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 Services

THE 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 Services
ii
iii

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-1
Table 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-1
Table 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 (IC50x)

and Rodent Acute Oral LD50 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 INTRODUCTION
Table 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 References
vii

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

FG 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 studies
List 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

OD540 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 A31
List of Acronyms/Abbreviations

x
xi

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, NC
Acknowledgements

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 Center

John 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 6/01
Acknowledgements

xiv
xv

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 LD50 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. Both
Preface

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. EPA
Using 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., LD50 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 LD50 values (the

median lethal dose, i.e., the dose that produces

lethality in 50% of the animals tested) and in vitro

cytotoxicity IC50 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 LD50 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 LD50 is not a

biological constant, but is influenced by many

factors (Klaassen and Eaton, 1991). For most

purposes, the LD50 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 IC50 values

averaged for each of 347 chemicals [i.e., one IC50x

value/chemical from multiple reports in the

literature]) paired with 347 in vivo acute oral LD50

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 (IC50x) and rodent acute oral

LD50 values for 347 chemicals.

The heavy line shows the fit of the data to a linear regression model, log (LD50) = 0.435 x

log (IC50x) + 0.625; r=0.67. The other lines show the empirical FG = ± log 5 acceptance

interval for the prediction model (Spielmann et al., 1999), which is based on the anticipated

precision of LD50 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 LD50 for that chemical.

The LD50 predicted from the RC regression

equation should then be used to choose the most

appropriate starting dose for the in vivo assay.

The LD50 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 LD50 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 LD50 (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 LD50 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 LD50 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 primary

cells, 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 IC50X 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 (R2=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 cytotoxicity
Using 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 IC50 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

8
Using 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 LD50 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 LD50

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 LD50 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 LD50 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 LD50 values.) The regression

equation from the candidate test is calculated by

linear regression (least square method) using the

candidate IC50 values and the corresponding LD50

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 IC50 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 LD50

starting dose, the IC50 (in mmol/l) for the trial

chemical is entered into the regression equation to
Using In Vitro Data to Estimate In Vivo Starting Doses for Acute Toxicity

10

calculate an LD50 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: Cytotoxicity test

Test 10 - 20 reference chemicals (low - high cytotoxicity) taken

from the RC, e.g. in the 3T3-Neutral Red Uptake test

Step 2: Linear regression analysis

 Use IC50 values and RC LD50 values to calculate regression

log (LD50) = a x log (IC50) +b

Step 3: Comparison of regressions

Compare resulting regression with RC regression

log (LD50) = 0.435 x log (IC50) + 0.625

Is regression parallel and within F G range ?

Step 4: Use test to predict starting dose for

 UDP or ATC

YES

NO:

Use recommended cells

 and protocol to better

tune test sensitivity

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 (IC50x/LD50)

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

IC50x

(mmol/

liter)

LD50

(mmol/

kg b.w.)

LD50

(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-Phenylenediamine0.05 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 LD50 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

IC50 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 (LD50) = 0.435 x log (IC50x) + 0.625

New NHK NRU regression:

log (LD50) = 0.498 x log (IC50) + 0.551 

[R2 = 0.9356]

New 3T3 NRU regression:

log (LD50) = 0.506 x log (IC50) + 0.475 

[R2 = 0.9848]

As expected, due to selection of reference

chemicals with data points close to the RC

regression, the determination coefficients (R2) 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

IC50x (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 IC50 /LD50 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

IC50x (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 IC50 /LD50 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

14
Using 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 chemicalsa

Substance CAS No. NR50

 b

(mg/ml)

Interlaboratory

reproducibilityc

(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.

bNR50= 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 IC50 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 (R2 = 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

IC50 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 1

Lab 2

y = 0.9365x - 0.0243

R

2

 = 0.7748

mg/ml

mg/ml
Using 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 sets

containing 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 NRU50 [i.e.,

concentration reducing NRU to 50 % of control

value] from one laboratory in these trials was 4.4

μg/ml; twelve years later the same laboratory has

an average NRU50 for SLS of 4.4 +/- 0.97 μg/ml).

