## Table of Contents (HTML) 

### NIH Publication No: 01-4500



# Guidance Document on Using In Vitro Data to Estimate In Vivo Starting Doses for Acute Toxicity

Based on Recommendations from an International Workshop Organized by the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM)

and the National Toxicology Program (NTP) Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM)

National Institute of Environmental Health Sciences

National Institutes of Health U.S. Public Health Service Department of Health and Human ServicesTHE INTERAGENCY COORDINATING COMMITTEE ON THE VALIDATION OF ALTERNATIVE METHODS AND THE NTP INTERAGENCY CENTER FOR THE EVALUATION OF ALTERNATIVE TOXICOLOGICAL METHODS

The Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) was established in 1997 by the Director of the National Institute of Environmental Health Sciences (NIEHS) to implement NIEHS directives in Public Law 103-43. P.L. 103-43 directed NIEHS to develop and validate new test methods, and to establish criteria and processes for the validation and regulatory acceptance of toxicological testing methods. P. L. 106-545, the ICCVAM Authorization Act of 2000, established ICCVAM as a permanent committee. The Committee is composed of representatives from 15 Federal regulatory and research agencies and programs that generate, use, or provide information from toxicity test methods for risk assessment purposes. The Committee coordinates cross-agency issues relating to development, validation, acceptance, and national/international harmonization of toxicological test methods.

The National Toxicology Program Interagency Center for the Evaluation of Alternative Toxicological Methods(Center) was established in 1998 to provide operational support for the ICCVAM, and to carry out committeerelated activities such as peer reviews and workshops for test methods of interest to Federal agencies. The Center and ICCVAM coordinate the scientific review of the validation status of proposed methods and provide recommendations regarding their usefulness to appropriate agencies. The NTP Center and ICCVAM seek to promote the validation and regulatory acceptance of toxicological test methods that will enhance agencies’ abilities to assess risks and make decisions, and that will refine, reduce, and replace animal use. The ultimate goal is the validation and regulatory acceptance of new test methods that are more predictive of human and ecological effects than currently available methods.

Additional Information Additional information can be found at the ICCVAM/Center Website: http://iccvam.niehs.nih.gov and in the publication: Validation and Regulatory Acceptance of Toxicological Test Methods, a Report of the ad hoc Interagency Coordinating Committee on the Validation of Alternative Methods (NIH Publication No. 97-3981, or you may contact the Center at telephone 919-541-3398, or by e-mail at iccvam@niehs.nih.gov . Specific questions about ICCVAM and the Center can be directed to the ICCVAM Co-chairs:

Dr. William S. Stokes, NIEHS, EC-17, P.O. Box 12233 Research Triangle Park, NC, 27709; 919-541-7997

stokes@niehs.nih.gov

Dr. Richard N. Hill, US EPA, MC-7101, 401 M Street, SW Washington, DC, 20460; 202-260-2894 hill.richard@epa.gov

ICCVAM Agencies and Programs Agency for Toxic Substances and Disease Registry Consumer Product Safety Commission Department of Agriculture Department of Defense Department of Energy Department of Interior Department of Transportation Environmental Protection Agency Food and Drug Administration National Cancer Institute National Institute of Environmental Health Sciences National Institutes of Health, Office of the Director National Institute of Occupational Safety and Health National Library of Medicine Occupational Safety and Health Administration

# Guidance Document on Using In Vitro Data to

Estimate In Vivo Starting Doses for Acute Toxicity

Based on Recommendations from an International Workshop Organized by the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) and the National Toxicology Program (NTP) Interagency Center for the Evaluation of

Alternative Toxicological Methods (NICEATM)

National Toxicology Program P.O. Box 12233 Research Triangle Park, NC 27709

August 2001 NIH Publication No. 01-4500

National Institute of Environmental Health Sciences

National Institutes of Health US Public Health Service Department of Health and Human Servicesiiiii 

## Table of Contents

List of Tables

............................................................................................................................................. v List of Figures

........................................................................................................................................... v List of Acronyms/Abbreviations

........................................................................................................... vii Workshop Breakout Group

.................................................................................................................... xi Acknowledgements

................................................................................................................................. xii Preface

..................................................................................................................................................... xv

1.0 Introduction

...................................................................................................................................... 1 1.1 Purpose and Scope of this Guidance Document

........................................................................... 1 1.2 The Correlation Between Basal Cytotoxicity and Acute Lethality

............................................... 1 1.3 In Vitro Determination of Starting Dose for In Vivo Tests

........................................................... 3 1.4 Determination of In Vitro Test Performance Characteristics

........................................................ 4

2.0 Elements of A Standard Test For Basal Cytotoxicity

................................................................... 5 2.1 Selection of Cell Lines / Cells

....................................................................................................... 5 2.2 Recommended Measurement Endpoints for Basal Cytotoxicity

.................................................. 6 2.3 Recommendations for Cytotoxicity Test Protocols

....................................................................... 7

3.0 Procedure for Qualifying a Cytotoxicity Test for use with the Registry of Cytotoxicity Prediction Model

.................................................................................... 9 3.1 Procedure to Determine Whether a Candidate Cytotoxicity Test Can Use the RC Prediction Model

........................................................................................................ 9 3.2 Recommended Reference Chemicals from the RC for Test Qualification

................................. 10 3.3 Results Obtained with the Recommended Reference Chemicals in Two Standard Tests for Basal Cytotoxicity with Human and Rodent Cells

....................................... 12

4.0 Recommended Basal Cytotoxicity Tests: BALB/C 3T3 And Normal Human Keratinocyte (NHK) Neutral Red Uptake (NRU) Tests

............................................... 15 4.1 Validation Status of the 3T3 NRU Test

...................................................................................... 15 4.2 Reliability of the 3T3 NRU Test

................................................................................................. 15 4.3 Validation Status of the NHK NRU Test

.................................................................................... 18 4.4 Reliability of the NHK NRU Test

............................................................................................... 19

5.0 Conclusion

...................................................................................................................................... 19 Acknowledgement

................................................................................................................................ 19

6.0 References

....................................................................................................................................... 21

Appendices

................................................................................................................................................. .

A. Registry Of Cytotoxicity

........................................................................................................... A-1

B. List of Test Protocols for Basal Cytotoxicity, European Centre for the Validation Of Alternative Methods (ECVAM) Scientific Information System (SIS)

.............. B-1Table of Contents

iv

C. Standard Operating Procedure (SOP) for the BALB/c 3T3 Neutral Red Uptake Cytotoxicity Test - A Test for Basal Cytotoxicity

........................................................ C-1

1.0 Standard Operating Procedure (SOP) for the BALB/c 3T3 Neutral Red Uptake Cytotoxicity Test - A Test for Basal Cytotoxicity

........................................ C-3 1.1 Background and Introduction

....................................................................................... C-3 1.2 Rationale

....................................................................................................................... C-3 1.3 Basic Procedure

............................................................................................................ C-3 1.4 Test Limitations

............................................................................................................ C-4 1.5 Material

........................................................................................................................ C-4 1.6 Methods

........................................................................................................................ C-6 1.7 Data Analysis

............................................................................................................. C-10 1.8 Prediction Model

........................................................................................................ C-11 1.9 References

.................................................................................................................. C-11

D. Standard Operating Procedure (SOP) for the Normal Human Keratinocyte Neutral Red Uptake Cytotoxicity Test - A Test for Basal Cytotoxicity

................................... D-1

1.0 Standard Operating Procedure (SOP) for the BALB/c 3T3 Neutral Red Uptake Cytotoxicity Test - A Test for Basal Cytotoxicity

............................................... D-3 1.1 Background and Introduction

....................................................................................... D-3 1.2 Rationale

....................................................................................................................... D-3 1.3 Basic Procedure

............................................................................................................ D-3 1.4 Test Limitations

............................................................................................................ D-3 1.5 Material

........................................................................................................................ D-4 1.6 Methods

........................................................................................................................ D-6 1.7 Data Analysis

............................................................................................................... D-9 1.8 Prediction Model

........................................................................................................ D-10 1.9 References

.................................................................................................................. D-10

E. 96-Well Plate Configuration

...................................................................................................... E-1

F. Decimal Geometric Concentration Series

.................................................................................. F-1

G. Standard Test Reporting Template

............................................................................................ G-1Table of Contents

v 

## List of Tables

1. Recommended reference chemicals for evaluating a cytotoxicity test for use with the RC prediction model

....................................................................................... 11

2. Interlaboratory reproducibility of the 3T3 NRU cytotoxicity test determined according to ISO 5725 in 12 laboratories for 29 chemicals

......................................... 15



## List of Figures

1. Registry of Cytotoxicity Regression Between Cytotoxicity (IC 50x ) and Rodent Acute Oral LD 50 Values for 347 Chemicals

................................................................... 3

2. Procedure for Evaluating a Cytotoxicity Test for Tiered In Vitro / In Vivo Testing for Acute Oral Toxicity Testing (Slightly Modified after Spielmann et. al., 1999)

............................................................................ 10

3. Regression Obtained by Testing the Recommended Reference Chemicals from the RC with Human Keratinocytes in the NHK NRU Cytotoxicity Test

.......................................................................................................... 13

4. Regression Obtained by Testing the Recommended Reference Chemicals from the RC with Mouse Fibroblasts in the BALB/c 3T3 NRU Cytotoxicity Test

............................................................................................................. 13

5. Interlaboratory Comparability of the 3T3 NRU Cytotoxicity Test for 147 Test Chemicals in 2 Different Laboratories per Chemical

.................................................. 17

List of Tables List of Figures List of Acronyms/Abbreviations Workshop Breakout Groups Acknowledgements Preface Executive Summary

1.0 INTRODUCTIONTable of Contents

vi

1.1 Purpose and Scope of this Guidance Document 1.2 The Correlation Between Basal Cytotoxicity and Acute Lethality 1.3 In Vitro Determination of Starting Dose for In Vivo Tests 1.4 Determination of In Vitro Test Performance Characteristics 2.0 ELEMENTS OF A STANDARD TEST FOR BASAL CYTOTOXICITY 2.1 Selection of Cell Lines / Cells 2.2 Recommended Measurement Endpoints for Basal Cytotoxicity 2.3 Recommendations for Cytotoxicity Test Protocols 3.0 PROCEDURE FOR QUALIFYING A CYTOTOXICITY TEST FOR USE WITH THE REGISTRY OF CYTOTOXICITY PREDICTION MODEL 3.1 Procedure to Determine Whether a Candidate Cytotoxicity Test Can Use the Registry of Cytotoxicity Prediction Model 3.2 Recommended Reference Chemicals from the RC for Test Qualification 3.3 Results Obtained with the Recommended Reference Chemicals in Two Standard Tests for Basal Cytotoxicity with Human and Rodent Cells 4.0 Recommended basal cytotoxicity TESTs: BALB/C 3T3 and Normal human keratinocyte (NHK) NEUTRAL RED UPTAKE (NRU) TESTs

4.1 Validation Status of the 3T3-NRU Test 4.2 Reliability of the 3T3-NRU Test 4.3 Validation Status of the NHK-NRU Test 4.4 Reliability of the NHK-NRU Test 5.0 CONCLUSION ACKNOWLEDGEMENT 6.0 REFERENCES

Appendices A. Registry Of Cytotoxicity B. List Of Test Protocols For Basal Cytotoxicity, European Centre For The Validation Of Alternative Methods (Ecvam) Scientific Information System (Sis) C. Standard Operating Procedure (Sop) For The Balb/C 3t3 Neutral Red Uptake Cytotoxicity Test - A Test For Basal Cytotoxicity 1.0 STANDARD OPERATING PROCEDURE (SOP) FOR THE BALB/C 3T3 NEUTRAL RED UPTAKE CYTOTOXICITY TEST - A TEST FOR BASAL CYTOTOXICITY

1.1 Background and Introduction 1.2 Rationale 1.3 Basic Procedure 1.4 Test Limitations 1.5 Material

1.6 Methods

1.7 Data Analysis 1.8 Prediction Model 1.9 Referencesvii 

## List of Acronyms/Abbreviations

ATC Acute Toxic Class ATCC American Type Culture Collection BALB/c Inbred strain of mouse BgVV Federal Institute for Health Protection of Consumers

and Veterinary Medicine (Germany) BMFT Ministry of Research and Technology (Germany) BSS Balanced Saline Solution b.w. Body weight CAS Chemical Abstract Service CHO Chinese hamster ovary cell line (epithelial) CFR Code of Federal Regulations CMF-HBSS Calcium/Magnesium-Free Hanks’ Balanced Salt (Saline) Solution COLIPA The European Cosmetic, Toiletry and Perfumery Industry CS Calf serum (bovine) CTFA The Cosmetic, Toiletry, and Fragrance Association (USA) CTLU Cytotoxicology Laboratory, Uppsala DMEM Dulbecco’s Modification of Eagle’s Medium without L-Glutamine DMSO Dimethyl Sulfoxide ECACC European Collection of Cell Cultures EC/HO European Commission/British Home Office ECVAM European Centre for the Validation of Alternative Methods EDTA Ethylenediaminetetraacetic Acid EPA Environmental Protection Agency ETOH Ethanol EZ4U Non-radioactive cell proliferation and cytotoxicity assay (Biomedica Gruppe) FBS Fetal Bovine Serum FDP Fixed-Dose Procedure F G Empirical linear-shaped prediction interval FL Fluorescein Leakage FRAME Fund for the Replacement of Animals in Medical Experiments GLP Good Laboratory Practice Regulations h Hour(s) HBSS Hanks’ Balanced Salt (Saline) Solution HEL-30 Murine keratinocyte cell line Hepa-1 Mouse hepatoma cell line (epithelial) HEPES N-2-Hydroxyethylpiperazine-N'-2-ethanesulfonic acid HET-CAM Hen’s Egg Test-Chorioallantoic Membrane HTD Highest tolerated dose IC50 Inhibitory concentration estimated to affect endpoint in question by 50% ICCVAM Interagency Coordinating Committee on the Validation of Alternative Methods ICH International Congress for Harmonization IIVS Institute for In Vitro Sciences INVITTOX ECVAM database ISO 5725 A program for analysis and reporting of proficiency tests and method

evaluation studiesList of Acronyms/Abbreviations

viii

ISO International Standards Organization KB Kenacid Blue KGM Keratinocyte Growth Medium L929 Mouse fibrosarcoma cell line (fibroblast) LD50 Dose producing lethality in 50% of the animals LDH Lactate Dehydrogenase MDCK Madin Darby canine kidney cells MEIC Multicentre Evaluation of In Vitro Cytotoxicity MEMO MEIC Monographs, CTLU MIT-24 Metabolic Inhibition Test MSDS Material Safety Data Sheet MTS 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-

(4-sulfophenyl)-2H-tetrazolium MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide NBCS New-born Calf Serum (bovine) NICEATM NTP Interagency Center for the Evaluation of Alternative Toxicological Methods NIEHS National Institute of Environmental Health Sciences/NIH NIH National Institutes of Health/DHHS NIOSH National Institute for Occupational Safety and Health NHEK Normal Human Epidermal Keratinocytes NHK Normal Human Keratinocytes NR Neutral Red dye NR50 Mean concentration of test substance reducing viability of cells to 50% of the

viability of controls NRU50 The concentration of the test article which inhibits the uptake of neutral red by

50% NRR Neutral Red Release NRU Neutral Red Uptake NTP National Toxicology Program OD 540 Optical density at 540nm OECD Organization for Economic Cooperation and Development PBS Phosphate Buffered Saline PC Positive Control PHOTO-32 Software for concentration response analysis from 96-well plates PT Phototoxicity Test RC Registry of Cytotoxicity/ZEBET RTECS Registry of Toxic Effects of Chemical Substances SIS ECVAM Scientific Information System SLS Sodium Lauryl Sulfate SOP Standard Operating Procedures TG 420 Test Guideline 420 (Acute Oral Toxicity - Fixed Dose Method) [OECD] TG 423 Test Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method) [OECD] TG 425 Test Guideline 425 (Acute Oral Toxicity: Up-and-Down Procedure) [OECD]List of Acronyms/Abbreviations

ix TNS Trypsin Neutralizing Solution UDP Up-and-Down Procedure V79 Chinese hamster lung fibroblast cell line VC Vehicle Control XTT Sodium 3,3-{1-[(phenylamino)carbonyl]-3,4-tetrazolium}-bis(4-methoxy-6-

nitro)benzene sulfonic acid hydrate ZEBET German Centre for the Documentation and Validation of Alternative Methods (at

BgVV) 3Rs Refinement, Reduction, and Replacement (of Animal Use) 3T3 BALB/c mouse fibroblast cells 3T3 A31 BALB/c mouse fibroblast cells – clone A31List of Acronyms/Abbreviations

xxi 

## Acknowledgements

Drs. Fentem, Curren, and Liebsch are acknowledged for their significant contributions to the guidance document. They along with the following scientists were invited to serve on the Breakout Group 1 panel for the International Workshop on In Vitro Methods for Assessing Acute Toxicology, October 17-20, 2000.



### Workshop Breakout Group 1

In Vitro Screening Methods for Assessing Acute Toxicity

Julia Fentem, Ph.D. (Co-Chair); (Co-Author) Unilever Research Shambrook Bedforshire, United Kingdom

Charles Tyson, Ph.D. ( Co-Chair) SRI International Menlo Park, CA

Robert Combes

, Ph.D. Fund for the Replacement of Animals in Medical Experiments (FRAME) Nottingham, United Kingdom

Rodger Curren

, Ph.D. (Co-Author) Institute for In Vitro Sciences, Inc. Gaithersburg, MD

Elke Genschow, Ph.D. German Centre for the Documentation and Validation of Alternative Methods (at BgVV) (ZEBET) Berlin, Germany Alan Goldberg, Ph.D. Johns Hopkins University Center for Alternatives to Animal Testing Baltimore, MD

A. Wallace Hayes

, Ph.D., D.A.B.T., D.A.T.S. The Gillette Company Boston, MA

Manfred Liebsch

, Ph.D. (Co-Author) German Centre for the Documentation and Validation of Alternative Methods (at BgVV) (ZEBET) Berlin, Germany

Lennart Romert

, Ph.D. Swedish National Chemicals Inspectorate Solna, Sweden

Noriho Tanaka, Ph.D. Hatano Research Institute Kanagawa, Japan

ICCVAM Agency Participants

Kailash Gupta

, D.V.M., Ph.D. U.S. CPSC, Bethesda, MD

Kenneth Hastings

, Ph.D. U.S. FDA/CDER, Rockville, MD

Richard N. Hill, M.D., Ph.D. U.S. EPA/OPPT, Washington, D.C.

Abdullah Khasawinah, Ph.D. U.S. EPA/OPP/HED, Arlington, VA

Elizabeth Margosches

, Ph.D. U.S. EPA/OPPT/OPPTS, Washington, D.C. Philip Sayre

, Ph.D. U.S. EPA/OPPT, Washington, D.C.

Leonard Schechtman

, Ph.D. U.S. FDA/CDER, Rockville, MD

Suhair Shallal, Ph.D. U.S. EPA/OPP/HED, Arlington, VA

William S. Stokes

, D.V.M., D.A.C.L.A.M. NIEHS, Research Triangle Park, NCAcknowledgements

xii



### ICCVAM Organizing Committee for the International Workshop on In Vitro Methods for Assessing Acute Systemic Toxicity

Consumer Product Safety Commission (CPSC) Kailash Gupta, D.V.M., Ph.D.

Department of Defense (DOD)John Frazier, Ph.D., (Organizing Committee cochair) Department of Transportation (DOT) George Cushmac, Ph.D.

Environmental Protection Agency (EPA) Richard Hill, M.D., Ph.D. (ICCVAM Co-Chair) Angela Auletta, Ph.D. Elizabeth Margosches, Ph.D. Karen Hamernik, Ph.D.Philip Sayre, Ph.D. (Organizing Committee cochair) Maurice Zeeman, Ph.D.

Food and Drug Administration (FDA) Leonard Schechtman, Ph.D. Melvin Stratmeyer, Ph.D. Ronald Brown, Ph.D. Thomas Collins, Ph.D. Peter Goering, Ph.D. Stephen Hundley, Ph.D.

National Institute of Environmental Health Sciences (NIEHS) Jerrold Heindel, Ph.D. William Stokes, D.V.M. (ICCVAM Co-Chair) Errol Zeiger, Ph.D., J.D.

National Institute for Occupational Safety and Health (NIOSH) Kenneth Weber, Ph.D.

National Library of Medicine (NLM) Vera Hudson, M.S.L.S.

Occupational Safety and Health Administration (OSHA) Surender Ahir, Ph.D.



### National Toxicology Program (NTP) Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM)

Integrated Laboratory Systems (ILS), Inc. Bradley Blackard, M.S.P.H. Sue Brenzel Ashlee Duncan, M.S. Thomas Goldworthy, Ph.D. Christina Inhof, M.S.P.H. Linda Litchfield Barry Margolin, Ph.D. National Institute of Environmental Health Sciences (NIEHS) Loretta Frye Debbie McCarley William S. Stokes, D.V.M., D.A.C.L.A.M. (Director)

Judy Strickland, Ph.D., D.A.B.T. Michael Paris Barbara Shane, Ph.D., D.A.B.T. Raymond Tice, Ph.D.Acknowledgements

xiii 

### Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM)

Agency for Toxic Substances and Disease Registry (ATSDR) *William Cibulas, Ph.D.

Consumer Product Safety Commission (CPSC) *Marilyn L. Wind, Ph.D. Susan Aitken, Ph.D. Kailash C. Gupta, D.V.M., Ph.D.

Department of Defense (DOD) *Harry Salem, Ph.D., U.S. Army Edgewood Chemical Biological CenterJohn M. Frazier, Ph.D., U.S. Air Force, WrightPatterson AFB

Department of Energy (DOE) *Marvin Frazier, Ph.D.

Department of Interior (DOI) *Barnett A. Rattner, Ph.D.

Department of Transportation (DOT) *James K. O’Steen George Cushmac, Ph.D.

Environmental Protection Agency (EPA) *Richard N. Hill, M.D., Ph.D. (Co-Chair) Office of Pollution Prevention and Toxics +Angela Auletta, Ph.D Philip Sayre, Ph.D. Office of Pesticides Programs +Karen Hamernik, Ph.D. Amy Rispin, Ph.D. Office of Research and Development +Harold Zenick, Ph.D. Suzanne McMaster OECD Test Guidelines Program Maurice Zeeman, Ph.D.

Food and Drug Administration (FDA) *Leonard M. Schechtman, Ph.D. Suzanne C. Fitzpatrick, Ph.D., D.A.B.T.

Center for Drug Evaluation and Research +Joseph DeGeorge, Ph.D. Joseph F. Contrera, Ph.D. Abby C. Jacobs, Ph.D. Center for Devices and Radiological Health

+ Mel E. Stratmeyer, Ph.D. Raju G. Kammula, D.V.M., Ph.D., D.A.B.T. Center for Biologics Evaluation and Research +Patrick G. Swann Center for Food Safety and Nutrition +David G. Hattan, Ph.D. Victor P. Frattali Center for Veterinary Medicine

+ Devaraya Jagannath, Ph.D. Louis T. Mulligan, D.V.M. National Center for Toxicological Research +William T. Allaben, Ph.D. Office of Regulatory Affairs +Atin Datta, Ph.D.

National Cancer Institute (NCI)

* David Longfellow, Ph.D. Yung-Pin Liu, Ph.D.

National Institute of Environmental Health Sciences (NIEHS) *William S. Stokes, D.V.M., D.A.C.L.A.M. (Co-Chair) John R. Bucher, Ph.D., D.A.B.T. Rajendra S. Chhabra, Ph.D., D.A.B.T

National Institute for Occupational Safety and Health (NIOSH) *Doug Sharpnack, D.V.M., D.A.C.V.P. +Paul Nicolaysen, V.M.D.

