ICCVAM recommends that in vitro basal cytotoxicity test methods should be considered before using animals for acute oral systemic toxicity testing and used where determined appropriate. Data from these test methods should be used in a weight-of-evidence approach for determining starting doses for in vivo studies. Appropriate use of the methods can reduce the number of animals required for each toxicity test. ICCVAM concluded that the in vitro test methods are not sufficiently accurate to replace animals for regulatory hazard classification purposes.
ICCVAM's recommendations are provided in the ICCVAM Test Method Evaluation Report: In Vitro Cytotoxicity Test Methods for Estimating Starting Doses for Acute Oral Systemic Toxicity Tests (NIH Publication No. 07-4519). Letters communicating the ICCVAM recommendations to federal agencies and responses from the agencies can be found below.
The BALB/c 3T3 neutral red uptake and the normal human keratinocyte (NHK) neutral red uptake assays were tested in a validation study conducted by NICEATM and the European Centre for the Validation of Alternative Methods (ECVAM, now known as EURL ECVAM). The NICEATM/ECVAM In Vitro Cytotoxicity Validation Study generated in vitro cytotoxicity data to predict rodent in vivo LD50 values and starting doses for acute oral systemic toxicity test methods. These in vitro tests used rodent (mouse fibroblast [3T3]) and human (NHK) cells.
IC50 values were generated by testing 72 chemicals in the in vitro basal cytotoxicity test methods. The IC50 values were then used to predict starting doses for acute oral systemic toxicity tests such as the up-and-down procedure and the acute toxic class method, using IC50-LD50 regression formulas developed from the Registry of Cytotoxicity. The IC50 values and human lethal and sublethal blood concentrations were used by ECVAM to develop a preliminary human prediction model.
The objectives of the validation study were to:
An independent scientific review panel convened in May 2006 reviewed the study and concluded that these in vitro cytotoxicity test methods should be considered for use in a weight-of-evidence approach to determine starting doses for acute oral systemic toxicity test methods, thereby reducing overall animal use requirements. However, the panel concluded that these in vitro cytotoxicity test methods could not be used to determine the hazard classification of chemicals. ICCVAM concurred with both of the panel's conclusions.
The validation study was used as the basis for the European Union's ACuteTox project. The aim of ACuteTox was to develop a battery of in vitro tests sufficiently robust and powerful to replace in vivo tests used for determining the acute toxicity of chemicals and products.
A publication subsequent to the validation study confirmed that animal use could be reduced by using the 3T3 in vitro basal cytotoxicity test to determine the starting dose for the acute toxic class method for acute oral toxicity. Schrage et al. (2011) estimated that use of the 3T3 test to determine starting doses reduced the minimum number of animals needed for the acute toxic class tests by 18% (150/834) compared with use of the default starting dose. Use of expert judgment and the 2000 mg/kg limit dose to determine starting doses provided further reductions in minimum animal use.