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NIH Publication No: 07-4518

BACKGROUND REVIEW DOCUMENTIn Vitro Cytotoxicity Test Methods for Estimating
Acute Oral Systemic Toxicity

Volume 1 of 2
Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM)
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

In Vitro Cytotoxicity Test Methods
for Estimating Acute Oral Systemic Toxicity
Background Review Document
Volume 1 of 2
Prepared by
The National Toxicology Program (NTP) Interagency Centerfor the Evaluation of Alternative Toxicological Methods (NICEATM)

National Institute of Environmental Health Sciences (NIEHS)
P.O. Box 12233
Mail Drop: EC-17
Research Triangle Park, NC 27709

November 2006
NIH Publication No. 07-4518
National Institute of Environmental Health Sciences
National Institutes of Health
U.S. Public Health Service
Department of Health and Human Services

This document is available electronically at:
http://iccvam.niehs.nih.gov/methods/invitro.htm

Printed: 11/30/2006

In Vitro Cytotoxicity Test Methods BRD Table of Contents

November 2006

TABLE OF CONTENTSAPPENDICES.................................................................................................................. xiii
LIST OF FIGURES .......................................................................................................... xvi
LIST OF TABLES...........................................................................................................xvii
LIST OF ACRONYMS AND ABBREVIATIONS ........................................................... xx
ACKNOWLEDGMENTS................................................................................................ xxv
PREFACE........................................................................................................................xxix
EXECUTIVE SUMMARY............................................................................................ xxxiii
1.0

INTRODUCTION AND RATIONALE FOR THE USE OF IN VITRO
NEUTRAL RED UPTAKE CYTOTOXICITY TEST METHODS TO
PREDICT STARTING DOSES FOR IN VIVO ACUTE ORALTOXICITY TESTING .........................................................................................1-3
1.1

Historical Background and Rationale for the Use of In Vitro
Cytotoxicity Assays to Predict Starting Doses for Rodent Acute
Oral Toxicity Tests....................................................................................1-4
1.1.1
The Multicentre Evaluation of In Vitro Cytotoxicity (MEIC)
Program........................................................................................1-4
1.1.2
An International Evaluation of Selected In Vitro Toxicity
Test Systems for Predicting Acute Systemic Toxicity ...................1-6
1.1.3
The Registry of Cytotoxicity (RC) ................................................1-7
1.1.4
The ZEBET Initiative to Reduce Animal Use ...............................1-9
1.1.5
The International Workshop on In Vitro Methods forAssessing Acute Systemic Toxicity ...........................................1-10
1.1.6
The NICEATM/ECVAM In Vitro NRU Cytotoxicity Study .......1-11
1.1.6.1
Study Design .............................................................1-12

1.2

Regulatory Rationale and Applicability for the Use of In Vitro
Cytotoxicity Test Methods to Predict Starting Doses for Acute
Oral Toxicity Testing in Rodents............................................................1-14
1.2.1
Current Regulatory Testing Requirements for Acute Oral
Toxicity ......................................................................................1-14
1.2.1.1
Test Methods for Assessing Acute Oral Toxicity ........1-16
1.2.2
Intended Regulatory Uses for In Vitro Cytotoxicity Test
Methods......................................................................................1-16
1.2.3
Similarities and Differences in the Endpoints of In Vitro
Cytotoxicity Test Methods and Rodent Acute Oral Toxicity
Test Methods ..............................................................................1-17
1.2.4
Use of In Vitro Cytotoxicity Test Methods in the Overall
Strategy of Hazard Assessment ...................................................1-18i

In Vitro Cytotoxicity Test Methods BRD Table of Contents

1.3

2.0

November 2006

Scientific Basis for the In Vitro NRU Test Methods .............................. 1-18
1.3.1
Purpose and Mechanistic Basis of In Vitro NRU Test
Methods ..................................................................................... 1-19
1.3.2
Similarities and Differences in the Modes/Mechanisms of
Action for In Vitro NRU Test Methods Compared with theSpecies of Interest ...................................................................... 1-20
1.3.3
Range of Substances Amenable to the In Vitro NRU Test
Methods ..................................................................................... 1-20

TEST METHOD PROTOCOL COMPONENTS OF THE 3T3 AND NHK
IN VITRO NRU TEST METHODS .....................................................................2-3
2.1

Basis for Selection of In Vitro NRU Cytotoxicity Test Methods..............2-4
2.1.1
Guidance Document Rationale for Selection of In Vitro NRU
Cytotoxicity Test Methods............................................................2-4
2.1.2
Guidance Document Rationale for Selection of Cell Types...........2-4

2.2

Overview of the 3T3 and NHK NRU Test Methods ................................2-5
2.2.1
The 3T3 NRU Test Method ..........................................................2-6
2.2.1.1
Initiating and Subculturing 3T3 Cells..........................2-6
2.2.1.2
Preparation of Cells for 96-well Plate Assays .............2-8
2.2.1.3
Reference Substance Application.................................2-8
2.2.2
The NHK NRU Test Method .......................................................2-8
2.2.2.1
Initiating and Subculturing NHK Cells........................2-8
2.2.2.2
Preparation of Cells for 96-well Plate Assays .............2-8
2.2.2.3
Reference Substance Application.................................2-9
2.2.3
Measurement of NRU in the 3T3 and NHK Test Methods ............2-9

2.3

Descriptions and Rationales of the 3T3 and NHK NRU Test
Methods.....................................................................................................2-9
2.3.1
Materials, Equipment, and Supplies..............................................2-9
2.3.1.1
3T3 Cells.....................................................................2-9
2.3.1.2
NHK Cells................................................................. 2-10
2.3.1.3
Tissue Culture Materials and Supplies ...................... 2-10
2.3.1.4
Cell Culture Materials .............................................. 2-10
2.3.1.5
Equipment................................................................. 2-10
2.3.1.6
Culture Medium ........................................................ 2-11
2.3.2
Reference Substance Concentrations/Dose Selection .................. 2-12
2.3.2.1
Range Finder Test..................................................... 2-12
2.3.2.2
Definitive Test ........................................................... 2-12
2.3.3
NRU Endpoints Measured .......................................................... 2-13
2.3.3.1
NRU and Measurement ............................................. 2-13
2.3.3.2
Determination of IC50, IC20, and IC80 Values .............. 2-13
2.3.4
Duration of Reference Substance Exposure ................................ 2-14
2.3.5
Known Limits of Use ................................................................. 2-14
2.3.5.1
Solubility/Precipitation/Volatility.............................. 2-14
2.3.5.2
Biokinetic Determinations ......................................... 2-15
2.3.5.3
Organ-Specific Toxicity............................................. 2-15

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2.3.5.4

2.3.6
2.3.7

2.3.8
2.3.9
2.3.102.3.11
2.3.122.3.132.3.14The Role of Cytotoxicity Tests in an In Vitro
Battery Approach for Possible Replacement of In
Vivo Acute Toxicity Testing .......................................2-15
Basis of the Response Assessed ..................................................2-16
Appropriate Positive, Vehicle, and Negative Controls.................2-16
2.3.7.1
Positive Control ........................................................2-16
2.3.7.2
Vehicle Control .........................................................2-16
2.3.7.3
Negative Control .......................................................2-17
Acceptable Ranges of Control Responses ...................................2-17
2.3.8.1
Vehicle Controls as a Quality Control Tool ...............2-18
Nature of Experimental Data Collected.......................................2-18
2.3.9.1
NRU OD Measurements ............................................2-18
2.3.9.2
Information and Data Collected ................................2-18
Data Storage Media ....................................................................2-19
Measures of Variability ..............................................................2-19
Methods for Analyzing NRU Data..............................................2-20
Decision Criteria for Classification of Reference Substances ......2-20
Information and Data Included in the Test Report.......................2-20

2.4

Proprietary Components of the In Vitro NRU Test Methods................2-22

2.5

Basis for the Number of Replicate and Repeat Experiments for the
3T3 and NHK NRU Test Methods..........................................................2-22

2.6

Basis for Modifications to the 3T3 and NHK NRU Test Method
Protocols ..................................................................................................2-22
2.6.1
Phase Ia: Laboratory Evaluation Phase .......................................2-22
2.6.1.1
NR Dye Crystals........................................................2-23
2.6.1.2
3T3 Cell Growth........................................................2-23
2.6.1.3
NHK Cell Growth......................................................2-23
2.6.1.4
Vehicle Control OD Limits ........................................2-24
2.6.1.5
Precipitate Formation ...............................................2-24
2.6.1.6
Dilution Factor .........................................................2-24
2.6.1.7
Test Acceptance Criteria ...........................................2-25
2.6.2
Phase Ib: Laboratory Evaluation Phase .......................................2-25
2.6.2.1
NR Crystal Formation ...............................................2-25
2.6.2.2
Heating of Reference Substance Solutions .................2-26
2.6.2.3
Growth of Untreated Cells.........................................2-27
2.6.2.4
Correction of Reference Substance OD Values ..........2-27
2.6.2.5
Laboratory Error Rates .............................................2-28
2.6.2.6
Resultant Protocol Changes for Phase II ...................2-28
2.6.2.7
Test Acceptance Criteria ...........................................2-29
2.6.3
Phase II: Laboratory Qualification Phase ....................................2-29
2.6.3.1
Testing of Volatile Reference Substances ...................2-29
2.6.3.2
Atypical Concentration-Responses ............................2-31
2.6.3.3
Hill Function.............................................................2-33
2.6.3.4
Insoluble Reference Substances .................................2-33
2.6.3.5
Inadequate Cell Growth in NHK Medium..................2-34
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2.6.3.6

Performance Standards for Media to Support
NHK Growth............................................................. 2-34
2.6.3.7
Test Acceptance Criteria for Phase II........................ 2-35
2.6.4 Phase III: Laboratory Testing Phase .............................................. 2-35
2.6.4.1
Required Cytotoxicity Values .................................... 2-35
2.6.4.2
Revisions to Data Analysis Procedures ..................... 2-35

3.0

2.7

Differences Between the 3T3 and NHK NRU Protocols for the
Validation Study and the Guidance Document Standard Protocols...... 2-36

2.8

Overview of the Solubility Protocol........................................................ 2-37

2.9

Basis of the Solubility Protocol............................................................... 2-38
2.9.1
Initial Solubility Protocol Development...................................... 2-38
2.9.2
Basis for Modification of the Phase II Protocol........................... 2-39

2.10

Components of the Solubility Protocol................................................... 2-40
2.10.1 Medium, Supplies, and Equipment Required .............................. 2-40
2.10.1.1 Medium and Chemical Supplies ................................ 2-40
2.10.1.2 Equipment................................................................. 2-40
2.10.1.3 Procedures................................................................ 2-40
2.10.2 Data Collection........................................................................... 2-41
2.10.3 Variability in Solubility Measurements....................................... 2-41
2.10.4 Solubility Issues During the Testing of the Reference
Substances.................................................................................. 2-41
2.10.5 Analysis of Solubility Data......................................................... 2-41

2.11

Summary ................................................................................................. 2-42

REFERENCE SUBSTANCES USED FOR VALIDATION OF THE 3T3
AND NHK NRU TEST METHODS ....................................................................3-3
3.1

Rationale for the 72 Reference Substances Selected for Testing ............3-3
3.1.1
Reference Substance Selection Criteria.........................................3-3
3.1.2
Candidate Reference Substances...................................................3-4
3.1.3
Selection of Reference Substances for Testing..............................3-5

3.2

Characteristics of the Selected Reference Substances .............................3-6
3.2.1
Source Databases Represented by the Selected Reference
Substances....................................................................................3-6
3.2.2
Chemical Classes Represented by the Selected Reference
Substances.................................................................................. 3-28
3.2.3
Product/Use Classes Represented by the Selected Reference
Substances.................................................................................. 3-28
3.2.4
Toxicological Characteristics of the Selected Reference
Substances.................................................................................. 3-28
3.2.4.1
Corrosivity ................................................................ 3-28
3.2.4.2
Toxicity Targets ........................................................ 3-32
3.2.4.3
Metabolism ............................................................... 3-32
3.2.5
Selection of Reference Substances for Testing in Phases Ib
and II.......................................................................................... 3-32
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In Vitro Cytotoxicity Test Methods BRD Table of Contents

3.2.6

4.0

5.0

November 2006

Unsuitable and Challenging Reference Substances .....................3-34

3.3

Reference Substance Procurement, Coding, and Distribution..............3-34
3.3.1
Exceptions..................................................................................3-35

3.4Reference Substances Recommended by the Guidance Document........3-35

3.5

Summary .................................................................................................3-36

RODENT ACUTE ORAL LD50 REFERENCE VALUES USED TO
ASSESS THE ACCURACY OF THE 3T3 AND NHK NRU TESTMETHODS ...........................................................................................................4-3
4.1

Methods Used to Obtain Rodent Acute Oral LD50 Reference
Values ........................................................................................................4-3
4.1.1
Identification of Candidate Rodent Acute Oral LD50
Reference Data .............................................................................4-3
4.1.2
Criteria Used to Select Candidate Rodent Acute Oral Data for
Determination of LD50 Reference Values......................................4-5
4.1.2.1
Final Exclusion Criteria ..............................................4-6
4.1.2.2
Assumptions Regarding Materials, Animals, andMethods ......................................................................4-6
4.1.2.3
Calculation Reference LD50 Values .............................4-7
4.1.2.4
Use of Rat and Mouse Data.........................................4-7

4.2

Final Rodent Acute Oral LD50 Reference Values ....................................4-7

4.3

Relevant Toxicity Information for Humans.............................................4-8

4.4

Accuracy and Reliability of the Rodent Acute Oral LD50 Reference
Values ......................................................................................................4-13
4.4.1
Variability Among the Acceptable LD50 Values..........................4-13
4.4.2
Comparison of Rodent Acute Oral LD50 Reference Values
with the Corresponding RC LD50 Values ...................................4-14
4.4.3
Comparison of the Variability Among Acceptable LD50
Values to Those Obtained in Other Studies .................................4-15

4.5

Summary .................................................................................................4-16

3T3 AND NHK NRU TEST METHOD DATA AND RESULTS .......................5-3
5.1

Study Timeline and Participating Laboratories ......................................5-3
5.1.1
Statements of Work (SOW) and Protocols ....................................5-3
5.1.2
Study Timeline .............................................................................5-4
5.1.3
Participating Laboratories.............................................................5-4

5.2

Coded Reference Substances and GLP Guidelines..................................5-5
5.2.1
Coded Reference Substances ........................................................5-5
5.2.2
Lot-to-Lot Consistency of Reference Substances ..........................5-5
5.2.3
Adherence to GLP Guidelines.......................................................5-5

5.3

3T3 and NHK NRU Test Method Protocols.............................................5-5
5.3.1
Phase Ia: Laboratory Evaluation Phase .........................................5-6v

In Vitro Cytotoxicity Test Methods BRD Table of Contents

5.3.2
5.3.3
5.3.4

November 2006

5.3.1.1
Protocol Changes and the Effect on the Data ..............5-6
Phase Ib: Laboratory Evaluation Phase.........................................5-6
5.3.2.1
Protocol Changes and the Effect on the Data ..............5-7
Phase II: Laboratory Qualification Phase ......................................5-7
5.3.3.1
Protocol Changes and the Effect on the Data ..............5-8
Phase III: Main Validation Phase..................................................5-9
5.3.4.1
Protocol Changes and the Effect on the Data ..............5-9

5.4

Data Used to Evaluate Test Method Accuracy and Reliability ............. 5-10
5.4.1
PC Data...................................................................................... 5-10
5.4.1.1
Phase Ib PC Data Acceptance Limits ........................ 5-12
5.4.1.2
Phase II PC Data Acceptance Limits......................... 5-12
5.4.1.3
Phase III PC Data Acceptance Limits ....................... 5-12
5.4.2
Reference Substance Data .......................................................... 5-12

5.5

Statistical Approaches to the Evaluation of 3T3 and NHK Data.......... 5-26
5.5.1
Statistical Analyses for Phase Ia Data......................................... 5-27
5.5.1.1
Outlier Determination for Replicate Well
Concentration Data................................................... 5-27
5.5.1.2
Curve Fit Criteria ..................................................... 5-27
5.5.1.3
Reproducibility Analyses for PC IC50 Values............. 5-27
5.5.2
Statistical Analyses for Phase Ib Data......................................... 5-28
5.5.2.1
Outlier Determination for Replicate Well
Concentration Data................................................... 5-28
5.5.2.2
Reproducibility Analyses of the Reference
Substance IC50 Values ............................................... 5-28
5.5.3
Statistical Analyses for Phase II Data ......................................... 5-28
5.5.3.1
Outlier Determination for Replicate Well
Concentration Data................................................... 5-28
5.5.3.2
Reproducibility Analyses of the Reference
Substance IC50 Values ............................................... 5-28
5.5.3.3
Comparison of 3T3 and NHK Test Results with
the RC Millimole Regression ..................................... 5-29
5.5.4
Statistical Analyses for Phase III Data ........................................ 5-29
5.5.4.1
Outlier Determination for Replicate Well
Concentration Data................................................... 5-29
5.5.4.2
Reproducibility Analyses of the PC IC50 Data ........... 5-29
5.5.4.3
Reproducibility Analyses of the Reference
Substance
IC50 Values................................................................ 5-30
5.5.4.4
Comparison of 3T3 and NHK Test Results with
the RC Millimole Regression ..................................... 5-31
5.5.5
Summary of the Data Used for Statistical Analyses .................... 5-32

5.6

Summary of NRU Test Results............................................................... 5-32

5.7

Availability of Data ................................................................................. 5-38

5.8

Solubility Test Results ............................................................................ 5-38

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5.8.1
5.8.2
5.9
6.0

November 2006

Solubility Data............................................................................5-43
Solubility and Volatility Effects in the Cytotoxicity Tests...........5-43

Summary .................................................................................................5-44

ACCURACY OF THE 3T3 AND NHK NRU TEST METHODS ......................6-3
6.1

Accuracy of the 3T3 and NHK NRU Test Methods for Predicting
Rodent Acute Oral Toxicity......................................................................6-5
6.1.1
Linear Regression Analyses for the Prediction of Rat Acute
Oral LD50 Values from In Vitro IC50 Values..................................6-5
6.1.2
Comparison of Combined-Laboratory 3T3 and NHK
Regressions to the RC Millimole Regression ................................6-8

6.2

Analysis of Outlier Substances for the RC Millimole Regression ...........6-9
6.2.1
Identification of Outlier Substances ............................................6-10
6.2.2
Evaluation of Outlier Substances ................................................6-12
6.2.2.1
Physical Characteristics............................................6-12
6.2.2.2
Chemical Class .........................................................6-12
6.2.2.3
Solubility...................................................................6-13
6.2.2.4
Metabolism................................................................6-13
6.2.2.5
Mechanism of Toxicity...............................................6-13

6.3

Improving the Prediction of In Vivo Rat Oral LD50 Values from In
Vitro IC50 Data .........................................................................................6-17
6.3.1
The RC Rat-Only Millimole Regression .....................................6-17
6.3.2
The RC Rat-Only Weight Regression .........................................6-17

6.4

Accuracy of the 3T3 and NHK NRU Test Methods for Predicting
GHS Acute Oral Toxicity Categories .....................................................6-18
6.4.1
Prediction of GHS Acute Oral Toxicity Category by the RC
IC50 Values Using the RC Millimole Regression.........................6-20
6.4.2
Prediction of GHS Acute Oral Toxicity Category by the 3T3
and NHK NRU Test Methods Using the RC Rat-Only
Millimole Regression..................................................................6-22
6.4.2.1
In Vitro – In Vivo Concordance Using the RC
Rat-Only Millimole Regression..................................6-22
6.4.2.2
Discordant Substances in the Prediction of GHS
Acute Oral Toxicity Category by the 3T3 andNHK NRU Test Methods and the RC Rat-Only
Millimole Regression.................................................6-24
6.4.3
Prediction of GHS Acute Oral Toxicity Category by the 3T3
and NHK NRU Test Methods Using the RC Rat-Only
Weight Regression......................................................................6-24
6.4.3.1
In Vitro – In Vivo Concordance Using the RC
Rat-Only Weight Regression......................................6-25
6.4.3.2
Discordant Substances in the Prediction of GHS
Acute Oral Toxicity Category by the 3T3 andNHK NRU Test Methods and the RC Rat-Only
Weight Regression.....................................................6-27
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6.4.4

7.0

November 2006

Summary of the Regressions Evaluated ...................................... 6-27

6.5

Correlation of NRU Concentration-Response Slope with Rat
Lethality Dose-Response Slope............................................................... 6-28

6.6

Strengths and Limitations of the Use of In Vitro Cytotoxicity Test
Methods with the IC50–LD50 Regressions for Prediction of Rodent
Acute Oral Toxicity................................................................................. 6-31
6.6.1
In Vitro Cytotoxicity Methods .................................................... 6-31
6.6.2
Use of Mole-Based vs. Weight-Based Regressions for thePrediction of Toxicity for Low and High Molecular Weight
Substances.................................................................................. 6-31

6.7

Salient Issues of Data Interpretation...................................................... 6-34

6.8

Comparison of NRU Test Results to Established PerformanceStandards ................................................................................................ 6-34

6.9

Summary ................................................................................................. 6-40

RELIABILITY OF THE 3T3 AND NHK TEST METHODS ............................7-3
7.1

Reference Substances Used to Determine the Reliability of the 3T3
and NHK Test Methods ............................................................................7-3

7.2

Reproducibility Analyses for the 3T3 and NHK Test Methods ..............7-5
7.2.1
Comparison of Laboratory-Specific IC50-LD50 Linear
Regression Analyses to the Mean Laboratory Regression .............7-5
7.2.2
ANOVA Results for the 3T3 and NHK NRU Test Methods .........7-5
7.2.2.1
Differences Among the IC50 Values inLaboratories Using the 3T3 NRU Test Method............7-5
7.2.2.2
Differences Among the IC50 Values inLaboratories Using the NHK NRU Test Method........ 7-21
7.2.3
CV Results for the 3T3 and NHK NRU Test Methods ................ 7-21
7.2.3.1
Reproducibility of Intralaboratory CV Values ........... 7-21
7.2.3.2
Reproducibility of Interlaboratory CV Values ........... 7-34
7.2.3.3
Variation of CV with Chemical Property ................... 7-34
7.2.3.4
Results of the Intralaboratory CV Analysis................ 7-35
7.2.3.5
Results of the Interlaboratory CV Analysis ................ 7-37
7.2.4
Comparison of Maximum to Minimum IC50 Values Using
Laboratory Means ...................................................................... 7-39
7.2.5
Comparison of Maximum:Minimum IC50 Ratios with the
Maximum:Minimum LD50 Ratios............................................... 7-40
7.2.6
Normalization of Reference Substance IC50 Values Using
SLS IC50 Values ......................................................................... 7-42

7.3

Historical Positive Control (PC) Data ................................................... 7-44
7.3.1
ANOVA and Linear Regression Results for the 3T3 NRU
Test Method .............................................................................. 7-46
7.3.1.1
Variation of the SLS IC50 Values with Time ............... 7-46
7.3.1.2
Comparison of SLS IC50 Values Among theLaboratories ............................................................. 7-46
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7.3.2

8.0

9.0

November 2006

ANOVA and Linear Regression Results for the NHK NRU
Test Method ...............................................................................7-48
7.3.2.1
Variation of SLS IC50 Values with Time.....................7-48
7.3.2.2
Comparison of SLS IC50 Values Among theLaboratories..............................................................7-48

7.4

Laboratory Concordance for Solvent Selection .....................................7-50

7.5

Summary .................................................................................................7-52

3T3 AND NHK NRU TEST METHOD DATA QUALITY ................................8-3
8.1

Compliance with Good Laboratory Practice Regulations.......................8-3
8.1.1
Guidelines Followed for Cytotoxicity Testing...............................8-3
8.1.1.1
Good Laboratory Practices .........................................8-3
8.1.1.2
Spirit of GLP ...............................................................8-3
8.1.1.3
Good Cell Culture Practices (GCCP)..........................8-4
8.1.2
Quality Assurance (QA) for NRU Cytotoxicity Test Data.............8-4
8.1.2.1
Coded Reference Substances .......................................8-4
8.1.2.2
Solubility Testing and Data Review .............................8-5
8.1.2.3
NRU Cytotoxicity Test Tallies......................................8-5
8.1.3
Guidelines Followed for Rodent Acute Oral LD50 Data
Collection .....................................................................................8-6
8.1.3.1
Rodent Acute Oral LD50 Values Used in theRegistry of Cytotoxicity (RC).......................................8-6
8.1.3.2
Rodent Acute Oral LD50 Values Collected by
NICEATM from Other Sources ....................................8-6

8.2

Results of Data Quality Audits .................................................................8-6
8.2.1
QA Statements..............................................................................8-6
8.2.2
QA Statements from the Laboratories ...........................................8-7
8.2.2.1
BioReliance QA Statements .........................................8-7
8.2.2.2
FAL QA Statements .....................................................8-7
8.2.2.3
ECBC QA Statements ..................................................8-7
8.2.2.4
IIVS QA Statements .....................................................8-8
8.2.2.5
Other QA Information .................................................8-8

8.3

Effect of Deviations or Non-compliance with GLPs ................................8-9
8.3.1
Laboratory Error Rates .................................................................8-9
8.3.2
Failure Rates for Definitive and PC Tests ...................................8-10
8.3.3
Intralaboratory Reproducibility...................................................8-11
8.3.4
Prediction of GHS Acute Oral Toxicity Categories .....................8-12

8.4

Availability of Laboratory Notebooks....................................................8-12

8.5

Summary .................................................................................................8-13

OTHER SCIENTIFIC REPORTS AND REVIEWS OF IN VITROCYTOTOXICITY TEST METHODS AND THEIR ABILITY TO
PREDICT IN VIVO ACUTE TOXICITY AND OTHER TOXICEFFECTS..............................................................................................................9-3ix

In Vitro Cytotoxicity Test Methods BRD Table of Contents

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November 2006

9.1

Relevant Studies........................................................................................9-3
9.1.1
Correlation of NRU Cytotoxicity Values with Rodent
Lethality.......................................................................................9-3
9.1.1.1
Peloux et al. (1992).....................................................9-4
9.1.1.2
Fautrel et al. (1993) ....................................................9-5
9.1.1.3
Roguet et al. (1993).....................................................9-5
9.1.1.4
Rasmussen (1999) .......................................................9-6
9.1.1.5
Creppy et al. (2004) ....................................................9-7
9.1.2
Use of In Vitro Cytotoxicity Data to Reduce the Use of
Animals in Acute Oral Toxicity Testing .......................................9-7
9.1.2.1
Halle et al. (1997): Animal Savings with the ATC
Method Using Cytotoxicity Data .................................9-7
9.1.2.2
Spielmann et al. (1999): Animal Savings Using
Cytotoxicity Data with the UDP ..................................9-9
9.1.2.3
EPA (2004): U.S. EPA HPV Challenge Program
Submission ................................................................ 9-10
9.1.3 Other Evaluations of 3T3 or NHK NRU Methods.......................... 9-10
9.1.3.1
Draize Eye Irritation ................................................. 9-11
9.1.3.2
Predicting Human Lethal Blood Concentrations(LC) .......................................................................... 9-13

9.2

Independent Scientific Reviews.............................................................. 9-13
9.2.1
In Vitro Acute Toxicity Testing for the Classification andLabeling of Chemicals................................................................ 9-13
9.2.1.1
Seibert et al. (1996): ECVAM Workshop 16 .............. 9-13
9.2.2
Use of In Vitro Cytotoxicity Data for Estimation of Starting
Doses for Acute Oral Toxicity Testing ....................................... 9-14
9.2.2.1
ICCVAM (2001a): Estimation of Animal Savings
Using Cytotoxicity Data with the ATC Method .......... 9-14
9.2.2.2
ICCVAM (2001a): Estimation of Animal Savings
Using Cytotoxicity Data with the UDP ...................... 9-16
9.2.3
Validation of 3T3 NRU Assay for Phototoxicity......................... 9-17
9.2.3.1
NHK NRU Phototoxicity Assay.................................. 9-17

9.3

Studies Using In Vitro Cytotoxicity Test Methods with Established
Performance Standards .......................................................................... 9-18
9.3.1
Guidance Document (ICCVAM 2001b)....................................... 9-18
9.3.2
King and Jones (2003)................................................................ 9-19
9.3.3
A-Cute-Tox Project: Optimization and Pre-Validation of an
In Vitro Test Strategy for Predicting Human Acute Toxicity(Clemedson 2005) ...................................................................... 9-19

9.4

Summary ................................................................................................. 9-20

ANIMAL WELFARE CONSIDERATIONS (REFINEMENT,REDUCTION, AND REPLACEMENT) ........................................................... 10-3
10.1

Use of 3T3 and NHK NRU Test Methods to Predict Starting Doses
for Rodent Acute Oral Toxicity Assays.................................................. 10-4x

In Vitro Cytotoxicity Test Methods BRD Table of Contents

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November 2006

10.2

Reduction and Refinement of Animal Use for the UDP ........................10-4
10.2.1 In Vivo Testing Using the UDP...................................................10-4
10.2.1.1 Main Test ..................................................................10-4
10.2.1.2 Limit Test ..................................................................10-5
10.2.2 Computer Simulation Modeling of the UDP ...............................10-6
10.2.3 Animal Savings in the UDP When Using 3T3- and NHKBased Starting Doses ..................................................................10-7
10.2.3.1 The Effect of the Dose-Mortality Slope on Animal
Use............................................................................10-7
10.2.3.2 Mean Animal Use for UDP Simulations –Comparison of Regressions and Predictions fromthe 3T3 and NHK NRU Test Methods ........................10-9
10.2.3.3 Animal Savings in the UDP by GHS Acute Oral
Toxicity Category Using 3T3- and NHK-Based
Starting Doses ...........................................................10-9
10.2.4 Refinement of Animal Use for the UDP When Using 3T3and NHK-Based Starting Doses ................................................10-15
10.2.5 Accuracy of the UDP Outcomes Using the IC50-Based
Starting Doses ..........................................................................10-16

10.3

Reduction and Refinement of Animal Use in the ATC Method ..........10-16
10.3.1 In Vivo Testing Using the ATC Method....................................10-16
10.3.1.1 Main Test ................................................................10-17
10.3.1.2 Limit Test ................................................................10-17
10.3.2 Computer Simulation Modeling of the ATC Method ................10-17
10.3.3 Animal Savings for the ATC Method When Using 3T3- andNHK-Based Starting Doses ......................................................10-18
10.3.3.1 The Effect of Dose-Mortality Slope on Animal
Use..........................................................................10-18
10.3.3.2 Mean Animal Use for ATC Simulations –Comparison of Regressions and Predictions fromthe 3T3 and NHK NRU Test Methods ......................10-20
10.3.3.3 Animal Savings in the ATC Method by GHS Acute
Oral Toxicity Category Using the 3T3- and NHKBased Starting Doses...............................................10-22
10.3.4 Refinement of Animal Use in the ATC Method When Using
3T3- and NHK-Based Starting Doses........................................10-26
10.3.5 Accuracy of the ATC Method Outcomes Using the IC50Based Starting Doses ................................................................10-27

10.4

The Impact of Accuracy on Animal Savings........................................10-28

10.5

The Impact of Prevalence on Animal Savings......................................10-30

10.6

Summary ...............................................................................................10-31

PRACTICAL CONSIDERATIONS ..................................................................11-3
11.1

Transferability of the 3T3 and NHK NRU Test Methods .....................11-3
11.1.1 Facilities and Major Fixed Equipment ........................................11-3
xiIn Vitro Cytotoxicity Test Methods BRD Table of Contents

11.1.2

11.1.3

November 2006

11.1.1.1 Facility Requirements ............................................... 11-3
11.1.1.2 Cell Culture Laboratory............................................ 11-4
11.1.1.3 Major Equipment ...................................................... 11-4
Availability of Other Necessary Equipment and Supplies ........... 11-4
11.1.2.1 General Equipment ................................................... 11-4
11.1.2.2 Cell Culture Materials and Supplies.......................... 11-5
11.1.2.3 Cell Cultures............................................................. 11-5
Problems Specific to the NHK NRU Test Method ...................... 11-5

11.2

3T3 and NHK NRU Test Method Training Considerations.................. 11-6
11.2.1 Required Training and Expertise ................................................ 11-6
11.2.1.1 Specific Training and Expertise Needed .................... 11-6
11.2.1.2 General Laboratory Expertise Needed ...................... 11-7
11.2.2 Training Requirements to Demonstrate Proficiency.................... 11-7
11.2.2.1 Proficiency with GLP-Compliance ............................ 11-8
11.2.3 Personnel Needed to Perform the In Vitro NRU Test Methods.... 11-8

11.3

Cost Considerations ................................................................................ 11-8
11.3.1 3T3 and NHK NRU Test Methods.............................................. 11-8
11.3.1.1 Equipment Costs ....................................................... 11-8
11.3.1.2 Costs for Cell Cultures and Supplies ......................... 11-8
11.3.1.3 Commercial Testing .................................................. 11-9
11.3.2 Rodent Acute Oral Toxicity Testing ......................................... 11-11

11.4

Time Considerations for Performing the 3T3 and NHK NRU Tests .. 11-12
11.4.1 The 3T3 NRU Test Method ...................................................... 11-12
11.4.2 The NHK NRU Test Method.................................................... 11-12
11.4.3 Prequalification of NHK Medium............................................. 11-12
11.4.4 In Vivo Testing ......................................................................... 11-12
11.4.5 The Limit Test.......................................................................... 11-13

11.5

Summary ............................................................................................... 11-14

12.0

REFERENCES ................................................................................................... 12-1

13.0

GLOSSARY........................................................................................................ 13-1

xii

In Vitro Cytotoxicity Test Methods BRD Table of Contents

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APPENDICES
Appendix A NICEATM/ECVAM Validation Study Management.............................A-1
Appendix B Validation Study Test Method Protocols (Phase III) .............................. B-1
B1
Test Method Protocol for the BALB/c 3T3 Neutral Red Uptake (NRU)
Cytotoxicity Test........................................................................................ B-3
B2
Test Method Protocol for the Normal Human Epidermal Keratinocyte(NHK) Neutral Red Uptake Cytotoxicity Test .......................................... B-25
B3
Test Method Protocol for Solubility Determination (Phase III)................. B-47
B4
Test Method Procedure for Prequalification of Normal Human
Epidermal Keratinocyte Growth Medium (Phase III)................................ B-59
Appendix C Validation Study Test Method Protocols (Phases Ia, Ib, and II)............C-1
C1
Test Method Protocol for the BALB/c 3T3 Neutral Red Uptake
Cytotoxicity Test (Phase Ia) ....................................................................... C-3
C2
Test Method Protocol for the Normal Human Epidermal Keratinocyte(NHK) Neutral Red Uptake Cytotoxicity Test (Phase Ia).......................... C-23
C3
Test Method Protocol for the BALB/c 3T3 Neutral Red Uptake
Cytotoxicity Test (Phase Ib) ..................................................................... C-41
C4
Test Method Protocol for the Normal Human Epidermal Keratinocyte(NHK) Neutral Red Uptake Cytotoxicity Test (Phase Ib) ......................... C-63
C5
Test Method Protocol for the BALB/c 3T3 Neutral Red Uptake
Cytotoxicity Test (Phase II)...................................................................... C-85
C6
Test Method Protocol for the Normal Human Epidermal Keratinocyte(NHK) Neutral Red Uptake Cytotoxicity Test (Phase II) ........................ C-109
Appendix D SAS Codes ................................................................................................D-1
D1
SAS Code for ANOVA and Contrasts ........................................................D-3
D2
SAS Code for Regression Comparisons......................................................D-7
Appendix E Neutral Red Dye Experiments ................................................................. E-1
E1
Institute for In Vitro Sciences (IIVS) Assessment of Protocol Variablesin the NICEATM/ECVAM Evaluation of Cytotoxicity Assays................... E-5
E2
Neutral Red (NR) Dye Experiments – 3T3 Cells – IIVS........................... E-13
E3
Neutral Red (NR) Dye Experiments – NHK Cells – IIVS......................... E-19
E4
Neutral Red (NR) Dye Experiments – 3T3 Cells – ECBC ........................ E-25
Appendix F Reference Substance Information ........................................................... F-1
F1
NRU Test Information for the 72 Reference Substances ............................. F-3
F2
Chemical, Physical, and Biological Information from the Literature forthe 72 Reference Substances .................................................................... F-11
F3
Candidate Reference Substances .............................................................. F-27
Appendix G Statement of Work (SOW).......................................................................G-1
G1
A Validation Study For In Vitro Basal Cytotoxicity Testing .......................G-3
G2
Procedures for Acquisition, Preparation, Solubility Testing, andDistribution of Test Chemicals for a Validation Study for In Vitro
Basal Cytotoxicity Testing .......................................................................G-57

xiii

In Vitro Cytotoxicity Test Methods BRD Table of Contents

November 2006

Appendix H Rat and Mouse Oral LD50 Database........................................................H-1
H1
Rat and Mouse Oral LD50 Database ........................................................... H-3
H2
Evaluation of the Candidate Reference Oral LD50 Data ............................ H-39
Appendix I
I1
I2
I3
I4

In Vitro NRU Data .................................................................................... I-1
3T3 NRU Reference Substance Data........................................................... I-3
NHK NRU Reference Substance Data .......................................................I-59
3T3 NRU Positive Control (SLS) Data.....................................................I-111
NHK NRU Positive Control (SLS) Data ..................................................I-127

Appendix J
J1
J2
J3
J4
J5
J6
J7

LD50 and Toxicity Category Predictions ..................................................J-1
3T3 NRU Predictions: RC Millimole Regression ........................................ J-5
NHK NRU Predictions: RC Millimole Regression .................................... J-11
3T3 NRU Predictions: RC Rat-Only Millimole Regression....................... J-17
NHK NRU Predictions: RC Rat-Only Millimole Regression..................... J-21
3T3 NRU Predictions: RC Rat-Only Weight Regression ........................... J-25
NHK NRU Predictions: RC Rat-Only Weight Regression......................... J-29
Comparison of Millimole Regression with Weight Regression
Regarding Prediction of Toxicity (LD50) for Low or High Molecular
Weight Chemicals..................................................................................... J-34

Appendix K
K1
K2
K3
K4

IC50 and LD50 Data for Regressions ........................................................K-1
IC50 and LD50 Values Used for Laboratory-Specific Regressions ............... K-3
IC50 and LD50 Values Used for Combined-Laboratory Regressions.......... K-17
RC IC50 and LD50 Values for RC Substances with Rat Oral LD50 Data..... K-23
Individual Laboratory LD50 Predictions: RC Rat-Only Millimole
Regression ............................................................................................... K-33

Appendix L Outlier Information ................................................................................. L-1
L1
Outlier Characterization for the 3T3 and NHK NRU Test Methods
with the RC Millimole Regression ............................................................. L-3
L2
Discordant Substances for GHS Acute Oral Toxicity Category
Predictions Using the 3T3 and NHK NRU Test Methods and RC RatOnly Regressions ....................................................................................... L-9
L3
Analysis of Outliers by Halle (1998, 2003) for the RC Millimole
Regression ............................................................................................... L-17
Appendix M Acute Oral Toxicity Test Guidelines ...................................................... M-1
M1
OECD UDP Test Guideline ...................................................................... M-3
M2
EPA UDP Test Guideline........................................................................ M-31
M3
OECD ATC Method Test Guideline ....................................................... M-71
M4
OECD FDP Test Guideline ..................................................................... M-87
M5
OECD Guidance on Acute Oral Toxicity Testing...................................M-103
Appendix N UDP/ATC Simulation Modeling Results................................................. N-1
N1
UDP Simulation Results Using Starting Doses One Default Dose
Lower than the LD50 Predicted by the 3T3 and NHK NRU IC50 and the
RC Rat-Only Millimole Regression – 5000 mg/kg Upper Limit Dose ........ N-3

xiv

In Vitro Cytotoxicity Test Methods BRD Table of Contents

N2
N3
N4

November 2006

UDP Simulation Results Using Starting Doses One Default Dose
Lower than the LD50 Predicted by the 3T3 and NHK NRU IC50 and the
RC Rat-Only Weight Regression – 5000 mg/kg Upper Limit Dose ..........N-13
ATC Simulation Results Starting at the Next Fixed Dose Below theLD50 Predicted by the 3T3 and NHK NRU IC50 and the RC Rat-Only
Millimole Regression - 2000 mg/kg Upper Limit Dose ............................N-23
ATC Simulation Results Starting at the Next Fixed Dose Below theLD50 Predicted by the 3T3 and NHK IC50 and the RC Rat-Only
Weight Regression – 2000 mg/kg Upper Limit Dose................................N-33

Appendix O Federal Register Notices ...........................................................................O-1
O1
70FR14473 - Request for Nominations for an Independent Peer Review
Panel To Evaluate In Vitro Testing Methods for Estimating Acute Oral
Systemic Toxicity and Request for In Vivo and In Vitro Data .....................O-3
O2
69FR61504 - Availability of Updated Standardized In Vitro
Cytotoxicity Test Method Protocols for Estimating Acute Oral
Systemic Toxicity; Request for Existing In Vivo and In Vitro Acute
Toxicity Data ...........................................................................................O-5
O3
69FR11448 - Notice of the Availability of Agency Responses to
ICCVAM Test Recommendations for the Revised Up-and-Down
Procedure for Determining Acute Oral Toxicity and In Vitro Methods
for Assessing Acute Systemic Toxicity ......................................................O-7
O4
66FR49686 - Report of the International Workshop on In Vitro
Methods for Assessing Acute Systemic Toxicity; Guidance Document
on Using In Vitro Data to Estimate In Vivo Starting Doses for Acute
Toxicity: Notice of Availability and Request for Public Comment .............O-9
O5
65FR57203 - Notice of an International Workshop on In Vitro
Methods for Assessing Acute Systemic Toxicity, co-sponsored by
NIEHS, NTP and the U.S. Environmental Protection Agency (EPA):
Workshop Agenda and Registration Information......................................O-11
O6
65FR37400 - Notice of an International Workshop on In Vitro
Methods for Assessing Acute Systemic Toxicity, co-sponsored by
NIEHS, NTP and the U.S. Environmental Protection Agency (EPA):
Request for Data and Suggested Expert Scientists ....................................O-15
Appendix P In Vitro Cytotoxicity Test Methods and the High Production
Volume (HPV) Challenge Program......................................................... P-1
P1
Supplemental Acute Toxicity Protocol ....................................................... P-3
P2
Office of Pollution Prevention and Toxics (OPPT) Letters to
Manufacturers/Importers ............................................................................ P-9
Appendix Q Additional UDP Simulation Modeling Results........................................Q-1
Q1
UDP Simulation Results for the RC Rat-Only Millimole Regression
Starting at the LD50 Predicted by the 3T3 and NHK NRU IC50 –5000 mg/kg Upper Limit Dose .................................................................. Q-3
Q2
UDP Simulation Results for the RC Rat-Only Weight Regression
Starting at the LD50 Predicted by the 3T3 and NHK NRU IC50 LD50 –5000 mg/kg Upper Limit Dose .................................................................Q-13
xv

In Vitro Cytotoxicity Test Methods BRD List of Figures

November 2006

LIST OF FIGURES
Figure 1-1
Figure 1-2
Figure 2-1
Figure 2-2
Figure 2-3
Figure 2-4
Figure 2-5
Figure 2-6
Figure 2-7
Figure 3-1
Figure 4-1
Figure 5-1
Figure 5-2
Figure 5-3
Figure 5-4
Figure 6-1
Figure 6-2
Figure 6-3
Figure 6-4
Figure 6-5
Figure 6-6
Figure 6-7
Figure 6-8
Figure 7-1
Figure 7-2
Figure 7-3
Figure 7-4
Figure 7-5

RC Millimole Regression for In Vitro Cytotoxicity (IC50x) and Rat andMouse Acute Oral LD50 Values for 347 Chemicals .....................................1-9
NICEATM/ECVAM Validation Study Phases .......................................... 1-13
Major Steps in the Performance of the NRU Test Methods .........................2-7
3T3 NRU OD for SLS as a Function of NR Concentration andDuration.................................................................................................... 2-26
SLS IC50 Values for Each NR Concentration and Incubation Duration(3T3 NRU) ............................................................................................... 2-27
Representative Concentration-Response for 2-Propanol in a 3T3 NRU
Range Finder Test..................................................................................... 2-30
Representative Concentration-Response for Aminopterin in a NHK
NRU Range Finder Test............................................................................ 2-31
Representative Concentration-Response for Aminopterin in a NHK
NRU Definitive Test ................................................................................. 2-32
Flow Chart for Determination of Reference Substance Solubility in
Medium, DMSO, or ETOH....................................................................... 2-38
The Fifty-Eight (58) Selected RC Reference Substances on the RC
Millimole Regression................................................................................ 3-27
Correlation of LD50 Values With the Reference LD50 Values for the
58 RC Chemicals ...................................................................................... 4-14
Reference Substance IC50 Results for the 3T3 NRU Test Method byLaboratory ................................................................................................ 5-24
Reference Substance IC50 Results for the NHK NRU Test Method byLaboratory ................................................................................................ 5-25
RC IC50 Values vs 3T3 NRU IC50 Values for 56 Substances in
Common ................................................................................................... 5-37
RC IC50 Values vs NHK NRU IC50 Values for 57 Substances in
Common .................................................................................................. 5-38
Combined-Laboratory 3T3 and NHK NRU Regressions .............................6-7
Regression for 47 RC Substances Using RC Data .......................................6-8
Regression for 47 RC Substances with the 3T3 and NHK Regressions........6-9
RC Rat-Only Millimole Regression (a) and RC Rat-Only Weight
Regression (b)........................................................................................... 6-19
Correlation of Dose-Mortality Slope to Hill Function Slope...................... 6-30
In Vitro – In Vivo Regressions for Phases Ib and II for ECBC ................... 6-37
In Vitro – In Vivo Regressions for Phases Ib and II for FAL...................... 6-38
In Vitro – In Vivo Regressions for Phases Ib and II for IIVS...................... 6-39
Mean Laboratory and Laboratory-Specific 3T3 and NHK NRU
Regressions.................................................................................................7-7
Frequency of Maximum:Minimum 3T3 NRU IC50 Ratios ......................... 7-39
Frequency of Maximum:Minimum NHK NRU IC50 Ratios....................... 7-40
Comparison of Maximum:Minimum NRU IC50 Ratios to Maximum:
Minimum LD50 Ratios .............................................................................. 7-41
SLS IC50 for Each Laboratory and Study Phase......................................... 7-45
xvi

In Vitro Cytotoxicity Test Methods BRD List of Tables

November 2006

LIST OF TABLES
Table ES-1
Table 1-1
Table 1-2
Table 2-1
Table 2-2
Table 2-3
Table 2-4
Table 2-5
Table 3-1
Table 3-2
Table 3-3
Table 3-4
Table 3-5
Table 3-6
Table 3-7
Table 3-8
Table 4-1
Table 4-2
Table 4-3
Table 4-4
Table 5-1
Table 5-2
Table 5-3
Table 5-4
Table 5-5
Table 5-6
Table 5-7
Table 5-8
Table 5-9
Table 5-10
Table 5-11
Table 6-1
Table 6-2
Table 6-3

Datasets Used for Validation Study Analyses.......................................... xxxvi
Summary of Current U.S. Legislation for Using Acute Toxicity Data
for Product Labeling .................................................................................1-14
Regulatory Classification Systems for Acute Oral Toxicity .......................1-15
Measured VC OD540 Values and Targets...................................................2-17
Refeeding/No Refeeding Data for the NHK NRU Test Method.................2-24
Error Rates in Phase Ib by Laboratory and Test.........................................2-28
Cell Seeding Densities ..............................................................................2-37
Comparison of Concentrations Tested in the Various Solubility
Protocols...................................................................................................2-39
GHS Classification Scheme for Acute Oral Toxicity ...................................3-3
Reference Substances Used in the 3T3 and NHK NRU Test Methods
Validation Study - Sorted by Toxicity .........................................................3-7
Distribution of Candidate Substances and Reference Substances by
Source and Toxicity Category ...................................................................3-26
Selected Substances: Distribution of RC Chemicals and RC Outliersby Toxicity Category.................................................................................3-26
Distribution of Chemical Class for the 72 Reference Substances by
Toxicity Category .....................................................................................3-29
Distribution of Product/Use Class for the 72 Reference Substances by
Toxicity Category .....................................................................................3-31
Reference Substances Metabolized to Active Metabolites .........................3-32
Reference Substances Tested in Phases Ib and II .......................................3-33
Internet-Accessible Databases Searched for LD50 Information.....................4-3
Rodent Acute Oral Reference LD50 Values Listed by GHS Category...........4-9
GHS Category Matches for the Rodent Acute Oral LD50 Initial andReference Values ......................................................................................4-12
Maximum:Minimum LD50 Ratios by GHS Toxicity Category ...................4-13
Validation Study Timetable.........................................................................5-4
Reference Substances Affected by the Stopping Rule................................5-10
Positive Control (PC) IC50 Results by Study Phase....................................5-11
3T3 NRU Test Method IC50 and Hill Slope Data by Laboratory................5-14
NHK NRU Test Method IC50 and Hill Slope Data by Laboratory..............5-19
Key to Validation Study Reference Substances .........................................5-26
Datasets Used for Validation Study Analyses............................................5-32
Comparison of 3T3 and NHK NRU IC50 Geometric Means.......................5-33
Frequency of 3T3:NHK IC50 Ratios for Reference Substances ..................5-36
Solubility Test Results (mg/mL)................................................................5-39
Reference Substances with Precipitate (PPT) and Volatility Issues............5-43
Datasets Used for Accuracy Analyses .........................................................6-4
Linear Regression Analyses of the 3T3 and NHK NRU and Rat Acute
Oral LD50 Test Results ................................................................................6-6
Outlier Substances for the RC and the 3T3 and NHK NRU Methods
When the RC Millimole Regression is Used..............................................6-10
xvii

In Vitro Cytotoxicity Test Methods BRD List of Tables

Table 6-4
Table 6-5
Table 6-6
Table 6-7
Table 6-8
Table 6-9
Table 6-10
Table 6-11
Table 6-12
Table 7-1
Table 7-2
Table 7-3
Table 7-4
Table 7-5
Table 7-6
Table 7-7
Table 7-8
Table 7-9
Table 7-10
Table 7-11
Table 7-12
Table 7-13
Table 7-14
Table 8-1
Table 8-2

November 2006

Substances With Mechanisms of Toxicity Not Expected to Be Activein the 3T3 or NHK Cells in Culture........................................................... 6-15
Linear Regression Analyses to Improve the Prediction of Rodent
Acute Oral LD50 Values from In Vitro NRU IC50 Using the RC
Database ................................................................................................... 6-17
Prediction of GHS Acute Oral Toxicity Category by the RC IC50
Values and the RC Millimole Regression .................................................. 6-21
Prediction of GHS Acute Oral Toxicity Category by the 3T3 and NHK
NRU Test Methods and the RC Rat-Only Millimole Regression ............... 6-23
Prediction of GHS Acute Oral Toxicity Category by the 3T3 and NHK
NRU Test Methods and the RC Rat-Only Weight Regression ................... 6-26
Comparison of Regressions and In Vitro NRU Test Methods for Their
Performance in Predicting GHS Acute Oral Toxicity Categories............... 6-28
Reference Substances with Dose-Mortality and NRU Hill Slopes ............. 6-29
Over- and Under- Prediction of Toxicity for Low and High Molecular
Weight Substances Using RC Rat-Only Weight and Millimole
Regressions............................................................................................... 6-33
Linear Regressions for 11 Substances Tested in Phases Ib and II............... 6-36
Reference Substances Excluded from Reproducibility Analyses
Because of Insufficient Cytotoxicity ...........................................................7-4
Number of Reference Substances Tested vs Number of Reference
Substances Yielding IC50 Values from Each Laboratory, by GHS
Acute Oral Toxicity Category .....................................................................7-4
Interlaboratory Reproducibility of the IC50 Values from the 3T3 NRU
Test Method................................................................................................7-8
Reference Substances with Significant ANOVA Differences Among
Laboratories for the 3T3 NRU Test Method.............................................. 7-20
Reproducibility of the IC50 Values from the NHK NRU Test Method ....... 7-22
Reference Substances with Significant ANOVA Differences Among
Laboratories for the NHK NRU Test Method............................................ 7-34
Summary of CV Results for the 3T3 and NHK NRU Test Methods .......... 7-34
Intralaboratory CV by Chemical Characteristics for the 3T3 and NHK
NRU Test Methods ................................................................................... 7-36
Interlaboratory 3T3 and NHK NRU Test Method CV Values Sorted by
Chemical Characteristics........................................................................... 7-38
CV Values for 3T3 and NHK NRU Test Method IC50 Values andNormalized IC50 Values ............................................................................ 7-43
ANOVA Results for the SLS IC50 Values in the 3T3 NRU Test
Method ..................................................................................................... 7-47
Linear Regression Analysis of SLS IC50 Values Over Time ...................... 7-48
ANOVA Results for the SLS IC50 Values in the NHK NRU Test
Method ..................................................................................................... 7-49
Solvent Determinations by Laboratory...................................................... 7-51
SMT-Recommended Documentation for FAL.............................................8-4
Phase III Error Rates in the Transfer of Data to the EXCEL® Template..... 8-10

xviii

In Vitro Cytotoxicity Test Methods BRD List of Tables

Table 8-3
Table 8-4
Table 8-5
Table 8-6
Table 9-1
Table 9-2
Table 9-3
Table 9-4
Table 10-1
Table 10-2
Table 10-3
Table 10-4
Table 10-5
Table 10-6
Table 10-7
Table 10-8
Table 10-9
Table 10-10
Table 11-1
Table 11-2
Table 11-3
Table 11-4

November 2006

Definitive Test and Positive Control (PC) Test Failure Rates in Phase
III..............................................................................................................8-10
Combined Definitive and Positive Control (PC) Test Success Rates forthe 3T3 and NHK Methods in Phase III.....................................................8-11
CV Values for Definitive Tests .................................................................8-11
GHS Acute Oral Toxicity Category Predictions by Laboratory..................8-13
Rat Acute Oral LD50 Ranges for Test Substances Used in Previous In
Vitro NRU Cytotoxicity Studies and the NICEATM/ECVAM Study ..........9-4
Chemical Classes Represented by the Substances Used in Published
Studies for Correlation of In Vitro NRU Cytotoxicity with Rodent
Acute Lethality ...........................................................................................9-4
EU Classes of Acute Oral Toxicity..............................................................9-8
Correct Identification of In Vivo Phototoxicants by the NHK NRU
Phototoxicity Assay ..................................................................................9-18
Change in Animal Use with Dose-Mortality Slope for the UDP ................10-8
Mean Animal Use in the UDP Using Starting Doses Based on the 3T3
and NHK NRU Test Methods with the Different Regressions .................10-10
Animal Use for the UDP by GHS Acute Oral Toxicity Category Using
Starting Doses Based on the 3T3 and NHK NRU Test Methods with
the RC Rat-Only Millimole Regression ...................................................10-12
Animal Use for the UDP by GHS Acute Oral Toxicity Category Using
Starting Doses Based on the 3T3 and NHK NRU Test Methods with
the RC Rat-Only Weight Regression .......................................................10-14
Animal Deaths in the UDP Using Starting Doses Based on the 3T3 andNHK NRU Test Methods ........................................................................10-15
Change in Animal Use with Dose-Mortality Slope in the ATC Method ...10-19
Animal Use for the ATC Method Using Starting Doses Based on the
3T3 and NHK NRU Test Methods with the Different Regressions ..........10-21
Animal Savings for the ATC Method by GHS Acute Oral Toxicity
Category Using Starting Doses Based on the 3T3 and NHK NRU Test
Methods with the RC Rat-Only Millimole Regression.............................10-23
Animal Savings for the ATC Method by GHS Acute Oral Toxicity
Category Using Starting Doses Based on the 3T3 and NHK NRU Test
Methods with the RC Rat-Only Weight Regression.................................10-25
Animal Deaths for the ATC Method Using Starting Doses Based on
the 3T3 and NHK NRU Test Methods.....................................................10-27
Costs for Major Laboratory Equipment .....................................................11-9
Costs for Cell Culture Materials and Commercial Laboratory In Vitro
Cytotoxicity Testing................................................................................11-10
Commercial Prices for Conducting In Vivo Acute Rat Toxicity Testing...11-11
Comparison of Time Needed for In Vitro and In Vivo Testing .................11-14

xix

In Vitro Cytotoxicity Test Methods BRD List of Acronyms and Abbreviations

November 2006

LIST OF ACRONYMS AND ABBREVIATIONS
A-CUTE-TOX
ADME
ANOVA
ASTDR
ASTM
ATC
ATCC
ATWG
BBB
BPE
BRD
o
C
CAS
CASRN
CCOHS
CDER
CESARS
CFU
CHRIS
CI
CICADSCIS
CNSCOLIPA
CPSC
CSF
CTFA
CV
DART®/ETIC
DEA
DHHS
DIMDI
DNA
DMEM
DMSO
D-PBS
DOD
DOT
EC
EC50

A-Cute-Tox Project (EU Research & Development Integrated Project)
Absorption, distribution, metabolism, and elimination
Analysis of varianceAgency for Toxic Substances and Disease Registry
American Society for Testing and Materials
Acute Toxic Class method
American Type Culture Collection
Acute Toxicity Working Group
Blood:brain barrier
Bovine pituitary extract
Background Review Document
Degrees Celsius
Chemical Abstracts Service
Chemical Abstracts Service Registry Number
Canadian Centre for Occupational Health and Safety (CCOHS)
U.S. FDA Center for Drug Evaluation and Research
Chemical Evaluation Search and Retrieval System
Colony forming units
Chemical Hazard Response
Confidence interval
Concise International Chemical Assessment Documents
ILO Occupational Safety and Health Information Centre
Central nervous system
The European Cosmetic Toiletry and Perfumery Association
U.S. Consumer Product Safety Commission
Colony stimulating factor
Cosmetic, Toiletries and Fragrance Association
Coefficient of variation
Developmental and Reproductive Toxicology/Environmental
Teratology Information Center
U.S. Drug Enforcement Administration
U.S. Department of Health and Human Services
Deutsches Institut fur Medizinische Dokumentation undInformation (The German Institute for Medical Documentation andInformation)
Deoxyribose nucleic acid
Dulbecco’s Modification of Eagle’s Medium
Dimethyl sulfoxide
Dulbecco’s phosphate buffered saline
U.S. Department of Defense
U.S. Department of Transportation
European Commission
Concentration of a substance that produces 50% of the maximum
possible response for that substancexx

In Vitro Cytotoxicity Test Methods BRD List of Acronyms and Abbreviations

ECBC
ECETOC
EC/HO
ECVAM
EDIT
EHC
EHS
EPA
ERG
ETOH
EU
EXTONET
FAL
FAO
FB1
FDA
FDP
FIFRA
FR
FRAME
GABA
GCCP
GHS
GLP
gm
HBSS
HPV
hr
HSDB
HSG
HTD
IARC
IC20
IC50
IC80
ICCVAM
ICSC
ID
ID50
IIVS
ILO
i.m.
INVITOXX

November 2006

U.S. Army Edgewood Chemical Biological Center
European Centre for Ecotoxicology and Toxicology of Chemicals
European Commission/British Home Office
European Centre for the Validation of Alternative Methods
Evaluation-guided development of new in vitro tests
Environmental Health Criteria
EPA’s Extremely Hazardous Substance list
U.S. Environmental Protection Agency
Emergency Response Guidebook
Ethanol (Ethyl alcohol)
European Union
The Extension Toxicology Network
FRAME Alternatives Laboratory
UN Food and Agriculture Organization
Fumonisin B1
U.S. Food and Drug Administration
Fixed Dose Procedure
U.S. Federal Insecticide, Fungicide, and Rodenticide Act
Federal Register
Fund for the Replacement of Animals in Medical Experiments
Gamma amino butyric acid
Good cell culture practices
Globally Harmonized System (of Classification and Labeling ofChemicals)
Good Laboratory Practices
Grams
Hanks’ balanced salt solution
High Production Volume
Hour(s)
Hazardous Substances Data Bank
Health and Safety Guides
Highest tolerated doseInternational Agency for Research on Cancer
Concentration producing 20% inhibition of the endpoint measured
Concentration producing 50% inhibition of the endpoint measured
Concentration producing 80% inhibition of the endpoint measured
Interagency Coordinating Committee for the Validation of Alternative
Methods
International Chemical Safety Cards
Insufficient data
Index of cytotoxicity; dose producing a 50% reduction in protein value
Institute for In Vitro Sciences
International Labour Organisation
Intramuscular
In Vitro Techniques in Toxicology (ERGATT FRAME ECVAM Data
bank)

xxi

In Vitro Cytotoxicity Test Methods BRD List of Acronyms and Abbreviations

IOM
i.p.
IPCS
IRAG
IRPTC
ISO
IUCLID
i.v.
JECFA
JMPR
KBM®
kg
Kow
L
LC
LD50
LDH
MAS
MEIC
MeSH®
µL
µm
µM
mg
MIT
mL
mM
MMAS
mmol
MPE
MSDS
MTT
N
NA
NADH
NC
NCS
NCTR
n.d.
NHK
NICEATM
NIEHS
NIH
NIOSH
NLM

November 2006

Institute of Medicine
Intraperitoneal
International Programme on Chemical Safety
Interagency Regulatory Alternatives GroupInternational Register of Potentially Toxic Chemicals
International Standards Organization
International Uniform Chemical Information Database
Intravenous
Joint Expert Committee on Food Additives
Joint Meeting on Pesticide Residues
Keratinocyte basal medium
Kilogram
Octanol-water partition coefficient
Liter
Lethal blood concentration
Dose that produces lethality in 50% of test animals
Lactate dehydrogenase
Maximum average Draize score
Multicentre Evaluation of In Vitro Cytotoxicity
Medical Subject Heading
Microliters
Micrometers
Micromoles
Milligram
Metabolic inhibition test
Milliliter
Millimolar
Modified maximum average score
Millimoles
Mean photo effect
Material Safety Data Sheets
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
Number (of substances)
Not applicable
Nicotine adenine dinucleotide (reduced)
Not calculated
Newborn calf serum
U.S. FDA National Center for Toxicological ResearchNot detectable
Normal human epidermal keratinocytes
National Toxicology Program Center for the Evaluation of Alternative
Toxicological Methods
U.S. National Institute of Environmental Health Sciences
U.S. National Institutes of Health
U.S. National Institute for Occupational Safety and HealthNational Library of Medicine

xxii

In Vitro Cytotoxicity Test Methods BRD List of Acronyms and Abbreviations

NR
NRU
NTP
OAT
OD
OD540
OECD
OHM/TADS
OPP
OPPTS
ORD
OSHA
OTA
PBS
PC
PDS
pg
PG
PIF
PIMS
pK
PLS
PPIS
PPT
QA
QC
R2
rs
RC
REACH
RTECS®
RTK NET
SD
SIDS
SIS
SLS
SMT
SOP
3T3
TESS
TG
TRI
TSCA
UDP
UN

November 2006

Neutral red
Neutral red uptake
U.S. National Toxicology Program
Organic anionic transporters
Optical density
Optical density (absorbance) at a wavelength of 540 nm
Organisation for Economic Co-operation and Development
EPA Oil and Hazardous Materials/Technical Assistance Data
SystemU.S. EPA Office of Pesticide Programs
EPA Office of Prevention, Pesticides and Toxic Substances
U.S. EPA Office of Research and Development
U.S. Occupational Safety and Health Administration
Ochratoxin A
Phosphate buffered saline
Positive control
Pesticide Data Sheets
Picogram
Packing group
Photoinhibition factor
Poisons Information Monographs
Acid/base dissociation constantPartial Least Squares (analysis)
EPA Pesticide Product Information System
Precipitate
Quality assurance
Quality control
Coefficient of determination
Spearman correlation coefficient
Registry of Cytotoxicity
Registration, evaluation, authorisation and restriction of chemicals
Registry of Toxic Effects of Chemical Substances
The Right-to-Know Network
Standard deviation
OECD Screening Information Data Sets
Scientific Information Service
Sodium lauryl sulfate
Study management teamStandard operating procedure
BALB/c mouse fibroblasts, clone A31 (ATCC # CCL-163)
Toxic Exposure Surveillance System
Test guideline
U.S. EPA Toxics Release Inventory
Toxic Substances Control Act
Up-and-Down Procedure
United Nations

xxiii

In Vitro Cytotoxicity Test Methods BRD List of Acronyms and Abbreviations

UNEP
USP
UV
VC
WHO
XTT
ZEBET

November 2006

United Nations Environment Programme
U.S. Pharmacopoeia
Ultraviolet (light)
Vehicle control
World Health Organization
2,3-Bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5carboxanilide
Zentralstelle zur Erfassung und Bewertung von Ersatz- undErgänzungsmethoden zum Tierversuch (German Center forDocumentation and Evaluation of Alternative Methods to Animal
Experiments)xxiv

In Vitro Cytotoxicity Test Methods BRD Acknowledgements

November 2006

ACKNOWLEDGMENTS
The following individuals are acknowledged for their contributions to the in vitro acutetoxicity test method review process.

Interagency Coordinating Committee on the Validation of Alternative
Methods (ICCVAM) Acute Toxicity Working Group (ATWG)

Consumer Product Safety Commission(CPSC)

Food and Drug Administration (FDA)
Leonard Schechtman, Ph.D. (ICCVAM Chair)
Kenneth Hastings, Ph.D.
Abigail Jacobs, Ph.D.
David Morse, Ph.D.
Thomas Umbreit, Ph.D.

Kailash Gupta, D.V.M., Ph.D.
Cassandra Prioleau, Ph.D.
Marilyn Wind, Ph.D. (ATWG Chair,
ICCVAM Vice Chair)
Department of Energy (DOE)

National Institute for Occupational
Safety & Health (NIOSH)

Po-Yung Lu, Ph.D.
Steven Reynolds, Ph.D.
Environmental Protection Agency(EPA)

National Institute of Environmental
Health Sciences (NIEHS)

Karen Hamernik, Ph.D.
Masih Hashim, Ph.D.
Marianne Lewis
Elizabeth Margosches, Ph.D.
Deborah McCallJohn Redden, Ph.D.
Amy Rispin, Ph.D.

Rajendra Chhabra, Ph.D., D.A.B.T.
William Stokes, D.V.M., D.A.C.L.A.M.
(ICCVAM Executive Director; NICEATM
Director)
Raymond Tice, Ph.D. (NICEATM Deputy
Director)
European Centre for the Validation of
Alternative Methods (ECVAM)
Liaisons
Silvia Casati, Ph.D.
Pilar Prieto, Ph.D.

xxv

In Vitro Cytotoxicity Test Methods BRD Acknowledgements

November 2006

National Toxicology Program (NTP) Interagency Center for the
Evaluationof Alternative Toxicological Methods (NICEATM)

Dave Allen, Ph.D.
ILS, Inc.

Linda Litchfield
ILS, Inc.

Bradley Blackard, M.S.P.H.
ILS, Inc.

Deborah McCarley
NIEHS

Sue Brenzel
ILS, Inc.

Michael Paris
ILS, Inc.

Thomas Burns, M.S.
ILS, Inc.

William Stokes, D.V.M., D.A.C.L.A.M.
(Director)
NIEHS

Patricia Ceger, M.S.
ILS, Inc.

Judy Strickland, Ph.D., D.A.B.T.
ILS, Inc.

Jeffery Charles, Ph.D., M.B.A., D.A.B.T.
ILS, Inc.

Raymond Tice, Ph.D. (Deputy Director)
NIEHS

Neepa Choksi, Ph.D.
ILS, Inc.

Jim Truax, M.A.
ILS, Inc.

Frank Deal, M.S.
ILS, Inc.

xxvi

In Vitro Cytotoxicity Test Methods BRD Acknowledgements

November 2006

Participants in the In Vitro Cytotoxicity Validation StudyNICEATM gratefully acknowledges the generous contributions of the individuals who
directly participated in the NICEATM/ECVAM Validation Study. Their time and efforts are
greatly appreciated.
BioReliance Corp.
(Chemical Distribution)
Martin Wenk, Ph.D. – Principal
Investigator

Constella Group (Statistical Analyses)
Patrick Crockett, Ph.D.
Eric Harvey, Ph.D.
Wendell Jones, Ph.D.
Robert Lee, M.S.
Jessica L. Matthews, M.S.
Michael Riggs, Ph.D.
Janine Wilcox
Nicole Williams

Institute for In Vitro Sciences (IIVS)(Lead Laboratory – Protocols)
Hans Raabe, M.S. – Study Director
Greg Mun – Laboratory Manager
Angela Sizemore – Research Technician
Gregory O. Moyer – Research Technician
John Harbell, Ph.D. – Scientific Director

Statistical Consultant
Joseph Haseman, Ph.D.

U.S. Army Edgewood Chemical
Biological Center (ECBC)(Testing Laboratory)
Cheng Cao, Ph.D. – Study Director
Janna Madren-Whalley – ResearchTechnician
Chundakkadu Krishna, Ph.D. – ResearchTechnician
James J. Valdes, Ph.D. – Scientific
AdvisorNIEHS
Grace Kissling, Ph.D. – Contract Project
Officer
Molly Vallant - Contract Project Officer
Study Management Team (SMT)NICEATM
William Stokes, D.V.M., D.A.C.L.A.M.
(NIEHS) – Director, NICEATM
Raymond Tice, Ph.D. (NIEHS) – Deputy
Director, NICEATM – Advisor
Judy Strickland, Ph.D., D.A.B.T. (ILS,
Inc.) – Project Coordinator
Michael Paris (ILS, Inc.) – Assistant
Project Coordinator
Jeffrey Charles, Ph.D., D.A.B.T. (ILS,
Inc.) – Advisor

FRAME Alternatives Laboratory (FAL)
University of Nottingham, UK(Lead Laboratory – Software)
Richard Clothier, Ph.D. – Study Director
Nicola Bourne – Research Technician
Monika Owen – Research Technician
Rachel Budworth – Research Technician

ECVAM
Thomas Hartung, Ph.D., M.D. – Head of
Unit from 2002
Silvia Casati, Ph.D. – Task Leader
Michael Balls, D. Phil.– Head of Unit until
June 2002

xxvii

In Vitro Cytotoxicity Test Methods BRD Acknowledgements[This Page Intentionally Left Blank]xxviiiNovember 2006

In Vitro Cytotoxicity Test Methods BRD Preface

November 2006

PREFACEThe Institute of Medicine estimates that more than 4 million poisonings occur annually in the
United States (Institute of Medicine [IOM] 2004). In 2001, 30,800 deaths placed poisoning
as the second leading cause of injury-related death behind automobile accidents (42,433
deaths) (IOM 2004). In order to ensure that all potentially hazardous substances have proper
warning labels, regulatory agencies require determination of acute toxicity hazard potentialof substances and products. This determination for oral acute toxicity hazard is currently
made using a test that requires laboratory rats. Historically, lethality estimated by the LD50
(i.e., the dose of a test substance that produces death in 50% of the animals tested) has been a
primary toxicological endpoint in acute toxicity tests.
The conventional LD50 acute oral toxicity in vivo test method has been modified in various
ways to reduce and refine1 animal use in toxicity testing (OECD 2001a, c, d, e; EPA 2002a).
Most recently, the LD50 was replaced, for hazard classification testing purposes, with the
UDP, based on an Interagency Coordinating Committee on the Validation of Alternative
Methods (ICCVAM) technical evaluation and formal ICCVAM recommendations (ICCVAM
2000, 2001c). This method now reduces animal use by over 70% compared to the previous
method.
In 1999, at the request of the U.S. Environmental Protection Agency (EPA) Office ofPesticides, Prevention, and Toxic Substances, ICCVAM reviewed the validation status of invitro methods for estimating acute oral toxicity. This request was based on studies publishedin recent years that showed a correlation between in vitro and in vivo acute toxicity. In vitro
cytotoxicity methods have been evaluated as another means to reduce and refine the use ofanimals and these methods may be helpful in predicting in vivo acute toxicity. Since moving
the starting dose closer to the LD50 reduces the number of animals necessary for the acuteoral systemic toxicity test, the use of in vitro cytotoxicity assays to predict a starting doseclose to the LD50 may reduce animal use.
In October of 2000, the International Workshop on In Vitro Methods for Assessing Acute
Systemic Toxicity sponsored by the National Toxicology Program (NTP), the National
Institute of Environmental Health Sciences (NIEHS) and the EPA was convened in
Arlington, VA. The Organizing Committee invited 33 expert scientists from academia,
industry, and government agencies to participate in the Workshop. Invited scientific experts
and ICCVAM agency scientists were assigned to one of four Breakout Groups and preparedrecommendations on the following:• In Vitro Screening Methods for Assessing Acute Toxicity• In Vitro Methods for Toxicokinetic Determinations• In Vitro Methods for Predicting Organ Specific Toxicity• Chemical Data Sets for Validation of In Vitro Acute Toxicity Test Methods

1

A reduction alternative is a new or modified test method that reduces the number of animals required. A
refinement alternative is a new or modified test method that refines procedures to lessen or eliminate pain or
distress in animals or enhances animal well-being (ICCVAM 2003).
xxix

In Vitro Cytotoxicity Test Methods BRD Preface

November 2006

Workshop participants concluded that none of the proposed in vitro methods had beenformally evaluated for reliability and relevance, and that their usefulness and limitations forgenerating information to meet regulatory requirements for acute toxicity testing had notbeen adequately assessed. However, an in vitro approach proposed by the German Center forDocumentation and Evaluation of Alternative Methods to Animal Experiments (ZEBET) was
recommended for rapid adoption so that data could be generated to establish its usefulness
with a large number of chemicals (ICCVAM 2001a). In addition, a separate GuidanceDocument on Using In Vitro Data to Estimate In Vivo Starting Doses for Acute Toxicity(ICCVAM 2001b) was prepared to provide sample cytotoxicity protocols and instructions forusing in vitro data to predict starting doses for acute in vivo systemic toxicity tests.
ICCVAM, which is charged with coordinating the technical evaluations of new, revised, andalternative test methods with regulatory applicability (ICCVAM Authorization Act of 2000,
Public Law 106-545; available: http://iccvam.niehs.nih.gov/about/PL106545.pdf), agreed
that in vitro basal cytotoxicity test methods should have a high priority for evaluation. The
NTP Center for the Evaluation of Alternative Toxicological Methods (NICEATM)
collaborated with the European Centre for the Validation of Alternative Methods (ECVAM),
a component of the European Commission’s Joint Research Centre, to further characterize
the usefulness of in vitro cytotoxicity assays as predictors of starting doses for acute oral
lethality assays. NICEATM and ECVAM designed a multi-laboratory validation study toevaluate the performance of two standardized in vitro basal cytotoxicity test methods using
72 reference substances with the ZEBET approach of using the Registry of Cytotoxicity (RC)regression model. Based on the procedures described in the Guidance Document (ICCVAM
2001b), the validation study used two mammalian cell types (i.e., BALB/c 3T3 mouse
fibroblasts [3T3] and primary normal human epidermal keratinocytes [NHK]) for in vitro
basal cytotoxicity test methods with a neutral red uptake (NRU) cell viability endpoint topredict starting doses for acute oral systemic toxicity test methods. The inclusion of humancells in the validation study also implements another workshop recommendation, that ofevaluating whether cytotoxicity in human or rodent cells can be used to predict human acutetoxicity.
The objectives identified for the validation study were to:• Further standardize and optimize the in vitro NRU basal cytotoxicity protocols
using 3T3 and NHK cells to maximize test method reliability (intralaboratory
repeatability, intra- and inter-laboratory reproducibility)• Assess the accuracy of the two standardized in vitro 3T3 and NHK NRU basal
cytotoxicity test methods for estimating rodent oral LD50 values across thefive United Nations (UN) Globally Harmonized System of Classification andLabelling of Chemicals (GHS; UN 2005) categories of acute oral toxicity, as
well as unclassified toxicities• Estimate the reduction and refinement in animal use achievable from using thein vitro 3T3 and NHK NRU basal cytotoxicity test methods to identify starting
doses for in vivo acute oral toxicity tests, assuming that no other information
were availablexxx

In Vitro Cytotoxicity Test Methods BRD Preface•

November 2006

Develop high quality in vivo acute oral lethality and in vitro NRU cytotoxicity
databases that can be used to support the investigation of other in vitro test
methods necessary to improve the prediction of in vivo acute oral lethality

Scientists assembled for the ICCVAM-sponsored scientific peer review panel meeting(“Panel”) on May 23, 2006 independently assessed the usefulness and limitations of the invitro basal cytotoxicity test methods to predict starting doses for acute oral systemic toxicity
test methods. The Background Review Document (BRD) on the two in vitro NRU test
methods prepared by NICEATM and provided to the peer review panel and the publiccontains:
1. Comprehensive summaries of the data generated in the validation study
2. An analysis of the accuracy and reliability of the test method protocols
3. Related information characterizing the potential animal savings produced byusing the in vitro basal cytotoxicity test methods as adjuncts to specific acutesystemic toxicity test methods
The Panel also evaluated draft test method performance standards, protocols, and draft
ICCVAM recommendations for test method uses and future studies. The public was invited
to provide comments on the BRD and other documents and to attend the Panel meeting. Prior
to the Panel meeting, public comments provided about the documents were provided to the
Panel for their consideration. The BRD can be obtained from the ICCVAM/NICEATM Web
site (http://iccvam.niehs.nih.gov) or by contacting NICEATM.
Following the conclusion of the Panel meeting, the ICCVAM and its Acute Toxicity
Working Group (ATWG) considered the Panel report, the performance standards for the useof in vitro basal cytotoxicity test methods to predict starting doses for acute systemic toxicity
test methods, and any public comments in preparation of its final test method
recommendations for these in vitro basal cytotoxicity test methods. These recommendations
will be made available to the public and provided to the U.S. Federal agencies forconsideration, in accordance with the ICCVAM Authorization Act of 2000 (Public Law 106545).
On behalf of the ICCVAM, we gratefully acknowledge the many contributions of all who
participated in the in vitro cytotoxicity validation study and those who assisted in the
preparation of the documents evaluated at the peer review meeting. We extend a special
thanks to the participating laboratory Study Directors and scientists who worked diligently toprovided critical data and information. We also thank the ECVAM scientists who
participated in the management of the validation study and who provided valuable
information, comments, and opinions throughout the study. The efforts of the ATWG
members were instrumental in assuring a complete and informative BRD. The efforts of the
NICEATM staff in coordinating the validation study, providing timely distribution ofxxxi

In Vitro Cytotoxicity Test Methods BRD Preface

November 2006

information, and preparing the various documents are acknowledged and appreciated. We
especially acknowledge Dr. Judy Strickland and Mr. Michael Paris for their coordination of
the validation study and preparation of the BRD and other documents.
William S. Stokes, D.V.M. D.A.C.L.A.M.
RADM, U.S. Public Health Service
Director, NICEATM
Executive Director, ICCVAM
Leonard Schechtman, Ph.D.
Deputy Director for Washington Operations
National Center for Toxicological Research
U.S. Food and Drug Administration
Chairman, ICCVAM

xxxii

In Vitro Cytotoxicity Test Methods BRD Executive Summary

November 2006

EXECUTIVE SUMMARY
This Background Review Document (BRD) reports the results of a validation study,organized and managed by the National Toxicology Program (NTP) Interagency Center for
the Evaluation of Alternative Toxicological Methods (NICEATM) and the European Centre
for the Validation of Alternative Methods (ECVAM), conducted to characterize two in vitro
basal cytotoxicity tests for determining starting doses for rodent acute oral toxicity assays. In
conducting this validation study, the protocols for two in vitro neutral red uptake (NRU)
assays using BALB/c mouse fibroblast 3T3 cells (3T3) and normal human epidermal
keratinocytes (NHK) were standardized and optimized, and the LD50 values for the reference
substances were refined. The accuracy and reliability of the two in vitro NRU test methods
were determined using 72 reference substances of various toxicities. Computer simulations
were used to estimate the potential reduction in animal usage that could be accomplished by
the use of either of these in vitro test systems. One outcome of this effort has been thegeneration of high quality in vivo lethality and in vitro cytotoxicity reference databases thatwill be useful in the development of other in vitro toxicity tests.
The validation study showed that the 3T3 and NHK NRU test methods are not sufficiently
accurate as stand-alone methods to correctly predict rodent acute oral toxicity. However,based on computer simulations for the reference substances tested in this study, the use ofeither of these two in vitro basal cytotoxicity test methods for the selection of starting doses
for rodent acute oral toxicity testing has the potential to reduce the number of animals used
per test and, in some cases, the number of substance-induced animal deaths.
Introduction and Rationale
Although in vitro basal cytotoxicity test methods are not currently regarded as suitablereplacements for rodent acute oral toxicity tests (Spielmann et al. 1999; ICCVAM 2001a),such methods have been examined as a possible approach to reduce and refine2 the use ofanimals for such testing. An international Multicentre Evaluation of In Vitro Cytotoxicity(MEIC) was initiated in 1983 to evaluate the relationship between in vitro cytotoxicity andacute human toxicity. Tests of 50 substances in 61 in vitro assays by multiple laboratories led
to the identification of a battery of three human cell line assays whose cytotoxicity responses
were highly correlated to human lethal blood concentrations (Bondesson et al. 1989;
Clemedson et al 1996, 1996a; Ekwall et al. 1998a, 1998b, 2000). The Registry ofCytotoxicity (RC), initially published in 1998, is a database of 347 substances that currently
consists of acute oral toxicity data from rats and mice and in vitro cytotoxicity data fromstudies using various mammalian cell types with a number of different toxic endpoints (Halle
1998, 2003). A regression formula, the RC millimole regression, constructed from these data
was proposed by ZEBET, the German National Centre for the Documentation and Evaluationof Alternative Methods to Animal Experiments, as a method to reduce animal use byidentifying the most appropriate starting doses for acute oral toxicity tests (Halle 1998, 2003;
Spielmann et al. 1999).
2

A reduction alternative is a new or modified test method that reduces the number of animals required. A
refinement alternative is a new or modified test method that refines procedures to lessen or eliminate pain or
distress in animals, or enhances animal well-being (ICCVAM 2003).
xxxiiiIn Vitro Cytotoxicity Test Methods BRD Executive Summary

November 2006

These, and other, initiatives to use in vitro cytotoxicity test methods to reduce animal use inacute toxicity testing were evaluated at the International Workshop on In Vitro Methods forAssessing Acute Systemic Toxicity, in October 2000 (“Workshop 2000”; ICCVAM 2001a).
This workshop was organized by the U.S. Interagency Coordinating Committee on the
Validation of Alternative Methods (ICCVAM) and NICEATM. Pursuant to this workshop,
ICCVAM recommended (ICCVAM 2001a) further evaluation of the use of in vitro
cytotoxicity data as one of the approaches that could be used to estimate the starting doses forrodent acute oral toxicity studies. The recommendations are based on preliminary
information suggesting that this approach could reduce the number of animals used in suchstudies (i.e., reduction), minimize the number of animals that receive lethal doses (i.e.,
refinement), and avoid underestimating hazard. To assist in the adoption and implementationof the ZEBET approach, the Guidance Document on Using In Vitro Data to Estimate In Vivo
Starting Doses for Acute Toxicity (hereafter referred to as Guidance Document; ICCVAM
2001b) was prepared by ICCVAM with the assistance of the workshop participants.
In its recommendations for further evaluations, ICCVAM concurred with the Workshop 2000
recommendation that near-term validation studies should focus on two standard basal
cytotoxicity assays: one using a human cell NHK system and one using a rodent cell (3T3)system. Historical data for in vitro cytotoxicity testing using mouse 3T3 cells are available(e.g., Balls et al. 1995; Brantom et al. 1997; Gettings et al. 1991, 1994a, 1994b; Spielmann etal. 1991, 1993, 1996), as are historical data for in vitro basal cytotoxicity testing using NHK
cells (e.g., Gettings et al. 1996; Harbell et al. 1997; Sina et al. 1995; Willshaw et al. 1994).
NICEATM, in partnership ECVAM, designed an international, multi-laboratory validation
study to evaluate the reduction or refinement in animal use that could result from using
cytotoxicity data from the 3T3 and NHK NRU test methods to estimate starting doses for tworodent acute oral toxicity test methods, the Up-and-Down Procedure (UDP; OECD 2001a;
EPA 2002a) and the Acute Toxic Class (ATC) method (OECD 2001d). The NRU protocols,
as presented in the Guidance Document, were the initial basis of the NICEATM/ECVAM
validation study protocols. These protocols were originally derived from the BALB/c 3T3
Cytotoxicity Test, INVITTOX Protocol No. 46 (available at the FRAME-sponsored
INVITTOX database [http://embryo.ib.amwaw.edu.pl/invittox/]), the 3T3 cell studies byBorenfreund and Puerner (1984, 1985) and the rat epidermal keratinocyte study of Heimann
and Rice (1983). A detailed description of the 3T3 and NHK NRU test method protocols
used in the NICEATM/ECVAM validation study is provided in Section 2.
Protocol Components
Many protocol components used in the validation study are similar for the 3T3 and NHK
cells. The following procedures are common to both cell types:• Testing was performed in four phases (Ia, Ib, II, and III)• Preparation of reference substances and positive control• Cell culture environment conditions• Determination of test substance solubility• Configuration of 96-well plates for testing samples• 48-hour exposure to test substance• Range finder and definitive testingxxxiv

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Microscopic evaluation of cell cultures for toxicity
Measurement of NRU
Data analysis

The main differences in the test methods for the two cell types are:
• The conditions of propagation of the cells in culture• The cell growth medium components• The volumes of reference substance added to the 96-well plateThree laboratories participated in testing the 72 reference substances in both cell types:• ECBC: The U.S. Army Edgewood Chemical Biological Center (Edgewood,MD)• FAL: Fund for the Replacement of Animals in Medical Experiments
Alternatives Laboratory (Nottingham, UK)• IIVS: The Institute for In Vitro Sciences (Gaithersburg, MD)
BioReliance Corporation (Rockville, MD) procured and distributed the coded reference
substances and performed solubility tests prior to their distribution to the testing laboratories,
but did not perform any of the in vitro tests.
Validation Reference Substances
The 72 reference substances were selected to represent: (1) the complete range of in vivo
acute oral toxicity (encompassing all five GHS acute oral toxicity categories as well as lower
toxicities [GHS; UN 2005]); (2) the types of substances regulated by various regulatory
authorities; and (3) substances with human toxicity data and/or human exposure potential. To
ensure that the complete range of toxicity was covered, 12 substances were selected for eachof the five acute oral toxicity categories, with an additional 12 substances with lower
toxicities (i.e., LD50 >5000 mg/kg). A discussion of the characteristics and sources of thereference substances can be found in Section 3. The selected reference substances had thefollowing characteristics:• 58 (81%) of the 72 substances were also included in the RC, and 38% (22/58)
of these were outliers with respect to the RC millimole regression.
• 27 (35%) of the substances were pharmaceuticals, 17 (22%) were pesticides, 8(10%) were solvents, and 5 (6%) were food additives. The remaining
substances were used for a variety of manufacturing and consumer products.
The number of assigned uses (77) is greater than the number of selected
substances because some of the substances have more than one use.
• 57 (79%) were organic compounds and 15 (21%) were inorganic; wellrepresented classes of organic compounds included heterocyclics, carboxylic
acids, and alcohols.
• 22 (31%) substances were known, or expected to have, toxicologically active
metabolites.
• Many of the selected substances had multiple target organs/effects; including
neurological, liver, kidney, and cardiovascular effects.

