|Background Review Document||Recommendations and Agency Responses|
|Test Method Evaluation Report||Validation Study Information|
Recommendations and Agency Responses
In February 2008, ICCVAM forwarded recommendations on the use of in vitro test methods for estimating starting doses for acute oral systemic toxicity tests. ICCVAM recommended that the in vitro basal cytotoxicity test methods tested in a validation study conducted by NICEATM and the European Centre for the Validation of Alternative Methods (ECVAM, now known as EURL ECVAM) should be considered before using animals for acute oral systemic toxicity testing, and that the methods should be used where determined appropriate. Data from the test methods should be used in a weight-of-evidence approach for determining starting doses for in vivo studies. Using these in vitro methods where appropriate is expected to 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 report 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).
The recommendations were communicated to Federal agencies in letters from Dr. Samuel H. Wilson, Acting Director, NIEHS, to each agency head. Links to these letters and responses from the agency heads or their designees can be found below.
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. The in vitro tests evaluated used rodent (mouse fibroblast [3T3]) and human (normal human epidermal keratinocyte [NHK]) cells.
The objectives of the validation study were to:
- 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
Upon conclusion of the study, an independent scientific review panel evaluated the validation status of the 3T3 and NHK basal cytotoxicity test methods in May 2006 for use as adjuncts to in vivo acute oral systemic toxicity tests for the purpose of determining starting doses. ICCVAM concurred with the peer review evaluation 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 (i.e., the Up-and-Down Procedure and the Acute Toxic Class method) to reduce the use of animals required for these methods. Additionally, ICCVAM agreed with the panel that these in vitro cytotoxicity test methods could not be used to determine the hazard classification of chemicals.
The validation study results were used as the basis for the ACuteTox project, which was funded
by the European Union through ECVAM.
The aim of ACuteTox was to develop a battery of in vitro tests sufficiently robust and powerful
enough to replace in vivo tests used for determining the acute toxicity of chemicals and
Visit the ACuteTox website
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 the minimum number of animals to be used in acute toxic class method tests for 203 substances when a number of different options were used to select starting doses. The 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 (34% [288/834]) and the 2000 mg/kg limit dose (38% [315/834] to determine starting doses provided further reductions in minimum animal use.
Schrage A, Hempel K, Schulz M, Kolle SN, van Ravenzwaay B, Landsiedel R. 2011. Refinement and
reduction of acute oral toxicity testing: a critical review of the use of cytotoxicity data. 2011. Altern Lab Anim. 39: 273-295.
View reference on PubMed
Final Background Review Document (BRD): In Vitro Cytotoxicity Test Methods for Estimating Acute Oral Systemic Toxicity (NIH Publication 07–4518)
Transmittal Letters and Agency Responses
- National Institute of Environmental Health Sciences (NIEHS)
- Agency for Toxic Substances and Disease Registry (ATSDR)
- Consumer Products Safety Commission (CPSC)
- Environmental Protection Agency (EPA)
- Food and Drug Administration (FDA)
- National Cancer Institute (NCI)
- National Institutes of Health (NIH)
- National Institute for Occupational Safety and Health (NIOSH)
- National Library of Medicine (NLM)
- Occupational Safety and Health Administration (OSHA)
- U.S. Department of Agriculture
- U.S. Department of Defense
- U.S. Department of Energy
- U.S. Department of the Interior
- U.S. Department of Transportation