Triglia et al. (1989) also reported interlaboratory

variability for 12 compounds replicated in four

laboratories. The interlaboratory CVs for the

NRU50 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. NRU50

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 NRU50 values for

SLS tested in four different keratinocyte isolates

were nearly identical at 66.7, 67.5, 70.9 and 73.4

μg/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 IC50s and in vivo LD50s 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

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

21

6.0 REFERENCES

Bagley, D., K.A. Booman, L.H. Bruner, P.L. Casterton, J. Demetrulias, J.E. Heinze, J.D. Innis, W.C.

McCormick, D.J. Neun, A.S. Rothenstein, and R.I. Sedlak. 1994. The SDA alternatives program phase

III: Comparison of in vitro data with animal eye irritation data on solvents, surfactants, oxidizing agents,

and prototype cleaning products. J. Toxicol.–Cutan. Ocul. 13: 127-155.

Balls, M., P.A. Botham, L.H. Bruner, and H. Spielmann. 1995. The EC/HO international validation

study on alternatives to the Draize eye irritation test for classification and labelling of chemicals. Toxicol.

In Vitro 9: 871-929.

Brantom P.G., L.H. Bruner, M. Chamberlain, O. DeSilva, J. Dupuis, L.K. Earl, D.P. Lovell, W.J.W. Pape,

M. Uttley, D.M. Bagley, F.W. Baker, M. Bracher, P. Courtellemont, I. Declercq, S. Freeman, W. Steiling,

A.P. Walker, G.J. Carr, N. Dami, G. Thomas, J. Harbell, P.A. Jones, U. Pfannenbecker, J.A. Southee, M.

Tcheng, H. Argembeaux, D. Castelli, R. Clothier, D.J. Esdaile, H. Itigaki, K. Jung, Y. Kasai, H. Kojima,

U. Kristen, M. Larnicol, R.W. Lewis, K. Marenus, O. Moreno, A. Peterson, E.S. Rasmussen, C. Robles,

and M. Stern. 1997. A summary report of the COLIPA international validation study on alternatives to

the Draize rabbit eye irritation test. Toxicol. In Vitro 11: 141-179.

Clemedson, C, E. McFarlane-Abdulla, M. Andersson, F.A. Barile, M.C. Calleja, C. Chesné, R. Clothier,

M. Cottin, R. Curren, E. Daniel-Szolgay, P. Dierickx, M. Ferro, G. Fiskesjö, L. Garza-Ocanas, M.J.

Gómez-Lechón, M. Gülden, B. Isomaa, J. Janus, P. Judge, A. Kahru, R.B. Kemp, G. Kerszman, U.

Kristen, M. Kunimoto, S. Kärenlampi, K. Lavrijsen, L. Lewan, H. Lilius, T. Ohno, G. Persoone, R.

Roguet, L. Romert, T. Sawyer, H. Seibert, R. Shrivastava, A. Stammati, N. Tanaka, O. Torres Alanis, J.-

U. Voss, S. Wakuri, E. Walum, X. Wang, F. Zucco, and B. Ekwall. 1996. MEIC evaluation of acute

systemic toxicity. Part I. Methodology of 68 in vitro toxicity assays used to test the first 30 reference

chemicals. ATLA 24 (Suppl. 1): 249-272.

Curren, R., L. Bruner, A. Goldberg, and E. Walum. 1998. 13th meeting of the Scientific Group on

Methodologies for the Safety Evaluation of Chemicals (SGOMSEC): Validation and acute toxicity

testing. Environ. Health Persp. 106: (Suppl. 2). 419-425.

Dickson, F.M., J.N. Lawrence, and D.J. Benford. 1993. Surfactant-induced cytotoxicity in cultures of

human keratinocytes and a commercially available cell line (3T3). Toxicol. In Vitro 7:381-384.

Eagle, H. and G.E. Foley. 1956. The cytotoxic action of carcinolytic agents in tissue culture. Am. J.

Med. 21: 739-745.

Ekwall, B., F.A. Barile, A. Castano, C. Clemedson, R.H. Clothier, P. Dierickx, Ba. Ekwall, M. Ferro, G.

