Comparison of in vitro and in vivo clastogenic potency based on benchmark dose analysis of flow cytometric micronucleus data

Abstract

The application of flow cytometry as a scoring platform for both in vivo and in vitro micronucleus (MN) studies has enabled the efficient generation of high quality datasets suitable for comprehensive assessment of dose-response. Using this information, it is possible to obtain precise estimates of the clastogenic potency of chemicals. We illustrate this by estimating the in vivo and the in vitro potencies of seven model clastogenic agents (melphalan, chlorambucil, thiotepa, 1,3-propane sultone, hydroxyurea, azathioprine and methyl methanesulfonate) by deriving BMDs using freely available BMD software (PROAST). After exposing male rats for 3 days with up to nine dose levels of each individual chemical, peripheral blood samples were collected on Day 4. These chemicals were also evaluated for in vitro MN induction by treating TK6 cells with up to 20 concentrations in quadruplicate. In vitro MN frequencies were determined via flow cytometry using a 96-well plate autosampler. The estimated in vitro and in vivo BMDs were found to correlate to each other. The correlation showed considerable scatter, as may be expected given the complexity of the whole animal model versus the simplicity of the cell culture system. Even so, the existence of the correlation suggests that information on the clastogenic potency of a compound can be derived from either whole animal studies or cell culture-based models of chromosomal damage. We also show that the choice of the benchmark response, i.e. the effect size associated with the BMD, is not essential in establishing the correlation between both systems. Our results support the concept that datasets derived from comprehensive genotoxicity studies can provide quantitative dose-response metrics. Such investigational studies, when supported by additional data, might then contribute directly to product safety investigations, regulatory decision-making and human risk assessment.

Figure 1.

Fitted curves for in vivo MN dose–response data from seven clastogens studied in rats. The horizontal-to-vertical dashed lines represent the BMD concentrations that correspond to a BMR = 200%, that is a 3-fold induction of MN (=distance of 0.48 on log10-scale). For each chemical, the small symbols represent the individual data points and the large symbol represents the geometric mean of these observations.

Figure 2.

Fitted curves for in vitro MN responses from seven clastogens studied in human TK6 cells. The horizontal-to-vertical dashed lines represent the BMD concentrations that correspond to a BMR = 200%, that is a 3-fold induction of MN (=distance of 0.48 on log10-scale). For each chemical, the small symbols represent the individual data points and the large symbol represents the geometric mean of these observations.

Figure 3.

In vivo MN versus in vitro MN responses for seven clastogens. BMD200 is the BMD corresponding to a BMR = 200%, that is a 3-fold induction of MN. Confidence intervals for each chemical are shown as the horizontal and vertical lines at each data point. The correlation between these two methods is represented by the diagonal lines that encompass the confidence intervals of all chemicals studied.

Figure 4.

BMD confidence intervals for in vitro (upper panel) or in vivo (lower panel) MN systems corresponding to a BMR = 200%, that is a 3-fold induction of MN.