National Institutes of Health (NIH) *Margaret D. Snyder, Ph.D.

National Library of Medicine (NLM) *Vera Hudson, M.S.L.S.

Occupational Safety and Health Administration (OSHA)

* Surender Ahir, Ph.D.

* Principal Agency Representatives + Principal Program Representatives 6/01Acknowledgements

xivxv 

## Preface

Acute systemic toxicity testing is conducted to determine the relative health hazard of chemicals and various products. Substances found to cause lethality in animals at or below prescribed doses are labeled to identify their hazard potential. While acute toxicity testing is currently conducted using animals, studies published in recent years have shown a correlation between in vitro and in vivo acute toxicity. These studies suggest that in vitro methods may be helpful in predicting in vivo acute toxicity.

An International Workshop on In Vitro Methods for Assessing Acute Systemic Toxicity was convened on October 17-20, 2000, to review the validation status of available in vitro methods for predicting acute toxicity, and to develop recommendations for future research and development efforts that might further enhance the use of in vitro assessments of acute systemic toxicity. The Workshop was organized by the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) and the National Toxicology Program (NTP) Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM). The U.S. Environmental Protection Agency (U.S. EPA), the NTP, and the National Institute of Environmental Health Sciences (NIEHS sponsored the workshop. Breakout Groups, comprised of invited scientific experts and ICCVAM agency scientists, developed conclusions and recommendations for four topics:

• In Vitro Screening Methods for Assessing Acute Toxicity;

• In Vitro Methods for Toxicokinetic Determinations;

• In Vitro Methods for Predicting Organ Specific Toxicity; and

• Chemical Data Sets for Validation of In Vitro Acute Toxicity Test Methods.

The Breakout Group that addressed the first topic,

“ In Vitro Screening Methods,” was charged with evaluating the current validation status of basal cytotoxicity methods, and recommending whether and how these methods might be used to reduce and refine animal use for acute toxicity testing. The Group concluded that in vitro cytotoxicity data could be useful in estimating starting doses for in vivo acute toxicity testing, which will reduce the number of animals required for such determinations. Their conclusions were based on several studies but primarily those by Drs. Horst Spielmann and Willi Halle, and their colleagues at the German Centre for the Documentation and Evaluation of Alternatives to Testing in Animals. Halle compiled a Registry of Cytotoxicity containing in vivo acute toxicity data and in vitro cytotoxicity data for 347 chemicals. These data were used to construct a regression model to estimate LD 50 values from cytotoxicity data. They subsequently proposed that using these estimates as starting doses for in vivo acute toxicity studies such as the Up-and-Down Procedure or the Acute Toxic Class method could reduce the number of animals used by as much as 30 percent. In addition, the Group recommended that this guidance document be prepared to provide practical guidance on how to generate and use basal cytotoxicity data to predict starting doses for in vivo acute toxicity assays. Drs. Manfred Liebsch, Rodger Curren, and Julia Fentem volunteered to draft this document and after the Workshop they worked with NICEATM to develop it. This guidance document has been reviewed by ICCVAM, the ICCVAM Workshop Organizing Committee, and those participating in the Breakout Group on In Vitro Screening Methods.

The workshop results have been published as the Report on the International Workshop on In Vitro Methods for Assessing Acute Systemic Toxicity (NIH Publication No. 01-4499). The Organizing Committee and ICCVAM developed test recommendations to forward with these publications to Federal agencies for their consideration in accordance with Public Law 106- 545. The ICCVAM recommendations are provided in the Workshop Report. BothPreface

xvi

publications are available at the ICCVAM/NICEATM website (http://iccvam.niehs.nih.gov), or a copy may be requested from NICEATM at P.O. Box 12233, MD EC-17, Research Triangle Park, NC 27709 (mail), 919-541-3398 (phone), 919-541-0947 (fax), or NICEATM@niehs.nih.gov (email).

On behalf of the ICCVAM, we gratefully acknowledge the efforts of the Breakout Group on In Vitro Screening Methods for their comprehensive evaluation of existing data and methods that served as the impetus for this guidance document. We extend our sincere appreciation to the contributing authors, Drs. Manfred Liebsch, Rodger Curren, and Julia Fentem, for their considerable efforts and contributions to this document. The efforts of the NICEATM staff in coordinating the preparation and publication of the document are acknowledged and appreciated, particularly those of Dr. Judy Strickland and Mr. Michael Paris, who worked diligently with the authors to produce the final version.

William S. Stokes, D.V.M. Co-Chair, ICCVAM NIEHS

Richard N. Hill, M.D., Ph.D. Co-Chair, ICCVAM U. S. EPAUsing In Vitro Data to Estimate In Vivo Starting Doses for Acute Toxicity

1 1.0 INTRODUCTION

1.1 Purpose and Scope of this Guidance Document

This guidance document describes how to use in vitro cytotoxicity tests to estimate starting doses for acute oral lethality assays. Development of this document was recommended by participants in the International Workshop on In Vitro Methods for Assessing Acute Systemic Toxicity, held October 17-20, 2000, in Arlington, VA, U.S.A. The Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) and the National Toxicology Program Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM) convened the workshop to evaluate the validation status of available in vitro methods for assessing acute toxicity. A workshop breakout group reviewed the use of in vitro screening methods to estimate acute in vivo toxicity (i.e., LD 50 values) and recommended the development of this guidance document which was written by three of its members.

This introduction summarizes background information about the correlation between in vitro cytotoxicity and acute lethality, explains the purpose of using in vitro cytotoxicity assays to predict starting doses for in vivo acute lethality assays, and describes a general approach for evaluating in vitro test performance. Chapter 2 describes the basic elements of in vitro assays for basal cytotoxicity and describes what investigators should consider before applying the results of these assays to their own situations. Chapter 3 describes the use of the Registry of Cytotoxicity (RC) prediction model to evaluate a candidate cytotoxicity assay. The RC prediction model is a regression analysis of LD 50 values (the median lethal dose, i.e., the dose that produces lethality in 50% of the animals tested) and in vitro cytotoxicity IC 50 values (i.e., concentration at which cell viability is inhibited by 50%) for 347 chemicals. Chapter 4 describes two candidate tests recommended for use with this method: neutral red uptake (NRU) assays using the mouse fibroblast cell line BALB/c 3T3 and normal human keratinocytes (NHK). Appendix A contains the RC data in spreadsheet format. Appendix B contains a list of test protocols for basal cytotoxicity from the Scientific Information System (SIS) of the European Centre for the Validation of Alternative Methods (ECVAM). Appendices C-G provide detailed stand-alone protocols for BALB/c 3T3 and NHK NRU assays, additional guidance for implementing the protocols, and a standard template for data collection.

1.2 The Correlation between Basal Cytotoxicity and Acute Lethality

Acute oral toxicity testing is typically the first step in identifying and characterizing the hazards associated with a particular chemical. Information derived from acute toxicity tests in laboratory animals (mainly rodents) is used for several purposes, including: (a) hazard classification and labeling of chemicals in accordance with national and international regulations (e.g., 49 Code of Federal Regulations [CFR] 173; 16 CFR 1500; 29 CFR 1910; 40 CFR 156; Organisation for Economic Co-operation and Development [OECD], 1998a); (b) risk assessments pertaining to the acceptability of acute exposures in the workplace, at home, and upon accidental release; (c) clinical diagnosis, treatment and prognosis of acute human poisoning cases; and (d) design (e.g., dose-setting,identification of potential target organs) of longerterm (e.g., 28-day) toxicity studies. Historically, lethality estimated by the LD 50 test has been a primary toxicological endpoint in acute toxicity tests, although more detailed toxicological information is sometimes collected. More recently, the conventional test procedure has been modified in various ways to refine and reduce animal use (OECD, 1992, 1996, 1998b). Aiding the acceptance of these alternative methods has been the recognition that the LD 50 is not a biological constant, but is influenced by many factors (Klaassen and Eaton, 1991). For most purposes, the LD 50 only needs to be characterized “within an order of magnitude range,” according to Klaassen and Eaton (1991).

The use of cell cultures in vitro as alternatives to predict acute lethality in vivo has been under study for almost 50 years (Pomerat and Leake, 1954; Eagle and Foley, 1956; Smith et al., 1963).Using In Vitro Data to Estimate In Vivo Starting Doses for Acute Toxicity

2 Numerous demonstrations of strong correlations between cytotoxicity in vitro and animal lethality in vivo exist. (For reviews see Phillips et al., 1990, and Garle et al., 1994). Recently, several major international in vitro initiatives have been directed toward reducing the use of laboratory animals for acute toxicity testing (Curren et al., 1998; Ekwall et al., 2000; Ohno et al., 1998a, 1998b, 1998c; Seibert et al., 1996; Spielmann et al., 1999). The status of these initiatives was reviewed at the International Workshop on In Vitro Methods for Assessing Acute Systemic Toxicity, jointly sponsored by the National Institute of Environmental Health Sciences (NIEHS), the National Toxicology Program (NTP), and the U.S. Environmental Protection Agency (U.S. EPA). Conclusions and recommendations from the workshop are published in the Report of the International Workshop on In Vitro Methods for Assessing Acute Systemic Toxicity (NIEHS, 2001).

The RC has made a major contribution to the knowledge of the correlation between in vitro cytotoxicity and in vivo lethality (Halle and Spielmann, 1992; Halle, 1998). The most recent RC compilation (Halle, 1998) contains in vitro cytotoxicity information (1,912 single IC 50 values averaged for each of 347 chemicals [i.e., one IC 50x value/chemical from multiple reports in the literature]) paired with 347 in vivo acute oral LD 50 values (mmol/kg) for rats (282 values) or mice (65 values) from the National Institute for Occupational Safety and Health (NIOSH) Registry of Toxic Effects of Chemical Substances (RTECS). (See Appendix A for the RC data.) Criteria for data to be included in the RC database are fully described by Halle (1998) and briefly described by Spielmann et al. (1999). The combination of rat and mouse data was justified, since it yielded a regression that was not significantly different from those obtained with either rat data or mouse data alone. The RC data clearly demonstrate a strong relationship between in vitro cytotoxicity and acute lethality in rodents (Figure 1).Using In Vitro Data to Estimate In Vivo Starting Doses for Acute Toxicity

3 Figure 1. Registry of Cytotoxicity regression between cytotoxicity (IC 50x ) and rodent acute oral

LD 50 values for 347 chemicals. The heavy line shows the fit of the data to a linear regression model, log (LD 50 ) = 0.435 x log (IC 50x ) + 0.625; r=0.67. The other lines show the empirical F G = ± log 5 acceptance interval for the prediction model (Spielmann et al., 1999), which is based on the anticipated precision of LD 50 values from rodent studies Halle (1998).

1.3 In Vitro Determination of Starting Dose for In Vivo Tests

Spielmann et al. (1999) have proposed – as an initial step – that the relationship found with the RC regression be used with in vitro data to predict starting doses for subsequent in vivo acute lethality assays. They suggest that before initiating any in vivo lethality assay for a chemical, an in vitro cytotoxicity assay should be conducted to estimate the LD 50 for that chemical. The LD 50 predicted from the RC regression equation should then be used to choose the most appropriate starting dose for the in vivo assay. The LD 50 estimate from the RC regression is based on molar amounts of the chemical, specifically a value in mmol/kg. This value must first be converted to a weight measurement expression, such as mg/kg, before using conventional LD 50 dosing calculations. Using this estimate should make the conduct of in vivo assays much more efficient and result in savings both in the number of animals and in the amount of time required to obtain the final results. The workshop report (NIEHS, 2001) includes a discussion of the potential number of animals saved, based on several currently available in vivo protocols, e.g., protocols that use new sequential dosing methods such as the Acute Toxic Class method (ATC, OECD TG 423; OECD, 1996) and the Up-and-Down Procedure (UDP, OECD TG 425; OECD, 1998b). In these tests, using the 

### Regression between cytotoxity (IC 50x 

### ) and
acute oral LD 50 

### values

0.0001 0.001 0.01 0.1 1 10 100 1000

0.00001 0.001 0.1 10 1000 IC50x (mmol/l)LD50 (mmol/kg)Using In Vitro Data to Estimate In Vivo Starting Doses for Acute Toxicity

4 fewest animals possible depends upon the correct choice of starting dose since, on average, the number of consecutive dosing steps is minimal if the starting dose is close to the true toxicity class (ATC) or to the true LD 50 (UDP).

1.4 Determination of In Vitro Test Performance Characteristics

Before the results obtained with any in vitro cytotoxicity test are used with the RC regression to generate an expected LD 50 value, the performance characteristics of the new method should be determined and compared with those of the RC information as discussed in Section 3.1. Section 3.2 suggests a set of reference chemicals that should be tested with the candidate in vitro cytotoxicity method. The resultant regression line should then be compared with that of the current RC regression line. If the line falls within the + log 5 boundaries indicated in Figure 1, then the regression parameters of the RC may be used to predict the LD 50 starting dose. Section 3.3 describes experimental trials, using two different cell types, performed after the workshop with the set of recommended reference chemicals. These experimental trials are included as examples of how to determine test performance for any in vitro test for basal cytotoxicity and to confirm the applicability of the test for use with the RC regression.Using In Vitro Data to Estimate In Vivo Starting Doses for Acute Toxicity

5 2.0 ELEMENTS OF A STANDARD TEST FOR BASAL CYTOTOXICITY

It is likely that many different in vitro cytotoxicity methods could be used to help select the in vivo starting dose for an acute lethality assay. Two decades of experience indicate that in vitro basal cytotoxicity data determined in various primarycells, as well as in various permanent nondifferentiated finite or transformed cell lines, generally show comparable cytotoxic concentrations of the same xenobiotic, regardless of the type of toxic endpoints investigated. The RC data

, which consist of information from many different in vitro methods that vary in both cell type and cytotoxicity endpoint (i.e., specific protocol), indicated that exceptions to this “rule” were observed only for those chemicals (some insecticides, neurotropic chemicals, and chemicals requiring metabolic activation) that require specific cell types to express their toxicity (Halle, 1998). Thus, a recommendation cannot be made for the "most relevant" or "most typical" in vitro test for basal cytotoxicity.

Currently the ECVAM SIS lists 20 different test protocols for basal cytotoxicity. (Appendix B and http://www.ivtip.org/protocols.html#basalcyto.) Several in vitro tests listed in the SIS as “specific” for a certain purpose, such as prediction of eye and skin irritancy, in fact provide only basal cytotoxicity information.

Nonetheless, since the responsiveness of all cell culture test systems to xenobiotics can be influenced significantly by test design and culture conditions, there is a consensus among in vitro toxicologists to give preference to protocols that are highly responsive. For example, while increasing exposure times (e.g., from 1 hour [h] up to 48 h) will usually increase the responsiveness of the test, an increase in serum in the culture medium (e.g., from 5% up to 20%) will generally decrease the responsiveness of a cytotoxicity test.

2.1 Selection of Cell Lines / Cells

Analyses performed before or during the workshop (NIEHS, 2001) did not reveal significant differences between the basal cytotoxicity results obtained using permanent mammalian cell lines, primary human cells, or using the IC 50X approach of Halle and Spielmann (Halle, 1998; Spielmann et al., 1999; Halle and Spielmann, 1992). Thus, primary cells, as well as many currently available mammalian cell lines could be used, provided they are of sufficient quality to assure reproducibility over time. However, rodent (i.e., rat or mouse) or human cells are expected to be most useful for this approach. Established rodent cell lines are recommended because: 1) it is assumed that rodent cells would give the best prediction of rodent acute lethality, and 2) the use of a standard cell type for this in vitro cytotoxicity technique will hasten the generation of a database that can be used to analyze the usefulness of this approach. There are also arguments for utilizing human cell lines to assess basal cytotoxicity. For example, an analysis of the RC rodent acute lethality data relative to cytotoxicity data generated using human cell lines in the MEIC program showed that both were highly correlative (R 2 =0.90) (NIEHS, 2001). A long-term advantage of using human cells is that the human cell cytotoxicity data derived from this approach can be added to human toxicity databases to facilitate the development of methods that may later predict acute human lethality.

Of the rodent cell lines used for basal cytotoxicity, the mouse fibroblast cell line BALB/c 3T3 A31 is probably the most frequently used. Thus, a stable background of historical data exists, including data from controlled and blinded validation studies (Gettings et al., 1991, 1992, 1994a, 1994b; Spielmann et al., 1991, 1993, 1996; Balls et al., 1995; Brantom et al., 1997). Other rodent cell lines that have been used in basal cytotoxicity assays are described by Clemedson et al. (1996).

Of the human cells used for basal cytotoxicity, NHK or fibroblasts are probably the cells most frequently used with good results in validation studies (Willshaw et al., 1994; Sina et al., 1995; Gettings et al., 1996; Harbell et al., 1997).

Fish cell lines or invertebrate cell lines are not recommended for determining basal cytotoxicityUsing In Vitro Data to Estimate In Vivo Starting Doses for Acute Toxicity

6 (Ekwall et al., 1998). Although, according to the concept of basal cytotoxicity, they are expected to show failure of the same basic cell functions as mammalian cells would show at comparable chemical concentrations, it is not easy to create test designs that are highly responsive to xenobiotics. For example, due to doubling times of up to several days, the responsive growth inhibition protocol cannot be used easily.

Highly differentiated cells may not give the best prediction of acute lethality for the large variety of chemicals likely to be tested for acute toxicity (Ekwall et al., 1998). For example, to eliminate the possibility of metabolic activation or inactivation of chemicals, neither hepatocyte nor hepatoma cytotoxicity data were included in the RC. This does not preclude the use of hepatocytes in future studies, however, either to estimate cytotoxicity or to investigate the effect of metabolism or cell-specific toxicity (Seibert et al., 1996). Hepatocytes are essential to investigations of metabolism-mediated toxicity that will be required to meet the longer-term goal of replacing in vivo acute lethality testing with in vitro methods (Seibert et al., 1996).

Whether rodent or human cells are used, they should be capable of active division (population doubling time of approximately 30 h or less) so that chemicals that exert their toxicity primarily during cell division will be adequately detected in these relatively short-term assays. As described in Section 2.3, chemical exposure should last at least one full cell cycle.

Finally, selection of a cell line always should be made in the context of the intended cytotoxicity endpoint to be measured. For example, if NRU is the intended measurement endpoint, the cells used must possess a significant amount of lysosomes to incorporate neutral red dye. Embryonic stem cells, for example, do not contain the requisite organelles, and NRU cannot be used to determine cytotoxicity in these cells.

Both the mouse and human cells mentioned above are easily obtainable from commercial sources. Cytotoxicity data from both the BALB/3T3 A31 cell line and NHK cells are presented in Section 3.3 of this document as examples of how to qualify new cytotoxicity protocols for use with the RC method for predicting starting doses for acute lethality assays in vivo

.

2.2 Recommended Measurement Endpoints for Basal Cytotoxicity

Many measurement endpoints for cytotoxicity are well established and have been used to assess basal cytotoxicity. For inclusion of IC 50 values in the RC, the following endpoints were accepted as sufficiently characteristic of basal cytotoxicity (Spielmann et al., 1999; Halle, 1998):

1) Inhibition of cell proliferation:

• Cell number

• Cell protein

• DNA content, DNA synthesis

• Colony formation

2) Cell viability - metabolic markers:

• Metabolic inhibition test (MIT-24)

• Mitochondrial reduction of tetrazolium salts into insoluble dye (MTT test), or, more recently, into soluble dye (MTS test or XTT test [e.g., “EZ4U”]).

3) Decreased cell viability - membrane markers:

• NRU into cell lysosomes

• Trypan Blue exclusion

• Cell attachment, cell detachment

4) Differentiation markers

• Functional differentiation within cell islets

• Morphological differentiation within cell islets

• Intracellular morphology

Markers of the release of intracellular components, such as the enzyme lactate dehydrogenase (i.e., LDH release test), or of dye introduced into the cells previous to chemical exposure (e.g., fluorescein leakage [FL] test or Neutral Red Release [NRR] test) were not considered to be characteristic for basal cytotoxicity because they specifically detect damage of the outer cell membrane and generally are associated with short-term chemical exposure. A chemical that specifically damages only cell membranes, however, will be detected correctly in one of the tests for basal cytotoxicity listed above.Using In Vitro Data to Estimate In Vivo Starting Doses for Acute Toxicity

7 2.3 Recommendations for Cytotoxicity Test Protocols

Since the RC was constructed with data from many different in vitro protocols, a number of different in vitro cytotoxicity protocols might produce correlations with in vivo acute lethality similar to the correlation produced by the RC. It is strongly suggested, however, that any proposed in vitro protocol incorporate the following conditions:

(a) Use a cell line (or primary cells) that divides rapidly with doubling times of less than 30 h under standard culture conditions, preferably with normal serum types, e.g., calf serum (CS), newborn calf serum (NBCS), or serum-free medium. (b) Use only cells in the exponential phase of growth. Never use cells immediately after thawing them from frozen stock. Allow cells to grow 1-2 passages before they are used in the cytotoxicity test. (c) The chemical exposure period should be at least the duration of one cell cycle, i.e., 24 – 72 h (Riddell et al., 1986). (d) Initial seeding should be done at a density that allows rapid growth throughout the exposure period. (e) Use appropriate positive and vehicle control materials for which cytotoxicity, or lack of cytotoxicity, has been well characterized by the performing laboratory. (f) Use solvents only at levels previously shown not to cause cytotoxicity to the cell system over the entire period of the
assay. (g) Use a measurement endpoint that is well established and that has good interlaboratory reproducibility. Give preference to endpoints that determine either cell proliferation or cell viability (e.g., NRU, MTT, XTT). Simple endpoints such as total protein content are not recommended, as they may under-predict the toxicity of certain test chemicals by staining dead cells. (h) The protocol should be compatible with 96-well plates and apparatus such as spectrophotometers that allow a quick and precise measurement of the endpoint. (i) Complete a detailed concentration-response experiment using a progression factor that yields graded effects between no effect and total cytotoxicity. Any desired toxicity measure can be derived from a well-designed concentration-response experiment. Experiments that seek to detect only a marker concentration, such as the highest tolerated dose or the lowest cytotoxic dose, are characterized by a lack of information and a low level of accuracy.Using In Vitro Data to Estimate In Vivo Starting Doses for Acute Toxicity

8Using In Vitro Data to Estimate In Vivo Starting Doses for Acute Toxicity

9 3.0 PROCEDURE FOR QUALIFYING A CYTOTOXICITY TEST FOR USE WITH THE REGISTRY OF CYTOTOXICITY (RC) PREDICTION MODEL

Workshop participants agreed that tests of basal cytotoxicity were sufficiently predictive for the rodent LD 50 such that cytotoxicity tests could be used to predict a starting dose for an in vivo lethality assay (NIEHS, 2001). This section discusses how to proceed. Theoretically, any in vitro test capable of determining basal cytotoxicity could be used to determine the best estimate of a starting dose for acute testing in the UDP (OECD, 1998b), the ATC method (OECD, 1996), or the Fixed Dose Procedure (FDP; TG 420, OECD, 1992). In addition, if the LD 50 predicted from cytotoxicity is high (i.e., 2,000 mg/kg b.w.), a range-finding study for the ATC or FDP may not be necessary, as testing could be initiated using the limit test of 2,000 or 5,000 mg/kg.

