In Vitro Methods for Assessing
Acute Systemic Toxicity:

Draft Background Review Document (BRD)
March 17, 2006



Note: This information is distributed solely for the purpose of pre-dissemination peer review under applicable information quality guidelines. It has not been formally disseminated by NIH. It does not represent and should not be construed to represent any NIH determination or policy.

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APPENDICES

  • Appendix A: NICEATM/ECVAM Validation Study Management [PDF]

  • Appendix B: Validation Study Test Method Protocols (Phase III) [PDF]
    1. Test Method Protocol for the BALB/c 3T3 Neutral Red Uptake (NRU) Cytotoxicity Test [PDF]
    2. Test Method Protocol for the Normal Human Epidermal Keratinocyte (NHK) Neutral Red Uptake (NRU) Cytotoxicity Test [PDF]
    3. Test Method Protocol for Solubility Determination (Phase III) [PDF ] Chemicals
    4. Test Method Procedure for Prequalification of Normal Human Epidermal Keratinocyte Growth Medium (Phase III) [PDF]

  • Appendix C: Validation Study Test Method Protocols (Phases Ia, Ib, and II) [PDF]
    1. Test Method Protocol for the BALB/c 3T3 Neutral Red Uptake Cytotoxicity Test (Phase Ia) [PDF]
    2. Test Method Protocol for the Normal Human Epidermal Keratinocyte (NHK) Neutral Red Uptake Cytotoxicity Test (Phase Ia) [PDF]
    3. Test Method Protocol for the BALB/c 3T3 Neutral Red Uptake Cytotoxicity Test (Phase Ib) [PDF]
    4. Test Method Protocol for the Normal Human Epidermal Keratinocyte (NHK) Neutral Red Uptake Cytotoxicity Test (Phase Ib) [PDF]
    5. Test Method Protocol for the BALB/c 3T3 Neutral Red Uptake Cytotoxicity Test (Phase II) [PDF]
    6. Test Method Protocol for the Normal Human Epidermal Keratinocyte (NHK) Neutral Red Uptake Cytotoxicity Test (Phase II) [PDF]

  • Appendix D: Relevant Publications [PDF]
    1. Guidance Document on Using In Vitro Data to Estimate In Vivo Starting Doses for Acute Toxicity. NIH Publication No. 01-4500 [PDF]
    2. Spielmann H, Genschow E, Liebsch M, Halle W. 1999. Determination of the starting dose for acute oral toxicity (LD50) testing in the up and down procedure (UDP) from cytotoxicity data. Altern Laboratory Anim 27:957-966. [Reference Only]

  • Appendix E: Neutral Red Dye Experiments [PDF]
    1. Assessment of Protocol Variables in the NICEATM/ECVAM Evaluation of Cytotoxicity Assays [PDF]
    2. Neutral Red (NR) Dye Experiments - 3T3 Cells - IIVS [PDF]
    3. Neutral Red (NR) Dye Experiments - NHK Cells - IIVS [PDF]
    4. Neutral Red (NR) Dye Experiments - 3T3 Cells - ECBC [PDF]

  • Appendix F: Reference Substances Information[PDF]
    1. NRU Test Information for the 72 Reference Chemicals
    2. Chemical, Physical, and Biological Information from the Literature for the 72 Reference Chemicals

  • Appendix G: Statement of Work (SOW) [PDF]
    1. A Validation Study For In Vitro Basal Cytotoxicity Testing [PDF]
    2. Procedures for Acquisition, Preparation, Solubility Testing, and Distribution of Test Chemicals for a Validation Study for In Vitro Basal Cytotoxicity Testing [PDF]

  • Appendix H: Rat and Mouse Oral LD50 Database [PDF]
    1. Rat and Mouse Oral LD50 Database [PDF]
    2. Evaluation of the Candidate Reference Data [PDF]

