In Vitro Cytotoxicity Test Methods: Background Review Document

November 2006
NIH Publication No. 07-4518

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Complete Volumes 1 and 2

Volume 1 of 2: Sections 1-13 
Volume 2 of 2: Appendices A - Q

Main Body

  • Title Page
  • Front Matter
    Includes: Table of Contents, List of Tables, List of Figures, Acronyms and Abbreviations, Acknowledgments, Preface, Executive Summary
  • Section 1: Introduction and Rationale for the Use of In Vitro Neutral Red Uptake Cytotoxicity Test Methods to Predict Starting Doses for In Vivo Acute Oral Toxicity Testing
  • Section 2: Test Method Protocol Components of the 3T3 and NHK In Vitro NRU Test Methods
  • Section 3: Reference Substances Used for Validation of the 3T3 and NHK NRU Test Methods
  • Section 4: Rodent Acute Oral LD50 Reference Values Used to Assess the Accuracy of the 3T3 AND NHK NRU Test Methods
  • Section 5: 3T3 and NHK NRU Test Method Data and Results
  • Section 6: Accuracy of the 3T3 and NHK NRU Test Methods
  • Section 7: Reliability of the 3T3 and NHK Test Methods
  • Section 8: 3T3 and NHK NRU Test Method Data Quality
  • Section 9: Other Scientific Reports and Reviews of In Vitro Cytotoxicity Test Methods and Their Ability to Predict In Vivo Acute Toxicity and Other Toxic Effects
  • Section 10: Animal Welfare Considerations (Refinement, Reduction, and Replacement)
  • Section 11: Practical Considerations
  • Section 12: References
  • Section 13: Glossary

Appendix A - NICEATM/ECVAM Validation Study Management

Appendix B - Validation Study Test Method Protocols (Phase III)

  • Appendix B1 - Test Method Protocol for the Balb/C 3T3 Neutral Red Uptake Cytotoxicity Test
  • Appendix B2 - Test Method Protocol for the Normal Human Epidermal Keratinocyte Neutral Red Uptake Cytotoxicity Test
  • Appendix B3 - Test Method Protocol for Solubility Determination
  • Appendix B4 - Test Method Procedure for Prequalification of Normal Human Epidermal Keratinocyte Growth Medium (Phase III)

Appendix C - Validation Study Test Method Protocols (Phases IA, IB, and II)

  • Appendix C1 - Test Method Protocol for the Balb/C 3T3 Neutral Red Uptake Cytotoxicity Test (Phase Ia)
  • Appendix C2 - Test Method Protocol for the Normal Human Epidermal Keratinocyte Neutral Red Uptake Cytotoxicity Test (Phase Ia)
  • Appendix C3 - Test Method Protocol for the Balb/C 3T3 Neutral Red Uptake Cytotoxicity Test (Phase Ib)
  • Appendix C4 - Test Method Protocol for the Normal Human Epidermal Keratinocyte Neutral Red Uptake Cytotoxicity Test (Phase Ia)
  • Appendix C5 - Test Method Protocol for the Balb/C 3T3 Neutral Red Uptake Cytotoxicity Test (Phase II)
  • Appendix C6 - Test Method Protocol for the Normal Human Epidermal Keratinocyte Neutral Red Uptake Cytotoxicity Test (Phase II)

Appendix D - SAS Codes

Appendix E - Neutral Red Dye Experiments

Appendix F - Reference Substance Information

Appendix G - Statement of Work

  • Appendix G1 - A Validation Study for In Vitro Basal Cytotoxicity Testing
  • Appendix G2 - Procedures for Acquisition, Preparation, Solubility Testing, and Distribution of Test Chemicals for a Validation Study for In Vitro Basal Cytotoxicity Testing

Appendix H - Candidate Reference Oral LD50 Data

Appendix I - In Vitro Neutral Red Uptake Data

  • Appendix I1 - 3T3 Neutral Red Uptake Reference Substance Data
  • Appendix I2 - Normal Human Epidermal Keratinocyte Neutral Red Uptake Reference Substance Data
  • Appendix I3 - 3T3 Neutral Red Uptake Positive Control (SLS) Data
  • Appendix I4 - Normal Human Epidermal Keratinocyte Neutral Red Uptake Positive Control (SLS) Data

