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Background Review Document (BRD)

In Vitro Cytotoxicity Test Methods for Estimating Acute Oral Systemic Toxicity

November 2006
NIH Publication No. 07-4518

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Volume 1 of 2: Sections 1-13

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View sections 3 to 5 [PDF]
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View sections 8 to 13 [PDF]

 Volume 2 of 2: Appendices A - Q [PDF]

Table of Contents

  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

    • Historical Background and Rationale for the Use of In Vitro Cytotoxicity Assays to Predict Starting Doses for In Vivo Acute Oral Toxicity Tests
      • The Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) Program
      • An International Evaluation of Selected In Vitro Toxicity Test Systems for Predicting Acute Systemic Toxicity
      • The Registry of Cytotoxicity (RC)
      • The ZEBET Initiative to Reduce Animal Use
      • The International Workshop on In Vitro Methods for Assessing Acute Systemic Toxicity
      • The NICEATM/ECVAM In Vitro NRU Cytotoxicity Validation Study

    • Regulatory Rationale and Applicability for the Use of In Vitro Cytotoxicity Test Methods to Predict Starting Doses for Acute Oral Toxicity Testing in Rodents
      • Current Regulatory Testing Requirements for Acute Oral Toxicity
      • Intended Regulatory Uses for In Vitro Cytotoxicity Test Methods
      • Similarities and Differences in the Endpoints of In Vitro Cytotoxicity Test Methods and Rodent Acute Oral Toxicity Test Methods
      • Use of In Vitro Cytotoxicity Test Methods in the Overall Strategy of Hazard Assessment

    • Scientific Basis for the In Vitro NRU Test Methods
      • Purpose and Mechanistic Basis of In Vitro NRU Test Methods
      • Similarities and Differences in the Modes/Mechanisms of Action for In Vitro NRU Test Methods Compared with the Species of Interest
      • Range of Substances Amenable to the In Vitro NRU Test Methods


  2. TEST METHOD PROTOCOL COMPONENTS OF THE 3T3 AND NHK IN VITRO NRU TEST METHODS

    • Basis for Selection of In Vitro NRU Cytotoxicity Test Methods
      • Guidance Document Rationale for Selection of In Vitro NRU Cytotoxicity Test Methods
      • Guidance Document Rationale for Selection of Cell Types

    • Overview of the 3T3 and NHK NRU Test Methods
      • The 3T3 NRU Test Method
      • The NHK NRU Test Method
      • Measurement of NRU in the 3T3 and NHK Test Methods

    • Descriptions and Rationales of the 3T3 and NHK NRU Test Methods
      • Materials, Equipment, and Supplies
      • Reference Substance Concentrations/Dose Selection
      • NRU Endpoints Measured
      • Duration of Reference Substance Exposure
      • Known Limits of Use
      • Basis of the Response Assessed
      • Appropriate Vehicle, Positive, and Negative Controls
      • Acceptable Ranges of Control Responses
      • Nature of Experimental Data Collected
      • Data Storage Media
      • Measures of Variability
      • Methods for Analyzing NRU Data
      • Decision Criteria for Classification of Reference Substances
      • Information and Data Included in the Test Report

    • Proprietary Components of the In Vitro NRU Test Methods

    • Basis for the Number of Replicate and Repeat Experiments for the 3T3 and NHK NRU Test Methods

    • Basis for Modifications to the 3T3 and NHK NRU Test Method Protocols
      • Phase Ia: Laboratory Evaluation Phase
      • Phase Ib: Laboratory Evaluation Phase
      • Phase II: Laboratory Qualification Phase
      • Phase III: Laboratory Testing Phase

    • Differences Between the 3T3 and NHK NRU Protocols for the Validation Study and the Guidance Document Standard Protocols

    • Overview of the Solubility Protocol

    • Basis of the Solubility Protocol
      • Initial Solubility Protocol Development
      • Basis for Modification of the Phase II Protocol

    • Components of the Solubility Protocol
      • Medium, Supplies, and Equipment Required
      • Data Collection
      • Variability in Solubility Measurement
      • Solubility Issues During the Testing of the Reference Substances
      • Analysis of Solubility Data