Fiskesjö, L. Garza-Ocanas, M.J. Gómez-Lechón, M. Gülden, T. Hall, B. Isomaa, A. Kahru, G. Kerszman,

U. Kristen, M. Kunimoto, S. Kärenlampi, L. Lewan, A. Loukianov, T. Ohno, G. Persoone, L. Romert,

T.W. Sawyer, H. Segner, R. Shrivastava, A. Stammati, N. Tanaka, M. Valentino, E. Walum, and F.

Zucco. 1998. MEIC evaluation of acute systemic toxicity. Part VI. Prediction of human toxicity by rodent

LD50 values and results from 61 in vitro tests. ATLA 26 (Suppl. 2): 617-658.

Ekwall, B., B. Ekwall, and M. Sjostrom. 2000. MEIC evaluation of acute systemic toxicity: Part VIII.

Multivariate partial least squares evaluation, including the selection of a battery cell line tests with a good

prediction of human acute lethal peak blood concentrations for 50 chemicals. ATLA 28 (Suppl. 1): 201-

234.
Using In Vitro Data to Estimate In Vivo Starting Doses for Acute Toxicity

22

Garle, M., J.H. Fentem, and J.R. Fry. 1994. In vitro cytotoxicity tests for the prediction of acute toxicity

in vivo. Toxicol. In Vitro 8: 1303-1312.

Gettings, S.D., D.M. Bagley, M. Chudkowski, J.L. Demetrulias, L.C. Dipasquale, C.L. Galli, R. Gay,

K.L. Hintze, J. Janus, K.D. Marenus, M.J. Muscatiello, W.J.W. Pape, K.J. Renskers, M.T. Roddy, and R.

Schnetzinger. 1992. Development of potential alternatives to the Draize eye test - The CTFA evaluation

of alternatives program - Phase II - Review of materials and methods. ATLA 20: 164-171.

Gettings, S.D., D.M. Bagley, J.L. Demetrulias, L.C. Dipasquale, K.L. Hintze, M.G. Rozen, J.J. Teal, S.L.

Weise, M. Chudkowski, K.D. Marenus, W.J.W. Pape, M.T. Roddy, R. Schnetzinger, P.M. Silber, S.M.

Glaza, and P.J. Kurtz. 1991. The CTFA evaluation alternatives program: An evaluation of in vitro

alternatives to the Draize primary eye irritation test. (Phase I) Hydro-alcoholic Formulations; (Part 2)

Data analysis and biological significance. In Vitro Toxicol. 4: 247-288.

Gettings, S.D., L.C. Dipasquale, D.M. Bagley, P.L. Casterton, M. Chudkowski, R.D. Curren, J.L.

Demetrulias, P.I. Feder, C.L. Galli, R. Gay, S.M. Glaza, K.L. Hintze, J. Janus, P.J. Kurtz, R.A. Lordo,

K.D. Marenus, J. Moral, M. Muscatiello, W.J.W. Pape, K.J. Renskers, M.T. Roddy, and M.G. Rozen.

1994a. The CTFA evaluation of alternatives program: An evaluation of in vitro alternatives to the Draize

primary eye irritation test. (Phase II) Oil/water emulsions. Food Chem. Toxicol. 32: 943-976.

Gettings, S.D., K.L. Hintze, D.M. Bagley, P.L. Casterton, M. Chudkowski, R.D. Curren, J.L.

Demetrulias, L.C. Dipasquale, L.K. Earl, P.I. Feder, C.L. Galli, R. Gay, S.M. Glaza, V.C. Gordon, J.

Janus, P.J Kurtz, R.A. Lordo, K.D. Marenus, J. Moral, W.J.W. Pape, K.J. Renskers, L.A Rheins, M.T.

Roddy, M.G. Rozen, J.P. Tedeschi, and J. Zyracki. 1994b. The CTFA evaluation of alternatives

program: Phase III (surfactant-based formulations). World Congress on Alternatives and Animal Use in

the Life Sciences, Baltimore, Maryland, USA, Nov. 14-19, 1993. In Vitro Toxicol. 7 (2): 166.

Gettings, S.D., R.A Lordo, K.L. Hintze, D.M. Bagley, P.L. Casterton, M.Chudkowski, R.D. Curren, J.L.

Demetrulias, L.C. Dipasquale, L.K. Earl, P.I. Feder, C.L. Galli, S.M. Glaza, V.C. Gordon, J. Janus, P.J.

