Analysis of variability in the rabbit skin irritation assay

The in vivo rabbit test is the benchmark against which new approach methodologies for skin irritation are usually compared. No alternative method offers a complete replacement of animal use for this endpoint for all regulatory applications. Variability in the animal reference data may be a limiting factor in identifying a replacement. We established a curated data set of 2624 test records, representing 990 substances, each tested at least twice, to characterize the reproducibility of the in vivo assay. Methodological deviations from guidelines were noted, and multiple data sets with differing tolerances for deviations were created. Conditional probabilities were used to evaluate the reproducibility of the in vivo method in identification of U.S. Environmental Protection Agency or Globally Harmonized System hazard categories. Chemicals classified as moderate irritants at least once were classified as mild or non-irritants at least 40% of the time when tested repeatedly. Variability was greatest between mild and moderate irritants, which both had less than a 50% likelihood of being replicated. Increased reproducibility was observed when a binary categorization between corrosives/moderate irritants and mild/non-irritants was used. This analysis indicates that variability present in the rabbit skin irritation test should be considered when evaluating nonanimal alternative methods as potential replacements. •Built a curated data set of 2624 in vivo skin irritation test results encompassing 990 substances.•Conditional probabilities were used to analyze the reproducibility of hazard classifications.•Reproducibility is best at the ends of the classification spectrums, corrosive or non-irritant.•Mild and moderate irritant results are far less reproducible, below 50% in most cases.•Variability in in vivo tests must be considered when benchmarking new approaches.