    • Summary

  3. REFERENCE SUBSTANCES USED FOR VALIDATION OF THE 3T3 AND NHK NRU TEST METHODS

    • Rationale for the 72 Reference Substances Selected for Testing
      • Reference Substance Selection Criteria
      • Candidate Reference Substances
      • Selection of Reference Substances for Testing

    • Characteristics of the Selected Reference Substances
      • Source Databases Represented by the Selected Reference Substances
      • Chemical Classes Represented by the Selected Reference Substances
      • Product/Use Classes Represented by the Selected Reference Substances
      • Toxicological Characteristics of the Selected Reference Substances
      • Selection of Reference Substances for Testing in Phases Ib and II
      • Unsuitable and Challenging Reference Substances

    • Reference Substance Procurement, Coding, and Distribution
      • Exceptions

    • Reference Substances Recommended by the Guidance Document

    • Summary

  4. RODENT ACUTE ORAL LD50 REFERENCE VALUES USED TO ASSESS THE ACCURACY OF THE 3T3 AND NHK NRU TEST METHODS

    • Methods Used to Obtain Rodent Acute Oral LD50 Reference Values
      • Identification of Candidate Rodent Acute Oral LD50 Reference Data
      • Criteria Used to Select Candidate Rodent Acute Oral Data for Determination of LD50 Reference Values

    • Final Rodent Acute Oral LD50 Reference Values

    • Relevant Toxicity Information for Humans

    • Accuracy and Reliability of the Rodent Acute Oral LD50 Reference Values
      • Variability Among the Acceptable LD50 Values
      • Comparison of Rodent Acute Oral LD50 Reference Values with the Corresponding RC LD50 Values
      • Comparison of the Variability Among Acceptable LD50 Values to Those Obtained in Other Studies

    • Summary


  5. 3T3 AND NHK NRU TEST METHOD DATA AND RESULTS

    • Study Timeline and Participating Laboratories
      • Statements of Work (SOW) and Protocols
      • Study Timeline
      • Participating Laboratories

    • Coded Reference Substances and GLP Guidelines
      • Coded Reference Substances
      • Lot-to-Lot Consistency of Reference Substances
      • Adherence to GLP Guidelines

    • 3T3 and NHK NRU Test Method Protocols
      • Phase Ia: Laboratory Evaluation Phase
      • Phase Ib: Laboratory Evaluation Phase
      • Phase II: Laboratory Qualification Phase
      • Phase III: Main Validation Phase

    • Data Used to Evaluate Test Method Accuracy and Reliability
      • PC Data
      • Reference Substance Data

    • Statistical Approaches to the Evaluation of 3T3 and NHK Data
      • Statistical Analyses for Phase Ia Data
      • Statistical Analyses for Phase Ib Data
      • Statistical Analyses for Phase II Data
      • Statistical Analyses for Phase III Data
      • Summary of the Data Used for Statistical Analyses

    • Summary of NRU Test Results

    • Availability of Data

    • Solubility Test Results
      • Solubility Data
      • Solubility and Volatility Effects in the Cytotoxicity Tests

    • Summary


  6. ACCURACY OF THE 3T3 AND NHK NRU TEST METHODS

    • Accuracy of the 3T3 and NHK NRU Test Methods for Predicting Rodent Acute Oral Toxicity
      • Linear Regression Analyses for the Prediction of Rat Acute Oral LD50 Values from In Vitro IC50 Value
      • Comparison of Combined-Laboratory 3T3 and NHK Regressions to the RC Millimole Regression

    • Analysis of Outlier Substances for the RC Millimole Regression
      • Identification of Outlier Substances
      • Evaluation of Outlier Substances

    • Improving the Prediction of In Vivo Rat Oral LD50 Values from In Vitro IC50 Data
      • The RC Rat-Only Millimole Regression
      • The RC Rat-Only Weight Regression