Appendix J - LD50 and Toxicity Category Predictions

  • Appendix J1 - 3T3 NRU Predictions: RC Millimole Regression
  • Appendix J2 - NHK NRU Predictions: RC Millimole Regression
  • Appendix J3 - 3T3 NRU Predictions: RC Rat-Only Millimole Regression
  • Appendix J4 - NHK NRU Predictions: RC Rat-Only Millimole Regression
  • Appendix J5 - 3T3 NRU Predictions: RC Rat-Only Weight Regression
  • Appendix J6 - NHK NRU Predictions: RC Rat-Only Weight Regression
  • Appendix J7 - Comparison of Millimole Regression with Weight Regression Regarding Prediction of Toxicity (LD50) for Low or High Molecular Weight Chemicals

Appendix K - IC50 and LD50 Data for Regressions

  • Appendix K1 - IC50 and LD50 Values Used for Laboratory-Specific Regressions
  • Appendix K2 - IC50 and LD50 Values Used for Combined-Laboratory Regressions
  • Appendix K3 - RC IC50 and LD50 Values for RC Substances with Rat Oral LD50
  • Appendix K4 - Individual Laboratory LD50 Predictions: RC Rat-Only Millimole Regression

Appendix L - Outlier Information

  • Appendix L1 - Outlier Characterization for the 3T3 and NHK NRU Test Methods with the RC Millimole
  • Appendix L2 - Test Method Protocol for the Normal Human Epidermal Keratinocyte Neutral Red Uptake Cytotoxicity Test (Phase Ia)
  • Appendix L3 - Test Method Protocol for the Balb/C 3T3 Neutral Red Uptake Cytotoxicity Test (Phase Ib)

Appendix M - Acute Oral Toxicity Test Guidelines

Appendix N - UDP/ATC Simulation Modeling Results

  • Appendix N1 - UDP Simulation Results Using Starting Doses One Default Dose Lower than the LD50 Predicted by the 3T3 and NHK NRU IC50 and the RC Rat-Only Millimole Regression - 5000 mg/kg Upper Limit Dose
  • Appendix N2 - UDP Simulation Results Using Starting Doses One Default Dose Lower than the LD50 Predicted by the 3T3 and NHK NRU IC50 and the RC Rat-Only Weight Regression - 5000 mg/kg Upper Limit Dose
  • Appendix N3 - ATC Simulation Results Starting at the Next Fixed Dose Below the LD50 Predicted by the 3T3 and NHK NRU IC50 and the RC Rat-Only Millimole Regression - 2000 mg/kg Upper Limit Dose
  • Appendix N4 - ATC Simulation Results Starting at the Next Fixed Dose Below the LD50 Predicted by the 3T3 and NHK NRU IC50 and the RC Rat-Only Weight Regression - 2000 mg/kg Upper Limit Dose

Appendix O - Federal Register Notices

  • 70 FR 14473 - Request for Nominations for an Independent Peer Review Panel to Evaluate In Vitro Testing Methods for Estimating Acute Oral Systemic Toxicity and Request for In Vivo and In Vitro Data
  • 69 FR 61504 - Availability of Updated Standardized In Vitro Cytotoxicity Test Method Protocols for Estimating Acute Oral Systemic Toxicity; Request for Existing In Vivo and In Vitro Acute Toxicity Data
  • 69 FR 11448 - Notice of Availability of Agency Responses to ICCVAM Test Recommendations for the Revised Up-and-Down Procedure for Determining Acute Oral Toxicity and In Vitro Methods for Assessing Acute Systemic Toxicity
  • 66 FR 49686 - Report of the International Workshop on In Vitro Methods for Assessing Acute Systemic Toxicity; Guidance Document on Using In Vitro Data to Estimate In Vivo Starting Doses for Acute Toxicity; Notice of Availability and Request for Public Comment
  • 65 FR 57203 - Notice of an International Workshop on In Vitro Methods for Assessing Acute Systemic Toxicity: Workshop Agenda and Registration Information
  • 65 FR 37400 - Notice of an International Workshop on In Vitro Methods for Assessing Acute Systemic Toxicity; Request for Data and Suggested Expert Scientists

Appendix P - In Vitro Cytotoxicity Test Methods and the High Production Volume (HPV) Challenge Program

Appendix Q - UDP Simulation Results for the RC Rat-Only Millimole Regression Starting at the LD50 Predicted by the 3T3 and NHK NRU IC50 - 5000 mg/kg Upper Limit Dose