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Table ES-1 reports the number of substances that were tested and the number of substancesused for the various analyses performed.
Table ES-1

Datasets Used for Validation Study Analyses1
Use

TestingComparison of laboratory IC50-LD50
regressions to one another
Comparison of combined-laboratory IC50LD50 regressions to a regression
calculated with RC data
Prediction of GHS accuracy using IC50
values in IC50-LD50 regressions;prediction of starting doses for acute oral
toxicity test (UDP and ATC) simulations
Reproducibility of acceptable rat oral
LD50 values
Reproducibility of IC50 values

3T3
NRU1
72

NHK
NRU1
72

47

51

47

47

67

68

NA

NA

64

68

Characteristics of Dataset
Substances tested
RC substances with IC50 values from alllaboratories and reference rat oral LD50
valuesRC substances with IC50 values for bothtest methods from all laboratories and rat
oral reference LD50 values
Substances with IC50 values from at least
one laboratory
62 substances with more than one
acceptable rat oral LD50 value
Substances with IC50 values from alllaboratories

Abbreviations: RC=Registry of Cytotoxicity; 3T3=BALB/c 3T3 fibroblasts; NHK=Normal human epidermal keratinocytes;
NRU=Neutral red uptake; NA=Not applicable.
1
Number of substances.

Rodent Acute Oral Toxicity Reference Data
Because the 3T3 and NHK NRU test methods are intended to be used as adjuncts to rodent
acute oral toxicity test methods, the LD50 values from rodent acute oral toxicity tests are themost appropriate reference data for evaluating the in vitro IC50 values (i.e., the test chemical
concentration that reduces cell viability by 50%). Rodent acute oral LD50 reference data forthe 72 reference substances were obtained from the literature. It was not possible to limit thedata to studies conducted under Good Laboratory Practice (GLP) guidelines (OECD 1998;
EPA 2003a, 2003b; FDA 2003) because only 2% of the published data retrieved were fromsuch studies. Although mouse toxicity data were initially considered for inclusion in the
database, the accuracy analyses were restricted to rat data. A total of 459 acute rodent oral
LD50 values were identified for the reference substances. Reference LD50 values for each
substance were identified by excluding studies with the following characteristics:• Feral rats
• Rats <4 weeks of age• Anesthetized rats
• Test substance administered in food or capsule• LD50 reported as a range or an inequality
For substances with multiple LD50 values (i.e., from different sources), the rodent reference
LD50 values for use in the validation study were determined by calculating a geometric meanof the available values for each reference substance. The reference LD50 values for 19 (26%)
of the 72 substances varied sufficiently from the initial LD50 values that came from the RC

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database and other summary sources, that the substances were reclassified into different GHS
categories.
The reliability of the calculated rat acute oral LD50 reference values was assessed bycomparison to other evaluations of the performance of rodent acute oral toxicity tests. For the
62 reference substances that had more than one LD50 value, the maximum:minimum ratios
ranged from 1.1 to 25.9, with most below an order of magnitude.
Test Method Accuracy
Although the 3T3 and NHK NRU test methods are not intended to be used as replacements
for rodent acute oral toxicity tests, they were evaluated for their ability to correctly predictthe reference LD50 values (i.e., accuracy3). The rationale for evaluating the accuracy of LD50
predictions is that the current acute oral toxicity test methods (i.e., UDP, ATC, and Fixed
Dose Procedure [FDP; OECD 2001c]) call for starting doses to be placed as close as possible
and just below the true LD50. When the starting dose is close to the true LD50 for a test
substance, fewer animals are needed. When the starting dose is below the true LD50, there is
reduced pain and suffering because doses tend to be lower, and the test outcome bias is moreconservative (i.e., higher toxicity). Regression models developed using IC50 and LD50 values
were used to derive estimated LD50 values from 3T3 or NHK NRU IC50 values.
A number of different analyses were performed in an attempt to improve the estimation of
the rat acute oral LD50. IC50-LD50 regressions (in millimole units) were calculated for each invitro cytotoxicity test method and participating laboratory using the 3T3 and NHK IC50
values. Because the regressions for each NRU test method among laboratories were notsignificantly different from one another (for each NRU test method, p >0.5), the regression
for each NRU test method was based on data pooled across the laboratories. This combinedlaboratory regression was then compared to the RC data using a regression based on RC IC50
and LD50 data for the 47 substances common to the validation study and the RC, with rat
acute oral LD50 reference values, and with both 3T3 and NHK IC50 values produced by all
three participating laboratories. The statistical comparison of slope and intercept(simultaneously) using an F test showed that neither the 3T3 regression nor the NHK
regression was significantly different from the RC regression for the 47 substances (p =0.61
and 0.76 respectively). These outcomes support use of the RC millimole regression.
Reference substances that fit the RC millimole regression poorly (i.e., outliers) were
evaluated to determine whether there were relationships between their outlier status and their
physical or chemical characteristics. Because the IC50-LD50 regressions for the 3T3 and NHK
NRU test methods yielded results that were not different from the RC regression for 47
substances, the RC millimole regression was preferred for analysis of outliers because it was
based on a much larger data set and because it had established acceptance limits (Halle 1998,
2003). Certain chemical structural classes, boiling points, molecular weights, and log Kow
values were related with outliers, but solubility in the 3T3 or NHK medium and the cells’lack of xenobiotic metabolic capability did not correlate with outlier status. Because these in3

Accuracy is the agreement between a test method result and an accepted reference value (ICCVAM 2003).

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vitro NRU test methods are based upon basal cytotoxicity, the mechanism of toxicity was
also considered as a characteristic to explain the presence of outliers. Twenty-two reference
substances were neurotoxic or cardiotoxic and were not expected to be active in the 3T3 andNHK cell cultures. Of these 22 substances, 13 (59%) were outliers (i.e., they fit the RC
millimole regression poorly) using the 3T3 NRU and 12 (55%) were outliers using the NHK
NRU. These substances represented 13/28 (46%) and 12/31 (39%) of the outliers for the 3T3
and the NHK NRU test methods, respectively. More information on the outlier analysis ispresented in Section 6.2.
The potential variation produced by combining the LD50 values of two rodent species in the
RC millimole regression was eliminated by developing a regression based solely on RC
substances with rat LD50 data (i.e., the RC rat-only millimole regression). The RC rat-only
data were also converted to a weight basis for an additional regression, the RC rat-only
weight regression, for applicability to mixtures or to substances for which molecular weightis unknown.
The accuracy of the in vitro NRU test methods when used with each of the IC50-LD50
regressions was characterized by determining the proportion of reference substances forwhich their GHS categories (based on rat acute oral LD50 data) were correctly predicted. The
accuracy of the RC rat-only millimole regression was 31% (21/67 reference substances) and
29% (20/68 reference substances) with the 3T3 and the NHK NRU test methods,
respectively. The accuracy of the RC rat-only weight regression was similar, 31% with the
3T3 NRU test method (21/67 reference substances) and 31% with the NHK NRU test method(21/68 reference substances). The poor accuracy is due, in part, to the skewness of thereference substance set with respect to the fit of the reference substances to the regressions
and to the differences between cell cultures and whole animal exposures. Each regression
showed a general trend to underpredict the toxicity of the most toxic chemicals, and to
overpredict the toxicity of the least toxic chemicals. A detailed discussion of the accuracy
analyses is presented in Section 6.4.
Test Method Reliability
Reproducibility is the consistency of individual test results obtained in a single laboratory(intralaboratory reproducibility) or among different laboratories (interlaboratory
reproducibility) using the same protocol and test samples. Reproducibility was evaluatedusing results from the 64 reference substances tested in 3T3 cells and the 68 substancestested in NHK cells that yielded replicate IC50 values in all three laboratories. Intra- andinter-laboratory reproducibility of the 3T3 and NHK NRU IC50 data was assessed using
analysis of variance (ANOVA), coefficient of variation (CV) analysis, comparison of thelaboratory-specific IC50-LD50 regressions, and comparison of maximum:minimum mean
laboratory IC50 values. Reproducibility was generally better with the NHK NRU test method.
Although ANOVA results for the positive control (sodium lauryl sulfate [SLS]) IC50 values
from the 3T3 NRU test method indicated that there were significant differences amonglaboratories (p =0.006) but not between study phases within laboratories (p >0.01), the data
show (see Figure 7-5) that laboratory means and standard deviations from each testing phaseoverlap , and that the IC50 was stable between testing phases. The interlaboratory CV values
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for the various study phases ranged from 2 to 16%. ANOVA results for the SLS IC50 fromthe NHK NRU test method showed significant differences among laboratories (p <0.001) andamong study phases within laboratories (p ≤0.001). The use of a different cell culture method
at FAL was responsible for SLS IC50 differences among the laboratories in Phases Ia and Ib.
After harmonization of culture methods across laboratories, the laboratory means andstandard deviations were similar for Phases II and III (see Figure 7-5). Interlaboratory CV
values for the NHK NRU for Phases Ia and Ib, were 39% and 21%, respectively.
Interlaboratory CV values for Phases II and III were 31% and 8%, respectively. The linear
regression analyses of the SLS IC50 over time (within each laboratory) for both NRU test
methods indicated that IC50 values generated over the 2.5-year duration of the study werestable.
For the reference substances, the similarity among the laboratories’ LD50 predictions (viaregression) from IC50 values (see Figure 7-1) was considered significant with respect to thereproducibility analyses because these in vitro NRU test methods are proposed for use indetermining starting doses for acute oral toxicity tests using the predicted LD50. ANOVA
showed significant laboratory differences for 23 substances with the 3T3 NRU test method(see Table 7-4) and six substances with the NHK NRU test method (see Table 7-6). Mean
intralaboratory CV values were 26% for both NRU test methods, but the NHK NRU test
method had a lower mean interlaboratory CV (28% vs. 47%). An analysis to determine therelationship, if any, between reference substance attributes and interlaboratory CV indicatedthat chemical class, physical form, solubility, and volatility had little effect. The CV seemed
to be related instead to the GHS hazard category, the IC50, and boiling point (see Section
7.2.3). However, the usefulness of these relationships is not known. Mean interlaboratory CV
values were larger for substances in the most toxic GHS hazard categories than for
substances in the other toxicity categories, especially with the 3T3 NRU test method. The
mean interlaboratory CV for substances in the LD50 ≤5 mg/kg (72%) and 5 < LD50 ≤50
mg/kg (78%) categories were larger than the mean overall interlaboratory CV (47%) with the
3T3 NRU test method. When the NHK NRU test method was used, the mean interlaboratory
CV was 37% for substances with LD50 ≤5 mg/kg, and 41% for substances with 5 < LD50 ≤50
mg/kg, and the mean overall interlaboratory CV was 28%. A Spearman correlation analysis
indicated that the IC50 was inversely correlated to interlaboratory CV for both the 3T3 (p=0.015) and NHK (p =0.014) NRU test methods, and that boiling point was positively
correlated to interlaboratory CV (p =0.007) for the 3T3 but not the NHK (p =0.809) NRU
test method.
The maximum:minimum mean laboratory IC50 ratios for the 3T3 NRU test method ranged
from 1.1 to 21.6, with 37 of 64 (58%) reference substances having ratios less than 2.5. The
maximum:minimum mean laboratory IC50 ratios for the NHK NRU test method ranged from
1.0 to 107.6, with 58 of 68 (85%) reference substances having ratios less than 2.5.
Data Quality
The laboratories reported no significant deviations from the protocols, and deviations that did
occur were acknowledged and addressed by the Study Directors. Tests that had deviations
affecting the data were rejected by the Study Directors and repeated. The computation of test
method and data collection errors showed that the non-GLP laboratory consistently had thexxxix

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highest error rate and the lowest intralaboratory reproducibility for IC50 results; however, thelaboratory’s GHS acute oral toxicity category predictions were comparable to that for theother laboratories.
An electronic copy of all data for the validation study can be obtained from NICEATM uponrequest by mail, fax, or e-mail to Dr. William S. Stokes, NICEATM, NIEHS, P. O. Box12233, MD EC-17, Research Triangle Park, NC, 27709, (phone) 919-541-2384, (fax) 919541-0947, (e-mail) niceatm@niehs.nih.gov.
Other Scientific Reports and Reviews
3T3 and NHK NRU methods have been evaluated for purposes other than the prediction ofstarting doses for acute toxicity studies (e.g., ocular irritancy; human lethal bloodconcentrations, in vivo phototoxicity). In vitro NRU cytotoxicity test methods using various
cell types have been evaluated for their correlation with rodent lethality endpoints (e.g.,
rat/mouse intravenous[i.v.], intraperitoneal [i.p.], and oral toxicity). Peloux et al. (1992) andFautrel et al. (1993) showed good correlations (r =0.88) of in vitro cytotoxicity with rodent
i.p./i.v. and i.v. toxicity data, respectively. A 3T3 NRU test method has been validated by
ECVAM for the identification of in vivo phototoxic potential.
No in vitro test methods have been validated for the prediction of acute oral toxicity.
Estimations of animal savings using in vitro cytotoxicity data to estimate starting doses for
the UDP did not use actual in vitro cytotoxicity data. Instead, animal savings were estimatedby assuming that the in vivo starting dose equals the true LD50, which is an approach thatassumes that cytotoxicity data can perfectly predict in vivo lethality. These theoreticalpredictions of animal savings in the UDP ranged from 25-40% (ICCVAM 2001a), as
compared with the average animal savings of 5.3-7.8% predicted using computer simulation
modeling of the UDP for the reference substances tested in the NICEATM/ECVAM study.
Halle et al. (1997) used the in vitro cytotoxicity data in the RC to determine that an animalsavings of 32% can be attained for the ATC method by using the LD50 predicted by the RC
millimole regression as the starting dose. For the reference substances tested in the
NICEATM/ECVAM validation study, most of which were a poor fit to the RC millimole
regression, the average animal savings for the ATC, as determined by computer simulation
modeling, was 4.8-10.2%.
Animal Welfare Considerations: Reduction, Refinement, and Replacement
Computer models were used to simulate testing of the reference substances using to the UDP
and ATC test methods. In principle, animal savings with the FDP could be estimated eventhough death is not the primary endpoint, but the validation study did not include thisanalysis. The number of animals that would be used, and the number of animals that would
survive or die during the UDP or ATC procedure, were determined for the default starting
doses and compared with those when starting dose was based on LD50 values determinedfrom IC50 values for each reference substance using the RC rat-only regressions.
Computer simulation of UDP testing showed that, for the reference substances used in thisvalidation study, using the 3T3 or NHK NRU test methods and the RC rat-only millimole
regression to identify the starting dose resulted in the use of fewer animals per test by anxlIn Vitro Cytotoxicity Test Methods BRD Executive Summary

November 2006

average of 5.3% (0.50 animals) to 6.6% (0.53 animals), depending upon the assumed
mortality-response slope and in vitro NRU test method used. The RC rat-only weight
regression predicted mean animal savings of 6.0% (0.56 animals) to 7.8% (0.62 animals).
When substances were grouped by GHS acute oral toxicity category, there were no animalsavings for substances in the 50 <LD50 ≤300 mg/kg category because the default starting
dose is in this range. The greatest animal savings were observed for substances with 2000 <LD50 ≤5000 mg/kg and LD50 >5000 mg/kg because the limit test, which would be used forsuch substances, uses fewer animals that the main test. Animal savings for these toxicity
categories using the RC rat-only millimole regression ranged from 11.3% (1.21 animals) to
20.3% (1.58 animals) per test. Use of the RC rat-only weight regression produced animalsavings of 12.8% (1.38 animals) to 21.0% (1.63 animals) per test. Although the use of the
3T3 and NHK NRU test methods to estimate starting doses for the simulated UDP decreased
the numbers of animals used per test, it did not change the numbers of animals that died.
Computer simulation of ATC testing showed that, for the reference substances used in thisvalidation study, using the 3T3 or NHK NRU test methods and the RC rat-only millimole
regression to identify the starting dose resulted in a savings of 4.8% (0.51 animals) to 7.3%
(0.80 animals) per test, depending upon the assumed mortality-response slope and the in vitro
NRU test method used. The use of the RC rat-only weight regression produced animalsavings of 8.6% (0.91 animals) to 10.2% (1.09 animals). When substances were grouped by
GHS acute oral toxicity category, there were no animal savings for substances in the 300 <LD50 ≤2000 mg/kg category because this category contains the default starting dose for the
ATC method. Animal savings were highest for substances with 5 < LD50 ≤50 mg/kg and
LD50 >5000 mg/kg. The mean animal savings for both in vitro NRU test methods for
substances with 5 < LD50 ≤50 mg/kg ranged from 9.8% (1.15 animals) to 11.4% (1.33
animals) per test for the RC rat-only millimole regression. The greatest reduction in animal
use would be for substances with LD50 >5000 mg/kg because the limit test used fewer
animals than the main test. Animal savings for these substances ranged from 17.1%, (2.03
animals) to 22.2% (2.66 animals) per test for the RC rat-only millimole regression. When the
RC rat-only weight regression was used, the mean animal savings with both in vitro NRU
test methods for substances with 5 < LD50 ≤50 mg/kg ranged from 10.8% (1.25 animals) to
13.0% (1.51 animals) per test. Mean animal savings for substances with LD50 >5000 mg/kg
ranged from 24.8% (2.94 animals) to 27.7% (3.33 animals) per test. The use of IC50 values toestimate starting doses for the ATC tests refined animal use by producing fewer animal
deaths by approximately 0.5 to 0.6 animals per test.
Simulations for the UDP and ATC method showed that the use of cytotoxicity results toestimate starting doses did not significantly alter the GHS categorizations compared with thecategories determined using default starting doses. This concordance was 97 to 99% for the
3T3 and NHK NRU test methods.
Practical Considerations
Practical issues with respect to the implementation of these in vitro NRU test methods
include the need for, and availability of, appropriate cell culture equipment, training andexpertise, cost, and time expenditure. The ECVAM Good Cell Culture Practice Task Force
Report 1 (Hartung et al. 2002) encourages the establishment of laboratory practices andxliIn Vitro Cytotoxicity Test Methods BRD Executive Summary

November 2006

principles that will reduce uncertainty in the development and application of in vitro test
methods.
All equipment and supplies are readily available, and the in vitro NRU test methods areeasily transferable to laboratories experienced with mammalian cell culture techniques. Muchof the training and expertise needed to perform the 3T3 and NHK NRU test methods arecommon to people with mammalian cell culture experience. Additional technical training
would not be intensive because these methods are similar in general performance to other invitro mammalian cell culture assays. GLP training should be provided to laboratory
personnel (including study directors and principal investigators) to ensure proper adherence
to test protocols and data documentation and verification procedures.
Prices for commercial in vitro NRU cytotoxicity testing to determine the IC50 for one
substance ranged from $1120 to $1850. It is not clear if the price of an in vivo test would be
reduced if it were preceded by an in vitro cytotoxicity test to set the starting dose. Thus, useof these test methods may not reduce the overall cost of rodent acute oral toxicity testing andmay increase the cost, but their use has the potential to reduce the number of animals and thetime needed for a study. The greatest savings in time and animals will occur if the IC50 data
determine that the rodent acute oral toxicity limit test should be performed, rather than themain test. Based on the cost and technical procedures associated with cell culture
maintenance, the 3T3 NRU test method is less expensive and less complicated to conduct
than the NHK NRU test method.

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1.0

INTRODUCTION AND RATIONALE FOR THE USE OF IN VITRO
NEUTRAL RED UPTAKE CYTOTOXICITY TEST METHODS TO
PREDICT STARTING DOSES FOR IN VIVO ACUTE ORALTOXICITY TESTING............................................................................................. 1-3

1.1

Historical Background and Rationale for the Use of In Vitro Cytotoxicity
Assays to Predict Starting Doses for Rodent Acute Oral Toxicity Tests ............ 1-4
1.1.1 The Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) Program ........ 1-4
1.1.2 An International Evaluation of Selected In Vitro Toxicity Test Systems
for Predicting Acute Systemic Toxicity......................................................... 1-6
1.1.3 The Registry of Cytotoxicity (RC) ................................................................ 1-7
1.1.4 The ZEBET Initiative to Reduce Animal Use ............................................... 1-9
1.1.5 The International Workshop on In Vitro Methods for Assessing Acute
Systemic Toxicity ....................................................................................... 1-10
1.1.6 The NICEATM/ECVAM In Vitro NRU Cytotoxicity Validation Study .... 1-11

1.2

Regulatory Rationale and Applicability for the Use of In Vitro
Cytotoxicity Test Methods to Predict Starting Doses for Acute Oral
Toxicity Testing in Rodents................................................................................... 1-14
1.2.1 Current Regulatory Testing Requirements for Acute Oral Toxicity ........... 1-14
1.2.2 Intended Regulatory Uses for In Vitro Cytotoxicity Test Methods............. 1-16
1.2.3 Similarities and Differences in the Endpoints of In Vitro Cytotoxicity
Test Methods and Rodent Acute Oral Toxicity Test Methods .................... 1-17
1.2.4 Use of In Vitro Cytotoxicity Test Methods in the Overall Strategy ofHazard Assessment ...................................................................................... 1-18

1.3

Scientific Basis for the In Vitro NRU Test Methods ........................................... 1-18
1.3.1 Purpose and Mechanistic Basis of In Vitro NRU Test Methods ................. 1-19
1.3.2 Similarities and Differences in the Modes/Mechanisms of Action for
In Vitro NRU Test Methods Compared with the Species of Interest .......... 1-20
1.3.3 Range of Substances Amenable to the In Vitro NRU Test Methods........... 1-20

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1-2

In Vitro Cytotoxicity Test Methods BRD Section 1

1.0

November 2006

INTRODUCTION AND RATIONALE FOR THE USE OF IN VITRO
NEUTRAL RED UPTAKE CYTOTOXICITY TEST METHODS TO
PREDICT STARTING DOSES FOR IN VIVO ACUTE ORAL TOXICITYTESTING

Poisoning is a more serious public health problem than generally recognized. The Institute ofMedicine (IOM) estimates that more than 4 million poisoning episodes occur annually in the
United States (IOM 2004). In 2001, poisoning (30,800 deaths) placed second behind
automobile accidents (42,433 deaths) as the leading cause of injury-related death (IOM
2004). To reduce the risk for accidental poisonings, various regulatory agencies in the UnitedStates (e.g., the Environmental Protection Agency [EPA], the Consumer Products Safety
Commission [CPSC]), require the testing of marketed products for acute oral toxicity inrodents. Increasing societal concerns about animal use have lead to the development andevaluation of alternative in vitro test methods that might refine, reduce, or replace acute oral
toxicity test methods1.
The purpose of this background review document (BRD) is to:• Describe a validation study organized and managed by the National
Toxicology Program (NTP) Interagency Center for the Evaluation of
Alternative Toxicological Methods (NICEATM) and the European Centre for
the Validation of Alternative Methods (ECVAM) to evaluate the ability of
two in vitro basal cytotoxicity test methods to predict starting doses for rodent
acute oral toxicity tests• Provide the results of an evaluation of the accuracy and reliability of the two
in vitro basal cytotoxicity test methods, as well as of the animal savings thatwould occur if these test methods were used to predict the starting dose.
The structure of the BRD follows the requested structure of the ICCVAM2 Guidelines for the
Nomination and Submission of New, Revised, and Alternative Test Methods (ICCVAM
2003).
This section provides:• A historical perspective of scientific efforts to develop and evaluate the abilityof in vitro cytotoxicity test methods to refine, reduce, or replace acute oral
toxicity test methods• A general review of reported correlations between in vitro cytotoxicity andacute oral lethality in rodents• The regulatory requirements for rodent acute oral toxicity testing• The scientific basis of using in vitro basal cytotoxicity test methods to predict
the starting doses for rodent acute oral toxicity assays
1

A reduction alternative is a new or modified test method that reduces the number of animals required. A
refinement alternative is a new or modified test method that refines procedures to lessen or eliminate pain or
distress in animals or enhances animal well-being (ICCVAM 2003).
2

The Interagency Coordinating Committee for the Validation of Alternative Methods
1-3

In Vitro Cytotoxicity Test Methods BRD Section 1•
1.1

November 2006

The intended regulatory uses and applicability of in vitro basal cytotoxicity
test methods

Historical Background and Rationale for the Use of In Vitro Cytotoxicity
Assays to Predict Starting Doses for Rodent Acute Oral Toxicity Tests

This section provides the historical background and rationale for the NICEATM/ECVAM
validation study by summarizing several major studies promoted by the European Union(EU) to investigate the properties and capabilities of cell-based methods to predict acutetoxicity. The Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) Program was initiatedin 1983 to compare in vitro methods to acute oral lethality in humans (Section 1.1.1). In
1992-1993, the Fund for the Replacement of Animals in Medical Experiments (FRAME)
conducted an international evaluation of selected in vitro toxicity test systems for predicting
acute systemic toxicity (Section 1.1.2). Dr. Willi Halle published a monograph regarding thedevelopment of the Registry of Cytotoxicity (RC) database to evaluate whether basal
cytotoxicity data could accurately predict acute oral lethality in rats and mice (Section 1.1.3).
ECVAM organized a workshop in 1994 to evaluate the use of in vitro data for theclassification and labeling of chemicals and reviewed the assessment of acute oral toxicity
using in vitro data. Workshop participants suggested that the use in vitro data to determine
starting doses for acute oral toxicity tests would reduce the use of animals. The German
Center for Documentation and Evaluation of Alternative Methods to Animal Experiments(ZEBET) then recommended that in vitro basal cytotoxicity data be used with the RC
millimole regression, which is referred to as the ZEBET approach (Section 1.1.4), to
determine starting doses for acute oral toxicity tests. Section 1.1.5 provides background on
an international workshop that reviewed and evaluated the EU studies above and Section
1.1.6 describes the NICEATM/ECVAM in vitro cytotoxicity validation study that expands
upon the EU studies.
1.1.1
The Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) Program
The Scandinavian Society for Cell Toxicology established the MEIC program in 1983 toinvestigate the ability of in vitro cytotoxicity test methods to predict acute oral lethality inhumans (Bondesson et al. 1989). MEIC was based on the following assumptions:• In vitro cell culture systems could be used to model in vivo acute oral toxicity.
• The basal cytotoxicity detected by these in vitro test methods is responsible
for a large proportion of in vivo toxic effects3.
The MEIC program was an open study that invited laboratories worldwide to participate in
testing 50 reference substances using laboratory-specific in vitro cytotoxicity assays.
Although the MEIC management team requested that all participating laboratories test
chemicals with high purity, no effort was made to assure that the substances tested werepurchased from the same supplier or were of the same purity (Clemedson et al. 1996a).
Minimal methodological directives were provided so as to maximize protocol diversity
among the 96 participating laboratories.
3

Basal, or general, cytotoxicity was described as toxicity resulting from interference with basic cellular
structures and functions, such as cell membranes, metabolism, ion regulation, and cell division that are common
to all human and animal cells.

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In Vitro Cytotoxicity Test Methods BRD Section 1

November 2006

The reference substances were selected to represent different chemical classes for whichreference acute oral lethality data existed in humans (i.e., lethal doses, kinetics, and lethal
blood/serum concentrations [LC]) and rodents (oral median lethal dose [LD50] values)(Bondesson et al. 1989). The MEIC management team collected human data from clinicaland forensic toxicology handbooks and case reports of human poisonings (Ekwall et al.
1998a). The resulting data were presented and analyzed in a series of 50 MEIC Monographs.
Rat and mouse oral LD50 data were collected from the Registry of Toxic Effects for
Chemical Substances (RTECS®)4.
The 50 reference substances were tested in as many as 61 different in vitro assays (Ekwall etal. 1998b). The metric of interest was the IC50 (i.e., the concentration that inhibited the
response measured by 50%) for the endpoint measured. Of the 20 test methods that usedhuman-derived cells, 18 used cell lines and two used primary cell cultures. Of the 21 test
methods that used mammalian (but other than human) cells, 12 used cell lines and nine usedprimary cell cultures. Eighteen test methods were ecotoxicological in nature and two usedcell-free systems. Cell viability and/or cell growth were the endpoints of choice in the
majority of the cell-based systems. The chemical exposure duration ranged from 5 minutes to
6 weeks, but most frequently was 24 hours (Clemedson et al. 1996).
The ability of the in vitro IC50 data to predict human acute oral lethality was assessed using
human LC values compiled from three different data sets (Ekwall et al. 2000):• Clinically measured acute lethal serum concentrations• Acute LC values measured post-mortem• Peak LC values derived from approximate LC50 curves over time after
exposure
A partial least squares (PLS) analysis indicated that the IC50 data generated from as many as
61 test methods predicted the three sets of LC data well (R2=0.77, 0.76, and 0.83, Q2=0.74,
0.72, and 0.81, respectively, where R2 is the determination coefficient and Q2 is the predictedvariance according to cross-validation in the PLS model used). A two component PLS modelusing rat and mouse oral LD50 values less accurately predicted human LC values (R2=0.65,
Q2=0.64). These results suggested that in vitro basal cytotoxicity assays might be more
effective in estimating human acute oral lethality than rodent acute oral toxicity test methods.
Because the MEIC study showed that the in vitro test methods with the best predictivity
generally used human cell lines (Ekwall et al. 1998b), the MEIC management team identified
a battery of in vitro assays using three human cell lines that had maximal performance forpredicting peak acute LC values in humans (R2=0.79 and Q2=0.76) (Ekwall et al. 2000).
However, it was concluded that improvements in the prediction of human acute oral lethality
were necessary before in vitro cytotoxicity assays could replace animal tests. To adjust forlethality produced by mechanisms other than basal cytotoxicity, the Evaluation-guided
Development of New In Vitro Tests (EDIT) program was proposed to address targeted
4

RTECS® was originally published by the U.S. National Institute for Occupational Safety and Health (NIOSH)
and is currently licensed to MDL Information Systems, Inc.
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In Vitro Cytotoxicity Test Methods BRD Section 1

November 2006

development of in vitro test methods for other endpoints, including biokinetics (e.g., gut
absorption, distribution, clearance), biotransformation, and target organ toxicity (Clemedson
et al. 2002).
1.1.2

An International Evaluation of Selected In Vitro Toxicity Test Systems for
Predicting Acute Systemic Toxicity
FRAME organized an international collaborative study conducted in 1992 - 1993 to evaluate
the prediction of rodent acute oral lethality by in vitro test methods (Fentem et al. 1993)5.
The objective of the study was to identify in vitro systems and strategies that could be used
for the classification and labelling of new chemicals, thereby reducing, and possibly
replacing, the use of animals for acute oral toxicity testing.
The 42 substances tested in the study comprised a diverse group of organic and inorganic
chemical classes, including surfactants, pharmaceuticals, and pesticides (Fentem et al. 1993).
In vitro toxicity assays using different mammalian cell lines, exposure periods, and toxicity
endpoints were evaluated, including:• Two cell proliferation assays (total protein in mouse BALB/c 3T3 fibroblast
cells and MTT6 reduction in Chinese hamster fibroblastoid V79 cells after a
72-hour exposure period)• Two cytolethality assays (MTT reduction in V79 cells and lactate
dehydrogenase [LDH] release from primary rat hepatocytes after a 24-hour
exposure period)• A cell function assay (myotube contractility inhibition in rat skeletal muscle
cells)The resulting in vitro IC50 data were linearly regressed against the lowest available rat or
mouse oral LD50 values for each test substance. There were no significant differences among
the IC50-LD50 regressions for the different in vitro test methods.
A subset of 26 to 40 of the 42 test substances, based on the availability of European Union(EU) hazard classification data, was used to evaluate two approaches for using in vitro IC50
data to classify chemicals into the four hazard categories used by the EU for acute oral
toxicity labelling (Fentem et al. 1993). One approach used the IC50 values obtained from thefive different in vitro test methods for each test substance to predict the LD50 value andhazard category from the IC50-LD50 regression. The accuracy of hazard classification for thefive in vitro tests was from 43 to 65%. The other approach used toxicokinetic parameters for
31 to 38 substances to convert the IC50 values to effective dose (i.e., ED50) values. Hazard
classification accuracy was 43 to 55%.

5

The collaborative study was conducted by the Institute of Toxicology, Kiel, Germany; the University of
Nottingham, United Kingdom; and the Gesellschaft für Strahlen- und Umweltforschung, Neuherberg, Germany(Society for Radiological and Environmental Research, which later changed its name to Center for
Environmental and Health Research [Forschungszentrum für Umwelt und Gesundheit])
6
MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide is metabolized by the mitochondrial
succinate dehydrogenase of proliferating cells to yield a purple formazan reaction product.
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In Vitro Cytotoxicity Test Methods BRD Section 1

November 2006

In addition, to ensure that a variety of toxic mechanisms were evaluated during in vitro
testing, the lowest predicted LD50 or ED50 from the results of a battery of three tests: a cellproliferation assay (total protein for 3T3 cells); a cytotoxicity/cytolethality assay using
primary rat hepatocytes (LDH release); and the rat skeletal muscle cell contractility assay,
was used also. The lowest predicted LD50 or ED50 of the three tests was then used to predict
toxicity classification. The accuracy of classification using this approach was 48% for the
ED50 and 45% for the predicted LD50 values.
Based on the results obtained, a battery of in vitro tests was recommended for classifying
chemicals for their acute lethal potency in rodents (Fentem et al. 1993). The first order test in
the battery measures basal cytotoxicity. This study observed no major differences in theperformances of the in vitro test methods that measure inhibition of cell growth regardless ofthe cell line (V79, 3T3-L1, or BALB/c 3T3), exposure duration (24-72 hours), or endpoint
measurement technique (MTT reduction, neutral red uptake [NRU], or protein
concentration). The second order test in the battery assesses hepatocyte-specific toxicity andthe role of biotransformation in cytotoxic activity. Co-cultures of rodent hepatocytes withproliferating cells such as 3T3 cells were recommended because the use of hepatocytes alone
would not indicate that a chemical requires bioactivation to produce its toxic effects. The
third order test in the battery detects chemicals that interfere with electrically excitable
membranes at non-cytotoxic concentrations (e.g., a contractility assay using primary culturesof rat muscle cells) (Fentem et al. 1993).
1.1.3
The Registry of Cytotoxicity (RC)The RC is a database of acute oral LD50 values for rats and mice obtained from RTECS®, andpublished IC50 values from in vitro cytotoxicity assays that used a variety of cell lines andcytotoxicity endpoints for substances with known molecular weights (Halle 1998, 2003). The
main purpose for compiling the RC was to evaluate, using data from substances with a wide
range of rodent acute oral toxicities, whether basal cytotoxicity (averaged over various cell
types, cell lines, and/or toxicity endpoints) accurately predicted acute oral lethality in rats andmice. The RC currently contains data for 347 different substances (Halle 1998, 2003) andefforts are underway to increase the number to 500 (ICCVAM 2001a). The RC does notcontain data on chemical mixtures.
The RC contains cytotoxicity data for substances that met the following criteria (Halle 1998,
2003):• At least two different IC50 values needed to be available, from studies using
either different cell types, different cell lines, or different cytotoxicity
endpoints• Data had to be generated using mammalian cells only (although data fromstudies using hepatocytes or related cells were excluded)• The chemical exposure duration had to be at least 16 hours, with no upper
limit
The following cytotoxicity endpoints were accepted:• Cell proliferation: cell number; cell protein; DNA content; DNA synthesis;
3
H-thymidine intake; colony formation

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In Vitro Cytotoxicity Test Methods BRD Section 1•••

November 2006

Cell viability/metabolic indicators: metabolic inhibition test (MIT-24);
mitochondrial reduction of tetrazolium salts into an insoluble (MTT) or
soluble (2,3-bis(2-methoxy-4-nitro-5- sulfophenyl)-2H-tetrazolium-5carboxanilide [XTT]) dye
Cell viability/membrane indicators: NRU; trypan blue exclusion; cellattachment; cell detachment
Differentiation indicators, such as functional and/or morphological changes
among and within cells

IC50 values (1,912) for 347 substances were obtained from 157 original publications (Halle
1998, 2003). The two to 32 IC50 values for each substance were averaged as geometric means
to produce one IC50x value for each substance. The rodent LD50 values used in the RC wereobtained from RTECS®. For the first 117 substances, designated as the training data set (RCI), LD50 values were not revised when subsequent issues of RTECS® reported lower values7.
For the most recent 230 substances, designated as the verification set (RC-II), the LD50
values were taken from the 1983/84 RTECS® publication. Whenever obtainable, oral LD50
data from rats were used (282 values). If rat data were unavailable, LD50 data from mice wereused (65 values). Combining rat and mouse data in the regression was deemed to be justified
when separate regressions for the mouse and rat LD50 values against the IC50x values did not
result in significant differences between the slopes and intercepts of the two regressions(Halle 1998, 2003).
To develop a model for the prediction of acute oral LD50 values from IC50x values, Halle(1998, 2003) calculated a linear regression from pairs of the log-transformed IC50x values (inmM) and log transformed rodent oral LD50 values (in mmol/kg) (see Figure 1-1). Molar
concentrations were used to allow for a comparison among chemicals based on the number ofmolecules rather than formula weights. The regression, referred to here as the RC millimole
regression, has the following formula:log LD50 (mmol/kg) = 0.435 x log IC50x (mM) + 0.625
To identify an acceptability range for practical use and research purposes, the acceptableprediction interval for the LD50 was empirically defined as approximately one-half an orderof magnitude on either side of the best-fit linear regression (i.e., ± log 5, or ±0.699) (Halle
1998, 2003). This interval was based on eight linear regressions calculated for in vitro
mammalian cell cytotoxicity data using various endpoints and oral LD50 values from rat,mouse, or rat and mouse from five publications. The prediction interval approximates thepredicted LD50 range for the eight regressions across about eight orders of magnitude of IC50
values. When this approach was used, 73% (252/347) of the RC substances fall within theprediction interval.

7

RTECS® published the lowest LD50 reported for a substance and updates the information periodically.
1-8

In Vitro Cytotoxicity Test Methods BRD Section 1

Figure 1-1

November 2006

RC Millimole Regression for In Vitro Cytotoxicity (IC50x)
and Rat and Mouse Acute Oral LD50 Values for 347 Chemicals

103

102

101

100

10-1

10-2

10-3

10-4
10-5

10-4

10-3

10-2

10-1

100

101

102

103

IC50x (mM)

Abbreviations: RC=Registry of Cytotoxicity; IC50x=Geometric mean (of multiple endpoints and cell types) test
substance concentration that reduces cell viability by 50%; LD50=Dose producing death in 50% of the animals
tested.
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 thinner lines show the empirical prediction interval (± log 5, or ±0.699) that is based on the
anticipated precision for the prediction of LD50 values from cytotoxicity data (Halle 1998, 2003).

1.1.4
The ZEBET Initiative to Reduce Animal Use
ECVAM organized a workshop in 1994 to evaluate the use of in vitro data for theclassification and labeling of chemicals (Seibert et al. 1996). Workshop participants reviewed
information on the assessment of acute oral toxicity using in vitro data and concluded that,for in vitro data to be used most effectively, the following information would be necessary:• The active concentration in vitro (i.e., the actual concentration available to thecultured cells)• The in vitro concentrations that produce basal cytotoxicity, hepatocyte
toxicity, and selective cytotoxicity (i.e., effects on cell-specific functions suchas transport processes or cell-to-cell communication)• The effect of biokinetic processes on acute oral toxicity in rodents• In vitro tests that provide the physicochemical parameters needed to estimate
equivalent body doses from in vitro data

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In Vitro Cytotoxicity Test Methods BRD Section 1

November 2006

The concept that in vitro data could be used to determine the starting doses for rodent acuteoral toxicity tests, so as to reduce the number of animals used, was first discussed at thisworkshop (Seibert et al. 1996). At that time, draft Organisation for Economic Co-operation
and Development (OECD) sequential rodent acute oral toxicity test guidelines (TGs) wereavailable; these included the:• Acute Toxic Class method (ATC; OECD draft Test Guideline [TG] 423[ICCVAM 2001a])• Up-and-Down Procedure (UDP; OECD draft TG 425 [ICCVAM 2001a])• Fixed Dose Procedure (FDP; OECD draft TG 420 [ICCVAM 2001a])
Final OECD TGs now exist for these rodent acute oral toxicity tests. The number of animals
needed depends upon the choice of the starting dose because the number of consecutive
dosing steps, and thus the number of animals used, is reduced as the starting dose moreclosely approximates the true toxicity class for the ATC or the FDP, or the true LD50 for the
UDP.
The ZEBET approach involves using an IC50 value from an in vitro basal cytotoxicity testwith the RC millimole regression to predict an LD50 value for use as a starting dose for the
ATC or UDP (Spielmann et al. 1999). Using simulation results performed to evaluate thedraft UDP test method, ZEBET predicted that the use of in vitro cytotoxicity assays topredict a starting dose equivalent to the LD50 had the potential to reduce animal use in the
UDP by 25-40%, depending upon the slope of the concentration response curve and thestopping rule applied (Spielmann et al. 1999; ICCVAM 2001a).
1.1.5

The International Workshop on In Vitro Methods for Assessing Acute Systemic
Toxicity
In 2000, the U.S. National Institute of Environmental Health Sciences (NIEHS), the NTP,
and the EPA jointly sponsored an International Workshop on In Vitro Methods for Assessing
Acute Systemic Toxicity (hereafter known as Workshop 2000). This workshop evaluated:• The ZEBET approach using the RC millimole regression to estimate LD50
values and set starting doses for in vivo testing• A testing strategy proposed by the European Center for the Validation of
Alternative Methods (ECVAM) (Siebert et al. 1996)• Other initiatives for reducing animal use in rodent acute oral toxicity testingby using in vitro cytotoxicity test methods (ICCVAM 2001a)
The Workshop 2000 participants concluded that no in vitro cytotoxicity test methods (orbattery of in vitro cytotoxicity test methods) existed that could replace the current in vivo
acute oral toxicity test methods (ICCVAM 2001a). Furthermore, they concluded that none of
the in vitro models reviewed had been adequately evaluated for reliability and relevance, andtheir usefulness and limitations for generating information for acute toxicity testing had notbeen assessed. However, there was agreement that: (1) in the near-term, in vitro basal
cytotoxicity test methods would be useful for estimating the starting dose for rodent acuteoral toxicity studies, and (2) further development, optimization, and validation of in vitro test
methods that considered target organ specificity and in vivo factors like adsorption,
distribution, metabolism, and excretion (ADME) that modulate the lethality of a xenobiotic
were needed (ICCVAM 2001a). Furthermore, the approach proposed by ZEBET (i.e., the use
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In Vitro Cytotoxicity Test Methods BRD Section 1

November 2006of in vitro basal cytotoxicity test methods to predict the starting dose for the sequential rodent
acute oral toxicity test methods) (Halle 1998, 2003; Spielmann et al. 1999) was
recommended for rapid adoption so that data could be generated to establish its usefulness
with a larger number of substances (ICCVAM 2001a). To assist in the adoption andimplementation of the ZEBET approach, several workshop participants prepared the
Guidance Document on Using In Vitro Data to Estimate In Vivo Starting Doses for Acute
Toxicity (hereafter referred to as Guidance Document; ICCVAM 2001b).
The Guidance Document recommended testing 10 to 20 RC substances (of high purity) fromthe RC in a candidate in vitro basal cytotoxicity assay to be used for predicting starting doses
for acute oral lethality tests (ICCVAM 2001b). The substances were to cover a wide range of
toxicities and fit the RC prediction model (i.e., the linear regression line) as closely as
possible. The in vitro test methods recommended and provided as examples were NRU
assays using 3T3 and normal human epidermal keratinocytes (NHK) cells. The IC50 resultsfrom testing the selected substances would be used to calculate a regression against the LD50
values used by the RC. If the resulting regression were parallel to the RC millimole
regression and within the ± log 5 (i.e., ±0.699) prediction interval for the RC, the GuidanceDocument recommended using the in vitro cytotoxicity assay to predict starting doses for
LD50 assays. If the regression from the in vitro assay did not meet these criteria, then the
Guidance Document advised either (a) adjusting the slope or (b) using the NRU protocols
offered in the Guidance Document (considered the most efficient approach).
Based on the conclusions and recommendation of the Workshop 2000 participants, ICCVAM
subsequently recommended that near-term validation studies should focus on two in vitro
basal cytotoxicity assays: one using human cells and one using rodent cells. Human cells areof interest because a principal aim of rodent acute oral toxicity testing is to predict potential
lethality in humans, while rodent cells may be a better predictor of lethality in rats and mice
(ICCVAM 2001a).
1.1.6
The NICEATM/ECVAM In Vitro NRU Cytotoxicity Validation Study
In response to the ICCVAM recommendation, NICEATM and ECVAM designed an
independent8 multi-laboratory validation study to evaluate in vitro basal cytotoxicity,
measured as NRU, as a predictor of acute oral lethality in rodents and potentially in humans.
Based on historical in vitro cytotoxicity data for mouse BALB/c 3T3 fibroblast cells (e.g.,
Balls et al. 1995; Brantom et al. 1997; Gettings et al. 1991, 1994a, 1994b; Spielmann et al.
1991, 1993, 1996) and NHK cells (e.g., Gettings et al. 1996; Harbell et al. 1997; Sina et al.
1995; Willshaw et al. 1994), it was decided that these two cells types should be the focus of
this validation effort.
The primary aim of this validation study was to determine if the NRU IC50 concentration of atest substance in either 3T3 or NHK cells could be used to estimate the rodent LD50, as ameans for predicting the starting doses for rodent acute oral toxicity studies. A secondary aim
was to determine the extent to which the NRU IC50 in either 3T3 or NHK cells could be used
8

“Independent” is used here to indicate that neither NICEATM nor ECVAM, nor its members, had a monetaryinterest in the test methods.

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In Vitro Cytotoxicity Test Methods BRD Section 1

November 2006

to estimate the blood serum concentrations associated with acute oral lethality in humans.
This evaluation will be the focus of a future ECVAM report.
The specific objectives for this validation study were to:• Further standardize and optimize the in vitro NRU basal cytotoxicity protocols
using 3T3 and NHK cells to maximize test method reliability (intralaboratory
repeatability, intra- and inter-laboratory reproducibility)• Assess the accuracy of the two standardized in vitro 3T3 and NHK NRU basal
cytotoxicity test methods for estimating rodent oral LD50 values across thefive United Nations (UN) Globally Harmonized System of Classification andLabelling of Chemicals (GHS; UN 2005) categories of acute oral toxicity, as
well as unclassified toxicities• Estimate the reduction and refinement in animal use achievable from using thein vitro 3T3 and NHK NRU basal cytotoxicity test methods to identify starting
doses for in vivo acute oral toxicity tests, assuming that no other information
were available• Develop high quality in vivo acute oral lethality and in vitro NRU cytotoxicity
databases that can be used to support the investigation of other in vitro test
methods necessary to improve the prediction of in vivo acute oral lethality
1.1.6.1 Study Design
The planning phase of the validation study included the selection of reference substances fortesting, which is described in Section 3, and the identification of rodent oral LD50 values forthe reference substances, which is described in Section 4. The validation study proceeded inseveral phases (see Figure 1-2) so that the Study Management Team (SMT) could evaluate
the reproducibility of results after each phase and refine the protocols, if necessary, before
proceeding to the next phase. The resulting NRU data collected were used to evaluate linear
regression formulas for the prediction of LD50 values from IC50 values (see Section 6).
Computer simulation modeling of acute oral toxicity test outcomes was then performed todetermine potential animal savings using the NRU-predicted starting doses compared withthe default starting dose for the UDP and the ATC (see Section 10). Study management andstudy participant information is provided in Appendix A.

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In Vitro Cytotoxicity Test Methods BRD Section 1

Figure 1-2

November 2006

NICEATM/ECVAM Validation Study Phases
Phase Ia: Laboratory Evaluation•••

Development of a positive control database for each laboratory
Perform at least 10 replicate NRU tests of the positive control substance (sodium laurylsulfate [SLS]) with each cell type.
Calculate mean IC50 value ±2 standard deviations for each cell type for each laboratory.
Establish acceptance criteria for positive control performance in future assays.
⇓
Phase Ib: Laboratory Evaluation••

Limited substance testing to demonstrate the reliability of the protocol
Each laboratory tests the same three coded substances three times with each cell type.
There was one substance each from low, medium, and high GHS toxicity categories.
Refine protocols and repeat, if necessary, until acceptable intra- and inter-laboratory
reproducibility is achieved.
⇓
Phase II: Laboratory Qualification

•
•
•
•

Evaluation of protocol refinements
Each laboratory tests nine coded substances covering the range of GHS toxicity
categories, with three replicate tests per substance in each test method.
Assure that corrective actions taken in Phase I have achieved the desired results.
Further refine protocols and re-test, if necessary, to achieve acceptable reliability.
Finalize protocols for Phase III.
⇓
Phase III: Laboratory Testing Phase•

Test of optimized protocols
Each laboratory tests 60 coded substances in three replicate tests using the finalized
protocol for each test method.

__________________________________________________________________________
Abbreviations: NRU=Neutral red uptake; GHS=Globally Harmonized System of Classification and Labelling ofChemicals (UN 2005)

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1.2

November 2006

Regulatory Rationale and Applicability for the Use of In Vitro Cytotoxicity
Test Methods to Predict Starting Doses for Acute Oral Toxicity Testing in
Rodents

1.2.1
Current Regulatory Testing Requirements for Acute Oral Toxicity
The major regulatory need for acute oral toxicity testing is for the hazard classification andlabeling of products, which is intended to alert handlers and consumers to potential toxicity
hazards. The LD50 values from acute oral toxicity tests using rodents are used to placesubstances in various toxicity categories that, in turn, invoke the associated hazard phrases tobe used on product labels. Table 1-1 shows the current U.S. legislation requiring the use of
acute oral toxicity testing for product labeling, and the substances regulated. Table 1-2
shows the statutory test protocol requirements and classification systems used by each U.S.
regulatory agency. Also included in this table is the UN Harmonized Integrated
Classification System for Human Health and Environmental Hazards of Chemical Substancesand Mixtures, which provides guidance to regulatory agencies on the use of the GHS (UN2005) as an internationally comprehensible system for hazard communication (OECD
2001b).
Table 1-1

Summary of Current U.S. Legislation for Using Acute Toxicity Data for
Product Labeling

Legislation(Year of Initial Enactment)
Federal Insecticide, Fungicide andRodenticide Act (FIFRA; 1947)
Federal Hazardous Substances Act (1964)
Occupational Safety and Health Act (1970)
Federal Hazardous Material Transportation
Act (1975)

U.S. Regulatory
AgencySubstances Regulated

EPA

Pesticides

CPSC
OSHA

Household products
Workplace materials

DOT

Transported substances

Abbreviations: EPA=U.S. Environmental Protection Agency; CPSC=U.S. Consumer Product Safety Commission;
FIFRA=Federal Insecticide, Fungicide, and Rodenticide Act; OSHA=U.S. Occupational Safety and Health Administration;
DOT=U.S. Department of Transportation.
Note: The U.S. Food and Drug Administration (FDA) does not require data for from acute lethality testing, and discourages
the use of animals for such testing (FDA 1993).

In addition to classification and labeling, acute oral toxicity test results may be used for:• Establishing dosing levels for repeated dose toxicity studies or other toxicity
studies• Identifying potential target organs• Providing information related to the mode of toxic action• Aiding in the diagnosis and treatment of toxic reactions• Providing information for comparison of toxicity and dose response among
substances in a specific chemical or product class• Aiding in the standardization of biological products• Aiding in judging the consequences of single, high accidental exposures in theworkplace, home, or from accidental release• Serving as a standard for evaluating alternatives to animal tests

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In Vitro Cytotoxicity Test Methods BRD Section 1

Table 1-2

November 2006

Regulatory Classification Systems for Acute Oral Toxicity

Regulatory Agency(Authorizing Act)
EPA (FIFRA)CPSC (Federal Hazardous
Substances Act)

Animals
Use current
EPA orOECD
protocolWhite rats,
200-300 g

OSHA (Occupational
Safety and Health Act)
DOT (Federal Hazardous
Material Transportation
Act)

Albino rats,
200-300 g
Male andfemale young
adult albinoratsOECD Guidance for Useof GHS (2001b)

Protocols not
specifiedEndpoint
Death

1

Death1 within 14 daysfor ≥ half of a group of≥10 animals
Death1, duration not
specified.
Death1 within 14 daysof half the animals
tested. Number ofanimals tested must be
sufficient for
statistically valid
results.
Not specified

Classification
I - LD50 ≤50 mg/kg
II - 50 < LD50 ≤500 mg/kg
III - 500 < LD50 ≤5000 mg/kg
IV - LD50 >5000 mg/kg
Highly toxic - LD50 ≤50 mg/kg
Toxic - 50 mg/kg < LD50 <5 g/kg
Highly toxic - LD50 ≤50 mg/kg
Toxic - 50 < LD50 <500 mg/kg
Packing Group 1 - LD50 ≤5 mg/kg
Packing Group II - 5 < LD50 ≤50 mg/kg
Packing Group III - LD50 <500 mg/kg (liquid)
LD50 <200 mg/kg (solid)

I - LD50 ≤5 mg/kg
II - 5 < LD50 ≤50 mg/kg
III - 50 < LD50 ≤300 mg/kg
IV - 300 < LD50 ≤2000 mg/kg
V - 2000 < LD50 ≤5000 mg/kg
Unclassified - LD50 >5000 mg/kg

Abbreviations: EPA=U.S. Environmental Protection Agency; OECD=Organisation for Economic Co-operation and Development; LD50=Dose
producing death in 50% of the animals tested; CPSC=U.S. Consumer Product Safety Commission; FIFRA=Federal Insecticide, Fungicide, andRodenticide Act; OSHA=U.S. Occupational Safety and Health Administration; DOT=U.S. Department of Transportation; GHS=Globally
Harmonized System of Classification and Labelling of Chemicals (UN 2005).
1
Guidance Document on the Recognition, Assessment and Use of Clinical Signs as Humane Endpoints for Experimental Animals Used in Safety
Evaluation calls for humane killing of moribund animals (OECD 2000). Moribund animals that are humanely euthanized are accepted as deaths.

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1.2.1.1 Test Methods for Assessing Acute Oral Toxicity
The current internationally recognized test methods for acute oral toxicity testing are the FDP(OECD 2001c), the ATC (OECD 2001d), and UDP (OECD 2001a; EPA 2002a) (seeAppendix M for test method guidelines). Information on toxic doses and signs of acutetoxicity and target organs can be obtained using any of these three methods. All threemethods are sequential tests in which the outcome of testing one or more animals at the firstdose is used to determine the second dose that should be tested. The FDP differs from the
UDP and ATC in that it involves using more animals per dose, and the primary endpoint ofinterest is evident toxicity9 rather than lethality. Both the FDP and the ATC methods provide
a range estimate of the LD50 for classification purposes. The UDP generally provides a point
estimate of the LD50 with a confidence interval (EPA 2002a).
Each of the test method guidelines includes a limit test in which up to five or six animals aretested at the limit, or upper bound, dose depending on the dose chosen (OECD 2001a, c, d, e;
EPA 2002a). The limit test can be performed using 2000 or 5000 mg/kg, depending on theregulatory need.
1.2.2
Intended Regulatory Uses for the In Vitro Cytotoxicity Test Methods
In vitro cytotoxicity test methods currently cannot serve as replacements for acute oral
toxicity tests in animals. However, such test methods can be used as adjuncts for rodent acuteoral toxicity tests. The current test guidelines for acute oral toxicity tests recommend using
information from structurally-related substances and the results of any other toxicity tests(EPA 2002b), including in vitro cytotoxicity test method (OECD 2001a, c, d; EPA 2002a) toselect the starting in vivo dose. The 3T3 and NHK NRU test methods may be used as part ofthis weight-of-evidence approach to select starting doses in order to reduce and refine the useof animals for acute oral toxicity testing.
Section 10 presents computer simulation analyses that characterize the extent of animal
reduction and refinement that may occur by using the in vitro NRU test methods to estimate
the starting doses for the UDP and the ATC method, by estimating the numbers of animals
used and the numbers of animal that die. These simulations determined (1) the numbers ofanimals used when using the default starting dose and, (2) the number of animals used whenusing a starting dose determined from the in vitro NRU test methods. These calculations
determined the reduction in animal use that can be achieved when using the in vitro NRU test
methods. To characterize the extent of refinement produced using the NRU-determined
starting dose, the number of animals that would have died with the NRU-determined starting
dose was compared with the number of animals that would have died when using the default
starting dose. Because there is a lack of information for specific substances about the dose atwhich evident toxicity occurs in relationship to the LD50, the FDP will not be consideredfurther in this document. However, the use of in vitro cytotoxicity data to determine starting
doses may also reduce the use of animals in the FDP.

9

Evident toxicity is a general term describing clear signs of toxicity following administration of the test
substance, such that the next highest fixed dose would result in the development of severe toxic signs, andprobably mortality (ICCVAM 2000).
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1.2.3

Similarities and Differences in the Endpoints of the In Vitro Cytotoxicity Test
Methods and Rodent Acute Oral Toxicity Test Methods
The endpoint measured in the in vitro NRU test methods is cell death. Neutral red dye istaken up and accumulated only by live cells; the primary measure of interest is the IC50 (i.e.,
the test substance concentration that causes a 50% inhibition of NRU). In contrast, theendpoint measured in acute oral toxicity assays is usually animal morbidity or death. Cell
death and animal death may have similar mechanistic bases because all cells, regardless ofwhether they are in animals or in vitro cell cultures, have similar cellular mechanisms; forexample, energy production and maintenance of cell membrane integrity.
Death of an animal death and death of a cultured cell due to toxicity both involve interferencewith vital cell processes or physical injury. Cell death in a culture system involves the deathof a single cell type, but through mechanisms that also operate in the animal. In contrast,
cellular injury in an animal, if sufficiently widespread or in a critical process, can lead to
injury or loss of function of other cell types in a tissue not directly affected by the treatment,
resulting in organ failure. Major organ system failures (e.g., liver and kidney failure),gastrointestinal corrosion, and bone marrow depression, can be fatal. Examples ofmechanisms leading to such organ failures are disruption of membrane structure or function,
inhibition of mitochondrial function, disturbance of protein turnover, and disruption ofenergy production (Gennari et al. 2004). Alternatively, the tissue injury could affect nonexposed vital organs or tissues through interference with homeostatic signaling mechanisms(Gennari et al. 2004). For example, respiratory depression leading to death may be due todepression of the central nervous system (CNS) rather than a direct assault on the respiratory
system itself.
Animal and cell culture systems are also different with respect to how a substance or toxicant
is delivered to the cell and how it is distributed within the cell, metabolized, and excreted.
After oral administration, animals must absorb the toxicant from the gastrointestinal tract,which involves the passage through membranes, many of which are selective with respect towhat molecules they will allow to pass. The toxicant may or may not be bound to serum
proteins, thereby reducing its availability to the target organ. The toxicant may be
metabolized before, during, and/or after its distribution to the target organs, or the toxicant or
its metabolites may be excreted before reaching the target organ or reacting with its
components. As a consequence, the most critical target organs may not be exposed to theactive metabolite, or be exposed for only a limited time or to a relatively small fraction of theadministered dose.
In contrast, in a cell culture system, the test substance is applied directly to the target cells
and the only membranes that must be passed are those of the target cell and its subcellularorganelles. No absorption and distribution by other cellular systems is required. Cell culture
systems may or may not include serum proteins, which could reduce the availability of
toxicant to the target site. For example, the 3T3 cell culture medium includes serum while theNHK cell culture medium does not. 3T3 and NHK cells have little to no capacity tometabolize xenobiotic compounds, and added cell-free metabolic activation systems, such as
rat liver homogenates, may not accurately mimic all phases of in vivo metabolism. Excretionfrom the cell culture milieu is not a consideration because anything excreted from the cell

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remains in the culture medium and is available to the other cells in the culture. As a result,the cells in culture (as opposed to cells in an animal) may be exposed to a test substance forthe entire duration of the test protocol.
Animals and cell culture systems may also differ with respect to the target on which a
toxicant acts. If a toxicant acts in a specialized organ system in vivo, it may not produce a
toxic effect by the same mechanism in cultured cells that are derived from a tissue different
from the target organ. For example, a substance that affects a neuroreceptor-mediatedpathway in animals would not be expected to produce a similar toxicity in 3T3 or NHK cells,
which are derived from fibroblasts and skin cells, respectively, and do not contain similar
neuroreceptors; if toxicity is seen in these cell cultures, it may be from a different mechanism
or in a different concentration relationship than in vivo. Even if a neurotoxin were applied toneuronal cells in culture, the cultured cells may not respond in the same way as neuronalcells in an animal because cells in culture, especially cell lines, may not retain the samefunctionalities as cells in vivo.
1.2.4

Use of In Vitro Cytotoxicity Test Methods in the Overall Strategy of Hazard
Assessment
In the overall strategy of hazard or safety assessment, the intended regulatory use of the invitro NRU test methods is to reduce and refine the use of animals in current acute toxicity
assays. The in vitro systems would serve as adjuncts to the in vivo test methods but are notintended as replacements for the rodent acute oral toxicity test methods. For the OECD
alternative acute oral toxicity assays (the ATC and UDP), the number of animals used
depends on the starting dose. The number of dosing steps (and animals) is reduced if thestarting dose is close to the true toxicity class (ATC) or the true LD50 (UDP) (Spielmann etal. 1999; ICCVAM 2001b).
As noted earlier, Spielmann et al. (1999) and the Guidance Document (ICCVAM 2001b)
suggest that the RC millimole regression analysis be used with in vitro cytotoxicity data to
predict starting doses for the ATC and UDP. The RC millimole regression cannot be applied
to unknown substances or to mixtures (e.g., product formulations) because such materials
cannot be assigned molecular weights. Therefore, the NICEATM/ECVAM validation study
also evaluated the classification accuracy and the reduction in animal use associated with aregression based on weight units (with IC50 in µg/mL and LD50 in mg/kg) (see Section 10).
This regression would potentially be appropriate for predicting the starting dose for mixturesand undefined substances.
1.3

Scientific Basis for the In Vitro NRU Test Methods

Cytotoxicity has been defined as the adverse effects resulting from interference withstructures and/or processes essential for cell survival, proliferation, and/or function (Ekwall1983). Ekwall (1983) described the concept of "basal cell functions" (mitochondrial activity,
plasma membrane integrity, etc.) that virtually all cells possess and suggested that, for mostsubstances, toxicity is a consequence of non-specific alterations in those cellular functions,which may then lead to adverse effects on organ-specific functions and/or death of theorganism. These effects may involve the integrity of membranes and the cytoskeleton,
cellular metabolism, the synthesis and degradation or release of cellular constituents or
products, ion regulation, and cell division.
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Ekwall (1983) and others (e.g., Grisham and Smith 1984) concluded that, because the actionsof substances that produce injury and death are ultimately exerted at the cellular level, in
vitro cytotoxicity assays might be useful for the prediction of acute lethality potency, as well.
Considerable research has been undertaken to develop and evaluate in vitro tests for use as
screens and as potential replacements for rodent LD50 tests, and numerous groups havereported good agreement between in vitro cytotoxicity and animal lethality (see reviews by
Phillips et al. 1990; Garle et al. 1994; Guzzie 1994). However, none of the proposed in vitro
models have been evaluated in any formal studies for reliability and relevance, and theirusefulness and limitations for generating information to meet regulatory requirements for
acute toxicity testing data have not been assessed.
1.3.1
Purpose and Mechanistic Basis of the In Vitro NRU Test Methods
A number of basal cytotoxicity endpoints can be used to measure cell death or interferencewith cell proliferation. The NRU test methods were chosen for the NICEATM/ECVAM
validation study because they were recommended in the Guidance Document for the purposeof obtaining cytotoxicity information to determine starting doses for rodent acute oral
toxicity assays (ICCVAM 2001b). Both the 3T3 and NHK NRU test methods werereproducible in previous validation studies (ICCVAM 2001b). In addition, both cell types areeasily obtainable from commercial sources and the Guidance Document provided
preliminary evidence that these assays could reproduce the RC millimole regression.
Additionally, the assays can be automated and they require no radioactivity or highly
dangerous reagents (see Section 2 for protocol discussion and Appendix B for protocols).
Neutral red is a weakly cationic water-soluble supravital dye that stains living cells(Borenfreund and Puerner 1985). It readily diffuses through the plasma membrane andconcentrates in lysosomes where it electrostatically binds to the anionic lysosomal matrix.
Toxicants can alter the cell surface or the lysosomal membrane to cause lysosomal fragility
and other adverse changes that gradually become irreversible. Thus, cell death and/or
inhibition of cell growth decreases the amount of neutral red retained by the culture.
Borenfreund and Puerner (1985) were the first to publish a protocol for the NRU assay using
3T3 cells as a method to objectively quantify toxicity previously assessed by subjective,
visual observation. The NRU assay, which was standardized for a 96-well plate format,
correlated two measurements of toxicity from the exposure of 3T3 cells to six surfactants: (1)a visual morphological evaluation of the cells under an inverted phase microscope, and (2) aquantitative measurement of NRU. The visual evaluation was designed to identify the highest
concentration of toxicant that causes only minimal morphological changes (i.e., the highest
tolerated dose [HTD]). Because Borenfreund and Puerner (1985) found that the HTD in the
NRU test was comparable to the concentration that produced 10% inhibition (i.e., the IC10)compared with the controls, the IC10 value was deemed to be a good index for comparing therelative toxicities of experimental agents. The assay was described as a rapid, reliable,
inexpensive, and reproducible in vitro test method for screening potentially toxic agents(Borenfreund and Puerner 1985). Furthermore, the authors suggested that the test method
was a good candidate for inclusion in a battery of assays for toxicity screening with the
purpose of reducing the use of animals for toxicity tests.

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1.3.2

Similarities and Differences in the Modes/Mechanisms of Action for the In Vitro
NRU Test Methods Compared with the Species of Interest
Although the ultimate species of interest for acute oral toxicity concerns is humans, labelingand hazard identification requirements are based on rodents. There are differences between
humans and rodents in terms of absorption, distribution, metabolism, excretion, and the
intrinsic sensitivity of target organs to xenobiotic compounds. The differences are largely
substance-specific and quantitative, although there are a number of substances where thehuman may produce metabolites not seen in the rodent and vice versa. In vitro cytotoxicity
studies have also noted differences in sensitivity between human cells and other mammaliancells (Clemedson et al. 1996b). It is important to note that, for certain chemicals, there canalso be large differences in sensitivity among different human cell types and cell lines(Clemedson et al. 1996b, 1998a, b).
Because of the differences in sensitivity between humans and rodents, it might be likely that
cultured human cells would predict human lethality better than cultured rodent cells and that
cultured rodent cells would predict rodent lethality better than human cells. Ekwall et al.
(1998b) showed that in vitro cytotoxicity test methods using human cell lines generally
predicted human toxicity more accurately than did test methods using nonhuman mammaliancells.
In addition to being derived from different species, there are several other differences
between 3T3 and NHK cells, all of which may contribute to differences in sensitivity.
• 3T3 cells are an immortal line, while the NHK cells are primary cells.
• The cells originate from different tissues; 3T3 cells are derived fromembryonic fibroblasts, while the NHK cells are isolated from neonatal
foreskin tissue.
• NHK cells grow more slowly in culture than the 3T3 cells (i.e., after seeding
into 96-well plates, NHK cells require 48-72 hours for growth to theappropriate confluence while 3T3 cells require approximately 24 hours; see
Appendix B).
• NHK cells have greater ability to metabolize xenobiotic compounds, in thatthey exhibit minimal cytochrome P450 activity (Babich et al. 1991), whereas
3T3 cells have practically no ability to metabolize xenobiotic compounds(INVITTOX 1991).
1.3.3
Range of Substances Amenable to the In Vitro NRU Test Methods
The in vitro NRU test methods can be applied to a wide range of substances as long as they
can be dissolved in the cell culture medium or in a nontoxic solvent (at the concentration
used), and do not react with the culture medium. Although these test methods may to be
applicable to mixtures, none were evaluated in this validation study. The toxicity of
substances that act by mechanisms not expected to be active in 3T3 or NHK cells (e.g., thosethat are specifically neurotoxic or cardiotoxic) will likely be underpredicted by these test
methods. Therefore, until more appropriate cell lines are developed, the results from basal
cytotoxicity testing with such substances may not be relevant for predicting in vivo effects.

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Insoluble substances or those unstable in aqueous environments are not compatible with thetest systems. Volatile substances may yield acceptable results if CO2 permeable plastic film
is used to seal the test plates. Testing for corrosive substances is unnecessary since there is no
regulatory requirement for acute oral toxicity testing for known corrosives. The 3T3 NRU
test method may underestimate the toxicity of substances that are highly bound to serum
proteins because the culture medium contains 5% serum during substance exposure. The
toxicity of substances that specifically affect lysosomes may be overestimated because they
may affect NRU binding, and therefore, retention, in the cell. Red substances (and othercolored substances) that absorb light in the optical density range of NR may interfere withthe test if they remain inside the cell in sufficient amounts after washing and are soluble in
the NR solvent.

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2.0

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

2.1

Basis for Selection of In Vitro NRU Cytotoxicity Test Methods .........................2-4
2.1.1 Guidance Document Rationale for Selection of In Vitro NRU
Cytotoxicity Test Methods ..........................................................................2-4
2.1.2 Guidance Document Rationale for Selection of Cell Types .........................2-4

2.2

Overview of the 3T3 and NHK NRU Test Methods ............................................2-5
2.2.1 The 3T3 NRU Test Method.........................................................................2-6
2.2.2 The NHK NRU Test Method ......................................................................2-8
2.2.3 Measurement of NRU in the 3T3 and NHK Test Methods...........................2-9

2.3

Descriptions and Rationales of the 3T3 and NHK NRU Test Methods..............2-9
2.3.1 Materials, Equipment, and Supplies ............................................................2-9
2.3.2 Reference Substance Concentrations/Dose Selection.................................2-12
2.3.3 NRU Endpoints Measured.........................................................................2-13
2.3.4 Duration of Reference Substance Exposure ...............................................2-14
2.3.5 Known Limits of Use ................................................................................2-14
2.3.6 Basis of the Response Assessed.................................................................2-16
2.3.7 Appropriate Vehicle, Positive, and Negative Controls ...............................2-16
2.3.8 Acceptable Ranges of Control Responses..................................................2-17
2.3.9 Nature of Experimental Data Collected .....................................................2-18
2.3.10 Data Storage Media...................................................................................2-19
2.3.11 Measures of Variability.............................................................................2-19
2.3.12 Methods for Analyzing NRU Data ............................................................2-20
2.3.13 Decision Criteria for Classification of Reference Substances.....................2-20
2.3.14 Information and Data Included in the Test Report .....................................2-20

2.4

Proprietary Components of the In Vitro NRU Test Methods ...........................2-22

2.5

Basis for the Number of Replicate and Repeat Experiments for the
3T3 and NHK NRU Test Methods .....................................................................2-22

2.6

Basis for Modifications to the 3T3 and NHK NRU Test Method
Protocols..............................................................................................................2-22
2.6.1 Phase Ia: Laboratory Evaluation Phase......................................................2-22
2.6.2 Phase Ib: Laboratory Evaluation Phase......................................................2-25
2.6.3 Phase II: Laboratory Qualification Phase...................................................2-27
2.6.4 Phase III: Laboratory Testing Phase ..........................................................2-35

2.7

Differences Between the 3T3 and NHK NRU Test Method Protocols
for the Validation Study and the Guidance Document Standard Protocols .....2-36

2.8

Overview of the Solubility Protocol ...................................................................2-37

2.9

Basis of the Solubility Protocol...........................................................................2-38
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2.9.1 Initial Solubility Protocol Development .................................................... 2-38
2.9.2 Basis for Modification of the Phase II Protocol ......................................... 2-39
2.10

Components of the Solubility Protocol .............................................................. 2-40
2.10.1 Medium, Supplies, and Equipment Required............................................. 2-40
2.10.2 Data Collection ......................................................................................... 2-41
2.10.3 Variability in Solubility Measurement....................................................... 2-41
2.10.4 Solubility Issues During the Testing of the Reference Substances ............. 2-41
2.10.5 Analysis of Solubility Data ....................................................................... 2-41

2.11

Summary............................................................................................................. 2-42

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2.0

November 2006

TEST METHOD PROTOCOL COMPONENTS OF THE 3T3 AND NHK IN
VITRO NRU TEST METHODSThe Guidance Document (ICCVAM 2001b) recommended that the following be incorporated
into any in vitro cytotoxicity protocol used to predict rodent acute oral lethality:• A cell line (or primary cells) that divides rapidly (e.g., with a doubling time of<24 hours)• An initial seeding density that allows for exponential cell growth throughout
the exposure period• An exposure period that spans at least one cell cycle• Appropriate positive control (PC) and vehicle control (VC) substances forwhich toxicity and lack of toxicity, respectively, has been well characterized
by the performing laboratory• Solvents that are used only at concentrations that do not cause significant
toxicity to the cell system over the entire period of the assay• A well-established, quantifiable cytotoxicity endpoint that has goodinterlaboratory reproducibility• Tests that are compatible with at least 96-well plates and equipment (e.g.,spectrophotometric microplate reader) that allow a quick and precise
measurement of the endpoint of interest• Use of a progression factor in the concentration-response experiment thatyields graded effects between 0% and 100% cytotoxicity
Section 2.1 provides the basis for the selection of the in vitro 3T3 and NHK NRU test
methods. Section 2.2 provides descriptions of the NRU protocols applicable to this validation
study. Section 2.3 provides details for performing the 3T3 and NHK NRU test methods andexplains the rationales for the various test method components, and Section 2.4 describes any
3T3 and NHK NRU test method proprietary aspects. Section 2.5 discusses the basis for the
replicate and repeat tests conducted during validation of these two test methods. Section 2.6
details the modifications and revisions made during the first two phases of the validation
study which contributed to the development of the final protocol used in Phase III. Section
2.7 describes the differences between the protocols used in this study and the protocols
outlined in the Guidance Document. Sections 2.8, 2.9, and 2.10 provide details on thesolubility protocol evaluated during the validation study and used to identify the appropriate
solvent for dissolving the reference substances.
The 3T3 and NHK NRU test method protocols were provided to the three laboratories thatparticipated in the validation study (see Section 5.6.3 for additional laboratory information).
These were:• The U.S. Army Edgewood Chemical Biological Center (ECBC)• The FRAME Alternatives Laboratory (FAL)• The Institute for In Vitro Sciences (IIVS)
A fourth laboratory (BioReliance Corporation, Rockville, MD) was used to procure anddistribute the coded reference substances, and to perform solubility tests on the validation
study reference substances prior to their distribution to the participating laboratories.

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In Vitro Cytotoxicity Test Methods BRD Section 2

2.1

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Basis for Selection of the In Vitro NRU Cytotoxicity Test Method

As stated in Section 1, in agreement with the recommendations of the Workshop 2000
participants (ICCVAM 2001a), ICCVAM made the following recommendations andforwarded them to U.S. Federal agencies along with the Workshop 2000 Report (ICCVAM
2001a) and Guidance Document (ICCVAM 2001b).
“ICCVAM concurs with the Workshop recommendation that near-term
validation studies should focus on two standard cytotoxicity assays: one
using a human cell system and one using a rodent cell system. Since themurine BALB/c 3T3 cytotoxicity assay has been evaluated for only alimited number of chemical classes, there is merit in determining its
usefulness with a broader array of chemical classes. Cell lines establishedfrom the rat rather than the mouse might also be considered, as most acuteoral toxicity testing is conducted in this species. Human cell lines should
also be considered since one of the aims of toxicity testing is to makepredictions of potential toxicity in humans. Future validation studies
should therefore compare rodent and human in vitro data with one another,with rodent in vivo data, and with human in vivo data. Correlations
between in vitro and in vivo data might help in selecting cytotoxicity
assays for further evaluation”. (ICCVAM 2001a)
Based on this recommendation and the Guidance Document recommendation, NICEATM
and ECVAM selected the 3T3 and NHK NRU basal cytotoxicity test methods for validation.
2.1.1

Guidance Document Rationale for Selection of In Vitro NRU Cytotoxicity Test
Methods
The Guidance Document (ICCVAM 2001b) provided the basic approach for the use of invitro NRU basal cytotoxicity test methods to predict starting doses for rodent acute oral
toxicity assays using the RC millimole regression. The 3T3 and NHK NRU test method
protocols used in the validation study were derived from those proposed in the GuidanceDocument.
2.1.2
Guidance Document Rationale for Selection of Cell Types
The Workshop 2000 participants (ICCVAM 2001a) concluded that there were no significant
differences between the basal cytotoxicity results obtained using permanent mammalian celllines, primary human cells (e.g., NHK cells), or the IC50x approach of Halle and Spielmann(Halle 1998, 2003; Spielmann et al. 1999; Halle and Spielmann 1992). Further, the GuidanceDocument recommended that in vitro basal cytotoxicity test methods not use hepatocytes (orrelated metabolically competent cells) or other types of highly differentiated cells because
they may not give the best prediction of acute lethality for the large variety of chemicals
likely to be tested (ICCVAM 2001b). However, it was recognized that, ultimately, simple
predictive systems (in vitro or in silico) would be needed for early identification of those
substances likely to be metabolized to more toxic or less toxic species than the parent
chemical as well as those that were likely to exhibit cell-specific toxicity (e.g., Fentem et al.
1993; Seibert et al. 1996; Curren et al. 1998; Ekwall et al. 1999).