    • Accuracy of the 3T3 and NHK NRU Test Methods for Predicting GHS Acute Oral Toxicity Category
      • Prediction of GHS Acute Oral Toxicity Category by the 3T3 and NHK NRU Test Methods Using the RC Millimole Regression
      • Prediction of GHS Acute Oral Toxicity Category by the 3T3 and NHK NRU Test Methods Using the RC Rat-Only Millimole Regression
      • Prediction of GHS Acute Oral Toxicity Category by the 3T3 and NHK NRU Test Methods Using the RC Rat-Only Weight Regression
      • Summary of the Regressions Evaluated

    • Correlation of NRU Concentration-Response Slope with Rat Lethality Dose-Response Slope

    • Strengths and Limitations of the In Vitro Cytotoxicity Test Methods with the IC50–LD50 Regressions for Prediction of Rodent Acute Oral Toxicity
      • In Vitro Cytotoxicity Methods
      • Use of Mole-Based vs. Weight-Based Regressions for the Prediction of Toxicity for Low and High Molecular Weight Substances

    • Salient Issues of Data Interpretation

    • Comparison of NRU Test Results to Established Performance Standards

    • Summary


  7. RELIABILITY OF THE 3T3 AND NHK TEST METHODS

    • Reference Substances Used to Determine the Reliability of the 3T3 and NHK Test Methods

    • Reproducibility Analyses for the 3T3 and NHK Test Methods
      • Comparison of Laboratory-Specific IC50-LD50 Linear Regression Analyses to the Mean Laboratory Regression
      • ANOVA Results for the 3T3 and NHK Test Methods
      • CV Results for the 3T3 and NHK Test Methods
      • Comparison of Maximum to Minimum IC50 Values Using Laboratory Means
      • Comparison of Maximum:Minimum IC50 Ratios with the Maximum:Minimum LD50 Ratios
      • Normalization of Reference Substance IC50 Values Using SLS IC50 Values

    • Historical Positive Control Data
      • ANOVA and Linear Regression Results for the 3T3 NRU Test Method
      • ANOVA and Linear Regression Results for the NHK NRU Test Method

    • Laboratory Concordance for Solvent Selection

    • Correlation of NRU Concentration-Response Slope with Rat

    • Summary


  8. 3T3 AND NHK NRU TEST METHOD DATA QUALITY

    • Compliance with Good Laboratory Practice Regulations
      • Guidelines Followed for Cytotoxicity Testing
      • Quality Assurance (QA) for NRU Cytotoxicity Test Data
      • Guidelines Followed for Rodent Acute Oral LD50 Data Collection

    • Results of Data Quality Audits
      • QA Statements
      • QA Statements from the Laboratories

    • Effect of Deviations or Non-compliance with GLPs
      • Laboratory Error Rates
      • Failure Rates for Definitive and PC Tests
      • Intralaboratory Reproducibility
      • Prediction of GHS Acute Oral Toxicity Categories

    • Availability of Laboratory Notebooks

    • Summary


  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

    • Relevant Studies
      • Correlation of NRU Cytotoxicity Values with Rodent Lethality
      • Use of In Vitro Cytotoxicity Data to Reduce the Use of Animals in Acute Oral Toxicity Testing
      • Other Evaluations of 3T3 or NHK NRU Methods

    • Independent Scientific Reviews
      • In Vitro Acute Toxicity Testing for the Classification and Labeling of Chemicals
      • Use of In Vitro Cytotoxicity Data for Estimation of Starting Doses for Acute Oral Toxicity Testing
      • Validation of 3T3 NRU Assay for Phototoxicity

    • Studies Using In Vitro Cytotoxicity Test Methods with Established Performance Standards
      • Guidance Document (ICCVAM 2001b)
      • King and Jones (2003)
      • A-Cute-Tox Project: Optimization and Pre-Validation of an In Vitro Test Strategy for Predicting Human Acute Toxicity (Clemedson 2005)

    • Summary


  10. ANIMAL WELFARE CONSIDERATIONS (REFINEMENT, REDUCTION, AND REPLACEMENT)

    • Use of 3T3 and NHK NRU Test Methods to Predict Starting Doses for Rodent Acute Oral Toxicity Assays