Before using a candidate in vitro cytotoxicity test to predict starting doses, the correlation between the in vitro test and the in vivo test must be established quantitatively. This can be achieved either by (1) in vitro testing of a large number of chemicals with known LD 50 values and deriving a regression formula based on the correlation between in vivo and in vitro data, or by (2) testing a smaller number of chemicals and applying Halle's RC prediction model (i.e., regression formula), which is derived from the correlation of in vivo and in vitro data for 347 chemicals (Halle, 1998; Spielmann et al., 1999). In the latter case, in vitro data for a small number of reference chemicals from the RC are compared with in vitro data from the RC to determine the adequacy of the test method.

Section 3.1 explains this procedure. Section 3.2 provides a set of 11 recommended reference chemicals from the RC. Section 3.3 presents experimental data from testing these 11 reference chemicals in the NRU cytotoxicity assay with both NHK cells and BALB/c 3T3 cells. 3.1 Procedure to Determine Whether a Candidate Cytotoxicity Test Can Use the RC Prediction Model

To determine whether predicted LD 50 values from a basal cytotoxicity method can be used as starting doses for routine testing of acute oral toxicity with the ATC or the UDP methods, Spielmann et al. (1999) suggested a procedure which is shown in Figure 2. Ten to twenty reference chemicals are selected from the RC (Halle, 1998) and tested in a standardized cytotoxicity test (Figure 2, Step 1). A promising candidate would be the BALB/c 3T3 NRU test (see Appendix C for the Standard Operating Procedure [SOP]), which has been highly reproducible in several validation studies (Gettings et al., 1991, 1992, 1994a, 1994b; Spielmann et al., 1991, 1993, 1996; Balls et al., 1995; Brantom et al., 1997). An alternative test, less frequently used, which has also yielded good results in validation studies, is the NHK NRU assay (Willshaw et al., 1994; Sina et al., 1995; Gettings et al., 1996; Harbell et al., 1997).

To allow comparison of the regression obtained with the candidate test (Figure 2, Step 2), selected reference chemicals should cover the entire range of cytotoxicity and be as close as possible to the RC regression line. (Section 3.2 presents a table with 11 reference chemicals from the RC and their corresponding LD 50 values.) The regression equation from the candidate test is calculated by linear regression (least square method) using the candidate IC 50 values and the corresponding LD 50 values from the RC (given in Table 1 in Section 3.2). The resulting regression is then compared with the RC regression (Figure 2, Step 3).

If the regression line obtained with the candidate cytotoxicity test parallels the RC regression and is within the + log 5 interval, then the test is considered suitable to generate IC 50 data to use with the RC regression for estimating starting doses (Figure 2, Step 4). The rationale for using the RC regression rather than the regression from the candidate cytotoxicity test is that the RC regression is based on data from 347 chemicals, while the candidate regression is based on data from only 10-20 chemicals. To predict an LD 50 starting dose, the IC 50 (in mmol/l) for the trial chemical is entered into the regression equation toUsing In Vitro Data to Estimate In Vivo Starting Doses for Acute Toxicity

10 calculate an LD 50 in mmol/kg b.w. Multiplying by the molecular weight of the trial chemical transforms the mmol/kg b.w. value into mg/kg b.w.

If the regression from the candidate test shows a significantly higher or lower slope than the RC regression, then it may be possible to adjust the candidate cytotoxicity test to a higher or lower slope. ( Note : This option was added post hoc publication of Spielmann et al., 1999.) However, a more efficient approach is likely to be to use one of the recommended cell lines (Section 2.1) and protocols (e.g., Appendices C and D). These are expected to produce results similar to the RC data. Two examples are given in Section 3.3.



### Step 1: C y t otox i c i ty te s t

T es t 1 0 - 2 0 r ef er e n c e c h e m ic a l s ( l o w - h i g h cyt o tox i c i ty ) t a ke n f r o m t h e R C

, e. g

. in th e 3T 3

- N e u t r al R ed U p t a ke t est



### Step 2 : Linea r r egr e s s ion ana l y s i s

U se I C 50 va lu es a n d RC L D 50 va lu es t o c al cu l a t e r e gr ess i o n l og (L D 50 ) = a x lo g (I C 50 ) + b



### Step 3 : C ompa r i s on of r egre s s i on s

C o m p ar e r es ul t i n g r e g re ssi o n wi t h RC r e g r ess io n

l og (L D 50 ) = 0 .4 3 5 x l o g (I C 50 ) + 0 .6 2 5 Is re g r ess i o n p a r a ll e l a n d w i t h i n F G r a n g e ?



### Step 4 : U se te s t to p r edi c t s t art i ng do s e fo r UD P o r A T C Y E S N O: Us e r e c om m ende d c e lls

an d pr o to c o l to b ette r tun e te s t s en si t ivi t y

Figure 2. Procedure for evaluating a cytotoxicity test for tiered in vitro / in vivo testing for acute oral toxicity testing (slightly modified from Spielmann et al., 1999).

3.2 Recommended Reference Chemicals from the RC for Test Qualification

To compare a regression obtained from a candidate cytotoxicity test with the RC, 11 reference chemicals (Table 1) from Halle's RC (Halle, 1998) were selected using the following criteria: • Cytotoxicity range must cover 5 - 6 logs from low to high toxicity.

• Chemical data points (IC 50x /LD 50 ) must be very close to the RC regression line.Using In Vitro Data to Estimate In Vivo Starting Doses for Acute Toxicity

11 • Chemicals must be available internationally, preferably from one supplier.

• Available purity of chemicals must be 95%. • Handling of chemicals must be acceptable with regard to sufficient solubility, low volatility, and safe use (e.g., avoid the use of known carcinogens).

Table 1. Recommended reference chemicals for evaluating a cytotoxicity test for use with the RC prediction

model

Chemical Name IC 50x

( mmol/ liter) LD 50 (mmol/ kg b.w.) LD 50

(mg/ kg b.w.) Molecular

Weight (g) CAS

Number SigmaAldrich Puchase # Purity Possible hazards;

risk phrases from MSDS
Sodium dichromate (VI) dihydrate 0.00093 0.19 49.8 298.0 7789-12-0 S9791 99.5% Very toxic, corrosive, possible carcinogen.

Cadmium II chloride 0.0064 0.48 88.0 183.3 10108-64-2 C2544 >99.0% May cause cancer. Harmful if swallowed. Prolonged exposure through inhalation or skin contact may cause serious health damage.p-Phenylenediamine 0.05 0.74 80.0 108.16 106-50-3 P6001 N/A Toxic, irritant, possible mutagen.

DL-Propranolol HCl 0.12 1.59 470.4 295.84 3506-09-0 P0884 N/A Toxic.

Trichlorfon 0.27 1.75 450.5 257.44 52-68-6 T5015 N/A Toxic by inhalation. May cause sensitization by skin
contact. Ibuprofen 0.52 4.89 1008.9 206.31 15687-27-1 I4883 N/A Harmful if swallowed. Possible risk of harm to unborn child. Nalidixic acid 1.5 5.81 1349.4 232.26 389-08-2 N8878 N/A Possible risk of harm to unborn child. May cause sensitization by inhalation, skin
contact. Salicylic acid 3.38 6.45 890.9 138.13 69-72-7 S6271 >99.0% May cause harm to unborn child. Harmful if swallowed. Irritating to eyes, respiratory system, skin. Antipyrene 11.6 9.56 1799.7 188.25 60-80-0 A5882 N/A Irritant.

Dimethyl formamide 114 38.3 2800.1 73.11 68-12-2 D8654 >99.8% Irritant, teratogen.

Glycerol 624 137 12,691.1 92.11 56-81-5 G8773 >99% Irritating to eyes, skin.Using In Vitro Data to Estimate In Vivo Starting Doses for Acute Toxicity

12 3.3 Results Obtained with the Recommended Reference Chemicals in Two Standard Tests for Basal Cytotoxicity with Human and Rodent Cells

The approach of using the RC regression (i.e., the RC prediction model) to estimate an LD 50 using data from a qualified cytotoxicity test was based on experience with comparable data obtained with various basal cytotoxicity tests (provided they followed the principles described previously). To convince even skeptical readers that cytotoxicity data for a small number of well selected reference chemicals would provide a candidate regression sufficiently comparable to the RC regression, the Institute for In Vitro Sciences (IIVS, Gaithersburg, MD) subsequently tested the 11 reference chemicals recommended in Section 3.2 using two candidate NRU test protocols (see SOPs in Appendices C and D). The cells used in this exercise were NHK obtained commercially from Clonetics Corp (Walkersville, MD, USA) and mouse BALB/c 3T3 clone A31 cells. Each of the 11 reference chemicals was tested in three independent test trials with each of the two cell types.

The outcomes of the experiments are shown in Figure 3 for the NHK and in Figure 4 for the BALB/c 3T3. Both figures depict the RC regression ± log 5 interval (black lines) and the 11 reference chemicals (triangles). Other chemicals from the RC were omitted for clarity. The new IC 50 values (means of the three trials) obtained with the NHK NRU test (Figure 3), or 3T3 NRU test (Figure 4) are shown (squares), as well as the new linear regression lines determined from these data (gray dashed line). The new regression lines obtained with NHK and 3T3 cells are within the ± log 5 interval of the RC, and, though slightly steeper, are almost parallel to the RC regression function. Thus, intercepts and regression coefficients of the experimentally obtained new regressions do not differ significantly from the literature-based RC regression equation:

RC regression: log (LD 50 ) = 0.435 x log (IC 50x ) + 0.625 New NHK NRU regression: log (LD 50 ) = 0.498 x log (IC 50 ) + 0.551 [R 2 = 0.9356]

New 3T3 NRU regression: log (LD 50 ) = 0.506 x log (IC 50 ) + 0.475 [R 2 = 0.9848]

As expected, due to selection of reference chemicals with data points close to the RC regression, the determination coefficients (R 2 ) of the new NHK and 3T3 regressions are very high.

In conclusion, by testing only 11 well selected reference chemicals from the RC, both the NHK NRU and 3T3 NRU tests yielded regression equations very close to the regression equation of the RC. Thus, both candidate cytotoxicity tests met the acceptance criteria of a test for basal cytotoxicity defined by Spielmann et al. (1999).Using In Vitro Data to Estimate In Vivo Starting Doses for Acute Toxicity

13 0.00010 0.00100 0.01000 0.10000 1.00000 10.00000 100.00000 1000.00000

0.00001 0.0001 0.001 0.01 0.1 1 10 100 1000 IC 50x (mmol/l)LD50(mmol/kg) Dimethylformamide
Salicylic acid

Sodium Bichromate VI p-Phenylenediamine Trichlorfon Glycerol Cadmium Chloride

Ibuprofen Antipyrine

Nalidixic acid Propranolol HCl

Figure 3. Regression obtained by testing the recommended reference chemicals from the RC with

human keratinocytes in the NHK NRU cytotoxicity test Figure shows the RC prediction regression (black bold line) ± log 5 interval (black thin lines) and the 11 reference chemicals (triangles). The new IC 50 /LD 50 points obtained with the NHK NRU test are shown (black squares) with the new linear regression line determined from these data (gray dashed line).

0.00010 0.00100 0.01000 0.10000 1.00000 10.00000 100.00000 1000.00000

0.00001 0.0001 0.001 0.01 0.1 1 10 100 1000 IC 50x (mmol/l)LD50(mmol/kg) Dimethylformamide Salicylic acid

Sodium Bichromate VI Propranolol HCl

p-Phenylenediamine Trichlorfon Glycerol Cadmium Chloride

Ibuprofen Antipyrine

Nalidixic acid

Figure 4. Regression obtained by testing the recommended reference chemicals from the RC with

mouse fibroblasts in the BALB/c 3T3 NRU cytotoxicity test. Figure shows the RC prediction regression (black bold line) ± log 5 interval (black thin lines) and the 11 reference chemicals (triangles). The new IC 50 /LD 50 points obtained with the BALB/c 3T3 NRU test are shown (black squares) with the new linear regression line determined from these data (gray dashed line).Using In Vitro Data to Estimate In Vivo Starting Doses for Acute Toxicity

14Using In Vitro Data to Estimate In Vivo Starting Doses for Acute Toxicity

15 4.0 RECOMMENDED BASAL CYTOTOXICITY TESTS: BALB/C 3T3 AND NORMAL HUMAN KERATINOCYTE (NHK) NEUTRAL RED UPTAKE (NRU) TESTS

4.1 Validation Status of the 3T3 NRU Test

The BALB/c 3T3 NRU test is probably the cytotoxicity test that has been used most frequently in formal validation programs, all of which were aimed at evaluation of cytotoxicity in predicting eye irritancy. Large-scale studies to be mentioned here are Phases I, II, and III of the Cosmetic, Toiletry, and Fragrance Association (CTFA) validation program (Gettings et al., 1991, 1992, 1994a, 1994b); the German eye irritation validation study (Spielmann et al., 1991, 1993, 1996); the European Commission/British Home Office (EC/HO) eye irritation validation study (Balls et al., 1995); and the European Cosmetic Toiletry and Perfumery Association (COLIPA) eye irritation study (Brantom et al., 1997). The 3T3 NRU Phototoxicity Test is a modification of the BALB/c 3T3 NRU test and involves a shorter chemical exposure and the additional application of light. The 3T3 NRU Phototoxicity Test has been fully validated (Spielmann et al., 1998a,b) and has gained regulatory acceptance.

For the purpose of evaluating the NRU test, and specifically the BALB/c 3T3 NRU test, as a standard test for basal cytotoxicity, all results available from these studies regarding the reliability (reproducibility within and between laboratories and over time) should be used to avoid wasting resources in repeating the establishment of reliability. Section 4.2 contains an example of establishing reliability of the BALB/c 3T3 NRU test from one of these studies.

4.2 Reliability of the 3T3 NRU Test

To establish interlaboratory reproducibility in the first phase of the German eye irritation validation study (Spielmann et al., 1991), 32 chemicals were tested in 12 laboratories using two tests: the hen’s egg test-chorioallantoic membrane (HET-CAM) and the BALB/c 3T3 NRU test. (NRU tests using 3T3 cells were done in accord with the SOP presented in Appendix C.) Five independent repeat tests were conducted per laboratory. Of these 32 chemicals, three compounds [n-hexane,aluminum hydroxide, and di-(2-ethylhexyl)phthalate] showed unacceptably high interlaboratory variability. For the other 29 chemicals, interlaboratory variability was acceptable (Table 2). Interlaboratory reproducibility was assessed with a standard procedure recommended by ISO 5725 (a program for analysis and reporting of proficiency tests and method evaluation studies). ISO 5725 describes reproducibility as an estimate of the limit below which the absolute value of the difference between two results determined in two different laboratories can be expected to fall, with a probability of 95%. The value tabulated in the far right column in Table 2 represents the span of about four standard deviations.Using In Vitro Data to Estimate In Vivo Starting Doses for Acute Toxicity

16 Table 2. Interlaboratory reproducibility of the 3T3 NRU cytotoxicity test determined according to ISO 5725

in 12 laboratories for 29 chemicals a

Substance CAS No. NR 50 b

(mg/ml) Interlaboratory

reproducibility c (mg/ml) Dimethylsulphoxide 67-68-5 44.06 18.36 Propylene glycol 57-55-6 36.27 25.40 Acetone 67-64-1 18.41 14.74 Ethanol 64-17-5 18.01 14.69 Acetonitrile 75-056-8 13.72 15.38 Sodium chloride 7647-14-5 7.74 3.66 Thiourea 62-56-6 6.41 5.49 2-Butoxyethanol 111-76-2 5.43 8.73 Nicotinamide 98-92-0 5.36 5.78 Glutamic acid 56-86-0 4.84 2.01 Lactic acid 598-82-3 4.16 1.56 Pyridine 110-86-1 3.71 4.78 Benzoic acid 65-85-0 3.09 1.67 Isobenzoic furano dione 85-44-9 2.47 0.63 Cyclohexanol 108-93-0 1.89 2.07 Toluene 108-88-3 1.72 3.96 Salicylic acid 69-72-7 1.63 2.04 Tin(II) chloride 7772-99-9 1.55 2.35 Nitrobenzene 98-95-3 1.39 1.33 Tetrachlorethene 127-18-4 1.08 2.35 Aniline 62-53-3 1.07 1.25 EDTA-Na salt 13235-36-4 0.95 0.50 Ascorbic acid 50-81-7 0.49 0.81 Phenol 108-95-2 0.35 0.74 Acrylamide 79-06-1 0.29 0.19 Copper (II) sulfate 7758-98-7 0.10 0.05 Sodium lauryl sulfate 151-21-3 0.093 0.09 2-Propane-1-ol 107-18-6 0.05 0.06 Benzalkonium chloride 8001-54-5 0.01 0.01

a From Spielmann et al., 1991.

b NR 50 = mean concentration of test substance reducing the viability of cells to 50% of the viability of controls.

c ISO 5725 describes reproducibility as an estimate of the limit below which the absolute value of the difference between two results determined in two different laboratories can be expected to fall, with a probability of 95%.Using In Vitro Data to Estimate In Vivo Starting Doses for Acute Toxicity

17 The second phase of the German eye irritation validation study was a blind trial for database development and involved the testing of 150 chemicals (Spielmann et al., 1993, 1996). Each chemical was assigned at random to two of the 12 total laboratories, since reproducibility of the BALB/c 3T3 NRU test was not an issue at this stage of the study. The final publication (Spielmann et al., 1997) on this phase focused on predictivity and test strategies for identification of severe eye irritants. The data from this publication have been re-analyzed for the present guidance document in the following way: since each chemical was tested in two different laboratories, the IC 50 values obtained in two laboratories were plotted against each other, as shown in Figure 5 for 147 of the 150 chemicals. (Three chemicals had to be excluded because they were not tested according to the SOP.) Note that "Lab 1" represents the total of all participating laboratories, as does "Lab 2". Thus, Figure 5 does not show the comparability of results between two given laboratories. Rather, it shows the comparability of data obtained under routine conditions between randomly selected laboratories performing the BALB/c 3T3 NRU test according to the same SOP.

Results of the correlation analysis shown in Figure 5 are quite promising, since the linear correlation line (black) deviates only slightly from the ideal line (gray line at 45° angle). The linear correlation coefficient of r = 0.88 (R 2 = 0.775) shows a sufficient comparability of the data. Outliers, where data of the two laboratories differed by more than 1 log, occurred for less than 10% of the chemicals. A predominant reason for these interlaboratory deviations, discussed in Spielmann et al. (1997), was that one laboratory had used an adequate solvent for a test chemical, while the other laboratory had tested the chemical in media at concentrations above the aqueous solubility of the chemical. Thus, concentrations reported by the second laboratory were nominal rather than actual. As a consequence of this experience, later validation studies (Spielmann et al., 1998a,b) emphasized guidance for the use of solvents.Using In Vitro Data to Estimate In Vivo Starting Doses for Acute Toxicity

18 Figure 5. Interlaboratory comparability of the 3T3 NRU cytotoxicity test for 147 test chemicals in 2

different laboratories per chemical. (Note: see text for explanation of the term "two laboratories per chemical".)

4.3 Validation Status of the NHK NRU Test

Although the NHK NRU test has been used less frequently in validation studies than has the BALB/c 3T3 NRU, the NHK NRU has been evaluated in several studies for its ability to predict eye irritation potential as reflected by Draize scores. It was used in Phases I, II, and III of the CTFA evaluation program (hydroalcoholic formulations, oil-and-water emulsions and surfactants and surfactant-containing formulations) (Gettings et al., 1991, 1994,1996); Phase III of the Soap and Detergent Manufacturers study using primarily neat surfactants and surfactant-containing formulations 

# IC 50



# 3T3 NRU

0,0001 0,001 0,01 0,1 1 10 100

0,0001 0,001 0,01 0,1 1 10 100
Lab 1Lab 2 

### y = 0.9365x - 0.0243
R 2 

### = 0.7748

mg/m l mg/m lUsing In Vitro Data to Estimate In Vivo Starting Doses for Acute Toxicity

19 (Bagley et al., 1994); as well as an independent study of surfactants and surfactant-containing formulations (Triglia et al., 1989). Many of these studies were subsequently reviewed by the Interagency Regulatory Alternatives Group, as part of a workshop review to evaluate the results of voluntary data submissions of in vitro methods to predict Draize scores (Harbell et al., 1997).

Gettings et al. (1996) evaluated the results of 34 different in vitro assays in testing 25 surfactantbased formulations for the prediction of Draize scores. The in vitro tests were ranked by discordance and separation index (i.e., the ability of the test to rank the toxicity of the 25 chemicals with the same relative rank as the Draize test). The NHK NRU test was not among the in vitro tests with the lowest discordance and highest separation index. Triglia et al. (1989), testing 12 surfactant-based formulations, suggested that sensitivity and specificity of the NHK NRU were sufficient for the test to be used as a screening tool as part of a battery of in vitro tests. Likewise, Harbell et al. (1997), in evaluating six data setscontaining 9-45 surfactant or surfactantcontaining materials, concluded that the NHK NRU had sufficient performance in predicting Draize scores that the assay could be used as a screen or adjunct over the range of toxicities found in personal care and household products.

4.4 Reliability of the NHK NRU Test

The reliability of the NHK NRU assay has been less well documented than that of the 3T3 NRU assay; however several reports have described the intralaboratory and interlaboratory variability of the test. Triglia et al. (1989) reported that 10 cytotoxicity trials in a single laboratory using the surfactant sodium lauryl sulfate (SLS) at five different concentrations produced coefficients of variation (CVs) <18% for all but the lowest concentration. (The average NRU 50 [i.e., concentration reducing NRU to 50 % of control value] from one laboratory in these trials was 4.4 μ g/ml; twelve years later the same laboratory has an average NRU 50 for SLS of 4.4 +/- 0.97 μ g/ml). Triglia et al. (1989) also reported interlaboratory variability for 12 compounds replicated in four laboratories. The interlaboratory CVs for the NRU 50 means ranged from 19% - 60%. More recently, as part of the Interagency Regulatory Alternatives Group evaluation, Harbell et al. (1997) analyzed data from two laboratories that tested 22 materials in a blind fashion. NRU 50 values for these materials showed an excellent interlaboratory correlation of 0.99. Dickson et al. (1993) also reported on variability for the NHK NRU assay and found that the NRU 50 values for SLS tested in four different keratinocyte isolates were nearly identical at 66.7, 67.5, 70.9 and 73.4 μ g/ml. Dickson et al. (1993) used a 24 h exposure rather than the 48 h exposure used for the other tests described in Sections 4.3 and 4.4.

5.0 CONCLUSION

This document provides guidance for using in vitro basal cytotoxicity assays to reduce the number of animals required for the conduct of in vivo lethality assays. The recommended approach takes advantage of the relationship between in vitro IC 50 s and in vivo LD 50 s derived from the RC for 347 chemicals (Halle and Spielmann, 1992; Halle, 1998). Detailed protocols for two recommended NRU assays, one using a rodent cell line, BALB/c 3T3 cells, and one using primary human cells, NHK, are included. Guidance is also provided for qualifying these tests, or any other in vitro cytotoxicity assay, for use with the RC regression to predict the starting dose for lethality assays.