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In Vitro Cytotoxicity Test Methods BRD Section 2

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Established rodent cell lines were recommended for validation because (ICCVAM 2001b):• It was assumed that such cells would give the best prediction of rat and mouseacute oral lethality (i.e., like correlates with like).
• The use of a readily available, easy to culture, immortalized cell line for invitro cytotoxicity testing would accelerate the development of a database thatcan be used to analyze the usefulness of this approach.
Human cells also offer potential advantages. As determined in the MEIC project, the in vitro
test methods with the best predictivity for peak acute LC values in humans generally usedhuman cell lines (Ekwall et al. 1998b). Thus, a long-term advantage of using human cells isthat in vitro human cell cytotoxicity data can be added to human toxicity databases tofacilitate the development of test methods that may better predict acute oral human lethality.
3T3, an immortalized mouse fibroblast cell line, and NHK, primary human cells, wereselected as representative rodent and human cells, respectively, for the NICEATM/ECVAM
validation study. Historical data for the 3T3 NRU test were available from a variety ofstudies, including controlled and blinded validation studies, indicating the reliability of thistest method (Gettings et al. 1991, 1992, 1994a, 1994b; Spielmann et al. 1991, 1993, 1996;
Balls et al. 1995; Brantom et al. 1997). NHK cells have also been used in validation studies
for basal cytotoxicity test methods with good results (Willshaw et al. 1994; Sina et al. 1995;
Gettings et al. 1996; Harbell et al. 1997).
2.2

Overview of the 3T3 and NHK NRU Test Methods

The Guidance Document (ICCVAM 2001b) includes a proposed 3T3 NRU test method
protocol based on the 3T3 Cytotoxicity Test (INVITTOX Protocol No. 46; available from the
FRAME-sponsored INVITTOX database [http://embryo.ib.amwaw.edu.pl/invittox/]), which
in turn was based on the Borenfreund and Puerner (1985) protocol, as elaborated on in
Spielmann et al. (1991, 1996). This protocol was updated based on experience obtained
during the validation of the 3T3 NRU Phototoxicity Test (INVITTOX Protocol No. 78; alsoavailable at the FRAME INVITTOX database). The RC millimole regression for predictionof acute oral rat and mouse toxicity (Halle 1998, 2003; Spielmann et al. 1999) was included
as the prediction model (ICCVAM 2001b; see Section 1.1.2).
The NHK NRU protocol provided in the Guidance Document was based on the protocol usedby IIVS, which was based on a NRU protocol of Borenfreund and Puerner (1984) and a rat
epidermal keratinocytes protocol (Heimann and Rice 1983). Formulations for the media andsolutions, and general NHK cell culture techniques, correspond to Clonetics products fromthe CAMBREX Corporation.
The protocol components for the 3T3 and NHK NRU test methods used in this validation
study are similar (see Figure 2-1). The nature of the NRU response is described in Section
1.3.1. Figure 2-1 provides an overview to the major steps for performance of the in vitro
NRU test methods. The following procedures are common to both cell types:•
Preparation of substances and the PC•
Cell culture environmental conditions•
Determination of test substance solubility•
96-well plate configuration for testing samples
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In Vitro Cytotoxicity Test Methods BRD Section 2

•
•
•
•

November 2006

Range finder and definitive tests
Microscopic evaluation of cell cultures for toxicity based on morphologicalalterations
Procedures for measurement of NRU
Data analysis procedures

The main protocol differences between the two cell lines are:•
The conditions of propagation of the cells in culture (e.g., time needed toreach appropriate confluence)•
The growth media components•
The volumes of substances applied to the 96-well plates•
The number of cell divisions undergone by each cell line during exposure to atest substance
2.2.1

The 3T3 NRU Test Method

2.2.1.1 Initiating and Subculturing 3T3 Cells
Each laboratory initially prepared a large pool of 3T3 cells (described further in Section
2.3.1.1), cryogenically preserved multiple ampules of these cells in liquid nitrogen, andperiodically removed an ampule when needed. Although the NRU protocols used for eachstudy phase provided cell culture density guidelines for subculturing the cells, eachlaboratory refined the final seeding density to achieve optimal growth.
Cryopreserved 3T3 cells were thawed, resuspended in a culture medium containing
Dulbecco’s Modification of Eagle’s Medium (DMEM) supplemented with non-heatinactivated 10% newborn calf serum (NCS), transferred into tissue culture flasks (25 or 7580 cm2), and incubated at 37 °C ±1 °C, 90% ±5% humidity, and 5.0% ±1% CO2/air. Whencells reached 50 to 80% confluence (as estimated from a visual inspection of cell density),
they were removed from the flask by trypsinization. A single-cell suspension was added to
new flasks for propagation and the cells were passaged/subcultured at least two times1 before
seeding into 96-well plates for testing. This study did not evaluate the potential effects thatcell passage number may have on the performance of the 3T3 NRU test method.

3T3 cells were maintained in culture for approximately two months (approximately 18 passages) and used for
the NRU test. The Guidance Document (ICCVAM 2001b) did not provide a rationale for using 18 passages as
the limit, but it was probably recommended to maintain homogeneity of the 3T3 cell population (i.e., decrease
the potential of the population to drift genetically). The more passages the cells undergo, the more likely their
response to chemical stress may change.
1

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Figure 2-1

November 2006

Major Steps in the Performance of the NRU Test Methods(1) Cells (3T3 or NHK) are seeded into 96-well plates to form a sub-confluent

monolayer; plates are incubated at 37 ºC (24 hours for 3T3 cells; 48-72 hours
for NHK cells)⇓(2) Culture medium is removed (3T3 cells only)⇓(3) Reference substances in the appropriate solvents are added to the cells; cells
are exposed for 48 hours at 37 ºC over a range of eight (8) concentrations⇓(4) Cells are evaluated microscopically for toxicity based on morphologicalappearance⇓(5) Treatment medium is removed; cells are washed once with Dulbecco’s
Phosphate Buffered Saline (D-PBS); Neutral Red (NR) dye medium is added(3T3 cells: 25 µg/mL NR dye; NHK cells: 33 µg/mL NR dye); plates are
incubated for 3 hours at 37 ºC⇓(6) NR medium is discarded; cells are washed once with D-PBS; NR desorbing
fixative is added to the wells⇓(7) Plates are shaken for 20 minutes at room temperature⇓(8) NR absorption is measured at optical density (OD) 540 ±10 nm⇓(9) NRU is calculated as a percent of vehicle control values to define IC20, IC50,
and IC80 concentrations (µg/mL)2_________________________________________________________________________
Abbreviations: NRU=Neutral red uptake; 3T3=BALB/c 3T3 fibroblasts; NHK=Normal human epidermal
keratinocytes; IC20, IC50, IC80=Substance concentration that reduces cell viability by 20, 50, and 80%,
respectively.

IC50 values are used for estimating the LD50 value of a reference substance. The IC20 and IC80 values were
determined for possible use in estimating human lethal concentrations in blood.
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2.2.1.2 Preparation of Cells for 96-well Plate Assays
3
After subculturing the cells, 100 µL of the cell suspension (2.0 – 3.0x10 cells/well) wereplaced in the appropriate wells and 100 µL of cell-free culture medium were dispensed into
the 36 peripheral wells (blanks). The peripheral wells were in rows 1 and 8 and columns 1
and 12 (See Figure 1 in Appendix B1 or B2). Peripheral wells were used only for blanks
because they may be subjected to more evaporation than interior wells. The GuidanceDocument authors (and the SMT and Study Directors) concluded that such conditions would
ultimately affect cell growth in these wells. One plate was prepared for each reference
substance. The cells were incubated for 24 ±2 hours at 37 ºC and checked visually to be surethat approximately a 50% confluent monolayer was present at the time of substanceapplication.
2.2.1.3 Reference Substance Application
After the appropriate incubation period to achieve a half-confluent monolayer, the medium
was removed and 50 µL of culture medium with 10% NCS were added to each well. Then,
50 µL treatment medium containing the appropriate substance concentrations were added fora final concentration of 5% NCS. The cells were then incubated at 37 ºC for 48 ±0.5 hours.
At the end of the incubation period, the cells were microscopically evaluated for changes inmorphology and their appearance was documented (as per Visual Observation Codes in the
protocol) prior to measurement of NRU.
2.2.2

The NHK NRU Test Method

2.2.2.1 Initiating and Subculturing NHK Cells
Cryopreserved NHK cells (ampules of cryopreserved cells were obtained from CAMBREX
Corporation and stored in liquid nitrogen until needed) were thawed, resuspended in serumfree keratinocyte complete growth medium (see Section 2.3.1.4 for components of themedium), transferred into tissue culture flasks (25 cm2 without fibronectin-collagen coating),
and incubated at 37 °C ±1 °C, 90% ±5% humidity, and 5.0% ±1% CO2/air. When the cells
reached 50 to 80% confluence (as estimated from a visual inspection of cell density), they
were removed from the flask by trypsinization and prepared for subculturing into the 96-wellplates. Care was taken to prevent the keratinocyte cultures from becoming 100% confluent as
this may lead to cell differentiation, which would alter the intrinsic sensitivity of these cells
to cytotoxic substances. To minimize potential sources of experimental variability, thelaboratories used the same lot of Clonetics® cells throughout the validation study, the samebrand of growth medium and supplements (and concentrations of supplements), and cells
were not used beyond their second passage. The protocols for each study phase provided cell
culture density guidelines, but each laboratory refined the final seeding densities to achieve
appropriate growth.
2.2.2.2 Preparation of Cells for 96-well Plate Assays
3
After subculturing the cells, 125 µL of the cell suspension (2.0 – 2.5x10 cells/well) wereplaced in the appropriate wells and 125 µL of cell-free culture medium were dispensed into
the peripheral wells (blanks). One plate per reference substance was prepared. The cells wereincubated at 37 ºC for 48-72 hours and checked to be sure that cultures were at 20 to 50%
confluence at the start of exposure to the reference substance.

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2.2.2.3 Reference Substance Application
To add the reference substances, 125 µL of culture medium containing the appropriate
reference substance concentrations were added to the existing 125 µL of culture medium inthe test wells. The cells were then incubated at 37 ºC for 48 ±0.5 hours. At the end of the
exposure period, the cells were microscopically evaluated for changes in morphology andtheir appearance was documented (as per Visual Observation Codes in the protocol [seeAppendices B1 and B2]) prior to measurement of their NRU.
2.2.3
Measurement of NRU in the 3T3 and NHK Test Methods
The treatment medium was removed from the 96-well plates, the cells were rinsed withphosphate buffered saline (PBS), and 250 µL NR dye medium was added to the wells (25 µg
NR/mL for 3T3 cells; 33 µg NR/mL for NHK cells). The plates were then incubated (37 °C±1 °C, 90% ±5% humidity, and 5.0% ±1% CO2/air) for three hours. After incubation, the NR
medium was removed, the cells were rinsed with PBS, and 100 µL of the desorb solution
were applied. The plates were shaken on a microtiter plate shaker for 20 to 45 minutes toextract NR from the cells and to form a homogeneous solution. The optical density (OD) of
the resulting colored solution was measured (within 60 minutes of adding the desorb
solution) at 540 nm ±10 nm (OD540) in a spectrophotometric microtiter plate reader, using theblank wells as reference. Data from the plate reader were transferred to a Microsoft®
EXCEL® (Microsoft Corporation, Redmond, WA, USA) spreadsheet template (hereafter
know as EXCEL® template) designed by the SMT and the testing laboratories for statisticalanalyses.
2.3

Descriptions and Rationales of the 3T3 and NHK NRU Test Methods

The protocols used in Phases I, II, and III of the validation study (Appendices B and C) are
modifications of the protocols reported in the Guidance Document (ICCVAM 2001b). The
participating laboratories provided comments and recommendations during the developmentof these protocols. The following information is specific to the protocols used in thisvalidation study.
2.3.1

Materials, Equipment, and Supplies

2.3.1.1 3T3 Cells
The CCL-163, 3T3 BALB/c mouse fibroblast, cell line, clone 31 from the American Type
Culture Collection (ATCC), Manassas, VA, USA, was used. The 3T3 cells, an immortalized
mouse fibroblast cell line, were procured from the ATCC by IIVS at passage 64. IIVS
cultured the cells to expand their number and cryogenically preserved them as a pool atpassage number 69. ECBC and FAL received frozen ampules of cells at passage number 69
from IIVS, propagated the cells, and cryopreserved multiple ampules of cells at a slightly
higher passage number to establish their working cell banks for use throughout the study.
Each laboratory determined the doubling time for the 3T3 cell line prior to NRU testing inPhase Ia as required by the protocol in Appendix C1. The following doubling times werereported: 18.6 hours by ECBC; 17 hours by FAL; and 17 hours by IIVS. No other doublingtime measurements were made. The extent of cell confluence was monitored during the study
to identify when the cultures were in exponential growth.

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2.3.1.2 NHK Cells
A single lot of pooled donor, primary neonatal foreskin keratinocyte (NHK) cells (Clonetics®
# CC-2507; lot # 1F0490N) from CAMBREX Bio Science Walkersville, Inc., Walkersville,
MD, USA, was used throughout the validation study. Keratinocytes from other sources
would be acceptable if they meet the growth requirements identified in the protocols. Eachlaboratory determined the doubling time for the NHK cells prior to testing in Phase Ia (asrequired by the protocol in Appendix C2). The following doubling times were reported: 21hours by ECBC; 10 hours by FAL; and 15.8 hours by IIVS. No other doubling timemeasurements were made. The extent of cell confluence was monitored during the study toidentify when the cultures were in exponential growth.
2.3.1.3 Tissue Culture Materials and Supplies
The 3T3 and NHK NRU test methods require general tissue culture materials and supplies(see Appendices B1 and B2 [protocols] for formulations, and concentrations of solutions andmedia). Both test methods used the same materials for solubility testing (Section 2.8.1).
Freshney (2000) provides information on all aspects of cell culture, including materials,
supplies, and equipment needed. The following materials were needed for both test methods:• Trypsin (0.05%)• PBS• Hanks’ Balanced Salt Solution (HBSS) without Ca2+ and Mg2+
• NR dye• Glacial acetic acid
• Dimethyl sulfoxide (DMSO) [analytical grade]• Ethanol (ETOH) [100% non-denatured for test substance preparation]• Distilled water
2.3.1.4 Cell Culture Materials
Laboratory items needed include the following:• Sterile, disposable tissue culture plasticware (e.g., 25 cm2,75-80 cm2 flasks;
multiwell/microtiter [96-well] plates; petri dishes) [Note: The laboratories inthis study used tissue culture plasticware from various suppliers.]• Cryogenic ampules• Pipettes, pipettors, pipette tips
• Multichannel solution reservoirs• Centrifuge tubes• Microporous sterilization filters• General plastic containers• Glass tubes (for preparation of substance dilutions)2.3.1.5 Equipment
Performance of the NRU tests requires a laboratory equipped with a designated cell culturearea. Essential equipment for cell culture work and the NRU test methods include:• Incubator (37 ºC ±1 ºC, 90% ±5% humidity, 5.0% ±1% CO2/air)• Laminar flow clean bench/cabinet (standard: "biological hazard")• Water bath (37 ºC ±1 ºC)• Inverted phase contrast microscope (with 10X to 40X objectives)• Centrifuge (capable of 220 x g)
• Laboratory balance (capable of measuring to 10 mg)
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•
•
•
•
•
•
•
•
•
•
•
•
•

November 2006

Spectrophotometer for reading 96-well plates (i.e., microtiter plate reader)equipped with 540 nm ±10 nm filter
Shaker for microtiter plates
Cell counter or hemocytometer
Pipetting aid (e.g., vacuum pipettor unit)
Pipettes, pipettors (multi-channel and single channel, multichannel repeater
pipette)Waterbath sonicator
Refrigerator
Freezer (to at least -70 ºC)
Cryostorage container (and liquid nitrogen supply)
Magnetic stirrer
Antistatic bar ionizer
Personal computer
Osmometer
pH meter

2.3.1.6 Culture Medium
For 3T3 Cells
DMEM containing high glucose (4.5 gm/L) and supplemented with NCS, L-glutamine,
penicillin, and streptomycin was used for the 3T3 cells. Heat-inactivated serum was not usedin this study. Heat-inactivation of serum is often used to destroy heat-labile components suchas complement factors, and microbial contaminants such as mycoplasma (Hyclone® 1996;
Mediatech, Inc. 2006). However, some heat-labile complement factors can also beinactivated by the standard cell culture practice of warming serum-containing medium to 37°C prior to use, and mycoplasma can be eliminated by filtering the medium (e.g., using 0.1µm pore-size rated filters). Heating serum to 56 °C (heat-inactivation temperature) can
destroy other heat-labile components such as growth factors, vitamins, amino acids, andhormones. Loss of these components can diminish the capacity of the serum to promote
attachment of cells to culture vessel surfaces and to support cell growth. An additional
confounding factor is that the procedure for heat-inactivation is highly precise, and deviationfrom the basic protocol can create additional issues such as protein denaturation and serum
turbidity.
For NHK Cells
Although the contents of the NHK basal culture medium are proprietary, the formulation isbased on a commercially available, non-proprietary basal medium (MCDB 153 medium
formulation [Tsao et al. 1982]; e.g., MCDB 153 medium - SIGMA-ALDRICH productnumber #M 7403 http://www.sigmaaldrich.com/sigma/datasheet/m7403dat.pdf). The
laboratories recommended this medium for use with the CAMBREX Clonetics® NHK cells
because they all had access to this supplier. Other media are acceptable for NHK NRU
testing if the performance standards prescribed in the media prequalification protocol are met(see Appendix B4 and Section 2.6.3.5).
The serum-free culture medium used for NHK cells was Clonetics® keratinocyte basal
medium (KBM®) supplemented with KBM® SingleQuots® (epidermal growth factor, insulin,hydrocortisone, antimicrobial agents, bovine pituitary extract [BPE]) and Calcium SingleQuots®
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(calcium) [all from CAMBREX Corporation] to make keratinocyte complete growth medium.
Although the keratinocyte complete growth medium is a defined serum-free medium, it
contains BPE collected from bovine pituitary glands. BPE contains growth factors andhormones, and is added to serum-free medium as a mitogenic supplement. Variability in thecomposition of the BPE could be a factor in cell growth kinetics. However, it is suggested
that the undefined BPE components could be replaced with defined growth supplements,
such as insulin, epidermal growth factor, and fibroblast growth factor, without adversely
affecting the cellular proliferation rates and general physiology of human keratinocytes (LifeTechnologies, Inc. 1997).
2.3.2
Reference Substance Concentrations/Dose Selection
Each laboratory weighed and dissolved the reference substances on the same day as the startof the exposure period. The highest concentration of dissolved reference substance was
identified using the solubility protocol and designated as the 2X stock solution. All reference
substance dilutions for an assay were serially derived from this stock solution (see GuidanceDocument for serial dilution methods).
2.3.2.1 Range Finder Test
A range finder 3T3 or NHK NRU test was performed to determine the concentrations of areference substance to be used for the definitive (concentration-response) test (see Section
2.3.2.2). The range finder test used eight concentrations of the reference substance preparedby diluting the stock solution using log intervals to cover a large concentration range (e.g.,
1:10, 1:100, 1:1000, etc.; up to eight orders of magnitude). The highest concentrations
applied to the cells were 10 mg/mL for substances dissolved in culture medium and 1 mg/mL
in medium for substances dissolved in DMSO, unless precluded by solubility. ETOH was not
used as a solvent for any of the substances in the validation study (see Sections 2.8, 2.9, and2.10).
If the range finder test did not produce cytotoxicity, then a second range finder test was
conducted at higher concentrations (e.g., the highest concentration would be >10 mg/mL if in
medium, >1 mg/mL if in DMSO) unless precluded by solubility. If the substance being tested
was insoluble or poorly soluble, then more stringent solubility procedures were employed toincrease the stock concentration (to the maximum concentration specified in Appendices B1
and B2). If the range finder test produced a biphasic dose-response curve3 for NR uptake, theconcentrations selected for the definitive tests covered the response range that included thelowest concentration that reduced viability by 50% (see Section 2.6.3.2).
2.3.2.2 Definitive Test
The concentration-response determination is referred to as the definitive test because it isused to determine the IC50 value of the substance being tested. The concentration closest to
the calculated IC50 value in the range finder test served as the midpoint of the eight
concentrations tested in the definitive test. In the absence of other information (e.g.,knowledge of the slope of the toxic response), the recommended dilution factor was 1.47
(6√10), which divides a log interval into six equidistant steps (e.g., 10, 14.7, 21.5, 31.6, 46.4,
68.1, 100). The Guidance Document considered a progression factor of 1.21 (12√10) to be the
A biphasic dose-response curve is a dose-response in which cytotoxicity increases (as dose increases),plateaus, and then increases again.
3

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smallest factor practically achievable, and this was the lowest required concentration interval.
The PC was tested similarly to the reference substances in the definitive test and the same
recommended dilution factors were used (dilution factor at the discretion of the Study
Director).
A definitive test was considered successful if it met all of the test acceptance criteria outlinedin the NRU protocols. Definitive tests were repeated as per the protocols if the test failed tomeet all of the test acceptance criteria. Section 2.5 addresses the basis for replicate testing.
If minimal or no cytotoxicity was observed in the range finder test, the maximum
concentration for the definitive test was determined as follows:• For Substances Prepared in NHK or 3T3 Medium: A review of the RC
chemicals used in this validation study showed that, among water-soluble
chemicals, glycerol had the highest reported IC50 value (57 mg/mL). To
capture this value, and that of other relatively non-toxic chemicals, the highest
concentration of a substance applied to the cells in the definitive test waseither 100 mg/mL (using 200 mg/mL 2X stock) or the maximum soluble dose
if the substance was not soluble at that concentration.
• For Substances Prepared in DMSO: Based on the maximum concentration of
DMSO that could be added to culture medium without causing cytotoxicity(i.e., 0.5%), the highest concentration of a substance that could be applied tothe cells in the definitive test was 2.5 mg/mL. In the event that the reference
substance was not soluble at this concentration, the highest soluble
concentration was used.
2.3.3

NRU Endpoints Measured

2.3.3.1 NRU and Measurement
After cells were exposed to the reference substance or the controls (PC; VC) for 48 hours,
they were washed and incubated with the NR dye at 37 ºC for an additional three hours. The
dye was eluted from the cells using a desorb solution and the OD of the resulting solutions
were measured using a spectrophotometric microtiter plate reader. Because NR is absorbed
by healthy cells, the amount of dye eluted, as measured by the spectrophotometer, is
proportional to NRU and thus to the number of live cells present at culture termination. The
OD data from the spectrophotometer were recorded on the EXCEL® template. Relative cell
viability for each reference substance and the PC was determined using six replicate wells(six wells [minimum of four scorable] in the 96-well plate) per concentration. Cells treatedwith the VC were considered to have 100% cell viability (i.e., the mean OD of the VC wells= 100% viability). Cell viability in other test wells was computed in reference to the meanVC OD value (i.e., [well OD/mean VC OD] x 100 = % viability).
2.3.3.2 Determination of IC50, IC20, and IC80 Values
IC50 values were determined from the concentration-response curve using a Hill function,
which is a four parameter logistic mathematical model relating the concentration of a
substance to the response (typically following a sigmoidal shape). Modifications to the Hillfunction used in later phases of the study are described in Section 2.6.3.

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Data from the EXCEL® template were transferred to a template designed by the SMT for
GraphPad PRISM® 3.0, a commercially available statistical software (GraphPad Software,Inc., San Diego, CA, USA – hereafter known as PRISM® template). The PRISM® templateused the Hill function to calculate the IC50, IC20, and IC80 concentrations, reported as µg/mLof reference substance in solution. IC20 and IC80 data were collected for potential use indesigning a prediction model for estimating human lethal blood concentrations.
2.3.4
Duration of Reference Substance Exposure
The SMT and laboratory representatives reevaluated the reference substance exposure
duration recommended in the Guidance Document (ICCVAM 2001b) before initiating thestudy. The Guidance Document recommended an exposure of 24 hours for the 3T3 cells and48 hours for the NHK cells. However, Riddell et al. (1986) showed large differences incytotoxicity for 3T3 cells in response to some chemicals, depending on whether the exposure
duration was 24 or 72 hours. Although the toxicity induced by substances that damage, forexample, cell membranes is likely to be observed in a relatively shorter time, the toxic effectsof substances that interfere with cell functions/processes specifically relating to DNA
replication (e.g., protein and nucleic acid synthesis) and cell division (e.g., mitotic spindle
formation) are more pronounced after longer exposure periods. This occurs because cells areaffected only at certain phases of the cell cycle.
IIVS conducted studies to evaluate the effect of exposure durations of 24, 48, and 72 hours
and of 48 and 72 hours on the sensitivity of 3T3 cells and NHK, respectively, to six
chemicals selected from the list in Riddell (1986). Because the closest fit to the RC millimole
regression occurred when a 48-hour exposure duration was used, this exposure duration was
selected for use with both cell types in the validation study (Curren et al. 2003) (seeAppendix E).
2.3.5

Known Limits of Use

2.3.5.1 Solubility/Precipitation/Volatility
In vitro test methods cannot be used for substances that cannot be dissolved in media,
DMSO, or ETOH at a sufficiently high concentration to induce cytotoxicity in excess of
50%. Also, chemicals that are unstable or exothermic in water cannot be adequately tested
with these in vitro test methods (as well as in vivo methods).
Precipitation of a test substance in the dosing solution or in the culture medium after thesubstance to be tested has been added can affect the concentration-response and thus reduce
the accuracy of the calculated IC50. Some reference substances used in the validation study
had precipitates in their medium/DMSO 2X concentrations prior to dilution for application to
the test wells. Precipitates were also observed for some substances in a number of test wells
after addition of the media/DMSO 1X solutions (see Section 5.8 and Table 5-11) to thecultures and/or at the end of the exposure period.
Volatility was detected for a number of reference substances during the range finder tests byobservance of cross contamination (i.e., high cytotoxicity) in VC wells. Plate sealers wereused during the definitive tests to control volatility (see Section 2.6.3 – Testing Volatile
Reference Substances), and could be used during the range finder tests if the Study Director
suspected that the reference substance might be volatile. The use of plate sealers required
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additional laboratory training, and some volatile substances were difficult to test even withthe use of plate sealers. Furthermore, some test substances (e.g., organic solvents) may reactchemically with the plastic in the sealers.
2.3.5.2 Biokinetic Determinations
The Workshop 2000 report (ICCVAM 2001a) discussed the role of chemical biokinetics invivo vis-a-vis acute toxicity, as illustrated in the following quote:“Results obtained from in vitro studies in general are often not directly
applicable to the in vivo situation. One of the most obvious differences
between the situation in vitro and in vivo is the absence of processes
regarding absorption, distribution, metabolism and excretion (i.e.,
biokinetics) that govern the exposure of the target tissue in the intact
organism. The concentrations to which in vitro systems are exposed may notcorrespond to the actual situation at the target tissue after in vivo exposure. In
addition, the occurrence of metabolic activation and/or saturation of specificmetabolic pathways or absorption and elimination mechanisms may also
become relevant for the toxicity of a compound in vivo. This may lead tomisinterpretation of in vitro data if such information is not taken into
account. Therefore, predictive studies on biological activity of compounds
require the integration of data on the mechanisms of action with data on
biokinetic behavior.”
The 3T3 and NHK NRU test methods do not account for biokinetics.
2.3.5.3 Organ-Specific Toxicity
The Workshop 2000 report also addressed concerns about the in vitro prediction of organspecific toxicity, and identified the organ systems for which failure after acute exposure
could lead to lethality (i.e., liver, central nervous system, kidney, heart, lung, andhematopoietic system) (ICCVAM 2001a). Each organ system was reviewed individually.
Although the 3T3 and NHK NRU test methods do not assess organ-specific toxicity, they
may be useful in a test method battery such as that proposed by the Workshop 2000
participants (see Section 2.3.5.4).
2.3.5.4

The Role of Cytotoxicity Tests in an In Vitro Battery Approach for Possible
Replacement of In Vivo Acute Toxicity Testing
A five-step in vitro testing scheme was proposed for a test battery that may eventually bedemonstrated to be an adequate replacement for rodent acute oral toxicity test methods forregulatory purposes (ICCVAM 2001a).
Step 1: Perform a physico-chemical characterization and biokinetic modeling.
Step 2: Evaluate basal cytotoxicity using, for example, the 3T3 or NHK NRU test
methods.
Step 3: Evaluate the potential that metabolism will mediate the basal cytotoxicity
effect.
Step 4: Assess the test substance’s effect on energy metabolism.
Step 5: Assess the ability of the test substance to disrupt epithelial cell barrier
function.

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The Workshop 2000 participants suggested that implementation of the 5-step testing scheme
would require the following:• Identification of the most appropriate cell culture systems to use based on
accuracy, reproducibility, cost, and availability• Development of a standardized protocol for each test method used in each of
the five steps, and validation of each test method using that protocol• Development of prediction models for the relevant human toxic levels
required by regulatory agencies• Evaluation of the test battery using substances that are appropriate for all
endpoints, and then test sufficient substances to develop a prediction model• Validation of the entire testing scheme and the prediction model
2.3.6
Basis of the Response Assessed
Neutral red is a weakly cationic, water-soluble, supravital dye that stains living cells byreadily diffusing through the cell membranes and concentrating in lysosomes. The intensityof the dye desorbed from the cells in a culture is directly proportional to the number of living
cells. Cell death and/or growth inhibition decreases the amount of neutral red taken up by theculture (see Section 1.3.1).
2.3.7

Appropriate Positive, Vehicle, and Negative Controls

2.3.7.1 Positive Control
The Guidance Document recommended sodium lauryl sulfate (SLS; Chemical Abstracts
Service Registry Number [CASRN] 151-21-3) as an appropriate PC for in vitro cytotoxicity
test methods (ICCVAM 2001b), and historical data are available (e.g., Spielmann et al.
1991). A PC test plate was included with every 3T3 and NHK NRU test method assay andwas treated the same as any reference substance assay plate.
The historical mean PC IC50, standard deviation (SD), and acceptance limits, were
determined separately for each laboratory (see Table 5-3), based on their individual
historical databases (see Figure 1-2). The acceptable range for the PC IC50 was based on thestatistical approach recommended in the Guidance Document. In Phase Ib, the IC50 limits
accepted for the PC tests were within two SD of the historical mean PC IC50 value. In the
Phase II studies, the IC50 limits for PC tests were within 2.5 SD of the historical mean value(i.e., from Phases Ia and Ib). In Phase III, the IC50 limits used for the PC were within 2.5
standard deviations of the mean PC IC50 from Phases I and II. The exception to this was the
FAL NHK data, where only the Phase II data were used as the basis for establishing theacceptable PC range. The SLS data produced by FAL during Phase I was not used insubsequent historical database compilations because FAL used a modified cell culture
protocol in Phase II (see Section 2.6.2.6).
2.3.7.2 Vehicle Control
The VC consisted of complete DMEM (see Appendix B1) for 3T3 cells and complete
growth medium (Clonetics® KBM® with supplements [see Appendix B2]) for NHK cells
when the reference substances were dissolved in culture medium. For reference substancesdissolved in DMSO, the VC consisted of medium with the same amount of DMSO (0.5%[v/v]) as was applied to the 96-well test plate.

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2.3.7.3 Negative Control
Negative control cultures (i.e., those that were not exposed to the solvent) were not used inthis validation study. Neither DMSO, at the concentration used, nor the culture medium
affected the performance of the 3T3 and NHK NRU test methods.
2.3.8
Acceptable Ranges of Control Responses
The Guidance Document established an absolute value (i.e., uncorrected for blankabsorbance) range of the OD540 for the VC to indicate whether the cells seeded in the 96-wellplate had grown with a normal doubling time during the assay. A mean OD540 ≥0.3 wasrecommended as the acceptable range of VC responses and was made a test acceptancecriterion for both cell types at the start of the study. However, prior to Phase II, this was
rescinded as a test acceptance criterion. The protocols for Phases II and III provide a range of
OD values for use as guidance in future studies with these test methods (Table 2-1).
Table 2-1
Laboratory

Measured VC OD540 Values1 and Targets
Phase Ia

Phase Ib

Phase II

Phase III

3T3 NRU Test Method
Target Range2

0.3≤ OD ≤1.1

0.30≤ OD ≤0.80

0.103≤ OD ≤0.813

0.103≤ OD ≤0.813

ECBC

0.326 – 0.457

0.214 – 0.839

0.217 – 0.730

0.191 – 0.797

FAL

0.490 – 0.780

0.247 – 0.742

0.289 – 0.768

0.126 – 1.161

IIVS

0.336 – 0.538

0.319 – 0.598

0.307 – 0.578

0.256 – 0.544

NHK NRU Test Method
Target Range2

0.3≤ OD ≤1.1

0.60≤ OD ≤1.70

0.35 ≤ OD ≤ 1.50

0.205 ≤ OD ≤ 1.645

ECBC

0.863 – 2.312

0.788 – 1.282

0.139 – 1.175

0.114 – 1.344

FAL

0.484 – 1.698

0.146 – 1.706

0.110 – 1.292

0.183 – 1.347

IIVS

0.550 – 1.883

0.487 – 1.001

0.201 – 0.841

0.430 – 0.834

Abbreviations: VC=Vehicle control; OD540=Optical density at 540 nM; 3T3=BALB/c 3T3 fibroblasts; NRU=Neutral red
uptake; NHK=Normal human epidermal keratinocytes; ECBC=Edgewood Chemical Biological Center; FAL=Fund for
Replacement of Animals in Medical Experiments Alternatives Laboratory; IIVS=Institute for In Vitro Sciences.
1
Lowest to highest OD values for tests that meet test acceptance criteria.
2Ranges used for all laboratories. Ranges for Phases Ia and Ib were test acceptance criteria. Ranges for Phases II and III
were used as target ranges, rather than as test acceptance criteria.

In Phase III, 99.5% (914/919) of all 3T3 mean VC OD values and 97% (913/944) of all NHK
mean VC OD values were within the target ranges. Most OD values outside the ranges werefrom range finding tests and were usually the result of volatile reference substances affecting
the VC cells adjacent to the highest reference substance concentration wells.
The VC OD values had a tendency to be lower in Phases II and III as compared to Phases Ia
and Ib. Protocol revisions made throughout Phases Ia, Ib, and II (as listed below) most likely
contributed to the differences in the OD values. Possible explanations for changes in OD
values for the 3T3 cells include:• Some tests in Phases Ia and Ib exhibited NR crystals that caused higher OD
readings.
• Cell seeding densities were revised from 2.5 x 103 cells/well to a range of 2.0– 3.0 x 103 cells/well.

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Possible explanations for changes in OD values for the NHK cells include:• The minimum percent confluence of cells necessary before the reference
substance could be applied was reduced from 30% to 20% confluence.
• Cell growth was reduced in some tests in the later study phases as a result of
medium and supplement issues (e.g., certain lots of basal medium andmedium supplements for NHK cells did not provide optimum growth
conditions for the keratinocytes).
2.3.8.1 Vehicle Controls as a Quality Control Tool
To check for systematic cell seeding errors and reference substance volatility, VCs wereplaced both at the left side (row 2) and the right side (row 11) of the 96-well plate (seeFigure 1 in Appendix B1). Volatile reference substances generally affected the left side VC,
which was next to the highest reference substance concentration in the 96-well plate. The test
acceptance criterion for the VC was that the means for the left and the right set of VCs had tobe within 15% of the mean of all VCs. This criterion, which was adopted from the protocols
in the Guidance Document (ICCVAM 2001b), was used for reference substances and the PC
in all phases of the validation study.
2.3.9
Nature of Experimental Data Collected
Each laboratory maintained a study workbook to document all aspects of the study andincluded the raw data for all steps of each assay (e.g., cell growth, test substance treatment,
weighing and dilution of reference substances), as well as for all solubility studies.
2.3.9.1 NRU OD Measurements
At the conclusion of the NRU desorb step, the OD of the resulting colored solution in eachwell of the 96-well plates was measured at 540 ±10 nm in a spectrophotometric microtiterplate reader. Each laboratory followed its in-house Standard Operating Procedure (SOP) for
use of the microplate readers. These SOPs included instructions for operation and calibrationof the instruments. Critical specifications such as alignment, accuracy, reproducibility, andlinearity were included as standard parameters for review and routine calibration. Raw OD
data from the plate reader was electronically transferred to the EXCEL® template. The
template converted the raw data from each treatment well (six wells/reference substanceconcentration) to derived data by subtracting the mean blank OD value (two blankwells/reference substance concentration) from each reference substance well OD. There were
12 VC wells and 20 associated blank wells. The corrected VC OD values were used to
calculate the mean VC OD, which was then used to calculate relative viability (% of meanVC OD) in each test well for the reference substance or PC. The percent viability values
were then transferred to the PRISM® template to calculate the IC20, IC50, and IC80 values.
2.3.9.2 Information and Data Collected
Originals of the raw data (i.e., the Study Workbook and computer printouts of absorbancereadings from the plate reader) and copies of other raw data, such as instrument logs, werecollected and archived under the direction of the Study Director according to Good
Laboratory Practice (GLP)-compliant procedures.
The Study Director/technicians entered the following information into the EXCEL® template:•Testing identification for: test facility, chemical code, study number, 96-wellplate number, experiment number

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•

•
•
•
•
•

•

•

November 2006

Reference substance preparation: solvent used, solvent concentration in
dosing solutions, highest stock concentration, dilution factor, pH of 2X dosing
solutions, medium clarity/color, presence/absence of precipitate in 2X
solutions, PC concentration range
Cell line/type: cell supplier, lot number, cryopreserved passage number,passage number in assay
Cell culture conditions: medium, supplements, suppliers and lot numbers,
serum concentrations
Timeline: dates of cell seeding, dose application, OD540 determination
Raw data: OD values from each well from the microtiter plate reader
Test results: mean corrected OD540 value, Hill function R2 value, logs of IC20,
IC50, and IC80 (PRISM® template presents data as logs of the ICx; EXCEL®
converts values to µg/mL)
Test acceptance criteria: acceptable number of values on each side of the IC50
(i.e., number of points >0 and ≤50% viability, and >50 and <100% viability),
acceptable percent difference for the VCs, acceptable Hill function R2 value(coefficient of determination) and calculated IC50 concentration for the PC
Visual observations: protocol codes for cell culture conditions for all reference
substance concentrations (i.e., relative level of cell cytotoxicity, cellmorphology, presence of precipitate)

2.3.10
Data Storage Media
Raw and derived data from the NRU tests were saved in the EXCEL® template file format
provided by the SMT. All EXCEL® and PRISM® files were copied and transferred tocompact disks. NICEATM and the laboratories printed copies of all data sheets (stored at
NICEATM and at the testing facilities), and included copies in the laboratories’ final reports.
2.3.11
Measures of Variability
Each 96-well plate used in the NRU tests had three main measures of variability.
1) Each plate contained VCs on each end of the plate (columns 2 and 11) (seeFigure 1 in Appendix B1 for plate map). The difference between the mean
NRU OD for each VC column and mean of the pooled VC wells was used as atest acceptance criterion. The Study Director rejected the test if the difference
was greater than 15%, which indicated cross-contamination from a volatile
substance or possible cell seeding errors.
2) A mean relative viability was determined for each concentration of thesubstance tested along with the SD and coefficient of variation(%CV=SD/mean x 100).
3) Macros were included in the EXCEL® template to perform an outlier test
(Dixon and Massey 1981) on the data for the six replicate wells for eachconcentration. Outliers (i.e., individual well values that exceeded the 99%
confidence interval [CI] for the replicate wells) were highlighted and could be
excluded from the resulting analysis to improve curve fit. The Study Director
made the decision as to whether or not to remove outliers and provided ajustification for the decision.

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November 2006

Other test-to-test measures of variability were considered in this study.
• Each set of assays for reference substances included a PC plate. If the SLS PC
test did not meet test acceptance criteria, then the tests for the associatedreference substances were rejected. The SMT recommended testing amanageable number of definitive test plates (e.g., 4 to 6) with each PC to limit
the number of definitive NRU tests rejected for PC failure. In this validation
study, 4.2% of all definitive tests performed were rejected because the PC
failed (i.e., the PC IC50 was outside the acceptable confidence limits).
• SDs and CVs were determined for mean IC50 values from replicate tests.
Replicate testing included three definitive tests for each reference substance,each performed on a different day.
2.3.12
Methods for Analyzing NRU Data
Relative cell viability for each reference substance concentration was calculated using theODs of the six replicate values (minimum of four acceptable replicate wells) per test
concentration. Relative cell viability was expressed as a percentage of the mean VC OD.
Absolute OD data from the microtiter plate reader was transferred to the EXCEL® templatefor performance of these calculations. Where possible, the concentration range (eight
concentrations) tested for each reference substance ranged from no effect to 100% toxicity.
The IC20, IC50, and IC80 values were determined from the concentration-response curve using
the PRISM® template and applying a Hill function to the % viability data. The IC20 and IC80
values were calculated for potential use in the development of a human prediction model(reported elsewhere).
2.3.13
Decision Criteria for Classification of Reference Substances
The 3T3 and NHK NRU test methods will not be used to classify reference substances inhazard categories but rather to aid in setting the starting dose for sequential rodent acute oral
toxicity test methods (i.e., the UDP and ATC) (see Section 10 for an analysis of theestimated animal savings). The RC millimole regression procedure was used to predict arodent LD50 value from an NRU IC50 value. Section 6.3 addresses the accuracy of the 3T3
and NHK NRU test methods for predicting GHS hazard categories when used with IC50-LD50
regressions, calculated using a subset of the RC data (i.e., substances with rat oral LD50 data).
2.3.14
Information and Data Included in the Test Report
Test and Control Substances
With the exception of the PC, the laboratories tested coded substances and had
minimal information about the test substances’ properties (see Section 3.3 for thereference substance information provided to the laboratories). The following
describes the test and test substance information that should be included in an NRU
test method report.
• Chemical name(s) and synonyms, if known• The CASRN, if known• Formula weight, if known• Purity and composition of the substance or preparation (in percentage[s] byweight)• Physicochemical properties (e.g., physical state, volatility, pH, stability,
chemical class, water solubility)
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November 2006

Solubilization of the test/control substances (e.g., vortexing, sonication,
warming, grinding) prior to testing, if applicable
Information Concerning the Sponsor and the Test Facility• Name and address of the sponsor, test facilities, study director, andparticipating laboratory technicians• Justification of the test method and specific protocol used
Test Method Integrity• The procedure used to ensure the integrity (i.e., accuracy and reliability) of thetest method over time (e.g., use of the PC data)
Criteria for an Acceptable Test
• Acceptable VC differences between each column of wells and the mean ofboth columns• Acceptable concurrent PC ranges based on historical data (include thesummary historical data)• Number of toxic points on either side of the IC50 (i.e., number of points >0and ≤50% viability and >50 and <100% viability)
Test Conditions• Experiment start and completion dates• Details of test procedures used
• Test concentration(s) used and how they were derived• Cell type used and source of cells• Description of modifications made to the test procedure• Reference to historical data of the test model (e.g., solvent and PCs)
• Description of the evaluation criteria used
Results• Tabulation of data from individual test samples (e.g., IC50 values for thereference substance and the PC, absolute and derived OD readings, reported intabular form, including data from replicate repeat experiments as appropriate,
and the means and standard deviations for each experiment)
Description of Other Effects Observed• Cell morphology, precipitate, NR crystals, etc.
Discussion of the Results
Conclusion
Quality Assurance (QA) Statement for GLP-Compliant Studies• A statement describing all inspections and other QA activities during thestudy, and the dates results were reported to the Study Director. This
statement will also serve to confirm that the final report reflects the raw data.
•

During the validation study, the GLP-compliant laboratories, IIVS and ECBC, followed
additional reporting requirements provided in the relevant GLP guidelines (e.g., OECD 1998;
EPA 2003a, b; FDA 2003).
The SMT and laboratories developed standard forms for data collection (i.e., EXCEL® and
PRISM® templates). The solubility test form was derived from a standard form provided by
IIVS. The EXCEL® template was an adaptation of a template format presented in the
Guidance Document (ICCVAM 2001b).

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2.4

November 2006

Proprietary Components of the In Vitro NRU Test Methods

The only proprietary components used in these test methods are the NHK cells and the NHK
basal culture medium obtained from CAMBREX Clonetics®. All other components arereadily available through various scientific product suppliers.
Section 2.3.1.2 describes the NHK cells used in the study and provides the only commercial
source. All laboratories throughout the entire study used cells from the same lot. Procedures
used to verify the integrity of the NHK cells included comparison of positive control data
across laboratories and observations of cell growth throughout the study. If a laboratory
reported a problem with the cells, the SMT and Study Directors evaluated the testing
parameters to decide if the problem was cell-oriented or if other factors influenced the
problem. Section 2.6.3.5 provides information concerning the resolution of cell-related issues
and revisions made to the protocols to address such difficulties.
Section 2.10.1.1 and Appendices B2 and B4 provide information about the NHK growth
medium, supplements, and commercial source. Problems arose with the keratinocyte growth
medium during the study and resolutions and outcomes are addressed in Sections 2.6.3.5,
2.6.3.6, 5.3.4, and 11.1.2.2.
Although this study used proprietary components for the NHK NRU test method, cells andmedium from the commercial source used in the study are not required for implementation ofthis test method.
2.5

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

The study protocols required each laboratory to test each coded reference substance in atleast one range finding test using a log dilution factor, and in at least three definitive tests on
three different days using a smaller dilution factor than used in the range finding test. Assays
were performed over a number of days to evaluate day-to-day variation. Laboratories tested
each coded reference substance until three definitive tests met the test acceptance criteria.
Additional testing was often dictated by:• Chemical issues (low toxicity, volatility, insolubility, and precipitation)• PC failure• Technical difficulties such as NR crystal formation
A stopping rule for insoluble reference substances was incorporated into the protocols for
Phase III to limit the number of retests (see Appendices B1 and B2):
“If the most rigorous solubility procedures have been performed and theassay cannot achieve adequate toxicity to meet the test acceptance criteria
after three definitive tests, then the Study Director may end all testing forthat particular chemical.”
2.6

Basis for Modifications to the 3T3 and NHK NRU Test Method Protocols

2.6.1
Phase Ia: Laboratory Evaluation Phase
All protocol revisions were implemented during Phase Ia unless otherwise stated.

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2.6.1.1 NR Dye Crystals
NR dye crystals formed in the 96-well test plates when used at 50 µg/mL (OD values
measured in the blanks increased from ~ 0.05 to 0.10) in both NRU test procedures.
Troubleshooting efforts included incubating the NR medium overnight; centrifuging andfiltering the NR medium prior to application to the 96-well plates; and reducing theconcentration of NR dye. The laboratories performed tests using a reduced NR concentrationof 33 µg/mL. Since there were no quantitative differences in results between tests with 50µg/mL and tests with 33 µg/mL NR, the SMT accepted tests with both concentrations.
Protocol Revision: The NR dye concentration was reduced to 33 µg/mL for both cell types insubsequent test Phases.
2.6.1.2 3T3 Cell Growth
The growth rate of 3T3 cells (as determined by monolayer confluence) was slower than
expected. As a result, the cells required more time in culture to obtain the proper density after
seeding.
Protocol Revision: The 3T3 cells must be passaged 2-3 times after thawing before being used
for the test. The protocol also emphasized attainment of the appropriate percentage of cell
confluence (not more than 50% for 3T3 cells) required at the time the cells were exposed to
the reference substance, rather than using the time in culture as the guide.
2.6.1.3 NHK Cell Growth
The NHK cells had an additional growth problem that manifested as a ring of dead/dying
cells around the center of the wells. Troubleshooting efforts included evaluating various
brands of 96-well plates (laboratories were not required to use the same brand of plates) andeliminating the change of medium prior to reference substance treatment. All laboratories
participated in evaluating the effect of changing (i.e., refeeding) or not changing (i.e., norefeeding) the medium by performing a small study with the PC (SLS). Tests were
performed: 1) after refeeding the cells with fresh medium, and 2) by adding SLS to themedium already on the cells. Control ODs were generally higher in the tests in which themedium was not replenished, but sensitivity to SLS was generally unchanged (see Table 22). FAL was experiencing difficulties in NHK cell growth at this stage of the study which
may account for the difference in the refeeding and no refeeding SLS IC50 values. The SMT
accepted tests with refeeding and those without refeeding (for Phase Ia) as long as they met
the test acceptance criteria.
IIVS presented detailed information on the ring of dead cells issue (Raabe 2004). The
laboratory showed that the ring of cell death coincided with the formation of a meniscus
resulting from the residual medium left in the well after removal of the spent medium. The
problem was resolved by eliminating the removal of medium before applying test chemicalrather than requiring a standard brand of 96-well plates.
Protocol Revision: Step 2 of the NHK NRU test method was eliminated (change of medium
prior to addition of reference substance). The volume of medium (with cells) was changed
from 250 µL/well to 125 µL/well.

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Table 2-2

November 2006

Refeeding/No Refeeding Data for the NHK NRU Test Method
RefeedECBC
No Refeed

IIVS
RefeedNo Refeed

RefeedFAL
No Refeed

Number of Test Plates

4

4

6

6

2

4

Absolute OD1 for VC

0.265±0.151

0.621±0.3220.885±0.0571.12±0.0331.41±0.1271.24±0.430OD1 for SLS IC50

0.102
±0.0790.282±0.1650.415±0.0290.533±0.0170.696±0.0650.606±0.217SLS IC50 (µg/mL)1

3.33
±0.47

3.23
±0.61

3.41
±0.58

3.49
±0.39

6.21
±0.88

8.14
±0.40

Abbreviations: NHK=Normal human epidermal keratinocyte; NRU=Neutral red uptake; ECBC=Edgewood Chemical
Biological Center; IIVS=Institute for In Vitro Sciences; FAL=Fund for the Replacement of Animals in Medical Experiments
Alternatives Laboratory; VC=Vehicle control; OD=Optical density; SLS=Sodium lauryl sulfate.
Note: OD values for SLS IC50 were extrapolated from the concentration-response curve data
1
Mean ± standard deviation (uncorrected for blank absorbance

FAL, in contrast to the other two laboratories, used 80 cm2 culture flasks for culturing thethawed cells from the ampules of cryogenically-preserved pool of cells and encountereddifficulties in obtaining a satisfactory number of adhering NHKs.
Protocol Revision (FAL only): Culture flasks were coated with fibronectin-collagen to
promote cell adherence.
2.6.1.4 Vehicle Control OD Limits
In Phase I, the acceptable range of VC OD values designated in the protocols (0.3 ≤ OD
≤1.1) were frequently unattainable in both test methods. Despite this, the Study Directors
reported that the cells were adequately responsive. The SMT withdrew the VC OD limits as atest acceptance criterion.
Protocol Revision for Phase Ib: OD ranges were provided as guidelines for each cell typebased on OD data from all laboratories, a review of the concentration-response data, and theability of each test to pass the other test acceptance criteria. Each laboratory developed its
own VC OD acceptability range based on its historical data.
2.6.1.5 Precipitate Formation
During solubility testing, it was observed that some substances, when tested at the same
concentrations, precipitated in the 3T3 medium but not in the NHK medium. When a liquid
reference substance (i.e., 2-propanol) produced this effect, the precipitate was attributed tothe protein in the serum in the 3T3 medium rather than insolubility.
Protocol Revision: The reference substances were dissolved in 3T3 medium without NCS to
make the 2X solutions. The dissolved 2X reference substance was added to medium
containing 10% NCS to reach the final 5% NCS and 1X reference substance concentrations.
2.6.1.6 Dilution Factor
After a range finder test was performed, the definitive tests were to be performed using a6√10=1.47 dilution scheme centered on the IC50 that was calculated from the range finder. In
Phase Ia, the Study Directors, for various reasons related to the specific substance being
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November 2006

tested, sometimes deviated from this requirement and used other dilution factors. The SMT
agreed that the dilution factor requirements should be modified to allow more flexibility insetting up tests. The SMT accepted data generated using dilution factors other than therecommended 1.47 for definitive tests if all other test acceptance criteria were met. The useof smaller dilution factors generally increased the number of concentrations in the 10% to
90% viability range, which improved the precision of the IC50 calculation.
Protocol Revision: The 6√10=1.47 dilution scheme was a suggested starting range, rather
than a specific test acceptance criterion in subsequent test Phases.
2.6.1.7 Test Acceptance Criteria
The test acceptance criteria at the beginning of Phase Ia were:• The IC50 for SLS had to be within the 95% CI of the historical PC mean
established by the Test Facility (rescinded after commencement of Phase Ia)• The OD540 of the VCs (with blank subtracted) had to be ≥0.3 and ≤1.1
(rescinded after commencement of Phase Ia)• Mean OD values of the left and right VCs (columns 2 and 11 in the 96-welltest plate) must not differ by more than 15% from the mean of all VC OD
values• At least two cytotoxicity values, one on either side of the IC50 but between
10% and 90% viability, needed to be present (added after commencement ofPhase Ia)
• The Hill function curve fits (R2 >0.9 or 0.8 < R2 <0.9) were evaluated on acase by case basis for acceptability by the SMT (added after commencementof Phase Ia).
2.6.2
Phase Ib: Laboratory Evaluation Phase
All protocol revisions developed during Phase Ia were implemented during Phase Ib unlessotherwise stated.
2.6.2.1 NR Crystal Formation
FAL and ECBC routinely observed NR crystals forming in the 96-well test plates in the 3T3
NRU tests when 33 µg/mL NR was used. All laboratories tested 25 and 33 µg/mL NR
concentrations and 2- and 3-hour NR incubation periods to determine which NR
concentration and incubation period would provide optimal NRU measurements without
crystal formation. In addition to determining whether NRU had reached a plateau at theseconcentrations and incubation times, the laboratories also determined whether the response to
SLS differed under these conditions. Crystals were observed only at 33 µg/mL NR whenpresent for three hours. Figure 2-2 shows that the average OD results were similar for all NR
concentrations and incubation periods tested. Figure 2-3 shows that the SLS IC50 values
were equivalent at the different NR concentrations and incubation periods. To minimizechanges to the 3T3 protocol, the NRU concentration was lowered from 33 to 25 µg/mL,
while the NR incubation period was maintained at three hours. The NR concentration and the
incubation period for the NHK NRU test method remained at 33 µg/mL and three hours,
respectively.
Protocol Revision for Phase II: The NR concentration for the 3T3 NRU test method was
reduced to 25 µg/mL for the three-hour incubation period. Revised methods for preparation
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In Vitro Cytotoxicity Test Methods BRD Section 2

November 2006of the NR dye solution included filtration of the solution, maintenance of the solution at 37ºC prior to application to the cells, and application of the NR solution to the cells within 15
minutes after removing it from 37 ºC. Also, cells were observed during the NR incubation
period to monitor possible crystal formation.
2.6.2.2 Heating of Reference Substance Solutions
The laboratories had difficulty solubilizing arsenic trioxide, one of the reference substancesused in Phase Ib. Heating and mechanical applications for increasing the laboratory’s ability
to solubilize substances into culture medium were reviewed and revised.
Protocol Revision for Phase II: The duration range for heating a stock solution at 37 ºC (ifheating is needed) was increased from 5 to 10 minutes to 5 to 60 minutes.
Figure 2-2

3T3 NRU OD for SLS as a Function of NR Concentration and Duration

Abbreviations: OD=Optical density; NR=Neutral red; SLS=Sodium lauryl sulfate; 3T3=BALB/c 3T3
fibroblasts; NRU=Neutral red uptake; h=Hours.
Note: Error bars are one standard deviation.

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In Vitro Cytotoxicity Test Methods BRD Section 2

Figure 2-3

November 2006

SLS IC50 Values for Each NR Concentration and Incubation Duration(3T3 NRU)

Abbreviations: SLS=Sodium lauryl sulfate; IC50=Test substance concentration that reduces cell viability by
50%; NR=Neutral red; 3T3=BALB/c 3T3 fibroblasts; NRU=Neutral red uptake; ECBC=Edgewood Chemical
Biological Center; IIVS=Institute for In Vitro Sciences; FAL=Fund for the Replacement of Animals in Medical
Experiments Alternatives Laboratory.
Note: SLS range is mean IC50 value ± one standard deviation.

Protocol Revision for Phase II: The duration range for heating a stock solution at 37 ºC (ifheating is needed) was increased from 5 to 10 minutes to 5 to 60 minutes.
2.6.2.3 Growth of Untreated Cells
VC OD values were frequently lower than specified in the Phase I acceptance criteria. Phases
Ia and Ib incorporated the acceptance limits shown in Table 2-1 for the VC, but the limits
were rescinded as test acceptance criteria for Phase II because the laboratories frequently
failed to meet them even though cell growth and responsiveness to SLS was adequate.
Protocol Revision for Phase II: The specified VC OD range was eliminated as a test
acceptance criterion. The OD data (all laboratories combined) from the VCs for both cell
types was used to calculate OD ranges that would serve as guidelines for other tests (seeSection 2.2.9).
2.6.2.4 Correction of Reference Substance OD Values
Each reference substance concentration was applied to six treatment wells and to two cellfree wells (i.e., blank wells) used to generate the background OD540 values to adjust forpotential interference with the NR dye. The mean blank well OD (absolute OD) for eachreference concentration was subtracted from the reference substance concentration ODs toprovide the corrected OD for each replicate well.

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November 2006

2.6.2.5 Laboratory Error Rates
The SMT determined the Phase 1b error rates (number of tests with errors/total number oftests conducted) for each laboratory (Table 2-3) and compiled a list of the types of errors
encountered. The vast majority of errors were transcriptional and typographical errors in thedata sheets provided to the SMT.
Table 2-3

Error Rates1 in Phase Ib by Laboratory and Test
NRU Test Method

Laboratory
3T3

NHK

ECBC

1/9 (10%)

4/17 (23%)

FAL

42/45 (93%)

12/29 (41%)

IIVS

1/20 (5%)

1/20 (5%)

Abbreviations: NRU=Neutral red uptake; 3T3=BALB/c 3T3 fibroblasts; NHK=Normal human epidermal keratinocytes;
ECBC=Edgewood Chemical Biological Center; FAL=Fund for the Replacement of Animals In Medical Experiments
Alternatives Laboratory; IIVS=Institute for In Vitro Sciences
Note: Most errors were transcriptional and typographical and not technical.
1
Number of tests with errors/total number of tests (some data files had more than one error)

2.6.2.6 Resultant Protocol Changes for Phase II
Following the completion of Phases Ia and Ib, IIVS sponsored a weeklong laboratory training
exercise for the cytotoxicity testing laboratories to help standardize the level of training
among the technical staff and to identify any further 3T3 and NHK NRU protocol revisions
that might be needed. Protocol revisions made because of this exercise included:• Multi-channel repeater pipettes can be used for dispensing cells into the 96well plates and dispensing plate rinse solutions, NR medium, and desorb
solution but are not accurate enough to dispense the PC or the reference
substances to the treatment wells.
• Use of 8-channel reservoirs for applying dosing solutions to the wells so thatmulti-channel single delivery pipettes could be used
• Use of a standardized length of time that the HBSS rinse remains on the cellmonolayers in flasks during the cell subculture step
• Protection of plates from light during the shaking step for NR extraction; alllaboratories will cover plates with a light-impermeable barrier (e.g., aluminumfoil) during this step
• Allow plates to stand for at least five minutes after the shaking step iscomplete and eliminate any bubbles in media observed in the wells before
measuring the OD• Change the allowable seeding density range for 3T3 NRU test method from
2.5x103 cells/well to 2 – 3x103 cells/well• Change the NHK culture flask size used at FAL for start-up of cryopreserved
cells from 80 cm2 to 25 cm2 (the size the other laboratories had been using),and discontinue using a fibronectin-collagen coating.

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November 2006

2.6.2.7 Test Acceptance Criteria
The test acceptance criteria were revised as follows:• The IC50 for SLS (PC) should be within 2 SDs (approximately 95%) of thehistorical mean established by each laboratory in Phase Ia.
• The mean OD values of the left and right VCs (columns 2 and 11 in the 96well test plate) should not differ by more than 15% from the mean of all VC
OD values on that plate.
• At least one calculated cytotoxicity value should be between 10% and 50%
viability, and one value between 50% and 90% viability.
• The Hill function curve fit (R2 >0.9 or 0.8 < R2 <0.9) should be evaluated on acase-by-case basis for acceptability by the SMT.
• VC OD criteria were based on Phase Ia data (mean ± two SDs): 0.3 to 0.8 forthe 3T3 test method, and 0.6 to 1.7 for the NHK NRU test method(requirement for use of VC OD criteria as test acceptance criteria wasrescinded after commencement of Phase Ib)
2.6.3
Phase II: Laboratory Qualification Phase
All protocol and acceptance criteria revisions were implemented during Phase II unlessotherwise stated.
2.6.3.1 Testing of Volatile Reference Substances
When 2-propanol was tested in 3T3 and NHK cells, vapors from the highest concentration
wells contaminated the adjacent VC wells and also appeared to affect some lower
concentration wells (i.e., the wells exhibited unexpectedly reduced levels of NRU). An
example range finder concentration-response curve is shown in Figure 2-4. Such tests failed
the VC criterion. When lower concentrations were used to avoid contaminating the VC wells
adjacent to the highest concentration, the toxicity was inadequate to produce an IC50. To
address this problem, IIVS repeated their tests using film plate sealers, which isolated
individual wells from one another; this was sufficient to prevent the cross-wellcontamination, and acceptable results were obtained. Based on these data, the SMT
recommended to the other two laboratories that film plate sealers be used when testing 2propanol.
FAL had previous experience layering mineral oil on the culture media in a well to prevent
volatile substances from escaping, and provided 2-propanol test data where mineral oil had
been added to each well. The data showed that the average oil vs. film IC50 values were notsignificantly different. However, there was less variability in the NRU data when using thefilm sealer so the SMT recommended this methodology.
A >15% difference between the mean VC OD of all VC cells and the mean OD of each VC
columns on opposite ends of the test plate was used as a general indicator of substancevolatility in the test if the VC adjacent to the highest test concentration had a significantly
reduced OD value.
Protocol Revision: The SMT included the use of film sealers in the Phase III protocols when
testing suspected volatile compounds.

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In Vitro Cytotoxicity Test Methods BRD Section 2

Figure 2-4

November 2006

Representative Concentration-Response for 2-Propanol in a 3T3 NRU
Range Finder Test

A
B
C
D
E
F
G
H

1
Blank
Blank
Blank
Blank
Blank
Blank
Blank
Blank

2
Blank
VC1
VC1
VC1
VC1
VC1
VC1
Blank

A
B
C
D
E
F
G
H

1
0.000
0.002
-0.0010.003
0.003-0.004-0.0040.004

2-0.002
0.080
0.067
0.058
0.077
0.068
0.071
0.000

3
Blank
C1
C1
C1
C1
C1
C1
Blank

4
Blank
C2
C2
C2
C2
C2
C2
Blank

96-WELL PLATE MAP
5
Blank
C3
C3
C3
C3
C3
C3
Blank

6
Blank
C4
C4
C4
C4
C4
C4
Blank

7
Blank
C5
C5
C5
C5
C5
C5
Blank

8
Blank
C6
C6
C6
C6
C6
C6
Blank

9
Blank
C7
C7
C7
C7
C7
C7
Blank

10
Blank
C8
C8
C8
C8
C8
C8
Blank

CORRECTED ABSORBANCE (Sample OD540 - Mean Blank OD540)
3-0.001-0.001
0.004
0.003
0.001
-0.002
0.003
0.001

4
-0.001
0.070
0.059
0.056
0.067
0.050
0.053
0.001

5
0.000
0.124
0.109
0.110
0.106
0.110
0.122
0.000

6
-0.003
0.206
0.171
0.163
0.092
0.164
0.147
0.003

7
0.001
0.296
0.284
0.243
0.218
0.216
0.204
-0.001

8
0.002
0.389
0.334
0.271
0.252
0.289
0.226
-0.002

9
0.002
0.291
0.237
0.246
0.328
0.336
0.263
-0.00210-0.001
0.301
0.308
0.251
0.250
0.267
0.295
0.001

11
Blank
VC2
VC2
VC2
VC2
VC2
VC2
Blank

12
Blank
Blank
Blank
Blank
Blank
Blank
Blank
Blank

11
-0.002
0.343
0.337
0.283
0.290
0.281
0.330
0.001

12
-0.0030.002-0.0040.002
0.003-0.001-0.003-0.002Abbreviations: 3T3=BALB/c 3T3 fibroblasts; VC=Vehicle control; C1 to C8=Test substance concentrations (C1-highest concentration, C8lowest concentration); OD540=Optical density at 540 nm; A to H=Row identification.
Note: %Difference of the two VC columns from the average VC was 63%. The mean corrected optical density (OD) for VC1, adjacent to
the highest 2-propanol concentration, was 0.070, while that for VC2, adjacent to the lowest 2-propanol concentration, was 0.310. Setting themean VC OD to 100% viability shifted the toxicity curve such that lower concentrations of 2-propanol seemed to be less toxic to the cells
than the VCs (i.e., >100%).
Error bars are ±1 standard deviation.

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In Vitro Cytotoxicity Test Methods BRD Section 2

November 2006

2.6.3.2 Atypical Concentration-Responses
Atypical concentration-responses are defined for this study as response curves that differ
from a basic sigmoidal shaped curve. Curves that show a biphasic response as well as thosethat exhibited a plateau-like response at toxicity levels than 100% were considered atypical.
Two of the laboratories observed biphasic concentration-responses in the range finder tests
for aminopterin and colchicine. When the range finder tests produced a biphasic response(see Figure 2-5 for an example), the SMT advised the laboratories to focus the definitive
tests on the lowest concentrations that produced at least a 50% loss in viability. Although
doing so eliminated the biphasic response in the definitive tests, the highest tested
concentrations did not reduce cell viability to 0% (see Figure 2-6). This effect withcolchicine was very reproducible across laboratories in the NHK NRU test, but only FAL
achieved this biphasic type of response with colchicine in the 3T3 NRU test. Aminopterin
produced similar concentration-responses in the NHK NRU test at ECBC and FAL, but not
at IIVS. In the 3T3 NRU test, only FAL obtained a biphasic response with aminopterin.
Figure 2-5

Representative Concentration-Response for Aminopterin in a NHK NRU
Range Finder Test

Abbreviations: NHK=Normal human epidermal keratinocytes; NRU=Neutral red uptake.
Representative dose-response for aminopterin in a NHK range finder test. Laboratories were instructed tofocus the definitive tests on the lowest concentration that produced a 50% reduction in viability in the range
finder test.

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In Vitro Cytotoxicity Test Methods BRD Section 2

Figure 2-6

November 2006

Representative Concentration-Response for Aminopterin in a NHK
NRU Definitive Test

Abbreviations: NHK=Normal human epidermal keratinocytes; NRU=Neutral red uptake.
Note that the maximum reduction in cell viability plateaued at about 75%

Biphasic concentration-responses are not uncommon. Calabrese (2005) states that numerous
mechanistic explanations (including hormesis4) could account for biphasic response curves.
Such concentration-responses could be because the substance acts through more than one
mechanism of action (e.g., one mechanism that is active at low test substance concentrations
and other mechanism[s]) that are effective at higher concentrations). Conolly and Lutz
(2004) also provide examples of pharmacological and toxicological data sets of biologically
based mechanisms that could explain biphasic responses. These examples include:• Membrane receptor subtypes with opposite downstream effects• Receptor-mediated gene expression• Induction of DNA repair and “co-repair” of background DNA damage• Modulation of the cell cycle
Although non-linear responses could also be due to technical error (e.g., improper dosing,unacceptable media, contamination), the responses seen in this study were reproducible, andthere was no evidence to suggest that technical errors were involved. The SMT assumed thatthese responses were based on the chemicals’ mechanisms of action. For example, colchicine
binds to microtubular protein and interferes with function of mitotic spindles, which arrests
cell division (NLM 2003). Aminopterin blocks the use of folic acid by the cells, inhibiting
metabolism, RNA production, and protein synthesis, which is lethal during the S phase of thecell cycle by (NLM 2002). The variability of IC50 results for these substances among thelaboratories may be due to different levels of cell confluence in the cultures at the time oftreatment.

Hormesis is a dose-response characterized by a compound’s ability to produce an opposite effect at low doses
compared with its effect at high doses (e.g., stimulatory at low doses and inhibitory at high doses).
4

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In Vitro Cytotoxicity Test Methods BRD Section 2

November 2006

2.6.3.3 Hill Function
The Hill function used in the various phases of this study was defined as follows:

Y = Bottom +Top " Bottom
1+ 10 (logEC50"logX)HillSlope

where Y=response (i.e., % viability), X is the substance concentration producing the
response, Bottom is the minimum response (0% viability, maximum toxicity), Top is the
maximum response!(maximum viability), EC50 is the substance concentration at the response
midway between Top and Bottom, and HillSlope describes the slope of the curve. When
Top=100% viability and Bottom=0% viability, the EC50 is the equal to the IC50.
Responses that do not achieve 100% cytotoxicity with increasing substance concentration do
not fit the Hill function well. The R2 values from such tests often failed the acceptancecriterion. To obtain a better model fit, the Bottom parameter was estimated without
constraints (the previous practice was to use Bottom=0). However, when Bottom≠0, the EC50
reported by the Hill function was not the same as the IC50 because the Hill function relies on
EC50, which is defined as the point midway between the Top and Bottom responses. Thus,
the Hill function calculation using the Prism® software was rearranged to calculate the IC50
as follows:# Top " Bottom &log%"1($ Y " Bottom'
logIC 50 = logEC 50 "HillSlope

where IC50 is the concentration producing 50% toxicity, EC50 is the concentration producing
a response midway between the Top and Bottom responses; Top being the maximum! survival), Bottom is the minimum response (0% viability, maximum
response (maximum
toxicity), Y=50 (i.e., 50% response), and HillSlope describes the slope of the response. The
X from the standard Hill function equation is replaced, in the rearranged Hill function
equation, by the IC50.
IIVS performed the recalculations for their NHK NRU colchicine tests and the SMT
performed the necessary recalculations for the other laboratories. Tests that were recalculatedby the SMT are noted in the data summaries.
Protocol Revision: The protocol was revised to state that if a range finding test produces abiphasic response, then the concentrations selected for the subsequent tests should cover themost toxic dose-response range.
2.6.3.4 Insoluble Reference Substances
Lithium carbonate was insoluble in 3T3 medium. Only ECBC managed to expose 3T3 cells
to sufficient lithium carbonate to produce three tests that met the acceptance criteria.
Precipitate was reported for two of those tests at the three highest concentrations in the wells.
Because the third highest concentration, 510.2 µg/mL, was approximately the IC50 (average
was 564 µg/mL), the true IC50 for lithium carbonate may actually be lower than was
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In Vitro Cytotoxicity Test Methods BRD Section 2

November 2006

calculated, and therefore the LD50 value would be underestimated. However, the data werereproducible and were not discarded.
Protocol Revision for Phase III: The protocol was revised to allow an increase in thestirring/rocking duration in an incubator from one to three hours if cytotoxicity in the range
finder test was limited by solubility. Also, a Stopping Rule for Insoluble Chemicals wasadded (see Section 2.5 and Appendices B1 and B2) so that the laboratories would notcontinue repeated testing of insoluble substances in order to obtain three acceptable definitive
tests.
2.6.3.5 Inadequate Cell Growth in NHK Medium
IIVS and FAL had several NHK NRU test failures that were attributed to poor cell growth.
The SMT compiled KBM and SingleQuot lot numbers that the laboratories were using,along with the laboratory assessments of NHK cell growth. The information was used to
identify the lots that produced adequate growth. The SMT also obtained quality assurance
and quality control test results from CAMBREX Clonetics on the lots of KBM, but the
information provided was inadequate for determining how the medium would perform in the
NHK NRU test method.
Resolution: A protocol for prequalifying the medium was developed (see Appendix B4). For
Phase III, the SMT asked IIVS to prequalify new lots of KBM and SingleQuots for use by
all laboratories.
2.6.3.6 Performance Standards for Media to Support NHK Growth
A prequalification-of-medium protocol (Appendix B4) was developed and used by IIVS totest several different lots of medium and supplements to find combinations that maintained
the typical growth characteristics of the NHK cells used in this study. The laboratories thenreserved samples of the acceptable lots at CAMBREX so that testing would not beinterrupted due to unavailability of adequate materials.
Test Acceptance Criteria for Prequalifying Media Using SLS• The fit of the SLS dose-response to the Hill model should be R2 ≥0.85 (i.e.,from PRISM® software).
• The difference between the mean of all VCs and (a) the left mean VC, and (b)the right mean VC should be ≤15%.
• At least one concentration should exhibit >0% and ≤50% viability and at least
one should exhibit >50% and <100% viability.
• After meeting all other acceptability criteria, the SLS IC50 must be within thehistorical range (±2.5 SD) established by the laboratory.
Other Criteria for Prequalifying Media (for consideration by a Study Director)• General observations: rate of cell proliferation; percent confluence; number of
mitotic figures per field; colony formation; distribution of cells in the flask;
absence or presence of contamination• Cell morphology observations should include overall appearance (e.g., good,
fair, poor), and presence of abnormal cells

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In Vitro Cytotoxicity Test Methods BRD Section 2•••
2.6.3.7

November 2006

Mean corrected OD540 of the VCs (e.g., are the values high/low whencompared to historical data)
Cell morphology and confluence of the VC wells at the end of the 48-hourtreatment
Cell doubling time, as compared to the doubling time with the previous
batches of medium

Test Acceptance Criteria for Phase II• The IC50 for SLS (PC) should be within 2.5 SDs of the historical mean
established by the laboratory (Phases Ia and Ib)• Mean OD values of the left and right VCs (columns 2 and 11 in the 96-welltest plate) do not differ by more than 15% from the mean of all VC well OD
values. At least one calculated cytotoxicity value ≥10% and ≤50% viability
and at least one value >50% and ≤90% viability• R2 ≥0.90. The test fails if R2 <0.80. If the 0.80 ≤ R2 <0.90, the SMT evaluates
the model fit (Note: The Study Director makes this determination for nonvalidation studies.)

2.6.4
Phase III: Laboratory Testing Phase
The changes below were made in the Phase III protocols based on the data and results in
Phase II.
2.6.4.1 Required Cytotoxicity Values
Obtaining at least one calculated cytotoxicity value >0% and ≤50% viability and at least one
that is >50% and <100% viability may be difficult or unattainable for substances with steep
dose responses.
Protocol Revision: The test acceptance criterion was qualified so that tests with only one
concentration between 0 and 100% viability were acceptable if the smallest practical dilution
factor (i.e., 1.21) was used and all other test acceptance criteria were met.
Tests for three reference substances were accepted that met this new criterion in the 3T3
NRU test method: diquat dibromide (1/9 tests); epinephrine bitartrate (2/9 tests); 1,1,1trichloroethane (2/8 tests). No NHK tests required the use of these criteria (i.e., one point
between 0% and 100% viability at the lowest dilution factor).
2.6.4.2 Revisions to Data Analysis Procedures
The following revisions to data analysis procedures were made in Phase III NRU protocols:• If the Bottom parameter of the Hill function was fit to a value <0%, then theparameter was set to zero (0) for the IC calculations.
• If toxicity plateaued above 20% viability (i.e., toxicity was <80%), the IC80
was not determined. The IC20 and IC50 values were calculated from the rangeof available toxic responses.
• The requirement for substance dose-responses to fit the Hill equation with R2≥0.90 was rescinded. The Hill equation was used to characterize the shape ofthe response rather than to establish an acceptance criterion. The PC
acceptance criterion was modified to R2 ≥0.85.

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In Vitro Cytotoxicity Test Methods BRD Section 2

2.7

November 2006

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

As the validation study progressed through Phases I and II, the protocols provided in the
Guidance Document (ICCVAM 2001b) were optimized to address problems that wereencountered during the validation study phases. Changes to the Guidance Document
protocols are described below.
• 3T3 cell seeding density for 96-well plates was decreased from 1x104
cells/well to 2.0 – 3.0x103 cells/well.
• The calcium concentration in NHK medium was changed from 0.15 mM to
0.10 mM. The test laboratories had expressed concern that cell differentiation
would occur at the higher concentration and requested a lower concentration.
CAMBREX Clonetics®, the supplier of the NHK cells and NHK medium usedin this study, normally grows NHK cells in 0.15 mM calcium and has seen no
differentiation. The supplier agreed that the cells would grow well at 0.10 mM
but should not be cultured at concentrations <0.10 mM in order to avoid
morphological and growth rate changes (CAMBREX technical division,
personal communication).
• NHK cells were subcultured once prior to being distributed to the test wells,
rather than for three passages. The laboratories expressed concern about thepossibility of cell differentiation with subsequent passages in culture.
• The highest recommended final concentrations of DMSO and ETOH in theculture media were reduced from 1% to 0.5%. IIVS performed experimentswith both cell types to determine the concentration necessary to avoid solvent
toxicity. 3T3 cells were tested with 0.5, 1, and 2% ETOH and DMSO at 0.1,
0.2, 0.3, 0.4, 0.5, 1, and 2% concentrations. The 0.5% concentrations of bothsolvents were chosen as optimal because that concentration of ETOH
produced no toxicity. Although 0.5% DMSO produced slight toxicity (i.e.,cells were 91% viable as compared to the control cells; See Appendix E1),
this concentration was chosen by the SMT and laboratories as an acceptabletrade-off between slight toxicity and the ability to test substances at higher
concentrations, and was used throughout the study for all reference substancesthat needed solvents other than culture medium (see Curren et al. 2003).
DMSO was the preferred solvent if the test substance was not soluble inculture medium, and ETOH was not used in this study.
• The pH of the reference substance solutions was not adjusted with NaOH or
HCl regardless of whether solutions became acidic or basic (optimum
mammalian cell culture pH is approximately 7.4 [Freshney, 2000]) upon
addition of the test substance because some of the basal cytotoxicity produced
by test substances may be due to pH effects. See Appendix F1 for pH valuesof the reference substances in culture medium.
• The CO2 concentration in the incubator was reduced from 7.5% to 5.0%
because the laboratories were already set up to use 5% CO2, which is a typicaloptimum CO2 concentration for mammalian cell culture.
• Washing and fixing the cells with a formaldehyde solution prior to NR elution
from the cells was eliminated. Formaldehyde disposal was problematic in
FAL’s regulatory environment. The SMT and the laboratories agreed that the2-36

In Vitro Cytotoxicity Test Methods BRD Section 2••

Table 2-4

November 2006

use of formaldehyde was unnecessary because the NR desorb solution (1%glacial acetic acid, 50% ETOH, and 49% H2O) adequately fixed the cells to
the test plate (INVITTOX 1991).
Reference substance exposure time for the 3T3 cells was extended from 24hours to 48 hours (see Section 2.2.4 and Appendix E1).
Cell culture seeding densities for subculture were provided as guidance, rather
than as strict cell number ranges. The laboratories determined adequate celldensities (see Table 2-4) based on their own experience with the growth ofthe cells in the wells, and the time needed to reach the appropriate level ofconfluence needed for addition of the test substance, the VC, and PC.
Cell Seeding Densities1

ProtocolGuidance Document2

3T3 cells/cm2
subculture toflasks1.25x104

3T3 cells/well
96-well Plate
2.5x103

NHK cells/cm2
subculture toflasks
3.5x103

NHK cells/well
96-well Plate2 – 2.5x103

Phase Ia

0.42 – 1.68x10

42.5x103

2.5 – 9x103

2 – 2.5x103

Phase Ib

0.42 – 1.68x104

2.5x103

2.5 – 9x103

2 – 2.5x103

Phase II

0.42 – 1.68x104

2 – 3x103

2.5 – 9x103

2 – 2.5x103

Phase III0.42 – 1.68x104

2 – 3x103

2.5 – 9x103

2 – 2.5x103

Abbreviations: 3T3=BALB/c 3T3 fibroblasts; NHK=Normal human epidermal keratinocytes
1
Cell numbers determined by Coulter Counter or hemocytometer
2
ICCVAM (2001b)

2.8

Overview of the Solubility Protocol

The SMT, with assistance from the laboratories, developed a solubility protocol to provide
guidance for determining the most appropriate solvent for each test substance. The solubility
protocol was based on an EPA guideline (EPA 1998) that involved testing for solubility in aparticular solvent, beginning at a relatively high concentration and proceeding tosuccessively lower concentrations by adding more solvent as necessary for dissolution.
Testing stopped when, upon visual observation, the procedure produced a clear solution withno cloudiness or precipitate. The order of selection priority was culture medium, DMSO, andETOH. Each laboratory tested the solubility of each reference substance using this protocol
and provided the data to the SMT prior to initiating cytotoxicity testing. The SMT analyzed
the solubility data provided by BioReliance and each testing laboratory, and designated thesolvent to be used by all laboratories for each reference substance. This eliminated one
potential variable in the NRU test results among laboratories.
The solubility protocol used by the in vitro laboratories required the sequential testing ofreference substances in the various solvents at concentrations that would be equivalent to theconcentration that would be applied to the cell cultures. The solubility flow chart in Figure
2-7 shows, for example, that 2 mg/mL medium and 200 mg/mL DMSO or ETOH wereequivalent concentrations because they yielded 1 mg/mL in cell culture. Medium was diluted
by one-half when applied to cultures. The 0.5% [v/v] final concentrations were achieved bydiluting DMSO and ETOH by 200-fold. At each concentration, the following mixing
procedures were employed, as necessary, to completely dissolve the reference substance in
2-37

In Vitro Cytotoxicity Test Methods BRD Section 2

November 2006

the sequence: vortex (1 to 2 minutes); sonication (up to 5 minutes); warming to 37 °C (5 to
60 minutes [NRU protocols allow warming to be extended to three hours if cytotoxicity inthe range finder test was limited by solubility]). If the reference substance was still notdissolved, the next lower concentration, or a different solvent, was tested.
Figure 2-7

Flow Chart for Determination of Reference Substance Solubility in
Medium1, DMSO, or ETOH

Tier

1

Concentration in
3T3 and NHK
Media

Start Here20 mg/mL2

3

2 mg/mL

0.20 mg/mL

20 mg/mL

Incomplete
solubility

Concentration
on Cells

200 mg/mL

10 mg/mL

2 mg/mL

0.2 mg/mL

Incomplete
solubility

200 mg/mL

Concentration in
Ethanol5

Incomplete
solubility

Incomplete
solubility

Concentration in
DMSO

4Incomplete
solubility

Incomplete
solubility

1 mg/mL

20 mg/mL

0.1 mg/mL

Incomplete
solubility

Incomplete
solubility

2 mg/mL

0.01
mg/mL

Incomplete
solubility

Incomplete
solubility

0.2 mg/mL
End

0.001
mg/mL

Abbreviations: DMSO=Dimethyl sulfoxide; ETOH=Ethanol; 3T3=BALB/c 3T3 fibroblasts; NHK=Normal
human epidermal keratinocytes.
Note: DMSO is U.S.P. analytical grade. ETOH is U.S.P. analytical grade (100% non-denatured).
1
3T3 Medium - DMEM (Dulbecco’s Modification of Eagle’s Medium) with supplements; NHK medium KBM (Keratinocyte Basal Medium) with supplements (from CAMBREX Clonetics).

