Structure activity relationship (SAR)-based read-across often is an integral part of toxicological safety assessment, and justification of the prediction presents the most challenging aspect of the approach. It has been established that structural consideration alone is inadequate for selecting analogues and justifying their use, and biological relevance must be incorporated. Here we introduce an approach for considering biological and toxicological related features quantitatively to compute a similarity score that is concordant with suitability for a read-across prediction for systemic toxicity. Fingerprint keys for comparing metabolism, reactivity, and physical chemical properties are presented and used to compare these attributes for 14 case study chemicals each with a list of potential analogues. Within each case study, the sum of these nonstructural similarity scores is consistent with suitability for read-across established using an approach based on expert judgment. Machine learning is applied to determine the contributions from each of the similarity attributes revealing their importance for each structure class. This approach is used to quantify and communicate the differences between a target and a potential analogue as well as rank analogue quality when more than one is relevant. A numerical score with easily interpreted fingerprints increases transparency and consistency among experts, facilitates implementation by others, and ultimately increases chances for regulatory acceptance.

Butylated hydroxytoluene (BHT) is a synthetic antioxidant widely used in many industrial sectors. BHT is a well-studied compound for which there are many favorable regulatory decisions. However, a recent opinion by French Agency for Food, Environmental and Occupational Health and Safety (ANSES) hypothesizes a role for BHT in endocrine disruption (ANSES 2021). This opinion is based on observations in mostly rat studies where changes to thyroid physiology is observed. While enzymatic induction of Cytochrome P450 mediated thyroid hormone catabolism has been proposed as a mechanism, a causal relationship has not been proven. Other evidence proposed in the document include a read across argument to butylated hydroxyanisole (BHA), another Community Rolling Action Plan (CoRAP) -listed substance with endocrine disruption concerns. We tested the hypothesis that BHT is an endocrine disruptor by using a Next Generation Risk Assessment (NGRA) method. Four different cell lines: A549, HCC1428, HepG2 and MCF7 were treated with BHT and a series of BHT analogs at 5 different concentrations, RNA was isolated from cell extracts and run on the L1000 gene array platform. A toxicogenomics-based assessment was performed by comparing BHT’s unique genomic signature to a large external database containing signatures of other compounds (including many known endocrine disruptors) to identify if any endocrine disruption-related modes of action (MoAs) are prevalent among BHT and other compounds with similar genomic signatures. In addition, we performed a toxicogenomics-based structure activity relationship (SAR) assessment of BHT and a series of structurally similar analogues to understand if endocrine disruption is a relevant MoA for chemicals that are considered suitable analogs to BHT using the P&G read across framework (Wu et al., 2010). Neither BHT nor any of its analogs connected to compounds that had endocrine activity for estrogens, androgens, thyroid, or steroidogenesis activity.