ACKNOWLEDGEMENT

The authors wish to thank Dr. Elke Genschow with the Centre for the Documentation and Validation of Alternative Methods, Federal Institute for Health Protection of Consumers and Veterinary Medicine (Germany) for performing the correlation analysis for Figure 1 and the regression analyses for Figures 3 and 4.Using In Vitro Data to Estimate In Vivo Starting Doses for Acute Toxicity

20Using In Vitro Data to Estimate In Vivo Starting Doses for Acute Toxicity

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# APPENDIX A

Registry of Cytotoxicity List of 347 Chemicals (Sorted by IC50 and LD50 Values)Appendix A: Registry of CytotoxicityAppendix A

Registry of Cytotoxicity: List of 347 Chemicals Sorted by IC50 (mM)

RC No MEIC No Chemical IC 50x (mM) Oral Rat or Mouse LD50 (mmol/kg) Oral Rat or

Mouse LD50 (mg/kg) 2 Actinomycin D 0.0000081 0.0057 7.2 3 Aminopterin 0.000012 0.0068 3.0 132 Triphenyltin hydroxide 0.000049 0.12 44.0 6 Colchicine 0.000054 0.015 6.0 133 Cytochalasin D 0.000092 0.071 36.0 8 Digitoxin 0.00011 0.073 55.8 134 Rotenone 0.00013 0.33 130.2 9 Amethopterin 0.00014 0.3 136.4 10 Emetine 0.00016 0.14 67.3 135 2,3,7,8-Tetrachlorodibenzo-p-dioxin 0.0002 0.00035 0.1 11 Doxorubicin * HCl 0.00033 1.2 696.0 12 Puromycin 0.00033 1.43 674.4 136 Diethyldithiocarbamate sodium* 3H20 0.00039 6.66 1500.7 137 Triethyltin chloride 0.00046 0.021 5.1 138 Tributyltin chloride 0.00054 0.37 120.4 139 Retinol 0.00054 6.98 1999.8 140 6-Thioguanine 0.00057 0.96 160.5 13 Cycloheximide 0.00059 0.0071 2.0 141 Cytosine arabinoside 0.00068 12.9 3137.9 142 Methylmercury chloride 0.00071 0.23 57.7 143 Triethylene melamine 0.00078 0.005 1.0 14 Mitomycin C 0.00084 0.042 14.0 144 Sodium bichromate VI 0.00093 0.19 49.8 15 8-Azaguanine 0.0013 9.86 1500.1 145 Potassium chromate VI 0.0015 0.93 180.6 146 Potassium bichromate VI 0.002 0.65 191.2 16 Azaserine 0.002 0.98 169.7 147 Mitoxantrone 0.0024 1.32 586.8 148 Nitrogen mustard * HCl 0.0026 0.052 10.0 17 5-Fluorouracil 0.0026 1.77 230.3 149 Chromium VI trioxide 0.0027 0.8 80.0 150 Cis-platinum 0.0028 0.086 25.8 151 Hexachlorocyclopentadiene 0.0031 0.41 111.8 152 8-Hydroxyquinoline 0.0033 8.27 1200.6 18 Captan 0.0039 33.3 10009.6 153 26 Arsenic III trioxide 0.0042 0.1 19.8 154 Maneb 0.0042 16.9 4500.6 155 Benzalkonium chloride 0.0052 1.1 401.5 156 Stearyltrimethylammonium chloride 0.006 1.54 536.1 20 Cadmium II chloride 0.0064 0.48 88.0 157 38 Hexachlorophene 0.0079 0.15 61.0 21 6-Mercaptopurine 0.008 1.84 280.0 158 Dichlorophene 0.0083 10 2691.3 22 6 Digoxin 0.0085 0.023 18.0 23 Daraprim 0.0089 0.51 126.9 159 Hexadecyltrimethylammonium bromide 0.0089 1.12 408.3 25 Thio-TEPA 0.011 0.2 37.8 160 N-Methyl-N'-nitro-N-nitrosoguanidine 0.012 0.61 89.7 26 Kelthane 0.012 1.55 574.2 161 Silver I nitrate 0.013 0.29 49.3 27 Chlorpromazine 0.014 0.44 140.3 29 28 Mercury II chloride 0.015 0.0037 1.0 162 Chlorhexidine 0.015 18.2 9200.5 31 41 Chloroquine diphosphate 0.017 1.88 969.9 164 Oxatomide 0.019 3.31 1412.1 163 Cetyltrimethylammonium chloride 0.021 1.31 474.4 165 Isoproterenol * HCl 0.022 8.96 2219.8 166 Triisooctylamine 0.023 4.58 1620.2 33 p-Chloromercuribenzoic acid 0.024 0.07 25.0 167 p,p'-DDD 0.024 0.35 112.0 168 Dicoumarol 0.027 2.11 709.6 169 Epinephrine bitartrate 0.028 0.012 4.0 170 29 Thioridazine * HCl 0.029 0.88 358.2 35 Flufenamic acid 0.029 0.97 272.8 171 Fumagillin 0.031 4.36 1999.5 37 Aflatoxin B1 0.034 0.016 5.0 172 Nabam 0.035 1.54 394.8 173 39 Pentachlorophenol 0.036 0.19 50.6 174 Ambazone 0.038 3.16 749.9 175 Norepinephrine 0.039 0.12 20.3 176 Papaverine 0.045 0.96 325.8 177 Busulphan 0.046 0.0076 1.9 178 Salicylanilide 0.046 11.3 2409.7

A-1Appendix A

Registry of Cytotoxicity: List of 347 Chemicals Sorted by IC50 (mM)

RC No MEIC No Chemical IC 50x (mM) Oral Rat or Mouse LD50 (mmol/kg) Oral Rat or

Mouse LD50 (mg/kg) 179 Acrolein 0.047 0.82 46.0 180 p-Phenylenediamine 0.05 0.74 80.0 181 30 Thallium I sulfate 0.054 0.057 28.8 38 Imipramine * HCl 0.054 0.96 304.2 182 Triton X-100 0.055 2.78 1798.7 39 2,4-Dichlorophenol 0.055 3.56 580.3 183 5 Amitriptyline 0.056 1.15 319.1 184 Butylated hydroxytoluene 0.056 4.04 890.4 185 Heptachlor 0.059 0.11 41.1 186 Zineb 0.059 18.9 5211.3 40 Chlordan 0.06 1.12 458.9 41 Chloroquine sulfate 0.06 2.6 1086.8 42 p-Aminophenol 0.062 15.2 1658.9 187 4-Hexylresorcinol 0.064 2.83 549.9 43 Aldrin 0.067 0.11 40.1 44 Hydroxyzine * HCl 0.067 2.31 950.4 188 t-Butyl hydroquinone 0.069 4.81 799.6 189 Antimycin 0.07 0.45 112.6 45 Quinine * HCl 0.075 1.72 620.8 190 Chlorambucil 0.076 0.25 76.1 191 Dimenhydrinate 0.076 2.81 1320.8 192 1,3-Bis(2-chloroethyl)- 1-nitrosourea 0.078 0.093 19.9 193 5-Azacytidine 0.079 2.34 571.5 47 Naftipramide 0.084 3.45 1029.7 48 Mefenamic acid 0.087 3.27 789.1 49 Parathion 0.093 0.0069 2.0 194 p-Toluylendiamine 0.094 0.83 101.4 50 Trypan blue 0.095 6.43 6204.2 195 p,p'-DDA 0.099 2.1 590.4 196 40 VerapamilHCl 0.1 0.22 108.0 197 p,p'-DDE 0.1 2.77 880.9 51 Disulfoton 0.11 0.0073 2.0 198 Ioxynil 0.11 0.3 111.3 199 Cupric chloride 0.11 1.04 139.8 52 all-trans-Retinoic acid 0.11 6.66 2001.2 200 Dimethylaminoethyl methacrylate (polymer) 0.11 11.1 1745.4 53 43 Quinidine sulfate 0.12 1.08 456.3 54 23 Propranolol * HCl 0.12 1.59 470.4 201 13-cis-Retinoic acid 0.12 11.3 3395.4 202 Formaldehyde 0.12 26.6 798.8 55 Zinc II chloride 0.13 2.57 350.2 56 Manganese II chloride *4 H2O 0.13 7.5 1484.4 57 L-Dopa 0.13 9.03 1780.8 203 Thallium I acetate 0.14 0.13 34.2 204 Azathioprine 0.14 1.93 535.2 58 Dihydralazine sulfate 0.14 2.84 818.8 59 Tetracycline * HCl 0.14 13.4 6444.6 205 Versalide 0.15 1.22 315.3 60 Indomethacin 0.16 0.034 12.2 61 p,p'-DDT 0.16 0.32 113.4 62 Cobalt II chloride 0.16 0.62 80.5 206 Diquat dibromide 0.16 0.67 230.5 63 4 Diazepam 0.16 2.49 709.1 207 Dieldrin 0.18 0.12 45.7 64 Bendiocarb 0.18 0.8 178.6 208 Undecylenic acid 0.18 13.6 2506.6 209 Propylparaben 0.18 35.1 6325.7 65 Oxyphenbutazone 0.19 3.08 999.2 210 p-Nitrophenol 0.2 2.52 350.6 67 15 Malathion 0.2 2.68 885.4 211 Catechol 0.2 35.3 3887.2 68 2,4-Dinitrophenol 0.21 0.16 29.5 69 Secobarbital sodium 0.21 0.48 124.9 70 49 Atropine sulfate 0.22 0.92 622.7 212 p-Cresol 0.22 1.91 206.6 213 Ammonium persulfate 0.23 3.59 819.3 214 Thymol 0.23 6.52 979.6 71 Diphenhydramine * HCl 0.24 2.93 855.1 215 Chlorotetracycline 0.24 5.22 2500.0 72 Butylated hydoxyanisole 0.24 12.2 2199.3 216 Refortan 0.25 10.1 3162.3 73 Carbaryl 0.26 1.24 249.5 74 Nickel II chloride 0.27 0.81 105.0

A-2Appendix A

Registry of Cytotoxicity: List of 347 Chemicals Sorted by IC50 (mM)

RC No MEIC No Chemical IC 50x (mM) Oral Rat or Mouse LD50 (mmol/kg) Oral Rat or

Mouse LD50 (mg/kg) 75 Trichlorfon 0.27 1.75 450.5 76 Sodium dodecyl sulfate 0.27 4.45 1288.0 217 Amrinone 0.28 0.54 101.1 218 o-Phenylenediamine 0.31 9.89 1069.7 78 6-Methylcoumarin 0.31 10.5 1681.9 79 Phenylbutazone 0.32 1.22 376.3 80 2-Thiouracil 0.32 7.8 999.6 219 Hydralazine 0.33 0.56 89.7 81 27 Cupric sulfate * 5 H2O 0.33 1.2 299.6 238 Imidazolidinyl urea 0.36 9.34 2598.9 220 m-Dinitrobenzene 0.39 0.49 82.4 82 44 Diphenylhydantoin 0.39 0.79 199.3 221 2-Nitro-p-phenylenediamine 0.39 20.1 3078.5 222 Glibenclamide 0.4 6.58 3250.8 223 32 Lindane 0.41 0.26 75.6 224 n-Butyl benzoate 0.41 28.8 5133.6 225 Ammonium sulfide 0.42 3.29 168.2 226 Dodecylbenzene sodiumsulfonate 0.42 3.62 1261.6 227 46 Sodium oxalate 0.44 1.16 155.4 228 2,4,5-Trichlorophenoxyacetic acid 0.44 1.17 298.9 229 22 Dextropropoxyphene * HCl 0.49 0.22 82.7 230 42 Orphenadrine * HCl 0.49 1.39 425.2 231 Tween 80 0.49 19.1 25021.0 232 o-Cresol 0.52 1.12 121.1 233 Ibuprofen 0.52 4.89 1008.9 234 Phenylthiourea 0.54 0.02 3.0 235 25 Paraquat 0.54 0.31 57.7 83 Thiopental 0.55 2.48 601.1 84 Amobarbital 0.56 1.52 344.0 236 Hydrogen peroxide 90% 0.56 58.8 2000.4 85 Metamizol 0.58 21.5 7189.2 237 Beryllium II sulfate 0.61 0.78 82.0 239 m-Cresol 0.66 2.24 242.3 240 Pentoxifylline 0.66 4.98 1386.2 86 31 Warfarin 0.67 1.05 323.8 241 Sodium azide 0.71 0.69 44.9 87 Pentobarbital sodium 0.71 0.81 201.1 242 1,2,4-Trichlorobenzene 0.71 4.17 756.6 243 p-Anisidine 0.73 11.4 1404.1 244 Doxylamine succinate 0.75 1.21 470.1 88 Dibutyl phthalate 0.76 43.1 11998.2 89 16 2,4-Dichlorophenoxyacetic acid 0.77 1.67 369.1 90 Iproniazid 0.79 2.04 365.7 91 45 Chloramphenicol 0.79 10.5 3393.1 245 Resorcinol 0.8 2.73 300.6 246 37 Barium II nitrate 0.81 1.36 355.4 247 (+)-Thalidomide 0.81 1.55 400.3 92 Di(2-ethylhexyl)phthalate 0.84 79.4 31015.2 93 Sulfisoxazole 0.85 25.4 6790.2 248 m-Aminophenol 0.86 15.2 1658.9 94 Menthol 0.95 20.3 3172.9

249 3-Cyano-2-morpholino-5-(pyrid-4-yl)-pyridine

(Chemical 122) 0.96 1.3 346.2 250 Valproate sodium 1 10.2 1695.4 251 Scopolamine * HBr 1.08 3.3 1268.2 95 Salicylamide 1.08 13.8 1892.7 252 19 Potassium cyanide 1.12 0.15 9.8 96 Cygon 1.24 0.66 151.3 97 Phenacetin 1.27 9.21 1650.8 253 Isoxepac 1.33 0.74 198.5 254 Buflomedil 1.35 1.19 365.8 98 Methylparaben 1.42 11.5 1749.8 255 Sodium monochloroacetate 1.45 0.65 75.7 99 Nalidixic acid 1.5 5.81 1349.4 256 Tin II chloride 1.51 3.69 699.6 257 Isononylaldehyde 1.52 22.8 3243.8 100 L-Ascorbic acid 1.52 67.6 11907.1 101 Glutethimide 1.56 2.76 599.7 102 Acrylamide 1.61 2.39 169.9 258 Diethyl sebacate 1.63 56 14470.4 259 Methyl salicylate 1.7 5.83 887.1 260 Coumarin 1.71 2 292.3 103 18 Nicotine 1.79 0.31 50.3

A-3Appendix A

Registry of Cytotoxicity: List of 347 Chemicals Sorted by IC50 (mM)

RC No MEIC No Chemical IC 50x (mM) Oral Rat or Mouse LD50 (mmol/kg) Oral Rat or

Mouse LD50 (mg/kg) 104 Tolbutamide 1.81 9.62 2601.1 105 21 Theophylline 1.83 3.33 600.0 261 3 Ferrous sulfate 1.85 2.1 319.0 106 14 Sodium I fluoride 1.85 4.29 180.1 262 47 Amphetamine sulfate 1.97 0.15 55.3 107 2 Acetylsalicylic acid 2.27 5.55 999.9 108 Gibberellic acid 2.3 18.2 6304.7 109 Frusemide 2.33 7.86 2599.8 110 Acrylonitrile 2.42 1.54 81.7 263 Acetaldehyde 2.45 43.8 1929.8 111 Clofibric acid 2.61 5.82 1249.3 112 48 Caffeine 2.64 0.99 192.3 264 Chloral hydrate 2.65 2.9 479.7 113 1 Acetaminophen 2.71 15.9 2403.8 265 Streptomycin sulfate 2.73 0.34 495.6 114 Natulan * HCl 2.74 3.04 783.7 266 Potassium hexacyanoferrate III 2.82 9.02 2970.0 267 p-Hydroxybenzoic acid 2.92 15.9 2196.3 115 12 Phenol 3.01 4.4 414.1 268 1-Octanol 3.06 13.7 1784.6 116 Cyclophosphamide * H2O 3.12 0.34 94.9 269 Potassium I fluoride 3.13 4.22 245.2 117 Di(2-ethylhexyl)adipate 3.15 24.6 9117.7 270 Propionaldehyde 3.25 24.3 1411.6 271 Styrene 3.3 48 4999.7 272 Salicylic acid 3.38 6.45 890.9 273 Bromobenzene 3.46 17.2 2700.7 274 L-Cysteine 3.56 5.45 660.4 275 Nitrilotriacetic acid 3.61 7.69 1470.0 276 Ambuphylline 3.67 2.23 600.7 118 24 Phenobarbital 3.81 0.7 162.6 277 Potassium cyanate 4.14 10.4 843.6 278 Phenylephrine * HCl 4.16 1.72 350.3 279 Thioacetamide 4.17 4.01 301.3 280 Theophylline sodium acetate 4.19 2.22 582.2 281 1,2-Dibromomethane 4.2 0.62 107.8 119 Sodium salicylate 4.33 9.99 1599.5 282 (-)-Phenylephrine 4.45 2.09 349.5 283 Milrinone 4.77 0.43 90.8 120 5-Aminosalicylic acid 5.07 50.6 7749.4 121 Aminophenazone 5.39 4.32 999.3 284 Ammonium chloride 5.52 30.8 1647.8 122 Diethyl phthalate 5.52 38.7 8601.5 285 Caffeine sodium benzoate 5.67 2.54 859.4 286 Benzylpenicillin sodium 5.73 19.4 6914.2 287 Benzylalcohol 5.81 11.4 1232.9 288 1-Heptanol 6.25 28 3254.4 289 Tetrachloroethene 6.54 53.4 8854.8 290 Sodium sulfite 6.78 6.51 820.5 291 Aniline 6.9 4.72 439.6 292 Allylalcohol 6.94 1.1 63.9 293 Diisopropylamine dichloroacetate 7 7.39 1700.9 123 35 Isoniazid 7.49 4.74 650.1 294 Trichloroacetic acid 8.19 30.6 4999.4 295 2,5-Hexanedione 8.45 23.7 2705.6 124 Acetazolamide 8.49 19.3 4289.6 125 34 Carbon tetrachloride 8.51 18.2 2799.3 296 Homatropine methylbromide 9 3.24 1199.9 297 11 1,1,1-Trichloroethane 10.3 77.2 10298.5 298 Dichloroacetic acid 11.5 21.9 2823.8 299 Imidazole 11.5 27.6 1879.3 300 Antipyrine 11.6 9.56 1799.7 301 17 Xylene 12 40.5 4300.3 302 Nitrobenzene 12.2 5.2 640.2 303 Theophylline sodium 12.4 2.19 445.0 304 Calcium II chloride 12.4 9.01 999.9 305 n-Butanal 12.8 34.5 2488.1 306 Anisole 13.2 34.2 3698.7 307 2-Ethylbutanal 13.2 39.7 3977.1 308 33 Chloroform 13.4 7.61 908.4 309 Isobutanal 13.5 39 2812.7 126 Triethyl citrate 14.7 25.3 6990.9 310 Tributylamine 15.4 2.91 539.5

A-4Appendix A

Registry of Cytotoxicity: List of 347 Chemicals Sorted by IC50 (mM)

RC No MEIC No Chemical IC 50x (mM) Oral Rat or Mouse LD50 (mmol/kg) Oral Rat or

Mouse LD50 (mg/kg) 311 1-Hexanol 15.4 7.04 719.5 312 Benzoic acid 15.7 20.7 2528.1 313 Xanthinol nicotinate 15.8 32.5 14121.6 314 Saccharin 16.4 92.8 17000.0 315 Isobenzoic furano dione 17 27.1 4014.1 316 Toluene 17.1 54.3 5003.7 317 Barbital sodium 18.6 3.88 800.1 318 Trifluoroacetic acid 20.5 1.75 199.6 127 Dimethyl phthalate 23.4 35.5 6894.1 319 Methylpentinol 23.8 5.35 525.2 320 N,N-Dimethylacetamide 24.2 58.4 5089.0 321 Acetic acid 24.3 55.1 3309.3 322 1-Pentanol 24.9 34.4 3033.0 323 Urethan 25.9 28.1 2504.0 324 2-Butoxyethanol 26 12.5 1477.5 325 Cyclohexanol 26.3 20.6 2063.7 326 Halothane 31.1 28.8 5684.8 327 20 Lithium I sulfate 33.7 10.8 1187.4 328 36 Dichloromethane 34.9 18.8 1596.7 329 Sodium cyclamate 35.4 75.8 15254.0 330 Sulfuric acid 36 21.8 2138.1 331 Strontium II chloride 36.4 14.2 2251.0 332 1,4-Dioxane 38.1 47.7 4203.3 333 Lithium I chloride 38.6 17.9 758.8 334 Isobutanol 40.1 33.2 2461.4 335 Potassium hexacyanoferrate II 42.3 17.4 6409.6 336 Nicotinamide 44.4 28.7 3505.4 337 Pyridine 46.9 11.3 893.9 338 1-Butanol 52.5 10.7 793.3 339 1-Nitropropane 57.9 5.11 455.4 340 Diethylene glycol 62.1 139 14753.5 341 Lactic acid 66 41.4 3729.7 342 Piperazine 67.2 22.1 1904.1 343 Magnesium II chloride * 6 H2O 70.4 39.8 8092.5 344 13 Sodium chloride 75.9 51.3 2998.0 345 Sodium I bromide 77.4 33.4 3504.3 346 50 Potassium I chloride 82 34.9 2601.8 347 Thiourea 86 1.64 124.9 348 1-Propanol 96.5 89.8 5397.9 349 Ethyl methyl ketone 104 47.1 3396.9 350 Tetrahydrofurfuryl alcohol 111 24.5 2502.7 351 Dimethylformamide 114 38.3 2800.1 352 1,2,6-Hexanetriol 123 119 15969.8 353 Ethyl acetate 128 125 11015.0 128 10 2-Propanol 167 97.2 5842.7 354 1,3,5-Trioxane 213 8.88 800.0 355 D-Glucose 226 143 25765.7 356 2-Methoxyethanol 251 32.3 2458.4 129 Dimethyl sulfoxide 252 252 19691.3 357 Propylene glycol 342 263 20016.9 358 Acetonitrile 368 92.5 3798.1 130 9 Ethanol 379 304 14008.3 359 Acetone 444 168 9759.1 360 7 Ethylene glycol 555 138 8567.0 131 Glycerol 624 137 12619.1 361 8 Methanol 930 406 13012.3

Reference

Halle, W. 1998. Toxizitätsprüfungen in Zellkulturen für eine Vorhersage der akuten Toxizität (LD50) zur Einsparung von Tierversuchen. Life Sciences/Lebenswissenschaften, Volume 1, 94 pp., Jülich: Forschungszentrum Jülich.