2.9

Basis of the Solubility Protocol

The solubility protocol used by BioReliance, which tested solubility of the reference
substances prior to testing by the in vitro laboratories, is provided in Appendix G. The
protocol is based largely on information from the literature and Internet searches forsolubility procedures, the experience of the SMT and IIVS, and solubility and IC50
information from the RC chemicals database (Halle 1998, 2003). The only formal solubility
protocol discovered was the EPA Product Properties Test Guideline, OPPTS 830.7840 Water
Solubility Column Elution Method; Shake Flask Method (EPA 1998).
2.9.1
Initial Solubility Protocol Development
BioReliance evaluated the solubility of each reference substance in cell culture media at
2000, 400, and 200 mg/mL, and if not soluble at those concentrations, in DMSO and then
ETOH, at the same concentrations (initial protocol). It was apparent that these concentrations
were not low enough when the laboratory was unable to achieve solubility for arsenic
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In Vitro Cytotoxicity Test Methods BRD Section 2

November 2006

trioxide. The solubility protocol was revised twice to lower the range of concentrations tested(see Table 2-5). An extra tier of concentrations ≤1 mg/mL was added for poorly soluble andinsoluble substances. The protocol used by the laboratories was further revised to reduce thenumber of steps required (by testing in log units) and to test in tiers using concentrations thatreflected the concentrations anticipated in the cell cultures (see Figure 2-7).
Table 2-5

Comparison of Concentrations Tested in the Various Solubility Protocols

Solubility
Protocol Version

Concentrations Tested (mg/mL)
Step 1

Step 2

Step 3

Step 4

Step 5

Steps 6-10

BioReliance (1 )(4/26/02) andPhase Ia

2,000

400

200

NA

NA

NA

BioReliance (2nd)(9/17/02)

200

40

20

10

2

NA

BioReliance (3rd)(10/11/02)

200

40

20

10

2

1, 0.5, 0.25,
0.125, 0.05

Phases Ib, II, III for
cytotoxicity
laboratories

20
Medium

2 Medium
200 DMSO
200 ETOH

0.2 Medium
20 DMSO
20 ETOH

2 DMSO
2 ETOH

0.2 DMSO
0.2 ETOH

stNA

Abbreviations: DMSO=Dimethyl sulfoxide; ETOH=Ethanol; Medium=Cell culture medium; NA=Not applicable
Note: DMSO is U.S.P. analytical grade. ETOH is U.S.P. analytical grade (100% non-denatured).

In Phases Ib and II, the SMT used the data from BioReliance to select the solvents to be used
for testing the various chemicals. When it became apparent that the laboratories sometimes
obtained different solubility results than those reported by BioReliance, the SMT used thecytotoxicity results from the laboratories to determine the solvents to be used for Phase III
reference substances.
The final protocol provided a tiered approach for determining the 2X stock concentration foreach reference substance (see Figure 2-7). This protocol had the advantage of reducing thenumber of steps for testing (compared to that used by BioReliance) (see Appendix B3).
2.9.2
Basis for Modification of the Phase II Protocol
All three testing laboratories found arsenic trioxide (tested in Phase Ib) less soluble (seeTable 5-10) than was reported by BioReliance (BioReliance values: 0.25 mg/mL in 3T3
medium and 0.05 mg/mL in NHK medium). This chemical was not soluble using theprocedures in the initial solubility protocol. IIVS warmed the stock solution (at least 200µg/mL for 2X) for longer than the protocol specified (i.e., 30 to 50 minutes) but still had
persistent, small, undissolved particles. ECBC obtained a clear solution (highest 2X
concentration was 30 to 50 µg/mL), but found precipitated particles after the solution stood atroom temperature. Sonication time was increased to 15 to 30 minutes, and heating time toapproximately 30 minutes to get a finer suspension. This procedure achieved a morehomogeneous mixture, resulting in more uniform serial dilutions and a more even applicationof the reference substance to the cells. FAL stirred the suspension (approx. 20 to 90 µg/mL)in the CO2 incubator for 1.5 to 2 hours to get clear medium.

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In Vitro Cytotoxicity Test Methods BRD Section 2

November 2006

Protocol Revision for Phase II: The duration of the heating step was altered from 5 to 20
minutes to 5 to 60 minutes.
2.10

Components of the Solubility Protocol

2.10.1

Medium, Supplies, and Equipment Required

2.10.1.1 Medium and Chemical Supplies• 3T3 culture medium: DMEM without L-glutamine and containing Hanks’salts and high glucose [4.5gm/l]; L-glutamine, 200 mM; NCS• NHK culture medium: Keratinocyte Basal Medium without Ca++ (KBM®,
Clonetics® CC-3104); KBM® SingleQuots® medium supplements (Clonetics®
CC-4131): epidermal growth factor, insulin, hydrocortisone, bovine pituitary
extract; Calcium SingleQuots® (Clonetics® CC-4202); penicillin/streptomycin
solution (antimicrobial agents)• United States Pharmacopoeia (U.S.P.) analytical grade DMSO
• U.S.P. analytical grade (100%, non-denatured) ETOH
2.10.1.2 Equipment• Waterbath (37 °C)• Sonication apparatus• Vortex mixer• Micropipettors• Balance (capable of weighing 10 mg)• pH meter
2.10.1.3 Procedures
The Phase III solubility protocol required the dissolving of approximately 10 mg of reference
substance in approximately 0.5 mL medium (both 3T3 and NHK media were used) for a final
concentration of 20 mg/mL (see Appendix B3). In order, the mixture was vortexed for 1 to 2
minutes, sonicated for up to 5 minutes, and warmed to 37 °C for 5 to 60 minutes, asnecessary, to dissolve the substance. The endpoint for dissolution was a clear solution withno noticeable precipitate. If the reference substance was not soluble in medium at 20 mg/mL,then more medium was added to a concentration of 2 mg/mL (i.e., a total volume of approx.
5 mL) (Step 2). The mixing procedures were repeated as necessary to dissolve the reference
substance. If the reference substance did not dissolve, approximately 10 mg reference
substance was added to approximately 0.5 mL DMSO in an attempt to dissolve it at aconcentration of 200 mg/mL (Step 3). If the reference substance was not dissolved, the same
concentration was attempted in 100% ETOH (Step 4). Step 5 began in the same way, with
0.2 mg/mL medium and then progressed to 20 mg/mL DMSO, and then 20 mg/mL ETOH.
Determination of reference substance solubility was limited to visual observation of theresulting solution. If a solution appeared clear, then solubility testing ceased. If particles werevisible or if the solution appeared cloudy, then more stringent mixing and/or heatingprocedures were employed. If necessary, the solubility procedure proceeded to the next
solvent/concentration tier. The duration of the solubility test was dependent on theprocedures used to achieve solubility. Some reference substances were immediately
solubilized (e.g., liquids) and others required up to 60 minutes of heating and agitation orsonication.
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In Vitro Cytotoxicity Test Methods BRD Section 2

November 2006

2.10.2
Data Collection
All laboratories (including the reference substance distribution laboratory, BioReliance) useda worksheet designed to capture the solubility information for each reference substance. The
endpoint for each step was a visual observation of the solution, a documented comment
describing the observation, the concentration, and a conclusion of soluble or insoluble. Eachworksheet contained:• Reference substance code number and physical description• Solvent used (3T3 medium, NHK medium, DMSO, ETOH)• Amount of reference substance (mg) used in the initial stage• Volume of solvent added and final volume (mL)• Test substance concentration (µg/mL) in the solvent• pH and color of the solution• Mechanical procedures used (vortexing, sonication, heating), duration, andtemperature• Comments (soluble/insoluble at the particular concentration; visualobservations; reactivity with solvent)The solubility test information and data from the laboratories were transferred via email to
the SMT and stored on the NICEATM server and as hard-copy printouts. Each laboratory
also maintained electronic and hard-copy files of its data.
2.10.3
Variability in Solubility Measurements
Solubility determinations were not replicated because within-laboratory results were notexpected to vary. Comparison of the results to determine inter-laboratory concordance for the
72 reference substances (see Section 5.8 for results) provided a measure of variability among
the laboratories and information about the reproducibility of the solubility determinations(see Section 7.4).
2.10.4
Solubility Issues During the Testing of the Reference Substances
Substance solutions were monitored throughout all aspects of the test procedures, andobservations were documented. The lowest concentration of the substance in a 2X solutionthat contained observable precipitates, particles, globules, or oily droplets, was documented
in the EXCEL® template. After substance exposure, all wells of the 96-well test plates wereobserved microscopically and scored using a visual observation code. The code addressed
growth characteristics and the presence or absence of precipitates (see Appendix B [testmethod protocols] for the observation codes used). For solubility issues, the Study Directors
made determinations of test acceptance based on the recommended concentration levels andthe presence of precipitates, their scientific expertise, and test acceptance criteria.
2.10.5
Analysis of Solubility Data
During Phase III, the SMT used the solubility data from all laboratories to determine thesolvents to be used for each chemical (see Section 5.8 for solubility results and SMT
selections). If the solubility of an individual reference substance was different in 3T3
medium and NHK medium, the same solvent would be used for both test methods, rather
than having different solvents for each method. For example, if solubility in one culture
medium was ≥2 mg/mL and solubility in the other was <2 mg/mL, and the substance wassoluble in DMSO at 200 mg/mL, the SMT would select DMSO as the solvent for both test
methods (each test method using its respective culture medium).
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In Vitro Cytotoxicity Test Methods BRD Section 2

November 2006

Solubilizing sufficient reference substance to produce cytotoxicity was challenging for
relatively insoluble, low toxicity, substances such as lithium carbonate (in the 3T3 NRU test
method) but generally was not a problem for toxic substances that did not require as high aconcentration to kill cells. Some insoluble and highly toxic reference substances were
problematic, however, because the amount of powdered reference substance added to solvent
was very small, and laboratory personnel found it difficult to determine the presence ofsolute particles in solution. Arsenic trioxide is an example of such a solute (see Section
2.9.2).
2.11

Summary

The Guidance Document NRU protocols were used as the basis of the validation study
protocols. The SMT and participating laboratories made initial modifications to the protocols
prior to implementation of the study. Other protocol modifications were made after
commencement of testing and were the result of recommendations from the laboratories andthe SMT, based on their experience with the initial protocols. The resulting optimized
protocols were used in the main testing phase (Phase III) of the study.
The protocol components used in the validation study were similar for the 3T3 and NHK
cells. The following procedures were common to the NRU protocols for both cell types:• Testing was performed in four phases (Phases Ia, Ib, II, and III)• Preparation of reference substances and positive control• Cell culture environment conditions• Determination of test substance solubility• Configuration of 96-well plates for testing samples• 48-hour exposure to test substance• Range finder and definitive testing• Microscopic evaluation of cell cultures for toxicity• Measurement of NRU• Data analysis
The main differences in the test methods for the two cell types were:• The conditions of propagation of the cells in culture• The cell growth medium components• The volumes of reference substance added to the 96-well plate
A solubility protocol was developed which allowed the laboratories to identify the mostappropriate solvent and appropriate limit concentrations for each test substance.
Three laboratories participated in testing the 72 reference substances in both cell types andone additional laboratory procured and distributed the coded reference substances andperformed solubility tests prior to their distribution to the testing laboratories.

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In Vitro Cytotoxicity Test Methods BRD Section 3

November 2006

3.0

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

3.1

Rationale for the 72 Reference Substances Selected for Testing ........................3-3
3.1.1 Reference Substance Selection Criteria .......................................................3-3
3.1.2 Candidate Reference Substances .................................................................3-4
3.1.3 Selection of Reference Substances for Testing ............................................3-5

3.2

Characteristics of the Selected Reference Substances.........................................3-6
3.2.1 Source Databases Represented by the Selected Reference Substances .........3-6
3.2.2 Chemical Classes Represented by the Selected Reference Substances .......3-28
3.2.3 Product/Use Classes Represented by the Selected Reference
Substances ................................................................................................3-28
3.2.4 Toxicological Characteristics of the Selected Reference Substances ..........3-28
3.2.5 Selection of Reference Substances for Testing in Phases Ib and II.............3-32
3.2.6 Unsuitable and Challenging Reference Substances ....................................3-34

3.3

Reference Substance Procurement, Coding, and Distribution .........................3-34
3.3.1 Exceptions ................................................................................................3-35

3.4Reference Substances Recommended by the Guidance Document ...................3-35

3.5

Summary.............................................................................................................3-36

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In Vitro Cytotoxicity Test Methods BRD Section 3

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3.0

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

3.1

Rationale for the 72 Reference Substances Selected for TestingThis section describes the procedures used to select the 72 reference substances selected fortesting in Phase Ia of the validation study.
3.1.1
Reference Substance Selection Criteria
The SMT (see Appendix A) selected reference substances for testing using a process basedon general recommendations made by Workshop 2000 participants (ICCVAM 2001a). The
following criteria were used:• The toxicities of the reference substances should be evenly distributed acrossthe expected range of rodent LD50 values, using the GHS classification for
acute oral toxicity as a guide (UN 2005).
• The reference substances should cover a wide range of structural and use
classes, and be relevant to the needs of the various user communities.
• Substances with human toxicity data and/or human exposure potential (i.e.,
substances of interest to society) should be included. Substances with humanacute toxicity data were particularly important to ECVAM for determining therelationship of the NRU IC50 values to human blood/serum LC.
Table 3-1 shows the GHS scheme for classifying substances into six toxicity categories (fivewith measured LD50 ranges and an unclassified category with LD50 values greater than 5000
mg/kg) based on acute rodent oral LD50 values (UN 2005). The SMT used this scheme forthe classification of candidate substances to assure that the reference substances selected forthe validation study represented the full range of acute oral toxicity.
Table 3-1

GHS Classification Scheme for Acute Oral Toxicity

Category

LD50 (mg/kg)

1

LD50 ≤52

5 < LD50 ≤50

350 < LD50 ≤300

4300 < LD50 ≤2000

5

2000 < LD50 ≤5000

Unclassified

LD50 >5000

Abbreviations: UN=United Nations; GHS=Globally Harmonized System ofClassification and Labelling of Chemicals (UN 2005).
LD50=Dose that produces lethality in 50% of the test animals.

For the purposes of the initial toxicity classification, the rodent oral LD50 values for theindividual substances were obtained from readily available toxicological databases. Theserodent oral LD50 values were re-evaluated in Section 4 for the purpose of identifying themost appropriate reference LD50 values to use for the accuracy analyses (i.e., determine to3-3

In Vitro Cytotoxicity Test Methods BRD Section 3

November 2006

what extent there is agreement between a test method result and an accepted reference value[see Section 6.3]). Rat LD50 data were preferred because:• The current acute oral toxicity test guidelines recommend using rats (OECD2001a, c, d; EPA 2002a)• The majority of LD50 data used in the RC millimole regression were fromstudies using rats (282 rat data points and 65 mouse data points) (Halle 1998,
2003)• The great majority of acute oral systemic toxicity testing is performed withrats
Mouse oral LD50 values were used (10 substances) for the initial toxicity classification when
rat data were unavailable, however, mouse data were not used in the regression analyses
presented in Section 6. The toxicological databases, in order of preference, were:• The RC, which contains LD50 values that came largely from the 1983/84
RTECS® (Halle 1998, 2003). The RC is a database of acute oral LD50 values
for rats and mice obtained from RTECS® and IC50 values from in vitro
cytotoxicity assays using multiple cell lines and cytotoxicity endpoints forchemicals with known molecular weights.
• The current RTECS® (MDL Information Systems 2001, 2002)• The current Hazardous Substances Data Bank (HSDB; U.S. National Libraryof Medicine [NLM] 2001, 2002).
To insure that a wide range of structural and use classes were selected, reference substancesof interest to the various U.S. regulatory agencies, as determined from substance lists
received from the various agencies, were included. Substances with human toxicity data
and/or human exposure potential were chosen by mining publicly available databases (e.g.,
the NTP test database, the MEIC database) for potential candidates.
3.1.2
Candidate Reference Substances
The process of identifying the 72 reference substances started with the compilation of adatabase of 116 candidates. The intent of the SMT was to compile a database with at least 12
substances in each GHS toxicity category that also met the other selection criteria, and thento prioritize the substances within each category to select the 72 to be tested. As
recommended by Workshop 2000 (ICCVAM 2001a), the following publicly available
databases and other sources were used to identify candidate substances:• The MEIC program, which collected human toxicity data and in vitro toxicity
data from 61 test methods for 50 substances (Ekwall et al. 1998)• The EDIT program, which targeted development of in vitro test methods forendpoints other than basal cytotoxicity; includes 20 chemicals that are asubset of the MEIC chemicals• The RC (Halle 1998, 2003), which contains in vitro cytotoxicity and in vivo
rodent LD50 data for 347 substances• The Toxic Exposure Surveillance System (TESS) (Litovitz et al. 2000), which
compiles reports of toxic human exposures from poison control centersthroughout the United States• Pesticides recommended for consideration by the EPA Office of Pesticide
Programs (OPP)

3-4

In Vitro Cytotoxicity Test Methods BRD Section 3•••

November 2006

The Guidance Document (ICCVAM 2001b), which reported in vitro NRU
results for 11 RC substances using protocols similar to those to be used in thevalidation study
The U.S. NTP test database, which contains information on the toxicity of
substances relevant to human exposure (NTP 2002)
The EPA High Production Volume (HPV) Challenge Program list ofchemicals. The HPV is a voluntary testing program to provide the public witha complete set of baseline health and environmental effects data for eachchemical that is manufactured within or imported into the United States atamounts >1 million pounds/year (EPA 2000a)

The candidate substances from the list of 116 that were not selected as reference substances
to use in the validation study are listed in Appendix F3, grouped by GHS category, alongwith the rat or mouse oral LD50 value, the database(s) or other source(s) used to identify thesubstance as a potential candidate, and the type of product and/or use for the substance.
3.1.3
Selection of Reference Substances for Testing
Using the candidate substance database, 72 reference substances (12 GHS-unclassified
substances and 12 substances from each of the five GHS acute oral toxicity hazard
categories) were selected. This number of substances per GHS category was consideredadequate by the ICCVAM Acute Toxicity Working Group (ATWG), ICCVAM, ECVAM,
and the SMT to accurately evaluate the performance of these two in vitro NRU test methods
for identifying the starting dose for rodent acute oral toxicity tests across the range of toxic
levels that would be encountered during testing. The criteria used for prioritizing thecandidate substances were:• The availability of rodent acute oral toxicity data
• The availability of human acute oral toxicity data and/or relevance for human
exposure• The level of volatility (because the cells are exposed for 48 hours while
incubated at 37 °C in 96-well plates, volatilization from wells containing avolatile reference substance would affect the accuracy of the IC50 calculationand potentially contaminate other wells)• Not a controlled substance according to the U.S. Drug Enforcement Agency(DEA). Excluding substances that are listed in DEA Schedules I and II fromconsideration obviates the requirement for U.S. laboratories to obtain a DEA
license and adhere to the DEA substance storage and control procedures• Practical considerations such as cost and disposal
If more than 12 candidate substances in a GHS category met the above criteria, then selection
was based on two further considerations. One consideration was the distribution of substancetoxicities within each toxicity category so as to select substances that represented the entire
range of toxicity within each category. Another consideration, which applied only to
candidate substances selected from the RC database, was the fit of the toxicity to the RC
millimole regression. Substances with the best fit to the RC millimole regression werepreferentially selected to prevent the entire set of reference substances from having
proportionally more “outlier” substances (i.e., greater than one-half log from the RC
millimole regression) than the entire RC database.
3-5

In Vitro Cytotoxicity Test Methods BRD Section 3

November 2006

The final list of selected reference substances is sorted by GHS acute oral toxicity category inTable 3-2.
3.2

Characteristics of the Selected Reference Substances

The physical/chemical and toxicological information in Appendix F may be useful forcharacterizing the performance of the in vitro NRU test methods for various chemical types(e.g., chemical class, toxic effect class). Appendix F1 lists the reference substances inalphabetical order with information on the CASRN, purity, supplier, pH (of the highest
concentration tested in NRU), and concentrations tested. Appendix F2 provides thereference substances in alphabetical order, and information on physical/chemical
characteristics such as molecular weight, chemical class, water solubility, acid/basedissociation constant (pK), boiling point, and octanol-water partition coefficient (log Kow), a
measure of lipid solubility. Although test substance concentration and toxicity may beheavily influenced by molecular charge and surface activity (ICCVAM 2006), these
attributes were not characterized because this type of information is not readily available.
Appendix F2 also includes the major toxic effects attributed to each chemical, ability to pass
the blood:brain barrier (BBB), metabolic activation/inactivation (whether or not it ismetabolized, or the identification of the metabolites), and mechanism of lethality (where
known) for each of the reference substances. The remainder of this section summarizes
selected characteristics of the reference substances.
3.2.1
Source Databases Represented by the Selected Reference Substances
The primary sources of substances were well represented in the final list of reference
substances. Table 3-3 shows the distribution of reference substances by GHS category fromeach of the source lists. Forty-two (58%) of the 72 substances were MEIC chemicals (17 of
the 42 MEIC chemicals [40%] were also EDIT chemicals), 46 (64%) were involved inhuman poisonings as reported by TESS, 51 (71%) have been evaluated by the NTP, and 18(25%) are listed in the EPA’s HPV Challenge Program. Some substances were present inmore than one database.
The other major source of reference substances was the RC, which contributed 58 (81%) of
the 72 chemicals, as shown in Table 3-4. Because the RC millimole regression was used to
identify outlier substances (see Section 6.2), the fit of the RC substances to this regression
was relevant (Halle 1998, 2003). Halle (1998, 2003) defined outliers as those chemicals withlog IC50-log LD50 points that were >0.699 (i.e., log 5) from the RC millimole regression.
Table 3-4 shows the number of RC outliers selected for testing and the corresponding
number of outliers in the RC. Although the percentage of outliers in several GHS categories
is similar to the percentage in the RC, the total percentage of RC outliers in the set ofreference substances (i.e., 38% [22/58]) is greater than the percentage in the RC (i.e., 27%[95/347]). This occurred because the fit to the RC millimole regression was not the major
deciding factor during selection of the 72 reference substances.

3-6

In Vitro Cytotoxicity Test Methods BRD Section 3

Table 3-2

November 2006

Reference Substances Used in the 3T3 and NHK NRU Test Methods Validation Study - Sorted by Toxicity

GHS
Category1/Substance

RodentOral LD502(mg/kg)Source3

Product/Use4

Molecular
log Kow5
Weight (g/mol)

Chemical Class6

Molecular Structure

Cl——Hg

LD50 ≤5 mg/kg

Mercury II chloride

1

MEIC, EDIT,
RC (outlier),
TESS, NTP

Preservative;
Manufacturing;
Insecticide

271.500.22

Inorganic compound;
Mercury compound;
Chlorine compound

Triethylenemelamine1

RC (outlier),
NTP

Manufacturing;
Insect chemosterilant

204.23–0.54

Organic compound;
Heterocyclic
compound

Sodium selenate

2**

TESS, NTP

Feed additive

188.90NA

Inorganic compound;
Sodium compound;
Selenium compound

Busulfan2

RC (outlier),
NTP

Pharmaceutical(antineoplastic)

246.31–0.52

Organic compound;
Alcohol;
Acyclic hydrocarbon;
Sulfur compound

3-7

In Vitro Cytotoxicity Test Methods BRD Section 3

Table 3-2

November 2006

Reference Substances Used in the 3T3 and NHK NRU Test Methods Validation Study - Sorted by Toxicity

GHS
Category1/Substance

RodentOral LD502(mg/kg)Source3

Product/Use4

Cycloheximide2

RC (outlier),
NTP

Antibiotic
Fungicide2

RC (outlier),
EPA, NTP

Pesticide(insecticide)

Parathion2

RC (outlier),
EPA, NTP

Pesticide(insecticide)

Strychnine

2*MEIC, TESS,EPA

Pesticide(rodenticide)

Disulfoton

Molecular
log Kow5
Weight (g/mol)

0.55

Organic compound;
Heterocyclic
compound4.02

Organic compound;
Organophosphorouscompound;Sulfur compound

291.28

3.83

Organic compound;
Organophosphorouscompound;Sulfur compound

334.401.93

Organic compound;
Heterocyclic
compound281.40274.423-8

Chemical Class6

Molecular StructureIn Vitro Cytotoxicity Test Methods BRD Section 3

Table 3-2

November 2006

Reference Substances Used in the 3T3 and NHK NRU Test Methods Validation Study - Sorted by Toxicity

GHS
Category1/Substance

RodentOral LD502(mg/kg)**Molecular
log Kow5
Weight (g/mol)

Source3

Product/Use4

Chemical Class6

RC

Pharmaceutical(antineoplastic);
Pesticide(rodenticide)476.45NA

Organic compound;
Heterocyclic
compoundAminopterin

3

Phenylthiourea

3

RC (outlier),
NTP

Pesticide(rodenticide)

152.200.71

Organic compound;
Sulfur compound;
Urea

Epinephrine bitartrate

4**

RC (outlier),
NTP (HCl salt)

Pharmaceutical(adrenergic)

333.30–1.52

Organic compound;
Alcohol;
Amine

Physostigmine*

EHS

Pharmaceutical(anticholinesterase)NA

Organic compound;
Carboxylic acid;
Heterocyclic
compound5

275.40

3-9

Molecular StructureIn Vitro Cytotoxicity Test Methods BRD Section 3

Table 3-2

November 2006

Reference Substances Used in the 3T3 and NHK NRU Test Methods Validation Study - Sorted by Toxicity

GHS
Category1/Substance

RodentOral LD502(mg/kg)Source3

Molecular
log Kow5
Weight (g/mol)

Product/Use4

Chemical Class6

5 < LD50 ≤50 mg/kg

Colchicine

6**MEIC, RC,
TESS, NTP

Pharmaceutical(gout suppressant)399.45

1.03

Organic compound;
Polycyclic compound

Potassium cyanide

10

MEIC, EDIT,
RC (outlier),
TESS

Electroplating

65.12

NA

Inorganic compound;
Potassium compound;
Nitrogen compound

Dichlorvos

17*

TESS, EPA,
NTP, HPV

Pesticide(insecticide)220.981.43, 1.45

Organic compound;
OrganophosphorouscompoundDigoxin

18**

MEIC, EDIT,
RC (outlier),
TESS

Pharmaceutical(antiarrhythmic)

780.901.26

Organic compound;
Polycyclic compound;
Carbohydrate

3-10

Molecular StructureIn Vitro Cytotoxicity Test Methods BRD Section 3

Table 3-2

November 2006

Reference Substances Used in the 3T3 and NHK NRU Test Methods Validation Study - Sorted by Toxicity

GHS
Category1/Substance

RodentOral LD502(mg/kg)Source3

Product/Use4

Fenpropathrin

18*

EPA

Pesticide(insecticide)

Endosulfan

18*

TESS, EPA,
NTP

Pesticide(insecticide)

Arsenic III trioxide

20

MEIC, EDIT,
RC, TESS,EPA, NTP

Thallium I sulfate

29**

MEIC, EDIT,
RC (outlier),
TESS

Molecular
log Kow5
Weight (g/mol)

Chemical Class6

6.0 @
20° C

Organic compound;
Nitrile; Ester; Ether

406.91

3.83

Organic compound;
Heterocyclic
Compound;Sulfur compound

Pesticide(insecticide)

197.80NA

Inorganic compound;
Arsenical

Pesticide(rodenticide/insecticide)504.80NA

Inorganic compound;
Metal;
Sulfur compound

349.43

3-11

Molecular StructureIn Vitro Cytotoxicity Test Methods BRD Section 3

Table 3-2

November 2006

Reference Substances Used in the 3T3 and NHK NRU Test Methods Validation Study - Sorted by Toxicity

GHS
Category1/Substance

RodentOral LD502(mg/kg)Source3

Product/Use4

Sodium arsenite

41*

TESS, NTP

Pesticide(herbicide, insecticide,fungicide)129.90NA

Inorganic compound;
Arsenical;
Sodium compound

Triphenyltin hydroxide

44

RC, EPA, NTP,
HPV

Pesticide(fungicide/insecticide)367.02NA

Organic compound;
Organometallic
compound

Sodium dichromate
dihydrate50

RC, EPA, GD,
NTP

Oxidizing agent

298.00NA

Inorganic compound;
Sodium compound;
Chromium compound

50

MEIC, EDIT,
RC (outlier),
TESS, EPA,
NTP

Pharmaceutical(stimulant)

162.020

1.17

Organic compound;
Heterocyclic
compoundNicotine

Molecular
log Kow5
Weight (g/mol)

3-12Chemical Class6

Molecular StructureIn Vitro Cytotoxicity Test Methods BRD Section 3

Table 3-2

November 2006

Reference Substances Used in the 3T3 and NHK NRU Test Methods Validation Study - Sorted by Toxicity

GHS
Category1/Substance

RodentOral LD502(mg/kg)Source3

Molecular
log Kow5
Weight (g/mol)

Product/Use4

Chemical Class6

50 < LD50 ≤300 mg/kg

Paraquat

58

MEIC, EDIT,
RC (outlier),
TESS, EPA

Pesticide(herbicide)257.20–4.22 @
pH 7.4

Organic compound;
Heterocyclic
compoundHexachlorophene

61

MEIC, RC,
TESS, NTP

Disinfectant

406.91

6.91

Organic compound;
Cyclic hydrocarbon;
PhenolLindane76

MEIC, EDIT,
RC (outlier),
EPA, NTP

Pesticide(insecticide)290.803.72

Organic compound;
Halogenatedhydrocarbon

Cadmium II chloride

88

RC, TESS, GD,
NTPConsumer;Industrial products

183.31

NA

Inorganic compound;Cadmiumcompound3-13

Molecular StructureIn Vitro Cytotoxicity Test Methods BRD Section 3

Table 3-2

November 2006

Reference Substances Used in the 3T3 and NHK NRU Test Methods Validation Study - Sorted by Toxicity

GHS
Category1/Substance

RodentOral LD502(mg/kg)Source3

Product/Use4

Verapamil HCl

108

MEIC, EDIT,
RC (outlier),
TESS, NTP

Pharmaceutical(antiarrhythmic)

491.08

3.79

Organic compound;
Amine

Haloperidol

128*

MEIC, TESS

Pharmaceutical(antipsychotic)

375.903.36

Organic compound;
KetoneSodium oxalate

155

MEIC, EDIT,
RC, TESS, NTP

Paints;Cleaners

134.00NA

Organic compound;
Carboxylic acid;
Sodium compound

Phenobarbital

163

MEIC, RC(outlier), TESS,NTP

Pharmaceutical(anticonvulsant)232.23

1.47

Organic compound;
Heterocyclic
compoundMolecular
log Kow5
Weight (g/mol)

3-14

Chemical Class6

Molecular StructureIn Vitro Cytotoxicity Test Methods BRD Section 3

Table 3-2

November 2006

Reference Substances Used in the 3T3 and NHK NRU Test Methods Validation Study - Sorted by Toxicity

GHS
Category1/Substance

RodentOral LD502(mg/kg)Source3

Product/Use4

Sodium I fluoride

180

MEIC, RC,
TESS, EPA,
NTPElectroplating;Water fluoridation

41.99

NA

Inorganic compound;
Sodium compound;
Fluorine compound

Caffeine

192

MEIC, RC(outlier), TESS,NTP, HPV

Pharmaceutical(stimulant);
Food additive

194.20–0.07

Organic compound;
Heterocyclic
compoundDiquat dibromide

231

MEIC, RC,TESS

Pesticide(herbicide)362.10–3.05

Organic compound;
Heterocyclic
compoundCupric sulfate * 5 H2O

300

MEIC, RC,
TESS, EPA,
NTP

Pesticide(insecticide/fungicide)249.70NA

Inorganic compound;
Sulfur compound;
Metal

Molecular
log Kow5
Weight (g/mol)

3-15

Chemical Class6

Molecular StructureIn Vitro Cytotoxicity Test Methods BRD Section 3

Table 3-2

November 2006

Reference Substances Used in the 3T3 and NHK NRU Test Methods Validation Study - Sorted by Toxicity

GHS
Category1/Substance

RodentOral LD502(mg/kg)Source3

Product/Use4

Molecular
log Kow5
Weight (g/mol)

Chemical Class6

300 < LD50 ≤2000 mg/kg

Amitriptyline HCl

319

MEIC, EDIT,
RC, TESS

Pharmaceutical(antidepressant)313.905.04

Organic compound;
Polycyclic
compoundPhenol414

MEIC, RC,
TESS, EPA,
NTP, HPVDisinfectant

94.11

1.46

Organic compound;
Phenol

Propranolol HCl

470**

MEIC, RC,
TESS, GD

Pharmaceutical(antiarrhythmic)

295.803.09

Organic compound;
Alcohol; Amine;
Polycyclic compound

Chloral hydrate

479

MEIC, RC,
TESS, NTP

Pharmaceutical(sedative)

165.400.99

Organic compound;
Alcohol

3-16

Molecular StructureIn Vitro Cytotoxicity Test Methods BRD Section 3

Table 3-2

November 2006

Reference Substances Used in the 3T3 and NHK NRU Test Methods Validation Study - Sorted by Toxicity

GHS
Category1/Substance

RodentOral LD502(mg/kg)Source3

Product/Use4

Glutethimide

600

MEIC, RC,TESS

Pharmaceutical(sedative)

217.301.9

Organic compound;
Heterocyclic
compoundAtropine sulfate

623

MEIC, EDIT,
RC, TESS

Pharmaceutical(antimuscarinic)

694.801.83

Organic compound;
Heterocyclic
compoundValproic acid1695 **RC, MEIC,
TESS, NTP

Pharmaceutical(anticonvulsant)144.202.75

Organic compound;
Carboxylic acid;
LipidsMolecular
log Kow5
Weight (g/mol)

3-17

Chemical Class6

Molecular StructureIn Vitro Cytotoxicity Test Methods BRD Section 3

Table 3-2

November 2006

Reference Substances Used in the 3T3 and NHK NRU Test Methods Validation Study - Sorted by Toxicity

GHS
Category1/Substance

RodentOral LD502(mg/kg)Source3

Product/Use4

Meprobamate

794*

MEIC, TESS

Pharmaceutical(antidepressant)218.30NA

Organic compound;
Carboxylic acid

Acetylsalicylic acid

1000

MEIC, EDIT,
RC, TESS, NTP

Pharmaceutical(analgesic)

180.201.19

Organic compound;
Carboxylic acid;
PhenolLithium I carbonate

7

1187

MEIC, RC,
TESS, NTP (Clsalt)

Pharmaceutical(mood stabilizer)

73.89

NA

Inorganic compound;
Lithium compound;
Alkylies;
Carbon compound

Procainamide

1950*

MEIC, TESS

Pharmaceutical(antiarrythmic)

271.79NA

Organic compound;
Carboxylic acid;
Amide

Molecular
log Kow5
Weight (g/mol)

3-18

Chemical Class6

Molecular StructureIn Vitro Cytotoxicity Test Methods BRD Section 3

Table 3-2

November 2006

Reference Substances Used in the 3T3 and NHK NRU Test Methods Validation Study - Sorted by Toxicity

GHS
Category1/Substance

RodentOral LD502(mg/kg)Source3

Product/Use4

Carbamazepine

1957*

MEIC, TESS

Pharmaceutical(antiepileptic)

Molecular
log Kow5
Weight (g/mol)

236.30Chemical Class6

2.45

Organic compound;
Heterocyclic
compoundMolecular Structure2000 < LD50 ≤5000 mg/kg

Acetaminophen

2404

MEIC, EDIT,
RC, TESS, NTP

Pharmaceutical(analgesic)

151.200.8

Organic compound;
Amide

Potassium I chloride

2602

MEIC, RC,
TESS, NTP

Pharmaceutical(electrolyte);
Manufacturing

74.55

NA

Inorganic compound;
Potassium compound;
Chlorine compound

Boric aid

2660*

TESS, EPA,
NTP

Pesticide(insecticide)

61.83

NA

Inorganic compound;
Boron compound;
Acids

3-19

K+

Cl-

In Vitro Cytotoxicity Test Methods BRD Section 3

Table 3-2

November 2006

Reference Substances Used in the 3T3 and NHK NRU Test Methods Validation Study - Sorted by Toxicity

GHS
Category1/Substance

RodentOral LD502(mg/kg)Source3

Product/Use4

Carbon tetrachloride

2799

MEIC, RC,
TESS, NTP,
HPV

Solvent

153.82

2.83

Organic compound;
Halogenatedhydrocarbon

Dimethylformamide

2800

RC, GD, NTP,
HPV

Solvent

73.10

–1.01

Organic compound;
Amide;
Carboxylic acid

Sodium chloride

2998

MEIC, EDIT,
RC, TESS,EPA, NTP

Pharmaceutical(electrolyte);
Food additive

58.44

NA

Inorganic compound;
Sodium compound;
Chlorine compound

Citric Acid

3000*

EPA, NTP,
HPV

Food additive

192.10–1.72

Organic compound;
Carboxylic acid

Molecular
log Kow5
Weight (g/mol)

3-20

Chemical Class6

Molecular Structure

Na+

Cl-

In Vitro Cytotoxicity Test Methods BRD Section 3

Table 3-2

November 2006

Reference Substances Used in the 3T3 and NHK NRU Test Methods Validation Study - Sorted by Toxicity
RodentOral LD502(mg/kg)Source3

Product/Use4

Chloramphenicol

3393

MEIC, RC,
NTP

Pharmaceutical(antibiotic)

323.14

1.14

Organic compound;
Alcohol;
Cyclic hydrocarbon;
Nitro compound

Lactic acid

3730

RC, NTP, HPV

Food additive

90.08

–0.72

Organic compound;
Carboxylic acid

Acetonitrile

3798

RC, NTP, HPV

Solvent

41.05

–0.34

Organic compound;
NitrileXylene(mixed isomers)

4300

MEIC, RC,
TESS, NTP,
HPV

Solvent

106.17

3.12 – 3.2

Organic compound;
Cyclic hydrocarbon

GHS
Category1/Substance

Molecular
log Kow5
Weight (g/mol)

3-21

Chemical Class6

Molecular StructureIn Vitro Cytotoxicity Test Methods BRD Section 3

Table 3-2

November 2006

Reference Substances Used in the 3T3 and NHK NRU Test Methods Validation Study - Sorted by Toxicity

GHS
Category1/Substance

RodentOral LD502(mg/kg)Source3

Product/Use4

Trichloroacetic acid

4999

RC, NTPFixative

Molecular
log Kow5
Weight (g/mol)

163.40Chemical Class6

1.33

Organic compound;
Carboxylic acid

LD50 >5000 mg/kg

2-Propanol

5843

MEIC, RC,
TESS, EPA,
NTP, HPVDisinfectant

60.10

0.05

Organic compound;
Alcohol

Gibberellic acid

6305

RC, EPA, NTP

Plant growth regulator

346.380.24

Organic compound;
Polycyclic compound

Propylparaben

6326**RC (outlier),
NTP

Food additive

180.203.04

Organic compound;
Carboxylic acid;
Phenol

3-22

Molecular StructureIn Vitro Cytotoxicity Test Methods BRD Section 3

Table 3-2

November 2006

Reference Substances Used in the 3T3 and NHK NRU Test Methods Validation Study - Sorted by Toxicity

GHS
Category1/Substance

RodentOral LD502(mg/kg)Source3

Product/Use4

5-Aminosalicylic acid7749**RC (outlier),
NTP

Pharmaceutical(antibiotic)

153.101.32

Organic compound;
Carboxylic acid;
PhenolEthylene glycol

8567

MEIC, EDIT,
RC, TESS,
NTP, HPV

Antifreeze

62.07

–1.36

Organic compound;
Alcohol

Diethyl phthalate

8602

RC (outlier),
NTP, HPV

Plasticizer

222.202.47

Organic compound;
Carboxylic acid

NA

Inorganic compound;
Sodiumcompound;
Oxygen compound;
Chlorine compound

Sodium hypochlorite

8

8910

TESS, NTP

Molecular
log Kow5
Weight (g/mol)

Disinfectant

74.44

3-23

Chemical Class6

Molecular StructureIn Vitro Cytotoxicity Test Methods BRD Section 3

Table 3-2

November 2006

Reference Substances Used in the 3T3 and NHK NRU Test Methods Validation Study - Sorted by Toxicity

GHS
Category1/Substance

RodentOral LD502(mg/kg)Source3

Product/Use4

1,1,1-Trichloroethane

10298

MEIC, RC,
NTP, HPV

Solvent

133.41

2.49

Organic compound;
Halogenatedhydrocarbon

Dibutyl phthalate

11998

RC (outlier),
NTP, HPV

Plasticizer

278.30

4.9

Organic compound;
Carboxylic acid

Glycerol12691

RC, GD, NTP,
HPV

Solvent

92.09

-1.76

Organic compound;
Alcohol

Methanol

13012

MEIC, EDIT,
RC, TESS,
NTP, HPV

Solvent

32.04

–0.77

Organic compound;
Alcohol

Molecular
log Kow5
Weight (g/mol)

3-24

Chemical Class6

Molecular StructureIn Vitro Cytotoxicity Test Methods BRD Section 3

Table 3-2Reference Substances Used in the 3T3 and NHK NRU Test Methods Validation Study - Sorted by Toxicity

GHS
Category1/Substance

Ethanol

November 2006

RodentOral LD502(mg/kg)Source3

Product/Use4

14008

MEIC, RC(outlier), TESS,EPA, NTP,
HPV

Solvent

Molecular
log Kow5
Weight (g/mol)

46.07

–0.31

Chemical Class6

Molecular Structure

Organic compound;
AlcoholAbbreviations: GHS=Globally Harmonized System of Classification and Labelling of Chemicals (UN 2005); LD50=Dose that produces lethality in 50% of the test animals; K ow=Octanol:water partition
coefficient; EDIT=Evaluation-guided Development of New In vitro Test Batteries (substances in EDIT program are a subset of the MEIC substance set); EPA=Pesticides registered with theEnvironmental Protection Agency; EHS=EPA’s Extremely Hazardous Substance list; HPV=High Production Volume chemicals (i.e., those that are imported into or produced in the United States inamounts >1,000,000 lbs/year); GD=Guidance Document (ICCVAM 2001b); MEIC=Multicentre Evaluation of In Vitro Cytotoxicity; NA=Non applicable; NTP=National Toxicology Program;
RC=Registry of Cytotoxicity with the chemicals classified as regression outliers shown in parentheses; TESS=Toxic Exposure Surveillance System (Litovitz et al. 2000); HSDB=Hazardous Substances
Data Bank; RTECS®=Registry of Toxic Effects of Chemical Substances.
*
From RTECS® (MDL Information Systems 2002).
**
Mouse.
1
GHS category designation for the substance (e.g., LD50 <5 mg/kg)
2
LD50 data are from the Registry of Cytotoxicity (Halle 1998, 2003) and are for rats, unless otherwise noted. The LD50 values are rounded to the nearest whole number.
3
Sources used to identify candidate chemicals.
4
Product/use categories from HSDB (NLM 2002) or RTECS®(MDL Information Systems 2002). Pharmaceutical uses from Gilman et al. (1985) or Thomson PDR® (2004).
5
From HSDB (NLM 2001, 2002) or Material Safety Data Sheets.
6
Based on Medical Subject Heading [MeSH ®] descriptors (NLM 2005).
7
Mouse data for lithium sulfate (Halle 1998, 2003).
8
From HSDB (NLM 2002).

3-25

In Vitro Cytotoxicity Test Methods BRD Section 3

November 2006

Distribution of Candidate Substances and Reference Substances by Source1 and Toxicity Category

Table 3-3

GHS Category(mg/kg)
LD50 ≤5

Reference Substances/ MEIC Reference/
Candidate Substances MEIC Candidates
12/13
2/2

EDIT Reference/
EDIT Candidates
1/1

TESS Reference/
TESS Candidates
3/3

NTP Reference/
NTP Candidates
5/9

HPV Reference/
HPV Candidates
0/0

5 < LD50 ≤50

12/15

6/6

5/5

9/10

8/11

2/5

50 < LD50 ≤300

12/26

11/17

4/5

11/19

9/18

1/3

300 < LD50 ≤2000

12/38

12/29

3/5

12/27

5/23

1/5

2000 < LD50 ≤5000

12/12

6/6

2/2

6/6

12/12

6/6

LD50 >5000

12/12

5/5

2/2

5/5

12/12

8/8

Total

72/116

42/65

17/20

46/70

51/85

18/27

Abbreviations: GHS=Globally Harmonized System of Classification and Labelling of Chemicals (UN 2005); LD50=Dose that produces lethality in 50% of the test animals;
MEIC=Multicentre Evaluation of In Vitro Cytotoxicity; EDIT=Evaluation-Guided Development of In vitro Tests; TESS=Toxic Exposure Surveillance System; NTP=U.S.
National Toxicology Program; HPV=U.S. Environmental Protection Agency (EPA) High Production Volume program.
1
Substances may have been selected from more than one source (see Table 3-2 and Appendix F3).

Selected Substances: Distribution of RC Chemicals and RC Outliers1 by Toxicity Category

Table 3-4

GHS Category(mg/kg)RC Outliers/
Total Chemicals

LD50 ≤55 < LD50 ≤50
50 < LD50 ≤300
300 < LD50 ≤2000
2000 < LD50 ≤5000
LD50 >5000

10/11 (91%)
15/26 (58%)
24/70 (34%)
14/139 (10%)
12/57 (21%)
20/44 (45%)

Candidate
Substances
13
15
26
38
12
12

Total

95/347 (27%)

116

Candidate and Selected Substances
RC Reference /RC Reference Outliers/
RC Candidates
RC Reference Chemicals
9/10
8/9 (89%)
8/10
4/8 (50%)
11/18
5/11 (45%)
9/29
0/9 (0%)10/10
0/10 (0%)
11/11
5/11 (45%)
58/88

22/58 (38%)

Abbreviations: RC=Registry of Cytotoxicity; GHS=Globally Harmonized System of Classification and Labelling of Chemicals (UN 2005); LD50=Dose
that produces lethality in 50% of the test animals.
1
Chemicals falling outside the log 5 (i.e., > ±0.699) prediction interval for the RC millimole regression (Halle 1998, 2003).

3-26

In Vitro Cytotoxicity Test Methods BRD Section 3

November 2006

Among the 58 RC substances selected for use in the validation study, 22 (38%) were outliers
for the RC millimole regression. Toxicity1 was underpredicted for 17 (77%) of these outlier
substances and overpredicted (i.e., predicted LD50 was lower than measured in vivo LD50) for
the remaining five (23%). For the 95 outlier substances in the RC, the number of substances
for which toxicity was over- or under-predicted was approximately the same. Toxicity was
underpredicted for 49 (52%) outliers and overpredicted for 46 (48%) outliers (Halle 1998,
2003). Figure 3-1 shows the 58 RC chemicals selected for testing, in addition to the 289 RC
chemicals that were not selected, and the RC millimole regression. In the figure, the outliers
are those points outside the RC prediction interval. For the 58 RC substances selected fortesting, the majority (17/22) of the outliers are below the RC millimole regression line.
Figure 3-1

The Fifty-Eight (58) Selected RC Reference Substances on the RC
Millimole Regression

103

102

101

100

10-1

10-2

10-3

10-4
10-6

10-5

10-4

10-3

10-2

10-1

100

101

102

103

IC50x(mM)
Abbreviations: RC=Registry of Cytotoxicity; LD50=Dose that produces lethality in 50% of the test animals;
IC50=Test substance concentration that reduces cell viability by 50%.
The 58 RC chemicals tested in the NICEATM/ECVAM validation study are shown by *. The RC regression,log (LD50) = 0.435 x log (IC50x) + 0.625, is shown by the bold line. The lighter lines show the ± log 5 (i.e.,±0.699) prediction interval (Halle 1998, 2003). The open boxes represent the 289 chemicals not included in thevalidation study.

1

Toxicity is inversely proportional to LD50. High LD50 values reflect low toxicity and low LD50 values reflect
high toxicity
3-27

In Vitro Cytotoxicity Test Methods BRD Section 3

November 2006

3.2.2
Chemical Classes Represented by the Selected Reference Substances
Medical subject heading (MeSH®) descriptors from the NLM were used to determine
chemical class designations for the selected substances. Of the 72 reference substances, 57(79%) were organic and 15 (21%) were inorganic. The number of substances in the organic(79) and inorganic (31) subclasses is greater than the number of substances in each classbecause some of the substances are classified in more than one subclass. The most commonly
represented classes of organic compounds were heterocyclics (14/57, 25%), carboxylic acids(14/57, 25%), and alcohols (10/57, 18%). Table 3-5 shows the distribution of the substancesamong the GHS toxicity categories. The 14 heterocyclics were evenly distributed among thefirst four GHS toxicity categories for LD50 ≤2000 mg/kg with the majority of theheterocyclics (11/14) in the categories for LD50 <300 mg/kg. The majority of the carboxylic
acids (12/14) and alcohols (8/10) had an LD50 >300 mg/kg, while the majority of the
inorganics (10/15) had an LD50 <300 mg/kg.
3.2.3
Product/Use Classes Represented by the Selected Reference Substances
Product and use information was obtained from HSDB (NLM 2002) or RTECS® (MDL
Information Systems 2002). The number of assigned uses (77) is greater than the number of
selected substances because some of the substances have more than one use. Table 3-6
shows the distribution of products and uses of the selected substances according to their GHS
categories. Pharmaceutical (27/77; 35%) and pesticide (17/77; 22%) uses were observed
most frequently. The toxicity category of 300 < LD50 ≤2000 mg/kg had the highest numberof pharmaceuticals. Every toxicity category except LD50 >5000 mg/kg had at least four
substances with pharmaceutical uses. The majority of pesticides (16/17; 94%) had an LD50
<300 mg/kg. The next most frequent uses were as solvents (8/77; 10%) and food additives(5/77; 6%); LD50 >2000 mg/kg contained most of the substances with solvent (8/8; 100%)and food additive (4/5; 80%) uses.
3.2.4

Toxicological Characteristics of the Selected Reference Substances

3.2.4.1 Corrosivity
The intent of the SMT was to prioritize only those substances with low corrosivity because
guidelines for acute systemic toxicity testing indicate that corrosive or severely irritating
substances need not be tested (OECD 2001a, c, d). The UN and U.S. Department ofTransportation Packing Group (DOT PG) classification system was used to classify the
corrosivity hazard associated with the candidate substances. However, after substanceselection was completed and testing had begun, the SMT learned that the PG classification
system was also based on hazards other than corrosivity (e.g., dermal and inhalation toxicity,
flammability, etc.). Therefore, the selected substances were not actually prioritized bycorrosivity. Subsequent information on the corrosivity of the selected substances was
obtained from HSDB (NLM 2004) and the Material Safety Data Sheets (MSDS) provided
with the purchased substances. Seven substances that were not identified by the DOT PG
classification system had corrosive notations. The MSDS notations for lactic acid, sodium
hypochlorite, sodium oxalate, and trichloroacetic acid indicated that these substances should
carry a corrosive label. Chloral hydrate, mercury II chloride, and potassium cyanide werenoted by HSDB to be corrosive to eyes or skin.

3-28

In Vitro Cytotoxicity Test Methods BRD Section 3

Table 3-5

November 2006

Distribution of Chemical Class for the 72 Reference Substances by Toxicity Category

Chemical Class1

GHS Acute Oral Toxicity Category (mg/kg)
50 < LD50 ≤300 300 < LD50 ≤2000 2000 < LD50 ≤5000

LD50 >5000

Total

LD50 ≤55 < LD50 ≤50

Carboxylic acid

1

0

1

4

4

4

14

Heterocyclic compound

5

2

4

3

0

0

14

Alcohol

2

0

0

2

1

5

10

Phenol

0

0

1

2

0

2

5

Polycyclic compound

0

2

0

2

0

1

5

Sulfur compound

4

1

0

0

0

0

5

Amine

1

0

1

1

0

0

3

Cyclic hydrocarbon

0

0

1

0

1

1

3

Halogenated hydrocarbon
Organophosphorouscompound
Amide

0

0

1

0

1

1

3

2

1

0

0

0

0

3

0

0

0

1

2

0

3

Nitrile

0

1

0

0

1

0

2

Acyclic hydrocarbon

1

0

0

0

0

0

1

Carbohydrate

0

1

0

0

0

0

1

Ester

0

1

0

0

0

0

1

Ether

0

1

0

0

0

0

1

Ketone

0

0

1

0

0

0

1

Lipid

0

0

0

1

0

0

1

Nitro compound

0

0

0

0

1

0

1

Organometallic compound

0

1

0

0

0

0

1

Sodium compound

0

0

1

0

0

0

1

Urea

1

0

0

0

0

0

1

Total Organics

17

11

11

16

11

14

79

Organic

3-29

In Vitro Cytotoxicity Test Methods BRD Section 3

Table 3-5

November 2006

Distribution of Chemical Class for the 72 Reference Substances by Toxicity Category

Chemical Class1

GHS Acute Oral Toxicity Category (mg/kg)
50 < LD50 ≤300 300 < LD50 ≤2000 2000 < LD50 ≤5000

LD50 >5000

Total

LD50 ≤55 < LD50 ≤50

Sodium compound

1

2

1

0

1

1

6

Chlorine compound

1

0

1

0

2

1

5

Arsenical

0

2

0

0

0

0

2

Metal

0

1

1

0

0

0

2

Potassium compound

0

1

0

0

1

0

2

Sulfur compound

0

1

1

0

0

0

2

Acid

0

0

0

0

1

0

1

Alkalies

0

0

1

0

0

0

1

Boron compound

0

0

0

0

1

0

1

Cadmium compound

0

0

1

0

0

0

1

Carbon compound

0

0

0

1

0

0

1

Chromium compound

0

1

0

0

0

0

1

Fluorine compound

0

0

1

0

0

0

1

Lithium compound

0

0

0

1

0

0

1

Mercury compound

1

0

0

0

0

0

1

Nitrogen compound

0

1

0

0

0

0

1

Oxygen compound

0

0

0

0

0

1

1

Selenium compound

1

0

0

0

0

0

1

Total Inorganic

4

9

7

2

6

3

31

Inorganic

Abbreviations: GHS=Globally Harmonized System of Classification and Labelling of Chemicals (UN 2005).
1
Based on the Medical Subject Heading [MeSH®] descriptor (NLM 2005). Some substances are counted more than once because they appear in more than one subclassunder the organic or inorganic classes.

3-30

In Vitro Cytotoxicity Test Methods BRD Section 3

Table 3-6

November 2006

Distribution of Product/Use1 Class for the 72 Reference Substances by Toxicity Category

Product/Use Class1

GHS Acute Oral Toxicity Category (mg/kg)
50< LD50 ≤300 300< LD50 ≤2000 2000< LD50 ≤5000
0
0
0

5< LD50 ≤50
0Antifreeze

0

0

0

0

0

1

1

Consumer/industrial products

0

0

1

0

0

0

1

Disinfectant

0

0

1

1

0

2

4

Electroplating

0

2

0

0

0

0

2

Fluoridation

0

0

1

0

0

0

1

Feed additive

1

0

0

0

0

0

1

Fixative

0

0

0

0

1

0

1

Food additive

0

0

1

0

3

1

5

Manufacturing

1

0

0

0

1

0

2

Oxidizing agent

0

1

0

0

0

0

1

Paints, cleaners

0

0

1

0

0

0

1

Pesticide

5

7

4

0

1

0

17

Pharmaceutical

4

3

4

11

4

1

27

Plant growth regulator

0

0

0

0

0

1

1

Plasticizer

0

0

0

0

0

2

2

Preservative

1

0

0

0

0

0

1

Solvent

0

0

0

0

4

4

8

Antibiotic/fungicide

LD50 >5000
0

Total

LD50 ≤51

1

Abbreviations: GHS=Globally Harmonized System of Classification and Labelling of Chemicals (UN 2005).
1
Product/use information from Hazardous Substances Data Bank (NLM 2002) or Registry of Toxic Effects of Chemical Substances ([RTECS®], MDL Information Systems 2002).
Some substances are counted more than once because they appear in more than one use category.

3-31

In Vitro Cytotoxicity Test Methods BRD Section 3

November 2006

3.2.4.2 Toxicity Targets
As shown in Appendix F2, the most common toxicological effects in humans or rodents
were neurological (40 substances); 26 cause central nervous system (CNS) depression, sevenproduce CNS stimulation, four produce CNS affects such as encephalopathy, and three affectthe peripheral nervous system. Other common target systems include the liver (17substances), kidney (15 substances), and cardiovascular system (10 substances). No target
organ information was available for gibberellic acid. Among the 72 reference substances, 27
had more than one toxicity target.
3.2.4.3 Metabolism
Table 3-7 shows the 22 reference substances that are known or expected to produce
active/toxic metabolites in vivo. In contrast, dichlorvos, fenpropathrin, meprobamate,phenylthiourea, and sodium dichromate are rapidly metabolized to less toxic compounds.
Because the NHK and 3T3 cells have little (Babich 1991) or no (INVITTOX 1991)
metabolic capability, respectively, metabolites of these compounds would not be expected to
be present in vitro. Appendix F2 provides for more information on the metabolism(activation/inactivation) of the selected reference substances.
Table 3-7

Reference Substances Metabolized to Active Metabolites
Active Metabolites
Expected

Known to Have Active Metabolites
Acetaminophen

Carbamazepine

Digoxin

Methanol

Carbon tetrachloride

Acetonitrile

Chloral hydrate

Disulfoton

Parathion

Triethylenemelamine

Acetylsalicylic acid

Cycloheximide

Ethanol

Procainamide HCl

Valproic acid

Amitriptyline HCl

Dibutyl phthalate

Ethylene glycol

Verapamil HCl

Busulfan

Diethyl phthalate

Glutethimide

3.2.5
Selection of Reference Substances for Testing in Phases Ib and II
Based on the Guidance Document (ICCVAM 2001b) recommendation that 10 to 20
substances be tested to qualify candidate in vitro cytotoxicity tests for determining starting
doses for rodent acute oral toxicity assays, 12 reference substances were chosen from amongthe 72 reference substances for testing in Phases Ib and II (see Table 3-8). The criteria forchoosing these reference substances, in order of importance, were:• Two reference substances must be included from each of the five GHS
toxicity categories and the unclassified category.
• The log LD50 (mmol/kg) must be within the prediction interval (±0.699) of the
RC millimole regression. The Guidance Document (ICCVAM 2001b)
recommends that reference substances for evaluating an in vitro basal
cytotoxicity test to use with the RC millimole regression fit the regression as
closely as possible.
• MEIC chemicals must be included. Cytotoxicity data from these phases (andPhase III of this study), and the available human toxicity information for the
MEIC chemicals, could be used to build a prediction model for estimating

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human LC values. The Phase Ib reference substances arsenic trioxide andethylene glycol are also EDIT chemicals (subset of MEIC chemicals).
If more than two substances in a GHS category met the above criteria, reference substanceswere selected so that the LD50 was as close to the RC millimole regression as possible and/or
to represent the full range of toxicity in each GHS category.
Table 3-8

Reference Substances Tested in Phases Ib and II

Reference Substances

Aminopterin
Sodium selenate
Colchicine
Arsenic III trioxide

RC
MEIC
Reference Reference
No.
No.
LD50 ≤5 mg/kg
54-62-63NA13410-01-0
NANA
5 < LD50 ≤50 mg/kg

CASRN

64-86-81327-53-3

6
153

60
26

Rodent Oral
LD501
(mg/kg)Observed –Predicted
log LD502

3
1.63-0.652NA64
20-0.593-0.591

88
180

0.011
-0.1094704
11874,5-0.023-0.2564

2602
3393

0.085
0.441

5843
8567

0.396
0.321

50 < LD50 ≤300 mg/kg
Cadmium II chloride
Sodium I fluoride

10108-64-2
7681-49-4

81
106

NA
14

300 < LD50 ≤2000 mg/kg
DL-Propranolol HCl
Lithium I carbonate

350-60-90
544-13-2

54
3274

23
20

2000 < LD50 ≤5000 mg/kg
Potassium I chloride
Chloramphenicol
2-Propanol
Ethylene glycol

7447-40-7
56-75-7

346
50
91
45
LD50 >5000 mg/kg
67-63-0
128
10
107-21-1
360
7Abbreviations: CASRN=Chemical Abstracts Service Registry Number; RC=Registry of Cytotoxicity; MEIC=Multicentre
Evaluation of In Vitro Cytotoxicity; NA=Not applicable (i.e., substances not included in the RC and/or MEIC studies);
RTECS®=Registry of Toxic Effects of Chemical Substances.
1
From the RC (Halle 1998, 2003) unless otherwise indicated. Data are for rats unless otherwise indicated.
2
Available only for substances included in the RC. This figure characterizes the log LD50 deviation from the RC regression.
Outliers are > ±0.699 from the regression line.
3
RTECS® (MDL Information Systems 2002).
4
Mouse data.
5
For lithium sulfate.

Only nine of the 72 reference substances met all three criteria. In the most toxic category(i.e., LD50 ≤5 mg/kg), only one RC chemical, aminopterin, was within 0.699 of the RC
millimole regression. Sodium selenate was selected as the second reference substance in thiscategory even though its fit to the RC millimole regression was not known. Neither
aminopterin nor sodium selenate were MEIC chemicals. For the 50 < LD50 ≤300 mg/kg
category, cadmium chloride was selected over the MEIC chemicals cupric sulfate 5H2O,
diquat dibromide, sodium oxalate, and hexachlorophene because it fit the RC millimole
regression better than the four MEIC chemicals (the observed LD50 minus log predicted LD50
values were -0.534 to -0.337).

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November 2006

3.2.6
Unsuitable and Challenging Reference Substances
Several reference substances could not be adequately tested for cytotoxicity in 3T3 cells
and/or NHKs in from one to all three of the laboratories. The following reference substances
did not produce sufficient toxicity at soluble concentrations for calculation of an IC50 at thehighest concentrations tested under the testing conditions used in the study (see also Tables
5-2, 5-4, and 5-5):• Carbon tetrachloride (no 3T3 or NHK NRU IC50 data from ECBC, FAL, or
IIVS)• Xylene (no 3T3 or NHK NRU IC50 data from ECBC or FAL)
• Methanol (no 3T3 NRU IC50 data from ECBC, FAL, or IIVS; no NHK NRU
IC50 data from ECBC)• Lithium carbonate (no 3T3 NRU IC50 data from FAL or IIVS)• 1,1,1-Trichloroethane (no 3T3 NRU IC50 data from FAL or IIVS; no NHK
NRU IC50 data from ECBC)• Valproic acid (no 3T3 NRU IC50 data from ECBC or FAL; no NHK NRU
IC50 data from ECBC, FAL, or IIVS)
Other reference substances were difficult to test because of volatility or lack of toxicity, butthree acceptable tests could be obtained after a number of trials.
• Acetonitrile and 2-propanol were highly volatile and nontoxic, so that evenwith the use of film plate sealers, from one to seven tests failed the VC anddata points test acceptance criteria at each laboratory.
• Disulfoton failed at least one test in both test methods at ECBC and FAL
because of inadequate toxicity (i.e., an IC50 could not be detected) andinsolubility. All laboratories reported precipitate in the test plates for 3T3 andNHK NRU tests. IIVS had no failed tests in either test method.
• Dibutyl phthalate failed one 3T3 NRU test at ECBC and one NHK NRU testat FAL because of inadequate toxicity and solubility.
• Lindane failed one 3T3 NRU test at FAL because of inadequate toxicity andsolubility and one because of its volatility.
• Parathion failed one test because of inadequate toxicity and solubility in bothtest methods and one NHK NRU test because of volatility at FAL.
• Diethyl phthalate failed one NHK NRU test because of volatility at FAL.
• Digoxin (all laboratories), gibberellic acid (ECBC and FAL), and strychnine(ECBC and FAL) failed at least one 3T3 NRU test because of inadequate
toxicity and solubility.
3.3

Reference Substance Procurement, Coding, and Distribution

BioReliance collected information from the suppliers of the reference substances on theiranalytical purity, composition, and stability (see Appendix F1), tested the reference
substances for solubility, packaged them into 4 g aliquots for shipment to the testinglaboratories, and archived two additional samples. All reference substances were given arandom number code that was unique for each testing facility to conceal the identities fromthe testing laboratories. Approximately 100 g of the PC substance, SLS, was distributed,
uncoded, to each laboratory and one additional sample was archived.

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November 2006

Reference substances were packaged so as to minimize damage during transit, and shipped
under appropriate storage conditions and according to the appropriate regulatorytransportation procedures. Testing facilities were notified upon shipment in order to prepare
for receipt. With the exception of the PC substance which was shipped directly to the Study
Directors, the reference substances were shipped to the test facility Safety Officers.
Shipments were accompanied by a sealed information packet containing the appropriate
health and safety procedures (i.e., MSDS or equivalent documentation with information
regarding the proper protection for handling, procedures for dealing with accidental ingestion
or contact with skin or eyes, and for containing and recovering spills), and a code disclosure
key. Also provided was a data sheet giving a minimum of essential information needed by
the testing laboratory for each reference substance, including color, odor, physical state,weight or volume of sample, specific density for liquid reference substances, and storage
instructions. The shipment directed the Safety Officer to:• Notify BioReliance and the SMT upon receipt of reference substances• Retain the health and safety package and provide the coded reference
substances and chemical data sheets with minimum essential information to
the laboratory Study Director without revealing the identities of the test
substances• Notify the SMT if test facility personnel open the health and safety packet atany time, for any reason, during the study• Return the unopened health and safety package to BioReliance after testing iscompleted
3.3.1
Exceptions
The Safety Officer for ECBC required the information on reference substance codes before
the substances were shipped in order to satisfy the facility’s environmental procedures andrequirements. The reference substance codes were stored in a classified safe located in the
Safety Office which was in a building separate from the cytotoxicity testing laboratory, andwere to be opened only by the Safety Officer. The ECBC Safety Officer opened the sealed
health and safety packets for lithium carbonate and ethanol upon receipt of those substances
because the code information for these substances was not included in the list originallyprovided. ECBC cytotoxicity testing personnel did not have direct access to the reference
substance codes.
3.4

Reference Substances Recommended by the Guidance Document

The Guidance Document specifically recommended testing the following 11 substances tovalidate candidate in vitro basal cytotoxicity assays: sodium dichromate dihydrate, cadmium
chloride, p-phenylenediamine, DL-propranolol HCl, trichlorfon, ibuprofen, nalidixic acid,salicylic acid, antipyrene, dimethylformamide, and glycerol (ICCVAM 2001b). Of these 11
substances (see Appendix F3 and Section 3.1.2), five (sodium dichromate dihydrate,cadmium chloride, DL-propranolol HCl, dimethylformamide, and glycerol) were chosen for
testing after the candidate substances were prioritized as described in Section 3.1.3. The
seven that were not selected did not satisfy the selection criteria (e.g., not MEIC chemicals,
not identified as high exposure risk in TESS)

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3.5

November 2006

Summary

Seventy-two reference substances were selected for testing in the NICEATM/ECVAM
validation study. These substances were selected to represent: (1) the complete range of invivo acute oral LD50 values; (2) the types of substances regulated by the various regulatory
authorities; and (3) those with human toxicity data and/or human exposure potential. To
insure that the complete range of toxicity was covered, the GHS (UN 2005) was used to
select 12 substances for each acute oral toxicity category and 12 unclassified substances. The
set of selected reference substances had the following characteristics:• Thirty-five percent (27/77 uses) were pharmaceuticals, 22% (17/77 uses) werepesticides, 10% (8/77 uses) were solvents, and 6% (5/77 uses) were foodadditives. The remaining substances were used for a variety of manufacturing
and consumer products.
• In terms of relevance of the substances to human exposure, 58% (42/72) were
included in the MEIC study (substances chosen because of availability ofhuman lethality data), 24% (17/72) were included also in the EDIT program(EDIT substances are a subset of the MEIC substances), 64% (46/72) had
human exposure data reported by TESS, 71% (51/72) had been evaluated by
NTP, and 25% (18/72) were on the EPA HPV list.
• Eighty-one percent (58/72) of the substances were in the RC and 38% (22/58)
of these were outliers with respect to the RC millimole regression. The RC
millimole regression underpredicted the toxicity of 77% (17/22) of the outliers
and overpredicted the toxicity of 23% (5/22). For the 95 outlier substances in
the RC, however, the number of substances for which toxicity was over- or
under-predicted was approximately the same (i.e., toxicity was underpredicted
for 49 [52%] outliers and overpredicted for 46 [48%] outliers [Halle 1998,
2003]).
• Seventy-nine percent (57/72) were organic compounds and 21% (15/72) were
inorganic. The most commonly represented classes of organic compounds
were heterocyclics (25%, 14/57), carboxylic acids (25%, 14/57), and alcohols(18%, 10/57).
• Nineteen substances (26%, 19/72,) were known to have active metabolites andthree others were expected to have active metabolites based on their chemical
structures.
• Many of the substances produced toxicity in more than one organ system. The
most common target systems were neurological (40 substances), liver (17substances), kidney (15 substances), and cardiovascular (10 substances). No
target organ information was available for one substance (gibberellic acid).

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November 2006

4.0

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

4.1

Methods Used to Obtain Rodent Acute Oral LD50 Reference Values................. 4-3
4.1.1 Identification of Candidate Rodent Acute Oral LD50 Reference Data .......... 4-3
4.1.2 Criteria Used to Select Candidate Rodent Acute Oral Data for
Determination of LD50 Reference Values...................................................... 4-5

4.2

Final Rodent Acute Oral LD50 Reference Values ................................................. 4-7

4.3

Relevant Toxicity Information for Humans .......................................................... 4-8

4.4

Accuracy and Reliability of the Rodent Acute Oral LD50 Reference
Values ...................................................................................................................... 4-13
4.4.1 Variability Among the Acceptable LD50 Values ......................................... 4-13
4.4.2 Comparison of Rodent Acute Oral LD50 Reference Values with theCorresponding RC LD50 Values .................................................................. 4-14
4.4.3 Comparison of the Variability Among Acceptable LD50 Values toThose Obtained in Other Studies ................................................................. 4-15

4.5

Summary................................................................................................................. 4-16

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4-2

In Vitro Cytotoxicity Test Methods BRD Section 4

4.0

November 2006

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

The procedures and analyses presented in this section were designed to identify the mostaccurate rodent acute oral LD50 values for the 72 reference substances used in the validation
study. These values were needed to ensure that the reference substances were correctly
placed within the different GHS toxicity categories and to provide a data set against which tocompare the predicted LD50 values estimated using the IC50 data obtained from the 3T3 andNHK NRU test methods (see Section 6). The predicted LD50 values are used to determine thestarting dose for rodent acute oral toxicity tests and the more accurate the prediction, thefewer the number of rodents that would be used in an acute oral toxicity test (see Sections
1.0 and 1.2.2).
4.1

Methods Used to Obtain Rodent Acute Oral LD50 Reference Values

4.1.1
Identification of Candidate Rodent Acute Oral LD50 Reference Data
No animal testing was performed to obtain the rodent oral acute LD50 reference data for thisvalidation study. To identify reference data for the 72 substances, rat acute oral LD50 studies
were located using literature searches, secondary references, and electronic databasesearches. Literature searches were conducted in PubMed (U.S. NLM) and the Institute of
Scientific Information (ISI) Web of Science® (Thomson Scientific, Philadelphia, PA) using
each chemical name and “lethal dose 50” as search terms. Secondary references included
NTP technical reports, Toxicological Profiles from the Agency for Toxic Substances andDisease Registry (ATSDR), Cosmetic Ingredient Reviews by the Cosmetics Industry
Council, pesticide handbooks, the Merck Index, and various other summary sources. Table
4-1 lists the electronic databases searched to locate references for rat oral LD50 values. Rat
LD50 data were preferred because:• The current acute oral toxicity test guidelines recommend using rats (OECD2001a, c, d; EPA 2002a)• The majority of LD50 data used in the RC millimole regression were fromstudies using rats (282 rat data points and 65 mouse data points) (Halle 1998,
2003)• The majority of acute oral systemic toxicity testing is performed with rats
Table 4-1

Internet-Accessible Databases Searched for LD50 Information

Database/Source1
Agency for Toxic Substances and Disease Registry(ATSDR)
Center for Drug Evaluation and Research (CDER)
CHEMFINDER
Chemical Carcinogenesis Research Information
System (CCRIS); National Cancer Institute (NCI)
WebsiteSponsor(s)U.S. Department of Health and Human Services (DHHS)
U.S. Food and Drug Administration (FDA)
CambridgeSoft Corporation
NCI; National Institutes of Health (NIH); DHHS
Michigan Department of Natural Resources; Ontario
Ministry of the Environment; Canadian Centre for
Occupational Health and Safety (CCOHS)
CHEMpendium
U.S. Coast Guard

Chemical Evaluation Search and Retrieval System(CESARS)
Chemical Hazard Response (CHRIS)
4-3

In Vitro Cytotoxicity Test Methods BRD Section 4

Table 4-1

November 2006

Internet-Accessible Databases Searched for LD50 Information
Database/Source1

Sponsor(s)U.S. Environmental Protection Agency (EPA) Office ofPesticide Programs (OPP); California EPA Department of
Pesticide Regulation(CCOHS) CHEMpendiumChemical Ingredients Database
CHEMINDEX; CHEMINFOChemRTK High Production Volume (HPV)
Challenge Program; OPPT Chemical Fact Sheets;
Chemical Information Collection and Data
Development

EPA Office of Pollution Prevention and Toxics (OPPT)World Health Organization (WHO) International
Programme on Chemical Safety (IPCS); CCOHS;
International Labour Organisation (ILO) Occupational
Safety and Health Information Centre (CIS)
WHO IPCS; CCOHS; ILO; United Nations Environment
Programme (UNEP)
U.S. Consumer Product Safety Commission (CPSC)
Zentralstelle zur Erfassung und Bewertungvon Ersatz- und
Erganzungsmethoden zum Tierversuch (ZEBET) [German
Centre for the Documentation and Validation of Alternative
Methods]
EPA; The National Library of Medicine (NLM); The
National Institute of Environmental Health Sciences(NIEHS); National Center for Toxicological Research(NCTR)
Transport Canada; U.S. Department of Transportation(DOT); Secretariat of Communications and Transportationof Mexico

CIS Chemical Information
Concise International Chemical Assessment
Documents (CICADS)
Consumer Product Safety Commission Website
Deutsches Institut fur Medizinische Dokumentation
und Information (DIMDI) [The German Institute for
Medical Documentation and Information];
Registry of Cytotoxicity (RC)
Developmental and Reproductive
Toxicology/Environmental Teratology Information
Center (DART®/ETIC)
Emergency Response Guidebook (ERG 2000)
Environmental Health Criteria (EHC) monographs;
Health and Safety Guides (HSG); International
Agency for Research on Cancer (IARC)
European Centre for the Validation of Alternative
Methods (ECVAM) Scientific Information Service(ECVAM SIS)
HAZARDTEXT®; MEDITEXT®; INFOTEXT®;
SARATEXT®; REPROTEXT®; REPROTOX®
Integrated Risk Information System (IRIS)International Chemical Safety Cards (ICSC)
IPCS/EC Evaluation of Antidotes Series
International Uniform Chemical Information
Database (IUCLID)
Joint Expert Committee on Food Additives(JECFA); Joint Meeting on Pesticide Residues(JMPR); Pesticide Data Sheets (PDS)
Material Safety Data Sheets (MSDS)
Multicentre Evaluation of In Vitro Cytotoxicity(MEIC)The National MSDS Repository
National Toxicology Program (NTP) Chemical
Health and Safety Database
National Transportation Library
New Jersey Hazardous Substance Fact Sheets
Oil and Hazardous Materials/Technical Assistance

WHO IPCS; CCOHS
European Commission Joint Research Centre
TOMES Plus®, MICROMEDEX, Greenwood Village, CO
EPA Office of Research and Development (ORD)
WHO IPCS; CCOHS; Commission of the European Union(EU)
European Chemicals Bureau
WHO IPCS; CCOHS; Food and Agriculture Organization(FAO) of the United Nations
Interactive Learning Paradigms, Incorporated
Scandinavian Society for Cell Toxicology
MSDSSEARCH, Inc.
NIEHS
DOT
New Jersey Department of Health and Senior Services
EPA Office of Waste and Water Management

4-4

In Vitro Cytotoxicity Test Methods BRD Section 4

Table 4-1

November 2006

Internet-Accessible Databases Searched for LD50 Information

Database/Source1
Data System (OHM/TADS)
Organisation for Economic Co-operation andDevelopment (OECD) Screening Information Data
Sets (SIDS)
Pesticide Action Network Pesticide Database
Pesticide Product Information System (PPIS)
Poisons Information Monographs (PIMs)
Registry of Toxic Effects of Chemical Substances(RTECS®);NIOSH Pocket Guide to Chemical
Hazards
SCORECARD

Sponsor(s)IPCS; CCOHS; International Register of Potentially Toxic
Chemicals (IRPTC); UNEP
Pesticide Action Network North America
EPA Office of Pesticide Programs (OPP)
IPCS; CCOHS
National Institute for Occupational Safety and Health(NIOSH)
Environmental Defense
University of California, Davis; Oregon State University;
Michigan State University; Cornell University; Universityof Idaho

The EXtension TOXicology NETwork(EXTOXNET)

Office of Management and Budget Watch; Center for
Public Data access

The Right-to-Know Network (RTK NET)
Toxic Chemical Release Inventory (TRI);
GENE-TOX
Toxic Substances Control Act Test Submissions(TSCATS)TOXLINE®; Hazardous Substances Data Bank(HSDB); ChemIDplus

The National Library of Medicine (NLM)
EPA OPPTNLM (TOXNET)

Abbreviations: LD50=Dose lethal to 50% of the animals tested
1
Includes public and proprietary databases

A total of 195 references containing LD50 data retrieved through these searches werereviewed and evaluated. Information regarding the materials, animals, and methods used toderive the 491 LD50 values reported by these references were compiled and are provided inAppendix H1. Appendix H2 provides a narrative characterization and evaluation of theLD50 values.
4.1.2

Criteria Used to Select Candidate Rodent Acute Oral Data for Determination ofLD50 Reference Values
This effort was to designed to derive a set of high quality reference oral LD50 values fromdata that were collected using standardized protocols, accompanied by documentation
showing that established testing procedures were followed in compliance with national andinternational GLP guidelines (OECD 1998; FDA 2003; EPA 2003a,b). After a review of thecollected data, the SMT determined that a requirement for GLP compliance would eliminate
99% (452 of the 459 values remaining after exclusion of 30 duplicate values and twoerroneous values) of the oral LD50 values.
The SMT then considered limiting the selection of LD50 values to those from studies thatused the specifications for animals recommended by the current acute oral toxicity test
guidelines. The current guidelines recommend using young adult rats, 8 to 12 weeks of age,of a common laboratory strain (e.g., Sprague-Dawley) and the most sensitive sex (OECD2001a, c, d; EPA 2002a). Female animals are recommended if there is no information fromwhich to determine the most sensitive sex. A limited number of LD50 values were available
4-5

In Vitro Cytotoxicity Test Methods BRD Section 4

November 2006

from animals that fit this description; only 3% (14/459) of the oral LD50 values were
determined using 8 to 12 week old female laboratory rats. An additional 15 LD50 values wereobtained from female rats in an appropriate weight range (age not provided in the reference)for that age range (~ 176-250 g according to Charles River [http://www.criver.com], Harlan[http://www.harlan.com/us/index.htm], and Taconic Farms[http://www.taconic.com/anmodels/spragued.htm] websites). Thus, only 6% (29/459) of theacute oral LD50 values in the database, covering 21 of the 72 reference substances (29%),
were from studies that used the strain, sex, and age of rats recommended by current test
guidelines (OECD 2001a; EPA 2002a).
4.1.2.1 Final Exclusion Criteria
Because so few studies met the initial criteria (i.e., GLP compliance and use of animals
recommended by current acute oral toxicity test guidelines), the database was reviewed andevaluated to derive alternative criteria for the development of reference LD50 values. For thisevaluation, the SMT looked for commonalities among the data records that, when selected,
provided a comparable data set for each chemical. Review of the available data indicated thatthe majority of acute oral toxicity tests were conducted by gavage to unanesthetized, young
adult laboratory rats of both genders. Thus, the selection process was revised to exclude
studies that reflected the following, less typical, materials, animals, and methods in order tocompile a homogenous set of reference LD50 values for each chemical. The studies excluded
were those with:• Feral rats
• Rats <4 weeks of age• Anesthetized rats
• Test chemical administered in food or capsule• LD50 reported as a range or inequality
Data from feral rats were excluded because the health status and age of these animals was
uncertain. All laboratory rat strains/stocks were deemed acceptable on the assumption thatthey were healthy and provided with adequate care and housing during testing. Data fromneonates and weanlings were excluded because their sensitivity to chemical toxicity may
differ from that of adults. Four weeks was considered the minimum acceptable age because
rats are typically weaned at approximately three weeks of age (Barrow 2000). Data fromfeeding experiments or experiments that involved administration of the chemical in capsules
were also excluded because gavage is the most common mode of administration for acuteoral studies and the rate of gastrointestinal absorption for these other methods is likely to bedifferent (Nebendahl 2000). Because LD50 point estimates are required for the prediction
model, LD50 values reported as ranges or inequalities were unacceptable.
4.1.2.2 Assumptions Regarding Materials, Animals, and Methods
The level of detail for describing the materials, animals, and methods for the LD50 studies
varied greatly. For example, some studies reported only that white rats were used, whileothers provided complete information on stock/strain, gender, and age of animals. Details onother protocol components such as the number of animals tested per dose group, method ofadministration, doses administered, clinical signs, and times of death varied as well. In order
to use as much of the available data as possible, the following assumptions were made if astudy report did not state otherwise:
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In Vitro Cytotoxicity Test Methods BRD Section 4•••

November 2006

Rats were young adults of a common laboratory strain
Rats were not anesthetized
Oral route of administration was by gavage

4.1.2.3 Calculation of Reference LD50 Values
If a substance had multiple LD50 values after the application of the exclusion criteria, theoutliers at the 99% level (Dixon and Massey 1981) were excluded. A geometric mean and
95% confidence limits were calculated from the remaining values, and used as the reference
LD50. A geometric mean was used because it is the antilog of the mean of the logarithm of
the values and is less affected than the arithmetic mean by extreme values. The use of ageometric mean also corresponds with the approach used for the RC millimole regression toderive a single IC50 value from multiple IC50 values (Halle 1998, 2003), and with theapproach used to derive the IC50 value for each chemical for the in vitro - in vivo regressions
evaluated in the NICEATM/ECVAM validation study (see Section 6).
In addition to the statistical evaluation of outliers, an extreme value, which was not a
statistical outlier but was based on biological plausibility, was identified for trichloroacetic
acid. This chemical had five reported LD50 values ranging from 400-8900 mg/kg after
applying the exclusionary criteria. The lowest value (400 mg/kg) was rejected as biologicallyimplausible because up to 1000 mg/kg/day had been used in an oral chronic rodent
carcinogenicity study with no, or only minimal, toxicity (EPA 1996).
4.1.2.4 Use of Rat and Mouse Data
If no rat oral LD50 values could be found for a reference substance, mouse acute oral LD50
values were evaluated using the same approach as was used for rat values. Because an IC50LD50 regression model using only rat data was preferable, the three reference substances (i.e.,epinephrine bitartrate, colchicine, and propylparaben) for which mouse values only wereavailable were not used for the evaluations of accuracy (Section 6) or animal reduction(Section 10).
4.2

Final Rodent Acute Oral LD50 Reference Values

After the application of the exclusionary criteria, there were 385 acceptable rodent acute oral
LD50 values from which to calculate reference LD50 values. Table 4-2 shows the reference
LD50 value for each substance in descending order of toxicity, presented both as mg/kg andas mmol/kg. Data are presented as mmol/kg in order to be consistent with the RC approach.
The RC millimole regression used units of mmol/kg for the LD50 and mM for the IC50 (seeSection 1.1.3). Also shown for each substance are the 95% confidence limits around thegeometric mean, the ratio of the maximum to the minimum acceptable value, the number ofLD50 values used to calculate the reference value, the number of LD50 values available (notincluding duplicate values or erroneous values), and the LD50 value initially used for hazard
classification of the reference substance (see Table 3-2).
Table 4-2 lists the reference substances grouped by GHS acute oral toxicity category (UN2005) using the reference LD50 values that were derived as described above. The initialcategorization for this study, which used the LD50 values in the far right column of Table 4-2(i.e., values reported in Table 3-2, which come from the RC unless otherwise specified),
placed 12 substances in each toxicity category. Table 4-3 compares the number of substancesin each GHS toxicity category based on their reference LD50 values with the number in each
4-7

In Vitro Cytotoxicity Test Methods BRD Section 4

November 2006

category based on the initial LD50 values. The initial and reference LD50 values placed 53(74%) of the substances in the same GHS category. Nineteen substances (26%) werereclassified based on the reference LD50 values (this value is the sum of the numbers in the
discordant cells in Table 4-3). Compared with the initial LD50 value, the reference LD50
value was higher for 18 (25%) and lower for only one (1%) of the substances.
Of the 19 reference substances that were reclassified because of the reference LD50 values,
five substances originally assigned to the most toxic, LD50 ≤5 mg/kg, category (i.e.,aminopterin, mercury chloride, busulfan, parathion, and strychnine) were moved to the next,
less toxic, category (5< LD50 ≤50 mg/kg). In the 5< LD50 ≤50 mg/kg category, four
substances (dichlorvos, fenpropathrin, sodium dichromate dihydrate, and nicotine) moved to
the less toxic 50< LD50 ≤300 mg/kg category, and one (triphenyltin hydroxide) moved two
categories to 300< LD50 ≤2000 mg/kg. In the 50< LD50 ≤300 category, four substances(haloperidol, caffeine, copper sulfate pentahydrate, and sodium oxalate) moved to a lower
toxicity category (300< LD50 ≤2000 mg/kg). Only carbamazepine moved from the 300<
LD50 ≤2000 mg/kg category to the 2000< LD50 ≤5000 mg/kg category. In the 2000< LD50
≤5000 mg/kg category, citric acid, trichloroacetic acid and dimethylformamide moved to thenext lower toxicity category (LD50 >5000 mg/kg). In the LD50 >5000 mg/kg category, 5aminosalicylic acid moved to the higher toxicity, 2000< LD50 ≤5000 mg/kg category. This
was the only substance that moved to a more toxic category
4.3

Relevant Toxicity Information for Humans

The relevance of rodent acute oral LD50 data to human LC values was assessed by the MEIC
program (Ekwall et al. 1998b), which used mouse and rat oral LD50 data from RTECS®
(Ekwall et al. 1998a). Mean lethal doses in humans were collected primarily from handbooks
containing human clinical toxicity information (Ekwall et al. 1998a) supplemented, whennecessary, by an in-house compendium from the Swedish Poisons Information Centre.
Ekwall et al. (1998b) calculated least squares linear regressions for the prediction of themean human LC values by rat and/or mouse oral LD50 data for the 50 MEIC substances using
units of log mol/kg. They reported a correlation of R2 =0.607 for the rat oral LD50 predictionof mean human LC values and R2 =0.653 for the mouse oral LD50 prediction of mean human
LC values. It is important for comparisons of MEIC data with rodent LD50 values to note that
the MEIC human values are not lethal doses, and therefore not equivalent to LD50 values.
Many of the values (if not the majority) are blood concentrations that were associated withmorbidity or mortality, and usually do not reflect the actual dose consumed by the patient.
These are not necessarily the peak blood concentrations, but only the concentrations at thetime of ascertainment, which could have ranged from immediately after onset of medical
treatment to post-mortem. The MEIC organizers readily admitted that they could not relate
the blood concentrations to the administered dose.
The relevance of the NRU data collected in the NICEATM/ECVAM validation study to theprediction of human acute toxicity will be addressed elsewhere by ECVAM in a separate
evaluation.

