P30-49 Hazard assessment of endocrine disruption potential using new alternative methods: integration of in vitro thyroid peroxidase inhibition potency and metabolic fate of 3-amino-2,6-dimethylphenol explains its lack of thyroid toxicity in vivo

Endocrine Disruption (ED) is a toxicological hazard of regulatory importance defined by an endocrine mode of action, adverse effects in intact organisms or (sub)populations, and a biologically plausible link between the mode of action and the observed adversity. While the first criterion can be assessed using new alternative methods (NAMs), the others have historically required the use of animal models to contextualize any observed endocrine activities within the toxicokinetic fates of substances. We introduce an integrated hazard assessment using NAMs-generated toxicokinetic and endocrine data to avoid additional animal testing. For this purpose, we determined the toxicokinetic stability of the hair dye 3-amino-2,6-dimethylphenol (A162) using human hepatocytes (systemic metabolism) or keratinocytes (dermal metabolism) with identification of all significant metabolites. QSAR modeling was then applied to predict the potential of A162 or its metabolites for (anti-)estrogenic, anti-androgenic, thyroid- or steroidogenic activities. Thyroid peroxidase (TPO) inhibition was identified as the main effect of concern. The relevant A162 metabolites were then synthesized and tested for TPO inhibition activity using the in vitro tyrosine iodination assay. While the TPO inhibition potency of the parent A162 was in the range of the propylthiouracil standard, phase II mono-conjugation reduced this potency significantly. The di-conjugates failed to inhibit TPO, suggesting that nucleophilic amino and phenol groups are necessary for this activity. The in silico predictions generated for the A162 metabolites were mostly in line with the in vitro data suggesting a potential detoxification role of metabolism. Our data indicate that at systemically available levels of A162 parent and metabolites no TPO inhibition is likely to occur. This is supported by the results of a 13-week OECD 408 study in rats demonstrating that neither the thyroid follicular morphology nor the T3, T4 and TSH hormone levels were biologically altered by up to 630 mg/kg bw/d A162. Together these findings indicate a no relevant hazard from A162 and suggest the validity of our NAMs-based hazard assessment for the determination of ED for regulatory purposes.