  • Appendix I: In Vitro NRU Data [PDF]
    1. 3T3 Test Chemical Data [PDF]
    2. NHK Test Chemical Data [PDF]
    3. 3T3 Positive Control (SLS) Data [PDF]
    4. NHK Positive Control (SLS) Data [PDF]

  • Appendix J: LD50 and Toxicity Category Predictions [PDF]
    1. 3T3 NRU Predictions: RC Millimole Regression [PDF]
    2. 3T3 NRU Predictions: 3T3 Combined-Laboratory Regression [PDF]
    3. NHK NRU Predictions: RC Millimole Regression [PDF]
    4. NHK NRU Predictions: NHK Combined-Laboratory Regression [PDF]
    5. 3T3 NRU Predictions: RC Rat-Only Weight Regression [PDF]
    6. NHK NRU Predictions: RC Rat-Only Weight Regression [PDF]
    7. 3T3 NRU Predictions: RC Rat-Only Weight Regression Excluding Chemicals with Specific Mechanisms of Toxicity [PDF]
    8. NHK NRU Predictions: RC Rat-Only Weight Regression Excluding Chemicals with Specific Mechanisms of Toxicity [PDF]


  • Appendix K: [PDF]
    1. NRU Data Used for Laboratory-Specific Regressions [PDF]
    2. Geometric Mean of Laboratory NRU Data Used for Combined Regressions [PDF]
    3. RC Chemicals with Rat LD50 Data [PDF]

  • Appendix L: [PDF]
    1. Outlier Characterization for the 3T3 and NHK NRU Test Methods with the RC Millimole Regression [PDF]
    2. Discordant Chemicals for GHS Toxicity Category Predictions
      3. Using the 3T3 and NHK NRU Test Methods and Associated Regressions [PDF]
    3. Analysis of Outliers by Halle (1998) for the RC Millimole Regression [PDF]

  • Appendix M: Acute Systemic Toxicity Test Guidelines [PDF]
    1. OECD UDP Test Guideline [PDF]
    2. EPA UDP Test Guideline [PDF]
    3. OECD ATC Test Guideline [PDF]
    4. OECD FDP Test Guideline [PDF]

  • Appendix N: UDP/ATC Simulation Modeling Results [PDF]
    1. UDP Results for the RC Millimole Regression - Starting One Dose Lower Than Estimated LD50 - 5000 mg/kg Upper Limit [PDF]
    2. UDP Results for the RC Rat-Only Weight Regression - Starting One Dose Lower Than Estimated LD50 - 5000 mg/kg Upper Limit [PDF]
    3. UDP Results for the RC Rat-Only Weight Regression Excluding Chemicals with Specific Mechanisms of Toxicity - Starting One Dose Lower Than Estimated LD50 - 5000 mg/kg Upper Limit [PDF]
    4. ATC Results for the RC Millimole Regression - 2000 mg/kg Upper Limit [PDF]
    5. ATC Results for the RC Rat-Only Weight Regression - 2000 mg/kg Upper Limit [PDF]
    6. ATC Results for the RC Rat-Only Weight Regression Excluding Chemicals with Specific Mechanisms of Action - 2000 mg/kg Upper Limit [PDF]

  • 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 [PDF or or Word]
    1. Supplemental Acute Toxicity Protocol
    2. Office of Pollution Prevention and Toxics (OPPT) Letters to Manufacturers/Importers

  • Appendix Q: Additional UDP Simulation Modeling Results [PDF]
    1. UDP Results for the RC Millimole Regression - Starting at Estimated LD50 - 5000 mg/kg Upper Limit [PDF]
    2. UDP Results for the RC Rat-Only Weight Regression - Starting at Estimated LD50 - 5000 mg/kg Upper Limit [PDF]
    3. UDP Results for the RC Rat-Only Weight Regression Excluding Substances with Specific Mechanisms of Toxicity - Starting at Estimated LD50 - 5000 mg/kg Upper Limit [PDF]

  • Appendix R: [PDF]
    1. SAS Code for ANOVA and Contrasts
    2. SAS Code for Regression Comparisons
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