    • Reduction and Refinement of Animal Use for the UDP
      • In Vitro Testing Using the UDP
      • Computer Simulation Modeling of the UDP
      • Animal Savings in the UDP When Using 3T3- and NHK-Based Starting Doses
      • Refinement of Animal Use for the UDP When Using 3T3- and NHK-Based Starting Doses
      • Accuracy of the UDP Outcomes Using the IC50-Based Starting Doses

    • Reduction and Refinement of Animal Use in the ATC Method
      • In Vivo Testing Using the ATC Method
      • Computer Simulation Modeling of the ATC Method
      • Animal Savings for the ATC Method When Using 3T3- and NHK-Based Starting Doses
      • Refinement of Animal Use in the ATC Method When Using 3T3- and NHK-Based Starting Doses
      • Accuracy of the ATC Method Outcomes Using IC50-Based Starting Doses

    • The Impact of Accuracy on Animal Savings

    • The Impact of Prevalence on Animal Savings

    • Summary


  11. PRACTICAL CONSIDERATIONS

    • Transferability of the 3T3 and NHK NRU Test Methods
      • Facilities and Major Fixed Equipment
      • Availability of Other Necessary Equipment and Supplies
      • Problems Specific to the NHK NRU Test Method

    • 3T3 and NHK Test Method Training Considerations
      • Required Training and Expertise
      • Training Requirements to Demonstrate Proficiency
      • Personnel Needed to Perform the In Vitro NRU Test Methods

    • Cost Considerations
      • 3T3 and NHK NRU Test Methods
      • Rodent Acute Oral Toxicity Testing

    • Time Considerations for Performing the 3T3 and NHK NRU Tests
      • The 3T3 NRU Test Method
      • The NHK NRU Test Method
      • Prequalification of NHK Medium
      • In Vivo Testing
      • The Limit Test

    • Summary

  12. REFERENCES

  13. GLOSSARY

APPENDICES

APPENDIX A - NICEATM/ECVAM VALIDATION STUDY MANAGEMENT

APPENDIX B - VALIDATION STUDY TEST METHOD PROTOCOLS (PHASE III)

  1. Test Method Protocol for the BALB/c 3T3 Neutral Red Uptake (NRU) Cytotoxicity Test

  2. Test Method Protocol for the Normal Human Epidermal Keratinocyte (NHK) Neutral Red Uptake Cytotoxicity Test

  3. Test Method Protocol for Solubility Determination (Phase III)

  4. 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)

  1. Test Method Protocol for the BALB/c 3T3 Neutral Red Uptake Cytotoxicity Test (Phase Ia)

  2. Test Method Protocol for the Normal Human Epidermal Keratinocyte (NHK) Neutral Red Uptake Cytotoxicity Test (Phase Ia)

  3. Test Method Protocol for the BALB/c 3T3 Neutral Red Uptake Cytotoxicity Test (Phase Ib)

  4. Test Method Protocol for the Normal Human Epidermal Keratinocyte (NHK) Neutral Red Uptake Cytotoxicity Test (Phase Ib)

  5. Test Method Protocol for the BALB/c 3T3 Neutral Red Uptake Cytotoxicity Test (Phase II)

  6. Test Method Protocol for the Normal Human Epidermal Keratinocyte (NHK) Neutral Red Uptake Cytotoxicity Test (Phase II)

APPENDIX D - SAS CODES

  1. SAS Code for ANOVA and Contrasts

  2. SAS Code for Regression Comparisons

APPENDIX E - NEUTRAL RED DYE EXPERIMENTS

  1. Institute for In Vitro Sciences (IIVS) Assessment of Protocol Variables in the NICEATM/ECVAM Evaluation of Cytotoxicity Assays

  2. Neutral Red (NR) Dye Experiments - 3T3 Cells - IIVS

  3. Neutral Red (NR) Dye Experiments - NHK Cells - IIVS

  4. Neutral Red (NR) Dye Experiments - 3T3 Cells - ECBC

APPENDIX F - REFERENCE SUBSTANCE INFORMATION

  1. NRU Test Information of the 72 Reference Substances

  2. Chemical, Physical and Biological Information from the Literature for the 72 Reference Substances

  3. Candidate Reference Substances

APPENDIX G - STATEMENT OF WORK (SOW)

