Comprehensive interpretation of in vitro micronucleus test results for 292 chemicals: from hazard identification to risk assessment application

Abstract

Risk assessments are increasingly reliant on information from in vitro assays. The in vitro micronucleus test (MNvit) is a genotoxicity test that detects chromosomal abnormalities, including chromosome breakage (clastogenicity) and/or whole chromosome loss (aneugenicity). In this study, MNvit datasets for 292 chemicals, generated by the US EPA's ToxCast program, were evaluated using a decision tree-based pipeline for hazard identification. Chemicals were tested with 19 concentrations (n = 1) up to 200 µM, in the presence and absence of Aroclor 1254-induced rat liver S9. To identify clastogenic chemicals, %MN values at each concentration were compared to a distribution of batch-specific solvent controls; this was followed by cytotoxicity assessment and benchmark concentration (BMC) analyses. The approach classified 157 substances as positives, 25 as negatives, and 110 as inconclusive. Using the approach described in Bryce et al. (Environ Mol Mutagen 52:280-286, 2011), we identified 15 (5%) aneugens. IVIVE (in vitro to in vivo extrapolation) was employed to convert BMCs into administered equivalent doses (AEDs). Where possible, AEDs were compared to points of departure (PODs) for traditional genotoxicity endpoints; AEDs were generally lower than PODs based on in vivo endpoints. To facilitate interpretation of in vitro MN assay concentration-response data for risk assessment, exposure estimates were utilized to calculate bioactivity exposure ratio (BER) values. BERs for 50 clastogens and two aneugens had AEDs that approached exposure estimates (i.e., BER < 100); these chemicals might be considered priorities for additional testing. This work provides a framework for the use of high-throughput in vitro genotoxicity testing for priority setting and chemical risk assessment.

Keywords: Benchmark dose; Genotoxicity; IVIVE; Micronucleus; Point of departure; Toxicokinetics.

Fig. 1

Decision-making scheme for evaluating MN assay results (color figure online)

Fig. 2

Scatter plots of positive controls, vinblastine (− S9) and cyclophosphamide (+ S9), versus relative survival rate. Each plot represents increasing concentrations of test agents (1.25, 2.5, and 5 µM for cyclophosphamide, and 0.00625, 0.0125, and 0.025 µM for vinblastine). Wells with relative survival rates below 40% are colored in red, between 40 and 60% in orange, and above 60% in green. One outlier was identified for cyclophosphamide for each of 2.5 µM and 5 µM and eight outliers were identified for all three vinblastine concentrations (circled in red). Two outliers were not plotted for cyclophosphamide at 1.25 µM (color figure online)

Fig. 3

Overall distributions for solvent control (DMSO) %MN and %hypodiploid, ± S9. For the %MN and %hypodiploid + S9 distributions (A and C), nine outliers belonging to 20131009 Plate 2 are colored in red. Four data points from solvent control DMSO − S9 (hypo %) (D) were also removed (hypodiploid ranges from 15 to 132%). No clear outliers were observed in (B) (color figure online)

Fig. 4

POD ratio distribution between lowest traditional POD (i.e., across in vivo genotoxicity and cancer studies) and AEDs determined herein using in vitro MN data. The dashed line indicates where the traditional POD and AED are equivalent. Chemicals to the left of the dashed line have a higher AED than the traditional POD, and chemicals to the right have a lower AED than the traditional POD (i.e., are more conservative)

Fig. 5

Bioactivity Exposure Ratios (BERs) for compounds classified as clastogens based on the decision tree (yellow). A ExpoCast median exposure estimates (green), and B ExpoCast 95th percentile exposure estimates (green). Traditional genetic toxicology POD (blue) and cancer POD (black) from ToxValDB are plotted for comparison. The full list of chemicals and their respective POD values are in Supplemental File 8 (color figure online)

Fig. 6

Bioactivity Exposure Ratios (BERs) for compounds classified as aneugens based on the Bryce et al. (2011) approach (red). A ExpoCast median exposure estimates (green); B ExpoCast 95th percentile exposure estimates (green) (color figure online)