National Toxicology Program

National Toxicology Program
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Validation Study of In Vitro Cytotoxicity Test Methods

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.

Background

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. Read More

Validation Study Objectives
  • Further standardize and optimize 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 United Nations Globally Harmonized System of Classification and Labelling of Chemicals categories of acute oral systemic toxicity, 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 that can be used to support the investigation of other in vitro test methods necessary to improve the prediction of in vivo acute oral lethality

Conclusions of the Independent Peer Review

Peer review panel report

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.

Application to the Acute Toxic Class Method

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. Read More

Related Documents
Transmittal Letters and Agency Responses
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