The present publication surveys several applications of in silico (i.e., computational) toxicology approaches across different industries and institutions. It highlights the need to develop standardized protocols when conducting toxicity-related predictions. This contribution articulates the information needed for protocols to support in silico predictions for major toxicological endpoints of concern (e.g., genetic toxicity, carcinogenicity, acute toxicity, reproductive toxicity, developmental toxicity) across several industries and regulatory bodies. Such novel in silico toxicology (IST) protocols, when fully developed and implemented, will ensure in silico toxicological assessments are performed and evaluated in a consistent, reproducible, and well-documented manner across industries and regulatory bodies to support wider uptake and acceptance of the approaches. The development of IST protocols is an initiative developed through a collaboration among an international consortium to reflect the state-of-the-art in in silico toxicology for hazard identification and characterization. A general outline for describing the development of such protocols is included and it is based on in silico predictions and/or available experimental data for a defined series of relevant toxicological effects or mechanisms. The publication presents a novel approach for determining the reliability of in silico predictions alongside experimental data. In addition, we discuss how to determine the level of confidence in the assessment based on the relevance and reliability of the information.

The concept of thresholds in genotoxicity has been open for debate in the last decades. The micronucleus (MN) test contributed to a large extent in understanding the dose-response relationship for aneugens and clastogens. The threshold concept for aneuploidy is well accepted by the scientific community based on the data and for mechanistic reasons. The concept of threshold for clastogens is still challenging. Acceptance is based on a case-by-case basis together with thorough mechanistic understanding of the different steps from the mutagen-target interactions to MN formation for this class of genotoxicants. This review summarises the significant achievements in the assessment of threshold for genotoxins using the MN test and concludes with an overview of knowledge gaps and recommendations.

In recent years, experimental evidence has accumulated that supports the existence of sublinear dose-response relationships at low doses of DNA reactive mutagens. However, creating the in vivo data necessary to allow for a more detailed dose-response modeling with the currently available tools might not always be practical. The purpose of the current work was to evaluate the utility of the Pig-a gene mutation assay to rapidly identify dose response relationships for direct acting genotoxicants. The induction of mutations in the peripheral blood of rats was evaluated following 28 days of exposure down to low doses of the direct acting alkylating agents ethyl methane sulfonate (EMS) and ethylnitrosourea (ENU). Using statistical modeling based on the 28-day studies, a threshold for mutation induction for EMS was estimated to be 21.9 mg/kg, whereas for the more potent ENU the threshold was estimated to be 0.88 mg/kg. Comparing mutation frequencies from acute and sub-chronic dosing indicated less than additive dose-response relationships, further confirming the possibility of a thresholded dose-response relationship for both compounds. In conclusion, the work presented provides evidence that the Pig-a assay might be a practical alternative to other in vivo mutation assays when assessing dose-response relationships for direct acting mutagens and that an experimental approach using fractionated dosing could be used to substantiate a biological mechanism responsible for the observation of a sub-linear dose-response relationship.

N-Ethyl-N-nitrosourea (ENU) was evaluated as part of the Stage III trial for the rat Pig-a gene mutation assay. Groups of six- to eight-week-old male Sprague Dawley (SD) or Fischer 344 (F344) rats were given 28 daily doses of the phosphate buffered saline vehicle, or 2.5, 5, or 10 mg/kg ENU, and evaluated for a variety of genotoxicity endpoints in peripheral blood, spleen, liver, and colon. Blood was sampled predose (Day-1) and at various time points up to Day 57. Pig-a mutant frequencies were determined in total red blood cells (RBCs) and reticulocytes (RETs) as RBC(CD592-) and RET(CD592-) frequencies. Consistent with the results from a reference laboratory, RBC(CD592-) and RET(CD592-) frequencies increased in a dose and time-dependent manner, producing significant increases at all doses by Day 15, with similar frequencies seen in both rat strains. ENU also induced small but significant increases in % micronucleated RETs on Days 4 and 29. No significant increases in micronuclei were seen in the liver or colon of the ENU-treated SD rats. Hprt and Pig-a lymphocyte mutation assays conducted on splenocytes from Day 56 F344 rats detected two- to fourfold stronger responses for Hprt than Pig-a mutations. Results from the in vivo Comet assay in SD rats at Day 29 showed generally weak increases in DNA damage in all tissues evaluated. The results with ENU indicate that the Pig-a RET and RBC assays are reproducible, transferable, and complement other genotoxicity endpoints that could potentially be integrated into 28-day repeat dose rat studies.

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