Kurtz, K.D. Marenus, J. Moral, W.J. Pape, K.J. Renskers, L.A. Rheins, M.T. Roddy, M.G. Rozen, J.P.

Tedeschi, J. Zyracki. 1996. The CFTA evaluation of alternatives program: An evaluation of in vitro

alternatives to the Draize Primary Eye Irritation Test. (Phase III) Surfactant-based Formulations. Food

Chem.Toxicol. 34:79-117.

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.

Halle, W. and H. Spielmann. 1992. Two procedures for the prediction of acute toxicity (LD50) from

cytotoxicity data. ATLA 20: 40-49.

Harbell, J. W., S. W. Koontz, R.W. Lewis, D. Lovell and D. Acosta. 1997. IRAG working group 4. Cell

cytotoxicity assays. Interagency Regulatory Alternatives Group. Food Chem.Toxicol. 35:79-126.

Klaassen, C.D. and D.L. Eaton. 1991. Principles of toxicology. In: Casarett and Doull’s Toxicology:

The Science of Poisons, 4th Edition. M.O. Amdur, J. Doull, and C.D. Klaassen, [Eds.], pp. 16-17.

Pergamon Press, Inc., New York.

NIEHS (National Institute of Environmental Health Sciences). 2001. Report of the international

workshop on in vitro methods for assessing acute systemic toxicity. NIH Publication 01-4499. NIEHS,

Research Triangle Park, North Carolina.
Using In Vitro Data to Estimate In Vivo Starting Doses for Acute Toxicity

23

OECD (Organisation for Economic Co-operation and Development). 1998a. Harmonized Integrated

Hazard Classification System for Human Health and Environmental Effects of Chemical Substances as

Endorsed by the 28th Joint Meeting of the Chemicals Committee and the Working Party on Chemicals in

November 1998, Part 2, p.11. OECD, Paris. http://www.oecd.org/ehs/class/HCL6htm.

OECD (Organisation for Economic Co-operation and Development). 1998b. OECD Guideline for

Testing of Chemicals. 425. Acute Oral Toxicity - Up-and-Down Procedure. OECD. Paris. Updated in

2000: http://www.oecd.org//ehs/test/testlist.htm.

OECD (Organisation for Economic Co-operation and Development). 1996. OECD Guideline for Testing

of Chemicals. 423. Acute Oral Toxicity - Acute Toxic Class Method. OECD, Paris. Updated in 2000:

http://www.oecd.org//ehs/test/testlist.htm.

OECD (Organisation for Economic Co-operation and Development). 1992. OECD Guideline for Testing

of Chemicals. 420. Acute Oral Toxicity - Fixed Dose Method. OECD, Paris. Updated in 2000:

http://www.oecd.org//ehs/test/testlist.htm.

Ohno, T., M. Asakura, T. Awogi et al. 1998a. Validation study on five cytotoxicity assays by JSAAE. I.

Overview of the study and analyses of variations of ED50 values. AATEX 5: 1-38.

Ohno, T., Y. Futamura, A. Harihara et al. 1998b. Validation study on five cytotoxicity assays by

JSAAE. VI. Details of the LDH release assay. AATEX 5: 99-118.

Ohno, T., Y. Futamura, A. Harihara et al. 1998c. Validation study on five cytotoxicity assays by JSAAE.

VIII. Details of the neutral red uptake assay. AATEX 5: 131-145.

Phillips, J.C., W.B. Gibson, J. Yam, C.L. Alden, and G.C. Hard. 1990. Survey of the QSAR and in vitro

approaches for developing non-animal methods to supersede the in vivo LD50 test. Food Chem. Toxicol.

28: 375-394.

Pomerat, C.M. and C.D. Leake. 1954. Short term cultures for drug assays: general considerations. Ann.

New York Acad. Sci. 58: 1110-1128.

Riddell, R.J., D.S. Panacer, S.M. Wilde, R.H. Clothier, and M. Balls. 1986. The importance of exposure

period and cell type in in vitro cytotoxicity tests. ATLA 14: 86-92.

Seibert, H., M. Balls, J.H. Fentem, V. Bianchi, R.H. Clothier, P.J. Dierickx, B. Ekwall, M.J. Garle, M.J.