4-8

In Vitro Cytotoxicity Test Methods BRD Section 4

Table 4-2

November 2006

Rodent Acute Oral Reference LD50 Values Listed by GHS Category1

GHS Category1/
Reference Substance

Reference
Acute Oral
LD502,3(mg/kg)95%
ConfidenceInterval4(mg/kg)Cycloheximide
Phenylthiourea
Sodium selenate
Epinephrine bitartrate
Triethylenemelamine
Physostigmine
Disulfoton

2
3
3
4 (mouse)
4
5
5

NC
NC
NC
NC
1-25
NC
2-10

Parathion
Strychnine
Aminopterin
Potassium cyanide
Busulfan
Colchicine
Thallium I sulfate
Arsenic III trioxide
Endosulfan
DigoxinMercury II chloride
Sodium arsenite

6
6
7
7
12
15 (mouse)
25
25
28
28
40
44

3-12
NC
NC
5-10
NC
NC
NC
10-64
NC
NC
27-60
36-53

51

44-58

34.17-64.5

59
70
76
82
93
100
111

40-88
68-72
57-10068-98
65-13278-129NC

17-97.5
68-71
48.5-16456-21557-11588-125108-114

Sodium dichromate
dihydrate
Dichlorvos
Nicotine
Fenpropathrin
Hexachlorophene
Paraquat
LindaneVerapamil HCl

Reference
Reference
Acute Oral
Acute Oral
LD50 Range5
LD502(mg/kg)(mmol/kg)
LD50 ≤5 mg/kg (N =7)
1-2.5
0.0071130.0197
1.6-5.98
0.015940.01961-13
0.012050.0182
2.3-12.6
0.01825 < LD50 ≤50 mg/kg (N =12)
1.8-300.02092.35-16.2
0.0188
7
0.016
5-10
0.111
1.9-290.049
5.886-29
0.037525
0.049513-81.5
0.127
18-43
0.068
28
0.0362
12-92
0.148
36-53
0.336
50 < LD50 ≤300 mg/kg (N =12)

4-9

95%
ConfidenceInterval4(mmol/kg)

Maximum:
Minimum
Value6N

Initial Rodent
Acute Oral
LD503,7(mg/kg)

NC
NC
NC
NC
0.0037-0.12
NC
0.009-0.036

2.5
NC
3.7
NC
13.0
NC
5.5

3
1
2
1
4
1
6

2
3
28
4 (mouse)
158
20.010-0.041
NCNC
0.077-0.15
0.008-0.38
NCNC
0.050-0.32
NCNC
0.010-0.22
0.28-0.40

16.7
6.9
NC
2.0
15.3
4.9
NC
6.3
2.4
NC
7.7
1.5

10
3
1
7
4
3
1
5
2
1
10
5

2
28
3 (mouse)102
6 (mouse)
29 (mouse)
20
188
18 (mouse)
1
418

0.193

0.17-0.22

1.9

11

50

0.266
0.430
0.217
0.202
0.498
0.344
0.226

0.18-0.40
0.42-0.44
0.16-0.29
0.17-0.24
0.35-0.71
0.27-0.44
NC

5.7
1.0
3.4
3.8
2.0
1.4
1.1

9
4
9
19
5
4
2

178
50
188
61
58
76
108

In Vitro Cytotoxicity Test Methods BRD Section 4

Table 4-2

November 2006

Rodent Acute Oral Reference LD50 Values Listed by GHS Category1

Sodium I fluoride
Cadmium II chloride
Diquat dibromide
Phenobarbital

Reference
Acute Oral
LD502,3(mg/kg)
127
135
160
224

95%
ConfidenceInterval4(mg/kg)
92-17588-208NC
NC

Caffeine
Triphenyltin hydroxide
Haloperidol
Amitriptyline HCl
Propranolol HCl
Cupric sulfate ● 5 H2O
Phenol

310
329
330
348
466
474
548

256-374
208-520NCNCNC
269-836434-692

Lithium carbonate590

479-728

Glutethimide
Sodium oxalate
Chloral hydrate
Atropine sulfate
Valproic acid
Meprobamate
Acetylsalicylic acid
Procainamide HCl

600
633
638
819
995
1387
1506
1950

NC
NC
391-1040
641-1045NC
1291-1489
1224-1854
NC

Acetaminophen
Potassium I chloride
Carbamazepine
Boric aid
5-Aminosalicylic acid
Chloramphenicol
Acetonitrile
Lactic acid

2163
2799
2805
3426
3429
3491
3598
3639

GHS Category1/
Reference Substance

Reference
Reference
Acute Oral
Acute Oral
LD50 Range5
LD502(mg/kg)(mmol/kg)
64-2793.020
88-211
0.738
121-2310.466
162-3180.966
300 < LD50 ≤2000 mg/kg (N =16)
192-483
1.59
46.4-1200
0.896
128-8500.877
320-380
1.18
466
1.575
236.2-960
1.90
317-1500
5.82
525-710

7.98

600
2.76
558-7074.724
479-8633.86
600-1136
1.21
670-1480
6.91
1286-1522
6.35
616-2840
8.36
1950
8.286
2000 < LD50 ≤5000 mg/kg (N =11)
NC
1944-2404
14.3
NC
2600-3020
37.6
NC
1957-4025
11.9
2617-4486
2660-5140
55.4
NC
2800-4200
22.4
NC
2500-5000
10.8
2951-4375
1320-8120
87.6
NC
3543-3730
40.3

4-10

95%
ConfidenceInterval4(mmol/kg)
2.19-4.16
0.48-1.14
NC
NC

Initial Rodent
Acute Oral
LD503,7(mg/kg)
180
88
231
163

Maximum:
Minimum
Value6

N

4.4
2.4
1.9
2.0

12
5
3
3

1.32-1.93
0.57-1.42
NCNCNC
1.08-3.35
4.82-7.68

2.5
25.9
6.6
1.2
NC
4.1
4.7

10
15
2
2
1
6
14

6.5-9.9

1.4

4

NC
NC
2.36-6.29
0.95-1.54
NC
5.92-6.82
6.8-10.3
NC

NC
1.3
1.8
1.9
2.2
1.2
4.6
NC

1
211
4
7
2
6
1411
1

192
44
1288
319
470 (mouse)
300
414
1187 (mouse;
sulfate salt)600
155 (mouse)9479
623
1695 (mouse)
7948
1000
19508

NC
NC
NC
42.3-72.6
NCNC
71.9-107
NC

1.2
1.2
2.1
1.9
1.5
2.0
6.2
1.1

2
2
2
6
2
3
26
2

2404
2602
19578
26608
7749 (mouse)
3393
3798
3730

In Vitro Cytotoxicity Test Methods BRD Section 4

Table 4-2

November 2006

Rodent Acute Oral Reference LD50 Values Listed by GHS Category1

Carbon tetrachloride
Sodium chloride
XyleneReference
Acute Oral
LD502,3(mg/kg)
3783
4046
4667

95%
ConfidenceInterval4(mg/kg)3024-4732
2917-5623
1294-16827

2-Propanol
Trichloroacetic acid
Dimethylformamide
Citric Acid
Gibberellic acid
Propylparaben
Ethylene glycol
Methanol
Dibutyl phthalate
Diethyl phthalate
Sodium hypochlorite
Ethanol
1,1,1-Trichloroethane
Glycerol

5105
5229
5309
5929
6040
6332 (mouse)
7161
8710
8892
9311
10328
11324
12078
19770

4624-5636
2745-9961
3548-7925
NCNCNC
6266-8204
6223-12218
6180-12794
NCNC
8610-14894
10000-14588
10495-37154

GHS Category1/
Reference Substance

Reference
Reference
Acute Oral
Acute Oral
LD50 Range5
LD502(mg/kg)(mmol/kg)
2350-10054
24.6
3000-6140
69.3
1537-8620
43.9
LD50 >5000 mg/kg (N =14)
4500-5840
84.9
3320-8900
32.0
2800-7182
72.6
3000-11700
30.9
5780-6300
17.4
6332
35.1
4000-9900
115.4
5628-12880
272
7499-12436
31.9
8600-10100
41.9
8200-13000
62.8
7060-17775
245.7
9600-16000
90.5
12600-27650
215

95%
ConfidenceInterval4(mmol/kg)
20-31
50-96
12-15877-94
16.8-61.0
49-108NC
NC
NC
101-132
194-38122-46
NCNC
187-32375-109114-403

Maximum:
Minimum
Value6

N

4.3
2.0
5.6

15
5
4

Initial Rodent
Acute Oral
LD503,7(mg/kg)
2799
2998
4300

1.3
2.7
2.6
3.9
1.1
NC
2.5
2.3
1.7
1.2
1.6
2.5
1.7
2.2

6
4
6
2
2
1
16
6
4
2
2
8
6
4

5843
4999
2800
30008
6305
6326 (mouse)
8567
13012
11998
8602
891010
14008
10298
12691

Abbreviations: LD50=dose lethal to 50% of the animals tested; GHS=Globally Harmonized System of Classification and Labelling of Chemicals(UN 2005); N=Number of acceptable values used for geometric mean; NC=Not calculated.
1
Categorized using the reference oral LD50.
2
Based on a geometric mean of acceptable LD50 values from adult laboratory rats unless otherwise specified.
3
Values rounded to the nearest whole number.
4
For the geometric mean of the acceptable LD50 values, NC is used for substances with three acceptable values or less, which was considered
too few for calculation of a valid confidence interval.
5
Range of acceptable oral LD50 values.
6
Ratio of minimum acceptable LD50 to maximum acceptable LD50.
7Values rounded to the nearest whole number. Values are from the RC unless otherwise specified; rat data unless otherwise specified.
8
RTECS® (MDL Information Systems 2002).
9
RC reference for rat oral LD50 of 155 mg/kg is Shrivastava et al. (1992), which references Klinger and Kersten (1961). Klinger and Kersten(1961) indicate the value was determined by intraperitoneal administration to mice.
10
HSDB (NLM 2002).
11
An erroneous value obtained from the literature was not included.

4-11

In Vitro Cytotoxicity Test Methods BRD Section 4

Table 4-3

November 2006

GHS Category Matches for the Rodent Acute Oral LD50 Initial and Reference Values
Reference LD50 (mg/kg)

Initial LD50
(mg/kg1)

Total

Category
Match

Reference
LD50
Lower

Reference
LD50
Higher

LD50 ≤55 < LD50 ≤50

50 < LD50 ≤300

300 < LD50 ≤2000

2000 < LD50 ≤5000

LD50 >5000

LD50 ≤5

7

5

0

0

0

0

12

58%

0%

42% (5)

5 < LD50 ≤50

0

7

4

1

0

0

12

58%

0%

42% (5)50 < LD50 ≤300

0

0

8

4

0

0

12

67%

0%

33% (4)300 < LD50 ≤2000

0

0

0

11

1

0

12

92%

0%

8% (1)

2000 < LD50 ≤5000

0

0

0

0

9

3

12

75%

0%

25% (3)

LD50 >5000

0

0

0

0

1

11

12

92%

8%

0% (0)

Total

7

12

12

16

11

14

72

74%

1%

25% (18)

Abbreviations: GHS=Globally Harmonized System of Classification and Labelling of Chemicals (UN 2005); LD50=Dose lethal to 50% of animals tested.
Note: Shaded cells show the number of chemicals for which both LD50 categories agree.
1
Initial LD50 values were used for reference substance selection and were obtained from the RC (Halle 1998, 2003), RTECS® (MDL Information Systems
2002), and HSDB (NLM 2002) (see Table 3-2).

4-12

In Vitro Cytotoxicity Test Methods BRD Section 4

4.4

November 2006

Accuracy and Reliability of the Rodent Acute Oral LD50 Reference Values

Accuracy (concordance) is the closeness of agreement between a test method result and an
accepted reference value (in this case to the rodent acute oral LD50 measurement) (ICCVAM
2003). Because there are insufficient data to permit a comparison between rodent and humanlethal doses, the accuracy of rodent acute oral LD50 values for predicting the oral LD50 inhumans cannot be determined. Acute toxicity testing in rodents leads to a relative ranking of
the toxicity of chemicals for regulatory purposes, with the default assumption that the rodent
values and ranking are predictive of the human values and ranking.
The among laboratory reproducibility of the reference LD50 values determined in this section
may be judged by evaluating the range of acceptable LD50 values for each reference
substance and by comparing the values (and their variability) with the variability of LD50
values derived from controlled acute oral toxicity studies.
4.4.1
Variability Among the Acceptable LD50 Values
The variability among the acceptable rodent acute oral LD50 values used to calculate thereference LD50 value for each reference substance was assessed by calculating the ratio of the
maximum to the minimum value (see Table 4-2). For the 62 reference substances with morethan one acceptable LD50 value, the maximum:minimum ratio ranged from 1.1 to 25.9, with amean of 4.3 and a median of 2.2. The maximum:minimum ratios were greater than 10 for
four substances: triethylenemelamine, parathion, busulfan, and triphenyltin hydroxide. The
low LD50 values for triethylenemelamine, busulfan, and parathion may have contributed tothe high maximum:minimum ratios. The four LD50 values for triethylenemelamine ranged
from 1 to 13 mg/kg, the four values for busulfan ranged from 1.9 to 29 mg/kg, and the 10
values for parathion ranged from 1.8 to 30 mg/kg.
Table 4-4 shows the maximum:minimum LD50 ratios by toxicity category. The more toxic
substances (i.e., LD50 ≤50 mg/kg) tended to have higher maximum:minimum ratios thansubstances with lower toxicity (i.e., LD50 >50 mg/kg). This is anticipated because small dayto-day, or laboratory-to-laboratory variations in weighing and dosing the lower
concentrations would have a higher impact on the chemicals being administered in low doses
than those being administered in the high dose range.
Table 4-4

Maximum:Minimum LD50 Ratios by GHS Toxicity Category

GHS Category(LD50 in mg/kg)
LD50 ≤55 < LD50 ≤50
50 < LD50 ≤300
300 < LD50 ≤2000
2000 < LD50 ≤5000
LD50 >5000

Mean
Maximum:Minimum
LD50 Ratio
6.2
7.1
2.4
4.6
2.6
2.3

MedianMaximum:Minimum
LD50 Ratio
4.6
6.3
1.9
2.2
2.0
2.3

Range ofMaximum:Minimum
LD50 Ratio
2.5 – 13.0
2.0 - 16.7
1.1 - 5.7
1.2 - 25.9
1.2- 22.3
1.1 - 3.9

N
4
9
12
13
11
13

Abbreviations: LD50=Dose lethal to 50% of animals tested; GHS-Globally Harmonized System of Classification andLabelling of Chemicals (UN 2005); N=Number of chemicals with more than one acceptable LD50 value after application of
the exclusion criteria described in Section 4.1.2.

4-13

In Vitro Cytotoxicity Test Methods BRD Section 4

November 2006

4.4.2

Comparison of Rodent Acute Oral LD50 Reference Values with the Corresponding
RC LD50 Values
The correspondence of the rodent acute oral LD50 reference values with the RC LD50 values
for the 58 reference substances in common with the RC are shown on a log scale in Figure 41. Not surprisingly, a Spearman correlation analysis for the two sets of log transformed
values yielded a significant correlation (p <0.0001) with a correlation coefficient, rs, of 0.97.
Figure 4-1 shows that the LD50 reference values tended to be higher than the RC LD50
values. One factor in this difference is that the majority of LD50 values used in the RC werefrom the 1983/84 RTECS®, which contains the lowest LD50 value found for a particularchemical without regard to the available methodological information, without considerationof whether it is an outlier with respect to the other available values, and without scientificreview before publication. Thus, because the reference LD50 values are based on thegeometric mean from multiple studies, it is not surprising that these values tended to behigher than the single values in the RC database.
Correlation of LD50 Values With the Reference LD50 Values for the 58
RC Chemicals

Figure 4-1
100000

10000

1000
Triphenyltinhydroxide100

Mercury
chloride
Busulfan

10

1

0.1
0.1

1

10

100

1000

10000

100000Registry of Cytotoxicity LD50 (mg/kg)
Abbreviations: LD50=Dose lethal to 50% of animals tested; RC=Registry of Cytotoxicity.
The diagonal line shows the 1:1 relationship.

4-14

In Vitro Cytotoxicity Test Methods BRD Section 4

November 2006

When comparing the reference LD50 values to the RC values, the substances with the largest
differences were busulfan, triphenyltin hydroxide, and mercury chloride (see Figure 4-1).
• The LD50 reference value for busulfan was six times that of the RC value (12mg/kg vs. 1.9 mg/kg). The RC value (from 1983/84 RTECS®) was from apaper by Schmahl and Osswald (1970) in which they cited a rat oral LD50 of
1.86 mg/kg. The literature also contained rat oral LD50 values of 28 and 29
mg/kg for male and female Sprague-Dawley rats, respectively (Matsuno et al.
1971).
• The LD50 reference value for triphenyltin hydroxide was 7.5 times the RC
LD50 (329 mg/kg vs. 44 mg/kg). The 15 LD50 values used to determine thereference value included the RC value, and had a wide range, 44-1200 mg/kg.
Because of the large variation in the data, which was evenly distributed
throughout the range neither the highest nor the lowest values were outliers.
• The LD50 reference value for mercury chloride was 40 mg/kg, while the RC
value was 1 mg/kg. The RC value was from a summary document thatreported the rat oral LD50 as a range of 1-5 mg/kg (Worthing and Walker
1991). Because it was reported as a range, it was excluded from thecalculation of the reference value (see Section 4.1.2.1). The remaining 11
values ranged from 12 to 160 mg/kg. The highest value (160 mg/kg) was
considered an outlier when compared to the other 10 values and therefore
excluded from the reference value calculation.
4.4.3

Comparison of the Variability Among Acceptable LD50 Values to Those Obtainedin Other Studies
The variation seen here for 62 reference substances is not atypical, considering the results ofother studies that examined the variation among rodent acute oral LD50 values derived for thesame substance. For example, Weil and Wright (1967) showed that LD50 values varied by asmuch as five-fold for the 10 substances tested in eight laboratories using exactly the same
protocol. Another international study involving 65 participating laboratories in eight
countries that did not control the LD50 protocols among laboratories, reported
maximum:minimum ratios from 3.6 to 11.3 (with LD50 values ranging from 44 to 5420
mg/kg) for five substances (Hunter et al. 1979). The chemicals tested, and the LD50 ranges
were:• PCP144-523 mg/kg• Sodium salicylate
800-4150 mg/kg• Aniline
350-1280 mg/kg• Acetanilide
805-5420 mg/kg• Cadmium chloride
70-513 mg/kg
The results of a follow-on study in which the same substances were tested by 100
laboratories in 13 countries showed that adherence to a specific protocol reduced the range ofmaximum:minimum LD50 ratios from 3.6 to 11.3 to 2.4 to 8.4 (Zbinden and Flury-Roversi
1981).

1

Compound undefined in the publication.
4-15

In Vitro Cytotoxicity Test Methods BRD Section 4

November 2006

Although the LD50 data collected from the literature for the NICEATM/ECVAM validation
study used various rat strains, sexes, observation durations, and calculation methods for
estimating the LD50, the variation in LD50 values for individual substances was similar to thedata of the earlier cited studies. The current study found four of the 62 substances withmultiple LD50 values had maximum:minimum LD50 values higher than that reported by
Hunter et al. (1979) (i.e., >11.3), and three of those were in the highest toxicity category.
Hunter et al. (1979) also observed that the largest variation was associated with the more
highly toxic substances.
4.5

Summary

To enable the comparison of in vitro NRU data with rodent acute oral toxicity data, LD50
reference values for the 72 reference substances were calculated using data obtained from theliterature, database searches, and secondary references. Rat acute oral LD50 values werepreferred, but mouse acute oral LD50 values were collected for three substances with noavailable or acceptable rat data. The 491 LD50 values that were retrieved comprised 485 rat
LD50 values and six mouse values. It was not possible to identify a high quality data set
produced under GLP guidelines because only 3% of the data records were in GLP
compliance. Instead, as described in Section 4.1.2.1, a homogenous set of LD50 values foreach substance was identified by applying specific exclusion criteria related to the materials,
animals, and methods used for each study.
After analysis of the acceptable values for outliers, the remaining 385 values were used toderive rodent acute oral LD50 reference values by calculation of a geometric mean of thevalues for each substance. As a result of this procedure, the LD50 reference values for 19 of
the 72 reference substances were sufficiently different from the values that were used in the
RC and other summary sources, so that they were reclassified into different GHS oral
toxicity categories.
Because there is no reference standard against which to evaluate the accuracy of the rodent
acute oral toxicity test, the reliability of the LD50 reference values was assessed bycomparison to other evaluations of the performance of this test method. The
maximum:minimum ratio of the acceptable values for the 62 reference substances that had
more than one LD50 value ranged from 1.1 to 25.9, and the ratios for four of the substanceswere greater than one order of magnitude.

4-16

In Vitro Cytotoxicity Methods BRD Section 5

November 2006

5.0

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

5.1

Study Timeline and Participating Laboratories ................................................... 5-3
5.1.1 Statements of Work (SOW) and Protocols .................................................... 5-3
5.1.2 Study Timeline............................................................................................... 5-4
5.1.3 Participating Laboratories.............................................................................. 5-4

5.2

Coded Reference Substances and GLP Guidelines............................................... 5-5
5.2.1 Coded Reference Substances ......................................................................... 5-5
5.2.2 Lot-to-Lot Consistency of Reference Substances.......................................... 5-5
5.2.3 Adherence to GLP Guidelines ....................................................................... 5-5

5.3

3T3 and NHK NRU Test Method Protocols .......................................................... 5-5
5.3.1 Phase Ia: Laboratory Evaluation Phase.......................................................... 5-6
5.3.2 Phase Ib: Laboratory Evaluation Phase ......................................................... 5-6
5.3.3 Phase II: Laboratory Qualification Phase ...................................................... 5-7
5.3.4 Phase III: Main Validation Phase .................................................................. 5-9

5.4

Data Used to Evaluate Test Method Accuracy and Reliability ......................... 5-10
5.4.1 PC Data ........................................................................................................ 5-10
5.4.2 Reference Substance Data............................................................................ 5-12

5.5

Statistical Approaches to the Evaluation of 3T3 and NHK Data ...................... 5-26
5.5.1 Statistical Analyses for Phase Ia .................................................................. 5-27
5.5.2 Statistical Analyses for Phase Ib.................................................................. 5-28
5.5.3 Statistical Analyses for Phase II .................................................................. 5-28
5.5.4 Statistical Analyses for Phase III ................................................................. 5-29
5.5.5 Summary of the Data Used for Statistical Analyses.................................... 5-32

5.6

Summary of NRU Test Results............................................................................. 5-32

5.7

Availability of Data ................................................................................................ 5-38

5.8

Solubility Test Results ........................................................................................... 5-38
5.8.1 Solubility Data ............................................................................................. 5-43
5.8.2 Solubility and Volatility Effects in the Cytotoxicity Tests.......................... 5-43

5.9

Summary................................................................................................................. 5-44

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In Vitro Cytotoxicity Methods BRD Section 5

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[This Page Intentionally Left Blank]

5-2

In Vitro Cytotoxicity Methods BRD Section 5

5.0

November 2006

3T3 AND NHK NRU TEST METHOD DATA AND RESULTS

This section summarizes the IC50 results generated by testing 72 coded reference substances(see Section 3) in the 3T3 and NHK NRU test method protocols. These IC50 values wereused to evaluate the accuracy (also known as concordance - see Section 6) of the two in vitro
cytotoxicity test methods for predicting in vivo GHS acute oral toxicity categories and theirreliability (intra- and inter-laboratory reproducibility - see Section 7). The individual test
data for the passing and failing tests are provided in Appendix I for the reference substances
and the PC. The raw data for each test (in EXCEL® and PRISM® files) are available uponrequest from NICEATM on compact disk(s), as are the laboratory reports. Requests can bemade by mail, fax, or e-mail to Dr. William S. Stokes, NICEATM, NIEHS, P. O. Box 12233,
MD EC-17, Research Triangle Park, NC, 27709, (phone) 919-541-2384, (fax) 919-541-0947,(e-mail) niceatm@niehs.nih.gov.
Section 5.1 discusses the timeline for the validation study, the study participants, and theirroles in the study. Section 5.2 documents the use of coded reference substances and the GLP
compliance by the participating laboratories. Section 5.3 discusses the protocol revisions thatwere made during the study and the effect the revisions had on the results. Section 5.4
presents the IC50 data collected during each phase to assess the reliability and accuracy(relevance) of the NRU methods. Section 5.5 presents the statistical analyses performed.
Section 5.6 summarizes the results of IC50 comparisons of the 3T3 and NHK methods.
Section 5.7 offers information about the availability of all the data (e.g., raw OD data fromall tests, laboratory reports), and Section 5.8 presents the solubility test results for thereference substances from all laboratories.
5.1

Study Timeline and Participating Laboratories

5.1.1
Statements of Work (SOW) and Protocols
The SMT provided the laboratories with SOWs for each test method prior to initiation of
testing (see Appendix G), and proposed dates for completion of the various aspects of thestudy (e.g., transfer of data, provision of reports). The SOWs defined the following:• Project objectives• Management and key personnel• Required facilities, equipment, and supplies• Quality assurance requirements• Test phases and schedules• Products (e.g., reports) required• Report preparation
The SOW for BioReliance contained all of the above requirements, and also included
requirements for:• Reference substance acquisition, coding, preparation, and distribution• Solubility testing
The SMT, in consultation with the laboratories, prepared Test Method Protocols for eachphase of the study. Cytotoxicity testing in each phase of the validation study was initiated ineach laboratory when the SMT received a signed protocol specific for that phase from the
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In Vitro Cytotoxicity Methods BRD Section 5

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Study Director. Solubility testing for the Phases I and II substances was performed prior tocytotoxicity testing for those substances; most of the solubility testing for the Phase III
substances was performed toward the end of Phase II and during the early part of Phase III.
5.1.2
Study Timeline
The actual timeline of the study is shown in Table 5-1. The SMT modified the original
timeline presented in the SOWs because of a number of factors, such as, protocol revisions,side studies, difficulties with acquisition of medium, etc.
Table 5-1

Validation Study Timetable

Event
Receipt of SOW from SMT
Procurement of Test
Substances
Solubility Testing
Completed
Distribution of Reference
Substances
Phase Ia
Phase Ib
Phase II
Phase III
Initiation of Phase Ia
Completion of Phase Ia
Initiation of Phase Ib
Completion of Phase Ib
Initiation of Phase II
Completion of Phase II
Initiation of Phase III
Completion of Phase III

BioRelianceJun 2002

ECBC
Jun 2002

FAL
Jun 2002

IIVS
Jun 2002

Jul 2002 - Jan 2003

NA

NA

NA

Jul 2002 - Jan 2003

Dec 2003

Dec 2003

Jan 2004

NA

NA

NA

Aug 2002
Nov 2002
Dec 2002
May 2003
Jun 2003
Nov 2003
Dec 2003
Dec 2004

Aug 2002
Nov 2002
Dec 2002
May 2003
Jun 2003
Nov 2003
Dec 2003
Dec 2004

Aug 2002
Oct 2002
Dec 2002
May 2003
Jun 2003
Nov 2003
Dec 2003
Jan 2005

Jul 2002
Sep 2002
Nov 2002
Feb - Mar 2003
NA
NA
NA
NA
NA
NA
NA
NA

Abbreviations: ECBC=Edgewood Chemical Biological Center; FAL=Fund for the Replacement of Animals in Medical
Experiments Alternatives Laboratory; IIVS=Institute for In Vitro Sciences; SOW=Statement of Work; SMT=StudyManagement Team; NA=Not applicable.
Note: BioReliance distributed the reference substances and performed solubility testing. ECBC, FAL, and IIVS tested thereference substances for solubility and in vitro cytotoxicity.

5.1.3

Participating Laboratories• BioReliance Corporation
14920 Broschart Road
Rockville, Maryland 20850-3349
Study Director: Dr. Martin Wenk
• U.S. Army Edgewood Chemical Biological Center (ECBC)
Molecular Engineering Team
Aberdeen Proving Ground, MD 21010
Study Director: Dr. Cheng Cao

5-4

In Vitro Cytotoxicity Methods BRD Section 5

•

•

5.2

November 2006

Institute for In Vitro Sciences (IIVS)
21 Firstfield Road Suite 220
Gaithersburg, MD 20878
Study Director: Mr. Hans Raabe
Fund for the Replacement of Animals in Medical Experiments Alternatives
Laboratory (FAL)
Queens Medical Centre, University of Nottingham
Nottingham NG7 2UH
United Kingdom
Study Director: Dr. Richard Clothier

Coded Reference Substances and GLP Guidelines

5.2.1
Coded Reference Substances
BioReliance acquired 73 substances (72 reference substances and one PC substance) from
reputable commercial sources (see Appendix F1). All but eight of the reference substanceswere >99% pure (see Section 8.1.2.1). BioReliance coded each substance with a unique,random identification number when repackaging them into smaller units for distribution to
the laboratories. These units were given an additional code unique to the respective
cytotoxicity laboratories, so that they could be provided in a blinded fashion (see Section 3.4
for distribution procedures). The coded substance units were packaged and shipped such that
their identities were concealed; however, all laboratories knew the identity of the positive
control. The SMT revealed the codes for each phase after all laboratories had submitted theirdata and reports for that phase. The laboratories periodically required additional aliquots ofreference substance, and BioReliance provided these aliquots from the original stock ofreference substance in the same manner that the original aliquots were provided.
5.2.2
Lot-to-Lot Consistency of Reference Substances
Each substance was purchased as a single lot, and each laboratory received aliquots from thissame lot throughout the validation study. The reference substance suppliers provided
certificates of analysis for each lot, along with the MSDS documents containing substance,
physical, and safety and handling information.
5.2.3
Adherence to GLP Guidelines
BioReliance, ECBC, and IIVS, followed GLP procedures for all testing, with the exceptionof tests designed to resolve technical challenges (e.g., formation of NR crystals; use of film
plate sealers for volatile substances; slow growth of cells). The laboratories submitted all data
to their respective quality assurance units (as per GLP requirements) and copies of the data
were submitted to NICEATM. FAL followed most of the GLP guidelines, but did not employ
independent quality assurance reviews of laboratory procedures or documentation. The Study
Director for FAL performed all data reviews and provided copies to NICEATM. Hard copy
printouts and electronic versions of all data are available at NICEATM.
5.3

3T3 and NHK NRU Test Method Protocols

The protocols for the 3T3 and NHK NRU test methods used during Phase III laboratory
testing were the result of modifications and revisions to the Guidance Document (ICCVAM
2001b) protocols, the optimization of the protocols used in the laboratory evaluation Phases
Ia and Ib, and the laboratory qualification phase (Phase II) (see Section 2.6). Figure 1-2

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In Vitro Cytotoxicity Methods BRD Section 5

November 2006

provides an outline of the study phases, and identifies where repeated observations werecarried out to permit protocol evaluation and comparison. Sections 2.2 and 2.3 address thesimilarities and differences between the 3T3 and NHK protocols. The remaining subsectionsin Section 5.3 address the modifications to the protocols used in each phase, and how thosemodifications affected each data set.
5.3.1
Phase Ia: Laboratory Evaluation Phase
During Phase Ia, each laboratory established an historical database for the PC substance,
SLS. No reference substances were tested in this phase. Ten concentration-response tests
were performed using SLS and no more than two tests were performed/day. The resulting
data were used to calculate the acceptable response limits for the SLS IC50 for use during
Phase Ib testing.
Section 2.6.1 summarizes issues that occurred during Phase I and addresses protocol changes
made after the initiation of Phase Ia. The specific changes to the protocols for both cellsystems are summarized below, along with the impact these changes had on the test data.
Changes made in the protocols during Phase Ia were incorporated into the Phase Ib protocols.
5.3.1.1

Protocol Changes and the Effect on the Data
• NR Dye Crystals: Reduced the NR dye concentration for both cell types. No
subsequent tests failed because of NR crystal formation. The background OD
values decreased and this was not interpreted as a negative effect on the data.
• 3T3 Cell Growth: Modified cell culture conditions for 3T3 cells to improvecell growth characteristics. No apparent effect on the data was detected.
• NHK Cell Growth (96-well plates): Removed the cell culture refeeding step
performed prior to reference substance addition. Although the OD values forthe vehicle controls became higher, the SLS IC50 results were similar whether
or not the cells were re-fed.
• NHK Cell Growth (in culture flasks): FAL coated their culture flasks withfibronectin-collagen prior to seeding thawed cells. This may have affected the
SLS data from FAL because it had the highest SLS IC50 values of the threelaboratories (7.45 µg/mL vs. 4.03 µg/mL for ECBC and 3.68 µg/mL for
IIVS). The fibronectin-collagen coating procedure was eliminated, andsubsequent SLS data and IC50 results from FAL were comparable to the datafrom the other two laboratories.
• OD Limits: Eliminated the VC OD range as a test acceptance criterion. The
SMT decided to accept tests that had VC ODs outside the originally preset
range if all other test acceptance criteria were met. Test data were notadversely affected by relaxing this criterion.
• Dilution Factor: The SMT accepted data generated using dilution factors
other than the recommended 1.47 for definitive tests if all other test
acceptance criteria were met. The use of smaller dilution factors generally
increased the number of data points between 10 - 90% viability, and theprecision of the IC50 calculation was improved.

5.3.2
Phase Ib: Laboratory Evaluation Phase
Phase Ib was designed to determine whether the protocol revisions following Phase Ia wereeffective in improving intra- and inter-laboratory reproducibility, and to determine whether
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In Vitro Cytotoxicity Methods BRD Section 5

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the laboratories could obtain reproducible results when testing coded reference substances ofvarious toxicities. Three coded reference substances representing the full range of toxicity
were tested: arsenic trioxide (high toxicity: 5< LD50 ≤50 mg/kg), propranolol HCl (medium
toxicity: 300< LD50 ≤2000 mg/kg), and ethylene glycol (low toxicity: LD50 >5000 mg/kg)(see Section 3.3.5 for the selection of substances to be tested in Phases Ib and II). Because
Phase Ib was part of the laboratory evaluation phase, the SMT decided that three substances
would be sufficient, and that it was not necessary to represent all GHS acute oral toxicity
categories. Each substance was tested in all laboratories at least once in a range finding
experiment, and then in three, acceptable definitive tests performed on three different days.
Section 2.6.2 summarizes the technical challenges that arose during this phase and addresses
protocol changes made after initiation of Phase Ib. The specific changes made in the 3T3 andNHK protocols, along with the effect the changes had on the test data, are summarized
below.
5.3.2.1

Protocol Changes and the Effect on the Data
• NR Dye Crystals: Reduced the concentration of NR in the 3T3 method. The
OD values and SLS IC50 results were similar in four exploratory experiments
regardless of the NR concentration or NRU incubation time. The eliminationof NR crystals reduced the background OD values without affecting thesensitivity of the procedure.
• VC OD Range: Used new VC OD ranges for guidance (e.g., as target values toassess cell growth), rather than as a test acceptance criterion, for the remainderof the study. This increased the number of tests that met the acceptancecriteria. Relative toxicities did not change. The test data were not adversely
affected by the removal of this criterion.

5.3.3
Phase II: Laboratory Qualification Phase
The results from Phase II were used to determine whether the protocol revisions from Phase
Ib were effective in improving intra- and inter-laboratory reproducibility, and whether thelaboratories could obtain reproducible results when testing a larger set of substances covering
a wider range of physical/substance characteristics and toxicities. Nine coded reference
substances were tested: aminopterin, cadmium chloride, chloramphenicol, colchicine, lithium
carbonate, potassium chloride, 2-propanol, sodium fluoride, and sodium selenate. These
substances (with the exception of sodium selenate) are included in the RC, and were selected
because they fit the RC millimole regression line (i.e., they were within the acceptanceintervals established by Halle [1998, 2003]). The RC is a database of acute oral LD50 values
for rats and mice obtained from RTECS® and IC50 values from in vitro cytotoxicity assays
using multiple cell lines and cytotoxicity endpoints for substances with known molecularweights (Halle 1998, 2003). Sodium selenate was selected because of its high toxicity,
despite the fact that it was not in the RC, because there were no other substances in thehighest GHS acute oral toxicity category, other than aminopterin, that were within the RC
millimole regression acceptance intervals. Each laboratory tested each substance at least oncein a range finding experiment, and then in three acceptable definitive tests performed on
different days.

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In Vitro Cytotoxicity Methods BRD Section 5

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Section 2.6.2 summarizes the technical issues that arose during this phase and the protocol
changes made prior to Phase II. The specific changes made in the 3T3 and NHK NRU
protocols, along with the effect the changes had on the test data, are summarized below.
5.3.3.1

Protocol Changes and the Effect on the Data
• Blank Wells: Added reference substance to blank wells of the test plate todetermine if reference substance affected (i.e., increased OD values) compared
to medium-filled blank wells. There was no apparent effect on the test data as
there were no noticeable differences in OD values between blanks with culture
medium or culture medium and reference substance.
• VC OD Range: Eliminated the VC OD range as an acceptance criterion. There
was no apparent effect on test data from not restricting the OD values to a preset range.
• Harmonization of Laboratory Techniques: Made revisions to the Phase II
protocols as a result of the harmonization training by the testing laboratories(see Section 2.6.2.6). There was no apparent effect on the test data from IIVS
and ECBC, but there was an improvement in the FAL data quality (e.g., fewer
lost OD values due to cell seeding errors, more uniform OD values for six
replicate wells per reference substance).
• 3T3 Cell Seeding Density: Added a range of cell seeding densities to be usedby the laboratories. This optimized the cell confluence at the end of chemical
exposure and no apparent effects on the data were detected because of thismodification.
• NHK Cell Growth from Cryopreserved Stock Cells: Eliminated the use offibronectin-collagen coating of 80-cm2 flasks for the initial propagation of
NHK cells. By doing this, FAL achieved better cell growth, lower IC50 values
for the PC, and better agreement of the mean SLS IC50 values with those of
the other laboratories.
• Volatile Substances: Added the use of a CO2 permeable plate sealer to control
volatility (as identified by cross contamination of the control wells). The useof plate sealers for volatile substances was incorporated into the Phase III
protocols.
• R2 Acceptance Criterion: Relaxed the R2 criterion for the fit of the doseresponse data to the Hill function. Some tests that did not meet the original
criterion were accepted by the SMT after determining that even though thecurve fit was not optimum, it adequately conveyed the toxicity of thesubstance (i.e., an IC50 could be calculated with an adequate number of
toxicity points between 0 and 100% viability).
• Unusual Concentration-Response: Revised the Hill function calculation toaddress substances that produced a concentration-response in which toxicity
plateaued before reaching 0% viability. This modification allowed for a curve
fit to the Hill function for such substances, and thus a better estimation of their
IC50 values.
• PC IC50 Range: Expanded the SLS IC50 acceptable range, which resulted inadditional tests in Phase II being acceptable. Expanding the PC range reduced
the number of reference substance retests, and thereby qualified additional

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In Vitro Cytotoxicity Methods BRD Section 5

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definitive tests as acceptable because they would not fail simply because thePC was out of the pre-set range.
5.3.4
Phase III: Main Validation Phase
The purpose of Phase III was to generate high quality in vitro cytotoxicity data using the 3T3
and NHK NRU test methods with protocols that were optimized based on the experience andresults in Phases I and II. Sixty coded reference substances were tested; 46 of these were RC
substances that covered a broad range of toxicity. The reference substances in Phase III
spanned all five GHS toxicity categories and unclassified substances. Each substance wastested in each laboratory at least once in a range finding experiment, and then in three
acceptable definitive tests performed on different days.
Section 2.6.4 addresses protocol changes made before the initiation of Phase III. The specificchanges made in the 3T3 and NHK protocols, along with the effect the changes had on thetest data, are summarized below.
5.3.4.1

Protocol Changes and the Effect on the Data
• Prequalification of NHK Culture Medium: Included a protocol forprequalifying NHK culture medium and supplements. This prevented theparticipating laboratories from using medium and supplements that did notsupport adequate growth of the cells.
• Stopping Rule for Testing: Added this rule for reference substances that wereinsoluble (i.e., <200 µg/mL) and/or did not produce sufficient cytotoxicity forthe calculation of an IC50. This rule allowed testing to end for substances that
produced no IC50 data after three definitive tests. Substances for which an IC50
was not produced by one or more laboratories are presented in Table 5-2.
Carbon tetrachloride did not produce an IC50 in any of the laboratories ineither the 3T3 or the NHK NRU test methods, and methanol did not producean IC50 in the 3T3 NRU test method.
• Acceptable Range for Dose-Response Data Points: Modified the test
acceptance criterion for the number of data points required on the toxicity
curve. The criterion was changed from requiring a minimum of two points (atleast one >0% and ≤50% viability, and at least one >50% and <100%viability) to one point >0% and <100% viability, if the smallest practical
dilution factor (i.e., 1.21) was used, and all other test acceptance criteria were
met. This reduced the number of failed experiments for substances with very
steep concentration-response curves, without reducing the quality of the IC50
data. For the 3T3 NRU test method, diquat dibromide (1/9 definitive tests),
epinephrine bitartrate (2/9 definitive tests), and 1,1,1-trichloroethane (2/8
definitive tests) had such steep dose-responses that some acceptable tests met
these revised criteria. None of the NHK NRU tests needed the revised criteria.
• R2 Acceptance Criterion: Rescinded the R2 criterion for the fit of the Hillfunction. The SMT determined that the R2 criterion was best used tocharacterize the shape of the concentration-response curve rather than toestablish a criterion for test acceptability. This reduced the number of failed
experiments without affecting the calculation of the IC50 values as long as an

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In Vitro Cytotoxicity Methods BRD Section 5•••

Table 5-2

November 2006

adequate number of toxicity points between 0 and 100% viability wereobtained.
PC Acceptance Criteria: Modified the PC acceptance criterion for Hillfunction fit.
Hill Function Analysis: Altered the PRISM® template for the Hill function
analysis to perform calculations for ICx values in two ways: (1) constraining
Bottom parameter to zero, and (2) fitting the Bottom parameter. As a result of
the changes and efforts by the laboratories to use dilution schemes that
captured the entire concentration-response range, very few tests in Phase III
had R2 <0.9.
Biphasic Dose-Response in Range Finder Test: Provided guidance forproceeding with definitive testing when a biphasic dose-response wasobtained in the range-finder test. The definitive test was to focus on the lowest
concentrations that produced responses around 50% viability (See Section
2.6.3.2).
Reference Substances Affected by Stopping Rule1

Reference Substance
Carbon tetrachloride
Disulfoton
Gibberellic acid
Methanol
1,1,1-Trichloroethane
Valproic acid
XyleneTesting Stopped -- No IC50 Data
3T3 NRU Test Method
NHK NRU Test Method
ECBC
FAL
IIVS
ECBC
FAL
IIVS
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X

Abbreviations: 3T3=BALB/c 3T3 fibroblasts; NHK=Normal human epidermal keratinocytes; NRU=Neutral red uptake;
ECBC=Edgewood Chemical Biological Center; FAL=Fund for the Replacement of Animals in Medical Experiments
Alternatives Laboratory; IIVS=Institute for In Vitro Sciences.
1
Substances that did not provide sufficient cytotoxicity for the calculation of an IC50 in one or more laboratories (identifiedby X).

5.4

Data Used to Evaluate Test Method Accuracy and Reliability

This section first presents the acceptable PC data and IC50 results from each laboratory foreach phase of the validation study, and then presents the reference substance IC50 results andHill Slopes from each phase. The individual test data for both passing and failing tests areprovided in Appendix I for the PC and reference substances. Accuracy (concordance for theprediction of GHS acute oral toxicity category) and reliability assessments are provided in
Sections 6 and 7, respectively.
5.4.1
PC Data
A summary of the acceptable SLS data IC50 results used to calculate quality control
acceptance limits for each test method in each laboratory are provided in Table 5-3. The SLS
IC50 results were used to calculate acceptable limits for each laboratory to use in subsequent
study phases. One of the test acceptance criteria for each reference substance test was that theassociated SLS IC50 must be within the acceptance limits. The individual test data for bothpassing and failing PC tests are provided in Appendix I3 for the 3T3 and in Appendix I4 for
the NHK methods.
5-10In Vitro Cytotoxicity Methods BRD Section 5

Positive Control (PC)1 IC50 Results by Study Phase

Table 5-3
Study
Phase

November 2006

Mean
IC50
(µg/mL)

ECBC
Standard
Acceptance
Deviation
Limits(µg/mL)

N

Mean
IC50
(µg/mL)

FAL
Standard
Acceptance
Deviation
Limits(µg/mL)

N

Mean
IC50
(µg/mL)

IIVS
Standard
Acceptance
Deviation
Limits(µg/mL)N

3T3 NRU
Ia2
3

Ib

4

II

III

5

38.3

4.71

28.8 – 47.7

15

42.3

8.56

25.2 – 59.5

25

40.9

3.19

34.5 – 47.3

12

41.3

5.99

26.4 – 56.3

12

43.2

4.68

31.5 – 54.9

17

42.1

3.40

33.6 – 50.6

13

41.2

4.20

30.8 – 51.6

29

45.9

7.50

27.2 – 64.7

36

40.6

3.50

31.8 – 49.3

21

41.6

3.41

NA

65

41.1

6.23

NA

26

41.5

3.74

NA

22

4.03

1.32

1.40 – 6.67

15

7.45

3.07

1.34 – 13.6

18

3.68

0.555

2.57 – 4.79

30

NHK NRU
Ia2
3

Ib

4

II

III

5

6

3.65

0.98

1.22 – 6.10

11

5.35

2.32

0 – 11.1

15

3.57

0.59

2.10 – 5.04

17

3.59

1.41

0.07 – 7.11

22

3.20

1.05

0.57 – 5.82

15

3.78

0.73

1.94 – 5.61

26

3.03

0.75

NA

57

3.45

0.90

NA

35

3.12

0.53

NA

20

Abbreviations: 3T3=BALB/c 3T3 fibroblasts; NHK=Normal human epidermal keratinocytes; NRU=Neutral red uptake; ECBC=Edgewood Chemical Biological Center;
FAL=Fund for the Replacement of Animals in Medical Experiments Alternatives Laboratory; IIVS=Institute for In Vitro Sciences; N=Number of acceptable tests; NA=Not
applicable1
PC was sodium lauryl sulfate (SLS).
2
Values generated from Phase Ia data were used as acceptance criteria for Phase Ib tests; Acceptance limits = Mean ±2 X standard deviation.
3
Values generated from Phases Ia and Ib data were used as acceptance criteria for Phase II tests; Acceptance limits = Mean ±2.5 X standard deviation.
4
Values generated from Phases Ia, Ib, and II data were used as acceptance criteria for Phase III tests; Acceptance limits = Mean ±2.5 X standard deviation.
5
Values generated from Phase III test data.
6
Calculation of lower limits yielded a negative value, so that lower limit was set at 0 and later revised to 0.1 µg/mL.

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In Vitro Cytotoxicity Methods BRD Section 5

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5.4.1.1 Phase Ib PC Data Acceptance Limits
The SLS IC50 acceptance limits for Phase Ib testing were calculated using the Phase Ia data.
The data sets from each laboratory were examined for outliers using the method of Dixon
and Massey (1981), but none were identified. The acceptance limits for the SLS IC50 values
for each laboratory and test method were the mean ±2 SD.
5.4.1.2 Phase II PC Data Acceptance Limits
The IC50 values from the Phase Ia and Ib SLS tests were used to calculate laboratory-specificand test method-specific quality control acceptance limits for Phase II. Phase Ib tests that had
SLS IC50 values outside of the acceptance limits were considered acceptable if they met allother test acceptance criteria. For any day during which there was more than one SLS test(for any one method and laboratory), the IC50 values were averaged to better reflect day-today variation and avoid overweighting the overall mean with multiple values from a single
day. Outliers at the 99% level were removed and the remaining values were used to calculate
the mean ±2.5 SD acceptance limits. The acceptance limits were expanded from 2 SD in
Phase Ib to 2.5 SD for Phase II to allow for the fact that the SDs decrease as more data arecollected.
5.4.1.3 Phase III PC Data Acceptance Limits
The IC50 values from the Phase I and II SLS tests were used to calculate laboratory-specificand method-specific quality control acceptance limits for Phase III data. The SLS IC50 values
outside the acceptance limits were considered acceptable if the tests met all other acceptancecriteria. For any day for which there was more than one SLS test (for any one method andlaboratory), the IC50 values were averaged to better reflect day-to-day variation and avoid
overweighting the overall mean with multiples values from a single day. ANOVA was used
to compare the Phase Ia, Ib, and II data within each laboratory to determine whether the SLS
IC50 for each method and laboratory was changing over the course of the study. For PC datathat were not significantly different from phase to phase at p <0.05, the IC50 values were used
to calculate the mean ±2.5 SD as the acceptance limits for Phase III. The only significant
differences in SLS values seen between study phases (p <0.0002) were the FAL results forNHK. This difference was attributed to the changes in cell culture practices between Phases
Ib and II (see Section 5.3.3). Thus, only the Phase II SLS IC50 values were used to calculate
the acceptance limits for Phase III NHK data at FAL.
5.4.2
Reference Substance Data
Reference substance data and results from the individual 3T3 and NHK tests (both acceptableand unacceptable) from each laboratory are presented in Appendices I1 and I2. Tables 5-4
and 5-5 summarize the IC50 and Hill Slope data from the acceptable 3T3 and NHK tests,
respectively, for each reference substance and laboratory. The Hill Slope data are provided
for supplemental information on the concentration-response characteristics for each reference
substance, but were not used for reliability or accuracy analyses. These tables are organizedalphabetically by substance name and provide substance class (based on the NLM Medical
Subject Heading [MeSH index]), arithmetic mean IC50 and SD for each laboratory, arithmetic
mean Hill Slope and SD for each laboratory, and the number of tests used to produce themean values. Figure 5-1 graphically presents the 3T3 IC50 data from Table 5-4, and Figure
5-2 presents the NHK IC50 data from Table 5-5. The reference substances in Figures 5-1 and
5-2 are ordered by ascending IC50 (lowest value [most toxic] to highest value [least toxic])
using the 3T3 IC50 values from IIVS (the lead laboratory for the study). This allows a simple
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In Vitro Cytotoxicity Methods BRD Section 5

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comparison of each reference substance value from each laboratory. Table 5-6 provides thenumerical key to the reference substances in Figures 5-1 and 5-2.
Because of their low toxicity and/or low solubility, some substances were not sufficiently
toxic for calculation of an IC50 value. For the 3T3 NRU test method, no IC50 values wereobtained for carbon tetrachloride or methanol in any laboratory (see Table 5-4). ECBC was
the only laboratory that obtained IC50 values for lithium carbonate, and IIVS was the only
laboratory that obtained IC50 values for xylene. Only one acceptable test (and IC50 value) was
obtained for disulfoton at FAL, for 1,1,1-trichloroethane at ECBC, and for valproic acid at
IIVS. FAL did not achieve sufficient toxicity for the calculation of an IC50 for gibberellicacid in any 3T3 NRU tests performed. For the NHK NRU test method (see Table 5-5), there
was insufficient toxicity in all tests in all laboratories for a calculation of an IC50 for carbon
tetrachloride. Only one laboratory achieved sufficient toxicity for the calculation of an IC50
for 1,1,1-trichloroethane (ECBC) and xylene (IIVS). One laboratory, ECBC, failed toachieve sufficient toxicity for the calculation of an IC50 for methanol. All of these substances,with the exception of methanol, produced precipitate in the cell culture medium. The solvent
used for methanol was DMSO, and because the amount of DMSO that could be used in thecell culture was limited to 0.5%, the amount of DMSO that could be used to dissolve
methanol was also limited. The differences among laboratories regarding their ability toattain a high enough concentration to achieve an IC50 for some substances may be due to thediffering perceptions of the laboratory personnel regarding whether or not the substance wassufficiently dissolved, or differences in the techniques used to dissolve the substances.

5-13

In Vitro Cytotoxicity Methods BRD Section 5

Table 5-4

3T3 NRU Test Method IC50 and Hill Slope Data by Laboratory

Amide

Phase
in
which
TestedIII

Acetonitrile

NitrileAcetylsalicylic
acid

Carboxylic
Acid; Phenol

Aminopterin
5-Aminosalicylic
acid
Amitriptyline
HCl
Arsenic III
Trioxide
Atropine sulfate

Substance

ChemicalClass5

Acetaminophen

Boric acid

BusulfanCadmium II
chloride

November 2006

ECBC
2

FAL

1

IC50
µg/mL

SD
(IC50)N

Hill
Slope3

40.8

9.12

3

III

6433

129

III

646

Heterocyclic

II

Carboxylic
Acid; Phenol

2

IIVS

SD4

1

IC50
µg/mL

SD
(IC50)N

Hill
Slope3

-1.53

0.354

66.2

23.0

3

3

-2.29

0.648

9690

5634

61.5

3

-1.75

0.473

1234

0.005

0.001

3

-2.00

0.395

III

1467

203

3

-1.82

Polycyclic

III

6.03

1.38

3

Arsenical

Ib

2.41

0.782

Heterocyclic

III

54.1

III

Boron
compound;Acid
Alcohol;
Sulfurcompound;Acyclic
hydrocarbon
Cadmiumcompound;
Chlorinecompound

2

SD4

1

IC50
µg/mL

SD
(IC50)N

Hill
Slope3

SD4

-1.23

0.503

43.4

11.4

3

-1.55

0.165

3

-1.55

0.196

9330

1217

3

-2.63

0.245

298

3

-1.99

0.393

401

62.0

3

-1.31

0.167

0.012

0.005

3

-3.36

1.59

0.005

0.001

3

-1.46

0.198

0.267

2070

334

3

-2.33

0.809

1557

179

3

-1.64

0.326

-2.47

0.668

7.86

2.20

3

-2.98

0.446

7.81

1.38

3

-4.48

0.916

4

-1.94

0.204

1.04

0.070

4

-3.02

2.09

4.09

2.23

3

-1.62

0.285

29.6

3

-1.32

0.480

133

41.1

3

-2.20

0.695

70.0

5.7

3

-1.27

0.165

1497

484

3

-1.14

0.039

3987

693

3

-1.86

0.654

1202

581

3

-1.71

0.677

III

40.4

19.3

3

-0.515

0.003

321

180

3

-1.14

0.802

43.7

1.77

3

-0.627

0.164

II

0.480

0.066

3

-1.85

0.529

0.400

0.129

3

-3.05

0.743

0.817

0.427

3

-2.45

0.449

Caffeine

Heterocyclic

III

133

13.3

3

-1.11

0.097

157

81.7

3

-0.866

0.250

191

14.4

3

-1.27

0.077

Carbamazepine

Heterocyclic

III

83.0

12.0

3

-1.94

0.539

152

56.9

3

-3.50

1.27

91.8

11.0

3

-2.34

0.307

Carbontetrachloride

Halogenatedhydrocarbon

III

NA

NA

-

NA

NA

NA

NA

-

NA

NA

NA

NA

-

NA

NA

Chloral hydrate

Alcohol

III

151

15.6

3

-1.73

0.172

241

25.1

3

-2.16

0.597

170

19.9

3

-1.68

0.084

II

55.3

12.4

4

-0.779

0.057

273

82.2

4

-1.16

0.249

156

27.9

3

-0.952

0.036

III

473

138

3

-1.89

0.423

1148

143

4

-3.68

0.407

865

160

3

-2.51

0.530

Chloramphenicol

Citric acid

Alcohol;Nitro
compound;Cyclichydrocarbon
Carboxylic
acid

5-14

In Vitro Cytotoxicity Methods BRD Section 5

Table 5-4
Substance

November 2006

3T3 NRU Test Method IC50 and Hill Slope Data by Laboratory
ChemicalClass5

Phase
in
which
Tested
II

ECBC
2

FAL

1

IC50
µg/mL

SD
(IC50)N

Hill
Slope3

0.021

0.002

4

2

IIVS

SD4

1

IC50
µg/mL

SD
(IC50)N

Hill
Slope3

-1.69

0.049

0.093

0.042

3

2

SD4

1

IC50
µg/mL

SD
(IC50)N

Hill
Slope3

SD4

-1.61

1.80

0.028

0.0003

3

-1.69

0.255

Colchicine

Polycyclic

Cupric sulfate
pentahydrate

Sulfurcompound;
Metal

III

82.7

3.18

3

-4.85

0.700

123

54.0

4

-17.7

15.5

5.72

1.75

3

-5.71

1.14

Cycloheximide

Heterocyclic

III

0.125

0.057

3

-1.19

0.167

0.647

0.451

3

-1.53

0.128

0.109

0.025

3

-0.937

0.158

III

23.5

3.98

3

-3.37

1.27

191

94.5

4

-0.965

0.140

20.7

1.37

3

-2.62

0.283

III

9.83

3.42

3

-1.32

0.297

32.8

2.07

3

-3.42

1.00

18.3

2.09

3

-2.13

0.439

III

85.5

29.0

3

-1.11

0.340

147

37.8

3

-2.03

0.422

106

25.3

3

-2.35

0.824

Dibutyl phthalate
Dichlorvos
Diethyl phthalate

Carboxylicacid
Organophosphorous
Carboxylicacid
Polycyclic;

III

351

137

3

-2.11

2.05

892

319

3

-3.26

2.21

317

67.9

2

-3.04

1.52

Dimethylformamide

Amide;
Carboxylic
acid

III

5343

515

3

-1.96

0.087

5483

517

3

-1.80

0.143

4900

183

3

-1.87

0.102

Diquat dibromide
monohydrate

Heterocyclic

III

3.87

0.887

3

-1.59

0.197

36.1

35.5

3

-11.5

10.1

5.39

1.36

3

-3.00

0.784

III

137

74.9

3

-2.06

1.88

11200

NA

1

-1.22

NA

60.4

52.5

3

-2.23

1.08

III

5.27

3.01

3

-0.669

0.243

15.2

11.9

4

-0.762

0.221

3.61

1.53

3

-0.871

0.636

III

51.5

6.16

3

-5.99

3.08

63.4

6.63

3

-45.1

32.0

63.4

1.91

3

-4.74

1.51

Digoxin

Disulfoton

Endosulfan
Epinephrine
bitartrate

Carbohydrate

Organophosphorous;
SulfurcompoundHeterocyclic
SulfurcompoundAlcohol;
Amine

Ethanol

Alcohol

III

5360

1754

3

-1.33

0.104

8420

1205

3

-1.88

0.128

6413

345

3

-1.99

0.372

Ethylene glycol

Alcohol

Ib

18325

1658

4

-3.79

4.08

31650

7453

4

-1.70

0.166

25900

3081

3

-1.67

0.079

Fenpropathrin

Nitrile;Ester; Ether

III

22.6

2.41

3

-2.54

0.350

42.4

26.8

4

-1.44

0.645

16.7

2.03

3

-2.53

0.495

Gibberellic acid

Polycyclic

III

8027

908

3

-1.95

0.678

NA

NA

-

NA

NA

7657

745

3

-1.66

0.087

Glutethimide

Heterocyclic

III

167

7.00

3

-1.3

0.045

284

20.7

3

-1.47

0.131

125

9.25

4

-1.20

0.163

Glycerol

Alcohol

III

20000

2987

3

-2.02

0.273

38878

28238

4

-2.27

1.29

27833

10882

3

-1.87

0.306

Haloperidol

Ketone

III

5.32

0.649

3

-2.34

0.445

7.99

0.655

3

-4.99

0.378

5.47

0.654

3

-1.86

0.048

5-15

In Vitro Cytotoxicity Methods BRD Section 5

Table 5-4
Substance

Hexachlorophene
Lactic acid
Lindane
Lithium I
carbonate
Meprobamate
Mercury II
chloride

November 2006

3T3 NRU Test Method IC50 and Hill Slope Data by Laboratory
ChemicalClass5Cyclichydrocarbon
PhenolCarboxylic
acid
Halogenatedhydrocarbon
Alkalies;
Inorganiccarbon;LithiumcompoundCarboxylicacid
Mercury
compound;
Chlorinecompound

Phase
in
which
Tested

1

IC50
µg/mL

SD
(IC50)N

Hill
Slope3

III

5.02

2.41

3

III

2943

315

III

125

II

ECBC
2

FAL
2

IIVS

SD4

1

IC50
µg/mL

SD
(IC50)N

Hill
Slope3

-1.62

0.189

5.35

1.75

3

3

-4.13

1.54

3487

561

119

3

-0.737

0.231

266

564

67.6

3

-1.59

0.313

III

353

49.7

3

-1.16

III

3.45

0.177

3

-4.18

2

SD4

1

IC50
µg/mL

SD
(IC50)N

Hill
Slope3

SD4

-1.17

0.322

3.06

0.289

3

-1.66

0.217

3

-6.62

3.23

2790

259

3

-3.64

1.09

94.8

4

-1.26

1.283

90.4

111

5

-1.46

0.262

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

0.438

877

128

4

-1.32

0.270

386

9.02

3

-1.12

0.133

0.988

5.99

1.87

3

-4.34

1.11

3.51

0.120

3

-4.16

1.31

MethanolAlcohol

III

NA

NA

-

NA

NA

NA

NA

-

NA

NA

NA

NA

-

NA

NA

Nicotine

Heterocyclic

III

272

65.3

3

-1.58

0.357

412

136

3

-12.0

6.99

450

54.7

3

-49.6

70.9

Paraquat

Heterocyclic

III

21.3

7.29

3

-1.32

0.341

24.9

16.5

3-

-4.10

3.13

23.7

15.2

3

-1.92

0.581

Parathion

Organophosphorous;
Sulfurcompound

III

22.7

12.1

3

-1.89

1.33

141

98.7

4

-1.62

0.520

22.0

4.94

3

-1.55

0.562

Phenobarbital

Heterocyclic

III

634

134

3

-1.43

0.177

726

255

3

-1.84

0.851

476

111

4

-1.67

0.418

Phenol

III

50.2

10.9

3

-1.46

0.318

104

24.8

3

-1.55

0.205

58.1

6.78

3

-1.41

0.259

III

30.1

19.8

3

-0.781

0.218

239

65.8

3

-0.890

0.206

89.0

21.9

3

-1.40

0.127

III

28.2

14.9

3

-1.51

0.595

37.8

1.93

3

-7.22

1.04

20.4

6.71

4

-1.70

0.157

II

3352

468

4

-3.32

1.17

3842

1198

5

-4.31

2.27

3710

417

3

-2.87

0.147

PhenolPhenylthiourea
Physostigmine
Potassium I
chloride

Sulfurcompound;
Urea
Carboxylic
acid;
Heterocyclic
Potassiumcompound;
Chlorinecompound

5-16

In Vitro Cytotoxicity Methods BRD Section 5

Table 5-4
Substance

November 2006

3T3 NRU Test Method IC50 and Hill Slope Data by Laboratory
ChemicalClass5

Phase
in
which
Tested

1

IC50
µg/mL

SD
(IC50)N

Hill
Slope3

III

15.3

3.76

3

III

400

15.3

ECBC
2

FAL
2

IIVS

SD4

1

IC50
µg/mL

SD
(IC50)N

Hill
Slope3

-1.48

0.677

159

81.9

3

3

-12.4

1.91

431

4.73

2

SD4

1

IC50
µg/mL

SD
(IC50)N

Hill
Slope3

SD4

-1.03

0.152

18.9

0.950

3

-3.43

0.488

3

-45.6

18.4

497

39.3

3

-19.9

13.1

Procainamide
HCl

Potassiumcompound;NitrogencompoundCarboxylicacid; Amide

2-Propanol

Alcohol

II

2610

240

2

-1.80

0.001

3970

139

3

-1.65

0.241

4110

161

3

-1.93

0.160

Propranolol HCl

Alcohol

Ib

13.6

4.37

4

-2.54

0.627

13.5

6.85

4

-3.31

2.53

17.6

3.78

3

-3.45

1.44

III

20.9

3.33

3

-1.23

0.259

51.8

14.8

3

-1.45

0.442

17.1

2.10

3

-1.24

0.245

III

0.496

0.028

3

-1.43

0.087

1.44

0.819

3

-3.79

1.22

0.683

0.117

3

-1.90

0.535

III

4790

233

3

-1.55

0.182

4625

611

4

-2.67

0.620

4877

457

3

-2.03

0.366

III

0.603

0.087

3

-1.64

0.136

0.657

0.244

3

-5.01

1.51

0.547

0.092

3

-1.93

0.194

II

61.3

5.55

3

-5.06

1.50

96.1

17.7

3

-4.40

0.971

82.0

5.81

3

-2.73

0.850

III

823

108

3

-2.57

1.12

805

367

3

-4.13

3.05

2005

872

4

-3.20

0.279

III

42.0

17.3

3

-1.83

0.380

31.0

8.66

3

-3.11

0.367

49.5

26.3

4

-2.32

0.592

II

12.7

1.62

3

-1.59

0.217

54.2

10.4

3

-3.76

0.968

36.5

5.23

3

-1.65

0.112

Potassium
cyanidePropylparaben
Sodium arsenite

Sodium chloride
Sodiumdichromate
dihydrate
Sodium I fluoride

Sodiumhypochlorite

Sodium oxalate

Sodium selenate

Carboxylicacid; Phenol
Sodiumcompound;Arsenical
Sodiumcompound;
Chlorinecompound
Sodiumcompound;
Chromiumcompound
Sodiumcompound;Fluorinecompound
SodiumcompoundOxygencompound;
Chlorinecompound
Sodiumcompound;Carboxylic
acid
Sodiumcompound;Seleniumcompound

5-17

In Vitro Cytotoxicity Methods BRD Section 5

Table 5-4
Substance
Strychnine
Thallium I
sulfateTrichloroaceticacid
1,1,1-Trichloroethane
Triethylenemelamine
Triphenyltinhydroxide
Valproic acid
Verapamil HCl
XyleneNovember 2006

3T3 NRU Test Method IC50 and Hill Slope Data by Laboratory
ChemicalClass5

Phase
in
which
Tested
III

ECBC
2

FAL
IC50
µg/mL

SD
(IC50)N

Hill
Slope3

-2.51

0.728

124

20.3

3

3

-1.02

0.201

13.4

10.4

99.1

3

-1.66

0.118

1220

41100

NA

1

-2.38

NA

III

0.086

0.009

3

-0.567

III

0.026

0.004

3

III

547

67.1

Amine

III

32.2

Cyclichydrocarbon

III

NA

Heterocyclic
Organometallic
compoundCarboxylicacid; Lipids

SD(IC50)N

Hill
Slope3

389

80.9

3

III

2.81

0.671

III

762

III

IIVS

SD4

Sulfurcompound;
Metal
Carboxylic
acid
Halogenatedhydrocarbon

IC50
µg/mL

2

1

Heterocyclic

1

2

SD4

1

IC50
µg/mL

SD
(IC50)N

Hill
Slope3

SD4

-5.85

0.922

83.5

5.35

3

-6.49

2.12

4

-0.714

0.302

6.27

1.75

3

-0.752

0.081

72.1

3

-2.22

0.089

801

114

3

-1.77

0.130

21250

2357

3

-31.5

32.1

9827

180

3

-21.8

8.47

0.018

1.45

0.265

3

-1.88

1.04

0.169

0.049

3

-0.615

0.138

-1.66

0.257

0.026

0.021

3

-4.78

3.37

0.015

0.008

3

-1.46

0.149

3

-2.24

0.742

1807

175

3

-4.07

0.766

574

NA

1

-1.24

NA

5.82

3

-4.43

1.362

34.6

1.72

3

-29.1

18.6

38.9

4.20

3

-5.00

0.935

NA

-

NA

NA

NA

NA

-

NA

NA

724

87.1

3

-1.91

0.473

Abbreviations: ECBC=Edgewood Chemical Biological Center; FAL=Fund for the Replacement of Animals in Medical Experiments Alternatives Laboratory; IIVS=Institute for In Vitro Sciences; SD=Standard deviation;
N=Number of data points; NA=Not available (i.e., IC50 values or Hill Slope values could not be generated [see notes in Appendix I for more information])
1
Arithmetic mean.
2Standard deviation of IC50.
3Arithmetic Mean of Hill Slope values.
4Standard deviation of Hill Slope values.
5
Chemical class assigned is based on the classification of the National Library of Medicine’s Medical Subject Heading (MeSH), http://www.nlm.nih.gov/mesh/meshhome.html.

5-18

In Vitro Cytotoxicity Methods BRD Section 5

Table 5-5

November 2006

NHK NRU Test Method IC50 and Hill Slope Data by Laboratory
IC501
µg/mL

2

SD

Amide

Phase
in
which
TestedIII

558

Acetonitrile

NitrileIII

Acetylsalicylic
acid

Carboxylic
Acid; Phenol

Aminopterin
5-Aminosalicylic
acid
Amitriptyline
HCl
Arsenic III
Trioxide
Atropine sulfate

Substance

ChemicalClass5

Acetaminophen

Boric acid

BusulfanCadmium II
chloride

ECBC

FAL
SD

IC501
µg/mL

2

SD

-1.09

0.108

447

4

-2.61

0.424

19.9

3

-1.94

889

182

3

III

29.9

6.52

Polycyclic

III

10.8

Arsenical

Ib

Heterocyclic

SD

2

SD

N

Hill
Slope3

SD4

-1.09

0.646

571

79.0

3

-1.20

0.154

4

-5.95

3.34

9290

413

3

-2.79

0.306

98.3

3

-1.85

0.324

514

79.1

3

-1.97

0.083

545

42.2

3

-1.27

0.225

611

70.7

2

-1.72

0.547

0.806

78.2

42.3

3

-7.96

6.90

48.8

7.90

3

-3.66

0.629

-1.79

0.236

7.57

5.43

3

-1.43

0.479

10.9

1.04

3

-2.27

0.278

4

-2.67

0.470

2.55

1.92

6

-1.78

1.14

20.9

6.4

3

-2.02

0.338

10.5

3

-1.26

0.307

104

88.2

3

-2.90

3.48

83.2

21.0

3

-1.21

0.101

440

138

3

-1.19

0.233

517

378

3

-0.752

0.117

464

11

3

-1.33

0.194

III

253

68.2

3

-0.783

0.323

268

193

3

-1.50

0.357

313

37.2

3

-1.66

0.459

II

2.20

0.823

5

-4.01

1.25

1.88

1.22

3

-3.36

3.14

1.86

0.151

3

-4.65

1.38

N

80.7

3

10868

7824

III

631

Heterocyclic

II

Carboxylic
Acid; Phenol

Boron
compound;Acid
Alcohol;
Sulfurcompound;Acyclic
hydrocarbon
Cadmiumcompound;
Chlorinecompound

IIVS
IC501
µg/mL

Hill
Slope3N

Hill
Slope3

83.7

3

10153

1960

0.367

694

-2.03

0.375

3

-3.45

3.34

3

7.77

2.54

III

85.4

III

4

4

Caffeine

Heterocyclic

III

817

256

3

-1.44

0.504

591

186

3

-1.06

0.499

574

7.81

3

-1.28

0.117

Carbamazepine

Heterocyclic

III

66.1

8.4

3

-1.15

0.307

253

325

3

-2.57

2.53

63.9

5.27

3

-1.34

0.444

Carbontetrachloride

Halogenatedhydrocarbon

III

NA

NA

-

NA

NA

NA

NA

-

NA

NA

NA

NA

-

NA

NA

Alcohol

III

140

34.2

3

-1.55

0.378

159

50.1

3

-1.33

0.105

112

1.73

3

-1.42

0.123

II

318

142

3

-1.51

0.794

414

182

4

-1.16

0.091

367

79.7

3

-0.917

0.249

III

526

82.4

3

-1.62

0.158

312

51.6

4

-1.25

0.249

433

22.3

3

-1.62

0.080

Chloral hydrate

Chloramphenicol

Citric acid

Alcohol;Nitro
compound;Cyclichydrocarbon
Carboxylic
acid

5-19

In Vitro Cytotoxicity Methods BRD Section 5

Table 5-5
Substance

November 2006

NHK NRU Test Method IC50 and Hill Slope Data by Laboratory
ChemicalClass5

Phase
in
which
Tested
II

IC50
µg/mL

SD2

N

Hill
Slope3

SD4

IC50
µg/mL

SD2

N

Hill
Slope3

SD4

IC50
µg/mL

SD2

N

Hill
Slope3

SD4

0.005

0.002

3

-2.15

1.39

0.008

0.001

3

-3.16

1.96

0.008

0.002

3

-13. 8

11.0

ECBC
1

FAL
1

IIVS
1

Colchicine

Polycyclic

Cupric sulfate
pentahydrate

Sulfurcompound;
Metal

III

190

19.6

3

-6.16

3.16

195

12.5

3

-3.85

0.328

207

7.09

3

-5.69

0.871

Cycloheximide

Heterocyclic

III

0.053

0.012

3

-1.24

0.152

0.120

0.094

3

-0.850

0.388

0.071

0.013

3

-1.54

0.178

III

28.3

7.64

3

-1.40

0.295

47.4

34.3

3

-1.02

0.352

22.0

1.32

3

-1.33

0.197

III

8.56

2.28

3

-1.17

0.147

12.4

3.74

3

-2.29

2.33

12.2

0.416

3

-1.50

0.214

III

174

14.4

3

-2.21

0.358

71.5

67.3

3

-1.67

0.637

189

33.1

3

-1.97

0.242

III

0.00540.0007

3

-2.00

0.127

0.00010.00002

3

-1.38

0.684

0.004

0.0003

3

-4.59

1.73

Dibutyl phthalate
Dichlorvos
Diethyl phthalate
Digoxin

Carboxylicacid
Organophosphorous
Carboxylicacid
Polycyclic;
Carbohydrate

Dimethylformamide

Amide;
Carboxylic
acid

III

9353

155

3

-3.67

0.273

7817

100

3

-2.85

0.590

6397

202

3

-3.00

0.161

Diquat dibromide
monohydrate

Heterocyclic

III

3.59

0.825

3

-1.44

0.051

6.77

3.73

4

-1.38

0.488

3.84

0.313

3

-1.10

0.139

III

140

27.0

3

-1.65

1.15

808

213

3

-0.841

0.452

186

59.2

3

-0.836

0.209

III

3.44

0.573

3

-1.68

0.438

1.42

0.701

4

-1.19

0.369

2.19

0.437

3

-2.20

0.242

III

115

10.8

3

-7.37

2.10

81.7

28.4

3

-8.39

5.81

75.0

12.2

3

-4.90

2.81

Disulfoton

Endosulfan
Epinephrine
bitartrate

Organophosphorous;
SulfurcompoundHeterocyclic
SulfurcompoundAlcohol;
Amine

Ethanol

Alcohol

III

8290

390

3

-2.13

0.035

12013

2286

3

-1.82

0.635

10250

867

3

-2.29

0.185

Ethylene glycol

Alcohol

Ib

38000

4681

3

-3.22

0.650

49800

4371

3

-3.02

0.188

40000

5341

4

-2.56

0.444

Fenpropathrin

Nitrile;Ester; Ether

III

3.73

1.01

3

-1.42

0.486

2.23

0.616

3

-4.37

4.45

1.82

0.310

3

-1.78

0.617

Gibberellic acid

Polycyclic

III

2850

402

3

-2.45

0.372

2940

276

3

-5.90

2.69

2807

121

3

-3.30

1.104

Glutethimide

Heterocyclic

III

187

64.3

3

-1.47

0.616

170

24.1

3

-1.29

0.145

176

27.5

3

-1.54

0.237

Glycerol

Alcohol

III

34267

15399

3

-3.32

1.97

18023

8334

3

-1.62

0.521

29033

4596

3

-2.69

0.511

Haloperidol

Ketone

III

3.69

1.01

3

-0.964

0.206

3.72

1.81

3

-0.732

0.097

3.29

1.15

3

-0.840

0.100

5-20

In Vitro Cytotoxicity Methods BRD Section 5

Table 5-5
Substance

Hexachlorophene
Lactic acid
Lindane

Lithium I
carbonate
Meprobamate
Mercury II
chloride

November 2006

NHK NRU Test Method IC50 and Hill Slope Data by Laboratory
ChemicalClass5Cyclichydrocarbon
PhenolCarboxylic
acid
Halogenatedhydrocarbon
Alkalies;
Inorganiccarbon;LithiumcompoundCarboxylicacid
Mercury
compound;
Chlorinecompound

Phase
in
which
Tested

IC50
µg/mL

SD2

N

Hill
Slope3

SD4

IC50
µg/mL

SD2

N

Hill
Slope3

SD4

IC50
µg/mL

SD2

N

Hill
Slope3

SD4

III

0.027

0.004

3

-2.21

0.301

0.046

0.020

3

-2.91

0.662

0.021

0.002

3

-2.36

0.059

III

1290

52.9

3

-2.36

0.306

1320

60.8

3

-3.25

0.328

1313

138

3

-3.23

0.408

III

19.1

3.14

3

-3.02

0.969

23.2

7.09

3

-2.24

0.315

15.6

2.4

3

-2.61

0.265

II

411

119

3

-1.95

0.456

486

95.7

3

-1.78

1.31

535

31.6

3

-2.64

0.164

III

761

116

3

-1.90

0.695

163

189

3

-0.806

0.206

624

84.2

3

-2.04

0.170

III

6.87

1.04

3

-16.3

4.95

5.4

1.02

3

-17.8

13.1

5.35

0.09

3

-17.8

3.31

ECBC
1

FAL
1

IIVS
1

MethanolAlcohol

III

NA

NA

-

NA

NA

1133

213

3

-1.79

0.874

2100

226

3

-1.86

0.297

Nicotine

Heterocyclic

III

94.3

24.7

3

-0.654

0.092

134

78.4

3

-0.668

0.077

112

27.7

3

-0.7330.047

Paraquat

Heterocyclic

III

48.3

6.03

3

-1.04

0.158

96.6

37.2

3

-1.34

0.326

53.4

5.52

3

-1.47

0.034

Parathion

Organophosphorous;
Sulfurcompound

III

34.0

10.0

3

-1.60

0.640

31.2

11.9

3

-1.18

0.200

29.0

8.34

3

-1.85

0.956

Phenobarbital

Heterocyclic

III

693

180

3

-1.10

0.214

360

95.5

3

-0.976

0.229

381

69.9

3

-1.68

0.353

Phenol

III

59.1

21.4

3

-0.919

0.084

93.2

5.97

3

-1.15

0.209

80.8

5.12

3

-0.915

0.029

III

363

58

3

-1.55

0.726

401

83.6

3

-3.49

1.91

272

71.7

3

-1.00

0.053

III

164

5.51

3

-3.05

0.552

212

238

3

-3.81

2.44

139

8.74

3

-2.97

0.135

II

2560

432

3

-2.23

0.383

2287

631

3

-1.09

0.163

1990

161

3

-2.05

0.165

PhenolPhenylthiourea
Physostigmine
Potassium I
chloride

Sulfurcompound;
Urea
Carboxylic
acid;
Heterocyclic
Potassiumcompound;
Chlorinecompound

5-21

In Vitro Cytotoxicity Methods BRD Section 5

Table 5-5
Substance

November 2006

NHK NRU Test Method IC50 and Hill Slope Data by Laboratory
ChemicalClass5

Phase
in
which
Tested

IC50
µg/mL

SD2

N

Hill
Slope3

SD4

IC50
µg/mL

SD2

N

Hill
Slope3

SD4

IC50
µg/mL

SD2

N

Hill
Slope3

SD4

III

29.3

6.9

3

-1.21

0.241

89.0

100

3

-1.10

0.319

16.9

2.21

3

-1.37

0.154

III

1480

200

3

-3.56

0.813

1787

221

3

-4.22

1.57

2027

229

3

-4.42

0.459

ECBC
1

FAL
1

IIVS
1

Procainamide
HCl

Potassiumcompound;NitrogencompoundCarboxylicacid; Amide

2-Propanol

Alcohol

II

5263

583

3

-2.01

0.173

4273

1139

3

-2.31

0.211

7087

480

3

-3.01

0.406

Alcohol

Ib

38.3

4.54

3

-3.44

0.559

43.8

2.52

3

-2.72

1.461

28.6

3.28

4

-2.09

0.413

III

18.1

2.42

3

-1.18

0.122

18.6

2.84

3

-1.58

0.399

13.8

1.21

3

-1.20

0.065

III

0.79

0.248

3

-1.69

0.222

0.336

0.187

3

-1.54

0.317

0.470

0.066

3

-1.96

0.197

III

3583

263

3

-2.43

0.153

1118

1388

3

-1.96

0.371

3470

300

3

-2.47

0.208

III

0.784

0.113

3

-2.35

0.282

0.851

0.302

4

-3.52

1.49

0.576

0.100

3

-2.32

0.199

II

48.7

6.92

3

-2.50

0.263

39.7

9.61

3

-2.60

1.04

53.7

6.82

4

-2.71

0.150

III

1863

581

3

-5.19

1.14

1243

576

3

-2.78

1.27

1633

180

3

-3.86

0.211

III

355

54.9

3

-4.00

1.99

350

147

4

-6.10

6.40

360

94.6

3

-3.13

0.555

II

7.47

0.861

3

-1.78

0.529

16.1

9.55

3

-3.07

0.456

10.0

1.33

3

-1.75

0.226

Potassium
cyanidePropranolol HCl
Propylparaben
Sodium arsenite

Sodium chloride
Sodiumdichromate
dihydrate
Sodium I fluoride

Sodiumhypochlorite

Sodium oxalate

Sodium selenate

Carboxylicacid; Phenol
Sodiumcompound;Arsenical
Sodiumcompound;
Chlorinecompound
Sodiumcompound;
Chromiumcompound
Sodiumcompound;Fluorinecompound
SodiumcompoundOxygencompound;
Chlorinecompound
Sodiumcompound;Carboxylic
acid
Sodiumcompound;Seleniumcompound

5-22

In Vitro Cytotoxicity Methods BRD Section 5

Table 5-5
Substance
Strychnine
Thallium I sulfate
Trichloroaceticacid
1,1,1-Trichloroethane
Triethylenemelamine
Triphenyltinhydroxide
Valproic acid
Verapamil HCl
XyleneNovember 2006

NHK NRU Test Method IC50 and Hill Slope Data by Laboratory
Phase
in
which
Tested
III

IC50
µg/mL

SD2

N

Hill
Slope3

SD4

IC50
µg/mL

SD2

N

Hill
Slope3

SD4

IC50
µg/mL

SD2

N

Hill
Slope3

SD4

100

76.6

4

-1.30

0.729

52.5

28.0

3

-1.60

0.260

55.1

3.43

3

-1.47

0.466

III

0.198

0.100

3

-2.08

1.01

0.153

0.031

3

-2.64

0.639

0.127

0.020

3

-2.90

0.338

III

348

63.5

3

-1.36

0.241

541

150

3

-1.34

0.411

394

50.8

3

-1.48

0.103

III

8137

591

3

-14.0

6.08

NA

NA

-

NA

NA

NA

NA

-

NA

NA

III

1.69

0.950

3

-0.838

0.076

2.03

0.471

3

-1.37

0.471

2.13

0.480

3

-1.95

0.369

III

0.021

0.007

3

-2.46

0.698

0.007

0.007

3

-3.55

1.68

0.011

0.003

3

-3.34

0.396

III

468

116

3

-1.31

0.252

702

160

3

-1.83

0.455

430

71.5

3

-1.24

0.115

Amine

III

60.5

13.6

3

-1.72

0.238

79.4

33.9

3

-1.88

0.915

66.2

5.57

3

-2.53

0.221

Cyclichydrocarbon

III

NA

NA

-

NA

NA

NA

NA

-

NA

NA

486

185

3

-2.88

1.99

ChemicalClass5Heterocyclic
Sulfurcompound;
Metal
Carboxylic
acid
Halogenatedhydrocarbon
Heterocyclic
Organometallic
compoundCarboxylicacid; Lipids

ECBC
1

FAL
1

IIVS
1

Abbreviations: ECBC=Edgewood Chemical Biological Center; FAL=Fund for the Replacement of Animals in Medical Experiments Alternatives Laboratory; IIVS=Institute for In Vitro Sciences; SD=Standard deviation;
N=Number of data points; NA=Not available (i.e., IC50 values or Hill Slope values could not be generated [see notes in Appendix I for more information])
1
Arithmetic mean.
2Standard deviation of IC50.
3Arithmetic Mean of Hill Slope values.
4Standard deviation of Hill Slope values.
5
Chemical class assigned is based on the classification of the National Library of Medicine’s Medical Subject Heading (MeSH), http://www.nlm.nih.gov/mesh/meshhome.html.