A-5Appendix A: Registry of Cytotoxicity



### A-6Appendix A

Registry of Cytotoxicity: List of 347 Chemicals Sorted by LD50 (mg/kg)

RC No MEIC No Chemical IC 50x (mM) Oral Rat or Mouse LD50 (mmol/kg) Oral Rat or

Mouse LD50 (mg/kg) 135 2,3,7,8-Tetrachlorodibenzo-p-dioxin 0.0002 0.00035 0.1 29 28 Mercury II chloride 0.015 0.0037 1.0 143 Triethylene melamine 0.00078 0.005 1.0 177 Busulphan 0.046 0.0076 1.9 13 Cycloheximide 0.00059 0.0071 2.0 51 Disulfoton 0.11 0.0073 2.0 49 Parathion 0.093 0.0069 2.0 3 Aminopterin 0.000012 0.0068 3.0 234 Phenylthiourea 0.54 0.02 3.0 169 Epinephrine bitartrate 0.028 0.012 4.0 37 Aflatoxin B1 0.034 0.016 5.0 137 Triethyltin chloride 0.00046 0.021 5.1 6 Colchicine 0.000054 0.015 6.0 2 Actinomycin D 0.0000081 0.0057 7.2 252 19 Potassium cyanide 1.12 0.15 9.8 148 Nitrogen mustard * HCl 0.0026 0.052 10.0 60 Indomethacin 0.16 0.034 12.2 14 Mitomycin C 0.00084 0.042 14.0 22 6 Digoxin 0.0085 0.023 18.0 153 26 Arsenic III trioxide 0.0042 0.1 19.8 192 1,3-Bis(2-chloroethyl)- 1-nitrosourea 0.078 0.093 19.9 175 Norepinephrine 0.039 0.12 20.3 33 p-Chloromercuribenzoic acid 0.024 0.07 25.0 150 Cis-platinum 0.0028 0.086 25.8 181 30 Thallium I sulfate 0.054 0.057 28.8 68 2,4-Dinitrophenol 0.21 0.16 29.5 203 Thallium I acetate 0.14 0.13 34.2 133 Cytochalasin D 0.000092 0.071 36.0 25 Thio-TEPA 0.011 0.2 37.8 43 Aldrin 0.067 0.11 40.1 185 Heptachlor 0.059 0.11 41.1 132 Triphenyltin hydroxide 0.000049 0.12 44.0 241 Sodium azide 0.71 0.69 44.9 207 Dieldrin 0.18 0.12 45.7 179 Acrolein 0.047 0.82 46.0 161 Silver I nitrate 0.013 0.29 49.3 144 Sodium bichromate VI 0.00093 0.19 49.8 103 18 Nicotine 1.79 0.31 50.3 173 39 Pentachlorophenol 0.036 0.19 50.6 262 47 Amphetamine sulfate 1.97 0.15 55.3 8 Digitoxin 0.00011 0.073 55.8 235 25 Paraquat 0.54 0.31 57.7 142 Methylmercury chloride 0.00071 0.23 57.7 157 38 Hexachlorophene 0.0079 0.15 61.0 292 Allylalcohol 6.94 1.1 63.9 10 Emetine 0.00016 0.14 67.3 223 32 Lindane 0.41 0.26 75.6 255 Sodium monochloroacetate 1.45 0.65 75.7 190 Chlorambucil 0.076 0.25 76.1 149 Chromium VI trioxide 0.0027 0.8 80.0 180 p-Phenylenediamine 0.05 0.74 80.0 62 Cobalt II chloride 0.16 0.62 80.5 110 Acrylonitrile 2.42 1.54 81.7 237 Beryllium II sulfate 0.61 0.78 82.0 220 m-Dinitrobenzene 0.39 0.49 82.4 229 22 Dextropropoxyphene * HCl 0.49 0.22 82.7 20 Cadmium II chloride 0.0064 0.48 88.0 219 Hydralazine 0.33 0.56 89.7 160 N-Methyl-N'-nitro-N-nitrosoguanidine 0.012 0.61 89.7 283 Milrinone 4.77 0.43 90.8 116 Cyclophosphamide * H2O 3.12 0.34 94.9 217 Amrinone 0.28 0.54 101.1 194 p-Toluylendiamine 0.094 0.83 101.4 74 Nickel II chloride 0.27 0.81 105.0 281 1,2-Dibromomethane 4.2 0.62 107.8 196 40 VerapamilHCl 0.1 0.22 108.0 198 Ioxynil 0.11 0.3 111.3 151 Hexachlorocyclopentadiene 0.0031 0.41 111.8 167 p,p'-DDD 0.024 0.35 112.0 189 Antimycin 0.07 0.45 112.6 61 p,p'-DDT 0.16 0.32 113.4 138 Tributyltin chloride 0.00054 0.37 120.4 232 o-Cresol 0.52 1.12 121.1

A-7Appendix A

Registry of Cytotoxicity: List of 347 Chemicals Sorted by LD50 (mg/kg)

RC No MEIC No Chemical IC 50x (mM) Oral Rat or Mouse LD50 (mmol/kg) Oral Rat or

Mouse LD50 (mg/kg) 347 Thiourea 86 1.64 124.9 69 Secobarbital sodium 0.21 0.48 124.9 23 Daraprim 0.0089 0.51 126.9 134 Rotenone 0.00013 0.33 130.2 9 Amethopterin 0.00014 0.3 136.4 199 Cupric chloride 0.11 1.04 139.8 27 Chlorpromazine 0.014 0.44 140.3 96 Cygon 1.24 0.66 151.3 227 46 Sodium oxalate 0.44 1.16 155.4 140 6-Thioguanine 0.00057 0.96 160.5 118 24 Phenobarbital 3.81 0.7 162.6 225 Ammonium sulfide 0.42 3.29 168.2 16 Azaserine 0.002 0.98 169.7 102 Acrylamide 1.61 2.39 169.9 64 Bendiocarb 0.18 0.8 178.6 106 14 Sodium I fluoride 1.85 4.29 180.1 145 Potassium chromate VI 0.0015 0.93 180.6 146 Potassium bichromate VI 0.002 0.65 191.2 112 48 Caffeine 2.64 0.99 192.3 253 Isoxepac 1.33 0.74 198.5 82 44 Diphenylhydantoin 0.39 0.79 199.3 318 Trifluoroacetic acid 20.5 1.75 199.6 87 Pentobarbital sodium 0.71 0.81 201.1 212 p-Cresol 0.22 1.91 206.6 17 5-Fluorouracil 0.0026 1.77 230.3 206 Diquat dibromide 0.16 0.67 230.5 239 m-Cresol 0.66 2.24 242.3 269 Potassium I fluoride 3.13 4.22 245.2 73 Carbaryl 0.26 1.24 249.5 35 Flufenamic acid 0.029 0.97 272.8 21 6-Mercaptopurine 0.008 1.84 280.0 260 Coumarin 1.71 2 292.3 228 2,4,5-Trichlorophenoxyacetic acid 0.44 1.17 298.9 81 27 Cupric sulfate * 5 H2O 0.33 1.2 299.6 245 Resorcinol 0.8 2.73 300.6 279 Thioacetamide 4.17 4.01 301.3 38 Imipramine * HCl 0.054 0.96 304.2 205 Versalide 0.15 1.22 315.3 261 3 Ferrous sulfate 1.85 2.1 319.0 183 5 Amitriptyline 0.056 1.15 319.1 86 31 Warfarin 0.67 1.05 323.8 176 Papaverine 0.045 0.96 325.8 84 Amobarbital 0.56 1.52 344.0

249 3-Cyano-2-morpholino-5-(pyrid-4-yl)-pyridine

(Chemical 122) 0.96 1.3 346.2 282 (-)-Phenylephrine 4.45 2.09 349.5 55 Zinc II chloride 0.13 2.57 350.2 278 Phenylephrine * HCl 4.16 1.72 350.3 210 p-Nitrophenol 0.2 2.52 350.6 246 37 Barium II nitrate 0.81 1.36 355.4 170 29 Thioridazine * HCl 0.029 0.88 358.2 90 Iproniazid 0.79 2.04 365.7 254 Buflomedil 1.35 1.19 365.8 89 16 2,4-Dichlorophenoxyacetic acid 0.77 1.67 369.1 79 Phenylbutazone 0.32 1.22 376.3 172 Nabam 0.035 1.54 394.8 247 (+)-Thalidomide 0.81 1.55 400.3 155 Benzalkonium chloride 0.0052 1.1 401.5 159 Hexadecyltrimethylammonium bromide 0.0089 1.12 408.3 115 12 Phenol 3.01 4.4 414.1 230 42 Orphenadrine * HCl 0.49 1.39 425.2 291 Aniline 6.9 4.72 439.6 303 Theophylline sodium 12.4 2.19 445.0 75 Trichlorfon 0.27 1.75 450.5 339 1-Nitropropane 57.9 5.11 455.4 53 43 Quinidine sulfate 0.12 1.08 456.3 40 Chlordan 0.06 1.12 458.9 244 Doxylamine succinate 0.75 1.21 470.1 54 23 Propranolol * HCl 0.12 1.59 470.4 163 Cetyltrimethylammonium chloride 0.021 1.31 474.4 264 Chloral hydrate 2.65 2.9 479.7 265 Streptomycin sulfate 2.73 0.34 495.6 319 Methylpentinol 23.8 5.35 525.2

A-8Appendix A

Registry of Cytotoxicity: List of 347 Chemicals Sorted by LD50 (mg/kg)

RC No MEIC No Chemical IC 50x (mM) Oral Rat or Mouse LD50 (mmol/kg) Oral Rat or

Mouse LD50 (mg/kg) 204 Azathioprine 0.14 1.93 535.2 156 Stearyltrimethylammonium chloride 0.006 1.54 536.1 310 Tributylamine 15.4 2.91 539.5 187 4-Hexylresorcinol 0.064 2.83 549.9 193 5-Azacytidine 0.079 2.34 571.5 26 Kelthane 0.012 1.55 574.2 39 2,4-Dichlorophenol 0.055 3.56 580.3 280 Theophylline sodium acetate 4.19 2.22 582.2 147 Mitoxantrone 0.0024 1.32 586.8 195 p,p'-DDA 0.099 2.1 590.4 101 Glutethimide 1.56 2.76 599.7 105 21 Theophylline 1.83 3.33 600.0 276 Ambuphylline 3.67 2.23 600.7 83 Thiopental 0.55 2.48 601.1 45 Quinine * HCl 0.075 1.72 620.8 70 49 Atropine sulfate 0.22 0.92 622.7 302 Nitrobenzene 12.2 5.2 640.2 123 35 Isoniazid 7.49 4.74 650.1 274 L-Cysteine 3.56 5.45 660.4 12 Puromycin 0.00033 1.43 674.4 11 Doxorubicin * HCl 0.00033 1.2 696.0 256 Tin II chloride 1.51 3.69 699.6 63 4 Diazepam 0.16 2.49 709.1 168 Dicoumarol 0.027 2.11 709.6 311 1-Hexanol 15.4 7.04 719.5 174 Ambazone 0.038 3.16 749.9 242 1,2,4-Trichlorobenzene 0.71 4.17 756.6 333 Lithium I chloride 38.6 17.9 758.8 114 Natulan * HCl 2.74 3.04 783.7 48 Mefenamic acid 0.087 3.27 789.1 338 1-Butanol 52.5 10.7 793.3 202 Formaldehyde 0.12 26.6 798.8 188 t-Butyl hydroquinone 0.069 4.81 799.6 354 1,3,5-Trioxane 213 8.88 800.0 317 Barbital sodium 18.6 3.88 800.1 58 Dihydralazine sulfate 0.14 2.84 818.8 213 Ammonium persulfate 0.23 3.59 819.3 290 Sodium sulfite 6.78 6.51 820.5 277 Potassium cyanate 4.14 10.4 843.6 71 Diphenhydramine * HCl 0.24 2.93 855.1 285 Caffeine sodium benzoate 5.67 2.54 859.4 197 p,p'-DDE 0.1 2.77 880.9 67 15 Malathion 0.2 2.68 885.4 259 Methyl salicylate 1.7 5.83 887.1 184 Butylated hydroxytoluene 0.056 4.04 890.4 272 Salicylic acid 3.38 6.45 890.9 337 Pyridine 46.9 11.3 893.9 308 33 Chloroform 13.4 7.61 908.4 44 Hydroxyzine * HCl 0.067 2.31 950.4 31 41 Chloroquine diphosphate 0.017 1.88 969.9 214 Thymol 0.23 6.52 979.6 65 Oxyphenbutazone 0.19 3.08 999.2 121 Aminophenazone 5.39 4.32 999.3 80 2-Thiouracil 0.32 7.8 999.6 304 Calcium II chloride 12.4 9.01 999.9 107 2 Acetylsalicylic acid 2.27 5.55 999.9 233 Ibuprofen 0.52 4.89 1008.9 47 Naftipramide 0.084 3.45 1029.7 218 o-Phenylenediamine 0.31 9.89 1069.7 41 Chloroquine sulfate 0.06 2.6 1086.8 327 20 Lithium I sulfate 33.7 10.8 1187.4 296 Homatropine methylbromide 9 3.24 1199.9 152 8-Hydroxyquinoline 0.0033 8.27 1200.6 287 Benzylalcohol 5.81 11.4 1232.9 111 Clofibric acid 2.61 5.82 1249.3 226 Dodecylbenzene sodiumsulfonate 0.42 3.62 1261.6 251 Scopolamine * HBr 1.08 3.3 1268.2 76 Sodium dodecyl sulfate 0.27 4.45 1288.0 191 Dimenhydrinate 0.076 2.81 1320.8 99 Nalidixic acid 1.5 5.81 1349.4 240 Pentoxifylline 0.66 4.98 1386.2 243 p-Anisidine 0.73 11.4 1404.1 270 Propionaldehyde 3.25 24.3 1411.6

A-9Appendix A

Registry of Cytotoxicity: List of 347 Chemicals Sorted by LD50 (mg/kg)

RC No MEIC No Chemical IC 50x (mM) Oral Rat or Mouse LD50 (mmol/kg) Oral Rat or

Mouse LD50 (mg/kg) 164 Oxatomide 0.019 3.31 1412.1 275 Nitrilotriacetic acid 3.61 7.69 1470.0 324 2-Butoxyethanol 26 12.5 1477.5 56 Manganese II chloride *4 H2O 0.13 7.5 1484.4 15 8-Azaguanine 0.0013 9.86 1500.1 136 Diethyldithiocarbamate sodium* 3H20 0.00039 6.66 1500.7 328 36 Dichloromethane 34.9 18.8 1596.7 119 Sodium salicylate 4.33 9.99 1599.5 166 Triisooctylamine 0.023 4.58 1620.2 284 Ammonium chloride 5.52 30.8 1647.8 97 Phenacetin 1.27 9.21 1650.8 42 p-Aminophenol 0.062 15.2 1658.9 248 m-Aminophenol 0.86 15.2 1658.9 78 6-Methylcoumarin 0.31 10.5 1681.9 250 Valproate sodium 1 10.2 1695.4 293 Diisopropylamine dichloroacetate 7 7.39 1700.9 200 Dimethylaminoethyl methacrylate (polymer) 0.11 11.1 1745.4 98 Methylparaben 1.42 11.5 1749.8 57 L-Dopa 0.13 9.03 1780.8 268 1-Octanol 3.06 13.7 1784.6 182 Triton X-100 0.055 2.78 1798.7 300 Antipyrine 11.6 9.56 1799.7 299 Imidazole 11.5 27.6 1879.3 95 Salicylamide 1.08 13.8 1892.7 342 Piperazine 67.2 22.1 1904.1 263 Acetaldehyde 2.45 43.8 1929.8 171 Fumagillin 0.031 4.36 1999.5 139 Retinol 0.00054 6.98 1999.8 236 Hydrogen peroxide 90% 0.56 58.8 2000.4 52 all-trans-Retinoic acid 0.11 6.66 2001.2 325 Cyclohexanol 26.3 20.6 2063.7 330 Sulfuric acid 36 21.8 2138.1 267 p-Hydroxybenzoic acid 2.92 15.9 2196.3 72 Butylated hydoxyanisole 0.24 12.2 2199.3 165 Isoproterenol * HCl 0.022 8.96 2219.8 331 Strontium II chloride 36.4 14.2 2251.0 113 1 Acetaminophen 2.71 15.9 2403.8 178 Salicylanilide 0.046 11.3 2409.7 356 2-Methoxyethanol 251 32.3 2458.4 334 Isobutanol 40.1 33.2 2461.4 305 n-Butanal 12.8 34.5 2488.1 215 Chlorotetracycline 0.24 5.22 2500.0 350 Tetrahydrofurfuryl alcohol 111 24.5 2502.7 323 Urethan 25.9 28.1 2504.0 208 Undecylenic acid 0.18 13.6 2506.6 312 Benzoic acid 15.7 20.7 2528.1 238 Imidazolidinyl urea 0.36 9.34 2598.9 109 Frusemide 2.33 7.86 2599.8 104 Tolbutamide 1.81 9.62 2601.1 346 50 Potassium I chloride 82 34.9 2601.8 158 Dichlorophene 0.0083 10 2691.3 273 Bromobenzene 3.46 17.2 2700.7 295 2,5-Hexanedione 8.45 23.7 2705.6 125 34 Carbon tetrachloride 8.51 18.2 2799.3 351 Dimethylformamide 114 38.3 2800.1 309 Isobutanal 13.5 39 2812.7 298 Dichloroacetic acid 11.5 21.9 2823.8 266 Potassium hexacyanoferrate III 2.82 9.02 2970.0 344 13 Sodium chloride 75.9 51.3 2998.0 322 1-Pentanol 24.9 34.4 3033.0 221 2-Nitro-p-phenylenediamine 0.39 20.1 3078.5 141 Cytosine arabinoside 0.00068 12.9 3137.9 216 Refortan 0.25 10.1 3162.3 94 Menthol 0.95 20.3 3172.9 257 Isononylaldehyde 1.52 22.8 3243.8 222 Glibenclamide 0.4 6.58 3250.8 288 1-Heptanol 6.25 28 3254.4 321 Acetic acid 24.3 55.1 3309.3 91 45 Chloramphenicol 0.79 10.5 3393.1 201 13-cis-Retinoic acid 0.12 11.3 3395.4 349 Ethyl methyl ketone 104 47.1 3396.9 345 Sodium I bromide 77.4 33.4 3504.3 336 Nicotinamide 44.4 28.7 3505.4

A-10Appendix A

Registry of Cytotoxicity: List of 347 Chemicals Sorted by LD50 (mg/kg)

RC No MEIC No Chemical IC 50x (mM) Oral Rat or Mouse LD50 (mmol/kg) Oral Rat or

Mouse LD50 (mg/kg) 306 Anisole 13.2 34.2 3698.7 341 Lactic acid 66 41.4 3729.7 358 Acetonitrile 368 92.5 3798.1 211 Catechol 0.2 35.3 3887.2 307 2-Ethylbutanal 13.2 39.7 3977.1 315 Isobenzoic furano dione 17 27.1 4014.1 332 1,4-Dioxane 38.1 47.7 4203.3 124 Acetazolamide 8.49 19.3 4289.6 301 17 Xylene 12 40.5 4300.3 154 Maneb 0.0042 16.9 4500.6 294 Trichloroacetic acid 8.19 30.6 4999.4 271 Styrene 3.3 48 4999.7 316 Toluene 17.1 54.3 5003.7 320 N,N-Dimethylacetamide 24.2 58.4 5089.0 224 n-Butyl benzoate 0.41 28.8 5133.6 186 Zineb 0.059 18.9 5211.3 348 1-Propanol 96.5 89.8 5397.9 326 Halothane 31.1 28.8 5684.8 128 10 2-Propanol 167 97.2 5842.7 50 Trypan blue 0.095 6.43 6204.2 108 Gibberellic acid 2.3 18.2 6304.7 209 Propylparaben 0.18 35.1 6325.7 335 Potassium hexacyanoferrate II 42.3 17.4 6409.6 59 Tetracycline * HCl 0.14 13.4 6444.6 93 Sulfisoxazole 0.85 25.4 6790.2 127 Dimethyl phthalate 23.4 35.5 6894.1 286 Benzylpenicillin sodium 5.73 19.4 6914.2 126 Triethyl citrate 14.7 25.3 6990.9 85 Metamizol 0.58 21.5 7189.2 120 5-Aminosalicylic acid 5.07 50.6 7749.4 343 Magnesium II chloride * 6 H2O 70.4 39.8 8092.5 360 7 Ethylene glycol 555 138 8567.0 122 Diethyl phthalate 5.52 38.7 8601.5 289 Tetrachloroethene 6.54 53.4 8854.8 117 Di(2-ethylhexyl)adipate 3.15 24.6 9117.7 162 Chlorhexidine 0.015 18.2 9200.5 359 Acetone 444 168 9759.1 18 Captan 0.0039 33.3 10009.6 297 11 1,1,1-Trichloroethane 10.3 77.2 10298.5 353 Ethyl acetate 128 125 11015.0 100 L-Ascorbic acid 1.52 67.6 11907.1 88 Dibutyl phthalate 0.76 43.1 11998.2 131 Glycerol 624 137 12619.1 361 8 Methanol 930 406 13012.3 130 9 Ethanol 379 304 14008.3 313 Xanthinol nicotinate 15.8 32.5 14121.6 258 Diethyl sebacate 1.63 56 14470.4 340 Diethylene glycol 62.1 139 14753.5 329 Sodium cyclamate 35.4 75.8 15254.0 352 1,2,6-Hexanetriol 123 119 15969.8 314 Saccharin 16.4 92.8 17000.0 129 Dimethyl sulfoxide 252 252 19691.3 357 Propylene glycol 342 263 20016.9 231 Tween 80 0.49 19.1 25021.0 355 D-Glucose 226 143 25765.7 92 Di(2-ethylhexyl)phthalate 0.84 79.4 31015.2

Reference

Halle, W. 1998. Toxizitätsprüfungen in Zellkulturen für eine Vorhersage der akuten Toxizität (LD50) zur Einsparung von Tierversuchen. Life Sciences/Lebenswissenschaften, Volume 1, 94 pp., Jülich: Forschungszentrum Jülich.

A-11Appendix A: Registry of Cytotoxicity



### A-12

# APPENDIX B

List of Test Protocols for Basal Cytotoxicity

European Centre for the Validation of Alternative Methods (ECVAM) Scientific Information System (SIS)Appendix B: List of Test Protocols for Basal CytotoxicityAppendix B

B-1 

## Appendix B

List of Test Protocols for Basal Cytotoxicity European Centre for the Validation of Alternative Methods (ECVAM) Scientific Information System (SIS) http://www.ivtip.org/protocols.html#basalcyto

THE FRAME MODIFIED NEUTRAL RED UPTAKE CYTOTOXICITY TEST The cytotoxic effect of chemicals upon cells in culture is measured by cell viability (neutral red uptake) method. Topics: Basal Cytotoxicity. Contact: Dr. Richard H. Clothier, Queen's Medical Centre, UK Last update: September 1990. Protocol no: 3.

HUMAN LYMPHOCYTE CYTOTOXICITY ASSAY This method measures the leakage of DNA and lactate dehydrogenase (LDH, EC. 1.1.1 27) from lymphocytes into the surrounding medium as an indicator of cytotoxicity. This method also includes an assay of intracellular (mitochondrial) diaphorase as a measure of cellular activity (MTT assay). Topics: Basal Cytotoxicity. Contact: Prof. Jorgen Clausen, Roskilde University, DK. Last update: May 1991. Protocol no: 6.

THE USE OF MEMBRANE PERMEABILITY AS A MEASURE OF CYTOTOXICITY IN PERFUSED CELL CULTURESMembrane permeability of perfused cell cultures, as determined by the afflux of [3H]-2-deoxy-Dglucose-6-phosphate, is used as an indicator of the cytotoxic effect of chemicals. Topics: Basal Cytotoxicity. Contact: Dr. E. Walum, Bioscience Centre, SEK. Last update: June 1989. Protocol no: 9.

HEL-30 CYTOTOXICITY TEST The ability of cultured cells to synthesize protein is used to assess the effect of a test compound on cellular anabolic competence. Topics: Basal Cytotoxicity Contact: Dr. Marina Marinovich, Universita di Milano, I. Last update: April 1990. Protocol no: 14.

THE FRAME CYTOTOXICITY TEST (KENACID BLUE) The cytotoxic effect of chemicals upon cells in culture is measured by the change in total cell protein arising from the inhibition of cell proliferation (Kenacid Blue R dye binding method). Topics: Basal Cytotoxicity. Contact: Dr. Richard H. Clothier, Queen's Medical Centre, UK. Last update: July 1992. Protocol no: 15.

CYTOTOXICITY AND GENOTOXICITY IN PRIMARY CULTURES OF HUMAN HEPATOCYTES This test determines the cytotoxic and genotoxic effect of test compounds on primary cultures of human hepatocytes, by measuring cell viability, DNA damage, and unscheduled DNA synthesis. Topics: Basal Cytotoxicity, Mutagenicity. Contact: Prof. Giovanni Brambilla, University of Genoa, I. Last update: May 1992. Protocol no: 16.

MTT ASSAY This method outlines a simple assay to determine the viability/number of cells in culture, through the formation of a colored product (in a mitochondria-dependent reaction) to which the cell membrane isAppendix B: List of Test Protocols for Basal Cytotoxicity

B-2 impermeable. Topics: Basal Cytotoxicity. Contact: Dr. Rosanna Supine, Istituto Nadonale Tumori, I. Last update: April 1990. Protocol no:17.