Gómez-Lechón, L. Gribaldo, M. Gülden, M. Liebsch, E. Rasmussen, R. Roguet, R. Shrivastava, and E.

Walum. 1996. Acute toxicity testing in vitro and the classification and labeling of chemicals: The report

and recommendations of ECVAM Workshop 16. ATLA 24: 499-510.

Sina, J.F., D.M. Galer, R.G. Sussman, P.D. Gautheron, E.V. Sargent, B. Leong, P.V. Shah, R.D. Curren,

and K. Miller. 1995. A collaborative evaluation of seven alternatives to the Draize eye irritation test using

pharmaceutical intermediates. Fundam. Appl. Toxicol. 26: 20-31.

Smith, C.G., J.E. Grady, and J.I. Northam. 1963. Relationship between cytotoxicity in vitro and whole

animal toxicity. Cancer Chemoth. Rep. 30: 9-12.

Spielmann, H., M. Balls, J. Dupuis, W.J.W. Pape, G. Pechovitch, O. De Silva, H.-G. Holzhütter, R.

Clothier, P. Desolle, F. Gerberick, M. Liebsch, W.W. Lovell, T. Maurer, U. Pfannenbecker, J.M. Potthast,
Using In Vitro Data to Estimate In Vivo Starting Doses for Acute Toxicity

24

M. Csato, D. Sladowski, W. Steiling, and P. Brantom. 1998a. EU/COLIPA "In vitro phototoxicity"

validation study, results of phase II (blind trial), part 1: the 3T3 NRU phototoxicity test. Toxicol. In Vitro

12: 305-327.

Spielmann, H., M. Balls, J. Dupuis, W.J.W. Pape, O. De Silva, H.-G. Holzhütter, F. Gerberick, M.

Liebsch, W.W. Lovell, and U. Pfannenbecker. 1998b. A study on the phototoxic potential of UV filter

chemicals from Annex VII of EU Directive 76/768/EEC in the 3T3 NRU in vitro phototoxicity test.

ATLA 26: 679-705.

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.

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., S. Kalweit, M. Liebsch, T. Wirnsberger, I. Gerner, E. Bertram-Neis, K. Krauser, R.

Kreiling, H.G. Miltenburger, W. Pape, and W. Steiling. 1993. Validation study of alternatives to the

Draize eye irritation test in Germany: Cytotoxicity testing and HET-CAM test with 136 industrial

chemicals. Toxicol. In Vitro 7 (4): 505-510.

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.

Triglia, D., P.T. Wegener, J. Harbell, K. Wallace, D. Matheson, and C. Shopsis. 1989. Interlaboratory

validation study of the keratinocyte neutral red bioassay from Clonetics Corporation. In Alternative

Methods in Toxicology, Volume 7. A.M. Goldberg, ed., pp. 357-365. Mary Ann Liebert, Inc., New

York.

Willshaw, A., L. J. Moore, and M. Balls. 1994. In vitro alternatives for the detection of photoirritant

chemicals - the EEC COLIPA trial. Toxicol. In Vitro 8: 723-725.
APPENDIX A

Registry of Cytotoxicity

List of 347 Chemicals

(Sorted by IC50 and LD50 Values)
Appendix A: Registry of Cytotoxicity
Appendix 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-1
Appendix 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-2
Appendix 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-3
Appendix 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-4
Appendix 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-5
Appendix A: Registry of Cytotoxicity

A-6
Appendix 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-7
Appendix 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-8
Appendix 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-9
Appendix 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-10
Appendix 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-11
Appendix 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 Cytotoxicity
Appendix 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 CULTURES

Membrane 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 is
Appendix 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 been

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

C-2
Appendix 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 3T3

Neutral 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 KB50

values were close or even identical to the NR50

values, but in several cases where necrotic cells

were fixed to the plastic material of the plates and

then stained with KB, the KB50 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 test
Appendix 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

IC50 (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 IC50 may be

variable, especially when the toxicity of the

compound is fairly low. This can be

overcome if plates are sealed with CO2

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 % CO2/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 cm2, 25 cm2)

• 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 Ca2+

and Mg2+(for trypsinization)

• PBS with Ca2+ and Mg2+(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 H2O 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 H2O

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 % H2O
Appendix 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 used

provided 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 cm2 tissue culture grade flasks, at

37°C in a humidified atmosphere of 7.5 % CO2.