5-23

In Vitro Cytotoxicity Methods BRD Section 5

Figure 5-1

November 2006

Reference Substance IC50 Results for the 3T3 NRU Test Method by Laboratory

Abbreviations: ECBC=Edgewood Chemical Biological Center; FAL=Fund for the Replacement of Animals in Medical Experiments Alternatives
Laboratory; IIVS=Institute for In Vitro Sciences.
Points show the mean arithmetic IC50 (µg/mL) for each reference substance from each laboratory. Error bars show the standard deviation. Data were
sorted in ascending order of 3T3 IC50 values from IIVS (lead laboratory in the validation study). Table 5-6 provides the numerical key for reference
substance identification.

5-24

In Vitro Cytotoxicity Methods BRD Section 5

Figure 5-2

November 2006

Reference Substance IC50 Results for the NHK NRU Test Method by Laboratory

Abbreviations: ECBC=Edgewood Biological Center; FAL=Fund for the Replacement of Animals in Medical Experiments Alternatives Laboratory;
IIVS=Institute for In Vitro Sciences.
Points show the mean arithmetic IC50 (µg/mL) for each reference substance from each laboratory. Error bars show the standard deviation. Data were
sorted in ascending order of 3T3 IC50 values from IIVS (lead laboratory in the validation study). Table 5-6 provides the numerical key for reference
substance identification.

5-25

In Vitro Cytotoxicity Methods BRD Section 5

November 2006

Key to Validation Study Reference Substances1

Table 5-6No

Reference SubstanceNo

Reference SubstanceNo

Reference SubstanceNo

Reference Substance1

Aminopterin

19

Propylparaben

37

Strychnine

55

Citric acid2

Triphenyltin hydroxide20

Propranolol HCl

38

Phenylthiourea

56

Boric acid3

Colchicine

21

Dichlorvos

39

Lindane57

5-Aminosalicylic acid4

Cycloheximide

22

Potassium cyanide

40

Carbamazepine

58

Sodium hypochlorite

5

Triethylenemelamine

23

Physostigmine

41

Diethyl phthalate

59

Lactic acid6

Sodium dichromate
dihydrate

24

Dibutyl phthalate

42

Glutethimide

60

Potassium I chloride

7

Sodium arsenite

25

Parathion

43

Chloramphenicol

612-Propanol

8

Cadmium II chloride

26

Paraquat

44

Chloral hydrate

62

Sodium chloride

9

Hexachlorophene

27

Sodium selenate

45

Caffeine

63

Dimethylformamide

10

Mercury II chloride

28

Verapamil HCl

46

Digoxin64

Ethanol

11

Endosulfan

29

Acetaminophen

47

Meprobamate

65

Gibberellic acid12

Arsenic III trioxide

30

Busulfan48

Acetylsalicylic acid66

Acetonitrile

13

Diquat dibromide
monohydrate

31

Sodium oxalate

49

Nicotine

67

1,1,1-Trichloroethane

14

Haloperidol

32

Phenol50

Phenobarbital

68

Ethylene glycol

15

Cupric sulfate
pentahydrate

33

Disulfoton

51

Procainamide HCl

69

Glycerol16

Thallium I sulfate

34

Epinephrine bitartrate

52

Valproic acid70

Lithium I carbonate

17

Amitriptyline HCl

35

Atropine sulfate

53

Xylene71

Carbon tetrachloride

18

Fenpropathrin

36

Sodium I fluoride

54

Trichloroacetic acid72

MethanolAbbreviations: No=Number.
1
As used in Figures 5-1 and 5-2.

5.5

Statistical Approaches to the Evaluation of 3T3 and NHK Data

The statistical approaches used for data evaluation are reviewed in the following sections foreach phase of the validation study. Section 2.2.3 discussed the endpoint measurements forthe 3T3 and NHK test methods. The OD values of each of six replicate wells ([minimum offour] in the 96-well plate) per test concentration (eight concentrations/reference substance orPC) were used to determine relative cell viability in relation to the mean VC OD on the sameplate. The cell viability values calculated for the replicate wells for each concentration wereused to determine the concentration-response curve (percent viability vs. log concentration)for each test. The IC50 value was determined from fitting the curve to a Hill function.

5-26

In Vitro Cytotoxicity Methods BRD Section 5

November 2006

5.5.1
Statistical Analyses for Phase Ia Data
The laboratories reported the IC50 results for SLS in µg/mL. The SMT used the results fromthe acceptable tests to calculate means and SDs for each method at each laboratory.
5.5.1.1 Outlier Determination for Replicate Well Concentration Data
A test for outliers at the 99% level (Dixon and Massey 1981) was used to determine thepresence of outlier OD values among the six replicate wells for each reference substanceconcentration. The SMT applied the outlier test to the Phase Ia data when extreme values
were noted. Outliers were excluded from the data set, and the IC50 was recalculated. The raw
data files include all data provided by the laboratories, including the excluded outlier OD
values. Because the protocol required a minimum of four acceptable test wells per reference
substance concentration, no more than two wells of the six replicates could be excluded.
5.5.1.2 Curve Fit Criteria
After the completion of Phase Ia testing, a curve fit criterion was implemented for test
acceptance following a visual review of the fit of the OD data to the Hill function curve. The
SMT considered the fit of the concentration-response curve to the Hill function to be
acceptable when R2 >0.9. A fit of R2 <0.8 was considered unacceptable and the data fromthat test were rejected. Curves with a fit of 0.8 < R2 <0.9 were evaluated visually for
goodness of fit and accepted if the SMT concluded that there were sufficient data points
between 0 and 100% cytotoxicity, and a reasonable shape to the curve, to calculate areasonably accurate IC50 value. Each test with a curve fit in this range was analyzed on acase-by-case basis, and no standard pass/fail criterion was developed. [Note: The use of acurve fit criterion was reevaluated in Phases Ib and II, and was eliminated as a test
acceptance criterion for Phase III test results. An R2 value ≥0.85 was maintained as a test
acceptance criterion for the PC because its fit to the Hill function was well characterized.]
5.5.1.3 Reproducibility Analyses for PC IC50 Values
To evaluate reproducibility of the IC50 values for the PC for each test method, within andbetween the laboratories, the SMT considered the American Society of Testing and Materials(ASTM) Standard E691-99, Standard Practice for Conducting an Interlaboratory Study to
Determine the Precision of a Test Method (ASTM 1999). This method uses two statistics, hand k, to judge the consistency of means and variances between laboratories. However, aminimum of six laboratories is required for this type of analysis and the SMT decided that it
could not be appropriately applied to three laboratories. The variability of the PC IC50 results
obtained from each test and laboratory was assessed using CV analysis and one-way
ANOVA. Dividing the SD by the arithmetic mean IC50 value, and multiplying by 100
produced the CV. CV values were calculated for the acceptable tests within each laboratory
to determine intralaboratory reproducibility. To compare the variation among laboratories,
the CV was calculated using the arithmetic mean IC50 values from each of the threelaboratories. Although no criterion for an acceptable CV was determined for this study,
ECVAM recently used CV <30% as an acceptable range for both intra- and inter-laboratory
reproducibility (Zuang et al. 2002; Fentem et al. 2001). Although CV <30% was intended toreflect an acceptable maximum for normal biological variability, the range was not supported
by data.
For the ANOVA, IC50 values were first converted to mM units and then log-transformed toobtain normal distributions. One-way ANOVA was performed with SAS PROC GLM
5-27

In Vitro Cytotoxicity Methods BRD Section 5

November 2006

software (SAS Institute 1999; see Appendix D1 for example SAS code). A significance levelof p <0.01 was used to test results between the laboratories in order to be conservative withrespect to identifying laboratory differences.
5.5.2

Statistical Analyses of Phase Ib Data

5.5.2.1 Outlier Determination for Replicate Well Concentration Data
For consistency of replicate well concentration data, the SMT applied the same outlier test
used for the Phase Ia data (Dixon and Massey 1981) when extreme OD values were noted. If
the extreme value was an outlier at the 99% level, it was excluded from the data set, and theIC50 was recalculated. All data are available in the data files provided by the laboratories,
including the excluded outlier OD values.
5.5.2.2 Reproducibility Analyses of the Reference Substance IC50 Values
One-way ANOVA and CV analyses were used to assess method reproducibility within andamong laboratories. For the ANOVA, the IC50 values were first converted to mM units andthen log-transformed to obtain normal distributions. One-way ANOVA was performed withSAS PROC GLM (SAS Institute 1999; see Appendix D1 for example SAS code). A
significance level of p <0.01 was used to test results between the laboratories in order to beconservative with respect to identifying laboratory differences. When the ANOVA detectedsignificant differences among the laboratories, contrast analyses were performed todetermine which laboratory was different from the others. These analyses compared theresults of each laboratory with those of the other two laboratories. A significant difference in
response among the laboratories was indicated by p <0.01.
CV values were calculated for each reference substance by dividing the SD by the arithmetic
mean IC50 value and multiplying by 100. CV values were calculated for the acceptable tests
in each laboratory to determine intralaboratory reproducibility. To compare the variation
among laboratories, the CV was calculated using the arithmetic mean IC50 values from eachof the three laboratories.
As an additional approach to the assessment of interlaboratory reproducibility for each test
substance, the maximum:minimum IC50 ratios (i.e., the maximum arithmetic mean laboratory
IC50 value compared to the minimum arithmetic mean laboratory IC50 value) were calculated.
This approach is similar to the calculation of maximum:minimum LD50 ratios for examining
reproducibility of reference LD50 values (see Section 4.4.1).
5.5.3

Statistical Analyses of Phase II Data

5.5.3.1 Outlier Determination for Replicate Well Concentration Data
The Dixon and Massey (1981) outlier test was incorporated into the EXCEL® templates toassess the consistency of replicate well data for each reference substance concentration.
Outliers at the 99% level were highlighted and the Study Director was offered the option ofremoving the value from subsequent calculations (e.g., mean OD of the six replicates; %viability; IC50).
5.5.3.2 Reproducibility Analyses of the Reference Substance IC50 Values
The intra- and inter-laboratory reproducibility of the IC50 values were assessed using theacceptable tests to calculate the mean IC50, SD, and CV for each substance, method, and5-28

In Vitro Cytotoxicity Methods BRD Section 5

November 2006

laboratory, as described in Section 5.5.2.2. One-way ANOVAs and calculations ofmaximum:minimum IC50 ratios were performed as described in Section 5.5.2.2.
5.5.3.3 Comparison of 3T3 and NHK Test Results with the RC Millimole Regression
To compare the 3T3 and NHK test results for the reference substances to those of the RC
millimole regression, each IC50 value was transformed to mM units for the calculation ofgeometric mean IC50 values. The use of geometric means corresponded with the approach
used to obtain single IC50 values from multiple IC50 values for the RC millimole regression(Halle 1998, 2003). The log geometric mean IC50 values (in mM) of the 11 RC substancestested during Phases Ib and II (see Table 3-8) were used with the log RC LD50 values, after
transformation to log mmol/kg units (see Appendices J1 and J2), to calculate least squares
linear regressions for the data from each test method and laboratory. Each of thesemethod/laboratory regressions was compared to the RC millimole regression using an F testwith SAS PROC REG (SAS Institute 1999; see Appendix D2 for example SAS code). An F
test with a significance level of p <0.01 was used to determine whether the joint comparisonof slope and intercept indicated that the method/laboratory regressions were significantly
different from the RC millimole regression.
As an alternate analysis, a least squares linear regression using IC50 and LD50 values from the
RC was constructed for the 11 RC substances (the RC-11 regression) tested in Phases Ib andII. Each of these method/laboratory regressions was compared to the RC-11 regression using
an F test with SAS PROC GLM (SAS Institute 1999; see Appendix D2 for example SAScode) at a significance level of p <0.01. This was used to determine whether the comparisonsof slope and intercept indicated that the laboratory regressions were significantly different
from the RC-11 regression.
5.5.4

Statistical Analyses of Phase III Data

5.5.4.1 Outlier Determination for Replicate Well Concentration Data
The laboratories used the Dixon and Massey (1981) outlier test at the 99% level that wasincorporated into the EXCEL® templates to test for outlier values among replicate well dataat the different reference substance concentrations. The Study Director had the option of
excluding the outliers from the data set, which were highlighted by the template, andsubsequent calculations. All data are available in the data files provided by the laboratories,
including the outlier OD values.
5.5.4.2 Reproducibility Analyses of the PC IC50 Data
A number of analyses were performed to determine whether the SLS IC50 values werereproducible across study phases. The SLS IC50 values used to access variability weredifferent from those shown in Table 5-3. To get an assessment of the true variation of SLS
IC50 values, the reproducibility analyses included additional IC50 values from SLS tests that
did not meet the IC50 acceptance limits (see Table 5-3) for each laboratory and study phase if
they passed all other test acceptance criteria. If more than one SLS test was performed on asingle day (for any test method and laboratory), the IC50 values were averaged to determine asingle IC50 for the day. This prevented multiple data values from a single day from overly
influencing the mean for each phase. CV analyses were performed as described in Section
5.5.1 using the arithmetic mean SLS IC50 values for each method, laboratory, and study
phase.

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In Vitro Cytotoxicity Methods BRD Section 5

November 2006

For the remaining analyses of reproducibility, the IC50 values were first log-transformed toobtain normal distributions. One-way ANOVAs were performed with SAS PROC GLM(SAS Institute 1999; see Appendix D1 for example SAS code) for each method using study
phase and laboratory as individual variables. A significance level of p <0.01 was used to test
for a statistical difference among the laboratory and/or phase results.
To determine whether there was a linear time trend for the SLS IC50 data, linear regression
analyses using a least squares method were performed for each laboratory and method using
SAS PROC REG (SAS Institute 1999). Time was expressed as an index for each test. The
index number of each SLS test reflected its order of testing without respect to the time
lapsing between tests. For example, the first SLS test was assigned a time index of 1 and thesecond SLS test was assigned a time index of 2 whether it occurred the day after the first testor one week after the first test. The slopes of the linear regressions were judged to bestatistically significant at p <0.05, which indicated that the IC50 had changed significantly
over time.
5.5.4.3 Reproducibility Analyses of the Reference Substance IC50 Values
CV, one-way ANOVA analyses, and maximum:minimum IC50 ratios were performed toassess the intra- and/or inter-laboratory reproducibility of the Phase III reference substancedata, as described in Section 5.5.2.2. An additional evaluation to determine whether
normalizing the reference substance IC50 to the SLS IC50 would reduce interlaboratory
variability was performed using five substances (for each test method) for which the
ANOVAs indicated significant interlaboratory differences. The reference substance IC50
values were normalized to the SLS IC50 by calculating the reference substance IC50:SLS IC50
ratio. CVs were calculated for each substance using the mean ratios from each laboratory. To
determine whether this normalization reduced variability among the laboratories, the CVs forthe substance IC50:SLS IC50 ratios were compared to the CVs for the substance IC50.
In addition, the geometric mean IC50 values were used to calculate least squares linear
regression models after log transforming the data. Linear regressions were fit for eachmethod and laboratory using the log-transformed reference LD50 values from Table 4-2 (inmmol/kg), with log IC50 in mM. To detect differences among the linear regressions in eachlaboratory, two models were fit for each method. The first was a full model that included
effects for laboratory and interactions, and generated a regression line for each substance ineach laboratory, by test method. The second model, which was considered to be a reduced
model, assumed that one model fit all the laboratories. A goodness of fit F test was
performed to compare the full and reduced models for each method. A significance level of p
<0.01 was used to test whether the regressions among laboratories were significantly
different from one another. The following criteria were established for selection of data foruse in the regression analyses for each test method:• The substance was included in the RC• All three laboratories reported IC50 values• There was an associated rat oral reference LD50 value (see Table 4-2)
There were 47 reference substances that fit these criteria for the 3T3 and 51 test substancesthat fit the criteria for the NHK test methods.

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5.5.4.4 Comparison of 3T3 and NHK Results with the RC Millimole Regression
To determine whether the IC50 values determined in the validation study were significantly
different from the RC values, the laboratory-specific regression values for each method werecombined using the geometric means of the laboratory-specific geometric mean IC50 values
in mM and the reference LD50 in mmol/kg. Thus, there was one regression analysis withpooled laboratory data for the 3T3 NRU test method and another regression analysis (alsowith pooled data) for the NHK NRU test method. A third linear regression was calculatedusing the IC50 and LD50 values from the RC. The IC50 values and LD50 values were logtransformed for the regression calculations. The following criteria were established for theselection of substances to be used for the regression analyses:• The substance was included in the RC• All three laboratories reported IC50 values for both the 3T3 and NHK NRU
test methods• There was an associated rat oral reference LD50 value (see Table 4-2)
Forty-seven substances met these criteria. Two models were fit for each test method to detect
differences between the NRU regression and the 47 RC substance regression. The firstregression model was a full model that included effects for the RC and the NRU regression,and generated one regression line each for the RC and the NRU test method. The second(reduced) model assumed that a single model fit the combined RC and NRU IC50 data. The
RC regression for the 47 reference substances was compared to the combined laboratory
regression for each NRU test method using an F test to simultaneously compare slopes andintercepts. The NRU regressions were statistically different from the RC regressions if
p <0.01.
To assess the accuracy of the NRU methods and the associated IC50-LD50 regressions, apredicted LD50 was calculated for each reference substance using its laboratory geometricmean IC50 in two analyses:• The RC rat-only millimole regression calculated from the 282 RC substanceswith rat LD50 values, using units of mM for the IC50 and mmol/kg for the LD50
(see Section 6.4.2)• The RC rat-only weight regression calculated from the 282 RC substanceswith rat LD50 values, using units of µg/mL for the IC50 and mg/kg for the
LD50 (see Section 6.4.3)
The LD50 values predicted from the regression analyses were used to predict GHS acute oral
toxicity categories (see Section 6.4). The accuracy of the predictions was determined by
calculating the proportion of substances for which the predicted GHS toxicity category
matched the GHS toxicity category. The LD50 predictions from these regression models werealso used to determine starting doses for acute systemic toxicity test simulations for the
purpose calculating animal use and savings that would be achieved using the NRU test
methods. The simulation modeling methods, and results from the UDP and ATC methods,
are described in Section 10.

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November 2006

5.5.5
Summary of the Data Used for Statistical Analyses
Table 5-7 summarizes the number of substances that were tested and the number ofsubstances used for the various analyses performed to determine the accuracy and reliabilityof the in vitro NRU test methods.
Table 5-7

Datasets Used for Validation Study Analyses1
3T3 NRU
Test
Method1
72

NHK NRU
Test
Method1
72

47

51

47

47

67

68

Reproducibility of acceptable rat
oral LD50 values

NA

NA

Reproducibility of IC50 values

64

68Comparison of reproducibility of
IC50 values with reproducibility ofLD50 values

53

57

Use
TestingComparison of laboratory IC50LD50 regressions to one another
Comparison of combinedlaboratory IC50-LD50 regressions
to a regression calculated withRC data
Prediction of GHS accuracy using
IC50 values in IC50-LD50
regressions; prediction of starting
doses for acute oral toxicity test
(UDP and ATC) simulations

Characteristics of Dataset
Substances tested
RC substances with IC50 values
from all laboratories andreference rat oral LD50 values
RC substances with IC50 values
for both test methods from alllaboratories and rat oralreference LD50 values
Substances with IC50 values
from at least one laboratory
62 substances with more than
one acceptable rat oral LD50
value
Substances with IC50 values
from all laboratories
Substances with IC50 values
from all laboratories and morethan one acceptable rat oral LD50
value

Abbreviations: RC=Registry of Cytotoxicity; 3T3=BALB/c 3T3 fibroblasts; NHK=Normal human epidermal
keratinocytes; NRU=Neutral red uptake; NA=Not applicable.
1
Number of substances.

5.6

Summary of NRU Test Results

Table 5-8 shows the 3T3 and NHK IC50 values as geometric means of the geometric mean
laboratory values, as a basis to compare the 3T3 and NHK NRU IC50 values for eachreference substance. The substances in Table 5-8 are organized by ascending 3T3 NRU IC50
values (as was done for Figures 5-1 and 5-2). For each method, the table provides thegeometric mean IC50 (combined across laboratories) in µg/mL, the ratio of the geometricmean IC50 to the SLS IC50, and the 3T3 IC50:NHK IC50 ratios. Geometric means were used
for this comparison because they were used for both the IC50 and LD50 regression analyses(see Sections 5.5.3.3, 5.5.4.3, and 5.5.4.4). The 3T3 and NHK NRU IC50 values werecompared using the ratios of their geometric means. The IC50 values for each reference
substance were also compared to the IC50 for SLS using the ratio of reference substancegeometric mean IC50 to SLS geometric mean IC50.
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In Vitro Cytotoxicity Methods BRD Section 5

Table 5-8

November 2006

Comparison of 3T3 and NHK NRU IC50 Geometric Means
3T3 NRU

NHK NRU

Reference SubstanceGeometric
Mean1 IC50(µg/mL)

Ratio
Geometric
Mean IC50
to SLS IC50

Geometric
Mean1 IC50(µg/mL)

Ratio
Geometric
Mean IC50
to SLS IC50

IC50 Ratios
3T3:NHK

Carbon tetrachloride
MethanolAminopterin
Triphenyltin hydroxide
Colchicine
Cycloheximide
Triethylenemelamine
Cadmium II chloride
Sodium dichromate dihydrate
Sodium arsenite
Arsenic trioxide
Mercury II chloride
Hexachlorophene
Thallium I sulfate
Haloperidol
Endosulfan
Amitriptyline HCl
Diquat dibromide monohydrate
Propranolol
Dichlorvos
Paraquat
Fenpropathrin
Physostigmine
Propylparaben
Sodium selenate
Potassium cyanide
Verapamil HCl
Parathion
Sodium oxalate
Sodium lauryl sulfate (SLS)*
Cupric sulfate pentahydrate
Acetaminophen
Dibutyl phthalate
Epinephrine bitartrate
PhenolAtropine sulfate
BusulfanSodium I fluoride
Phenylthiourea
Carbamazepine

NA
NA
0.006
0.017
0.034
0.187
0.272
0.518
0.587
0.759
1.96
4.12
4.19
5.74
6.13
6.35
7.05
8.04
13.9
17.7
20.1
24.2
25.8
26.1
29
34.6
34.9
37.4
37.7
41.7
42.1
47.7
49.7
59
66.3
76
77.7
78
79
103

NA
NA
0.00010.0004
0.001
0.004
0.007
0.01
0.01
0.02
0.05
0.1
0.1
0.1
0.1
0.2
0.2
0.2
0.3
0.4
0.5
0.6
0.6
0.6
0.7
0.8
0.8
0.9
0.9
1.0
1.0
1.1
1.2
1.4
1.6
1.8
1.9
1.9
1.9
2.5

NA
15293
669
0.01
0.007
0.073
1.85
1.84
0.721
0.477
5.26
5.8
0.029
0.152
3.36
2.13
8.96
4.48
35.3
10.7
61.6
2.43
88.5
16.6
10.2
29
66.5
30.3
337
3.99
197
518
28.7
87.4
75
81.8
260
49.8
336
83.2

NA
383.2
167.7
0.003
0.002
0.02
0.5
0.5
0.2
0.1
1.3
1.5
0.01
0.04
0.8
0.5
2.2
1.1
8.8
2.7
15.4
0.6
22.2
4.2
2.6
7.3
16.7
7.6
84.5
1.0
49.4
129.8
7.2
21.9
18.8
20.5
65.2
12.5
84.2
20.9

NA
NA
0.00001
1.7
4.9
2.6
0.1
0.3
0.8
1.6
0.4
0.7
144.5
37.8
1.8
3.0
0.8
1.8
0.4
1.7
0.3
10.0
0.3
1.6
2.8
1.2
0.5
1.2
0.1
10.5
0.2
0.1
1.7
0.7
0.9
0.9
0.3
1.6
0.2
1.2

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In Vitro Cytotoxicity Methods BRD Section 5

Table 5-8

November 2006

Comparison of 3T3 and NHK NRU IC50 Geometric Means
3T3 NRU

Reference Substance
Diethyl phthalate
Lindane
Chloramphenicol
Disulfoton
Caffeine
Strychnine
Glutethimide
Chloral hydrate
Nicotine
Procainamide HCl
Digoxin
Meprobamate
Lithium I carbonate
Phenobarbital
Acetylsalicylic acid
XyleneCitric acid
Trichloroacetic acid
Valproic acid
Sodium hypochlorite
5-Aminosalicylic acid
Boric acid
Lactic acid
Potassium I chloride
2-Propanol
Sodium chloride
Dimethylformamide
Ethanol
Gibberellic acid
Acetonitrile
1,1,1-Trichloroethane
Ethylene glycol
GlycerolNHK NRU

Geometric
Mean1 IC50(µg/mL)

Ratio
Geometric
Mean IC50
to SLS IC50

Geometric
Mean1 IC50(µg/mL)

Ratio
Geometric
Mean IC50
to SLS IC50

IC50 Ratios
3T3:NHK

107
108
128
133
153
158
174
183
361
441
466
519
5622
573
676
7212
796
902
916
1103
1667
1850
3044
3551
3618
4730
5224
6523
78103
7951
17248
24317
24655

2.6
2.6
3.1
3.2
3.7
3.8
4.2
4.4
8.7
10.6
11.2
12.4
13.5
13.7
16.2
17.3
19.1
21.6
22.0
26.5
40.0
44.4
73.0
85.2
86.8
113.4
125.3
156.4
187.3
190.7
413.6
583.1
591.2

120
18.7
348
270
638
62.5
174
133
107
1741
0.001
357
468
448
605
4662
400
413
512
1502
46.7
421
1304
2237
5364
1997
7760
10018
2856
9528
81222
41852
24730

30.1
4.7
87.2
67.7
159.9
15.7
43.6
33.3
26.8
436.3
0.0003
89.5
117.3
112.3
151.6
116.8
100.3
103.5
128.3
376.4
11.7
105.5
326.8
560.7
1344.4
500.5
1944.92510.8715.82388.02035.610489.2
6198.0

0.9
5.8
0.4
0.5
0.2
2.5
1.0
1.4
3.4
0.3
466000.0
1.5
1.2
1.3
1.1
1.5
2.0
2.2
1.8
0.7
35.7
4.4
2.3
1.6
0.7
2.4
0.7
0.7
2.7
0.8
2.1
0.6
1.0

Abbreviations: 3T3=BALB/c 3T3 fibroblasts; NHK=Normal human epidermal keratinocytes; NRU=Neutral red uptake;
SLS=Sodium lauryl sulfate; NA=Not available.
Reference substances are ordered by 3T3 NRU IC50 values.
1
Geometric mean IC50 of the laboratory geometric mean values.
2
Data available from only one laboratory.
3
Data available from only two laboratories.
*Acceptable positive control (SLS) values from all study phases: N=293 for the 3T3 NRU and N=281 for the NHK NRU.

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November 2006

Table 5-8 shows that there are nine reference substances for which the 3T3 and NHK NRU
IC50 values differ by at least one order of magnitude (i.e., 3T3 IC50:NHK IC50 ≤0.1 or ≥10):
aminopterin, triethylenemelamine, hexachlorophene, thallium sulfate, fenpropathrin, sodium
oxalate, acetaminophen, digoxin, and 5-aminosalicylic acid. The IC50 values for SLS, also
differed by slightly more than one order of magnitude in the two NRU test methods (41.7µg/mL for 3T3 and 3.99 µg/mL for NHK). One test method was not more consistently
sensitive (i.e., produced lower IC50 values) than the other for these nine reference substances.
The 3T3 NRU test method was more sensitive than the NHK NRU test method for four of
the nine substances: aminopterin, triethylenemelamine, sodium oxalate, and acetaminophen.
The NHK NRU test method was more sensitive than the 3T3 NRU test method for five
substances: hexachlorophene, thallium sulfate, fenpropathrin, digoxin, and 5-aminosalicylic
acid. Despite the normalization procedure, the reference substance IC50:SLS IC50 ratios forthe two methods were still greater by at least one order of magnitude for six of the nine
substances (aminopterin, triethylenemelamine, hexachlorophene, sodium oxalate,acetaminophen, and digoxin) and the order of magnitude difference increased for all six
substances. A number of factors could potentially be responsible for these differences
between the 3T3 and NHK NRU IC50 values:• Cell culture conditions (i. e., the 3T3 treatment medium contains serum while
the NHK treatment medium does not; differences in cell density in thetreatment medium)• Differences in sensitivity between the fibroblast cell line and primary
keratinocytes• Differences in sensitivity between human and mouse cells• Differences in metabolic activity between the cell types
These factors may affect the results for some substances more than others. For example, asubstance that binds to serum proteins would be less available to the 3T3 cells (which haveserum in their growth medium) than to NHK cells (which are grown without serum). No
additional testing was performed to investigate the differences between the 3T3 and NHK
NRU IC50 values.
Two substances, digoxin and aminopterin, have IC50 values that differ by five orders ofmagnitude between the two NRU test methods. Digoxin was much more toxic to the NHK
cells and aminopterin was more toxic to the 3T3 cells. Both substances are known substrates
for organic anionic transporters (OAT) (ICCVAM 2006). Such transporters are important forin vivo toxicity responses in terms of the ability of challenge substances to be absorbed, reachtarget tissues, accumulate, or be excreted. The differential susceptibilities of the 3T3 andNHK cells may be explained by differential functioning of OAT between the cell types.
Although species and tissue differences in OAT have been reported (Sekine et al. 2000;
Miyazaki et al. 2004), the reason for these differential sensitivities is not known.
The 3T3 IC50:NHK IC50 ratios shown in Table 5-8 were used to determine the frequency
distributions shown in Table 5-9. These distributions indicate that the 3T3 and NHK NRU
IC50 values were within one order of magnitude of each other for 85% of the reference
substances (obtained by adding 38.9% and 45.8% for the 0.1 < IC50 ratio ≤1 and 1 < IC50
ratio <10 ranges). Ninety-three percent of the reference substances have 3T3 and NHK NRU
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In Vitro Cytotoxicity Methods BRD Section 5

November 2006

IC50 values within two orders of magnitude of each other (obtained by adding 4.2% each forthe 10 ≤ IC50 ratio ≤100 and 0 < IC50 ratio ≤0.1 ranges to the 85% above).
Table 5-9

Frequency of 3T3:NHK IC50 Ratios1 for Reference Substances

IC50 Ratio <0.00001

Number of
Substances
1% ofSubstances
1.4

0 < IC50 Ratio ≤0.1

3

4.2

0.1 < IC50 Ratio ≤1

28

38.9

1 < IC50 Ratio <10

33

45.8

10 ≤ IC50 Ratio <1003

4.2

100 ≤ IC50 Ratio <1000

1

1.4

IC50 Ratio ≥1000

1

1.4

Not Available

2

2.8

3T3:NHK IC50 Ratio Range

Abbreviations: 3T3=Neutral red uptake using BALB/c 3T3 fibroblasts; NHK= Neutral
red uptake using normal human epidermal keratinocytes.
Note: Compiled using reference substance data from Table 5-7.

Correlations of the mean IC50 values for the reference substances common to the RC
database with the IC50 values (i.e., geometric mean of IC50 values obtained from the literature
for various basal cytotoxicity endpoints and cell types) from the RC (Halle 1998, 2003) are
shown in Figure 5-3 (3T3 values) and Figure 5-4 (NHK values). Although the validation
study tested 58 RC substances in common with the RC, IC50 values were obtained for 56
substances using the 3T3 NRU test method and 57 substances using the NHK NRU test
method. Spearman correlation analyses of the log-transformed IC50 data (in mM) indicatedthat the NRU IC50 values were significantly correlated with the RC IC50x values (p<0.001, forboth the 3T3 and NHK NRU test methods). The Spearman correlation coefficient, rs, was
0.93 for the 3T3 values and 0.86 for the NHK values.

5-36

In Vitro Cytotoxicity Methods BRD Section 5

Figure 5-3
1000
100

November 2006

RC IC50 Values vs 3T3 NRU IC50 Values for 56 Substances in Common
rs = 0.93
n = 56

10
1
0.1
0.01
0.001
0.00010.00001
0.00001

0.0001

0.001

0.01

0.1

1

10

100

1000

Registry of Cytotoxicity IC50 (mM)

Abbreviations: RC=Registry of Cytotoxicity; 3T3=BALB/c 3T3 fibroblasts; NRU=Neutral red uptake;rs=Spearman correlation coefficient; n=Number of substances; mM=Millimolar.
The diagonal line indicates the predicted values for a 1:1 correspondence. No IC50 values were obtained forcarbon tetrachloride or methanol because of insufficient toxicity. The Registry of Cytotoxicity IC50 values aregeometric means of IC50 values obtained from the literature for various basal cytotoxicity endpoints and cell
types.

5-37

In Vitro Cytotoxicity Methods BRD Section 5

Figure 5-4
1000
100

November 2006

RC IC50 Values vs NHK NRU IC50 Values for 57 Substances in Common
rs = 0.86
n = 57

10
1
0.1
0.01
0.001
0.00010.00001
0.00001

0.0001

0.001

0.01

0.1

1

10

100

1000

Registry of Cytotoxicity IC50 (mM)

Abbreviations: RC=Registry of Cytotoxicity; NHK=Normal human epidermal keratinocytes; NRU=Neutral red
uptake; rs=Spearman correlation coefficient; n=Number of substances; mM=Millimolar.
The diagonal line indicates the predicted values for a 1:1 correspondence. No IC50 values were obtained formethanol because of insufficient toxicity. The Registry of Cytotoxicity IC50 values are geometric means of IC50
values obtained from the literature for various basal cytotoxicity endpoints and cell types.

5.7

Availability of Data

All data were provided to the SMT as electronic files and paper copies. The laboratories also
maintained copies of all raw data and the electronic files. The individual test data and IC50
results for both passing and failing tests are provided in Appendix I for the reference
substances and the PC.
5.8

Solubility Test Results

A solubility protocol (see Section 2-8 and Appendix B3) designed to identify the solventthat would provide the highest concentration of a reference substance for in vitro testing wasevaluated. Each laboratory performed solubility tests on all reference substances. However,to avoid the use of different solvents by the laboratories when testing the same substance,
which might increase the variability of the IC50 results among the laboratories, the SMT
assigned the solvents to be used (see Table 5-10). The objectives of the solubility testing
were to evaluate the utility and appropriateness of the solubility protocol, and to evaluate theconcordance among laboratories in selecting the solvents for each of the 72 reference
substances.

5-38

In Vitro Cytotoxicity Methods BRD Section 5

Table 5-10

November 2006

Solubility Test Results (mg/mL)
BioReliance1

Reference Substance

ECBC3

FAL3

IIVS3

2

3T34
NHK5
DMSO
Medium Medium

ETOH

SMT
Selection

3T34
NHK5
3T34
NHK5
3T34
NHK5
DMSO ETOHDMSO ETOHDMSO ETOH
Medium Medium
Medium Medium
Medium Medium

Phase I
Arsenic III trioxide

0.25

0.05

<2

<2

Medium

0.02560.0256<0.2<0.2

0.13560.1356<0.2<0.2<0.026<0.026<0.2<0.2

Ethylene glycol

400

400

NT

NT

Medium

20

20

NT

NT

20

20

NT

NT

20

20

NT

NT

Propranolol HCl

<2

10

200

20

DMSO

0.2

2

200

NT

20

20

200

NT

20

2

NT

NT

Aminopterin

2

2

NT

NT

DMSO

2.0

<2

200

NT

<2

2

200

NT

0.2

0.2

200

NT

Cadmium II chloride

<2

<2

200

<200

DMSO

<2

<2

200

NT

<2

<2

200

NT

<0.2

<0.2

20

<20

Chloramphenicol

2

2

400

<200

DMSO

2.0

<2

200

NT

<2

<2

200

NT

0.2

0.2

20

20

Colchicine

400

400

NT

NT

Medium

20

20

NT

NT

20

20

NT

NT

20

20

NT

NT

Lithium I carbonate

0.25

10

<2

NT

Medium

0.2

2.0

<20

<20

0.2

2

<200

<200

0.2

2

<2

<2

Potassium I chloride

200

200

NT

NT

Medium

20

20

NT

NT

20

20

NT

NT

20

20

NT

NT

2-Propanol

400

400

400

400

Medium

20

20

NT

NT

20

20

NT

NT

20

20

NT

NT

Sodium I fluoride

20

20

<200

<200

Medium

20

20

NT

NT

20

20

NT

NT

20

20

NT

NT

Sodium selenate

200

200

<200

<200

Medium

20

20

NT

NT

20

20

NT

NT

20

20

NT

NT

Acetaminophen

10

10

400

<200

DMSO

2

2

NT

NT

2

2

NT

NT

<2

<2

200

NT

Acetonitrile

400

400

400

400

Medium

20

20

NT

NT

20

20

NT

NT

20

20

NT

NT

Acetylsalicylic acid

10

10

400

200

DMSO

2

2

NT

NT

<2

<2

200

NT

2

2

NT

NT

5-Aminosalicylic acid

2

2

<200

<200

Medium

2

2

NT

NT

2

2

NT

NT

2

2

NT

NT

Amitriptyline HCl

200

200

NT

NT

DMSO

<2

<2

200

NT

<2

<2

200

NT

0.2

0.2

200

NT

Phase II

Phase III

5-39

In Vitro Cytotoxicity Methods BRD Section 5

Table 5-10

November 2006

Solubility Test Results (mg/mL)
BioReliance1

Reference Substance

ECBC3

FAL3

IIVS3

2

3T34
NHK5
DMSO
Medium Medium

ETOH

SMT
Selection

3T34
NHK5
3T34
NHK5
3T34
NHK5
DMSO ETOHDMSO ETOHDMSO ETOH
Medium Medium
Medium Medium
Medium Medium

Atropine sulfate

200

200

NT

NT

Medium

20

20

NT

NT

20

20

NT

NT

20

20

NT

NT

Boric aid

40

40

200

<200

Medium

20

20

NT

NT

20

20

NT

NT

2

2

NT

NT

Busulfan

<2

<2

40

<200

DMSO

<2

<2

200

NT

<2

<2

506

<200

<0.2

<0.2

20

<200

Caffeine

10

10

20

NT

Medium

20

20

NT

NT

20

20

NT

NT

20

20

NT

NT

Carbamazepine

<2

<2

40

<200

DMSO

0.2

0.2

20

20

<2

<2

200

NT

<0.2

<0.2

2

<20

Carbon tetrachloride

2

10

NT

NT

DMSO

20

20

NT

NT

<0.2

<0.2

2

NT

20

20

NT

NT

Chloral hydrate

400

400

NT

NT

Medium

20

20

NT

NT

20

20

NT

NT

20

20

NT

NT

Citric acid

400

400

NT

NT

Medium

20

20

NT

NT

20

20

NT

NT

20

20

NT

NT

Cupric sulfate
pentahydrate

1

0.5

<2

2

Medium

2

0.2

<200

<200

2

2

NT

NT

0.2

0.2

<200

NT

Cycloheximide

20

20

400

<200

Medium

20

20

NT

NT

20

20

NT

NT

2

2

NT

NT

Dibutyl phthalate

<2

<2

400

400

DMSO

<2

<2

200

NT

<2

<2

200

NT

<2

<2

200

NT

Dichlorvos

10

10

NT

NT

DMSO

2

2

NT

NT

<2

<2

200

NT

2

2

NT

NT

Diethyl phthalate

<2

<2

400

400

DMSO

<2

<2

200

NT

0.2

<0.2

200

NT

<2

<2

200

NT

Digoxin

0.05

0.05

200

< 200

DMSO

<2

<2

200

NT

<0.2

<0.2

200

NT

<2

<2

200

NT

Dimethylformamide

400

400

NT

NT

Medium

20

20

NT

NT

20

20

NT

NT

20

20

NT

NT

Diquat dibromide
monohydrate

200

200

NT

NT

Medium

20

20

NT

NT

20

20

NT

NT

20

20

NT

NT

Disulfoton

<2

<2

500

NT

DMSO

<2

<2

200

NT

<2

<2

200

NT

<2

<2

200

NT

Endosulfan

<0.05

<0.05

40

NT

DMSO

<0.2

<0.2

20

<200

<0.2

<0.2

2

<200

<0.2

<0.2

20

<200

Epinephrine bitartrate

400

400

NT

NT

Medium

20

20

NT

NT

20

20

NT

NT

2

2

NT

NT

Ethanol

200

200

NT

NT

Medium

20

20

NT

NT

20

20

NT

NT

20

20

NT

NT

5-40

In Vitro Cytotoxicity Methods BRD Section 5

Table 5-10

November 2006

Solubility Test Results (mg/mL)
BioReliance1

Reference Substance

ECBC3

FAL3

IIVS3

2

3T34
NHK5
DMSO
Medium Medium

ETOH

SMT
Selection

3T34
NHK5
3T34
NHK5
3T34
NHK5
DMSO ETOHDMSO ETOHDMSO ETOH
Medium Medium
Medium Medium
Medium Medium

Fenpropathrin

<20

<20

500

NT

DMSO

<2

<2

200

NT

<0.2

<0.2

200

NT

<2

<2

200

NT

Gibberellic acid

10

10

NT

NT

Medium

2

2

NT

NT

2

2

NT

NT

2

2

NT

NT

Glutethimide

<2

<2

500

NT

DMSO

<2

<2

200

NT

<2

<2

200

NT

<2

<2

200

NT

Glycerol

400

400

NT

NT

Medium

20

20

NT

NT

20

20

NT

NT

20

20

NT

NT

Haloperidol

<20

<20

40

NT

DMSO

<0.2

<0.2

20

<20

<0.2

<0.2

20

<20

<2

<2

20

<20

Hexachlorophene

0.05

<0.05

400

400

DMSO

<2

<2

200

NT

<2

<2

200

NT

<2

<2

200

NT

Lactic acid

200

200

NT

NT

Medium

20

20

NT

NT

20

20

NT

NT

20

20

NT

NT

Lindane

<0.05

<0.05

400

<200

DMSO

<2

<2

200

NT

<2

<2

200

NT

<0.2

<0.2

20

<200

1

1

200

NT

DMSO

2

2

200

NT

2

2

200

NT

<0.2

<0.2

200

NT

Mercury II chloride

0.125

0.125

400

<200

DMSO

<2

<2

200

NT

<2

<2

200

NT

<0.2

<0.2

200

NT

Methanol

40

40

400

400

DMSO

20

20

NT

NT

20

20

NT

NT

<2

<2

200

NT

Nicotine

400

400

NT

NT

Medium

20

20

NT

NT

20

20

NT

NT

20

20

NT

NT

Paraquat

400

400

NT

NT

Medium

20

20

NT

NT

20

20

NT

NT

20

20

NT

NT

Parathion

0.05

<0.05

400

400

DMSO

<2

<2

200

NT

<2

<2

200

NT

<2

<2

200

NT

Phenobarbital

2

2

200

<200

DMSO

2

2

NT

NT

<2

<2

200

NT

<2

<2

200

NT

Phenol

40

40

400

400

Medium

20

20

NT

NT

20

20

NT

NT

20

20

NT

NT

Phenylthiourea

2

2

400

<200

DMSO

2

<2

200

NT

20

20

NT

NT

<2

<2

200

NT

Physostigmine

2

2

400

200

DMSO

2

2

NT

NT

<2

<2

200

NT

<2

<2

200

NT

Potassium cyanide

400

400

NT

NT

Medium

20

20

NT

NT

20

20

NT

NT

20

20

NT

NT

Procainamide HCl

200

200

NT

NT

Medium

20

20

NT

NT

20

20

NT

NT

20

20

NT

NT

Meprobamate

5-41

In Vitro Cytotoxicity Methods BRD Section 5

Table 5-10

November 2006

Solubility Test Results (mg/mL)
BioReliance1

Reference Substance

ECBC3

FAL3

IIVS3

2

3T34
NHK5
DMSO
Medium Medium

ETOH

SMT
Selection

3T34
NHK5
3T34
NHK5
3T34
NHK5
DMSO ETOHDMSO ETOHDMSO ETOH
Medium Medium
Medium Medium
Medium Medium

Propylparaben

0.25

0.25

400

400

DMSO

<2

<2

200

NT

<2

<2

200

NT

<2

<2

200

NT

Sodium arsenite

400

400

NT

NT

Medium

20

20

NT

NT

20

20

NT

NT

20

20

NT

NT

Sodium chloride

200

200

NT

NT

Medium

20

20

NT

NT

20

20

NT

NT

20

20

NT

NT

Sodium dichromate
dihydrate

400

400

NT

NT

Medium

20

20

NT

NT

20

20

NT

NT

20

20

NT

NT

Sodium hypochlorite

200

200

NT

NT

Medium

20

20

NT

NT

20

20

NT

NT

20

20

NT

NT

Sodium oxalate

<0.05

20

0.125

<0.05

Medium

<0.2

20

0.2

<2

20

20

NT

NT

<0.2

<0.2

<0.2

<0.2

Strychnine

<2

<2

2

2

Medium

0.2

<0.2

2

2

0.2

0.2

<200

<200

<0.2

<0.2

<0.2

<0.2

Thallium I sulfate

1

0.5

<2

<2

Medium

0.2

0.2

<200

<200

<0.2

<0.2

<0.2

<0.2

0.2

0.2

<20

<200

Trichloroacetic acid

200

200

NT

NT

Medium

20

20

NT

NT

20

20

NT

NT

20

20

NT

NT

1,1,1-Trichloroethane

10

10

400

400

Medium

20

20

NT

NT

20

20

NT

NT

20

20

NT

NT

Triethylenemelamine

<2

<2

2

<20

DMSO

0.2

0.2

<200

<200

<0.2

<0.2

2

<2

<0.2

<0.2

<0.2

<0.2

Triphenyltin hydroxide <0.05

<0.05

10

<20

DMSO

<0.2

<0.2

2

<20

<0.2

<0.2

2

<200

<2

<2

2

<20

Valproic acid

10

2

NT

NT

DMSO

2

2

NT

NT

<2

<2

200

NT

2

<2

200

NT

Verapamil HCl

<0.05

0.25

200

NT

DMSO

<2

<2

200

NT

<2

<2

200

NT

<0.2

<0.2

20

NT

1

1

500

NT

DMSO

<2

<2

200

NT

2

<2

200

NT

<2

<2

200

NT

XyleneAbbreviations: 3T3=BALB/c 3T3 fibroblasts; NHK=Normal human epidermal keratinocytes; SMT=Study Management Team; ECBC=Edgewood Chemical Biological Center; FAL=Fund for the Replacement of Animals in
Medical Experiments Alternatives Laboratory; IIVS=Institute for In Vitro Sciences; DMSO=Dimethyl sulfoxide; ETOH=ethanol; NT=Not tested.
Note: Table sorted by study phase and alphabetical by substance.
1
The solubility protocol used was different from that used by the testing laboratories.
2
Solvents selected by the SMT for cytotoxicity testing. The BioReliance results were used to determine solvents for Phases I and II. Results from all laboratories were used to determine solvents for Phase III. 3T3 and NHK media
were treated as a single solvent. If a substance insoluble in one medium, and not the other, and soluble in DMSO, then DMSO was selected for use with both cell types.
3
Used protocol in Figure 2-7.
4Dulbecco’s Modification of Eagle’s Medium.
5
Keratinocyte Growth Medium (KGM from CAMBREX Clonetics®).
6
The results were obtained using a deviation from the standard protocol.
Laboratories agreed on solvent.
Laboratories did not agree on solvent. boldProtocol did not provide enough guideline information to select a single solvent.

5-42

In Vitro Cytotoxicity Methods BRD Section 5

November 2006

5.8.1
Solubility Data
BioReliance evaluated the solubility of the reference substances, first in media, then in
DMSO, and then in ETOH, at 400 and 200 mg/mL. Based on their experience, a solubility
protocol was developed for the testing laboratories. This revised protocol required testing at
lower concentrations, and use of the various solvents at concentrations that would beequivalent when applied to the cell cultures (see Table 2-5). The solubility flow chart(Figure 2-7) illustrates the tests for solubility in 3T3 and NHK medium, DMSO, and ETOH.
Table 5-10 provides the solubility test results.
5.8.2
Solubility and Volatility Effects in the Cytotoxicity Tests
The laboratories reported solubility results for the stock solutions of reference substance foreach 3T3 and NHK test. Prior to the addition of the NR dye medium, the laboratories visuallyobserved the test cultures and documented noticeable precipitate. Table 5-11 illustrates theexistence of solubility issues (in both the 3T3 and NHK NRU test methods) as evidenced by
the observation of precipitates with some reference substances. Sections 3.2.6 and 5.4.2
provide additional information on ability of the laboratories to achieve sufficient toxicity forthe calculation of an IC50 in the presence of limited solubility. Table 5-11 also notes thepresence of volatility, as indicated by the use of film plate sealers during incubation.
Table 5-11

Reference Substances with Precipitate (PPT) and Volatility Issues1
3T3 NRU Test Method

Reference Substances

Acetonitrile
Aminopterin
5-Aminosalicylic acidArsenic III trioxide
Cadmium II chloride
Carbamazepine
Carbon tetrachloride
Citric acid
Cupric sulfate pentahydrate
Dibutyl phthalate
Dichlorvos
Diethyl phthalate
DigoxinDimethylformamide
Disulfoton
Endosulfan
Ethanol
Fenpropathrin
Gibberellic acid
Glutethimide
Lindane
Lithium I carbonate
Nicotine
Parathion
PhenolPotassium I chloride
Potassium cyanide

PPT in2X Stock
Dilutions

PPT in1X Plate
Dilutions

PPT in
Stock
and
Plate
Dilutions

NHK NRU Test Method

Volatility

PPT in2X Stock
Dilutions

PPT in1X Plate
Dilutions

PPT in
Stock
and
Plate
Dilutions

X
X

Volatility

X
X

X
X

X
X

X
X
X

X
X
X

X

X
X

X

X

X
X
X
X

X

X
X
X

X
X

X

X

X

X
X
X

X

X

X
X

X
X

X
X

X
X

5-43

X

X

X

X

In Vitro Cytotoxicity Methods BRD Section 5

November 2006

Reference Substances with Precipitate (PPT) and Volatility Issues1

Table 5-11

3T3 NRU Test Method
Reference Substances

2-Propanol
Sodium arsenite
Sodium chloride
Sodium I fluoride
Sodium hypochlorite
Sodium oxalate
Strychnine
Trichloroacetic acid
1,1,1-Trichloroethane
Valproic acid
Verapamil HCl
Xylene

PPT in2X Stock
Dilutions

PPT in1X Plate
Dilutions

PPT in
Stock
and
Plate
Dilutions

NHK NRU Test Method

Volatility

PPT in2X Stock
Dilutions

PPT in1X Plate
Dilutions

PPT in
Stock
and
Plate
DilutionsX

Volatility

X
X

X
X
X

X
X
X
X

X
X
X

X
X

X
X
X

X

Abbreviations: 3T3=BALB/c 3T3 fibroblasts; NHK=Normal human epidermal keratinocytes; NRU=Neutral red uptake; PPT=Precipitate.
Note: Table sorted alphabetical by reference substance.
1
Results are based on at least one laboratory having precipitate or volatility issues with a substance. Volatility was denoted by the use ofplate sealers during testing. 2X stock dilutions are prepared for each of 8 test substance concentrations. 1X plate dilutions are the result of
diluting the 2X stock solutions with medium in the 96-well plates.

5.9

Summary
•
•
•

•

•

•

The BioReliance, ECBC, and IIVS laboratories performed the 3T3 and NHK
NRU tests in compliance with GLP guidelines.
The quality and consistency of the reference substances was maintained
during the study by the central purchase and distribution of individual lots ofreference substances to the testing laboratories.
Modifications and revisions made to the protocols during Phases I and II
contributed to the optimization of the final protocols used in Phase III of thestudy. As a general rule, the protocol changes enhanced the performance ofthe methods and allowed more tests to meet the acceptance criteria.
FAL improved the quality of its NHK data prior to Phase II testing by
modifying the methods used to propagate the cells. Positive control IC50 datain Phases II and III from FAL more closely resemble the data from the otherlaboratories.
Summary test data and IC50 results are presented in tabular and graphic
formats. Comparisons of 3T3 NRU IC50 values to NHK NRU IC50 values
show that the values for 85% of the reference substances are within one orderof magnitude of each other. Digoxin and aminopterin yielded differences of
up to five orders of magnitude when the IC50 values of the 3T3 and NHK
NRU test methods were compared.
Although each laboratory followed the same solubility protocol, theysometimes obtained different results. This may have been due to thesubjective judgment of whether or not solubility was achieved. Additionally,
the laboratories may have used solubility procedures that were beyond thelevel of detail in the solubility protocol.

5-44In Vitro Cytotoxicity Test Methods BRD Section 6

November 2006

6.0

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

6.1

Accuracy of the 3T3 and NHK NRU Test Methods for Predicting Rodent
Acute Oral Toxicity ................................................................................................. 6-5
6.1.1 Linear Regression Analyses for the Prediction of Rat Acute Oral LD50
Values from In Vitro IC50 Values .................................................................. 6-5
6.1.2 Comparison of Combined-Laboratory 3T3 and NHK Regressions
to the RC Millimole Regression .................................................................... 6-8

6.2

Analysis of Outlier Substances for the RC Millimole Regression ....................... 6-9
6.2.1 Identification of Outlier Substances............................................................. 6-10
6.2.2 Evaluation of Outlier Substances................................................................. 6-12

6.3

Improving the Prediction of In Vivo Rat Oral LD50 Values from In Vitro
IC50 Data ................................................................................................................. 6-17
6.3.1 The RC Rat-Only Millimole Regression ..................................................... 6-17
6.3.2 The RC Rat-Only Weight Regression.......................................................... 6-17

6.4

Accuracy of the 3T3 and NHK NRU Test Methods for Predicting GHS
Acute Oral Toxicity Categories ............................................................................ 6-18
6.4.1 Prediction of GHS Acute Oral Toxicity Category by the RC IC50
Values Using the RC Rat-Only Millimole Regression................................ 6-20
6.4.2 Prediction of GHS Acute Oral Toxicity Category by the 3T3 and NHK
NRU Test Methods Using the RC Rat-Only Millimole Regression............ 6-22
6.4.3 Prediction of GHS Acute Oral Toxicity Category by the 3T3 and NHK
NRU Test Methods Using the RC Rat-Only Weight Regression................ 6-24
6.4.4 Summary of the Regressions Evaluated ...................................................... 6-27

6.5

Correlation of NRU Concentration-Response Slope with Rat Lethality
Dose-Response Slope ............................................................................................. 6-28

6.6

Strengths and Limitations of the Use of In Vitro Cytotoxicity Test
Methods with the IC50–LD50 Regressions for Predictions of Rodent Acute
Oral Toxicity .......................................................................................................... 6-31
6.6.1 In Vitro Cytotoxicity Methods ..................................................................... 6-31
6.6.2 Use of Mole-Based vs. Weight-Based Regressions for the Predictionof Toxicity for Low and High Molecular Weight Substances..................... 6-31

6.7

Salient Issues of Data Interpretation.................................................................... 6-34

6.8

Comparison of NRU Test Results to Established Performance Standards...... 6-34

6.9

Summary................................................................................................................. 6-40

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In Vitro Cytotoxicity Test Methods BRD Section 6

6.0

November 2006

ACCURACY OF THE 3T3 AND NHK NRU TEST METHODSThis section discusses the accuracy of the 3T3 and NHK NRU test methods for predicting therodent acute oral toxicity (the LD50) of chemicals. Accuracy, the agreement between a test
result and an accepted reference value, is a critical component of the evaluation of thevalidation status of a method (ICCVAM 2003). Although the 3T3 and NHK NRU test
methods are not suitable as replacements for acute oral toxicity assays, the rationale for
evaluating the accuracy of LD50 predictions from the in vitro IC50 values is that the animalsavings produced by using these in vitro test methods to predict starting doses for acute oral
toxicity assays will be greatest when the starting dose is as close as possible to the “true”
LD50 value (see Section 10 for the evaluation of the potential reduction of animal use).
The ability of the 3T3 and NHK NRU test methods to correctly predict rodent acute oral
toxicity is based on the validity of the in vivo – in vitro (i.e., IC50-LD50) regression model.
The IC50-LD50 regression establishes the relationship between the in vitro IC50 values and theLD50 values that will be used to set the starting doses for the computer-simulated acute oral
toxicity assays in this study (see Section 10). The regressions generated by the threelaboratories for each NRU test method were not statistically different, and the data from the
3T3 and NHK NRU test methods were combined (using a geometric mean IC50 of the three
individual laboratory geometric mean IC50 values) into single regressions (see Section 6.1).
Only rat LD50 data were used for these regressions to reduce the variation that would be
produced by combining data from multiple species. Table 6-1 describes the datasets used forthe analyses in Sections 6.1 through 6.4.
To test the assumption in the Guidance Document that the RC millimole regression can beobtained using a basal cytotoxicity method with a single cell type and cytotoxicity endpoint(ICCVAM 2001b), the regressions for each NRU test method (3T3 and NHK) werecompared with regressions for the same substances that were calculated using the RC IC50
and LD50 values (see Section 6.1). Because the 3T3 and NHK regressions were notstatistically different from the RC regressions for the same chemicals, the RC data were used
to develop a regression to predict LD50 values from the NRU-generated IC50 values because
this regression was based on a larger number of substances than the NICEATM/ECVAM
regressions (see Section 6.3).
The RC millimole regression was used to identify outlier substances (i.e., those that did not
fit the regression within the established acceptance limits; see Section 6.2) tested in thevalidation study because:• Acceptance limits for the RC millimole regression had been established• The 3T3 and NHK NRU IC50 – rat oral LD50 regressions were notsignificantly different from the RC regressions calculated for the same
substances• Use of the RC regressions allow a comparison of the outlier substances
determined using RC data to those determined using the 3T3 and NHK data

6-3

In Vitro Cytotoxicity Test Methods BRD Section 6

Table 6-1

November 2006

Datasets Used for Accuracy Analyses1
3T3
NRU1

NHK
NRU1

Testing with NRU test methods

72

72

Comparison of laboratory IC50-LD50
regressions to one another47

51

Comparison of combined-laboratory
IC50-LD50 regressions to a regression
calculated with RC data

47

47

RC millimole regression

NANA

RC rat-only millimole regression

NANA

RC rat-only weight regression

NA

NA

Analysis of outliers for the RC
millimole regression

70

71

Substances with IC50 values from atleast one laboratory

67

68

Substances with IC50 values from atleast one laboratory and rat oral LD50
referene values

Use

Prediction of GHS accuracy using
IC50 values in RC rat-only regressions

Characteristics of Dataset
Substances tested; 58 substanceswere common to the RC
RC substances with IC50 values fromall laboratories and reference rat oralreference LD50 values
RC substances with IC50 values forboth test methods from alllaboratories and reference rat oral
LD50 values
RC IC50 (mM) and RC oral LD50
(mmol/kg) values for 347 substances(282 rat and 65 mouse LD50 values)
RC IC50 (mM) and RC oral LD50
values (mmol/kg) for 282 substanceswith rat oral LD50 data
RC IC50 (µg/mL) and RC oral LD50
values (mg/kg) for 282 substanceswith rat oral LD50 data

Abbreviations: RC=Registry of Cytotoxicity; 3T3=BALB/c 3T3 fibroblasts; NA=Not applicable; NHK=Normal
human epidermal keratinocytes; NRU=Neutral red uptake.
1
Number of substances.

To improve upon the RC millimole regression’s1 ability to accurately predict LD50 values
from IC50 values, and to also make this approach relevant to the testing of mixtures andsubstances without known molecular weights, two regressions were calculated (see Section
6.3). The first regression – the RC rat-only millimole regression – uses the 282 (of 347)
substances in the RC dataset that had reported rat LD50 values. The LD50 data for the
regression were limited to one species to decrease the variability in LD50 values that would
occur if the data from more than one species were combined. Rats were selected because they
are the preferred species for acute oral toxicity testing (EPA 2002b; OECD 2001a; OECD
2001d) (see Section 6.3.1). The RC rat-only millimole regression was transformed to one
based on weight units (mg/kg body weight for LD50 and µg/mL for IC50) in order to make theregression equation more generally applicable to the testing of mixtures and substances of
unknown molecular weights.
1

The RC millimole regression was created using rat and mouse oral LD50 values from RTECS® and IC50 values
from in vitro cytotoxicity assays using multiple cell lines and cytotoxicity endpoints for 347 substances withknown molecular weights (Halle 1998, 2003)
6-4

In Vitro Cytotoxicity Test Methods BRD Section 6

November 2006

The ability of the 3T3 and NHK NRU IC50 data to correctly predict rat acute oral LD50 values
based on using the RC rat-only millimole regression and the RC rat-only weight regression,
was evaluated by determining the extent to which the appropriate GHS acute oral toxicity
category was identified for each reference substance (see Section 6.4). The rationale for
evaluating the accuracy of LD50 predictions is that the acute oral toxicity test methods (i.e.,
UDP, FDP, and ATC) call for starting doses to be placed as close as possible and just below
the true LD50. When the starting dose is close to the true LD50 for a test substance, fewer
animals are needed. When the starting dose is below the true LD50, there is reduced pain andsuffering because doses tend to be lower, and the test bias is more conservative. Thisapproach permits an assessment of accuracy that is specific to each GHS hazard
classification category. The discordant reference substances from the predictions of GHS
category are presented in Appendix L2.
The remainder of Section 6 discusses physical, chemical, and biological, characteristics ofsubstances that may have an impact on the accuracy of the 3T3 and NHK methods.
6.1

Accuracy of the 3T3 and NHK NRU Test Methods for Predicting Rodent
Acute Oral Toxicity

The rat LD50 values provided in Section 4.2 are used as the reference values for assessing theability of the 3T3 and NHK test methods to accurately predict acute oral toxicity2. The
accuracy of the two in vitro cytotoxicity test methods is assessed in two ways: (1) by the
goodness of fit of the in vitro IC50 data to the rat LD50 data in linear regression analyses, and(2) by the concordance (i.e., extent of agreement) between the GHS acute oral toxicity
categories (UN 2005) assigned based on rat LD50 data and those predicted using in vitro IC50
values.
6.1.1

Linear Regression Analyses for the Prediction of Rat Acute Oral LD50 Values
from In Vitro IC50 Values
As described in Section 5.5.4.3, linear regressions for each laboratory and in vitro method
were calculated using log IC50 values (mM) versus the corresponding reference log LD50
values (mmol/kg) identified in Table 4-2. The reference substances used to calculate each of
the laboratory regressions met the following criteria for each test method:• The substance was included in the RC• All three laboratories reported IC50 values• There was an associated rat acute oral LD50 reference value (see Table 4-2).
There were 47 and 51 reference substances that fit these criteria for the 3T3 and NHK test
methods, respectively. The slopes for the all of the laboratory-specific regressions were
statistically significantly different from zero (p <0.0001), which indicates a significant
correlation between in vitro IC50 values and the corresponding rat acute oral LD50 values.
Comparison of the individual laboratory regressions to one another using the goodness of fit
2

Toxicity is inversely proportional to LD50. High LD50 values reflect low toxicity and low LD50 values reflect
high toxicity

6-5

In Vitro Cytotoxicity Test Methods BRD Section 6

November 2006

F-test for regression slopes and intercepts described in Section 5.5.4.3 indicated that thelaboratory-specific regressions for either NRU method were not significantly different fromone another. For the 3T3 method, p=0.605 for the slope comparisons and p=0.947 for theintercept comparisons. For the NHK method, p=0.792 for the slope comparisons and p=0.999
for the intercept comparisons.
Because the individual laboratory regressions were not significantly different, the laboratory
data were combined into a single regression for each method using the geometric mean of themean IC50 values determined by each laboratory for each substance (see the “Combinedlaboratory” regressions in Table 6-2 and Figure 6-1). The combined-laboratory 3T3
regression yielded a better fit to the reference LD50 data (R2=0.579) than the NHK regression(R2=0.463).
Table 6-2

Linear Regression Analyses of the 3T3 and NHK NRU and Rat Acute
Oral LD50 Test Results1

Laboratory

N

ECBC2
FAL2
IIVS2
Combined-laboratory3

47
47
47
47

ECBC2
FAL2
IIVS2
Combined-laboratory3

51
51
51
51

Slope
3T3 NRU
0.573
0.539
0.552
0.561
NHK NRU
0.491
0.428
0.483
0.470

Intercept

R2

0.541
0.373
0.507
0.475

0.613
0.519
0.586
0.579

0.412
0.407
0.416
0.413

0.480
0.422
0.478
0.463

Abbreviations: ECBC=Edgewood Chemical Biological Center; FAL=Fund for the Replacement of Animals in
Medical Experiments Alternatives Laboratory; IIVS=Institute for In Vitro Sciences; 3T3=BALB/c 3T3
fibroblasts; N=Number of substances used to calculate the regression; NHK=Normal human epidermal
keratinocytes; NRU=Neutral red uptake; R2=Coefficient of determination.
1
Log IC50 in mM; log LD50 in mmol/kg.
2
Regression based on a single point per substance (i.e., the geometric mean of the within laboratory replicate
IC50 values and the reference rat acute oral LD50 from Table 4-2).
3
Regression based on a single point per substance (i.e., the geometric mean of the geometric mean IC50 values
obtained for each laboratory and the reference rat acute oral LD50 from Table 4-2).

6-6

In Vitro Cytotoxicity Test Methods BRD Section 6

Figure 6-1

November 2006

Combined-Laboratory 3T3 and NHK NRU Regressions
3T3 NRU

1000

log LD50 = 0.561 log IC50 + 0.475
R2 = 0.579

100

10

1

0.1

0.01

0.001

0.00010.000001 0.00001

0.0001

0.001

0.01

0.1

1

10

100

1000

IC50 (mM)

NHK NRU
1000

log LD50 = 0.470 log IC50 + 0.413
R2 = 0.463
100

10

1

0.1

0.01

0.001

0.00010.000001 0.00001

0.0001

0.001

0.01

0.1

1

10

100

1000

IC50 (mM)

Abbreviations: 3T3=BALB/c 3T3 fibroblasts; NHK=Normal human epidermal keratinocytes; NRU=Netural red
2
uptake; R =Coefficient of determination.
Points show the geometric means of the laboratory geometric mean IC50 values and the reference rat acute oral
LD50 values (from Table 4-2) for 47 reference substances for the 3T3 and 51 reference substances for NHK
test methods. Solid lines show the combined-laboratory regressions for each method (see Table 6-2).

6-7

In Vitro Cytotoxicity Test Methods BRD Section 6

November 2006

6.1.2

Comparison of the Combined-Laboratory 3T3 and NHK Regressions to the RC
Millimole Regression
The validation study tested 58 RC substances using the 3T3 and NHK NRU test methods(see Figure 3-1). The resulting method regressions for each cell type were compared to the
RC regressions for the same substances to test the assumption in the Guidance Document that
the RC millimole regression can be obtained with a basal cytotoxicity test method using asingle cell type and endpoint (ICCVAM 2001b). The 47 substances used to calculate these
regressions met the following criteria:• The substance was included in the RC• All three laboratories reported IC50 values for both the 3T3 and NHK NRU
test methods• There was an associated rat oral reference LD50 value (see Table 4-2)
The regression calculated for the 47 substances using the RC IC50 and LD50 data is shown in
Figure 6-2. A graphic comparison of the RC regressions and the 3T3 and NHK combinedlaboratory regressions is in Figure 6-3. A statistical comparison of slope and intercept(simultaneously) using an F test showed that neither the 3T3 regression (p=0.612) nor the
NHK regression (p=0.759) was significantly different from the 47 RC substance regression.
Figure 6-2
1000

Regression for 47 RC Substances Using RC Data
log LD 50 = 0.640 log IC 50 + 0.262
R2 = 0.694

100

10

1

0.1

0.01

0.001

0.00010.000001 0.00001

0.0001

0.001

0.01

0.1

1

10

100

1000

IC50 (mM)
Abbreviations: RC=Registry of Cytotoxicity; R2=Coefficient of determination.
Points show the IC50 values and the reference rodent (rat and mouse) acute oral LD50 values from the RC for
47 reference substances. The dashed line shows the calculated regression.

6-8

In Vitro Cytotoxicity Test Methods BRD Section 6

Figure 6-3

November 2006

Regression for 47 RC Substances with the 3T3 and NHK Regressions

1000

RC Regression for 47 Substances

100

3T3 NRU Regression
NHK NRU Regression

10

1

0.1

0.01

0.001

0.00010.000001 0.00001

0.0001

0.001

0.01

0.1

1

10

100

1000

IC50 (mM)
Abbreviations: RC=Registry of Cytotoxicity; 3T3=BALB/c 3T3 fibroblasts; NHK=Normal human epidermal
keratinocytes; NRU=Neutral red uptake.
The regression for 47 RC substances using RC data is log LD50 = 0.640 log IC50 + 0.262 (R2=0.694). The
combined-laboratory 3T3 regression for the same 47 substances, is log LD50 = 0.561 log IC50 + 0.475 (R2 =0.579) (from Table 6-2). The combined-laboratory NHK regression for the same 47 substances, is log LD50 =0.471 log IC50 + 0.445 (R2 = 0.487).

6.2

Analysis of Outlier Substances for the RC Millimole Regression

The RC millimole regression and each in vitro NRU test method were used to identify
outliers among the reference substances tested in the validation study (i.e., those for which
the rodent LD50 was not accurately predicted by the in vitro IC50). The outlier substanceswere then evaluated to determine if they had common characteristics that could assist in
identifying the types of substances that are not suited for use in the 3T3 or NHK NRU test
methods for determining starting doses for acute oral toxicity assays.
The RC millimole regression was used to determine the outlier status of reference substances
because:• The RC millimole regression had associated acceptance limits (Halle 1998,
2003): a difference greater than 0.699 (or log 5) for log-observed LD50 (inmmol/kg) from the log-predicted LD50 identifies a substance as an outlier• The 3T3 and NHK IC50 – rat oral LD50 regressions were not significantly
different from the RC regressions calculated for the same substances

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In Vitro Cytotoxicity Test Methods BRD Section 6•

November 2006

Use of the RC millimole regression allows a comparison of the outlier
substances determined using RC IC50 values to those determined using the
3T3 and NHK NRU IC50 values.

6.2.1
Identification of Outlier Substances
For each in vitro NRU test method, the predicted LD50 values for the reference substanceswere determined using the geometric mean IC50 values of the three geometric mean
laboratory values in the RC millimole regression. Outliers were identified using the RC
method (Halle 1998): a difference greater than 0.699 (or log 5) for log-observed LD50 (inmmol/kg) minus the log-predicted LD50 identifies a substance as an outlier (see Appendix J1for the 3T3 NRU test method and Appendix J2 for the NHK NRU test method for thepredicted LD50 values). For the best comparison with the RC outlier results, the outlier
evaluation for the 3T3 and NHK NRU test methods used same observed LD50 values as thoseused in the RC database for the 58 reference substances that were included in the RC
database (see Table 3-2). For the non-RC substances, the observed values (in Table 3-2)
were obtained from other databases such as RTECS or Hazardous Substances Database(NLM 2002). The outlier analysis included all the reference substances that yielded IC50
values from at least one laboratory in the validation study whether the in vivo LD50 values
were from rats or mice. Thus, 70 substances were used for the 3T3 NRU outlier analysis and71 substances were used for the NHK NRU outlier analysis. Table 6-3 lists the outlier
substances for the RC millimole regression when using the RC IC50 values and the 3T3 andNHK NRU IC50 values.
Table 6-3

Outlier Substances for the RC and the 3T3 and NHK NRU Methods
When the RC Millimole Regression is Used1
Substances Included in the RC Identified as Outliers in:
RC

2

3T33

NHK4

Acetaminophen (+)
Arsenic III trioxide (–)

Arsenic III trioxide (–)
Aminopterin (–)

5-Aminosalicylic acid (+)
Busulfan (–)

5-Aminosalicylic acid (+)
Busulfan (–)

Caffeine (–)

Busulfan (–)
Caffeine (–)

Cycloheximide (–)

Cycloheximide (–)

Cycloheximide (–)

Dibutyl phthalate (+)

Dibutyl phthalate (+)

Dibutyl phthalate (+)

Diethyl phthalate (+)

Diethyl phthalate (+)

Digoxin (–)

Digoxin (–)

Disulfoton (–)

Disulfoton (–)

Disulfoton (–)

Epinephrine bitartrate (–)

Epinephrine bitartrate (–)

Epinephrine bitartrate (–)

Ethanol (+)

Ethanol (+)

Ethanol (+)

Lindane (–)

Lindane (–)

Mercury II chloride (–)

Mercury II chloride (–)

Mercury II chloride (–)
Methanol (+)

6-10

In Vitro Cytotoxicity Test Methods BRD Section 6

Table 6-3

November 2006

Outlier Substances for the RC and the 3T3 and NHK NRU Methods
When the RC Millimole Regression is Used1
Substances Included in the RC Identified as Outliers in:RC

2

Nicotine (–)

3T33
Nicotine (–)

Paraquat (–)

NHK4
Nicotine (–)
Paraquat (–)

Parathion (–)

Parathion (–)

Parathion (–)

Phenobarbital (–)

Phenobarbital (–)

Phenobarbital (–)

Phenylthiourea (–)

Phenylthiourea (–)

Phenylthiourea (–)

Potassium cyanide (–)

Potassium cyanide (–)

Potassium cyanide (–)

Propylparaben (+)

Propylparaben (+)

Propylparaben (+)
Sodium oxalate (–)

Thallium I sulfate (–)

Thallium I sulfate (–)

Triethylenemelamine (–)

Triethylenemelamine (–)

Triethylenemelamine (–)

Verapamil HCl (–)

Verapamil HCl (–)

1,1,1-Trichloroethane (+)
Verapamil HCl (–)

Xylene (+)
Outliers That Were Not Included in the RC
Dichlorvos (–)

Dichlorvos (–)

Endosulfan (–)

Endosulfan (–)

Fenpropathrin (–)

Fenpropathrin (–)

Physostigmine (–)

Physostigmine (–)

Sodium hypochlorite (+)

Sodium hypochlorite (+)

Sodium selenate (–)

Sodium selenate (–)

Strychnine (–)

Strychnine (–)

Abbreviations: RC=Registry of Cytotoxicity; 3T3=BALB/c 3T3 fibroblasts; NHK=Normal human epidermal keratinocytes;
NRU=Neutral red uptake; (–)=Toxicity was underpredicted by the IC50 and RC millimole regression (i.e., the LD50 value
predicted by the IC50 was higher than the in vivo LD50 value); (+)=Toxicity was overpredicted by the IC50 and RC millimole
regression (i.e., the LD50 value predicted by the IC50 was lower than the in vivo rodent LD50 value).
[Note: Empty cells indicate that the substance was not an outlier for that particular IC50 value.]1
Log LD50 (mmol/kg) = 0.435 log IC50 (mM) + 0.625. Log LD50 (mmol/kg) values for outlier substances were >0.699 fromthe RC millimole regression.
2
Using RC IC50 in the RC millimole regression for the 58 RC substances tested in the validation study.
3
Using the 3T3 NRU IC50 in the RC millimole regression for the 70 reference substances that yielded IC50 values from any
laboratory in the validation study.
4
Using the NHK NRU IC50 in the RC millimole regression the RC for the 71 reference substances that yielded IC50 values
from any laboratory in the validation study.
Bolded substances have active metabolites in vivo (see Table 3-7).
Substances that showed evidence of insolubility (i.e., precipitates) during testing (see Table 5-11) are identified by italics.