CYTOSKELETAL ALTERATIONS AS A PARAMETER FOR ASSESSMENT OF TOXICITY Changes in the balance of cytoskeletal proteins after exposure to test compounds can be detected by indirect immunofluorescence microscopy and quantitative biochemical methods. Topics: Basal Cytotoxicity. Contact: ECVAM SIS. Last update: July 1991. Protocol no: 24.

YEAST GROWTH RATE CYTOTOXICITY TEST The cytotoxic effect of chemicals upon yeast (Saccharomyces cerevisiae) cells in culture is determined by inhibition of cell proliferation, as measured by cell density. Topics: Basal Cytotoxicity. Contact: Dr. Ingolf Cascorbi, Institute of Clinical Pharmacology, D. Last update: January 1994. Protocol no: 33.

YEAST PLASMA MEMBRANE H+-ATPASE TOXICITY TEST The effect of chemicals on the activity of the plasma membrane-bound H+-ATPase, isolated from yeast (Saccharomyces cerevisiae) cells, is used as a measure of their toxicity. Topics: Basal Cytotoxicity. Contact: Dr. Ingolf Cascorbi, Humboldt-University, D. Last update: January 1994. Protocol no: 34.

CHINESE HAMSTER OVARY CELL NA+/K+-ATPASE TEST The effect of chemicals on the activity of the plasma membrane-bound Na+/K+ -ATPase isolated from Chinese Hamster Ovary (CHO) cells is used as a measure of their toxicity. Topics: Basal Cytotoxicity Contact: Dr. Ingolf Cascorbi, Humboldt-University, D. Last update: January 1994. Protocol no: 35.

CHINESE HAMSTER OVARY (CHO) CELL PROLIFERATION TEST The inhibition of CHO cell proliferation provides an overall assessment of the toxicity of the test substance. Topics: Basal Cytotoxicity Contact: Dr. Ingolf Cascorbi, Humboldt-University, D. Last update: January 1994. Protocol no: 36.

LS-L929 CYTOTOXICITY TEST This simple cell culture-based cytotoxicity test (in which cell viability is determined by uptake of the dyes ethidium bromide and fluorescein acetate) has been developed as a general test for acute toxicity. Topics: Basal Cytotoxicity, Eye Irritation. Contact: Dr. R.B. Kemp, University College of Wales, UK. Last update: July 1992. Protocol no: 38.

V79 CYTOTOXICITY/ TEST FOR MEMBRANE DAMAGE The cytotoxic effect of test chemicals in V79 cell culture can be determined by assessing damage to the plasma membrane as determined by a nucleic acid leakage assay. Topics: Basal Cytotoxicity. Contact: Prof. Vera Bianchi, University of Padova, I. Last update: June 1990. Protocol no: 39.

BALB/C 3T3 CYTOTOXICITY TEST The cytotoxic effect of chemicals upon Balb/c 3T3 cells in culture is measured by cell viability (Neutral Red Uptake) and total cell protein (Kenacid Blue R dye binding method). Topics. Basal Cytotoxicity, Eye Irritation. Contact: Dr. med. Horst Spielmann, ZEBET BgVV, D. Last update: January 1992. Protocol no: 46, German EGA Validation Study Protocol.

QUANTITATIVE VIDEO MICROSCOPY OF INTRACELLULAR MOTION AND MITOCHONDRIA-SPECIFIC FLUORESCENCE AVEC-DIC microscopy in combination with mitochondria-specific fluorescence allows a quantitative analysis of cell organelle dynamics and fine structure in cell cultures exposed to test compounds. Topics: Basal Cytotoxicity. Contact: Dr. Toni Lindl, Inst. f. Angewandte Zellkultur, D. Last update: April 1992. Protocol no: 52.Appendix B: List of Test Protocols for Basal Cytotoxicity

B-3 UV ABSORPTION AS AN APPROXIMATION FOR CELL NUMBER The absorption of UV at 260nm in a fixed volume of solubilized cells is proportional to the cell number, and therefore can be used as a simple means of obtaining a cell count. Cell counts obtained in this way can be combined with measurements of the inhibition of DNA synthesis ([3H]-thymidine incorporation) by test compounds, to produce an index of cytotoxicity. Topics: Basal Cytotoxicity. Contact: Dr. Ming J.W. Chang, Chang Gung Medical College, Rep. of China. Last update: September 1992. Protocol no: 58.

IN VITRO PREDICTION OF THE MAXIMUM TOLERATED DOSE The results of cytotoxicity tests in primary cultures of rat hepatocytes and in MDCK and McCoy cells can be used to predict the in vivo 4-wk maximum tolerated dose in rats and dogs. A correlation between in vitro cytotoxicity, as measured in this system, and LD50 values in rats and mice has also beenestablished. Topics: Basal Cytotoxicity, Acute Systemic Toxicity. Contact: Dr. R. Shrivastava, RLCERM, F. Last update: February, 1992. Protocol no: 66.

TWO-COMPARTMENT HUMAN TISSUE CYTOTOXICITY TEST The activating system (human liver microsomes) is separated by a semi-permeable membrane from the target cells (human mononuclear leukocytes or red cells) in order to identify cytotoxic metabolites that are capable of diffusing away from the site of production. Topics: Basal Cytotoxicity, Hepatotoxicity I Metabolism - Mediated Toxicity. Contact: Dr. M.D. Tingle, University of Liverpool, UK. Last update: January 1994. Protocol no: 73.

TETRAHYMENA ASSAY FOR MEMBRANE-STABILIZING ACTIVITY The effect of a test compound on lipid structure and protein ion channels in biological membranes can be determined by using video image analysis to assess its effect on the swimming speed of the ciliated protozoan, Tetrahymena pyriformis. Topics: Basal Cytotoxicity, Ecotoxicity, Aqueous contamination. Contact: Dr. S.L. Cassidy, Dow Corning Corporation, USA. Last update: February 1994. Protocol no: 76.

CYP1A1-INDUCING POTENCY AND CYTOTOXICITY TEST IN THE HEPA-1 MOUSE HEPATOMA CELL LINE This bioassay utilizes cultured Hepa-lclc7 (Hepa-l) mouse hepatoma cells to assess the CYP1A1-inducing potency or cytotoxicity of pure test chemicals or environmental samples. In the Hepa-l induction test, the CYP1A1-inducing potency of the test sample is detected as increased aryl hydrocarbon hydroxylase (AHH) and 7-ethoxyresorufin-O-deethylase (EROD) activities. In the Hepa-l cytotoxicity test, the effect of the sample on cell viability is measured. Topics: Basal Cytotoxicity, Ecotoxicity. Contact: Dr. Sirpa Kärenlampi, Dr. Riitta Torronen, Dr. Paivi Kopponen, University of Kuopio, FIN. Last update: October 1995. Protocol no: 112, MEIC Project Protocol.Appendix B: List of Test Protocols for Basal Cytotoxicity

B-4

# APPENDIX C

Standard Operating Procedure (SOP) for the BALB/c 3T3 Neutral Red Uptake Cytotoxicity Test

(A Test for Basal Cytotoxicity)Appendix C: SOP for the BALB/c 3T3 Neutral Red Uptake Cytotoxicity TestAppendix C

C-1 

## Appendix C Table of Contents

1.0 STANDARD OPERATING PROCEDURE (SOP) FOR THE BALB/C 3T3 NEUTRAL RED UPTAKE CYTOTOXICITY TEST - A TEST FOR BASAL CYTOTOXICITY

....................... 3 1.1 Background and Introduction

.................................................................................................. 3 1.2 Rationale

..................................................................................................................................... 3 1.3 Basic Procedure

......................................................................................................................... 3 1.4 Test Limitations

......................................................................................................................... 4 1.5 Material

...................................................................................................................................... 4 1.5.1 Cell Lines

............................................................................................................................ 4 1.5.2 Technical Equipment

.......................................................................................................... 4 1.5.3 Chemicals, Media, and Sera

............................................................................................... 4 1.5.4 Preparations

....................................................................................................................... 5 1.5.4.1 Media

............................................................................................................................ 5 1.5.4.2 Neutral Red (NR) Stock Solution

.................................................................................. 5 1.5.4.3 Neutral Red (NR) Medium

............................................................................................ 5 1.5.4.4 Ethanol/Acetic Acid Solution (NR Desorb)

.................................................................. 5 1.5.4.5 Preparation of Test Chemicals

..................................................................................... 6 1.6 Methods

...................................................................................................................................... 6 1.6.1 Cell Maintenance and Culture Procedures

........................................................................ 6 1.6.1.1 Routine Culture of BALB/C 3T3 Cells

......................................................................... 6 1.6.1.2 Cell Counting

............................................................................................................... 6 1.6.1.3 Subculture

..................................................................................................................... 6 1.6.1.4 Freezing

........................................................................................................................ 7 1.6.1.5 Thawing

........................................................................................................................ 7 1.6.2 Quality Check of Assay (I): Positive Control (PC)

............................................................. 7 1.6.3 Quality Check of Assay (II): Vehicle Control (VC)

............................................................ 7 1.6.4 Quality Check of Concentration-Response

......................................................................... 8 1.6.5 Concentrations of Test Chemical

........................................................................................ 8 1.6.5.1 Range Finder Experiment

............................................................................................ 8 1.6.5.2 Main Experiment

.......................................................................................................... 8 1.6.6 Test Procedure

.................................................................................................................... 8 1.7 Data Analysis

............................................................................................................................ 10 1.8 Prediction Model

...................................................................................................................... 11 1.9 References

................................................................................................................................. 11Appendix C: SOP for The BALB/c 3T3 Neutral Red Uptake Cytotoxicity Test

C-2Appendix C: SOP for The BALB/c 3T3 Neutral Red Uptake Cytotoxicity Test

C-3 1.0 STANDARD OPERATING PROCEDURE (SOP) FOR THE BALB/C 3T3 NEUTRAL RED UPTAKE CYTOTOXICITY TEST - A TEST FOR BASAL CYTOTOXICITY

1.1 Background and Introduction

The present in vitro SOP based on Borenfreund and Puerner (1985) was originally elaborated in 1990 by ZEBET (German National Centre for the Documentation and Evaluation of Alternatives to Animal Experimentation) in co-operation with participants of the German BMFT (Ministry of Research and Technology) sponsored "BGA (Federal Health Institute) eye irritation validation study" (Spielmann et al., 1991). The SOP was used in the second phase of the study, data base development, to assess the cytotoxicity of 150 test chemicals under blind conditions (Spielmann et al., 1996). The test had successfully undergone an interlaboratory assessment phase in which 35 chemicals were tested in 12 laboratories with five independent repeat tests per laboratory (Spielmann et al., 1991). The SOP was submitted in 1992 to INVITTOX, where it is still available as Protocol No. 46 (FRAME, 1992) and published in a methods handbook (Liebsch and Spielmann, 1995).

For the present purpose of being a recommended standard test for basal cytotoxicity, the protocol was refined by adding some paragraphs and appendices, none of which change the original method. The additions are based on experience made with a modification of the test, the 3T3Neutral Red Uptake Phototoxicity Test (3T3NRUPT), which has meanwhile gained regulatory acceptance. The additions cover test acceptance criteria and recommendations on the concentration series to be tested. The RC regression for prediction of acute oral systemic rodent toxicity (Halle, 1998; Spielmann et al., 1999) is included as the prediction model in Section 1.8 for the specific used described in this document. Two deletions were made with regard to the original SOP. The second endpoint, a cell protein staining with Kenacid Blue (KB), was deleted because it did not contribute to the test. For about 90% of the chemicals tested, the KB 50 values were close or even identical to the NR 50 values, but in several cases where necrotic cells were fixed to the plastic material of the plates and then stained with KB, the KB 50 values led to an under-prediction of cytotoxicity. The second deletion is the microscopic determination of the "highest tolerated dose" (HTD), since this measure turned out to be too subjective and yielded insufficient interlaboratory comparability in the validation study.

1.2 Rationale

The NRU cytotoxicity assay procedure is a cell survival/viability chemosensitivity assay based on the ability of viable cells to incorporate and bind neutral red (NR), a supravital dye. NR is a weak cationic dye that readily penetrates cell membranes by non-ionic diffusion and accumulates intracellularly in lysosomes. Alterations of the cell surface or the sensitive lysosomal membrane lead to lysosomal fragility and other changes that gradually become irreversible. Such changes brought about by the action of xenobiotics result in a decreased uptake and binding of NR. It is thus possible to distinguish between viable, damaged, or dead cells, which is the basis of this assay.

Healthy BALB/c 3T3 cells, when maintained in culture, continuously divide and multiply over time. A toxic chemical, regardless of site or mechanism of action, will interfere with this process and result in a reduction of the growth rate as reflected by cell number. Cytotoxicity is expressed as a concentration dependent reduction of the uptake of the vital dye, NR, after one day (= one cell cycle) of chemical exposure, thus providing a sensitive, integrated signal of both cell integrity and growth inhibition.

1.3 Basic Procedure

BALB/c 3T3 cells are seeded into 96-well plates and maintained in culture for 24 hours (h) (~ 1 doubling period) to form a semi-confluent monolayer (see Section 1.6.1 for more information on cell maintenance and culture procedures). They are then exposed to the testAppendix C: SOP for The BALB/c 3T3 Neutral Red Uptake Cytotoxicity Test

C-4 compound over a range of eight concentrations. After 24 h exposure, NRU is determined for each treatment concentration and compared to that determined in control cultures. For each treatment (i.e., concentration of the test chemical) the percent inhibition of growth is calculated. The IC 50 (a.k.a., the concentration producing 50% reduction of NR uptake) is calculated from the concentration-response and expressed as μg/ml or mmol/l.

1.4 Test Limitations

• Volatile chemicals tend to evaporate under the conditions of the test; thus the IC 50 may be variable, especially when the toxicity of the compound is fairly low. This can be overcome if plates are sealed with CO 2 permeable plastic film, which is impermeable to volatile chemicals.

• Other chemicals that are difficult to test include those that are unstable or explosive in
water.

• Due to low metabolic capacity of the BALB/c 3T3 cells, the test is likely to underestimate the toxicity of chemicals that require metabolic activation to a toxic intermediary or product.

• The in vivo toxicity of substances that specifically attack dividing cells may be overestimated.

• The toxicity of substances that bind to serum proteins may be underestimated. This is overcome to a certain extent by lowering the serum content from 10% to 5% during chemical exposure. Theoretically, serum-free media can be developed for any cell line, but does not yet exist for the BALB/c 3T3 cells.

• It is possible that low cell viability readings may result in those cases where a chemical has a relatively selective effect upon the lysosomes/endosomes of the cell. An example of this would be chloroquine sulfate, which alters the pH of lysosomes/endosomes, an effect that inhibits NRU.

• Red chemicals absorbing in the range of NR might interfere with the test, provided they are present in sufficient amounts within the cells after washing, and are soluble in the NR solvent. 1.5 Material

1.5.1 Cell Lines

BALB/c 3T3 cells, clone 31 (e.g., ECACC # 86110401, European Collection of Cell Cultures, Salisbury, Wiltshire SP4 OJG, UK; CCL-163, American Type Culture Collection [ATCC], Manassas, VA, USA)

1.5.2 Technical Equipment

• Incubator: 37oC, humidified, 7.5 % CO 2 /air

• Laminar flow clean bench (standard: "biological hazard")

• Water bath: 37oC

• Inverse phase contrast microscope

• Laboratory burner

• Centrifuge (optionally: equipped with microtiter plate rotor)

• Laboratory balance

• 96-Well plate photometer equipped with 540 nm filter

• Shaker for microtiter plates

• Cell counter or hemacytometer

• Pipetting aid

• Pipettes, 8-channel-pipettes, dilution block

• Cryotubes

• Tissue culture flasks (80 cm 2

, 25 cm 2 )

• 96-Well tissue culture microtiter plates (e.g., Nunc #

167 008)

1.5.3 Chemicals, Media, and Sera

• Dulbecco’s Modification of Eagle’s Medium (DMEM) without L-Glutamine (e.g., ICN-Flow Cat. No. 12-332-54)Appendix C: SOP for The BALB/c 3T3 Neutral Red Uptake Cytotoxicity Test

C-5 • L-Glutamine 200 mM (e.g., ICN-Flow #

16-801-49)

• New Born Calf Serum (NBCS) (e.g., Biochrom #

SO 125)

Note: Due to lot variability of NBCS, first check a lot for growth stimulating properties with 3T3 cells (20-25 hrs doubling time) and then reserve sufficient amount of NBCS.

• Trypsin/EDTA solution (e.g., ICN-Flo, #

16891-49)

• Phosphate buffered saline (PBS) without Ca 2+ and Mg 2+ (for trypsinization)

• PBS with Ca 2+ and Mg 2+ (for rinsing)

• Penicillin/streptomycin solution (e.g. ICN-Flow #

16-700-49)

• Neutral Red (NR)

• Dimethyl sulfoxide (DMSO), analytical grade

• Ethanol (ETOH), analytical grade

• Glacial acetic acid, analytical grade

• Distilled H 2 O or any purified water suitable for cell culture

1.5.4 Preparations

Note: All solutions (except NR stock solution, NR medium and NR desorb), glassware, etc., shall be sterile and all procedures should be carried out under aseptic conditions and in the sterile environment of a laminar flow cabinet (biological hazard standard).

1.5.4.1 Media

DMEM (buffered with sodium bicarbonate) supplemented with (final concentrations in DMEM are quoted): (A) for Freezing 20 % NBCS 7 - 10 % DMSO

(B) for Routine Culture 10 % NBCS 4 mM Glutamine 100 IU Penicillin 100 μg/ml Streptomycin

(C) for Treatment with Test Chemicals 5 % NBCS 4 mM Glutamine 100 IU Penicillin 100 μg/ml Streptomycin

Note: The serum concentration of treatment medium is reduced to 5%, since serum proteins may mask the toxicity of the test substance. Serum cannot be totally excluded because cell growth is markedly reduced in its absence.

Complete media should be kept at 4° C and stored for no longer than two weeks.

1.5.4.2 Neutral Red (NR) Stock Solution

0.4 g NR Dye 100 ml H 2 O

Make up prior to use and store dark at room temperature for up to two months.

1.5.4.3 Neutral Red (NR) Medium

1 ml NR Stock Solution 79 ml DMEM

Note: The NR medium should be incubated overnight at 37oC and centrifuged at 600 x g for 10 min (to remove NR crystals) before adding to the cells. Alternative procedures (e.g., Millipore filtering) can be used as long as they guarantee that NR medium is free of crystals.

1.5.4.4 Ethanol/Acetic Acid Solution (NR Desorb)

1 % Glacial acetic acid solution 50 % Ethanol 49 % H 2 OAppendix C: SOP for The BALB/c 3T3 Neutral Red Uptake Cytotoxicity Test

C-6 Prepare immediately prior to use. Do not store for longer than 1h.

1.5.4.5 Preparation of Test Chemicals

1. Depending on the solubility, dissolve test chemical either in sterile treatment medium, or ETOH, or DMSO, as appropriate - at 100- fold the desired final concentration in the case of solvents. Other solvents may be usedprovided they have been tested to be noncytotoxic at the final concentration used in the test. The final solvent concentration should be kept at a constant level of 1-2 % (v/v) in the vehicle controls and in all of the eight test concentrations. This means, the test chemical is dissolved in the solvent first, and then 1-2 part(s) of this stock solution is added to 98-99 parts of sterile pre-warmed (37°C) medium. Check carefully to determine whether the chemical is still dissolved after the transfer from solvent stock solution to medium, and reduce the highest test concentration, if necessary. 2. Measure the pH of the highest concentration of the test chemical. If strong acids or bases have changed the pH of the medium, they should be neutralized with 0.1N NaOH or 0.1N HCl. In this case, prepare highest concentration of the chemical in ~ 80% of final volume, measure pH, neutralize, and add medium to desired final volume. 3. Vortex mixing and/or sonication and/or warming to 37°C may be used, if necessary, to aid solubilization. The concentrations used for relatively insoluble chemicals should range from the soluble to the precipitating dose.

Note: Test chemical must be freshly prepared immediately prior to use. Preparation under red light may be necessary, if rapid photodegradation is likely to

occur.

1.6 Methods

1.6.1 Cell Maintenance and Culture Procedures

BALB/c 3T3 cells are routinely grown as a monolayer in 80 cm 2 tissue culture grade flasks, at 37°C in a humidified atmosphere of 7.5 % CO 2 . The cells should be examined on a daily basis under a phase contrast microscope, and any changes in morphology or their adhesive properties noted. Cells should be checked regularly for the absence of mycoplasma contamination and only used if none is found.



### 1.6.1.1 Routine Culture of BALB/C 3T3 Cells

• When cells approach confluence they should be removed from the flask by trypsinization:

— Decant medium, rinse cultures with ~5 ml PBS (without Ca 2+ , Mg 2+ ) per 25 cm 2 flask.

— Wash cells by gentle agitation to remove any remaining serum that might inhibit the action of the trypsin.

— Discard the washing solution.

— Add 1-2 ml trypsin-EDTA solution to the monolayer for a few seconds.

— Remove excess trypsin-EDTA solution and incubate the cells at 37°C.

— After 2-3 minutes (min), lightly tap the flask to detach the cells into a single cell suspension.



### 1.6.1.2 Cell Counting

After detaching the cells, add 0.1-0.2 ml of routine culture medium/cm flask, i.e., 2.5 ml for a 25 cm 2 flask. Disperse the monolayer by gentle trituration. It is important to obtain a single cell suspension for exact counting. Count a sample of the cell suspension obtained using a hemocytometer or cell counter.

1.6.1.3 Subculture

After determination of cell number, the culture can be sub-cultured into another flask or seededAppendix C: SOP for The BALB/c 3T3 Neutral Red Uptake Cytotoxicity Test

C-7 into a 96-well microtiter plate. BALB/c 3T3 cells are routinely passaged at a cell density of
~ 1 x 10 6 cells in 80 cm 2 flasks every 3-4 days (average doubling time is 20-24 h).

Note: It is important that cells have overcome the lag growth phase when they are used for the test.

1.6.1.4 Freezing

Stocks of BALB/c 3T3 cells can be stored in sterile, freezing tubes in liquid nitrogen. DMSO is used as a cryoprotective agent.

• Centrifuge trypsinized cells at 200 x g.

• Suspend the cells in routine culture medium, containing 20 % NBCS, at a concentration of 1-5x10 6 cells/ml.

• Aliquot 120-180 μl of cooled DMSO into freezing tubes and fill to 1.8 ml with the cell suspension.

• Place the tubes into a freezer at -80°C for 24 h. This gives a freezing rate of 1°C/min.

• Place the frozen tubes into liquid nitrogen for storage.

1.6.1.5 Thawing

Thaw cells by putting ampules into a water bath at 37°C. Leave for as brief a time as possible.

• Resuspend the cells and transfer into routine culture medium.

• Incubate at 37°C in a humidified 7.5 % CO 2 atmosphere.

• When the cells have attached to the bottom of the flask (this may take up to 4 h), decant the supernatant and replace with fresh medium. Culture as described above. • Passage two to three times before using the cells in a cytotoxicity test.

A fresh batch of frozen cells should be thawed out approximately every two months. This period resembles a sequence of about 18 passages.