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 Ca2+, Mg2+) per 25 cm2

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 cm2 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 seeded
Appendix 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 106 cells in 80 cm2 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 1-5x106 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 % CO2 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 (IC50) on the same plate

together with the test chemical. For the latter

procedure, the 95% confidence interval (CI) of the

IC50 of SLS has to be established and defined as

an acceptance criterion for test sensitivity in the

SOP.

The historical mean IC50 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 IC50 for

SLS is within the 95% CI

1.6.3 Quality Check of Assay (II): Vehicle

Control (VC)

The absolute value of optical density (OD540 of

NRU) obtained in the untreated vehicle control

indicates whether the 1×104 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 OD540

of VCs is 0.3
Appendix 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 IC50 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., 210 = 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., 610 = 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 1x105 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 IC50

mean has been determined, one need only include

that PC concentration in the test material plate.]

2. Incubate cells for 24 h (7.5% CO2, 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% CO2, 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 % C02 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

IC50.
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: 1x104 cells / 100 μl DMEM culture medium / well

Incubate (37°C / 7.5% CO2 / 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% CO2 / 24 h)

Microscopic evaluation of morphological alterations

Remove treatment medium

wash once with 150 μl PBS

Add 100 μl NR medium

Incubate (37°C / 7.5% CO2 / 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 IC50) 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 a

logistic regression) to the concentrationresponse data. Before using the IC50 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. An example is PHOTO32, which uses a nonmonotonous curve fitting

algorithm (Holzhütter and Quedenau, 1995) and

addresses the influence of variability on the IC50

by bootstrapping procedures performed on

concentration replicates (Holzhütter, 1997).

Before using the IC50 information in any

subsequent estimation of rodent LD50, be sure that

the IC50 data are expressed as mmol/l since the

prediction model described in this guidance

document is based on the relationship between the

LD50 (in mmol/kg) and the IC50 (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 LD50

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 (LD50 [mmol/kg]) = 0.435 x log (IC50 [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 (LD50) 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 Test
Appendix 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................................................................................................................................. 10
Appendix D: SOP for The NHK Neutral Red Uptake Cytotoxicity Test

D-2
Appendix 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 IC50

(a.k.a., NRU50, 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 IC50 may be

variable, especially when the toxicity of the

compound is fairly low. This can be

overcome if plates are sealed with CO2

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 the
Appendix 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

oC, humidified, 5 ± 1 %

CO2/air

• Laminar flow clean bench (standard:

"biological hazard")

• Water bath: 37o ± 1oC

• 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 cm2, 25 cm2)

• 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 teBuff 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 Ca2+

or Mg2+ (CMF-HBSS) (e.g., Invitrogen #

14170)

• Formaldehyde

• Calcium chloride

• Distilled H2O or any purified water suitable

for cell culture
Appendix 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 H2O

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% H2O

1.5.4.5 Ethanol/Acetic Acid Solution (NR

Desorb)

1 % Glacial acetic acid solution

50 % Ethanol

49 % H2O

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

oC for a 

minimum of 60 minutes (min) to a llow the

te mpera ture ofthe medium to equilibr ate.

• Aseptic ally re move the medium and repla ce

with fr esh K GM wa rmed to appr oxima tely

37o C.

• Unle ss other wise specified, the culture s a rethen

incubated at 37o ± 1oC and 5 ± 1%CO2 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 the 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/cm2 into flasks containing routine

pre-warmed culture medium.

• Incubate thec ultur es at 37o ± 1

oC 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

oC and 5 ± 1% CO2 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 a25 c m

2

 f la sk

is 50 to 80%c onf luent, remove theme 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 thec ell suspension to a centr ifugetube .

• 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 1.0x104c ells/ml

in K GM. Transfer the cells into flasks

containing pre-warmed growth medium at

3500 cells/cm2. 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 μl ce ll

suspension to thea 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

incubatorat 37o ± 1oC and 5 ± 1%CO2 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 5x105 to 2x106 cells

per ml. Aliquot to freezing vials.