When the RC millimole regression and the RC method of identifying outlier substances wereused (Halle 1998, 2003), there were 28 outliers for the 3T3 NRU test method and 31 for the
NHK NRU test method. The top part of Table 6-3 shows a comparison of the 22 RC
substances that were identified by the RC as outliers (see Table 3-2) and the RC reference
substances that were identified as outliers using either the 3T3 or NHK NRU IC50 values
with the RC millimole regression. For the 58 RC substances that were tested in the validation
6-11

In Vitro Cytotoxicity Test Methods BRD Section 6

November 2006

study, 18 of the 22 RC outliers also responded as outliers in both NRU test methods, butsome of the substances were outliers only in one of the two NRU test methods. The RC
regression outliers, 5-aminosalicylic acid, caffeine, paraquat, and 1,1,1-trichloroethane were
not outliers when 3T3 data were used, and the RC outliers, digoxin, lindane, thallium sulfate,and 1,1,1-trichloroethane, were not outliers when the NHK NRU test method was used. In
contrast the 3T3 NRU test method identified three substances as outliers that were not
identified by the RC: acetaminophen, arsenic trioxide, and diethyl phthalate, and the NHK
NRU test method identified six: aminopterin, arsenic trioxide, diethyl phthalate, methanol,
sodium oxalate, and xylene. Seven additional substances, that were not included in the RC
database, were identified as outliers using the NRU IC50 values in the RC millimole
regression: dichlorvos, endosulfan, fenpropathrin, physostigmine, sodium hypochlorite,
sodium selenate, and strychnine.
6.2.2
Evaluation of Outlier Substances
A number of physico-chemical and toxicologic characteristics were evaluated for theirfrequency of occurrence among the 28 and 31 outlier substances in the 3T3 and NHK NRU
test methods, respectively, to identify attributes that may have contributed their outlier status.
This section provides a summary of these analyses based on the RC millimole regression andoutlier criteria. The frequency of outliers versus the total number of reference substances foreach physico-chemical and toxicologic category examined is shown in Appendix L1.
6.2.2.1 Physical Characteristics
A number of physical characteristics were evaluated for their frequency of occurrence in theset of outlier substances versus the complete set of reference substances. The characteristics
chosen were those that were assumed to be readily available, or relatively easy to measure,for new substances that may be tested in these NRU assays. The characteristics examined
included chemical class, molecular weight, boiling point, IC50, pH, and log Kow (i.e., log
octanol:water partition coefficient). Unfortunately, these attributes were not available for all
substances. For example, log Kow was available for 50 of the 70 (71%) substances evaluatedfor the 3T3 NRU test method and for 51 of the 71 (72%) substances evaluated for the NHK
NRU test method. Boiling point was available for only 24 of 70 (34%) substances evaluatedfor the 3T3 NRU test method and for 25 of the 71 (35%) substances evaluated for the NHK
NRU test method. For substances with log Kow >3.00, 8/13 (62%) were outliers for both the
3T3 and NHK test methods. For molecular weights >400 g/mole, 4/7 (57%) substances were
outliers using the 3T3 NRU test method and 3/7 (43%) were outliers using the NHK NRU
test method. For substances with boiling points >200oC, 9/13 (69%) were outliers using the
3T3 NRU test method and 8/13 (62%) were outliers using the NHK NRU test method.
6.2.2.2 Chemical Class
Examination of outliers by chemical class for the RC millimole regression showed that all of
the chemical classes that contained at least three reference substances also contained at least
one outlier for one test method. Two classes contained 100% outliers for both test methods:
organophosphates (3/3) and organic sulfur compounds (5/5). The remaining classes withhigher frequencies of outliers included: 2/3 (67%) amines were outliers for both test methods,
7/14 (50%) heterocylics were outliers for the 3T3 NRU and 10/14 (71%) heterocyclics were
outliers for the NHK NRU, 2/5 (40%) chlorine compounds were outliers for both test
methods, 2/6 (33%) sodium compounds were outliers for both test methods, 3/9 (33%)
alcohols were outliers for the 3T3 NRU and 4/10 (40%) alcohols were outliers for the NHK
6-12

In Vitro Cytotoxicity Test Methods BRD Section 6

November 2006

NRU, and 4/14 (29%) carboxylic acids were outliers for the 3T3 NRU and 6/14 (43%)
carboxylic acids were outliers for the NHK NRU.
6.2.2.3 Solubility
Another attribute that may cause a substance to be an outlier is the lack of solubility in thetest system. Because the SMT expected the toxicity of insoluble substances to beunderpredicted in the in vitro assays, substances that formed precipitates in the tests werenoted and compared with the outlier substances. However, insolubility was not consistently
associated with the outlier substances for which toxicity was underpredicted. For example,
eight of the 22 (36%) underpredicted substances identified by applying the 3T3 results to the
RC millimole regression exhibited signs of insolubility in at least one laboratory. NHK
results showed that seven of 23 (30%) underpredicted substances exhibited signs ofinsolubility in at least one laboratory (see Table 5-11 for substances that had precipitates in
the assays). Additionally, there was evidence of insolubility in the 3T3 and NHK NRU test
methods of dibutyl phthalate and diethyl phthalate, but toxicity was overpredicted for bothsubstances, rather than underpredicted. This overprediction may be a characteristic of thephthalates, but more substances would have to be tested before a general rule could be
adopted.
There were 25 substances that showed evidence of insolubility in the 3T3 test method in atleast one laboratory, and 11 (44%) of these were outliers. Of the 24 substances showed
evidence of insolubility in at least one NHK laboratory, 11 (46%) were outliers.
6.2.2.4 Metabolism
It was anticipated that the toxicity of substances metabolized in vivo to active compounds(see Section 3.3.4.3 and Table 3-7) would be underpredicted in vitro by 3T3 and NHK cells,
which have little or no metabolic capability (Babich 1991; INVITTOX 1991). Of the 72
reference substances, 19 (26%) are known to have active metabolites in vivo, and 10 (45%)
of these were classified as outliers for 3T3. Of these 10 substances, which accounted for 36%
of the 28 outlier substances, the toxicity of six (60%) was underpredicted, while the toxicity
of four (40%) was overpredicted. Among the 31 outliers in the NHK NRU test method, nine(29%) are metabolized to active metabolites. Nine of the 19 substances known to produce
active metabolites in vivo were discordant for the NHK NRU test method. NHK cells
underpredicted the toxicity of five (56%) of these nine substances and overpredicted theother four (44%). These nine outlier substances accounted for 29% of the 31 outliers in the
NHK NRU test method. Thus, the fact that a substance has active metabolites that are notexpected to be produced in the in vitro tests does not necessarily indicate that its toxicity will
be underpredicted by in vitro basal cytotoxicity test methods.
Similarly, Halle (1998, 2003) noted that the RC substances that required metabolic activation
to produce in vivo toxicity were not necessarily outliers with respect to their fit to the RC
millimole regression. They found that eight (50%) of the 16 substances that required
metabolic activation to product toxicity were outliers (see Table L3-3 in Appendix L3).
6.2.2.5 Mechanism of Toxicity
Substances whose mechanisms of toxicity would not be detected in the 3T3 or NHK cells
would be expected to fit the RC millimole regression poorly. In particular, toxic mechanismsthat include, for example, specific actions on the central nervous system (CNS) or the heart
6-13

In Vitro Cytotoxicity Test Methods BRD Section 6

November 2006

are not expected to be active in the 3T3 or NHK cells. Neurotoxic mechanisms would
include, for example, cholinesterase inhibition, CNS nicotinic receptor blockade or
activation, or any activity other than membrane destabilization such as that produced by asolvent, or disturbance of energy utilization such as interruption of oxidative
phosphorylation. Representative cardiotoxic mechanisms would include calcium channel
blockage and beta-adrenergic receptor activation or blockage.
The 72 reference substances used to validate the 3T3 and NHK NRU test methods included
16 (22%) that had specific CNS toxicity (see Table 6-4). Of these 16 substances, 10 (63%)
were outliers in both in vitro NRU test methods. Three of the six (50%) reference substancesthat are cardiotoxic were outliers in the 3T3 NRU test method and two (33%) were outliers in
the NHK NRU test method. When all the reference substances with mechanisms that are notexpected to be active in the 3T3 and NHK cells (i.e., in Table 6-4) are summed, 13/22 (59%)
are outliers for the 3T3 NRU and 12/22 (55%) are outliers for the NHK NRU. These
substances represented 13/28 (46%) and 12/31 (39%) of the total outlier substances for the
3T3 and NHK NRU test methods, respectively. Halle (1998, 2003) reported similar findings
for the RC database (i.e., approximately half of the substances expected to be outliers basedon their mechanisms of toxicity were outliers) (see Appendix L3).

6-14

In Vitro Cytotoxicity Test Methods BRD Section 6

Table 6-4

November 2006

Substances With Mechanisms of Toxicity Not Expected to Be Active in the 3T3 or NHK Cells in Culture

Substance

Mechanism of Toxicity1

3T3 Outlier2

NHK Outlier2

No

No

No

Yes

No

No

No

No

Yes

Yes

Yes

Yes

Endosulfan

Neurotoxic
Antimuscarinic; anticholinergic action; competitive antagonism of anticholinesterase at cardiac
and CNS receptor sites.
Inhibition of phosphodiesterase leading to AMP accumulation; translocation of intracellular
Ca++; adenosine receptor antagonism; neurotoxic.
Therapeutically decreases firing of noradrenergic neurons.
Potentiation of GABAA receptor activity; inhibition of N-methyl-D-aspartate activity;
modulation of 5-hydroxytryptamine3 receptor-mediated depolarization of the vagas nerve3.
Inhibition of acetylcholinesterase resulting in acetylcholine accumulation in CNS and effector
organs.
Inhibition of acetylcholinesterase resulting in acetylcholine accumulation in CNS and effector
organs.
Affects brain neurotransmitter levels4.

Yes

Yes

Fenpropathrin

Delays closure of sodium channel causing persistent depolarization of membrane.

Yes

Yes

Glutethimide

CNS depression; anticholinergic activity.

No

No

Haloperidol

Blocks dopamine receptors.
CNS depression through inhibition of GABA receptor linked chloride channel at the picrotoxin
binding site, leading to blockade of chloride influx into neurons.
Cholinergic block causing polarization of CNS and PNS synapses.
Inhibition of acetylcholinesterase resulting in acetylcholine accumulation in CNS and effector
organs.
CNS depression through inhibition of GABA synapses; inhibits hepatic NADH cytochromeoxidoreductase.
Inhibition of acetylcholinesterase resulting in acetylcholine accumulation in CNS and effector
organs.
Increases glutamic acid in the CNS.

No

No

Yes

No

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

No

No

Yes

No

Atropine sulfate
Caffeine
Carbamazepine
Chloral hydrate
Dichlorvos
Disulfoton

LindaneNicotine
Parathion
Phenobarbital
Physostigmine
Strychnine

Amitriptyline HCl
Digoxin

Cardiotoxic
Blocks norepinephrine, 5-hydroxytryptamine, and dopamine presynaptic uptake; prevents
reuptake of heart norepinephrine.
Impairs ion transport and increases sarcoplasmic calcium by binding to Na+/K+ ATPase,
increasing automaticity of cardiac cells.
6-15

In Vitro Cytotoxicity Test Methods BRD Section 6

Table 6-4

November 2006

Substances With Mechanisms of Toxicity Not Expected to Be Active in the 3T3 or NHK Cells in Culture
Mechanism of Toxicity1

Substance
Epinephrine bitartrate

Adrenergic receptor stimulation.

Potassium chloride

Disturbs cardiac membrane potential and electrical activity.

Procainamide HCl
Verapamil HCl

5

Slows impulse conduction in the heart .
++Inhibition of transmembrane Ca flux in excitatory tissues; alpha-adrenergic blockade.

Abbreviations: NA=Not available or information not found; CNS=Central nervous system; GABA=Gamma aminobutyricacid; PNS=Peripheral nervous system; NADH=Nicotine adenine dinucleotide (reduced).
1
From Ekwall et al. (1998) or Hazardous Substances Data Bank (NLM 2001, 2002) unless otherwise noted.
2
As shown in Table 6-3.
3
EPA (2000b).
4
ATSDR (2000a).
5
Hardman et al. (1996).

6-16

3T3 Outlier2

NHK Outlier2

Yes

Yes

No

No

No

No

Yes

Yes

In Vitro Cytotoxicity Test Methods BRD Section 6

6.3

November 2006

Improving the Prediction of In Vivo Rat Oral LD50 Values from In Vitro IC50
Data

Because the 3T3 and NHK IC50 – rat oral LD50 regressions were not significantly different
from the RC regression for the same substances, the next step was an attempt to improve the
RC millimole regression for the prediction of LD50 values from IC50 values. Because thevalidation study provided results similar to the RC, and because the RC database has morethan 3.5 times the number of substances tested in the validation study, the RC rat data (282substances) were used to determine the relationship between IC50 and LD50. The RC data
were used to develop two new regressions, the RC rat-only millimole regression and the RC
rat-only weight regression. For reference, the original RC millimole regression, log LD50
(mmol/kg) = 0.435 x log IC50 (mM) + 0.625 (Halle 1998, 2003), is shown in Table 6-5.
6.3.1
The RC Rat-Only Millimole Regression
The first regression used the RC data for the 282 substances with rat LD50 data and theoriginal units of mM for IC50 and mmol/kg for LD50 (see Table 6-5 and Figure 6-9). Only rat
data were used because:• Rats and mice are not always equally sensitive to all substances• The majority of acute oral LD50 data used in the RC millimole regression werefrom studies using rats (282 rat data points versus 65 mouse data points)(Halle 1998, 2003)• Most acute oral toxicity testing is performed with rats.
The RC rat-only millimole regression is applicable to substances of known molecular weightthat are relatively pure.
Table 6-5

Linear Regression Analyses to Improve the Prediction of Rodent Acute
Oral LD50 Values from In Vitro NRU IC50 Using the RC Database1

Data Used
347 RC substances (282 rat and 65 mouse LD50
values) – millimole units2
282 RC substances with rat LD50 data – millimoleunits2282 RC substances with rat LD50 data – weight units4

Slope

Intercept

R2

0.435

0.625

0.4523

0.439

0.621

0.452

0.372

2.024

0.325

Abbreviations: NRU=Neutral red uptake; RC=Registry of Cytotoxicity; R2=Coefficient of determination.
1
Slopes of all regressions were significantly different (p <0.05) from zero at p <0.0001.
2
IC50 in mM; LD50 in mmol/kg.
3
Calculated from RC data (i.e., not reported by Halle [1998, 2003]).
4
IC50 in µg/mL; LD50 in mg/kg.

Table 6-5 shows that the RC millimole regression using only rat acute oral LD50 data wasessentially identical to the original regression that used both rat and mouse data. The slopechanged from 0.435 to 0.439 and the intercept changed from 0.625 to 0.621; these changes
were not statistically significantly different.
6.3.2
The RC Rat-Only Weight Regression
The second regression used the same RC rat acute oral LD50 data for the 282 substances but
was calculated using weight units rather than millimolar units (see Table 6-5 and Figure 66-17

In Vitro Cytotoxicity Test Methods BRD Section 6

November 20064b). Weight units (i.e., mg/kg for the LD50 and µg/mL for the IC50) were selected for theunits of measurement because• Millimole units are not applicable to mixtures and substances with unknown
structures or molecular weights.
• They are the most practical, i.e., hazard classification in all regulatory systems
is based on LD50 values expressed in mg/kg (see Table 1-2).
The RC rat-only weight regression is applicable for use with complex mixtures, substances
whose structures or molecular weights are unknown, and substances that are relatively
impure (i.e., mixtures that are primarily composed of a named substance).
6.4

Accuracy of the 3T3 and NHK NRU Test Methods for Predicting GHS Acute
Oral Toxicity Categories

Based on the correlations/regressions obtained between the 3T3 and NHK NRU IC50 values
and the rat LD50 values, it is clear that these in vitro methods are not suitable as replacements
for rodent acute oral toxicity tests. The use of in vitro methods to reduce animal use forrodent acute oral toxicity assays (i.e., to assist in determining the starting doses for in vivo
assays) also depends upon their accuracy for the prediction of LD50 values. However, thislatter (adjunct) use does not require the same precision in LD50 prediction as complete
replacement would.
The NRU-predicted LD50 values were determined using the in vitro NRU IC50 values in the
RC rat-only regressions presented in Table 6-5. The predicted LD50 values were used toassign each substance to a predicted GHS acute oral toxicity category (UN 2005). The
accuracy of the 3T3 and NHK NRU test methods for predicting GHS acute oral toxicity
categories was determined by comparison with categorization based on rat acute oral LD50
data. The rationale for evaluating the accuracy of LD50 predictions was that the animalsavings produced by using these in vitro NRU test methods to predict starting doses forrodent acute oral toxicity assays would be greatest when the starting dose is as close as
possible to the LD50. This approach was used because regulatory authorities use rodent acuteoral toxicity test results for hazard classification and labelling of products to protect handlers
and consumers.
The in vitro NRU test methods were evaluated for their ability to predict GHS acute oral
toxicity categories using the two regressions presented in Section 6.3, the RC rat-only
millimole regression and the RC rat-only weight regression. The same reference substanceswere evaluated for each regression. Sixty-seven and 68 substances were evaluated using the
3T3 and NHK NRU test methods, respectively. Of the original 72 reference substancestested, epinephrine bitartrate, colchicine, and propylparaben were excluded because they had
no rat acute oral LD50 reference data (see Table 4-2). Carbon tetrachloride and methanol
were excluded from the 3T3 evaluations because no laboratory attained sufficient toxicity inany test for the calculation of an IC50 (see Table 5-4). Carbon tetrachloride was excluded
from the NHK evaluations because no laboratory attained sufficient toxicity in any test forthe calculation of an IC50 (see Table 5-5).

6-18

In Vitro Cytotoxicity Test Methods BRD Section 6

Figure 6-4

November 2006

RC Rat-Only Millimole Regression (a) and RC Rat-Only Weight
Regression (b)

a.
1000

log LD 50 = 0.439 log IC 50 + 0.621
R2 = 0.452

100

10

1

0.1

0.01

0.001

0.00010.000001 0.00001

0.0001

0.001

0.01

0.1

1

10

100

1000

100

1000

10000

100000IC50 (mM)b.
100000log LD 50 = 0.372 log IC 50 + 2.024
R2 = 0.325

10000

1000

100

10

1

0.1
0.001

0.01

0.1

1

10

IC50 (µg/mL)

Abbreviations: RC=Registry of Cytotoxicity; R2=Coefficient of determination.
Regressions calculated using IC50 and rat oral LD50 datapoints for 282 substances from the RC (see Table 6-5).

6-19

In Vitro Cytotoxicity Test Methods BRD Section 6

November 2006

For comparison with the NRU test method results and RC rat-only regressions, Section 6.4.1
provides the accuracy analysis for the RC database used with the RC millimole regression.
Sections 6.4.2 and 6.4.3 provide the accuracy information for the 3T3 and NHK NRU test
methods for the RC rat-only millimole regression and RC-rat only weight regression,respectively. A summary of predictivity3 is provided for each predicted toxicity category,along with the percentage of substances whose toxicity was underpredicted or overpredicted.
6.4.1

Prediction of GHS Acute Oral Toxicity Category by the RC IC50 Values Using the
RC Millimole Regression
Table 6-6 shows the concordance of the observed (i.e., in vivo) and predicted GHS acute oral
toxicity categories (UN 2005) for the 347 RC IC50 values in the RC millimole regression, log
LD50 (mmol/kg) = 0.435 x log IC50 (mM) + 0.625 (Halle 1998, 2003). Accuracy is theagreement of the in vitro category predictions with those based on the 347 rodent (282 rat
and 65 mouse) oral LD50 values used in the RC database (Halle 1998, 2003). Substances forwhich the in vitro toxicity category prediction did not match the in vivo category wereconsidered discordant for the GHS acute oral toxicity category predictions.
The overall accuracy of the RC IC50 values for correctly predicting GHS acute oral toxicity
classification category using the RC millimole regression was 40% (140/347substances)(Table 6-6). Rodent acute oral toxicity was overpredicted for 34% (118/347) andunderpredicted for 26% (89/347) of the substances. For this analysis, with respect to thepredictions of each GHS category:• None (0%) of the 12 substances with LD50 <5 mg/kg (GHS Category I) was
correctly predicted.
•
•
•

•
•

Four (15%) of 26 substances in the 5 < LD50 ≤50 mg/kg category (GHS
Category II) were correctly predicted.
Twenty (29%) of 69 substances in the 50 < LD50 ≤300 mg/kg category (GHS
Category III) were correctly predicted.
Ninety-seven (69%) of 140 substances in the 300 < LD50 ≤2000 mg/kg
category (GHS Category IV) were correctly predicted. This toxicity category
was also predicted for 106 other substances (52%; 106/203) that did not fall inthis category. Thus, the overall predictivity for this category was 48% (97/203
substances predicted for this category matched the in vivo category).
Fourteen (25%) of the 56 substances in the 2000 < LD50 ≤5000 mg/kg
category (GHS Category V) were correctly predicted.
Five (11%) of the 44 substances with LD50 >5000 mg/kg (GHS Unclassified)were correctly predicted.

.

3

Proportion of correct in vivo category matches for all substances with in vitro predictions for a particular
category. Predictivity is one of the measures of test accuracy (ICCVAM 2003).
6-20

In Vitro Cytotoxicity Test Methods BRD Section 6

Table 6-6

November 2006

Prediction of GHS Acute Oral Toxicity Category by the RC IC50 Values and the RC
Millimole Regression1
Total

Accuracy

Toxicity
Overpredicted

Toxicity
Underpredicted

0

12

0%

0%

100%

IC50-Predicted GHS Category (mg/kg)3

In Vivo Rodent Oral
LD502 (mg/kg)

LD50 <5

5 < LD50 ≤50

50 < LD50 ≤300

300 < LD50 ≤2000

2000 < LD50 ≤5000

LD50 >5000

LD50 < 5

0

5

3

4

0

5 < LD50 ≤50

0

4

13

9

0

0

26

15%

0%

85%

50 < LD50 ≤300

0

9

20

38

2

0

69

29%

13%

58%

300 < LD50 ≤2000

0

4

24

97

14

1

140

69%

20%

11%

2000 < LD50 ≤5000

0

1

5

36

14

0

56

25%

75%

0%

LD50 >5000

0

0

1

19

19

5

44

11%

89%

0%

347

40%

34%

26%

Total

0

23

66

203

49

6

Predictivity

0%

17%

30%

48%

29%

83%

Category Overpredicted

0%

61%

45%

27%

39%

0%

Category Underpredicted

0%

22%

24%

25%

33%

17%

Abbreviations: GHS=Globally Harmonized System of Classification and Labelling of Chemicals (UN 2005); RC=Registry of Cytotoxicity. Shaded cells are those containing the correct
predictions; RTECS®=Registry of Toxic Effects for Chemical Substances®.
1
The RC millimole regression is log LD50 (mmol/kg) = log IC50 (mM) x 0.435 + 0.625. Numbers in table represent numbers of substances.
2
Rat (282 values) and mouse (65 values) oral LD50 values, mostly from the 1983/84 RTECS® that were converted to mmol/kg for used in the RC (Halle 1998, 2003).
3
IC50 values from the RC are geometric mean IC50 values from in vitro cytotoxicity assays using multiple cell lines and cytotoxicity endpoints (Halle 1998,
2003). GHS categories were predicted by using the IC50 values to calculate predicted LD50 values with the RC millimole regression equation. Predicted LD50
values in mmol/kg for each substance were converted to mg/kg and used to classify the substance in the appropriate predicted GHS acute oral toxicity category.

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In Vitro Cytotoxicity Test Methods BRD Section 6

November 2006

The highest accuracy, 69%, for the RC IC50 values in the RC millimole regression wereobtained for substances in the 300 < LD50 ≤2000 mg/kg category (GHS Category IV). The
lowest accuracy, 0%, was obtained for substances with LD50 <5 mg/kg (GHS Category I).
Although the 11% accuracy was low for substances with LD50 >5000 mg/kg (GHS
Unclassified), the highest predictivity, 83%, was obtained for substances in this group. The
RC millimole regression generally underpredicted toxicity for substances in the highest
toxicity (i.e., lowest LD50) categories and overpredicted for substances in the lowest toxicity(i.e., highest LD50) categories (see Table 6-6).
Rodent acute oral toxicity was overpredicted for 34% (118) and underpredicted for 26% (89)of the 347 RC substances. Thus, there was a total of were 207 discordant substances. GHS
category was overpredicted for 57% (118/207) of the discordant substances andunderpredicted for 43% (89/207) of the discordant substances.
6.4.2

Prediction of GHS Acute Oral Toxicity Category by the 3T3 and NHK NRU Test
Methods Using the RC Rat-Only Millimole Regression
Table 6-7 shows the concordance of the observed (i.e., in vivo) and predicted GHS acute oral
toxicity categories (UN 2005) for each in vitro test method using the geometric mean IC50
values (of the three laboratories) in the RC rat-only millimole regression, log LD50
(mmol/kg) = 0.439 x log IC50 (mM) + 0.621. Accuracy is the agreement of the in vitro
category predictions with those based on the rat acute oral LD50 reference values in Table 42. Substances for which the in vitro toxicity category prediction did not match the in vivo
category were considered discordant for the GHS acute oral toxicity category predictions.
6.4.2.1 In Vitro – In Vivo Concordance Using the RC Rat-Only Millimole Regression
The overall accuracy of the 3T3 NRU test method for correctly predicting GHS acute oral
toxicity classification category using the RC rat-only millimole regression was 31% (21/67
substances) (Table 6-7). Rat acute oral toxicity was overpredicted for 34% (23) andunderpredicted for 34% (23) of the substances. For this analysis, with respect to thepredictions of each GHS category:• None (0%) of the six substances with LD50 <5 mg/kg (GHS Category I) was
correctly predicted.
• One (9%) of 11 substances in the 5 < LD50 ≤50 mg/kg category (GHS
Category II) was correctly predicted.
• Five (42%) of 12 substances in the 50 < LD50 ≤300 mg/kg category (GHS
Category III) were correctly predicted.
• Thirteen (81%) of 16 substances in the 300 < LD50 ≤2000 mg/kg category(GHS Category IV) were correctly predicted. This toxicity category was also
predicted for 32 other substances (71%; 32/45) that did not fall in thiscategory. Thus, the overall predictivity for this category was 29% (13/45
substances predicted for this category matched the in vivo category).
• None (0%) of the 10 substances in the 2000 < LD50 ≤5000 mg/kg category(GHS Category V) were correctly predicted.
• Two (17%) of the 12 substances with LD50 >5000 mg/kg (GHS Unclassified)were correctly predicted.

6-22

In Vitro Cytotoxicity Test Methods BRD Section 6

Table 6-7

November 2006

Prediction of GHS Acute Oral Toxicity Category by the 3T3 and NHK NRU Test
Methods and the RC Rat-Only Millimole Regression1
Total

Accuracy

Toxicity
Overpredicted

Toxicity
Underpredicted

0

63

0%

0%

100%

4

3T3 -Predicted GHS Category (mg/kg)

Reference Rat Oral
LD502 (mg/kg)

LD50 <5

5 < LD50 ≤50

50 < LD50 ≤300

300 < LD50 ≤2000

2000 < LD50 ≤5000

LD50 >5000

LD50 < 5

0

2

0

4

0

5 < LD50 ≤50

0

1

6

3

1

0

11

9%

0%

91%

50 < LD50 ≤300

0

0

5

7

0

0

12

42%

0%

58%

300 < LD50 ≤2000

0

1

2

13

0

0

16

81%

19%

0%

2000 < LD50 ≤5000

0

0

0

10

0

0

105

0%

100%

0%

LD50 >5000

0

0

0

8

2

2

126,7

17%

83%

0%

67

31%

34%

34%

Total

Accuracy

Toxicity
Overpredicted

Toxicity
Underpredicted

Total

0

4

13

45

3

2

Predictivity

0%

25%

38%

29%

0%

100%

Category Overpredicted

0%

25%

15%

40%

67%

0%

Category Underpredicted

0%

50%

46%

31%

33%

0%

NHK -Predicted Toxicity Category (mg/kg)

Reference Rat Oral
LD502

LD50 <5

5 < LD50 ≤50

50 < LD50 ≤300

300 < LD50 ≤2000

2000 < LD50 ≤5000

LD50 >5000

LD50 <5

0

1

2

3

0

0

63

0%

0%

100%

5 < LD50 ≤50

0

2

5

3

1

0

114

18%

0%

82%

50 < LD50 ≤300

0

1

6

5

0

0

12

50%

8%

42%

300 < LD50 ≤2000

0

1

2

12

1

0

16

75%

19%

6%

2000 < LD50 ≤5000

0

0

0

10

0

0

105

0%

100%

0%

LD50 >5000

0

0

0

7

6

0

137

0%

100%

0%

Total

0

5

15

40

8

0

68

29%

40%

31%

Predictivity

0%

40%

40%

30%

0%

0%

Category Overpredicted

0%

40%

13%

43%

75%

0%

Category Underpredicted

0%

20%

47%

28%

25%

0%

Abbreviations: GHS=Globally Harmonized System of Classification and Labelling of Chemicals (UN 2005); 3T3=BALB/c 3T3 fibroblasts; NHK=Normal human epidermal
keratinocytes; NRU=Neutral red uptake; RC=Registry of Cytotoxicity. Shaded cells are those containing the correct predictions.
1
The RC rat-only millimole regression is log LD50 (mmol/kg) = log IC50 (mM) x 0.439 + 0.621. Numbers in table represent numbers of substances.
2
Reference rat oral LD50 values in mg/kg from Table 4-2.
3
Epinephrine bitartrate excluded because no rat reference acute oral LD50 was identified (see Table 4-2).
4Colchine excluded because no rat acute oral LD50 was identified (see Table 4-2).
5
Carbon tetrachloride excluded because no laboratory attained sufficient toxicity for the calculation of an IC50.
6Methanol excluded because no laboratory attained sufficient toxicity for the calculation of an IC50.
7Propylparaben excluded because no rat acute oral LD50 was identified (see Table 4-2).

6-23

In Vitro Cytotoxicity Test Methods BRD Section 6

November 2006

The overall accuracy of the NHK NRU test method for correctly predicting the GHS acuteoral toxicity classification, when the prediction was based on the RC rat-only millimole
regression, was 29% (20/68 substances) (see Table 6-7). Toxicity was overpredicted for 40%(27) and underpredicted for 31% (21) of the 68 substances. The pattern of concordance
between in vitro and in vivo results for the NHK NRU test method with the RC rat-only
millimole regression was similar to that for the 3T3 NRU test method with the exception thatnone of the substances with a toxicity of LD50 >5000 mg/kg were correctly predicted. For
this analysis, with respect to the predictions of each GHS category:• None (0%) of the six substances with LD50 <5 mg/kg (GHS Category I) werecorrectly predicted.
• Two (18%) of 11 substances in the 5< LD50 ≤50 mg/kg category (GHS
Category II) were correctly predicted.
• Six (50%) of 12 substances in the 50< LD50 ≤300 mg/kg category (GHS
Category III) were correctly predicted.
• 12 (75%) of 16 substances in the 300< LD50 ≤2000 mg/kg category (GHS
Category IV) were correctly predicted; however, this category was also
predicted for 28 (70%; 28/40) substances that did not match the category.
Thus, the overall predictivity for this category was 30% (12/40).
• None (0%) of the 10 substances in the 2000< LD50 ≤5000 mg/kg category(GHS Category V) were correctly predicted.
• None (0%) of the 13 substances with LD50 >5000 mg/kg (GHS Unclassified)were correctly predicted.
The RC rat-only millimole regression generally underpredicted toxicity for substances in thehighest toxicity (i.e., lowest LD50) categories and overpredicted toxicity for substances in thelowest toxicity (i.e., highest LD50) categories (see Table 6-7). Although substances at thevery low and high ends of the toxicity range were poorly predicted, those in the middle range(i.e., 300 < LD50 ≤2000 mg/kg) were predicted much better, with 75 to 81% accuracy. The
pattern of accuracy for the GHS categories was similar to the pattern seen with the RC IC50
and LD50 values and the RC millimole regression (see Table 6-6) (i.e., lowest accuracy forvery toxic and very nontoxic substances and highest accuracy for substances with 300 < LD50
≤2000 mg/kg).
6.4.2.2

Discordant Substances in the Prediction of GHS Acute Oral Toxicity Category by
the 3T3 and NHK NRU Test Methods and the RC Rat-Only Millimole Regression
Appendix L2 identifies the discordant substances, that is, those for which the in vitro
predicted GHS acute oral toxicity category did not match the GHS acute oral toxicity
category assigned based on the reference rat acute oral LD50 data in Table 4-2. Of the total
number of substances used for this evaluation (67 for 3T3, 68 for NHK), the 3T3 test method
underpredicted the GHS category for 23 (50%) and overpredicted for 23 (50%) of the 46
discordant substances. The NHK test method underpredicted toxicity for 21 (44%) andoverpredicted for 27 (56%) of the 48 discordant substances.
6.4.3

Prediction of GHS Acute Oral Toxicity Category by the 3T3 and NHK NRU Test
Methods Using the RC Rat-Only Weight Regression
Table 6-8 shows the concordances of the observed and predicted GHS acute oral toxicity
categories for each in vitro NRU method using the geometric mean IC50 values from the

6-24

In Vitro Cytotoxicity Test Methods BRD Section 6

November 2006

three laboratories and the RC rat-only weight regression (Table 6-5). The regression formulafor the RC rat-only weight regression was log LD50 (mg/kg) = log IC50 (µg/mL) x 0.372 +2.024. Accuracy is the agreement of the GHS acute oral toxicity category predictions madeusing the in vitro NRU data with those based on the reference rat acute oral LD50 values(Table 4-2).
6.4.3.1 In Vitro – In Vivo Concordance Using the RC Rat-Only Weight Regression
The overall accuracy of the 3T3 NRU test method with the RC rat-only weight regression
was 31% (21/67) (Table 6-8). The toxicity was overpredicted for 33% (24) andunderpredicted for 36% (22) of the substances. For this analysis, with respect to thepredictions of the GHS category:• None (0%) of the six substances with LD50 <5 mg/kg (GHS Category I) werecorrectly predicted.
• One (9%) of 11 substances in the 5< LD50 ≤50 mg/kg category (GHS
Category II) was correctly predicted.
• Four (33%) of 12 substances in the 50< LD50 ≤300 mg/kg category (GHS
Category II) were correctly predicted; however, because 10 other substanceswere also predicted to be in this category, the overall predictivity was 29%(4/14).
• Twelve (75%) of 16 substances in the 300< LD50 ≤2000 mg/kg category(GHS Category IV) were predicted correctly. Because a total of 40 substanceswere predicted to be in this category, the overall predictivity was 30% (12/40).
• Four (40%) of 10 substances in the 2000< LD50 ≤5000 mg/kg category (GHS
Category V) were correctly predicted; however, because a total of 11
substances were predicted to be in this category, the overall predictivity was
36% (4/11).
• None (0%) of the 12 substances with LD50 >5000 mg/kg (GHS Unclassified)were correctly predicted.
The overall accuracy of the NHK predictions using the RC rat-only weight regression was
31% (21/68) (see Table 6-8). The in vivo GHS toxicity categories were overpredicted for
37% (22) and underpredicted for 32% (25) of the substances. For this analysis, with respect
to the predictions of the GHS category:• None (0%) of the six substances with LD50 <5 mg/kg (GHS Category I) werecorrectly predicted.
• One (9%) of 11 substances in the 5 < LD50 ≤ 50 mg/kg category (GHS
Category II) was correctly predicted.
• Five (42%) of 12 substances in the 50 < LD50 ≤300 mg/kg category (GHS
Category III) were correctly predicted; however, because six other substanceswere also predicted to be in this category, the overall predictivity was 33%(3/9).
• Thirteen (81%) of 16 substances in the 300 < LD50 ≤2000 mg/kg category(GHS Category IV) were predicted correctly; however, because 29 othersubstances were also predicted to be in this category, the overall predictivity
was 31% (13/42).

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In Vitro Cytotoxicity Test Methods BRD Section 6

Table 6-8

November 2006

Prediction of GHS Acute Oral Toxicity Category by the 3T3 and NHK NRU
Test Methods and the RC Rat-Only Weight Regression1
3T3 -Predicted Toxicity Category (mg/kg)
Total

Accuracy

Toxicity
Overpredicted

Toxicity
Underpredicted

0

63

0%

0%

100%

0

0

114

9%

0%

91%

8

0

0

12

33%

0%

67%

3

12

0

0

16

75%

25%

0%

0

0

6

4

0

105

40%

60%

0%

0

0

0

5

7

0

126,7

0%

100%

0%

0

2

14

40

11

0

67

31%

33%

36%

Toxicity
Underpredicted
100%

Reference Rat Oral
LD502 (mg/kg)

LD50 <5

5 < LD50 ≤50

50 < LD50 ≤300

300 < LD50 ≤2000

2000 < LD50 ≤5000

LD50 >5000

LD50 <5

0

0

2

4

0

5 < LD50 ≤50

0

1

5

5

50 < LD50 ≤300

0

0

4

300 < LD50 ≤2000

0

1

2000 < LD50 ≤5000

0LD50 >5000
Total
Predictivity

0%

50%

29%

30%

36%

0%

Category Overpredicted

0%

50%

21%

28%

64%

0%

Category Underpredicted

0%

0%

50%

43%

0%

0%

NHK -Predicted Toxicity Category (mg/kg)
Reference Rat Oral
LD502 (mg/kg)

LD50 <5

5 < LD50 ≤50

50 < LD50 ≤300

300 < LD50 ≤2000

2000 < LD50 ≤5000

LD50 >5000

Total

Accuracy

LD50 <5

0

1

2

3

0

0

63

0%

Toxicity
Overpredicted
0%

5 < LD50 ≤50

0

1

5

5

0

0

114

9%

0%

91%

50 < LD50 ≤300

0

1

5

6

0

0

12

42%

8%

50%

300 < LD50 ≤2000

0

1

2

13

0

0

16

81%

19%

0%

2000 < LD50 ≤5000

0

0

0

9

1

0

105

10%

90%

0%

LD50 >5000

0

0

0

6

6

1

137

8%

92%

0%

68

31%

37%

32%

Total

0

4

14

42

7

1

Predictivity

0%

25%

36%

31%

14%

100%

Category Overpredicted

0%

50%

14%

36%

86%

0%

Category Underpredicted

0%

25%

50%

33%

0%

0%

Abbreviations: GHS=Globally Harmonized System of Classification and Labelling of Chemicals (UN 2005); 3T3=BALB/c 3T3 fibroblasts; NHK=Normal human epidermal
keratinocytes; NRU=Neutral red uptake; RC=Registry of Cytotoxicity.
1
The RC rat-only weight regression is log LD50 (mg/kg) = log IC50 (µg/mL) x 0.372 + 2.024.
2
Reference rat oral LD50 values in mg/kg from Table 4-2.
3
Epinephrine bitartrate excluded because no rat acute oral LD50 was identified (see Table 4-2).
4Colchine excluded because no rat acute oral LD50 was identified (see Table 4-2).
5
Carbon tetrachloride excluded because no laboratory attained sufficient toxicity for the calculation of an IC50.
6Methanol excluded because no laboratory attained sufficient toxicity for the calculation of an IC50.
7Propylparaben excluded because no rat acute oral LD50 was identified (see Table 4-2).

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In Vitro Cytotoxicity Test Methods BRD Section 6••

November 2006

One (10%) of 10 substances in the 2000 < LD50 ≤5000 mg/kg category (GHS
Category V) was correctly predicted.
One (8%) of 13 substances with LD50 >5000 mg/kg (GHS Unclassified) was
correctly predicted.

The RC rat-only weight regression generally underpredicted toxicity for substances in thehighest toxicity (i.e., lowest LD50) categories and overpredicted toxicity for substances in thelowest toxicity (i.e., highest LD50) categories (see Table 6-8). Although substances at thevery low and high ends of the toxicity range were poorly predicted, those in the middle range(i.e., 300 < LD50 ≤2000 mg/kg) were predicted much better, with 75 to 81% accuracy. The
pattern of accuracy for the GHS categories was similar to the pattern seen with the RC IC50
and LD50 values and the RC millimole regression (see Table 6-6) and with the NRU IC50 and
rat oral LD50 values and the RC rat-only millimole regression (see Table 6-7) (i.e., lowest
accuracy for very toxic and very nontoxic substances and highest accuracy for substanceswith 300 < LD50 ≤2000 mg/kg).
6.4.3.2

Discordant Substances in the Prediction of GHS Acute Oral Toxicity Category by
the 3T3 and NHK NRU Test Methods and the RC Rat-Only Weight Regression
Appendix L2 shows the substances for which the in vitro predicted GHS acute oral toxicity
category using the RC rat-only weight regression did not match those that were based on the
rat acute oral LD50 reference data. The two in vitro NRU test methods over- and underpredicted the GHS acute oral toxicity category for similar numbers of substances, comparedwith the GHS acute oral toxicity categories for the rat acute oral LD50 reference values inTable 4-2. The 3T3 NRU test method overpredicted the GHS acute oral toxicity category for
22 (48%) of 46 discordant substances, and underpredicted of 24 (52%) substances. The NHK
NRU test method overpredicted the GHS acute oral toxicity category for 25 (53%) of 47
discordant substances, and underpredicted 22 (47%) substances.
6.4.4
Summary of the Regressions Evaluated
Table 6-9 summarizes the regressions evaluated in Section 6.4 for accuracy in predicting the
GHS acute oral toxicity categories (UN 2005), and the proportion of over- or underpredictions. Prediction accuracy using the RC IC50 and LD50 values and the RC millimole
regression was higher that that for the NRU test methods with the RC rat-only regressions(i.e., 40% for the RC vs. 29% to 31% for the NRU test methods). Prediction accuracy wasslightly higher for the 3T3 NRU test method compared with the NHK NRU (i.e., 31% for
3T3 vs. 29% for NHK) using the RC rat-only millimole regression, and the same as the NHK
NRU test method (i.e., 31%) using the RC rat-only weight regression. The proportion of
discordant substances using the RC IC50 values and the RC millimole regression (60%) was
lower than that using the in vitro NRU test methods and the RC rat-only regressions (69% to71%). The proportion of discordant substances from the 3T3 test method, 69%, was the same
whether it was determined with the RC rat-only millimole regression or the RC rat-only
weight regression. The proportion of discordant substances for the NHK test method wasslightly lower with RC rat-only weight regression than with the RC rat-only millimole
regression (69% vs. 71%). The RC IC50 values and the RC millmole regression were
expected to perform better than the in vitro NRU methods and the RC rat-only regressions
since the IC50 and LD50 values used to evaluate the performance of the RC millimole
regression were exactly the same as those used to calculate the linear regression formula. The

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In Vitro Cytotoxicity Test Methods BRD Section 6

November 2006

NRU IC50 values and the reference oral LD50 values used to evaluate the RC rat-onlyregressions were different from those used to calculate the RC rat-only regressions.
Table 6-9

Comparison of Regressions and In Vitro NRU Test Methods for Their
Performance in Predicting GHS Acute Oral Toxicity Categories
N1

R2 Statistic

Accuracy

Discordant Substances2

RC millimole3

347

0.452

RC IC50 – 40%

RC IC50 – 207/347 (60%)

RC rat-only millimole3

282

0.452

RC rat-only weight3

282

0.325

3T3– 31%
NHK– 29%
3T3– 31%
NHK– 31%

3T3– 46/67(69%)
NHK– 48/68 (71%)
3T3– 46/67 (69%)
NHK– 47/68 (69%)

Regression

Abbreviations: GHS=Globally Harmonized System of Classification and Labelling of Chemicals (UN 2005);
3T3=BALB/c 3T3 fibroblasts; NHK=Normal human epidermal keratinocytes; NRU=Neutral red uptake;
RC=Registry of Cytotoxicity; R2=Coefficient of determination.
1
Number of substances used in regression.
2
Proportion of discordant substances.
3
From Table 6-5.

The accuracy of the GHS category predictions using the in vitro NRU test methods with the
RC rat-only regressions obtained for the reference substances from this validations study may
or may not be applicable to other substances. A number of reasons may explain the low
accuracy for the reference substances. One is the skewness of the substances selected fortesting with respect to fit to the RC millimole regression (see Figure 3-1). Table 3-4 showsthat 22 (38%) of the 58 RC substances selected for testing were known to poorly fit the RC
millimole regression (i.e., the predicted LD50 was outside the RC acceptance interval).
Toxicity was underpredicted for 17 (77%) of these outlier substances and overpredicted (i.e.,
predicted LD50 was lower than measured in vivo LD50) for the remaining five (23%). Table
6-3 shows that 40% (28/70 for 3T3) and 44% (31/71 for NHK) of the reference substancesthat yielded IC50 values were outliers. Other reasons for the low accuracy for GHS acute oral
toxicity prediction, such as those discussed in Section 1.2.3, include the major differences
between cell cultures and whole animals regarding the absorption, distribution (including
binding to serum proteins), availability, metabolism, and excretion of reference substances.
6.5

Correlation of NRU Concentration-Response Slope with Rat Lethality DoseResponse Slope

Because the slope calculations available for the NRU concentration-response curve analyses
were based on the Hill function, the SMT determined whether the Hill Slope correlated withthe rodent dose-mortality slope. If the two were correlated, the Hill Slope from the NRU test
methods could be used to estimate the dose-mortality slope, which could, in turn, be used toestimate the most appropriate dose progression for UDP testing in rodents. A more
immediate use for the validation study results, however, would be for the computer
simulation modeling of animal testing for the UDP and ATC acute oral toxicity methods(described in Sections 10.2 and 10.3).
Dose-mortality slope information was available for 22 of the 72 reference substances, as
shown in Table 6-10. Hill function slopes were available for 20 and 21 of the 22 substances
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In Vitro Cytotoxicity Test Methods BRD Section 6

November 2006

for the 3T3 and the NHK NRU test methods, respectively. The Hill function slopes weretransformed to absolute values because geometric means cannot be calculated for negative
numbers, and geometric mean Hill function slopes were calculated for the acceptable NRU
tests for each reference substance. When there was more than one dose-mortality slope
available for a substance, a geometric mean was calculated from the available values. The
absolute values of the geometric mean Hill function slopes are plotted against the geometricmean dose-mortality slopes in Figure 6-5. To determine whether there was a relationshipbetween the absolute value of the Hill Slope and the dose-mortality slope, Spearman
correlation analyses and least squares linear regression analyses were performed for eachmethod. Both analyses showed that the absolute value of the in vitro Hill function slope was
not related to the dose-mortality slope. The Spearman correlation analysis yielded
nonsignificant correlations for both in vitro NRU test methods (3T3 rs=-0.051 with p=0.831,
and NHK rs=-0.142 with p=0.541). Linear regression analyses for the prediction of dosemortality slope by the absolute value of the Hill function slope also showed that the slopes of
the regressions were not significantly different from zero (3T3 p=0.774, and NHK p=0.994).
Because there was no relationship between Hill function slope and dose-mortality slope, theHill function slope was not used to predict the dose-mortality slope for the simulation
modeling of animal testing for the UDP and ATC acute oral toxicity methods in Sections
10.2 and 10.3.
Table 6-10

Reference Substances with Dose-Mortality and NRU Hill Slopes

Reference Substance
Acetylsalicylic acid
Boric acid
Caffeine
Carbon tetrachloride
Dichlorvos
Dimethylformamide
Diquat dibromide
Ethanol
Ethylene glycol
GlycerolHexachorophene
Lactic acid
MethanolNicotine
Parathion
Potassium cyanide
Sodium arsenite
Sodium I fluoride
Trichloroacetic acid
Triethylene melamine
Valproic acid
XyleneCarbon tetrachloride

Dose-Mortality Slope1
1.45
7.70
6.27
2.06
1.24
1.11
16.57
4.57
38.38
8.90
12.84
4.04
8.53
3.00
1.31
14.50
7.60
1.26
20.97
2.10
1.20
9.60
2.06

3T3 Hill Slope2
1.658
1.511
1.069
NA
2.240
1.875
4.273
1.725
2.016
1.941
1.466
4.541
NA
11.019
1.551
1.931
2.317
3.952
1.883
0.963
2.467
1.871
NA

NHK Hill Slope2
1.906
1.083
1.215
NA
1.383
3.157
1.289
2.049
2.904
2.398
2.470
2.934
1.173
0.682
1.467
1.207
1.717
2.569
1.369
1.355
1.440
2.452
NA

Abbreviations: 3T3=BALB/c 3T3 fibroblasts; NHK=Normal human epidermal keratinocytes; NRU=Neutral red uptake;
NA=Not available.
1
Geometric mean if there was more than one value for each substance (from Appendix H2).
2
Geometric mean of absolute values from acceptable in vitro NRU tests.

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In Vitro Cytotoxicity Test Methods BRD Section 6

Figure 6-5

November 2006

Correlation of Dose-Mortality Slope to Hill Function Slope

a.
45
40
35
30
25
20
15
10
5
0

0

1

2

3

4

5

6

7

8

9

10

11

12

3T3 NRU Hill Slope

b.
45
40
35
30
25
20
15
10
5
0
0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

NHK NRU Hill Slope
Abbreviations: 3T3=BALB/c 3T3 fibroblasts; NHK=Normal human epidermal keratinocytes; NRU=Neutral red uptake.
Hill function slopes and dose-mortality slopes for the reference substances shown in Table 6-10 for (a) the 3T3 data and (b)the NHK data. The solid line indicates the theoretical, one-to-one correspondence of Hill function slope with dose-mortality
slope. Spearman’s correlation coefficients were rs=-0.051 (p=0.831) for the 3T3 and rs=-0.142 (p=0.541) for the NHK data.

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In Vitro Cytotoxicity Test Methods BRD Section 6

6.6

November 2006

Strengths and Limitations of the Use of In Vitro Cytotoxicity Test Methods
with the IC50-LD50 Regressions for Prediction of Rodent Acute Oral Toxicity

6.6.1
In Vitro Cytotoxicity Methods
The NRU basal cytotoxicity methods tended to underpredict the toxicity of the most toxic
substances and to overpredict the toxicity of the least toxic substances for each regression
evaluated. The 3T3 and NHK NRU test methods were best at predicting the toxicity of
substances with 300 < LD50 ≤2000 mg/kg. The accuracy of the in vitro prediction of thisGHS category using the RC rat-only millimole regression and the RC rat-only weight
regression was 75-81%. GHS toxicity categories of substances with higher or lower LD50
values were correctly predicted with less than 50% accuracy. The worst accuracy, 0%, wasobserved for:• Substances with LD50 ≤5 mg/kg in both in vitro test methods and regressions• Substances with 2000< LD50 ≤5000 mg/kg using 3T3 with the RC rat-only
millimole regression• Substances with 2000< LD50 ≤5000 mg/kg or LD50 >5000 mg/kg using NHKwith RC rat-only millimole regression• Substances with LD50 >5000 mg/kg using 3T3 with RC rat-only weight
regression
Some substances with low toxicity and low solubility could not be tested in the in vitro NRU
test methods because the concentration of dissolved substance was inadequate to obtain an
IC50 value. None of the laboratories obtained adequate toxicity in any of the 3T3 tests ofcarbon tetrachloride or methanol, and at least one laboratory failed to achieve adequate
toxicity with gibberellic acid or xylene. No laboratory achieved adequate toxicity in any of
the NHK experiments with carbon tetrachloride, and at least one laboratory could not achieveadequate toxicity with methanol, 1,1,1-trichloroethane, or xylene. Another limitation of useof the in vitro test methods is in the testing of substances that come out of solution byforming a film on the medium surface or plastic well wall (i.e., “film out”), and for
substances that etch the laboratory ware plastics (ICCVAM 2006). Substances that etch
plastics can be detected by looking for the presence of etched rings in the 96-well plates after
exposure. Some substances that produce films in medium also etch plastic.
The prediction of rodent acute oral toxicity (and the starting doses for acute oral toxicity
tests) by the in vitro NRU methods is expected to be poor for substances with mechanisms of
toxicity that are not effective in the 3T3 and NHK cells. Such toxic mechanisms include
specific, receptor-mediated actions on the CNS or the heart.
The evaluation of the 3T3 and NHK NRU test methods for predicting starting doses forrodent acute oral toxicity testing with its potential to reduce and refine animal use is provided
in Section 10.
6.6.2

Use of Mole-Based vs. Weight-Based Regressions for the Prediction of Toxicity
for Low and High Molecular Weight Substances
The ICCVAM ATWG expressed concern that the RC rat-only weight regression may lessaccurately predict the toxicity of low and high molecular weight substances than the RC ratonly millimole regression. Using the RC IC50 and LD50 values for the 282 RC substanceswith rat oral LD50 data, analyses were performed to:
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In Vitro Cytotoxicity Test Methods BRD Section 6•••

November 2006

Determine the difference in the over and under-prediction of rodent acute oral
toxicity (i.e., LD50) from IC50 values between low molecular weightsubstances (i.e., ≤100 g/mole) and substances with molecular weights >100g/moleDetermine the difference in the over and under-prediction of rodent acute oral
toxicity from IC50 values between high molecular weight substances (i.e.,≥400 g/mole) vs. substances with molecular weights <400 g/mole.
Compare the RC rat-only millimole regression with the RC rat-only weight
regression with respect to the over- and under-prediction of the toxicity of low
and high molecular weight substancesThis analysis used the RC data rather than the validation studies data because the RC
contains data for many more substances. The analysis assumes that the regressions either
underpredicted or overpredicted the toxicity of all of the substances evaluated. In otherwords, there was a difference between the LD50 predicted by the regression and the in vivo
LD50 used to calculate the regression even if it was a tiny fraction (i.e., no substances fit the
regression exactly). The complete analysis and discussion are presented in Appendix J7. Of
the 282 RC substances with rat acute oral LD50 values, there were 51 with molecular weights≤100 g/mole and 231 with molecular weights >100 g/mole. For the 51 substances withmolecular weight ≤100 g/mole, the RC rat-only millimole regression underestimated the
toxicity of 20/51 (39%) substances and overestimated the toxicity of 31/51 (61%) substances.
The RC rat-only weight regression underestimated the toxicity of 24/51 (47%) substancesand overestimated the toxicity of 27/51 (53%) substances. Fisher’s exact test indicated that
there was no difference between the millimole and weight regressions with respect to theunder or over-prediction of toxicity for the low molecular weight substances (two-tailed
p=0.549) (see Table 6-11).
For the 231 substances with molecular weights >100 g/mole, the RC rat-only millimole
regression underestimated the toxicity of 108/231 (47%) substances and overestimated the
toxicity of 123/231 (53%). The RC rat-only weight regression underestimated the toxicity of
101/231 (44%) substances and overestimated the toxicity of 130/231 (57%). Fisher’s exact
test indicated that there were no significant differences between the millimole and weight
regressions for the under- and over-prediction of toxicity for the 231 substances withmolecular weight >100 g/mole (two-tailed p=0.575). Fisher’s exact test also showed that
there were no significant differences in the under- and over-prediction of the toxicity of the
51 substances with molecular weight ≤100 g/mole compared to the under- and overprediction of the toxicity of the 231 with molecular weight >100 g/mole (two-tailed p=0.756
for the RC rat-only weight regression, and two-tailed p=0.355 for the RC rat-only millimole
regression).

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In Vitro Cytotoxicity Test Methods BRD Section 6

Table 6-11

November 2006

Over- and Under- Prediction of Toxicity for Low and High Molecular
Weight Substances Using RC Rat-Only Weight and Millimole
Regressions
Comparison

RC rat-only millimole vs. RC rat-only weight
regression
RC rat-only millimole vs. RC rat-only weight
regression
51 Low molecular weight (≤100 g/mole)substances vs. 231 other substances (>100g/mole)51 Low molecular weight (≤100 g/mole)substances vs. 231 other substances (>100g/mole)RC rat-only millimole vs. RC rat-only weight
regression
RC rat-only millimole vs. RC rat-only weight
regression
20 High molecular weight substances (≥400
g/mole) vs. 262 other substances (<400 g/mole)
20 High molecular weight substances (≥400
g/mole) vs. 262 other substances (<400 g/mole)For
Under- and over-prediction of toxicity
for 51 substances with molecularweight ≤100 g/mole
Under- and over-prediction of toxicity
for 231 substances with molecularweight >100 g/mole

Fisher’s Exact
Test1
0.549
0.575

RC rat-only millimole regression

0.355

RC rat-only weight regression

0.756

Under- and over-prediction of toxicity
for 20 substances with molecularweight ≥400 g/mole
Under- and over-prediction of toxicity
for 262 substances with molecularweight <400 g/mole

0.480
NT

RC rat-only millimole regression

0.362

RC rat-only weight regression

0.033

Abbreviations: RC=Registry of Cytotoxicity; NT=Not tested because the proportions were the same. Toxicity was
underpredicted for 121/262 (46%) substances and overpredicted for 141/262 (54%) substances.
1
P-values.

Of the 282 RC substances with rat acute oral LD50 values, there were 20 with molecularweights ≥400 g/mole and 262 with molecular weights <400 g/mole. The RC rat-only
millimole regression underestimated the toxicity of 7/20 (35%) of the ≥400 g/mole
substances and overestimated 13/20 (65%). The RC rat-only weight regression
underestimated the toxicity of 4/20 (20%) of the substances and overestimated 16/20 (80%).
Fisher’s exact test indicated that there were no differences between the millimole and weight
regressions for the under- and over-prediction of toxicity for the 20 high molecular weightsubstances (two-tailed p=0.4801).
For the remaining 262 substances with molecular weights <400 g/mole, both the RC rat-only
millimole and the RC rat-only weight regressions underestimated the toxicity of 121/262(46%) substances and overestimated 141/262 (54%). Thus, there were no statisticaldifferences in the under- and over-esimation of toxicity for the 262 substances withmolecular weights <400 g/mole regardless of which regression was used. Fisher’s exact test
also showed that there was no statistical difference in the under- and over-prediction of the
toxicity of substances with high molecular weight (≥400 g/mole) compared with the underand over-prediction the lower molecular weight substances using the RC rat-only millimole
regression (two-tailed p=0.362). In contrast the use of the RC rat-only weight regression,
resulted in a small but statistically significant difference in the under- and over-prediction of
6-33

In Vitro Cytotoxicity Test Methods BRD Section 6

November 2006

the toxicity of substances with high molecular weight (>400 g/mole) compared with theunder- and over-prediction of the toxicity of substances with lower molecular weight (twotailed p=0.033). The weight-based regression significantly overestimated the toxicity of thehigh molecular weight substances (compared with substances with lower molecular weight)
while the millimole regression did not.
6.7

Salient Issues of Data Interpretation

One of the most important considerations for the 3T3 and NHK NRU test methods, as forany test method, is the ability to generate good concentration-response results. In addition totechnical difficulties with these test methods, such as occasional poor cell growth and theformation of NRU crystals, this validation study yielded non-monotonic concentrationresponse curves for certain substances.
A number of substances produced non-monotonic concentration-response curves in the 3T3
and/or the NHK NRU range finding or definitive tests. Because the in vitro NRU test
methods, and the calculation of IC50 values from the resulting concentration curves, presume
that the toxic response is linear, the data from non-linear responses (e.g., biphasic curves), as
seen with aminopterin, do not always permit an IC50 determination by the standard Hillfunction analysis. In such cases, the lowest concentration that killed approximately 50% of
the cells in the range finding test was used to set the concentration range for the definitive
test. The definitive test used more closely spaced concentrations in an attempt to obtain amonotonic concentration-response curve. However, 100% toxicity (or 0%) viability wasoften unattainable in such definitive tests that exhibited a plateau of toxicity well over 0%
viability (e.g., 20%). Care must be used in the calculation of the IC50 for curves for which
toxicity plateaus to assure that the value reflects the concentration at 50% inhibition of the
VC value rather than simply the midpoint of the highest and lowest response.
Because of low toxicity and/or low solubility, some substances did not produce sufficient
toxicity for the calculation of an IC50 value. Carbon tetrachloride, methanol, xylene,
gibberellic acid, lithium carbonate, and 1,1,1-trichloroethane failed to yield acceptable IC50
results in at least one laboratory because of insufficient toxicity. All of these substances, withthe exception of methanol, produced precipitate in the cell culture medium.
6.8

Comparison of NRU Test Results to Established Performance Standards

The Guidance Document method of evaluating in vitro basal cytotoxicity assays forpredicting starting doses for rodent acute oral toxicity assays provides the existing
performance standard (ICCVAM 2001b) for the 3T3 and NHK NRU test methods. The
Guidance Document recommends testing 10 to 20 reference substances from the RC in an invitro basal cytotoxicity assay for predicting starting doses for rodent acute oral toxicity
testing (ICCVAM 2001b). These substances should cover a wide range of toxicity and fit the
RC millimole regression as closely as possible. The Guidance Document recommends using
the IC50 results for the selected reference substances from the candidate method to calculate a
new regression line with the LD50 values used by the RC. If the resulting regression isparallel to the RC millimole regression and within the ± log 5 (i.e., ± 0.699) prediction
interval for the RC, candidate assay may be considered effective for predicting starting doses
for substances in rodent acute oral toxicity assays.

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In Vitro Cytotoxicity Test Methods BRD Section 6

November 2006

One goal of the testing in Phases Ib and II of this study was to establish whether the resultsfrom the 3T3 and NHK NRU test methods were consistent with the RC millimole regression.
As discussed in Section 3.3.5, two of the major criteria for selecting the 12 coded substancestested from the 72 reference substances were:(a) Two substances must be included from each of the unclassified and classified
GHS acute oral toxicity categories, and(b) The substances must fit as closely to the RC millimole regression as possible.
Unfortunately, the SMT could not identify 12 substances that fit both criteria because there
was only one substance, aminopterin, in the LD50 <5 mg/kg category that fit the RC
millimole regression. The other substance chosen from that toxicity category was sodium
selenate. Because sodium selenate was not included in the RC, there was no indication of
how closely it would fit the RC millimole regression, and it was therefore not included in the
Phases Ib and II regression analyses. The other 10 substances selected for testing in Phases Ib
and II were colchicine, arsenic trioxide, cadmium chloride, sodium fluoride, propranolol,
lithium carbonate, potassium chloride, chloramphenicol, 2-propanol, and ethylene glycol.
The geometric mean log IC50 (mM) values from the 3T3 and NHK NRU test methods fromeach laboratory were used with the oral log rodent LD50 (mmol/kg) values from the RC (seeAppendices J1 and J2) for the least squares linear regression analyses (see Section 5.5.3.3)
for the substances tested in Phases Ib and II. The slopes for all regressions were significantly
different from zero at p <0.0001, which indicated that there was a significant relationshipbetween IC50 and LD50. The R2 values for the regressions from each laboratory, shown in
Table 6-12, show that the 3T3 NRU test method produced better-fitting regressions than thecorresponding NHK NRU test method (R2 = 0.940 to 0.953 vs. 0.577 to 0.621). The
relatively low R2 values for the NHK NRU test method were attributed to the much lower
toxicity of aminopterin in those cells (see Figures 6-6 to 6-8 and Tables 5-3 and 5-4). All
test method and laboratory-specific regressions were consistent with the RC millimole
regression. Table 6-12 shows that all joint comparisons of slopes and intercepts with the RC
millimole regression were not significant (i.e., p >0.01). The RC millimole regression slope
and intercept were used as constants for this comparison.
A graphic comparison of the IC50 regressions with the RC millimole regression as suggestedby the Guidance Document (ICCVAM 2001b) demonstrated that they were generally within
the RC millimole regression acceptance limits (see Figures 6-6, 6-7, and 6-8). According to
the Guidance Document (ICCVAM 2001b), in vitro basal cytotoxicity assays providing suchconsistency with the RC millimole regression are acceptable for predicting starting doses forrodent acute oral toxicity assays.
As an additional analysis, a regression for the 11 substances tested in Phases Ib and II (theRC-11 millimole regression), was calculated using the log RC IC50 (mM) and log LD50
(mmol/kg) values (see Table 6-12). Each of the laboratory regressions for each test method
was then compared to the RC-11 regression using an F test for a joint comparison of slope
and intercept. None of the regressions were significantly different from the RC-11 regression(p values ranged from 0.755 to 0.933).

6-35

In Vitro Cytotoxicity Test Methods BRD Section 6

Table 6-12

November 2006

Linear Regressions for 11 Substances Tested in Phases Ib and II
3T3 Regression1

Laboratory
ECBC
FAL
IIVS
Laboratory
ECBC
FAL
IIVS

Intercept
0.793
0.709
0.710

Slope

R2 Statistic

0.584
0.940
0.598
0.953
0.584
0.949
NHK Regression1

Intercept

Slope

R2 Statistic

0.401
0.429
0.373

0.530
0.548
0.549

0.577
0.621
0.590

Test Against RC
Regression2
0.040
0.024
0.041

Test Against RC-11
Regression3
0.829
0.909
0.933

Test Against RC
Regression2
0.620
0.569
0.538

Test Against RC-11
Regression3
0.805
0.853
0.755

Abbreviations: ECBC=Edgewood Chemical Biological Center; FAL=Fund for the Replacement of Animals in Medical
Experiments Alternatives Laboratory; IIVS=Institute for In Vitro Sciences; RC=Registry of Cytotoxicity; 3T3=BALB/c 3T3
fibroblasts; NHK=Normal human epidermal keratinocytes; R2=Coefficient of determination.
1
Laboratory and test method regressions were calculated after log transforming the NRU IC50 in mM and the RC LD50 in
mmol/kg for the 11 RC substances tested in study Phases Ib and II (shown in Figures 6-6 through 6-8).
2
Simultaneous comparison of slope and intercept with RC millimole regression: log LD50 (mmol/kg) = 0.435 x log IC50(mM) + 0.625; R2=0.452; the reported values are p values of the statistic.
3
Simultaneous comparison of slope and intercept with RC-11 regression (defined as a regression on the 11 substances): log
LD50 (mmol/kg) = 0.552 x log IC50 (mM) + 0.602; R2=0.971; the reported values are p values of the statistic.

6-36

In Vitro Cytotoxicity Test Methods BRD Section 6

Figure 6-6

November 2006

In Vitro – In Vivo Regressions1 for Phases Ib and II for ECBC
3T3 ECBC

1000

log LD50 = 0.584 log IC50 + 0.793
R2 = 0.940

Potassium chloride

100

Chloramphenicol

Lithium carbonate
2-Propanol

10
Propranolol HCl

1

EthyleneglycolSodium fluoride

Cadmium chloride

0.1
Arsenic trioxide

0.01

Colchicine
Aminopterin

0.001
0.000001 0.00001

0.0001

0.001

0.01

0.1

1

10

100

1000

IC50 (mM)

NHK ECBC

1000

log LD50 = 0.530 log IC50 + 0.401
R2 = 0.577

2-Propanol

100

EthyleneglycolPotassium chloride
Lithium carbonate
Chloramphenicol

10

Sodium fluoride
Propranolol HCl

1
Cadmium chloride

0.1
Arsenic trioxide

0.01

Colchicine
Aminopterin

0.001
0.000001 0.00001

0.0001

0.001

0.01

0.1

1

10

100

1000

IC50 (mM)

Abbreviations: ECBC=Edgewood Chemical Biological Center; RC=Registry of Cytotoxicity; 3T3=Neutral red
uptake using BALB/c 3T3 fibroblasts; NHK= Neutral red uptake using normal human epidermal keratinocytes;
R2=Coefficient of determination.
1
Regressions of substances tested in study Phases Ib and II do not include sodium selenate because it was notincluded in the RC. Regressions were calculated using the NRU IC50 values and the RC LD50 values.
The solid lines show RC millimole regression (bold) and acceptance limits (lighter). The dashed shows the
ECBC regressions.

6-37

In Vitro Cytotoxicity Test Methods BRD Section 6

Figure 6-7
1000

November 2006

In Vitro – In Vivo Regressions1 for Phases Ib and II for FAL
3T3 FALlog LD50 = 0.598 log IC50 + 0.709
R2 = 0.953

2-Propanol

100

EthyleneglycolPotassium chloride

10

Chloramphenicol
Sodium fluoride

Propranolol HCl

1
Cadmium chloride

0.1
Arsenic trioxide

0.01

Colchicine
Aminopterin

0.001
0.000001 0.00001

0.0001

0.001

0.01

0.1

1

10

100

1000

IC50 (mM)
1000

NHK FALlog LD50 = 0.548 log IC50 + 0.429
R2 = 0.621

2-Propanol

100
Chloramphenicol

EthyleneglycolPotassium chloride

10
Sodium fluoride Lithium carbonate
Propranolol HCl

1

Cadmium chloride

0.1
Arsenic trioxide

0.01

Colchicine

0.001
0.000001 0.00001

Aminopterin

0.0001

0.001

0.01

0.1

1

10

100

1000

IC50 (mM)

Abbreviations: FAL=Fund for the Replacement of Animals in Medical Experiments Alternatives Laboratory
RC=Registry of Cytotoxicity; 3T3=Neutral red uptake using BALB/c 3T3 fibroblasts; NHK=Neutral red uptake
using normal human epidermal keratinocytes; R2=Coefficient of determination.
1
Regressions of substances tested in study Phases Ib and II do not include sodium selenate because it was notincluded in the RC. Regressions were calculated using the NRU IC50 values and the RC LD50 values.
The solid lines show RC millimole regression (bold) and acceptance limits (lighter). The dashed shows the FAL
regressions.
6-38

In Vitro Cytotoxicity Test Methods BRD Section 6

Figure 6-8
1000

November 2006

In Vitro – In Vivo Regressions1 for Phases Ib and II for IIVS
3T3 IIVS

log LD50 = 0.584 log IC50 + 0.710
R2 = 0.949

2-Propanol

100

EthyleneglycolChloramphenicol
Potassium chloride

10
Propranolol HCl

1

Sodium fluoride

Cadmium chloride

0.1

Arsenic trioxide

0.01

Colchicine
Aminopterin

0.001
0.000001 0.00001

0.0001

0.001

0.01

0.1

1

10

100

1000

IC50 (mM)
1000

NHK IIVS

log LD50 = 0.549 log IC50 + 0.373
R2 = 0.590

EthyleneglycolPotassium chloride

100

2-Propanol
Chloramphenicol

10
Sodium fluoride

Lithium carbonate

Propranolol HCl

1

Cadmium chloride

0.1

0.01

Arsenic trioxide

Colchicine
Aminopterin

0.001
0.000001 0.00001

0.0001

0.001

0.01

0.1

1

10

100

1000

IC50 (mM)

Abbreviations: IIVS=Institute for In Vitro Sciences; RC=Registry of Cytotoxicity; 3T3=Neutral red uptake
using BALB/c 3T3 fibroblasts; NHK=Neutral red uptake using normal human epidermal keratinocytes;
R2=Coefficient of determination.
1
Regressions of substances tested in study Phases Ib and II do not include sodium selenate because it was notincluded in the RC. Regressions were calculated using the NRU IC50 values and the RC LD50 values.
The solid lines show RC millimole regression (bold) and acceptance limits (lighter). The dashed shows the IIVS
regressions.
6-39

In Vitro Cytotoxicity Test Methods BRD Section 6

6.9

November 2006

Summary

The millimole regressions developed using the validation study IC50 and LD50 values were
not significantly different from the regressions for the same 47 RC substances using the RCdata (F test; p=0.612 for the 3T3 regression and p=0.759 for the NHK regression). Because
this validation study provided results similar to the RC, which has more than 3.5 times thenumber of substances, the 282 RC substances with rat LD50 values were used to determine
the relationship between the IC50 and LD50 data. One linear regression was developed using
millimole units for the measurement of substances, the RC rat-only millimole regression, andone was developed using weight units (which are more practical in a routine testing
situation), the RC rat-only weight regression. The RC rat-only millimole regression isapplicable to substances of known molecular weight while the RC rat-only weight regression
is applicable for use with complex mixtures, substances whose molecular weight is unknown.
Characteristics that seemed promising for characterizing the RC millimole regression outliers
were chemical class, boiling point, molecular weight, and log Kow. Different chemical classes
behaved differently with respect to being outliers; ranging from 5/5 (100%) for the organic
sulfur compounds for both test methods to 4/14 (29%) for carboxylic acids for the 3T3 NRU.
Of the reference substances with boiling points >200°C, 9/13 (69%) were outliers for the 3T3
NRU and 8/13 (62%) were outliers for the NHK NRU. With respect to molecular weights,
4/7 (57%) substances with molecular weight >400 g/mole were outliers using the 3T3 data,and 3/7 (43%) were outliers using the NHK data. When log Kow was used, 8/13 (62%)
substances with a log Kow >3 were outliers for both test methods.
The lack of fit of individual substances to the RC millimole regression was not consistently
related to insolubility or to the fact that the test method systems had little to no metabolic
capability. Of the substances that exhibited precipitation, 11/25 (44%) were outliers in the
3T3 NRU assays and 11/24 (46%) were outliers in the NHK NRU assays. However, althoughthe 3T3 and NHK cells have little to no metabolic capability, the toxicity of substances
known to produce active metabolites in vivo was not underpredicted by these assays. Of the
19 substances known to produce active metabolites in vivo, 10 (53%) were outliers in the 3T3
NRU test method; the toxicity of six (60%) was underpredicted while the toxicity of four(40%) overpredicted. These 10 substances accounted for 36% of the 28 outliers identified by
the 3T3 NRU test method. Similarly, nine (47%) of the 19 substances known to produce
active metabolites in vivo were outliers in the NHK NRU test method. Of these nine, the
NHK NRU test method underpredicted the toxicity of five (56%) and overpredicted four(44%). These nine outliers accounted for 29% of the 31 outliers identified by the NHK NRU
test method.
The examination of outliers based on mechanisms of toxicity showed that 10/16 (63%)
substances with specific neurotoxic mechanisms were outliers in both the 3T3 and NHK
NRU test methods. Three of the six (50%) cardiotoxic substances were outliers in the 3T3
NRU test method and two (33%) were outliers in the NHK NRU test method. When all thereference substances with mechanisms of toxicity that are not expected to be active in the
3T3 and NHK systems (i.e., in Table 6-3) were summed, 13/22 (59%) were outliers for the
3T3 NRU and 12/22 (55%) were outliers for the NHK NRU.

6-40

In Vitro Cytotoxicity Test Methods BRD Section 6

November 2006

The accuracy of the 3T3 and NHK NRU test methods for predicting the GHS acute oral
toxicity categories was 31% (21/67) and 29% (20/68), respectively, when used with the RC
rat-only millimole regression. The corresponding accuracy with the RC rat-only weight
regression was 31% for both methods (21/67 for 3T3, and 21/68 for NHK). Accuracy was
highest for substances in the 300< LD50 ≤2000 mg/kg range. The accuracies of theregressions, with respect to the GHS categories, were similar for both regressions (millimole
and weight) and all three laboratories.
• 0% for substances with LD50 ≤5 mg/kg (GHS Category I)• 9% to 18% for substances with 5< LD50 ≤50 mg/kg (GHS Category II)• 33% to 50% for substances with 50< LD50 ≤300 mg/kg (GHS Category III)
• 75% to 81% for substances with 300< LD50 ≤2000 mg/kg (GHS Category IV)• 0% to 40% for substances with 2000< LD50 ≤5000 mg/kg (GHS Category V)• 0% to 17% for substances with LD50 >5000 mg/kg (GHS Unclassified)
The overall accuracy for prediction of GHS category prediction using the RC IC50 and LD50
values and the RC millimole regression was higher that that for the NRU test methods with
the RC rat-only regressions (i.e., 40% for the RC vs. 29% to 31% for the NRU test methods
and RC rat-only regressions). However, the pattern of accuracy for the GHS categories was
similar. For all the accuracy analyses, the lowest accuracy was obtained for very toxic andvery nontoxic substances and highest accuracy was obtained for substances with 300 < LD50
≤2000 mg/kg.
The accuracy of GHS acute oral toxicity category predictions using the in vitro NRU test
methods with the RC rat-only regressions obtained for the reference substances may or may
not be broadly applicable to substances that might require acute oral toxicity testing. The
reasons for the low accuracy obtained in this validation study include: the differences
between cell cultures and whole animals regarding the absorption, distribution, availability,
metabolism, and excretion of reference substances, and the presence or absence of toxicity
targets; the skewness of the selection of substances for testing (with respect to fit to theregression); and the structure of the GHS acute oral toxicity categories.

6-41

In Vitro Cytotoxicity Test Methods BRD Section 6

November 2006

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6-42

In Vitro Cytotoxicity Test Methods BRD Section 7

November 2006

7.0

RELIABILITY OF THE 3T3 AND NHK NRU TEST METHODS ...................7-3

7.1

Reference Substances Used to Determine the Reliability of the 3T3 andNHK NRU Test Methods......................................................................................7-3

7.2

Reproducibility Analyses for the 3T3 and NHK NRU Test Methods.................7-5
7.2.1 Comparison of Laboratory-Specific IC50-LD50 Linear Regression
Analyses to the Mean Laboratory Regression..............................................7-5
7.2.2 ANOVA Results for the 3T3 and NHK NRU Test Methods ........................7-5
7.2.3 CV Results for the 3T3 and NHK NRU Test Methods...............................7-21
7.2.4 Comparison of Maximum to Minimum IC50 Values Using Laboratory
Means .......................................................................................................7-39
7.2.5 Comparison of Maximum:Minimum IC50 Ratios with the
Maximum:Minimum LD50 Ratios .............................................................7-40
7.2.6 Normalization of Reference Substance IC50 Values Using SLS
Values ......................................................................................................7-42

7.3

Historical Positive Control (PC) Data................................................................7-44
7.3.1 ANOVA and Linear Regression Results for the 3T3 NRU Test Method....7-46
7.3.2 ANOVA and Linear Regression Results for the NHK NRU Test
Method .....................................................................................................7-48

7.4

Laboratory Concordance for Solvent Selection.................................................7-50

7.5

Summary.............................................................................................................7-52

7-1

In Vitro Cytotoxicity Test Methods BRD Section 7

November 2006

[This Page Intentionally Left Blank]

7-2

In Vitro Cytotoxicity Test Methods BRD Section 7

7.0

November 2006

RELIABILITY OF THE 3T3 AND NHK NRU TEST METHODS

The reliability of the 3T3 and NHK NRU test methods was assessed by determining intraand inter-laboratory reproducibility. Intralaboratory reproducibility is the agreement of
results produced when people in the same laboratory perform the method using the same test
protocol at different times (ICCVAM 2003). Interlaboratory reproducibility is the agreementof results among different laboratories using the same protocol and reference substances.
Interlaboratory reproducibility indicates the extent to which a method can be successfullytransferred among laboratories. Repeatability, usually applied to results within a laboratory,
is the closeness of agreement between test results obtained when the procedure is performedon the same substance under identical conditions within a given time. This study was notdesigned to assess intralaboratory repeatability.
The interlaboratory reproducibility of the test results was assessed by comparing thelaboratory-specific IC50-LD50 regressions for the 3T3 and NHK NRU test methods to themean (i.e., across-laboratory mean) laboratory regressions (see Section 7.2.1). This
comparison is relevant because the 3T3 and NHK NRU test methods are intended for use
with IC50-LD50 regressions to determine starting doses for acute oral toxicity tests.
Interlaboratory reproducibility of the 3T3 and NHK NRU test methods was also determinedusing ANOVA, CV analysis, and comparison of maximum:minimum IC50 ratios calculatedusing laboratory mean values (see Sections 7.2.2, 7.2.3, and 7.2.4, respectively), as discussed
in Section 5.5.2.2. Inter- and intra-laboratory reproducibility of the PC (SLS) was determinedusing ANOVA, CV analysis, and/or linear regression over time (see Section 7.3). The extent
of laboratory concordance in selecting the solvent to be used for each test substance(described in Section 2.10) is provided in Section 7.4.
7.1

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

The validation study was designed for the purpose of using the IC50 results of 72 reference
substances (see Table 3-2) to determine the reliability of the IC50 values from the 3T3 andNHK NRU test methods. The number of reference substances used for the reproducibility
analysis was not the same as the number of reference substances used for the accuracy
analyses in Section 6.4. In the former case, only reference substances for which all threelaboratories reported replicate IC50 values were used, while in the latter case, substances with
rat acute oral LD50 data only and at least one laboratory reporting replicate IC50 values wereused. Table 7-1 lists the reference substances that failed to yield sufficient toxicity for the
calculation of an IC50 in each laboratory, and the number of remaining reference substanceswith replicate IC50 values. The laboratories obtained acceptable IC50 values for 66 to 68
reference substances using the 3T3 NRU test method, and for 69 to 70 substances using the
NHK NRU test method. When only reference substances with IC50 values from all threelaboratories are considered, 64 and 68 substances were available to evaluate the reliability of
the 3T3 and NHK NRU test methods, respectively. The substances that were excluded fromthe 3T3 reliability analysis were carbon tetrachloride, disulfoton, gibberellic acid, lithium
carbonate, methanol, 1,1,1-trichloroethane, valproic acid, and xylene. The substances thatwere excluded from the NHK reliability analysis were carbon tetrachloride, methanol, 1,1,1trichloroethane, and xylene.

7-3

In Vitro Cytotoxicity Test Methods BRD Section 7

Table 7-1

Laboratory

ECBC

FAL

IIVS

November 2006

Reference Substances Excluded from Reproducibility Analyses Becauseof Insufficient Cytotoxicity
3T3 NRU Test Method
Reference Substances
Lacking IC50 Results
Carbon tetrachloride
Methanol
1,1,1-Trichloroethane
XyleneCarbon tetrachloride
Disulfoton
Gibberellic acid
Lithium carbonate
Methanol
XyleneCarbon tetrachloride
Lithium carbonate
Methanol
Valproic acid

1

N

NHK NRU Test Method
Reference Substances
Lacking IC50 Results

N1

68

Carbon tetrachloride
Methanol
Xylene69

66

1,1,1-Trichloroethane
Carbon tetrachloride
Xylene69

68

Carbon tetrachloride
1,1,1-Trichloroethane

70

Abbreviations: 3T3=BALB/c 3T3 fibroblasts; NHK=Normal human epidermal keratinocytes; NRU= Neutral
red uptake; ECBC=Edgewood Chemical Biological Center; FAL=Fund for the Replacement of Animals in
Medical Experiments Alternatives Laboratory; IIVS=Institute for In Vitro Sciences; N=Number of substances.
2
Number of substances with replicate IC50 values.