1.6.2 Quality Check of Assay (I): Positive Control (PC)

Of the many chemicals backed by sufficient history or intra- and interlaboratory repeat tests (e.g., those shown in Section 3.2 of the report) Sodium Lauryl Sulfate (SLS, CAS # 151-21-3) is one of the most frequently tested, and is therefore recommended as a PC. If a laboratory has not built a historical database on SLS, it is recommended that SLS be tested in a full-scale concentration-response test (at 8 concentrations), according to Section 1.6.5.2, concurrently with each experiment. Once historical data prove reproducibility, the PC might be applied in just one concentration (IC 50 ) on the same plate together with the test chemical. For the latter procedure, the 95% confidence interval (CI) of the IC 50 of SLS has to be established and defined as an acceptance criterion for test sensitivity in the SOP.

The historical mean IC 50 of SLS (Spielmann et. al., 1991) is 0.093 mg/ml

.

The 95% CI is 0.070 - 0.116 mg/ml.

A test meets acceptance criteria, if the IC 50 for SLS is within the 95% CI

1.6.3 Quality Check of Assay (II): Vehicle Control (VC)

The absolute value of optical density (OD 540 of NRU) obtained in the untreated vehicle control indicates whether the 1 × 10 4 cells seeded per well have grown exponentially with normal doubling time during the two days of the assay.

A test meets acceptance criteria if the mean OD 540 of VCs is 0.3Appendix C: SOP for The BALB/c 3T3 Neutral Red Uptake Cytotoxicity Test

C-8 To check for systematic cell seeding errors, untreated VCs are placed both at the left side (row 2) and the right side (row 11) of the 96-well plate (see Appendix E):

A test meets acceptance criteria if the left and the right mean of the VCs do not differ by more than 15% from the mean of all VCs.

Checks for cell seeding errors may also be performed by examining each plate under a phase contrast microscope to assure that cell quantity is consistent. Microscopic evaluation obviates the need for two rows of VCs.

1.6.4 Quality Check of ConcentrationResponse

The IC 50 derived from the concentration-response should be backed by at least two, or if possible, three responses between 10 and 90% inhibition of NRU. If this is not the case, and the concentration progression factor can be easily reduced, reject the experiment and repeat it with a smaller progression factor.

1.6.5 Concentrations of Test Chemical

1.6.5.1 Range Finder Experiment

Test eight concentrations of the test chemical by diluting the stock solution with a constant factor (e.g., 2 10 = 3.16, see Appendix F), covering a large range, e.g.,:

1 3.16 10 31.6 100 316 1000 3160 μg/ml

1.6.5.2 Main Experiment

Depending on the slope of the concentrationresponse curve estimated from the range finder, the dilution/progression factor in the concentration series of the main experiment should be smaller (e.g., 6 10 = 1.47). Try to cover the relevant concentration range (between 10% and 90% effect) with at least three points of a graded effect, avoiding too many non-cytotoxic and/or 100%-cytotoxic concentrations. Experiments revealing less than three cytotoxic concentrations in the relevant range, shall be repeated, where possible, with a smaller dilution factor. (Taking into account pipetting errors, a progression factor of 1.21 is regarded the smallest factor achievable.)

1.6.6 Test Procedure

See Table C.1 for a flow chart of the test procedure. Appendix G contains a recommended template for documenting the relevant data generated by the BALB/c 3T3 NRU assay.

1st day after growing up the cells from frozen stock:

1. Prepare a cell suspension of 1x10 5 cells/ml in culture medium. Using a multi-channel pipette, dispense 100 μl culture medium only into the peripheral wells of a 96-well tissue culture microtiter plate (= blanks). In the remaining wells, dispense 100 μl of a cell suspension of 1x10 5 cells/ml (= 1x10 4 cells/well). Prepare one plate per chemical to be tested and one plate for the PC.

[Note: Individual plates for the PC are for establishing an historical database. Once an IC 50 mean has been determined, one need only include that PC concentration in the test material plate.]

2. Incubate cells for 24 h (7.5% CO 2 , 37oC) so that cells form a half-confluent monolayer. This incubation period assures cell recovery and adherence and progression to exponential growth phase.

3. Examine each plate under a phase contrast microscope to assure that cell growth is relatively even across the microtiter plate. This check is performed to identify experimental errors.

2nd day

1. After 24 h incubation, aspirate culture medium from the cells.Appendix C: SOP for The BALB/c 3T3 Neutral Red Uptake Cytotoxicity Test

C-9 2. Per well, add 100 μl of treatment medium containing either the appropriate concentration of test chemical, or the PC, or nothing but vehicle (VC).

3. Incubate cells for 24 h (7.5% CO 2 , 37oC).

3rd day

A) Microscopic Evaluation

After 24 h treatment, examine each plate under a phase contrast microscope to identify systematic cell seeding errors and growth characteristics of control and treated cells. Record changes in morphology of the cells due to cytotoxic effects of the test chemical, but do not use these records for the calculation of HTD or any other quantitative measure of cytotoxicity. Undesirable growth characteristics of control cells may indicate experimental error and may be cause for rejection of the assay.

B) Measurement of NRU

This method is based upon that of Ellen Borenfreund (Borenfreund and Puerner, 1985). The uptake of NR into the lysosomes/endosomes and vacuoles of living cells is used as a quantitative indication of cell number and viability.

1. Wash the cells with 150 μl pre-warmed PBS. Remove the washing solution by gentle tapping. Add 100 μl NR medium and incubate at 37°C in a humidified atmosphere of 7.5 % C0 2 for 3 h.

2. After incubation, remove the NR medium, and wash cells with 150 μl PBS.

3. Decant and blot PBS totally. (Optionally: centrifuge the reversed plate.)

4. Add exactly 150 μl NR Desorb (ETOH/acetic acid) solution to all wells, including blanks.

5. Shake microtiter plate rapidly on a microtiter plate shaker for 10 min until NR has been extracted from the cells and formed a homogeneous solution.

6. Measure the absorption of the resulting colored solution at 540 nm in a microtiter plate reader, using the blanks as a reference. Save raw data in a file format (e.g., ASCII, TXT, XLS) appropriate for further analysis of the concentration-response and calculation of IC 50 .Appendix C: SOP for The BALB/c 3T3 Neutral Red Uptake Cytotoxicity Test

C-10 Table C.1. 3T3 NRU Cytotoxicity Test: Flow Chart

TIME (h) PROCEDURE

00:00

23:00

24:00

48:00

51:00

51:40

51:50

1.7 Data Analysis

A calculation of cell viability expressed as NRU is made for each concentration of the test chemical by using the mean NRU of the six replicate values per test concentration. This value is compared with the mean NRU of all VC values (provided VCs have met the VC acceptance criteria). Relative cell viability is then expressed as percent of untreated VC. If achievable, the eight concentrations of each compound tested should span the range of no effect up to total inhibition of cell viability. Seed 96-well plates: 1x10 4 cells / 100 μl DMEM culture medium / well

Incubate (37°C / 7.5% CO 2 / 22-24 h)

Remove culture medium

Treat with 8 concentrations of test chemical in treatment medium (100 μl) (untreated vehicle control = treatment medium)

Incubate (37°C / 7.5% CO 2 / 24 h)

Microscopic evaluation of morphological alterations

Remove treatment medium

wash once with 150 μ l PBS Add 100 μl NR medium

Incubate (37°C / 7.5% CO 2 / 3 h)

Discard NR Medium Wash once with 150 μl PBS Add 150 μl NR desorbing fixative (ETOH/Acetic acid solution)

Shake plate for 10 min

Detect NR Absorption at 540 nm (i.e., cell viability)Appendix C: SOP for The BALB/c 3T3 Neutral Red Uptake Cytotoxicity Test

C-11 Where possible, the concentration of a test chemical reflecting a 50% inhibition of cell viability (i.e., the IC 50 ) is determined from the concentration-response. This can be done either by applying:

• A manual graphical fitting method. The use of probability paper with "x = log" and "y = probit" scales is recommended because in most cases the concentration-response function will become almost linear in the relevant range. Semi-log paper could also be used for this technique.

or

• any appropriate non-linear regression procedure (preferably a Hill function* or alogistic regression) to the concentrationresponse data. Before using the IC 50 for further calculations, the quality of the fit should be appropriately checked. (* = Hill functions are monotonous and sigmoidal in shape and represent an acceptable model for many dose response curves.)

More sophisticated programs specially developed for concentration-response analysis from 96-wellplates can also be used. An example is PHOTO32, which uses a nonmonotonous curve fitting algorithm (Holzhütter and Quedenau, 1995) and addresses the influence of variability on the IC 50 by bootstrapping procedures performed on concentration replicates (Holzhütter, 1997).

Before using the IC 50 information in any subsequent estimation of rodent LD 50 , be sure that the IC 50 data are expressed as mmol/l since the prediction model described in this guidance document is based on the relationship between the LD 50 (in mmol/kg) and the IC 50 (in mmol/l).

1.8 Prediction Model

In general, basal cytotoxicity is highly valuable information per se, which can be used in combination with other information, e.g., bioavailability, for many purposes in the process of safety or risk evaluation. For the purpose of this document, basal cytotoxicity is to be used to predict starting doses for in vivo acute oral LD 50 values in rodents. After testing the reference chemicals recommended in Section 3.2 of this guidance document and qualifying the test as described in Section 3.1 (see Section 3.3 of the report for examples with two different cell types), best estimates of starting doses for in vivo acute oral toxicity tests are predicted according to the following prediction model:

log (LD 50 [mmol/kg]) = 0.435 x log (IC 50 [mmol/l]) + 0.625

1.9 References

Borenfreund, E. and J.A. Puerner. 1985. Toxicity determination in vitro by morphological alterations and neutral red absorption. Toxicol. Lett. 24: 119-124.

FRAME. 1992. BALB/c 3T3 NRU Cytotoxicity Test, INVITTOX Protocol No.46, Nottingham. Internet address: http://embryo.ib.amwaw.edu.pl/invittox/.

Halle, W. 1998. Toxizitätsprüfungen in Zellkulturen für eine Vorhersage der akuten Toxizität (LD 50 ) zur Einsparung von Tierversuchen. Life Sciences/ Lebenswissenschaften, Volume 1, 94 pp., Jülich: Forschungszentrum Jülich.

Holzhütter, H.G. 1997. A general measure of in vitro phototoxicity derived from pairs of dose response curves and its use for predicting in vivo phototoxicity of chemicals. ATLA 25: 445-462.

Holzhütter, H.G. and J. Quedenau. 1995. Mathematical modelling of cellular responses to external signals. J. Biol. Syst. 3: 127-138.

Liebsch, M. and H. Spielmann. 1995. BALB/c 3T3 Cytotoxicity Test. In : Methods in Molecular Biology , Vol. 43, In Vitro Toxicity Testing Protocols. S. O'Hare, C.K. Atterwill, eds., pp. 177-187. Humana Press, Totowa, NJ.

Spielmann, H., E. Genschow, M. Liebsch, and W. Halle. 1999. Determination of the starting dose for acute oral toxicity (LD50) testing in the up and down procedure (UDP) from cytotoxicity data. ATLA 27: 957-966.Appendix C: SOP for The BALB/c 3T3 Neutral Red Uptake Cytotoxicity Test

C-12 Spielmann, H., S. Gerner, S. Kalweit, R. Moog, T. Wirnserberger, K. Krauser, R. Kreiling, H. Kreuzer, N.P. Luepke, H.G. Miltenburger, N. Müller, P. Murmann, W. Pape, B. Siegmund, J. Spengler, W. Steiling, and F.J. Wiebel. 1991. Interlaboratory assessment of alternatives to the Draize eye irritation test in Germany. Toxicol. In Vitro 5: 539-542.

Spielmann, H., M. Liebsch, S. Kalweit, F. Moldenhauer, T. Wirnsberger, H.G. Holzhütter, B. Schneider, S. Glaser, I. Gerner, W.J.W. Pape, R. Kreiling, K. Krauser, H.G. Miltenburger, W. Steiling, N.P. Luepke, N. Müller, H. Kreuzer, P. Mürmann, J. Spengler, E. Bertram-Neis, B. Siegemund, and F.J. Wiebel. 1996. Results of a validation study in Germany on two in vitro alternatives to the Draize eye irritation test, the HET-CAM test and the 3T3 NRU cytotoxicity test. ATLA 24: 741-858.Appendix C: SOP for The BALB/c 3T3 Neutral Red Uptake Cytotoxicity Test

C-13

# APPENDIX D

Standard Operating Procedure (SOP) for the Normal Human Epidermal Keratinocyte Neutral Red Uptake Cytotoxicity Test (A Test for Basal Cytotoxicity)Appendix D: SOP for the Normal Human Epidermal Keratinocyte Neutral Red Uptake Cytotoxicity TestAppendix D

D-1 

## Appendix D Table of Contents

1.0 STANDARD OPERATING PROCEDURE (SOP) FOR THE NORM AL HU MAN EP ID ERM AL KERA TIN

OC YTE NEUTRAL RED UPTAKE CYTOTOXICITY TEST - A TEST FOR BASAL CYTOTOXICITY

............................................................................................................... 3 1.1 Background and Introduction

.................................................................................................. 3 1.2 Rationale

..................................................................................................................................... 3 1.3 Basic Procedure

......................................................................................................................... 3 1.4 Test Limitations

......................................................................................................................... 3 1.5 Material

...................................................................................................................................... 4 1.5.1 Cell Lines

............................................................................................................................ 4 1.5.2 Technical Equipment

.......................................................................................................... 4 1.5.3 Chemicals, Media, and Sera

............................................................................................... 4 1.5.4 Preparations

....................................................................................................................... 5 1.5.4.1 Culture and Treatment Medium

..................................................................................... 5 1.5.4.2 Neutral Red (NR) Stock Solution

................................................................................... 5 1.5.4.3 Neutral Red (NR) Medium

............................................................................................. 5 1.5.4.4 Wash/Fix Solution

.......................................................................................................... 5 1.5.4.5 Ethanol/Acetic Acid Solution (NR Desorb)

.................................................................... 5 1.5.4.6 Preparation of Test Chemicals

...................................................................................... 5 1.6 Methods

...................................................................................................................................... 6 1.6.1 Cell Maintenance and Culture Procedures

........................................................................ 6 1.6.1.1 Receipt of Keratinocytes

................................................................................................ 6 1.6.1.2 Thawing Cryopreserved Keratinocytes

.......................................................................... 6 1.6.1.3 Subculturing the Keratinocytes

...................................................................................... 6 1.6.1.4 Freezing Keratinocytes

.................................................................................................. 7 1.6.2 Quality Check of Assay (I): Positive Control (PC)

............................................................. 7 1.6.3 Quality Check of Assay (II): Vehicle Control (VC)

............................................................ 7 1.6.4 Quality Check of Concentration-Response

......................................................................... 7 1.6.5 Concentrations of Test Chemical

........................................................................................ 7 1.6.5.1 Range Finder Experiment

.............................................................................................. 7 1.6.5.2 Main Experiment

............................................................................................................ 8 1.6.6 Test Procedure

.................................................................................................................... 8 1.7 Data Analysis

.............................................................................................................................. 9 1.8 Prediction Model

...................................................................................................................... 10 1.9 References

................................................................................................................................. 10Appendix D: SOP for The NHK Neutral Red Uptake Cytotoxicity Test

D-2Appendix D: SOP for The NHK Neutral Red Uptake Cytotoxicity Test

D-3 1.0 STANDARD OPERATING PROCEDURE (SOP) FOR THE NORM AL HU

MAN EPID

ER MAL KERA TIN OC YTE NEUTRAL RED UPTAKE CYTOTOXICITY TEST - A TEST FOR BASAL CYTOTOXICITY

1.1 Background and Introduction

This SOP, based on a NRU assay by Borenfreund and Puerner (1984) using epidermal keratinocytes (Heimann and Rice, 1983), was obtained from the Institute of In Vitro Sciences (IIVS). Formulations for the media and solutions correspond to Clonetics products by BioWhittaker, Inc. For the present purpose of being a recommended standard test for basal cytotoxicity, the protocol from IIVS was embellished by adding details on equipment, media and reagent components, and experimental procedure to make it easier for novice users to follow. For the specific purpose of this guidance document, the RC regression for prediction of acute oral systemic rodent toxicity (Halle, 1998; Spielmann et al., 1999) is included as the prediction model in Section 1.8.

1.2 Rationale

The NRU cytotoxicity assay procedure is a cell survival/viability chemosensitivity assay based on the ability of viable cells to incorporate and bind neutral red (NR), a supravital dye. NR is a weak cationic dye that readily penetrates cell membranes by non-ionic diffusion and accumulates intracellularly in lysosomes. Alterations of the cell surface or the sensitive lysosomal membrane lead to lysosomal fragility and other changes that gradually become irreversible. Such changes brought about by the action of xenobiotics result in a decreased uptake and binding of NR. It is thus possible to distinguish between viable, damaged, or dead cells, which is the basis of this assay.

Healthy normal human keratinocytes (NHK) cells,when appropriately maintained in culture in a subconfluent state, continuously divide and multiply over time. A toxic chemical, regardless of site or mechanism of action, will interfere with this process and result in cell death and/or a reduction of the growth rate as reflected by cell number. Cytotoxicity is expressed as a concentration dependent reduction of the uptake of the vital dye, NR, after two days of chemical exposure, thus providing a sensitive, integrated signal of both cell integrity and growth inhibition.

1.3 Basic Procedure

NHK cells are seeded into 96-well plates and maintained in culture until cells form a 30-50% confluent monolayer. They are then exposed to the test compound over a range of six to eight concentrations. After 48 hours (h) exposure, NRU is determined for each treatment concentration and compared to that determined in control cultures. For each treatment (i.e., concentration of the test chemical) the percent inhibition of growth is calculated. The IC 50 (a.k.a., NRU 50 , the concentration producing 50% reduction of NR uptake) is calculated from the concentration-response and expressed as μg/ml or mmol/l.

1.4 Test Limitations



### • Volatile chemicals tend to evaporate under the conditions of the test; thus the IC 50 may be variable, especially when the toxicity of the compound is fairly low. This can be overcome if plates are sealed with CO 2 permeable plastic film, which is impermeable to volatile chemicals.



### • Materials that are not readily soluble in serum-free aqueous media may be difficult to test, and their in vivo toxicity potentially underestimated.



### • Other chemicals that are difficult to test include those that are unstable or explosive in
water.



### • The in vivo toxicity of substances that specifically attack dividing cells may be overestimated.



### • It is possible that low cell viability readings may result in those cases where a chemical has a relatively selective effect upon theAppendix D: SOP for The NHK Neutral Red Uptake Cytotoxicity Test

D-4 lysosomes/endosomes of the cell. An example of this would be chloroquine sulphate, which alters the pH of lysosomes/endosomes, an effect that inhibits NRU.



### • Red chemicals absorbing in the range of NR might interfere with the test, provided they are present in sufficient amounts within the cells after washing, and are soluble in the NR solvent.

1.5 Material

1.5.1 Cell Lines

NHK cells (e.g., Clonetics #CC-2507 for cryopreserved cells or

Clonetics #CC-2607 for proliferating cells , BioWhittaker, Inc., USA)

1.5.2 Technical Equipment

• Incubator: 37o ± 1 o C , humidified, 5 ± 1 % CO 2 /air

• Laminar flow clean bench (standard: "biological hazard")

• Water bath: 37o ± 1 o C

• Inverse phase contrast microscope

• Centrifuge

• Laboratory balance

• 96-Well plate photometer equipped with 540 or 550 nm filter

• Shaker for microtiter plates

• Cell counter or hemocytometer

• Pipetting aid

• Pipettes, 8-channel-pipettes, dilution block

• Cryotubes

• Tissue culture flasks (80 cm 2

, 25 cm 2 ) • 96-Well tissue culture microtiter plates (e.g., Nunc # 167 008)

Note : Tissue culture flasks and microtiter plates should be prescreened to ensure that they adequately support the growth of NHK.

1.5.3 Chemicals, Media, and Sera



### • Ke ra tinoc yte G row

th Me dium (K GM) complete with epide

rma l growth f ac tor , insulin, hydr ocortisone

, a ntimicr obial agents and supplemented w ith bovine pituita ry extr act (e.g., Clonetics # CC-3001 )



### • HE PE S Buf fer ed Sa line Solution ( HE PESBSS) (e.g., Clonetics # CC-5022)



### • 0.025% Tr

ypsin/ED TA solution (e.g., Clonetics # CC-5012)



### • Tr ypsin N eutra

liz ing Solution (T

NS) (e.g., Clonetics # CC-5002)

• Phospha

te Buff ere d Saline ( PBS)

• 10% fetal bovine serum (FBS)

• Neutral Red (NR)

• Dimethyl sulfoxide (DMSO), analytical grade

• Ethanol (ETOH), analytical grade

• Glacial acetic acid, analytical grade

• Hanks' Balanced Saline Solution without Ca 2+ or Mg 2+ (CMF-HBSS) (e.g., Invitrogen # 14170)

• Formaldehyde

• Calcium chloride

• Distilled H 2 O or any purified water suitable for cell cultureAppendix D: SOP for The NHK Neutral Red Uptake Cytotoxicity Test

D-5 1.5.4 Preparations

Note : All solutions (except NR stock solution, NR medium and NR desorb), glassware, etc., shall be sterile and all procedures should be carried out under aseptic conditions and in the sterile environment of a laminar flow cabinet (biological hazard standard).

1.5.4.1 Culture and Treatment Medium

KGM supplemented with :
0.1 ng/ml Human recombinant

epidermal growth factor 5 g/ml Insulin 0.5 g/ml Hydrocortisone 50 g/ml Gentamicin 50 ng/ml Amphotericin B 0.15 mM Calcium 2 ml 7.5 mg/ml Bovine pituitary

extract

Complete media should be kept at 4°C and stored for no longer than two weeks

.

1.5.4.2 Neutral Red (NR) Stock Solution

0.4 g NR Dye 100 ml H 2 O

Make up prior to use and store dark at room temperature for up to two months.

1.5.4.3 Neutral Red (NR) Medium

1 ml NR Stock Solution 79 ml KGM

Note : The NR medium should be incubated overnight at 37oC and centrifuged at 600 x g for 10 min (to remove NR crystals) before adding to the cells. Alternative procedures (e.g., Millipore filtering) can be used as long as they guarantee that NR medium is free of crystals.

1.5.4.4 Wash/Fix Solution

0.5% Formaldehyde 1.0% Calcium chloride 98.5% H 2 O 1.5.4.5 Ethanol/Acetic Acid Solution (NR Desorb)

1 % Glacial acetic acid solution 50 % Ethanol 49 % H 2 O

Prepare immediately prior to use. Do not store for longer than 1 h.

1.5.4.6 Preparation of Test Chemicals

1. The test artic

le should be dissolved in KG M, de ioniz ed distilled wa te

r, ETOH

, DMSO,

ac etone , or other a ppr

opria te solvent. Other solvents may be used provided they have been tested to be non-cytotoxic at the final concentration used in the test. I f the solvent is

some thing othe

r tha n K GM, a 100X

conc entra te of ea ch de sired f

ina l conce ntr ation of test a rticle should be pre par ed . This 100X concentrated dosing solution is then diluted 1:100 directly into sterile pre-warmed (37°C) KGM. This should ensure that the final solvent concentration in culture wells should not exceed 1% (v/v) in the vehicle controls and in all of the eight test concentrations. Check carefully to determine whether the chemical is still dissolved after the transfer from solvent stock solution to medium, and reduce highest test concentration, if necessary. The sta bility of the test a rticle under the actua l expe rimental c onditions should be deter mined for eac h exper ime nt. 2. Measure the pH of the highest concentration of the test chemical. If strong acids or bases have changed the pH of the medium, they should be neutralized with 0.1N NaOH or 0.1N HCI. In this case, prepare highest concentration of the chemical in ~ 80% of final volume, measure pH, neutralize, and add KGM to desired final volume. 3. Vortex mixing and/or sonication and/or warming to 37°C may be used, if necessary, to aid solubilization. The concentrations used for relatively insoluble chemicals should range from the soluble to the precipitating dose.Appendix D: SOP for The NHK Neutral Red Uptake Cytotoxicity Test

D-6 Note : Test chemical must be freshly prepared immediately prior to use. Preparation under red light may be necessary, if rapid photodegradation is likely to

occur.

