EURL ECVAM Recommendation on the 3T3 Neutral Red Uptake Cytotoxicity Assay for Acute Oral Toxicity Testing

Acute oral toxicity is currently being assessed by a suite of refinement test methods based on thetraditional LD50 lethality test and is, besides skin sensitisation, the only remaining animal test requiredunder REACH Annex VII. In view of assessing the use of alternatives for this health endpoint, EURL ECVAMconducted a study on the 3T3 Neutral Red Uptake cytotoxicity test method addressing the method's capacityto support specifically the identification of substances not requiring classification as acute toxicants.Following independent scientific peer review of this study by EURL ECVAM's scientific advisory committee(ESAC) and having considered input from regulators, stakeholders, international partners and the generalpublic, EURL ECVAM concludes that the 3T3 NRU test method may prove a valuable component of a WoE orITS approach for supporting hazard identification and safety assessment in agreement with the EU CLPRegulation and international regulatory schemes implementing the upper threshold of UN GHS Category 4 asthe cut-off for non-classification of substances. In particular, data from the 3T3 NRU assay may constitutean information source within a WoE approach under the provisions of the REACH regulation (Annex XI, 1.2)potentially supporting conclusions on absence of acute oral toxicity of industrial chemicals.