• Insulate the vials and place into a –70oC

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 (IC50)

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 IC50 for

SLS is the acceptance criterion for test sensitivity.

A test meets acceptance criteria, if the IC50 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 (OD540 of

NRU) obtained in the untreated vehicle control

indicates whether the 0.8 to 1x104c 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

OD540 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 forthe de ter mination of

the IC50. 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., 210 = 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., 610 = 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 0.8-1x104c ells/ml in

KG M. Using a multi-channel pipette, dispense

250 μl ce ll suspe nsion to the appr opr 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 forblanks.] 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 IC50

mean has been determined, only that PC

concentration need be included in the test material

plate.]

2. Incubate cells (37 ± 1oC and 5 ± 1% CO2) 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

oC 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 cells
Appendix 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

oC in a humidified atmosphere of 5 ± 1%

CO2 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

IC50.

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 103 cells/250 μl KGM

culture medium/well

• Incubate (37o ± 1oC, 5 ± 1% CO2, 24-72 h)

TEST MATERIAL TREATMENT

(48:00 h)

• Remove culture medium/add fresh KGM culture medium

(125 μl/well)

• Treat cells with 6-8 concentrations of test material in

treatment medium (125 μl/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 μl/well NR medium

• Incubate (37o ± 1oC, 5 ± 1% CO2, 3 h)

NEUTRAL RED BIOASSAY

(0:20 hours)

• Discard NR medium

• Add 250 μl/well Wash/Fix solution for 2 min

• Remove Wash/Fix solution

• Add 100 μl/well NR Desorb (ETOH/acetic acid solution)

• Shake plate for 20 min

• Detect NR absorption at OD540-550

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 total
Appendix 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 (IC50) 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 a

logistic regression) to the concentrationresponse data. Before using the IC50 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 IC50 information in any

subsequent estimation of rodent LD50, be sure that

the IC50 data are expressed as mmol/l since the

prediction model described in this guidance

document is based on the relationship between the

LD50 (in mmol/kg) and the IC50 (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 LD50

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 (LD50 [mmol/kg]) = 0.435 x log (IC50 [mmol/l]) + 0.625

1.9 References

Bore nfr eund, E . a nd J. Puer ne r. 1984. Asimple

quantitativeproc edureusing monolaye r cultures f or

cytotoxic ity a ssa ys (H TD / N R- 90) . J. Tissue

CultureMeth. 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 (LD50) 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 reemedium

corr ela tebe 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 Configuration
Appendix E: 96-Well Plate Configuration
Appendix 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 C1 C2 C3 C4 C5 C6 C7 C8 VC b

C b VC C1 C2 C3 C4 C5 C6 C7 C8 VC b

D b VC C1 C2 C3 C4 C5 C6 C7 C8 VC b

E b VC C1 C2 C3 C4 C5 C6 C7 C8 VC b

F b VC C1 C2 C3 C4 C5 C6 C7 C8 VC b

G b VC C1 C2 C3 C4 C5 C6 C7 C8 VC b

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

VC = untreated VEHICLE CONTROL

(mean viability set to 100%)

C1 - C8 = 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 Series
Appendix F: Decimal Geometric Concentration Series
Appendix 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 IC50 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 (= 210) divides a log

into 2 equidistant steps, a factor of 2.15 (= 310)

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 Template
Appendix G: Standard Test Reporting Template
Appendix 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, CO2, 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 μg/ml)

Concentration series (specify in μmol/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: IC50 of concurrent SLS test (specify and _) IC50 = .............μg /ml _ ACCEPT _ REJECT

PC: specify where PC data are recorded:

TEST RESULTS

Chem. Conc.

(μmol/ml)

OD540

MEAN + SD

Viability (%)

MEAN SD

VC = ZERO 100

C1 =

C2 =

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

substance

NRU RESULT:

IC50 = ........................ μmol/ml [equals mmol/l]

C3 =

C4 =

C5 =

C6 =

C7 =

C8 =

PREDICTED LD50:

log LD50 = .....................mmol/kg b.w.

LD50 = ......................mmol/kg b.w.

LD50 = ......................mg/kg b.w.

PREDICTED STARTING DOSE: UDP

one step (factor 3.2) below LD50 =............mg/kg

Signature:.............................

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