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Validation Study of In Vitro Cytotoxicity Test Methods

ICCVAM has recommended that in vitro basal cytotoxicity test methods should be considered before using animals in acute oral systemic toxicity testing.

Data from these test methods should be used in a weight-of-evidence approach in determining starting doses for in vivo studies. Proper use of these methods can reduce the number of animals required for each toxicity test.

ICCVAM determined that the evaluated in vitro test methods are not accurate enough to replace animals for regulatory hazard classification purposes.

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 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 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.

Study Objectives

  • Further standardize and improve the in vitro basal cytotoxicity protocols to maximize test reliability (intra- and inter-laboratory reproducibility).
  • Assess the accuracy of the standardized in vitro cytotoxicity test methods for estimating rodent oral LD50 values across the five GHS acute oral systemic toxicity categories, as well as unclassified toxicities.
  • Estimate the reduction and refinement in animal use achievable from using the in vitro basal cytotoxicity test methods to identify starting doses for in vivo acute oral systemic toxicity tests.
  • Develop databases containing high quality data from in vivo acute oral lethality and in vitro basal cytotoxicity tests. These databases would be used to support the investigation of other test methods necessary to improve predictions of in vivo acute oral lethality.

Related Documents

Conclusions and Regulatory Acceptance

An independent scientific review panel gathered in May 2006. They 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, 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 agreed with both conclusions.

The validation study was used as the basis for the European Union's ACuteTox project. The aim was to develop a battery of in vitro tests sufficiently robust and powerful to replace in vivo tests used to determine the acute toxicity of chemicals and products.

Use of the in vitro starting dose procedure was accepted internationally in 2010 via OECD Guidance Document 129.

Application to the Acute Toxic Class Method

A publication after 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 using the 3T3 test to determine starting doses reduced the minimum number of animals needed for testing 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 even more reductions in animal use.

Transmittal Letters and Agency Responses