Validated Non-animal Alternatives
The OECD Test Guideline (TG) for Acute Oral Toxicity (TG 401) was deleted in 2001. It has been replaced primarily with reduction test methods. The two in vitro cytotoxicity methods presented here are not replacement methods, but rather supplemental methods for reducing the number of animals used in the in vivo assays.
The Neutral Red uptake (NRU) test with mouse 3T3 fibroblast cells (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. Links to the specific protocols recommended by ICCVAM are provided in the table above.
ICCVAM has recommended the 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 US agencies for consideration in 2007." The in vitro Neutral Red uptake (NRU) test methods were not determined as valid for determining the hazard classification of chemicals. The specific limitations on the uses of these in vitro methods are described in documents on the ICCVAM website.
US agencies responded to the ICCVAM recommendation in 2008. Regulatory agencies that concurred with the ICCVAM recommendation include: CPSC and EPA. As an example, recommendations by the CPSC were stated a follows:
"The staff recommends that the Commission accept the ICCVAM recommendations that the in vitro basal cytotoxicity test methods should be considered and used where appropriate as part of a weight-of-evidence approach to estimate the starting does for acute oral in vivo toxicity test methods but not to predict acute oral toxicity for the purpose of regulatory hazard classification. These alternative in vitro test methods encourage the reduction, refinement, or replacement of animals in testing and the data indicate that the methods are scientifically valid methods."
The FDA concurred with ICCVAM recommendations, but indicated they have no applicable acute toxicity test recommendations in their guidelines. A letter from OSHA indicates that this agency does not recommend test methods to manufacturers that submit data to them, but that they would accept data submitted using these methods. The DOT stated that: "Compliance with the HMR [DOT's Hazardous Materials Regulations] is the shipper's responsibility (49 CFR 173.22). The HMR do not preclude the use of in vitro cytotoxicity test methods..." However, DOT did not indicate any effort would be made to notify or recommend the use of these validated non-animal methods to manufacturers or shippers responsible for safety testing.
ECVAM has not provided an endorsement of the in vitro basal cytotoxicity test methods at this time. A draft OECD TG, "Using Cytotoxicity Tests to estimate Starting Doses for Acute Oral Systemic Toxicity Tests," is under review for these test methods.
The OECD Guidance Document (GD) 129, "Guidance Document on Using Cytotoxicity Tests to estimate Starting Doses for Acute Oral Systemic Toxicity Tests," was adopted in 2010.
A review of 3Rs approaches for acute systemic toxicity testing is available in this article in Toxicological Sciences.
Additional information on in vitro methods being developed for acute systemic toxicity testing can be found on AltTox at Toxicity Endpoints & Tests: Acute Systemic Toxicity.
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