Acute Systemic Toxicity

Last Updated: December 6, 2007

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Validated Non-animal Alternatives

Methods	Test Purpose	Validation Authority	OECD TG
In vitro basal cytotoxicity assay: Neutral red uptake (NRU) test with rodent cells (mouse 3T3 fibroblasts) (3T3 NRU assay)	Adjunct to in vivo acute oral toxicity tests for determining starting doses	ICCVAM
In vitro basal cytotoxicity assay: Neutral red uptake (NRU) test with human cells (normal human epidermal keratinocytes (NHK)) (NHK NRU assay)	Adjunct to in vivo acute oral toxicity tests for determining starting doses	ICCVAM

Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) has recommended these two in vitro basal cytotoxicity assays for acute systemic toxicity testing as follows: "Recommended as reduction alternatives to estimate the starting dose in the Up-and-Down Procedure (UDP) and Fixed Dose Procedure (FDP) for assessing acute oral toxicity; to be sent to U.S. agencies for consideration in 2007." The in vitro NRU test methods have not been validated for determining the hazard classification of chemicals. The specific limitations on the uses of these in vitro methods are described in the documents on the ICCVAM website.

The NRU test with mouse 3T3 fibroblasts (3T3 NRU assay) and the NRU test with human cells (NHK NRU assay) are cytotoxicity assays. Neutral red is a dye that is taken up by living cells. Alterations to cell membranes caused by toxic chemicals that are related to cytotoxicity and/or inhibition of cell growth decrease the amount of neutral red taken up by the cells. The protocols and basis for the selection of the assays and cell types are explained in the ICCVAM Background Review Document.

Additional information on in vitro methods being developed for acute systemic toxicity testing can be found in Toxicity Endpoints & Tests: Acute Systemic Toxicity.