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Regul Toxicol Pharmacol. 2016 Nov;81:407-420. doi: 10.1016/j.yrtph.2016.09.028. Epub 2016 Sep 28.

Tiered application of the neutral red release and EpiOcular™ assays for evaluating the eye irritation potential of agrochemical formulations.

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The Dow Chemical Company, Midland, MI, USA. Electronic address:
Dow AgroSciences LLC, Indianapolis, IN, USA.
Dow Chemical Services UK Ltd, Milton Park, Abingdon, Oxon, UK.
The Dow Chemical Company, Midland, MI, USA.


Agrochemical formulations have been underrepresented in validation efforts for implementing alternative eye irritation approaches but represent a significant opportunity to reduce animal testing. This study assesses the utility of the neutral red release assay (NRR) and EpiOcular™ assay (EO) for predicting the eye irritation potential of 64 agrochemical formulations relative to Draize data. In the NRR, formulations with an NRR50 value ≤ 50 mg/mL were categorized as UN GHS Cat 1 and those >250 mg/mL were classified as UN GHS Non Classified (NC). The accuracy, sensitivity, and specificity were 78, 85 and 76% and 73, 85 and 61% for identifying UN GHS 1 and NC formulations, respectively. Specificity was poor for formulations with NRR50 > 50 to ≤250 mg/mL. The EO (ET-40 method) was explored to differentiate formulations that were UN GHS 1/2 and UN GHS NC. The EO resulted in accuracy, sensitivity, and specificity of 65%, 58% and 75% for identifying UN GHS NC formulations. To improve the overall performance, the assays were implemented using a tiered-approach where the NRR was run as a first-tier followed by the EO. The tiered-approach resulted in improved accuracy (75%) and balanced sensitivity (73%) and specificity (77%) for distinguishing between irritating and non-irritating agrochemical formulations.


Agrochemical formulations; EpiOcular assay; Eye irritation; In vitro; Neutral red release assay; Ocular irritation

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