1.6 Methods

A good discussion of the techniques used in the multiple-well plate assays, such as those described in this section, is given by Harbell (2001).

1.6.1 Cell Maintenance and Culture Procedures

1.6.1.1 Receipt of Keratinocytes

Upon receipt of proliferating keratinocytes, the cultures will be observed microscopically for signs of distress (e.g., floating cells, excessive debris, or lack of mitotic figures). Cultures exhibiting these properties will be discarded. Then perform the following:

• De conta

minate the outside of the c ultur e f la sks with 70% ETO H.

• Incubate the c ultur

es at 37o ± 1 o C for a minimum of 60 minutes (min) to a llow the
te mpera ture of the medium to equilibr ate.

• Aseptic

ally re move the medium and repla ce with fr esh K GM wa

rmed to approxima

tely 37o C.

• Unle ss other

wise specified, the culture s a re then incubated at 37o ± 1 o C and 5 ± 1% CO 2 in air

. Upon re ce ipt of c ryopr

eserved ke

ra tinoc yte s, the

ce lls should be stored in liquid nitr ogen.

1.6.1.2 Thawing Cryopreserved Keratinocytes

• Thaw cells by putting ampules into a water bath at 37°C for as brief a time as possible. Do not thaw cells at room temperature or by hand. Seed t

he thaw ed ce lls into c ultur

e fla sks

as quic kly a s possible a nd with minimal ha ndling.

• Slowly (taking approximately 1-2 min) add 9 ml of KGM to the cells suspended in the cryoprotective solution and transfer 3500 cells/cm 2 into flasks containing routine pre-warmed culture medium.

• Incubate the

c ultur es at 37o ± 1 o C until the ce lls
atta ch to the fla sk, a t which time the KGM should be re moved a nd re pla ce d w ith f

re sh KG M.

• Unle ss other

wise specified, the ce lls should be incubated at 37o ± 1 o C and 5 ± 1% CO 2 in air and fed e ver

y 2-3 days until the

y are 50-80% conf lue nt.

1.6.1.3 Subculturing the Keratinocytes

• When the ker atinocyte culture in a 25 c m

2 f la sk is 50 to 80%

c onf luent, remove the

me dium and rinse the culture twic

e with 5 ml HEPES-BSS. Disc ard the wa shing solution.

• Add 2 ml trypsin/ED TA solution to eac h fla sk and remove a fter 15 to 30 sec onds. I nc uba te the fla sk at r oom temper ature for 3 to 7 min. When more than 50% of the c ells be come dislodged, r ap the fla sk shar ply a gainst the
pa lm of the ha nd.

• When most of the ce lls have become de ta che d fr om the sur

fa ce, r inse the f

lask with 5 ml of
room te mpera ture TN S.

• Then rinse the flask w ith 5 ml CMF-HBSS and
tr ansfe r the c ell suspension to a centr

ifuge tube

.

• Pe llet the c ells by ce ntrif

ugation for 5 min at appr oxima

tely 220 x g. Remove the
supe rna ta nt by aspiration.

• Re suspe

nd the ker atinocyte pe lle t by gentle trituration (to have single cells) in KGM. Count a sample of the cell suspension obtained using a hemacytometer (Trypan Blue exclusion) or cell counter.

• Pr epare a suspension of 0.8 to 1.0x10 4 c ells/ml in K GM. Transfer the cells into flasks containing pre-warmed growth medium at 3500 cells/cm 2 . The keratinocyte cultures may be sustained through approximately three passages.Appendix D: SOP for The NHK Neutral Red Uptake Cytotoxicity Test

D-7 For subculturing into 96-we ll plates, obta

in the cell suspension a s descr

ibe d above

. Add 250 μ l ce ll suspension to the

a ppr opria te we lls on eac

h 96- we ll plate. ( Note: evapora

tion of the medium c an be a pr oblem; the

re for e, the edge wells should re ceive 250 μl PBS. I ncuba

te the c ultur

es in a humidif

ie d incubator at 37o ± 1 o C and 5 ± 1%

CO 2 in air

.

1.6.1.4 Freezing Keratinocytes

• Harvest the cells as above and resuspend the single cells in cold freezing solution (e.g., 80% growth medium, 10% fetal bovine serum [FBS], 10% DMSO) at 5x10 5 to 2x10 6 cells per ml. Aliquot to freezing vials.

• Insulate the vials and place into a –70 o C freezer overnight (12-24 h).

• Place vials into liquid nitrogen for storage.

1.6.2 Quality Check of Assay (I): Positive Control (PC)

Of the many chemicals backed by sufficient history or intra- and interlaboratory repeat tests (e.g., those shown in Section 3.2 of the report) Sodium Lauryl Sulfate (SLS, CAS # 151-21-3 ) is one of the most frequently tested, and is therefore recommended as a PC. If a laboratory has not built a historical database on SLS, it is recommended that SLS be tested in a full-scale concentration-response test (at six to eight concentrations), according to Section 1.6.5.2, concurrently with each test article experiment. Once historical data prove reproducibility, the PC might be applied in just one concentration (IC 50 ) on the same plate together with the test chemical (also noted in Section 1.6.6). For the latter procedure, two standard deviations of the IC 50 for SLS is the acceptance criterion for test sensitivity.

A test meets acceptance criteria, if the IC 50 for SLS is within 2 standard deviations of the historical mean. 1.6.3 Quality Check of Assay (II): Vehicle Control (VC)

The absolute value of optical density (OD 540 of NRU) obtained in the untreated vehicle control indicates whether the 0.8 to 1x10 4 c ells/ml seeded in each well have grown exponentially with normal doubling time during the three to five days of the assay.

A test meets acceptance criteria if the mean OD 540 of VCs is 0.3

To check for systematic cell seeding errors, untreated VCs are placed both at the left side (row 2 ) and the right side (row 11 ) of the 96-well plate (see Appendix E):

A test meets acceptance criteria if the left and the right mean of the VCs do not differ by more than 15% from the mean of all VCs.

Checks for cell seeding errors may also be performed by examining each plate under a phase contrast microscope to assure that cell quantity is consistent. Microscopic evaluation obviates the need for two rows of VCs.

1.6.4 Quality Check of ConcentrationResponse

If possible, the te st ar tic le conc entra

tions for the de finitive a ssay will be chosen such that at le ast six
tr ea tme nts w ill be ava ilable for the de ter mination of the IC 50 . I dea

lly, tw o c oncentr

ations should r esult in e xpe cted surviva

ls lower than 50%, one

conc entra tion should r esult in a n expec ted survival of a ppr oxima te ly 50% a nd tw o conce ntr ations should re sult in expec

te d sur vivals gre ate

r tha n 50%.

1.6.5 Concentrations of Test Chemical

1.6.5.1 Range Finder Experiment

In this preliminary NR bioassay, six to eight decreasing concentrations of the test material are selected based upon the available information for the test material. The test article should be dissolved in KGM, water, DMSO, acetone,Appendix D: SOP for The NHK Neutral Red Uptake Cytotoxicity Test

D-8 ETOH, or other appropriate solvent. The maximum solvent concentration (other than water or KGM) should be 1%. One way to determine concentrations of the chemical to be tested is to dilute the stock solution several times by a constant factor (e.g., 2 10 = 3.16, see Appendix F), covering a large range, e.g.:

1 3.16 10 31.6 100 316 1000 3160 μg/ml

1.6.5.2 Main Experiment

Depending on the slope of the concentrationresponse curve estimated from the range finder, the dilution/progression factor in the concentration series of the main experiment should be smaller (e.g., 6 10 = 1.47) to avoid too many non-cytotoxic and/or 100%-cytotoxic concentrations. Experiments revealing less than three cytotoxic concentrations in the relevant range shall be repeated, where possible , with a smaller dilution factor. (Taking into account pipetting errors, a progression factor of 1.21 is regarded the smallest factor achievable.)

1.6.6 Test Procedure

See Table D.1 for a flowchart of the test procedure. Appendix G contains a template recommended for documenting the relevant data.

1st day after growing up the cells from frozen stock:

1. Prepare a suspe

nsion of 0.8-1x10 4 c ells/ml in KG M. Using a multi-channel pipette, dispense 250 μl ce ll suspe

nsion to the appropr

ia te we lls

on e ach 96-w

ell tissue culture microtiter pla te. [N ote: evapora

tion of the medium c an be a pr oblem; the

re for e, the edge wells should

re ce ive 250 μl PBS for

blanks.] Prepare one plate per chemical to be tested and one plate for the PC.

[Note: Individual plates for the PC are for establishing an historical database. Once an IC 50 mean has been determined, only that PC concentration need be included in the test material plate.] 2. Incubate cells ( 37 ± 1 o C and 5 ± 1% CO 2 ) until a 30-50% confluent monolayer is produced (~24-72 h). This incubation period assures cell recovery and adherence and progression to the exponential growth phase.

3. Examine each plate under a phase contrast microscope to assure that cell growth is relatively even across the microtiter plate. This check is performed to identify systematic cell seeding errors.

2nd day:

1. After 24-72 h incubation, remove culture medium from the cells by inverting the uncovered 96-well plates over a liquid discard container and then gently blotting the plates several times on sterile paper towels.

2. Immediately add 125 μl fresh KGM to each well. Add 125 μl of the test a rticle dilutions, positive contr

ol dilutions and solvent contr ol dilution to the a ppropriate w ells. W ells de signa te d a s bla nks r ec eive 125 μl K GM.

3. Incubate cells for 48 h ( 37 ± 1 o C and 5 ± 1% CO 2 ).

3rd day:

A) Microscopic Evaluation

After 48 h treatment, examine each plate under a phase contrast microscope to identify test chemical precipitate, systematic cell seeding errors and growth characteristics of control and treated cells. Record changes in morphology of the cells due to cytotoxic effects of the test chemical, but do not use these records for the calculation of any quantitative measure of cytotoxicity. Undesirable growth characteristics of control cells may indicate experimental error and may be cause for rejection of the assay.

B) Measurement of NRU

This method is based upon that of Ellen Borenfreund (Borenfreund and Puerner, 1985). The uptake of the NR into the lysosomes/endosomes and vacuoles of living cellsAppendix D: SOP for The NHK Neutral Red Uptake Cytotoxicity Test

D-9 is used as a quantitative indication of cell number and viability.

1. Remove the treatment medium and add 250 μl NR medium to each well, except for blanks, which receive 250 μl KGM. Incubate at 37 ± 1 o C in a humidified atmosphere of 5 ± 1% CO 2 for 3 h.

2. After incubation, decant the NR medium, and add 250 μl Wash/Fix solution to each well.

3. After 2 min, decant and add 100 μl NR Desorb solution to all wells, including blanks. 4. Shake microtiter plate rapidly on a microtiter plate shaker for a minimum of 20 min at room temperature.

5. Measure the absorption of the resulting colored solution at 540-550 nm in a microtiter plate reader, using the blanks as a reference. Save raw data in a file format (e.g., ASCII, TXT, XLS) appropriate for further analysis of the concentration-response and calculation of IC 50 .

Table D.1. NHK NRU Cytotoxicity Test: Flow Chart

ASSAY PHASE PROCEDURE CELL GROWTH (24:00-72:00 h) [30–50% monolayer confluency] 

### • Seed 96-well plates: 2.0 to 2.5 x 10 3 cells/250 μ l KGM culture medium/well



### • Incubate (37 o ± 1 o C, 5 ± 1% CO 2 , 24-72 h)

TEST MATERIAL TREATMENT

(48:00 h) 

### • Remove culture medium/add fresh KGM culture medium (125 μ l/well)



### • Treat cells with 6-8 concentrations of test material in treatment medium (125 μ l/well) [test material is 2X concentration before adding to wells] for 48 h treatment

PRELIMINARY NEUTRAL RED

BIOASSAY (3:00 hours) 

### • Microscopic evaluation of morphological alterations



### • Remove treatment medium and add 250 μ l/well NR medium



### • Incubate (37 o ± 1 o C, 5 ± 1% CO 2 , 3 h)

NEUTRAL RED BIOASSAY (0:20 hours) 

### • Discard NR medium



### • Add 250 μ l/well Wash/Fix solution for 2 min



### • Remove Wash/Fix solution



### • Add 100 μ l/well NR Desorb (ETOH/acetic acid solution)



### • Shake plate for 20 min



### • Detect NR absorption at OD 540-550

1.7 Data Analysis

A calculation of cell viability expressed as NRU is made for each concentration of the test chemical by using the mean NRU of the six to eight replicate values per test concentration. This value is compared with the mean NRU of all VC values (provided VCs have met the VC acceptance criteria). Relative cell viability is then expressed as percent of untreated VC. If achievable, the six to eight concentrations of each compound tested should span the range of no effect up to totalAppendix D: SOP for The NHK Neutral Red Uptake Cytotoxicity Test

D-10 inhibition of cell viability.

Where possible, the concentration of a test chemical reflecting a 50% inhibition of cell viability (IC 50 ) is determined from the concentration-response. This can be done either by applying the following:



### • a manual graphical fitting method. The use of probability paper with "x = log" and "y = probit" scales is recommended because in most cases the concentration-response function will become almost linear in the relevant range. Alternatively, semi-log paper could also be used for this technique.

or



### • any appropriate non-linear regression procedure (preferably a Hill function* or alogistic regression) to the concentrationresponse data. Before using the IC 50 for further calculations, the quality of the fit should be appropriately checked. (* = Hill functions are monotonous and sigmoidal in shape and represent an acceptable model for many dose response curves.)

More sophisticated programs specially developed for concentration-response analysis from 96-well plates can also be used.

Before using the IC 50 information in any subsequent estimation of rodent LD 50 , be sure that the IC 50 data are expressed as mmol/l since the prediction model described in this guidance document is based on the relationship between the LD 50 (in mmol/kg) and the IC 50 (in mmol/l).

1.8 Prediction Model

In general, basal cytotoxicity is highly valuable information per se , which can be used in combination with other information, e.g., bioavailability, for many purposes in the process of safety or risk evaluation. For the purpose of this document, basal cytotoxicity is to be used to predict starting doses for in vivo acute oral LD 50 values in rodents. After testing the reference chemicals recommended in Section 3.2 of this guidance document and qualifying the test as described in Section 3.1 (see Section 3.3 of the report for examples with two different cell types), best estimates of starting doses for in vivo acute oral toxicity tests are predicted according to the following prediction model:

log (LD 50 [mmol/kg]) = 0.435 x log (IC 50 [mmol/l]) + 0.625

1.9 References

Bore nfr eund, E . a nd J. Puer ne r. 1984. A simple quantitative

proc edure using monolaye r cultures f or cytotoxic ity a

ssa ys (H TD / N R- 90) . J. Tissue Culture Meth. 9(1): 7- 9.

Clonetics Normal Human K era tinoc yte Systems Instruc tions f or Use, AA -1000-4- Re

v.03/00. (http://w ww.clone tics.com).

Halle, W. 1998. Toxizitätsprüfungen in Zellkulturen für eine Vorhersage der akuten Toxizität (LD 50 ) zur Einsparung von Tierversuchen. Life Sciences/ Lebenswissenschaften, Volume 1, 94 pp., Jülich: Forschungszentrum Jülich.

Harbell, J.W. 2001. Development of multiple-well plate biological assays, In Protocols for Neural Cell Culture . S. Fedoroff and A Richardson, (Eds). Humana Press, Inc., Totowa, NJ, p.265- 275.

He imann, R. and R.H . Ric e. 1983. Polycyclic

ar omatic hydrocar

bon toxicity and induc tion of

me ta bolism in cultivated esophagea l a nd epidermal ke ra tinoc yte s. Ca nc er Re s. 43: 4856.

Shopsis, C. and B. Eng. 1988. In v itro ocular ir ritancy pr ediction: assays in se rum-f ree medium

corr ela te be tter with in v ivo

data. In Alte rnative Me thods in Tox icology

, Vol, 6, A.M. Goldber g (E d.), Ma ry Ann L ie

ber t, Inc. NY , p. 253.

Spielmann, H., E. Genschow, M. Liebsch, and W. Halle. 1999. Determination of the starting dose for acute oral toxicity (LD50) testing in the up and down procedure (UDP) from cytotoxicity data. ATLA 27: 957-966.

# APPENDIX E

96-Well Plate ConfigurationAppendix E: 96-Well Plate ConfigurationAppendix E

E-1 

### Appendix E

96-Well Plate Configuration

Note : The plate configuration shown below is a recommendation, based on experience in two validation studies. Other plate map designs are possible and are discussed by Harbell (2001). Plate configurations must be fixed in the SOP. To avoid errors, plate configurations should be kept constant if reader files have to be transferred to secondary software for computational concentration-response analysis.

Note : Since evaporation (during opening the door of the incubator) may take place in the peripheral wells, it is recommended to use these wells for blanks only. Since modern incubators are able to compensate the drop in humidity much quicker than older ones, columns 1 and 12 may be used for other purposes (e.g., two typical
concentrations of the PC), while cells A2-A11 and H2-H11 can be used for the blanks.



### 1 2 3 4 5 6 7 8 9 10 11 12

A b b b b b b b b b b b b

B b VC C 1 

### C 2 

### C 3 

### C 4 

### C 5 

### C 6 

### C 7 

### C 8 

### VC b

C b VC C 1 

### C 2 

### C 3 

### C 4 

### C 5 

### C 6 

### C 7 

### C 8 

### VC b

D b VC C 1 

### C 2 

### C 3 

### C 4 

### C 5 

### C 6 

### C 7 

### C 8 

### VC b

E b VC C 1 

### C 2 

### C 3 

### C 4 

### C 5 

### C 6 

### C 7 

### C 8 

### VC b

F b VC C 1 

### C 2 

### C 3 

### C 4 

### C 5 

### C 6 

### C 7 

### C 8 

### VC b

G b VC C 1 

### C 2 

### C 3 

### C 4 

### C 5 

### C 6 

### C 7 

### C 8 

### VC b

H b b b b b b b b b b b b

VC = untreated VEHICLE CONTROL

(mean viability set to 100%)

C 1 - C 8 = TEST CHEMICAL at eight concentrations

(C1 = lowest, C8 = highest)

b = BLANKS

(containing no cells, but treated with NR medium and with NR Desorb solution)Appendix E

E-2

# APPENDIX F

Decimal Geometric Concentration SeriesAppendix F: Decimal Geometric Concentration SeriesAppendix F

F-1 

### Appendix F Decimal Geometric Concentration Series

Note : Whereas geometric concentration series (as opposed to arithmetic concentration series) are regarded as a requirement in for any cytotoxicity assay that is based on concentration response analysis, the decimal geometric concentration series described below is just a recommendation.

In general dose-response relationships of many pharmacological or toxicological endpoints investigated have a nonlinear , often sigmoidal shape, which can be linearized to some extent by logarithmic transformation of the x-axis. This usually has to be done when IC 50 values are calculated either by regression analysis or by graphical estimation for the current NRU assay. If the concentration series is done with arithmetic progression steps, transformation of the x-axis will result in an unequal distribution of measurements. Therefore, the use of a geometric concentration series (= constant dilution / progression factor) is recommended. The simplest geometric series are dual geometric series , e.g., a factor of 2. These series have the disadvantage of numerical values that permanently change between logs of the series (e.g., log0 -2, 4, 8; log1- 16, 32, 64; log2- 128, 256, 512; log3- 1024, 2048,).

The decimal geometric series , first described by Hackenberg and Bartling (1959) for use in toxicological and pharmacological studies has the advantage that independent experiments with wide or narrow dose factors can be easily compared because they share identical concentrations. Furthermore, under certain circumstances, experiments can even be merged together:

EXAMPLE:

10 31.6 100 10 21.5 46.4 100 10 14.7 21.5 31.6 46.4 68.1 100 10 12.1 14.7 17.8 21.5 26.1 31.6 38.3 46.4 56.2 68.1 82.5 100

The dosing factor of 3.16 (= 2 10) divides a log into 2 equidistant steps, a factor of 2.15 (= 3 10) divides a decade into 3 steps. The factor of 1.47 (= 6 10) divides a log into 6 equidistant steps, and the factor of 1.21 (= 12 10) divides the log into 12 steps.

For an easier biometrical evaluation of several related concentration response experiments use decimal geometric concentration series rather than dual geometric series. The technical production of decimal geometric concentration series is simple. An example is given for factor 1.47:

Dilute 1 volume of the highest concentration by adding 0.47 volumes of diluent. After equilibration dilute 1 volume of this solution by adding 0.47 volumes of diluent...(etc.).

Reference:

Hackenberg, U. and H. Bartling. 1959. Messen und Rechnen im pharmakologischen Laboratorium mit einem speziellen Zahlensystem (WL24-System). Arch. Exp. Pathol. Pharmakol. 235: 437-463.Appendix F

F-2

# APPENDIX G

Standard Test Reporting TemplateAppendix G: Standard Test Reporting TemplateAppendix G

G-1 

### Appendix G Standard Test Reporting Template

This template is recommended to compile the data necessary to check the performance of a NRU test. Additional data, (e.g., temperature, CO 2 , and humidity of incubators, or temperature of refrigerators, calibration of scales and pipettes, etc.), are not included since GLP laboratories usually record these in master records for the whole laboratory.

TEST SUBSTANCE

Name CAS-No. (if known)

Laboratory Code Molecular Weight (gram)

Storage Conditions (tick _) _ deep frozen _ room temperature
_ refrigerated _ dark

Expiration date (if known)

PREPARATION OF TEST SUBSTANCE

Name of Solvent (if used)

Percent Solvent (v/v) present in all wells

Aids used to dissolve (tick _) _ magnetic stirrer _ ultra-sonication

_ vortex _ heating to .........°C

pH (measured at highest test concentration)

Was neutralization necessary? (tick _) _ NO _ YES, with HCl _ YES, with NaOH

Concentration series (specify in μ g/ml)

Concentration series (specify in μ mol/ml)

CELL LINE

Name: Supplier:

Total Passage No. (if known): No. of Passages after Thawing:

CELL CULTURE CONDITIONS

Name of Medium: Supplier: Lot No.:

Name of Serum: Supplier: Lot No.:

Serum Concentration During growth: ..............% During Exposure: ...........%Appendix G: Standard Test Reporting Template

G-2

TEST ACCEPTANCE CRITERIA

VC: mean absolute OD540 (specify and _) Mean OD = .............. _ ACCEPT _ REJECT

VC: diff. betw. columns 2 and 11 (specify and _) Difference = .........% _ ACCEPT _ REJECT

PC: IC 50 of concurrent SLS test (specify and _) IC 50 = ............. μ g /ml _ ACCEPT _ REJECT

PC: specify where PC data are recorded:

TEST RESULTS

Chem. Conc.
( μ mol/ml) OD540

MEAN + SD Viability (%)

MEAN SD

VC = ZERO 100

C1 =

C2 = Template reports trial No. ........ of the test

substance

NRU RESULT:

IC 50 = ........................ μ mol/ml [equals mmol/l]

C3 =

C4 =

C5 =

C6 =

C7 =

C8 = PREDICTED LD 50 : log LD 50 = .....................mmol/kg b.w.

LD 50 = ......................mmol/kg b.w.

LD 50 = ......................mg/kg b.w.

PREDICTED STARTING DOSE: UDP one step (factor 3.2) below LD 50 =............mg/kg
Signature:.............................

Date:.............................