Evaluation of Variability Across Rat Acute Oral Systemic Toxicity Studies

Abstract

Regulatory agencies rely upon rodent in vivo acute oral toxicity data to determine hazard categorization, require appropriate precautionary labeling, and perform quantitative risk assessments. As the field of toxicology moves toward animal-free new approach methodologies (NAMs), there is a pressing need to develop a reliable, robust reference data set to characterize the reproducibility and inherent variability in the in vivo acute oral toxicity test method, which would serve to contextualize results and set expectations regarding NAM performance. Such a data set is also needed for training and evaluating computational models. To meet these needs, rat acute oral LD50 data from multiple databases were compiled, curated, and analyzed to characterize variability and reproducibility of results across a set of up to 2,441 chemicals with multiple independent study records. Conditional probability analyses reveal that replicate studies only result in the same hazard categorization on average at 60% likelihood. Although we did not have sufficient study metadata to evaluate the impact of specific protocol components (e.g., strain, age, or sex of rat, feed used, treatment vehicle, etc.), studies were assumed to follow standard test guidelines. We investigated, but could not attribute, various chemical properties as the sources of variability (i.e., chemical structure, physiochemical properties, functional use). Thus, we conclude that inherent biological or protocol variability likely underlies the variance in the results. Based on the observed variability, we were able to quantify a margin of uncertainty of ± 0.24 log10 (mg/kg) associated with discrete in vivo rat acute oral LD50 values.

A framework for establishing scientific confidence in new approach methodologies

“…Recent publications have assessed the results of traditional animal test methods and have demonstrated a low level of reproducibility for a range of endpoints, including the LD 50 test in rats for acute oral toxicity (Karmaus et al 2022 ), Draize rabbit tests for eye irritation (Weil and Scala 1971 ; Adriaens et al 2014 ; Luechtefeld et al 2016 ; Barroso et al 2017 ), and skin irritation (Rooney et al 2021 ), the LLNA using mice for skin sensitization (Dumont et al 2016 ), the Uterotropic assay using rodents for estrogen receptor testing (Kleinstreuer et al 2016 ), the Hershberger assays using rodents for androgen receptor testing (Browne et al 2018 ), rodent cancer bioassays (Paparella et al 2017 ), and repeat-dose toxicity studies (Pham et al 2020 ).…”

“…Use of publicly accessible curated databases allows for further assessments of reproducibility of traditional animal test methods, including the evaluation of statistical variability and the limits of detection (Browne et al 2015 ; Mansouri et al 2016 , 2020 ; Barroso et al 2017 ; Judson et al 2018a ; Karmaus et al 2022 ). Manual curation of data can be time-consuming but allows curators to identify errors common in reporting (e.g., misplaced decimal points or incorrect units), and quantitative confidence intervals can be incorporated into in silico tools (Karmaus et al 2022 ). While curation of data may improve reproducibility, it will not increase the biological relevance to human health effects.…”

The Evolution of Regulatory Toxicology: Where is the Gardener?

“…However, less is known about the data quality from animal studies (see Kolle et al, 28 Adriaens et al, 29 Luechtefeld et al, 30 Barroso et al 31 and Karmaus et al). 32 At present, regulators are more familiar with, and accepting of, the uncertainties from animal studies than from studies involving the use of NAMs. Indeed, in October 2021, Björn Hansen told the European Parliament's environment committee that: "One of the advantages of the current regulatory system is that regulators have a good understanding of the uncertainties associated with animal testing.…”

A network pharmacology study to determine the integrated application of dietary plant-derived natural flavonoids and gut microbiota against nonalcoholic fatty liver disease

“…During drug development, the level of toxicity is of importance factor to be successful as therapeutic agents. The toxicity was veri ed by the following assays: hERG blockers [31], Human Hepato-Toxicity (H-HT) [32], Rat Oral Acute Toxicity [33], Carcinogenicity [34], Eye Corrosion [35], Respiratory Toxicity [36]. The toxicity parameters are assessed by using ADMETlab platform [37].…”