adequate prior in vitro information on the different enzymes involved in the metabolism of the compound and relative activity of different genotypic forms and their population frequency, it is possible to predict the exposure of a chemical or vulnerability to chemical interaction as a function of genotype with reasonable accuracy. For example, Partosch et al. demonstrated that incorporating the influence of polymorphic UGT2B15 into PBPK model significantly improved the predictions of bisphenol A concentration profile in different populations (Partosch et al., 2013). Thus, the enzyme kinetic parameters obtained and predicted clearance could further be used to build a PBPK model for describing the in vivo disposition of BNZ and the possible sites more prone to its toxic effects. Additionally, the putative metabolites found in our study, can be screened in urine so that they can serve as biomarkers of BNZ exposure for biomonitoring studies.
Conclusions In conclusion, since BNZ is metabolized by multiple CYPs, it would be difficult for any single agent to precipitate a significant chemical interaction via inhibition of the metabolic pathway of BNZ. Similarly, single enzyme genetic polymorphism (e.g. CYP3A4, 2C9, 2E1, 2C19, 2D6 and 1A2) is unlikely to have profound effect on the toxicokinetics of benzanthrone. Further, similar metabolite pattern in rat as observed in human in vitro test system and comparable in vivo high predicted clearance in both rat and human suggests that rat might be a reliable model species to study the toxicity of BNZ and extrapolation of toxicity data to human for health risk assessment.