  1. A Validation Study For In Vitro Basal Cytotoxicity Testing

  2. Procedures for Acquisition, Preparation, Solubility Testing, and Distribution of Test Chemicals for a Validation Study for In Vitro Basal Cytotoxicity Testing

APPENDIX H - RAT AND MOUSE ORAL LD50 DATABASE

  1. Rat and Mouse Oral LD50 Database

  2. Evaluation of the Candidate Reference Data

APPENDIX I - IN VITRO NRU DATA

  1. 3T3 NRU Reference Substance Data

  2. NHK NRU Reference Substance Data

  3. 3T3 NRU Positive Control (SLS) Data

  4. NHK NRU Positive Control (SLS) Data

APPENDIX J - LD50 AND TOXICITY CATEGORY PREDICTIONS

  1. 3T3 NRU Predictions: RC Millimole Regression

  2. NHK NRU Predictions: RC Millimole Regression

  3. 3T3 NRU Predictions: RC Rat-Only Millimole Regression

  4. NHK NRU Predictions: RC Rat-Only Millimole Regression

  5. 3T3 NRU Predictions: RC Rat-Only Weight Regression

  6. NHK NRU Predictions: RC Rat-Only Weight Regression

  7. 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

  1. IC50 and LD50 Values Used for Laboratory-Specific Regressions

  2. IC50 and LD50 Values Used for Combined-Laboratory Regressions

  3. RC IC50 and LD50 Values for RC Substances with Rat Oral LD50 Data

  4. Individual Laboratory LD50 Predictions: RC Rat-Only Millimole Regression

APPENDIX L - OUTLIER INFORMATION

  1. Outlier Characterization for the 3T3 and NHK NRU Test Methods with the RC Millimole Regression

  2. Discordant Substances for GHS Toxicity Category Predictions Using the 3T3 and NHK NRU Test Methods and RC Rat-Only Regressions

  3. Analysis of Outliers by Halle (1998, 2003) for the RC Millimole Regression

APPENDIX M - ACUTE SYSTEMIC TOXICITY TEST GUIDELINES

  1. OECD UDP Test Guideline

  2. EPA UDP Test Guideline

  3. OECD ATC Test Guideline

  4. OECD FDP Test Guideline

  5. OECD Guidance on Acute Oral Toxicity Testing

APPENDIX N - UDP/ATC SIMULATION MODELING RESULTS

  1. 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

  2. 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

  3. 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

  4. ATC Simulation Results Starting at the Next Fixed Dose Below the LD50 Predicted by the 3T3 and NHK IC50 and the RC Rat-Only Weight Regression - 2000 mg/kg Upper Limit Dose

APPENDIX O - FEDERAL REGISTER NOTICES

  1. 70FR14473 - 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

  2. 69FR61504 - 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

  3. 69FR11448 - Notice of the 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

  4. 66FR49686 - 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

  5. 65FR57203 - Notice of an International Workshop on In Vitro Methods for Assessing Acute Systemic Toxicity, co-sponsored by NIEHS, NTP and the U.S. Environmental Protection Agency (EPA): Workshop Agenda and Registration Information

  6. 65FR37400 - Notice of an International Workshop on In Vitro Methods for Assessing Acute Systemic Toxicity, co-sponsored by NIEHS, NTP and the U.S. Environmental Protection Agency (EPA): Request for Data and Suggested Expert Scientists

APPENDIX P - IN VITRO CYTOTOXICITY TEST METHODS AND THE HIGH PRODUCTION VOLUME (HPV) CHALLENGE PROGRAM

  1. Supplemental Acute Toxicity Protocol

  2. Office of Pollution Prevention and Toxics (OPPT) Letters to Manufacturers/Importers

APPENDIX Q - ADDITIONAL UDP SIMULATION MODELING RESULTS

  1. 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

  2. UDP Simulation Results for the RC Rat-Only Weight Regression Starting at the LD50 Predicted by the 3T3 and NHK NRU IC50 - 5000 mg/kg Upper Limit

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