Despite the fact that IC50 values were not obtained by all the laboratories for all reference
substances, Table 7-2 shows that the complete range of LD50 responses, as defined by the
GHS classification for acute oral toxicity in Table 3-1, was covered by the reference
substances for which replicate IC50 values were obtained. The 3T3 NRU IC50 values ranged
from 0.005 to 38,878 µg/mL, while the NHK values covered a larger range, from 0.00005 to
49,800 µg/mL (see Tables 5-4 and 5-5).
Table 7-2

Number of Reference Substances Tested vs Number of Reference
Substances Yielding IC50 Values from Each Laboratory, by GHS Acute
Oral Toxicity Category

GHS Category1(mg/kg)
LD50 ≤55 < LD50 ≤50
50 < LD50 ≤300
300 < LD50 ≤2000
2000 < LD50 ≤5000
LD50 >5000

Reference Oral
LD502
7
12
12
16
11
14

3T3 NRU Test
Method3
6
12
12
14
9
11

NHK NRU Test
Method3
7
12
12
16
9
12

Abbreviations: 3T3=BALB/c 3T3 fibroblasts; NHK=Normal human epidermal keratinocytes; NRU= Neutral
red uptake; GHS=Globally Harmonized System of Classification and Labelling of Chemicals (UN 2005).
1
GHS category for acute oral toxicity.
2
Number of reference substances tested in each category. Reference acute oral LD50 values from rats and mice
were generated after evaluating LD50 values located through literature searches and references from toxicity
databases such as RTECS® (from Table 4-2).
3
Number of reference substances with IC50 values from all three laboratories.
7-4

In Vitro Cytotoxicity Test Methods BRD Section 7

7.2

November 2006

Reproducibility Analyses for the 3T3 and NHK NRU Test Methods

The interlaboratory reproducibility of the 3T3 and NHK NRU IC50 values was assessed bycomparing the laboratory-specific IC50-LD50 linear regressions for each method to aregression calculated using the mean IC50 values of the laboratories. The interlaboratory
reproducibility of the 3T3 and NHK NRU test methods was also assessed using ANOVA,
CV analysis, and analysis of the laboratory mean maximum:minimum IC50 ratios, as
described in Section 5.5.2.2. Intralaboratory reproducibility was assessed using a CV
analysis.
7.2.1

Comparison of Laboratory-Specific IC50-LD50 Linear Regression Analyses to the
Mean Laboratory Regression
The comparisons of laboratory-specific IC50-LD50 linear regressions to the mean laboratory
regression for each method were made because the 3T3 and NHK NRU test methods areintended for use with IC50-LD50 regressions to determine starting doses for acute oral toxicity
tests. Laboratory-specific IC50-LD50 linear regressions were generated and displayed
graphically for each method using the 64 and 68 reference substances for the 3T3 and NHK
NRU test methods, respectively, as indicated in Section 7.1. The regressions used thegeometric mean IC50 values for each substance with the rodent acute oral LD50 reference
value (Table 4-2). To determine whether the laboratory-specific regressions weresignificantly different from one another, they were compared against the mean laboratory
regression for each NRU test method that was calculated using the geometric mean of thelaboratory mean IC50 values and the rodent acute oral LD50 reference values. The mean
laboratory regression for each NRU test method is in Figure 7-1 with 95% confidence limits,and shows that the laboratory-specific regressions were all within the 95% confidence limitsof the mean laboratory regression.
7.2.2
ANOVA Results for the 3T3 and NHK NRU Test Methods
The ANOVA was performed as discussed in Section 5.5.2.2. Because the sample sizes fromthis study were small, usually three observations per laboratory, there may be differences thatwere statistically significant only because there were too few observations within thelaboratories to adequately characterize variability or because the within-laboratory variability
was small.
7.2.2.1

Differences Among the IC50 Values in Laboratories Using the 3T3 NRU Test
MethodThe ANOVA results in Table 7-3 show that there were statistically significant (p <0.01)
differences among the laboratories for 23 of the 64 (36%) reference substances evaluated.
The p values from the contrast analyses, post-hoc tests to determine which laboratory wassignificantly different from the others at p <0.01 (see Section 5.5.2.2), are also provided inTable 7-3. The substances for which statistically significant ANOVA and contrast results
were obtained are listed in Table 7-4 along with columns showing the laboratory withsignificantly differing values from the other two laboratories. Because significant laboratory
differences may have resulted from the insolubility or volatility of the test substance, Table
7-4 also indicates whether any laboratory reported insolubility or volatility during conduct of
the test. Insolubility was suggested by the presence of precipitates in either the stock
solutions or in cell culture. Volatility was identified by the need for plate sealers to containvolatile contamination of lower concentration wells by higher concentrations. Insolubility
and volatility were reported for only six of the 23 chemicals showing significant
7-5

In Vitro Cytotoxicity Test Methods BRD Section 7

November 2006

interlaboratory variability. In contrast, 22 of the 41 substances that were classified as
generating interlaboratory reproducible data exhibited precipitates and/or volatility.
For the 23 substances that yielded significantly different results among laboratories, contrast
analyses indicated that the IC50 values produced by ECBC and FAL were frequently different
from the other laboratories. ECBC tended to report the lowest IC50 values (i.e., highest
toxicity) among the laboratories while FAL tended to report the highest values of the threelaboratories. ECBC reported significantly different results from the other two laboratories for
15 of the 23 substances; for 13 of the 15, ECBC’s mean value IC50 was the lowest among thelaboratories. FAL reported significantly different results from the other two laboratories for
20 of the 23 substances; for 18 of the 20, FAL’s IC50 value was the highest among thelaboratories. IIVS reported significantly different values for 11 of the 26 substances, with notendency toward highest or lowest IC50 values.

7-6

In Vitro Cytotoxicity Test Methods BRD Section 7

Figure 7-1

November 2006

Mean Laboratory and Laboratory-Specific 3T3 and NHK NRU
Regressions

a.
1000
100

Mean laboratory;: Log LD 50 = 0.582logIC 50 + 0.436
ECBC: Log LD50 = 0.543logIC 50 + 0.477
FAL: Log LD 50 = 0.547logIC 50 + 0.329
IIVS: Log LD50 = 0.532logIC 50 + 0.449

10
1
0.1
0.01
0.001
0.00010.000001 0.00001

0.0001

0.001

0.01

0.1

1

10

100

1000

1

10

100

1000

IC50 (mM)b.
1000

Mean laboratory: Log LD 50 = 0.488logIC 50 + 0.403
ECBC: Log LD50 = 0.466logIC 50 + 0.377

100

FAL: Log LD 50 = 0.429logIC 50 + 0.388
IIVS: Log LD50 = 0.463logIC 50 + 0.386

10
1
0.1
0.01
0.001
0.00010.000001 0.00001

0.0001

0.001

0.01

0.1

IC50 (mM)

Abbreviations: 3T3=BALB/c 3T3 fibroblasts; NRU=Neutral red uptake; NHK=Normal human epidermal keratinocytes.
Solid lines show the mean laboratory linear regressions for the 3T3 NRU (a) and the NHK NRU (b) test methods withdashed curved lines to show the 95% confidence limits of the regression. The regressions were calculated using 64 and 68
reference substances for the 3T3 and NHK NRU test methods, respectively, as described in Section 7.1. Regressions used
geometric mean IC50 values and reference acute oral LD50 values from Table 4-2.

7-7

In Vitro Cytotoxicity Test Methods BRD Section 7

Table 7-3

November 2006

Interlaboratory Reproducibility of the IC50 Values from the 3T3 NRU Test Method

Reference
Substance/Laboratory
Acetaminophen
ECBC
FAL
IIVS
Acetonitrile
ECBC
FAL
IIVS
Acetylsalicylic acid
ECBC
FAL
IIVS
5-Aminosalicylic acid
ECBC
FAL
IIVS
Aminopterin
ECBC
FAL
IIVS
Amitriptyline HCl
ECBC
FAL
IIVS
Arsenic trioxide

Arithmetic
Mean IC50(mg/mL)1
50.1
40.8
66.2
43.4
8484
6433
9690
9330
760
646
1234
401
1698
1467
2070
1557
0.007
0.005
0.012
0.005
7.23
6.03
7.86
7.81
2.51

Maximum:
Minimum
Mean IC50
Ratio2

Arithmetic
IntraLab%CV

1.6

Arithmetic
InterLab
%CV
28

22
35
26
1.5

21
2
58
13

3.1

56
10
24
16

1.4

19
14
16
12

2.4

54
20
46
23

1.3

14
23
28
18

3.9

61

7-8

Log
Arithmetic
Mean IC50(mg/mL)1
1.7
1.61
1.82
1.64
3.93
3.81
3.99
3.97
2.88
2.81
3.09
2.6
3.23
3.17
3.32
3.19
-2.14
-2.28
-1.93
-2.33
0.86
0.78
0.9
0.89
0.4

ANOVA
P3

Contrast
P4

0.171
NA
NA
NA
0.553
NA
NA
NA
<0.001
0.581<0.001<0.001
0.054
NA
NA
NA
0.036
NA
NA
NA
0.348
0.163
0.469
0.445
0.004

In Vitro Cytotoxicity Test Methods BRD Section 7

Table 7-3

November 2006

Interlaboratory Reproducibility of the IC50 Values from the 3T3 NRU Test Method

Reference
Substance/Laboratory
ECBC
FAL
IIVS
Atropine sulfate
ECBC
FAL
IIVS
Boric acid
ECBC
FAL
IIVS
Busulfan
ECBC
FAL
IIVS
Cadmium chloride
ECBC
FAL
IIVS
Caffeine
ECBC
FAL
IIVS
Carbamazepine
ECBC

Arithmetic
Mean IC50(mg/mL)1
2.41
1.04
4.09
85.6
54.1
133
70
2228
1497
3987
1202
135
40
321
43.7
0.565
0.48
0.4
0.817
161
133
157
191
109
83

Maximum:
Minimum
Mean IC50
Ratio2

Arithmetic
IntraLab%CV

Arithmetic
InterLab
%CV

33
7
52
2.5

49
55
31
8

3.3

69
32
17
48

8.0

119
48
56
4

1.4

39
14
32
53

1.4

18
10
52
7.5

1.8

35
14

7-9

Log
Arithmetic
Mean IC50(mg/mL)1
0.38
0.02
0.61
1.93
1.73
2.12
1.85
3.35
3.18
3.6
3.08
2.13
1.6
2.51
1.64
-0.25
-0.32
-0.4
-0.09
2.21
2.12
2.2
2.28
2.04
1.92

ANOVA
P3

Contrast
P4
0.527
0.002
0.006

0.049
NA
NA
NA
0.01
NA
NA
NA
0.002
0.012
<0.001
0.033
0.124
NA
NA
NA
0.481
NA
NA
NA
0.049
NA

In Vitro Cytotoxicity Test Methods BRD Section 7

Table 7-3

November 2006

Interlaboratory Reproducibility of the IC50 Values from the 3T3 NRU Test Method

Reference
Substance/Laboratory
FAL
IIVS
Carbon tetrachloride
ECBC
FAL
IIVS
Chloral hydrate
ECBC
FAL
IIVS
Chloramphenicol
ECBC
FAL
IIVS
Citric acid
ECBC
FAL
IIVS
Colchicine
ECBC
FAL
IIVS
Cupric sulfate pentahydrate
ECBC
FAL

Arithmetic
Mean IC50(mg/mL)1
152
91.8
NA
NA
NA
NA
187
151
241
170
161
55.3
273
156
829
473
1148
865
0.047
0.02
0.093
0.028
70.6
82.7
123

Maximum:
Minimum
Mean IC50
Ratio2

Arithmetic
IntraLab%CV

Arithmetic
InterLab
%CV

37
12
NA

NA
NA
NA
NA

1.6

25
10
10
12

4.9

67
22
30
18

2.4

41
29
13
19

4.7

85
11
45
1

21.6

85
4
44

7-10

Log
Arithmetic
Mean IC50(mg/mL)1
2.18
1.96
NA
NA
NA
NA
2.27
2.18
2.38
2.23
2.21
1.74
2.44
2.19
2.92
2.68
3.06
2.94
-1.33
-1.70
-1.03
-1.55
1.85
1.92
2.09

ANOVA
P3

Contrast
P4
NA
NA

NA
NA
NA
NA
0.004
0.008
0.002
0.181
<0.001<0.001
0.001
0.165
0.002
0.001
0.003
0.298
0.001
0.00280.00050.0914<0.0010.001<0.001In Vitro Cytotoxicity Test Methods BRD Section 7

Table 7-3

November 2006

Interlaboratory Reproducibility of the IC50 Values from the 3T3 NRU Test Method

Reference
Substance/Laboratory
IIVS
Cycloheximide
ECBC
FAL
IIVS
Dibutyl phthalate
ECBC
FAL
IIVS
Dichlorvos
ECBC
FAL
IIVS
Diethyl phthalate
ECBC
FAL
IIVS
Digoxin
ECBC
FAL
IIVS
Dimethylformamide
ECBC
FAL
IIVS

Arithmetic
Mean IC50(mg/mL)1
5.7
0.293
0.125
0.647
0.109
78.3
23.5
191
20.7
20.3
9.8
32.8
18.3
113
85.5
147
106
520
351
892
317
5242
5343
5483
4900

Maximum:
Minimum
Mean IC50
Ratio2

Arithmetic
IntraLab%CV

Arithmetic
InterLab
%CV

31
5.9

104
45
70
23

9.2

124
17
50
7

3.3

57
35
6
11

1.7

28
34
26
24

2.8

62
39
36
21

1.1

6
10
9
4

7-11

Log
Arithmetic
Mean IC50(mg/mL)1
0.76
-0.53
-0.9
-0.19
-0.96
1.89
1.37
2.28
1.32
1.31
0.99
1.52
1.26
2.05
1.93
2.17
2.03
2.72
2.54
2.95
2.5
3.72
3.73
3.74
3.69

ANOVA
P3

Contrast
P4<0.001

0.021
NA
NA
NA
<0.001
0.012<0.001
0.005
0.002
0.001
0.002
0.823
0.127
0.092
0.07
0.846
0.043
NA
NA
NA
0.296
NA
NA
NA

In Vitro Cytotoxicity Test Methods BRD Section 7

Table 7-3

November 2006

Interlaboratory Reproducibility of the IC50 Values from the 3T3 NRU Test Method

Reference
Substance/Laboratory

Arithmetic
Mean IC50(mg/mL)1

Diquat dibromide monohydrate
ECBC
FAL
IIVS
Disulfoton
ECBC
FAL
IIVS
Endosulfan
ECBC
FAL
IIVS
Epinephrine bitartrate
ECBC
FAL
IIVS
Ethanol
ECBC
FAL
IIVS
Ethylene glycol
ECBC
FAL
IIVS
Fenpropathrin

15.1
3.9
36.1
5.4
98.6
137
NA
60.4
8.02
5.3
15.2
3.6
59.4
51.5
63.4
63.4
6731
5360
8420
6413
25292
18325
31650
25900
27.2

Maximum:
Minimum
Mean IC50
Ratio2

Arithmetic
IntraLab%CV

9.3

Arithmetic
InterLab
%CV
120

23
98
25
2.3

55
55
NA
87

4.2

78
57
78
42

1.2

12
12
11
3

1.6

23
33
14
5

1.7

26
9
24
12

2.5

49

7-12

Log
Arithmetic
Mean IC50(mg/mL)1
1.18
0.59
1.56
0.73
1.99
2.14
NA
1.78
0.9
0.72
1.18
0.56
1.77
1.71
1.8
1.8
3.83
3.73
3.93
3.81
4.4
4.26
4.50
4.41
1.43

ANOVA
P3

Contrast
P4

0.017
NA
NA
NA
0.003
NA
NA
NA
0.046
NA
NA
NA
0.048
NA
NA
NA
0.075
NA
NA
NA
0.007
0.004
0.01
0.505
0.301

In Vitro Cytotoxicity Test Methods BRD Section 7

Table 7-3

November 2006

Interlaboratory Reproducibility of the IC50 Values from the 3T3 NRU Test Method

Reference
Substance/Laboratory
ECBC
FAL
IIVS
Gibberellic Acid
ECBC
FAL
IIVS
Glutethimide
ECBC
FAL
IIVS
Glycerol
ECBC
FAL
IIVS
Haloperidol
ECBC
FAL
IIVS
Hexachlorophene
ECBC
FAL
IIVS
Lactic acid
ECBC

Arithmetic
Mean IC50(mg/mL)1
22.6
42.4
16.7
7842
8027
NA
7657
192
167
284.3
125.3
28904
20000
38878
27833
6.26
5.3
8
5.5
4.48
5
5.3
3.1
3073
2943

Maximum:
Minimum
Mean IC50
Ratio2

Arithmetic
IntraLab%CV

Arithmetic
InterLab
%CV

11
63
12
1.0

3
11
NA
10

2.3

43
4
7
7

1.9

33
15
73
39

1.5

24
12
8
12

1.7

27
48
33
9

1.2

12
11

7-13

Log
Arithmetic
Mean IC50(mg/mL)1
1.35
1.63
1.22
3.89
3.9
NA
3.88
2.28
2.22
2.45
2.1
4.46
4.3
4.59
4.44
0.8
0.72
0.9
0.74
0.65
0.7
0.72
0.49
3.49
3.47

ANOVA
P3

Contrast
P4
NA
NA
NA

0.621
NA
NA
NA
<0.001
0.029<0.001<0.001
0.846
NA
NA
NA
0.006
0.03
0.002
0.061
0.174
NA
NA
NA
0.16
NA

In Vitro Cytotoxicity Test Methods BRD Section 7

Table 7-3

November 2006

Interlaboratory Reproducibility of the IC50 Values from the 3T3 NRU Test Method

Reference
Substance/Laboratory
FAL
IIVS
Lindane
ECBC
FAL
IIVS
Lithium carbonate
ECBC
FAL
IIVS
Meprobamate
ECBC
FAL
IIVS
Mercury chloride
ECBC
FAL
IIVS
Methanol
ECBC
FAL
IIVS
Nicotine
ECBC
FAL

Arithmetic
Mean IC50(mg/mL)1
3487
2790
161
125
266
90.4
NA
564
NA
NA
539
353
877
386
4.32
3.5
6
3.5
NA
NA
NA
NA
378
272
412

Maximum:
Minimum
Mean IC50
Ratio2

Arithmetic
IntraLab%CV

Arithmetic
InterLab
%CV

16
9
2.9

58
95
36
122

NA

NA
12
NA
NA

2.5

54
14
15
2

1.7

33
5
31
3

NA

NA
NA

1.7

25
24
33

7-14

Log
Arithmetic
Mean IC50(mg/mL)1
3.54
3.45
2.21
2.1
2.43
1.96
NA
2.75
NA
NA
2.73
2.55
2.94
2.59
0.64
0.54
0.78
0.54
NA
NA
NA
NA
2.58
2.43
2.61

ANOVA
P3

Contrast
P4
NA
NA

0.066

NA

NA
NA
NA
NA
NA
NA
NA

<0.001
NA
NA
NA
0.021

NA

NA
NA
NA
NA
NA
NA
NA

0.128
NA
NA

In Vitro Cytotoxicity Test Methods BRD Section 7

Table 7-3

November 2006

Interlaboratory Reproducibility of the IC50 Values from the 3T3 NRU Test Method

Reference
Substance/Laboratory
IIVS
Paraquat
ECBC
FAL
IIVS
Parathion
ECBC
FAL
IIVS
Phenobarbital
ECBC
FAL
IIVS
Phenol
ECBC
FAL
IIVS
Phenylthiourea
ECBC
FAL
IIVS
Physostigmine
ECBC
FAL
IIVS

Arithmetic
Mean IC50(mg/mL)1
450
23.3
21.3
24.9
23.7
61.8
22.7
141
22
612
634
726
476
70.9
50.2
104
58.1
119
30.1
239
89
28.8
28.2
37.8
20.4

Maximum:
Minimum
Mean IC50
Ratio2

Arithmetic
IntraLab%CV

Arithmetic
InterLab
%CV

12
1.2

8
34
67
64

6.4

111
53
70
22

1.5

21
21
35
23

2.1

Log
Arithmetic
Mean IC50(mg/mL)1
2.65
1.37
1.33
1.4
1.37
1.79
1.36
2.15
1.34
2.79
2.8
2.86
2.68

41
22
24
12

7.9

90
66
28
25

1.9

30
53
5
33

7-15

ANOVA
P3

Contrast
P4
NA

1
NA
NA
NA
0.014
NA
NA
NA
0.232
NA
NA
NA
0.011

1.7
2.02
1.76
2.08
1.48
2.38
1.95
1.46
1.45
1.58
1.31

NA
NA
NA
0.007
0.004
0.006
0.718
0.149
NA
NA
NA

In Vitro Cytotoxicity Test Methods BRD Section 7

Table 7-3

November 2006

Interlaboratory Reproducibility of the IC50 Values from the 3T3 NRU Test Method

Reference
Substance/Laboratory
Potassium chloride
ECBC
FAL
IIVS
Potassium cyanide
ECBC
FAL
IIVS
Procainamide HCl
ECBC
FAL
IIVS
2-Propanol
ECBC
FAL
IIVS
Propranolol HCl
ECBC
FAL
IIVS
Propylparaben
ECBC
FAL
IIVS
Sodium arsenite

Arithmetic
Mean IC50(mg/mL)1
3635
3352
3842
3710
64.3
15.3
159
18.9
443
400
431
497
3563
2610
3970
4110
14.9
13.6
13.5
17.6
29.9
20.9
51.8
17.1
0.873

Maximum:
Minimum
Mean IC50
Ratio2

Arithmetic
IntraLab%CV

1.1

Arithmetic
InterLab
%CV
7

14
31
11
10.4

127
25
52
5

1.2

11
4
1
8

1.6

23
9
4
4

1.3

16
32
51
21

3.0

64
16
29
12

2.8

55

7-16

Log
Arithmetic
Mean IC50(mg/mL)1
3.56
3.53
3.58
3.57
1.81
1.18
2.2
1.28
2.65
2.6
2.63
2.7
3.55
3.42
3.6
3.61
1.17
1.13
1.13
1.25
1.48
1.32
1.71
1.23
-0.06

ANOVA
P3

Contrast
P4

0.846
NA
NA
NA
<0.0010.001<0.001
0.006
0.007
0.008
0.396
0.003
0.001
<0.001
0.004
0.002
0.488
NA
NA
NA
0.001
0.045
<0.0010.003
0.028

In Vitro Cytotoxicity Test Methods BRD Section 7

Table 7-3

November 2006

Interlaboratory Reproducibility of the IC50 Values from the 3T3 NRU Test Method

Reference
Substance/Laboratory
ECBC
FAL
IIVS
Sodium chloride
ECBC
FAL
IIVS
Sodium dichromate dihydrate
ECBC
FAL
IIVS
Sodium fluoride
ECBC
FAL
IIVS
Sodium hypochlorite
ECBC
FAL
IIVS
Sodium oxalate
ECBC
FAL
IIVS
Sodium selenate
ECBC

Arithmetic
Mean IC50(mg/mL)1
0.5
1.4
0.7
4764
4790
4625
4877
0.602
0.603
0.657
0.547
79.8
61.3
96.1
82
1211
823
805
2005
40.8
42
31
49.5
34.5
12.7

Maximum:
Minimum
Mean IC50
Ratio2

Arithmetic
IntraLab%CV

Arithmetic
InterLab
%CV

6
57
17
1.1

3
5
13
9

1.2

9
14
37
17

1.6

22
9
18
7

2.5

57
13
46
44

1.6

23
41
28
53

4.3

60
13

7-17

Log
Arithmetic
Mean IC50(mg/mL)1
-0.3
0.15
-0.15
3.68
3.68
3.67
3.69
-0.22
-0.22
-0.18
-0.26
1.9
1.79
1.98
1.91
3.08
2.92
2.91
3.3
1.61
1.62
1.49
1.69
1.54
1.1

ANOVA
P3

Contrast
P4
NA
NA
NA

0.759
NA
NA
NA
0.822
NA
NA
NA
0.016
NA
NA
NA
0.04
NA
NA
NA
0.643
NA
NA
NA
<0.001<0.001In Vitro Cytotoxicity Test Methods BRD Section 7

Table 7-3

November 2006

Interlaboratory Reproducibility of the IC50 Values from the 3T3 NRU Test Method

Reference
Substance/Laboratory
FAL
IIVS
Strychnine
ECBC
FAL
IIVS
Thallium Sulfate
ECBC
FAL
IIVS
Trichloroacetic acid
ECBC
FAL
IIVS
1,1,1-Trichloroethane
ECBC
FAL
IIVS
Triethylenemelamine
ECBC
FAL
IIVS
Triphenyltin hydroxide
ECBC
FAL

Arithmetic
Mean IC50(mg/mL)1
54.2
36.5
199
389
124
83.5
7.5
2.8
13.4
6.3
928
762
1220
801
15538
NA
21250
9827
0.568
0.086
1.45
0.169
0.022
0.026
0.026

Maximum:
Minimum
Mean IC50
Ratio2

Arithmetic
IntraLab%CV

Arithmetic
InterLab
%CV

19
14
4.7

83
21
16
6

4.9

72
24
78
28

1.6

27
13
6
14

2.2

52
NA
11
2

16.9

135
11
18
29

1.7

29
17
81

7-18

Log
Arithmetic
Mean IC50(mg/mL)1
1.73
1.56
2.3
2.59
2.09
1.92
0.88
0.45
1.13
0.8
2.97
2.88
3.09
2.9
4.19
NA
4.33
3.99
-0.25
-1.07
0.16
-0.77
-1.66
-1.59
-1.59

ANOVA
P3

Contrast
P4<0.001
0.026<0.001<0.0010.018<0.001
0.165
NA
NA
NA
0.005
0.022
0.002
0.069
<0.001NA
NA
NA<0.001<0.001<0.001
0.002
0.688
NA
NA

In Vitro Cytotoxicity Test Methods BRD Section 7

Table 7-3

November 2006

Interlaboratory Reproducibility of the IC50 Values from the 3T3 NRU Test Method

Reference
Substance/Laboratory
IIVS
Valproic acid
ECBC
FAL
IIVS
Verapamil HCl
ECBC
FAL
IIVS
Xylene
ECBC
FAL
IIVS

Arithmetic
Mean IC50(mg/mL)1
0.015
1177
547
1807
NA
35.2
32
34.6
38.9
NA
NA
NA
724

Maximum:
Minimum
Mean IC50
Ratio2

Arithmetic
IntraLab%CV

Arithmetic
InterLab
%CV

55
3.3

76
12
10
NA

1.2

10
18
5
11

NA

NA
NA
NA
12

Abbreviations: 3T3=BALB/c 3T3 fibroblasts; NRU=Neutral red uptake; ECBC=Edgewood Chemical Biological Center;
FAL= Fund for the Replacement of Animals in Medical Experiments Alternatives Laboratory; IIVS=Institute for In Vitro
Sciences; NA=No acceptable IC50 results reported or calculation was not performed (e.g., for contrast results);
CV=Coefficient of variation.
1
Results reported on the same row with chemical names are the means of all the laboratories. Results reported on the samerow as laboratories are the laboratory means.
2
Maximum laboratory mean IC50 divided by minimum laboratory mean IC50.
3p <0.01 indicated statistical significance.
4
Contrasts were performed if ANOVA was significant (p <0.01) to determine which laboratory was different from the othertwo laboratories. Significant contrasts were denoted by p <0.01. No contrast tests were performed if only two laboratories
reported IC50 values.

7-19

Log
Arithmetic
Mean IC50(mg/mL)1
-1.83
3.07
2.74
3.26
NA
1.55
1.51
1.54
1.59
NA
NA
NA
2.86

ANOVA
P3

Contrast
P4NA<0.001
NA
NA
NA
0.23

NA

NA
NA
NA
NA
NA
NA
NA

In Vitro Cytotoxicity Test Methods BRD Section 7

Table 7-4

November 2006

Reference Substances with Significant ANOVA Differences Among
Laboratories for the 3T3 NRU Test Method
Significant Contrast Results1
FAL

IIVS

Insoluble/
Volatile2

H
L
H
H
H
H
H
H
H
H

L
H

Precipitate

L

L
L

H
H
H
H

Potassium cyanide

L

H

Procainamide HCl
2-Propanol
Propylparaben
Sodium selenate
Strychnine
Trichloroacetic acid
Triethylenemelamine

L
L

Reference Substance
Acetylsalicylic acidArsenic trioxide
Busulfan
Chloral hydrate
Chloramphenicol
Citric acidColchicine
Cupric sulfate pentahydrate
Dibutyl phthalate
Dichlorvos
Ethylene glycol
Glutethimide
Haloperidol
Meprobamate
Phenylthiourea

ECBC

L
L
L
L
M
L
L

M
H
H

L
H

L
L

Precipitate
Precipitate

M
M

Precipitate/Volatile

H
H
L

Volatile

L

Precipitate

H
H

L

Abbreviations: ANOVA=Analysis of variance; 3T3=BALB/c 3T3 fibroblasts; NRU=Neutral red uptake;
H=Laboratory reported the highest mean IC50; L=Laboratory reported the lowest mean IC50; M=Laboratory reported
a mean IC50 between the values of the other two laboratories; ECBC=Edgewood Chemical Biological Center; FAL=
Fund for the Replacement of Animals in Medical Experiments Alternatives Laboratory; IIVS=Institute for In Vitro
Sciences.
1
Laboratories significantly different from the other two at p <0.01.
2
From Table 5-11. Precipitate reported by at least one laboratory is indicated by “Precipitate”. Use of plate sealers
by at least one laboratory to prevent volatile contamination of control wells indicated by “Volatility”.

7-20

In Vitro Cytotoxicity Test Methods BRD Section 7

November 2006

7.2.2.2 Differences Among the IC50 Values in Laboratories Using the NHK NRU Test Method
The ANOVA results in Table 7-5 indicate that there were statistically significant (p <0.01)
laboratory differences for six of the 68 (9%) reference substances evaluated. These substances
are listed in Table 7-6 along with columns showing which laboratory’s IC50 values were
statistically significantly different from the other two (as indicated by the contrast results), andindications of insolubility or volatility during conduct of the assay. Insolubility was reported forthree of the six substances, but none of the six substances were volatile.
For the six substances that yielded significantly different IC50 values among the laboratories,
ECBC reported the highest IC50 value for four substances and the lowest for one, FAL reported
the lowest values for three substances and the highest for two, and IIVS reported the highest IC50
value for one substance and the lowest for two.
7.2.3
CV Results for the 3T3 and NHK NRU Test Methods
CV values were calculated as described in Section 5.5.2.2. Tables 7-3 and 7-5 provide the intraand inter-laboratory CV values for the individual reference substances. Table 7-7 summarizes
the CV values for each method and shows that median and mean values were often similar.
Median CV values were frequently lower than the corresponding means, which indicated thatlarge individual CV values skewed the CV distributions.
7.2.3.1 Reproducibility of Intralaboratory CV Values
Table 7-7 shows that the intralaboratory CV values and mean intralaboratory CV values were
the same, 26%, for both NRU test methods. The median intralaboratory CV values were alsosimilar: 23% and 24% for the 3T3 and the NHK NRU test method, respectively. Of the threelaboratories, FAL had the highest mean and median CV values and IIVS had the lowest for bothmethods.

7-21

In Vitro Cytotoxicity Test Methods BRD Section 7

Table 7-5

November 2006

Reproducibility of the IC50 Values from the NHK NRU Test Method

Reference
Substance/Laboratory
Acetaminophen
ECBC
FAL
IIVS
Acetonitrile
ECBC
FAL
IIVS
Acetylsalicylic acid
ECBC
FAL
IIVS
5-Aminosalicylic acid
ECBC
FAL
IIVS
Aminopterin
ECBC
FAL
IIVS
Amitriptyline HCl
ECBC
FAL
IIVS
Arsenic trioxide

Arithmetic
Mean IC50(mg/mL)1
526
558
447
571
10104
10868
10153
9290
613
631
694
514
52.3
29.9
78.2
48.8
682
889
545
611
9.76
10.8
7.57
10.9
10.4

Maximum:
Minimum
Mean IC50
Ratio2

Arithmetic
IntraLab%CV

1.3

Arithmetic
InterLab
%CV
13

15
19
14
1.2

8
72
19
4

1.4

15
3
14
15

2.6

47
22
54
16

1.6

27
20
8
12

1.4

19
31
72
10

8.2

91

7-22

Log
Arithmetic
Mean IC50(mg/mL)1
2.72
2.75
2.65
2.76
4
4.04
4.01
3.97
2.79
2.8
2.84
2.71
1.72
1.48
1.89
1.69
2.83
2.95
2.74
2.79
0.99
1.03
0.88
1.04
1.02

ANOVA
P3

Contrast
P4

0.181
NA
NA
NA
0.964
NA
NA
NA
0.060
NA
NA
NA
0.044
NA
NA
NA
0.025
NA
NA
NA
0.365
NA
NA
NA
<0.001In Vitro Cytotoxicity Test Methods BRD Section 7

Table 7-5

November 2006

Reproducibility of the IC50 Values from the NHK NRU Test Method

Reference
Substance/Laboratory
ECBC
FAL
IIVS
Atropine sulfate
ECBC
FAL
IIVS
Boric acid
ECBC
FAL
IIVS
Busulfan
ECBC
FAL
IIVS
Cadmium chloride
ECBC
FAL
IIVS
Caffeine
ECBC
FAL
IIVS
Carbamazepine
ECBC

Arithmetic
Mean IC50(mg/mL)1
7.77
2.55
20.9
91.9
85.4
104
83.2
473
440
517
464
278
253
268
313
1.98
2.2
1.88
1.86
661
817
591
574
128
66.1

Maximum:
Minimum
Mean IC50
Ratio2

Arithmetic
IntraLab%CV

Arithmetic
InterLab
%CV

33
75
31
1.3

13
12
85
25

1.2

8
31
73
2

1.2

11
27
72
12

1.2

10
37
65
8

1.4

21
31
32
1

4.0

85
13

7-23

Log
Arithmetic
Mean IC50(mg/mL)1
0.89
0.41
1.32
1.96
1.93
2.02
1.92
2.67
2.64
2.71
2.67
2.44
2.4
2.43
2.5
0.3
0.34
0.27
0.27
2.82
2.91
2.77
2.76
2.11
1.82

ANOVA
P3

Contrast
P4
0.694<0.0010.00060.988
0.89030.90690.98320.931
0.96920.7391
0.768
0.659
NA
NA
NA
0.733
NA
NA
NA
0.296
NA
NA
NA
0.432
NA

In Vitro Cytotoxicity Test Methods BRD Section 7

Table 7-5

November 2006

Reproducibility of the IC50 Values from the NHK NRU Test Method

Reference
Substance/Laboratory
FAL
IIVS
Carbon tetrachloride
ECBC
FAL
IIVS
Chloral hydrate
ECBC
FAL
IIVS
Chloramphenicol
ECBC
FAL
IIVS
Citric acid
ECBC
FAL
IIVS
Colchicine
ECBC
FAL
IIVS
Cupric sulfate pentahydrate
ECBC
FAL

Arithmetic
Mean IC50(mg/mL)1
253
63.9
NA
NA
NA
NA
137
140
159
112
366
318
414
367
424
526
312
433
0.007
0.005
0.008
0.008
197
190
195

Maximum:
Minimum
Mean IC50
Ratio2

Arithmetic
IntraLab%CV

Arithmetic
InterLab
%CV

129
8
NA

NA
NA
NA
NA

1.4

17
24
32
2

1.3

13
45
44
22

1.7

25
16
17
5

1.6

22
46
10
21

1.1

4
10
6

7-24

Log
Arithmetic
Mean IC50(mg/mL)1
2.4
1.81
NA
NA
NA
NA
2.14
2.15
2.2
2.05
2.56
2.5
2.62
2.56
2.63
2.72
2.49
2.64
-2.16
-2.28
-2.12
-2.09
2.29
2.28
2.29

ANOVA
P3

Contrast
P4
NA
NA

NA
NA
NA
NA
0.302
NA
NA
NA
0.750
NA
NA
NA
0.006
0.009
0.002
0.483
0.174
NA
NA
NA
0.374
NA
NA

In Vitro Cytotoxicity Test Methods BRD Section 7

Table 7-5

November 2006

Reproducibility of the IC50 Values from the NHK NRU Test Method

Reference
Substance/Laboratory
IIVS
Cycloheximide
ECBC
FAL
IIVS
Dibutyl phthalate
ECBC
FAL
IIVS
Dichlorvos
ECBC
FAL
IIVS
Diethyl phthalate
ECBC
FAL
IIVS
Digoxin
ECBC
FAL
IIVS
Dimethylformamide
ECBC
FAL
IIVS

Arithmetic
Mean IC50(mg/mL)1
207
0.082
0.053
0.12
0.071
32.6
28.3
47.4
22
11.1
8.56
12.4
12.2
145
174
71.5
189
0.003140.005380.00005
0.00398
7856
9353
7817
6397

Maximum:
Minimum
Mean IC50
Ratio2

Arithmetic
IntraLab%CV

Arithmetic
InterLab
%CV

3
2.3

43
22
78
19

2.2

41
27
73
6

1.4

20
27
30
3

2.6

44
8
94
18

107.6

88
13
36
7

1.5

19
2
1
3

7-25

Log
Arithmetic
Mean IC50(mg/mL)1
2.32
-1.09
-1.28
-0.92
-1.15
1.51
1.45
1.68
1.34
1.05
0.93
1.09
1.09
2.16
2.24
1.85
2.28
-2.5
-2.27
-4.29
-2.4
3.9
3.97
3.89
3.81

ANOVA
P3

Contrast
P4
NA

0.302
NA
NA
NA
0.408
NA
NA
NA
0.181
NA
NA
NA
0.049
NA
NA
NA
<0.001<0.001<0.001<0.001<0.001<0.0010.508<0.001In Vitro Cytotoxicity Test Methods BRD Section 7

Table 7-5

November 2006

Reproducibility of the IC50 Values from the NHK NRU Test Method

Reference
Substance/Laboratory

Arithmetic
Mean IC50(mg/mL)1

Diquat dibromide monohydrate
ECBC
FAL
IIVS
Disulfoton
ECBC
FAL
IIVS
Endosulfan
ECBC
FAL
IIVS
Epinephrine bitartrate
ECBC
FAL
IIVS
Ethanol
ECBC
FAL
IIVS
Ethylene glycol
ECBC
FAL
IIVS
Fenpropathrin

4.73
3.59
6.77
3.84
378
140
808
186
2.35
3.44
1.42
2.19
90.6
115
81.7
75
10184
8290
12013
10250
42600
38000
49800
40000
2.6

Maximum:
Minimum
Mean IC50
Ratio2

Arithmetic
IntraLab%CV

1.9

Arithmetic
InterLab
%CV
37

23
55
8
5.8

99
19
26
32

2.4

43
17
50
20

1.5

24
9
35
16

1.4

18
5
19
9

1.3

15
12
9
13

2.0

39

7-26

Log
Arithmetic
Mean IC50(mg/mL)1
0.67
0.56
0.83
0.58
2.58
2.15
2.91
2.27
0.37
0.54
0.15
0.34
1.96
2.06
1.91
1.88
4.01
3.92
4.08
4.01
4.63
4.58
4.7
4.6
0.41

ANOVA
P3

Contrast
P4

0.217
NA
NA
NA
<0.001
0.002<0.001
0.018
0.029
NA
NA
NA
0.119
NA
NA
NA
0.035
NA
NA
NA
0.063
NA
NA
NA
0.031

In Vitro Cytotoxicity Test Methods BRD Section 7

Table 7-5

November 2006

Reproducibility of the IC50 Values from the NHK NRU Test Method

Reference
Substance/Laboratory
ECBC
FAL
IIVS
Gibberellic Acid
ECBC
FAL
IIVS
Glutethimide
ECBC
FAL
IIVS
Glycerol
ECBC
FAL
IIVS
Haloperidol
ECBC
FAL
IIVS
Hexachlorophene
ECBC
FAL
IIVS
Lactic acid
ECBC

Arithmetic
Mean IC50(mg/mL)1
3.73
2.23
1.82
2866
2850
2940
2807
177
187
170
176
27108
34267
18023
29033
3.57
3.69
3.72
3.29
0.031
0.027
0.046
0.021
1308
1290

Maximum:
Minimum
Mean IC50
Ratio2

Arithmetic
IntraLab%CV

Arithmetic
InterLab
%CV

27
28
17
1.0

2
14
9
4

1.1

5
34
14
16

1.9

31
45
46
16

1.1

7
27
49
35

2.2

41
16
44
11

1.0

1
4

7-27

Log
Arithmetic
Mean IC50(mg/mL)1
0.57
0.35
0.26
3.46
3.45
3.47
3.45
2.25
2.27
2.23
2.24
4.43
4.53
4.26
4.46
0.55
0.57
0.57
0.52
-1.5
-1.57
-1.34
-1.67
3.12
3.11

ANOVA
P3

Contrast
P4
NA
NA
NA

0.862
NA
NA
NA
0.968
NA
NA
NA
0.200
NA
NA
NA
0.935
NA
NA
NA
0.097
NA
NA
NA
0.904
NA

In Vitro Cytotoxicity Test Methods BRD Section 7

Table 7-5

November 2006

Reproducibility of the IC50 Values from the NHK NRU Test Method

Reference
Substance/Laboratory
FAL
IIVS
Lindane
ECBC
FAL
IIVS
Lithium carbonate
ECBC
FAL
IIVS
Meprobamate
ECBC
FAL
IIVS
Mercury chloride
ECBC
FAL
IIVS
Methanol
ECBC
FAL
IIVS
Nicotine
ECBC
FAL

Arithmetic
Mean IC50(mg/mL)1
1320
1313
19.3
19.1
23.2
15.6
477
411
486
535
516
761
163
624
5.87
6.87
5.4
5.35
1616
NA
1133
2100
113
94.3
134

Maximum:
Minimum
Mean IC50
Ratio2

Arithmetic
IntraLab%CV

Arithmetic
InterLab
%CV

5
11
1.5

20
17
31
15

1.3

13
29
20
6

4.7

61
15
116
14

1.3

15
15
19
2

1.9

42
NA
19
11

1.4

17
26
59

7-28

Log
Arithmetic
Mean IC50(mg/mL)1
3.12
3.12
1.29
1.28
1.37
1.19
2.68
2.61
2.69
2.73
2.71
2.88
2.21
2.8
0.77
0.84
0.73
0.73
3.21
NA
3.05
3.32
2.05
1.97
2.13

ANOVA
P3

Contrast
P4
NA
NA

0.203
NA
NA
NA
0.295
NA
NA
NA
0.027
NA
NA
NA
0.120
NA
NA
NA
0.007
NA
NA
NA
0.700
NA
NA

In Vitro Cytotoxicity Test Methods BRD Section 7

Table 7-5

November 2006

Reproducibility of the IC50 Values from the NHK NRU Test Method

Reference
Substance/Laboratory
IIVS
Paraquat
ECBC
FAL
IIVS
Parathion
ECBC
FAL
IIVS
Phenobarbital
ECBC
FAL
IIVS
Phenol
ECBC
FAL
IIVS
Phenylthiourea
ECBC
FAL
IIVS
Physostigmine
ECBC
FAL
IIVS

Arithmetic
Mean IC50(mg/mL)1
112
66.1
48.3
96.6
53.4
31.4
34
31.2
29
478
693
360
381
77.7
59.1
93.2
80.8
346
363
401
272
172
164
213
139

Maximum:
Minimum
Mean IC50
Ratio2

Arithmetic
IntraLab%CV

Arithmetic
InterLab
%CV

25
2.0

40
13
39
10

1.2

8
30
38
29

1.9

39
26
27
18

1.6

22
36
6
6

1.5

19
16
21
26

1.5

22
3
112
6

7-29

Log
Arithmetic
Mean IC50(mg/mL)1
2.05
1.82
1.68
1.98
1.73
1.5
1.53
1.49
1.46
2.68
2.84
2.56
2.58
1.89
1.77
1.97
1.91
2.54
2.56
2.6
2.44
2.24
2.21
2.33
2.14

ANOVA
P3

Contrast
P4
NA

0.047
NA
NA
NA
0.845
NA
NA
NA
0.027
NA
NA
NA
0.094
NA
NA
NA
0.133
NA
NA
NA
0.623
NA
NA
NA

In Vitro Cytotoxicity Test Methods BRD Section 7

Table 7-5

November 2006

Reproducibility of the IC50 Values from the NHK NRU Test Method

Reference
Substance/Laboratory
Potassium chloride
ECBC
FAL
IIVS
Potassium cyanide
ECBC
FAL
IIVS
Procainamide HCl
ECBC
FAL
IIVS
2-Propanol
ECBC
FAL
IIVS
Propranolol HCl
ECBC
FAL
IIVS
Propylparaben
ECBC
FAL
IIVS
Sodium arsenite

Arithmetic
Mean IC50(mg/mL)1
2279
2560
2287
1990
45.1
29.3
89
16.9
1764
1480
1787
2027
5541
5263
4273
7087
36.9
38.27
43.8
28.6
16.8
18.1
18.6
13.8
0.532

Maximum:
Minimum
Mean IC50
Ratio2

Arithmetic
IntraLab%CV

1.3

Arithmetic
InterLab
%CV
13

17
28
8
5.3

86
24
112
13

1.4

16
14
12
11

1.7

26
11
27
7

1.5

21
12
6
11

1.3

16
13
15
9

2.4

44

7-30

Log
Arithmetic
Mean IC50(mg/mL)1
3.36
3.41
3.36
3.3
1.65
1.47
1.95
1.23
3.25
3.17
3.25
3.31
3.74
3.72
3.63
3.85
1.57
1.58
1.64
1.46
1.23
1.26
1.27
1.14
-0.27

ANOVA
P3

Contrast
P4

0.396
NA
NA
NA
0.340
NA
NA
NA
0.053
NA
NA
NA
0.033
NA
NA
NA
0.003
0.325
0.006
0.001
0.066
NA
NA
NA
0.061

In Vitro Cytotoxicity Test Methods BRD Section 7

Table 7-5

November 2006

Reproducibility of the IC50 Values from the NHK NRU Test Method

Reference
Substance/Laboratory
ECBC
FAL
IIVS
Sodium chloride
ECBC
FAL
IIVS
Sodium dichromate dihydrate
ECBC
FAL
IIVS
Sodium fluoride
ECBC
FAL
IIVS
Sodium hypochlorite
ECBC
FAL
IIVS
Sodium oxalate
ECBC
FAL
IIVS
Sodium selenate
ECBC

Arithmetic
Mean IC50(mg/mL)1
0.79
0.336
0.47
2724
3583
1118
3470
0.737
0.784
0.851
0.576
47.4
48.7
39.7
53.7
1580
1863
1243
1633
355
355
350
360
11.2
7.47

Maximum:
Minimum
Mean IC50
Ratio2

Arithmetic
IntraLab%CV

Arithmetic
InterLab
%CV

32
56
14
3.2

51
7
124
9

1.5

19
14
36
17

1.4

15
14
24
13

1.5

20
31
46
11

1.0

1
15
42
26

2.2

40
12

7-31

Log
Arithmetic
Mean IC50(mg/mL)1
-0.1
-0.47
-0.33
3.44
3.55
3.05
3.54
-0.13
-0.11
-0.07
-0.24
1.68
1.69
1.6
1.73
3.2
3.27
3.09
3.21
2.55
2.55
2.54
2.56
1.05
0.87

ANOVA
P3

Contrast
P4
NA
NA
NA

0.045
NA
NA
NA
0.258
NA
NA
NA
0.313
NA
NA
NA
0.313
NA
NA
NA
0.926
NA
NA
NA
0.134
NA

In Vitro Cytotoxicity Test Methods BRD Section 7

Table 7-5

November 2006

Reproducibility of the IC50 Values from the NHK NRU Test Method

Reference
Substance/Laboratory
FAL
IIVS
Strychnine
ECBC
FAL
IIVS
Thallium Sulfate
ECBC
FAL
IIVS
Trichloroacetic acid
ECBC
FAL
IIVS
1,1,1-Trichloroethane
ECBC
FAL
IIVS
Triethylenemelamine
ECBC
FAL
IIVS
Triphenyltin hydroxide
ECBC
FAL

Arithmetic
Mean IC50(mg/mL)1
16.1
10
69.3
100
52.5
55.1
0.16
0.198
0.153
0.127
427
348
541
394
NA
8137
NA
NA
1.95
1.69
2.03
2.13
0.013
0.021
0.007

Maximum:
Minimum
Mean IC50
Ratio2

Arithmetic
IntraLab%CV

Arithmetic
InterLab
%CV

59
13
1.9

39
76
53
6

1.6

23
51
20
16

1.6

24
18
28
13

NA

NA
7
NA
NA

1.3

12
57
23
23

3.0

55
32
106

7-32

Log
Arithmetic
Mean IC50(mg/mL)1
1.21
1
1.84
2
1.72
1.74
-0.8
-0.7
-0.82
-0.9
2.63
2.54
2.73
2.6
NA
3.91
NA
NA
0.29
0.23
0.31
0.33
-1.89
-1.68
-2.15

ANOVA
P3

Contrast
P4
NA
NA

0.364
NA
NA
NA
0.405
NA
NA
NA
0.134
NA
NA
NA
NA
NA
NA
NA
0.562
NA
NA
NA
0.088
NA
NA

In Vitro Cytotoxicity Test Methods BRD Section 7

Table 7-5

November 2006

Reproducibility of the IC50 Values from the NHK NRU Test Method

Reference
Substance/Laboratory
IIVS
Valproic acid
ECBC
FAL
IIVS
Verapamil HCl
ECBC
FAL
IIVS
Xylene
ECBC
FAL
IIVS

Arithmetic
Mean IC50(mg/mL)1
0.011
533
468
702
430
68.7
60.5
79.4
66.2
NA
NA
NA
486

Maximum:
Minimum
Mean IC50
Ratio2

Arithmetic
IntraLab%CV

Arithmetic
InterLab
%CV

32
1.6

28
25
23
17

1.3

14
22
42
8

NA

NA
NA
NA
38

Log
Arithmetic
Mean IC50(mg/mL)1
-1.96
2.73
2.67
2.85
2.63
1.84
1.78
1.9
1.82
NA
NA
NA
2.69

Abbreviations: NHK=Normal human epidermal keratinocytes; NRU=Neutral red uptake; ECBC=Edgewood Chemical Biological Center; FAL=
Fund for the Replacement of Animals in Medical Experiments Alternatives Laboratory; IIVS=Institute for In Vitro Sciences; NA=No acceptable
IC50 results reported or calculation was not performed (e.g., for contrast results); CV=Coefficient of variation.
1
Results reported on the same row with chemical names are the means of all the laboratories. Results reported on the same row as laboratories
are the laboratory means.
2
Maximum laboratory mean IC50 divided by minimum laboratory mean IC50.
3p <0.01 indicated statistical significance.
4
Contrasts were performed if ANOVA was significant (p <0.01) to determine which laboratory was different from the other two laboratories.
Significant contrasts were denoted by p <0.01. No contrast tests were performed if only two laboratories reported IC50 values.

7-33

ANOVA
P3

Contrast
P4
NA

0.081
0.331
0.032
0.135
0.624
NA
NA
NA
NA
NA
NA
NA

In Vitro Cytotoxicity Test Methods BRD Section 7

Table 7-6

November 2006

Reference Substances with Significant ANOVA Differences Among
Laboratories for the NHK NRU Test Method
Significant Contrast Results1

Reference Substance

ECBC

Arsenic trioxide
Citric acid
DigoxinDimethylformamide
Disulfoton
Propranolol HCl

FAL

IIVS

L
L
L

H

H
H
H
L

Solubility/
Volatility2
Precipitate
Precipitate

L
H
H

PrecipitateL

Abbreviations: ANOVA=Analysis of variance; NHK=Normal human epidermal keratinocytes; NRU=Neutral
red uptake; H=Laboratory reported the highest mean IC50; L=Laboratory reported the lowest mean IC50;
ECBC=Edgewood Chemical Biological Center; FAL= Fund for the Replacement of Animals in Medical
Experiments Alternatives Laboratory; IIVS=Institute for In Vitro Sciences.
1
Laboratories significantly different from the other two at p <0.01
2
From Table 5-11. Precipitate reported by at least one laboratory.

7.2.3.2 Reproducibility of Interlaboratory CV Values
The mean and median interlaboratory CV for the reference substances were lower in the
NHK NRU test method (mean=28%; median=21% vs. mean=47%; median=37% for 3T3(see Table 7-7).
Table 7-7

Summary of CV Results for the 3T3 and NHK NRU Test Methods

CV
Intralaboratory CV
ECBC
FAL
IIVS
Interlaboratory CV

3T3 NRU Test Method

NHK NRU Test Method

N

Mean

Median

Range

N

Mean

Median

Range

198
64
64
64
64

26%
23%
33%
21%
47%

23%
17%
31%
14%
37%

1-122%
2-95%
1-98%
1-122%
3-135%

204
68
68
68
68

26%
23%
43%
13%
28%

24%
20%
34%
13%
21%

1-129%
2-76%
1-129%
1-35%
1-91%

Abbreviations: CV=Coefficient of variation; 3T3=BALB/c 3T3 fibroblasts; NHK=Normal human epidermal keratinocytes;
NRU=Neutral red uptake; N=number of values; ECBC=Edgewood Chemical Biological Center; FAL= Fund for the
Replacement of Animals in Medical Experiments Alternatives Laboratory; IIVS=Institute for In Vitro Sciences.
Note: For the 3T3 method, the following substances were excluded because all laboratories did not obtain sufficient IC50
data: carbon tetrachloride; disulfoton; gibberellic acid; lithium carbonate; methanol; 1,1,1-trichloroethane; valproic acid; andxylene. For the NHK method, the following substances were excluded because all laboratories did not obtain sufficient IC50
data: carbon tetrachloride; methanol; 1,1,1-trichloroethane; and xylene.

7.2.3.3 Variation of CV with Chemical Property
To identify chemical characteristics that may be associated with high or low CV values, theirassociations were assessed for chemical class along with the following chemical attributes:
physical state (i.e., solid or liquid), solubility, volatility, molecular weight, log Kow, IC50, andboiling point. The CVs were also examined with respect to their association with the GHS
acute oral toxicity class (UN 2005). For categorical characteristics such as physical form,
solubility (i.e., precipitate/no precipitate), volatile/not volatile, and chemical class, the mean
7-34

In Vitro Cytotoxicity Test Methods BRD Section 7

November 2006

CV values and ranges for the groups were compared to one another and to the overall mean
CV and CV range for each method. No statistical analyses were performed for thesecomparisons. Spearman correlation analyses were performed for chemical characteristics
measured by continuous variables, such as molecular weight, log Kow, and IC50, and boilingpoint.
7.2.3.4 Results of Intralaboratory CV Analysis
The intralaboratory CV analysis (see Table 7-8) uses one mean intralaboratory CV for eachreference substance that was calculated from the intralaboratory CV values from eachlaboratory. There seemed to be little difference in CV values among the categoricalphysical/chemical/toxicological attributes. The mean intralaboratory CV values for solids andliquids were similar (26 vs. 23% for 3T3; 27 vs. 24% for NHK). The mean intralaboratory
CV values for reference substances for which precipitates were observed were similar tovalues for substances with no precipitates were observed (32 vs. 23% for 3T3; 24 vs. 27% forNHK). The mean intralaboratory CV values for substances that exhibited volatility weresimilar to those that did not (31 vs. 25% for 3T3; 27 vs. 26% for NHK). Similarly, thesubstances grouped by GHS acute oral toxicity category (UN 2005) had mean intralaboratory
CV values that were similar (20-33% for 3T3; 19-31% for NHK) to the overall mean CV
values (26% for both test methods). However, the mean intralaboratory CV values for both
NRU test methods tended to increase with decreasing LD50.
Mean intralaboratory CV values were calculated for the chemical classes that contained atleast three of the reference substances included in the reproducibility analyses (i.e., 64
substances for 3T3 and 68 substances for NHK). Reference substances in the amide chemicalclass had unusually low mean intralaboratory CV values for both the 3T3 (13%) and theNHK (10%) NRU test method compared with the overall mean CV (26% for both test
methods), but there were only three substances in this chemical class (acetaminophen,dimethylformamide, procainamide HCl). Organic sulfur compounds had a high mean
intralaboratory CV for the 3T3 test method (46%), but not for the NHK NRU test method(29%) compared with the overall mean intralaboratory CV for both test methods (26%). The
intralaboratory CV values for the remaining chemical classes were unremarkable comparedwith the overall mean intralaboratory CV values.
For the characteristics amenable to correlation analysis, none of the Spearman correlation
coefficients were large (absolute value of rs <0.6), but several were statistically significantly
different from zero (p <0.05). Molecular weight (p=0.016), IC50 (p=0.002), and boiling point(p=0.009) exhibited statistically significant correlations to intralaboratory CV for the 3T3 test
NRU method. The higher molecular weight substances had higher intralaboratory CV values
and the substances with lower IC50 values had higher intralaboratory CV values. The findingthat substances with higher boiling points had higher CV values was consistent with thecategorical analysis of volatility. The substances that exhibited volatile characteristics (i.e.,cross contamination of VC wells) in the 3T3 NRU test method had slightly higher mean
intralaboratory CV values (31%) than the substances that did not exhibit volatile
characteristics (25%).

7-35

In Vitro Cytotoxicity Test Methods BRD Section 7

Table 7-8

November 2006

Intralaboratory CV Values by Chemical Characteristics for the 3T3 andNHK NRU Test Methods

Class/Attribute
All chemicals
Chemical form
Solid
LiquidSolubility
Precipitate2
No precipitate
Volatility3
Volatile
Nonvolatile
Chemical Class
Alcohol
Amide
Amine
Carboxylic acid
Heterocyclic
Organophosphorous
Organic sulfur
PhenolPolycyclic
Inorganic
Inorganic chlorine
Inorganic sodium
GHS Acute Oral
Toxicity Class
LD50 ≤5 mg/kg
5 < LD50 ≤50
50 < LD50 ≤300
300 < LD50 ≤2000
2000 < LD50 ≤5000
LD50 >5000
Correlations
Molecular weight
Log Kow
IC50
Boiling point

N164

3T3 NRU Test Method
Range
Mean
1-122%
26%

N1
68

NHK NRU Test Method
Range
Mean
1-129%
26%

51
13

4-84
6-48

26
23

53
15

6-57
2-40

27
24

18
46

11-84
4-55

32
23

19
49

2-47
7-57

24
27

10
54

6-84
4-55

31
25

9
592

11-50
2-57

27
26

9
3
3
13
14
2
4
5
4
14
5
6

6-42
4-28
9-35
4-41
6-59
NA
36-59
14-30
19-35
9-43
9-33
9-34

22
13
18
18
31
NA
46
20
27
25
19
20

9
3
3
14
14
3
5
5
5
15
5
6

10-37
2-16
10-24
2-48
13-50
20-32
21-27
11-31
9-38
6-50
12-50
17-47

22
10
18
23
32
26
29
19
20
29
32
30

6
12
12
14
9
11
N
64
454
64
245

9-46
13-59
11-84
4-51
9-32
6-42
rs
0.301
0.121
-0.382
0.520

27
32
33
22
20
20
P value
0.016
0.430
0.002
0.009

7
12
12
16
9
12
N
68
484
68
245

20-40
12-50
17-37
6-57
7-50
2-40
rs
0.181
0.310
-0.346
0.226

30
31
25
25
30
19
P value
0.140
0.032
0.004
0.289

Abbreviations: CV=Coefficient of variation; 3T3=BALB/c 3T3 fibroblasts; GHS=Globally Harmonized System ofClassification and Labelling of Chemicals (UN 2005); NA=Not applicable because class had less than three observations;
NHK=Normal human epidermal keratinocytes; NRU=Neutral red uptake; N=Number of values; rs=Spearman correlation
coefficient; Kow=Octanol:water partition coefficient.
1
One intralaboratory CV for each chemical was calculated by averaging the CV values for each reference substance.
2
Identified by laboratory reports of precipitate in the stock reference substance solutions or in cell culture (see Table 5-11).
3
Identified by laboratory reports of using plate sealers to avoid contamination of the VC wells (see Table 5-11).
4
Number of reference substances with CV values and log Kow data.
5
Number of reference substances with CV values and boiling point data.

Among the IC50 values obtained using the NHK NRU test method, two of the characteristics
amenable to correlation analysis were statistically significantly different from zero, although
the correlation coefficients did not have large magnitudes (absolute value of rs <0.4). The log
7-36

In Vitro Cytotoxicity Test Methods BRD Section 7

November 2006

Kow (p=0.032) and IC50 (p=0.004) exhibited statistically significant correlations (p <0.05) tothe intralaboratory CV. Log Kow was positively correlated (i.e., higher log Kow values wereassociated with higher mean intralaboratory CV), but the IC50 was negatively correlated (i.e.,higher log IC50 values were associated with lower mean intralaboratory CV) to mean
intralaboratory CV.
7.2.3.5 Results of the Interlaboratory CV Analysis
Table 7-9 shows the analysis of the interlaboratory CV values. There seemed to be little
difference in interlaboratory CV values for most of the categorical physical/chemical
characteristics. The mean interlaboratory CV values for solids and liquids were similar (48%for solids vs. 42% for liquids for 3T3, and 28% for solids vs. 21% for liquids for NHK), as
were the values for substances for which precipitates were observed versus no precipitates(58% vs. 43% for 3T3, and 24% vs. 28% for NHK), and the values for substances thatexhibited volatile characteristics (51% for volatile substances vs. 46% for nonvolatile
substances for 3T3, and 32% for volatile substances vs. 26% for nonvolatile substances forNHK).
Mean interlaboratory CV values were calculated for the chemical classes that contained atleast three of the reference substances included in the reproducibility analyses (i.e., 64
substances for 3T3 and 68 substances for NHK). Reference substances in the amide chemicalclass had low mean interlaboratory CV values for both the 3T3 (15%) and the NHK (16%)
NRU test methods compared with the overall mean interlaboratory CV (47% and 28%,
respectively). Substances in the amine class also had low mean interlaboratory CV values forthe 3T3 NRU (13%), but not for the NHK NRU (20%). Organic sulfur compounds had
unusually high mean interlaboratory CV values for the 3T3 test method (100%), but not for
the NHK NRU (36%) compared with the overall mean interlaboratory CV (47% and 28%,
respectively). Because of the low number of reference substances in these classes, theseresults were deemed to not be significant.
Mean interlaboratory CV values tended to be large for chemicals in the most toxic GHS
acute oral toxicity categories, especially with the 3T3 NRU test method. The meaninterlaboratory CV for reference substances in the LD50 ≤5 mg/kg (72%) and 5 < LD50 ≤50
mg/kg (78%) classes were larger than the mean overall interlaboratory CV (47%,). For the
NHK NRU test method, the mean interlaboratory CV for chemicals in the 5 < LD50 ≤5 mg/kg(37%) and 5 < LD50 ≤50 mg/kg (41%) classes were larger than the mean overall
interlaboratory CV (28%).
For the characteristics amenable to correlation analysis, none of the correlation coefficientswere large (absolute value of rs <0.6), but IC50 (p=0.015) and boiling point (p=0.007)
exhibited statistically significant correlations (p <0.05) to interlaboratory CV in the 3T3 test
NRU method. There was a negative correlation between interlaboratory CV and IC50, but thecorrelation between boiling point and interlaboratory CV was positive. The positive
correlation of CV with boiling point was largely consistent with the categorical analysis ofvolatility. The substances that exhibited volatile characteristics in the 3T3 NRU test method
had slightly higher mean CV values than substances that did not exhibit volatile
characteristics (51% vs. 46%). Only the IC50 was significantly correlated (p=0.014) to7-37

In Vitro Cytotoxicity Test Methods BRD Section 7

November 2006

interlaboratory CV with a negative correlation (rs=-0.271) when the NHK NRU test method
was used.
Table 7-9

Interlaboratory 3T3 and NHK NRU Test Method CV Values Sorted by
Chemical Characteristics

Class/Attribute
All chemicals
Chemical Form
SolidsLiquids
Solubility
Precipitate2
No precipitate
Volatility
Volatile3
Nonvolatile
Chemical Class
Alcohol
Amide
Amine
Carboxylic acid
Heterocyclic
Organic sulfur
Organophosphorous
PhenolPolycyclic
Inorganic
Inorganic chlorine
Inorganic sodium
GHS Acute Oral
Toxicity Class
LD50 ≤5 mg/kg
5 < LD50 ≤50
50 < LD50 ≤300
300 < LD50 ≤2000
2000 < LD50 ≤5000
LD50 >5000
Correlations
Molecular weight
Log Kow
IC50
Boiling point

3T3 NRU Test Method

NHK NRU Test Method

N

Range

Mean

N

Range

Mean

641

3-135%

47%

681

1-91%

28%

51
13

3-135
6-124

48
42

53
15

1-91
1-44

28
21

18
46

7-127
3-135

58
43

19
49

1-91
1-88

24
28

10
54

21-127
3-135

51
46

9
59

8-86
1-91

32
26

9
3
3
13
14
4
2
5
4
14
5
6

12-119
6-28
10-16
6-124
8-135
78-119
NA
19-64
14-85
3-127
3-127
3-60

38
15
13
38
57
100
NA
41
44
50
45
34

9
3
3
14
14
5
3
5
5
15
5
6

11-31
13-19
14-24
1-61
5-85
8-99
8-99
15-47
2-88
4-91
10-86
15-51

20
16
20
26
32
36
42
28
30
30
35
32

6
12
12
14
9
11
N
64
454
64
225

12-13533-127
8-120
11-85
3-69
6-124
rs
0.245
0.151
-0.304
0.563

72
78
37
35
29
41
P value
0.051
0.324
0.015
0.007

7
12
12
16
9
12
N
68
484
68
255

12-99
8-91
10-41
1-61
1-85
2-44
rs
0.169
0.210
-0.297-0.051

37
41
26
20
27
23
P value
0.168
0.151
0.014
0.809

Abbreviations: CV=Coefficient of variation; 3T3=BALB/c 3T3 fibroblasts; GHS=Globally Harmonized System ofClassification and Labelling of Chemicals (UN 2005); NA=Not applicable because class had less than three observations;
NHK=Normal human epidermal keratinocytes; NRU=Neutral red uptake; N=Number of values; rs=Spearman correlation
coefficient; Kow=Octanol:water partition coefficient.
1
One intralaboratory CV for each chemical was calculated by averaging the CV values for each reference substance.
2
Identified by laboratory reports of precipitate in the stock reference substance solutions or in cell culture (see Table 5-11).
3
Identified by laboratory reports of using plate sealers to avoid contamination of the VC wells (see Table 5-11).
4
Number of reference substances with CV values and log Kow data.
5
Number of reference substances with CV values and boiling point data.

7-38

In Vitro Cytotoxicity Test Methods BRD Section 7

November 2006

7.2.4
Comparison of Maximum to Minimum IC50 Values Using Laboratory MeansInterlaboratory reproducibility was also compared by calculating maximum to minimum
mean IC50 values using the laboratory means from each method, so that the reproducibility of
the IC50 values could be compared with the reproducibility of the reference LD50 values
derived in Section 4.2. The Figure 7-2 frequency histogram for the 3T3 NRU test method
maximum:minimum mean IC50 values shows that approximately half (37) of the 64 reference
substances produced ratios less than 2.5-fold of each other, and only nine chemicals hadratios greater than 5.5-fold, including one substance (cupric sulfate pentahydrate) that had aratio of 22.
Figure 7-2

Frequency of Maximum:Minimum 3T3 NRU IC50 Ratios

25
20
15
10
5
0

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

Maximum:Minimum 3T3 NRU IC50
Abbreviations: 3T3=BALB/c 3T3 fibroblasts; NRU=Neutral red uptake.
Bars show the number of substances with maximum:minimum 3T3 NRU IC50 ratios within ±0.5 units of the barlabel (e.g., the first bar indicates that there were 14 reference substances for which the laboratory mean
maximum:minimum 3T3 NRU IC50 ratios were 0.5 to1.4). The analysis includes 64 reference substances.
Carbon tetrachloride, disulfoton, gibberellic acid, lithium carbonate, methanol, 1,1,1-trichloroethane, valproic
acid, and xylene were excluded because not all laboratories obtained IC50 values.

The Figure 7-3 frequency histogram for the maximum:minimum mean IC50 values for the
NHK NRU test method shows that ratios of 58 of the 68 chemicals were less than 2.5-fold of
one another. The highest ratio of 108 for digoxin is not shown in the figure. Comparison of
Figures 7-2 and 7-3 shows that the interlaboratory reproducibility of the NHK NRU test
method was better than that for the 3T3 NRU test method based on the distribution of the low
maximum:minimum IC50 ratios.

7-39

In Vitro Cytotoxicity Test Methods BRD Section 7

Figure 7-3

November 2006

Frequency of Maximum:Minimum NHK NRU IC50 Ratios

30

20

10

0

1

2

3

4

5

6

7

8

9

Maximum:Minimum NHK NRU IC50
Abbreviations: NHK=Normal human epidermal keratinocytes; NRU=Neutral red uptake.
Bars show the number of substances with maximum:minimum NHK NRU IC50 ratios within ±0.5 units of thebar label (e.g., the first bar indicates that there were 30 reference substances for which the laboratory mean
maximim:minimum NHK NRU IC50 ratios were 0.5 to 1.4). The analysis includes 68 reference substances.
Carbon tetrachloride, methanol, 1,1,1-trichloroethane, and xylene were excluded because not all laboratories
obtained IC50 values. The maximum:minimum IC50 for digoxin of 108 was excluded from this figure.

7.2.5

Comparison of the Maximum:Minimum IC50 Ratios with the Maximum:Minimum
LD50 Ratios
To compare the reproducibility of the NRU IC50 values with that of the LD50 values, themaximum:minimum IC50 ratios for each method (shown in Tables 7-3 and 7-5) werecompared with the maximum:minimum LD50 ratios reported in Table 4-2. This analysis
excluded reference substances for which fewer than three laboratories reported IC50 values,
and reference substances for which fewer than two acceptable acute oral LD50 values wereidentified. As a result, there were 53 substances analysed for the 3T3 NRU test method and
57 for the NHK NRU test method. The following substances were excluded from bothanalyses because fewer than two acceptable LD50 values could be identified: aminopterin;colchicine; digoxin; epinephrine bitartrate; gluthethimide; phenylthiourea; physostigmine;
procainamide HCl, propranolol HCl; propylparaben; and thallium sulfate. Carbon
tetrachloride, disulfoton, gibberellic acid, lithium carbonate, methanol, 1,1,1-trichloroethane,
valproic acid, and xylene, were excluded from the 3T3 analysis, and carbon tetrachloride,methanol, 1,1,1-trichloroethane, and xylene, were excluded from the NHK analysis, because
fewer than three laboratories reported IC50 values.
Figure 7-4 shows that the maximum:minimum LD50 ratios tend to be larger than either the
3T3 NRU IC50 or NHK NRU IC50 ratios because there are more points below the theoretical
one-to-one correspondence line than above the line. The difference between the LD50
maximum:minimum values and the NRU IC50 maximum:minimum values is more striking
for the NHK since there are fewer points above the line for the NHK graph (Figure 7-4b)
than for the 3T3 graph (Figure 7-4a).
7-40

In Vitro Cytotoxicity Test Methods BRD Section 7

Figure 7-4

November 2006

Comparison of Maximum:Minimum NRU IC50 Ratios to
Maximum:Minimum LD50 Ratios

a.
30
25
20
15
10
5
0

0

5

10

15

20

25

30

Maximum:Minimum LD 50

b.
30
25
20
15
10
5
0

0

5

10

15

20

25

30

Maximum:Minimum LD50
Abbreviations: 3T3=BALB/c 3T3 fibroblasts; NRU=Neutral red uptake; NHK=Normal human epidermal
keratinocytes. Comparison of maximum:minimum ratios of IC50 and LD50 for 53 reference substances for the
3T3 NRU test method (a) and 57 reference substances for the NHK NRU test method (b). Solid lines show thetheoretical one to one correspondence of maximum:minimum IC50 to maximum:minimum LD50.

7-41

In Vitro Cytotoxicity Test Methods BRD Section 7

November 2006

7.2.6
Normalization of Reference Substance IC50 Values Using SLS IC50 Values
As an alternate analysis for reproducibility, IC50 values for reference substances werenormalized to those of the corresponding SLS IC50values. This approach was tested using
five reference substances for each test method to determine whether such normalization
would reduce the variability, measured using CV values, of the results. The reference
substances selected for this evaluation were those for which the ANOVA indicatedstatistically significant differences among the laboratories. Because there were a number ofreference substances that met this criterion for the 3T3 NRU test method, the substanceswere selected so as to cover a wide range of rodent acute oral toxicity. One reference
substance was selected from each GHS category with the exception of the 50 ≤ LD50 <300
mg/kg category. There was no substance represented by this category because there were six
acute oral toxicity categories and only five substances were used for this assessment. The
reference substances, shown in Table 7-10, were busulfan, chloramphenicol, meprobamate,propylparaben, and triethylenemelamine. Because there were only six reference substanceswith significant ANOVAs in the NHK NRU test method, the last five reference substances inTable 7-5 (citric acid, digoxin, dimethylformamide, disulfoton, and propranolol HCl) wereselected for this analysis.
Millimolar units were used for the IC50 values in this analysis since the mole is the mostappropriate unit for measuring and comparing biological activity. The IC50 value (in mM) for
each reference substance was normalized to the corresponding SLS IC50 value (in mM) by
dividing the SLS IC50 by the reference substance IC50. Intra- and inter-laboratory CV values
were calculated for both the IC50 values and for the SLS IC50:reference substance IC50 ratios
to determine whether this type of normalization would reduce the interlaboratory CV values.
Table 7-10 shows that the mean intralaboratory CV of the IC50 values for the five substancesused in the 3T3 evaluation was 22% and the interlaboratory CV was 88%. Normalizing thereference substance IC50 values to the SLS IC50 yielded a slightly higher intralaboratory CV
of 25% and a lower interlaboratory CV of 65%. The mean intralaboratory CV of the IC50
values for the five substances used in the NHK evaluation was 14% and the interlaboratory
CV was 50%. Normalizing the reference substance IC50 values to the SLS IC50 yielded aslightly higher intralaboratory CV of 16% and a higher interlaboratory CV of 61%. Whenthe normalization ratios are examined for each chemical-by-laboratory combination (Table
7-10), nine CVs increased, five decreased, and one remained the same for the 3T3 NRU test
method, and eight increased, six decreased, and one remained the same for the NHK NRU
test method. Thus, for the reference substances used in this analysis, normalizing thereference substance IC50 to the concurrent SLS IC50 did not reduce the overall variability ofthe measurements, as measured by the CV values.

7-42

In Vitro Cytotoxicity Test Methods BRD Section 7

Table 7-10

CV Values for 3T3 and NHK NRU Test Method IC50 Values andNormalized IC50 Values

Reference
Substance

IC50
(mM)1

Busulfan
ECBC
FAL
IIVS
Chloramphenicol
ECBC
FAL
IIVS
Meprobamate
ECBC
FAL
IIVS
Propylparaben
ECBC
FAL
IIVS
Triethylenemelamine
ECBC
FAL
IIVS
Mean

0.548
0.163
1.30
0.177
0.498
0.171
0.845
0.483
2.47
1.62
4.02
1.77
0.166
0.116
0.287
0.0949Citric Acid
ECBC
FAL
IIVS
Digoxin
ECBC
FAL
IIVS
Dimethylformamide
ECBC
FAL
IIVS
Disulfoton
ECBC
FAL
IIVS
Propranolol HCl
ECBC

November 2006

0.002780.000421
0.007100.000827

2.21
2.74
1.62
2.25
4.02E-06
6.89E-06
6.53E-08
5.10E-06

IntraLab
CV2 (%)

3T3 NRU Test Method
119
0.677
48
1.05
56
0.109
4
0.877
67
0.725
22
0.847
30
0.844
18
0.483
54
0.071
14
0.085
15
0.039
2
0.088
64
1.16
16
1.29
29
0.535
12
1.65
135
191
11
354
18
21.4
29
197
22
88
NHK NRU Test Method
25
0.00587
160.005317
0.007650.0047
88
62378
13
1264
36
183479
7
2389

107
128
107
87.5
1.38
0.509
2.94
0.679
0.125
0.129

InterLab
CV3 (%)SLS IC50:
Substance
IC504

19
2
1
3
99
19
26
32
21
12

7-43

0.000110.000070.000130.000130.0140
0.022
0.005
0.015
0.09470.081

IntraLab CV
SLS IC50:
Substance
IC505 (%)

InterLabCVSLS IC50:
Substance
IC506 (%)
74

70
53
9
29
30
22
21
39
23
29
3
49
20
22
9
87
11
24
23
25

65
26

14
28
16
168
10
44
26
31
7
1
19
61
6
5
20
20
15

In Vitro Cytotoxicity Test Methods BRD Section 7

Table 7-10

November 2006

CV Values for 3T3 and NHK NRU Test Method IC50 Values andNormalized IC50 Values

Reference
Substance

IC50
(mM)1

IntraLab
CV2 (%)

FAL
IIVS
Mean

0.148
0.09676
11
14InterLab
CV3 (%)SLS IC50:
Substance
IC504
0.087
0.116

50

IntraLab CV
SLS IC50:
Substance
IC505 (%)
25
9
16

InterLabCVSLS IC50:
Substance
IC506 (%)

61

Abbreviations: 3T3=BALB/c 3T3 fibroblasts; ECBC=Edgewood Chemical Biological Center; FAL= Fund for the
Replacement of Animals in Medical Experiments Alternatives Laboratory; IIVS=Institute for In Vitro Sciences; NA=No
acceptable IC50 results reported or calculation was not performed (e.g., for contrast results); CV=Coefficient of variation.
1
Results reported on the same row with reference substance names are the arithmetic means of all the laboratories. Results
reported on the same row as laboratories are the arithmetic laboratory means.
2
CV for IC50 values from the acceptable tests within each laboratory.
3
CV calculated using the arithmetic mean IC50 values from each laboratory.
4
Concurrent SLS IC50 in mM divided by the reference substance IC 50. Results reported on the same row with reference
substance names are the arithmetic means of all the laboratories. Results reported on the same row as laboratories are thearithmetic laboratory means.
5
CV for SLS IC50:reference substance IC50 values within each laboratory.
3
CV calculated using the mean SLS IC50:reference substance IC50 values from each laboratory.

7.3

Historical Positive Control (PC) Data

The reproducibility of the PC (SLS) data was assessed by CV analysis, ANOVA, and linear
regression over time, as described in Section 5.5.4.2. To obtain an assessment of the truevariation of SLS IC50 values, the reproducibility analyses also included IC50 values from SLS
tests that failed the test acceptance criterion for the IC50 acceptance limits determined foreach study phase. Therefore, the values used for this analysis included some that were notincluded in Table 5-3. These additional SLS tests, however, passed all other test acceptancecriteria. If more than one SLS test was performed in a single day (for each method andlaboratory), the IC50 values were averaged to determine a single IC50 for the day so that themultiple results from that day would not overly influence the average.
Figure 7-5 shows the average SLS IC50 values for each method, laboratory, and study phase.
The SLS IC50 for the 3T3 test method (Figure 7-5a) was relatively consistent over the entire
period of the study (approximately 2.5 years). The intralaboratory CV values for theindividual study phases ranged from 5% to 24% (Figure 7-5a). With the exception of thePhase Ib CV at FAL, the CV values for each laboratory and phase were less than 20%. Theinterlaboratory CV values were even smaller, 6% in Phases Ia and Ib, 10% in Phase II, and
2% in Phase III.
Figure 7-5b shows that the SLS IC50 for the NHK NRU test method tended to vary withtime, but, with the exception of the values from FAL, there appeared to be no consistent
trend. The IC50 values from FAL, which changed their cell culture methods after Phase Ib(see Section 5.3.3.1), tended to decrease over time. Although the change in cell culture
methods reduced the magnitude of the IC50, the variability (as evidenced by the
intralaboratory CV values shown in Figure 7-5b) remained relatively high (CV ≥34% for all
FAL study phases).
7-44

In Vitro Cytotoxicity Test Methods BRD Section 7

Figure 7-5

November 2006

SLS IC50 for Each Laboratory and Study Phasea

3T3 NRU Test Methodb

NHK NRU Test Method

Abbreviations: 3T3=BALB/c 3T3 fibroblasts; NHK=Normal human epidermal keratinocytes; NRU=Neutral red
uptake; ECBC=Edgewood Chemical Biological Center; FAL= Fund for the Replacement of Animals in Medical
Experiments Alternatives Laboratory; IIVS=Institute for In Vitro Sciences; N=Number of values.
Note: Bars show mean SLS IC50 values. Error bars show standard deviation. Percent values above error bars are
intralaboratory CVs.

7-45

In Vitro Cytotoxicity Test Methods BRD Section 7

November 2006

The CV values for all the laboratories and study phases show that the SLS IC50 values in the
NHK NRU test method are more variable within laboratories than the corresponding 3T3
SLS IC50 values. The CV values for the SLS IC50 for the NHK NRU test method ranged from11 to 51%, with nine of the 12 values greater than 20%. The interlaboratory CV values,
which were also greater than those for the 3T3 NRU test method, were 39% in Phase Ia, 21%
in Phase Ib, 31% in Phase II, and 8% in Phase III.
7.3.1

ANOVA and Linear Regression Results for the 3T3 NRU Test Method

7.3.1.1 Variation of SLS IC50 Values with Time
Table 7-11 shows the SLS ANOVA results from the 3T3 test method. When the IC50 values
in each laboratory were compared, there were no statistically significant differences (p <0.01)
among study phases for any laboratory. Table 7-12 shows that the slopes of the linear
regressions of the IC50 values over time (expressed as index values) were significantly
different from zero for ECBC and FAL (p=0.001 and 0.012, respectively), but, because theslopes were so small (0.000204 and -0.000324), and in different directions, these differenceswere considered to be unimportant, regardless of the statistical conclusions. The slope of the
IIVS regression of SLS IC50 over time was not significantly different from zero (p=0.651;
Table 7-12), which was consistent with the ANOVA analysis (Table 7-11), and showed thatSLS IC50 from IIVS did not vary with study phase (p=0.854). The ANOVA analysis, withstudy phase as the factor (with laboratories combined), showed that the 3T3 NRU IC50 values
from all the laboratories were consistent over time (p=0.304).
7.3.1.2 Comparison of SLS IC50 Values Among the Laboratories
When all study phases from each laboratory were combined, ANOVA, with laboratory as thefactor, showed that the SLS IC50 values in the 3T3 NRU test method differed significantly
among the laboratories (p <0.006) (Table 7-11). However, as can be seen in Figure 7-5a, theindividual laboratory SDs overlap one another.

7-46

In Vitro Cytotoxicity Test Methods BRD Section 7

Table 7-11

November 2006

ANOVA Results for the SLS IC50 Values in the 3T3 NRU Test Method

ECBC
Log Mean IC50
SD
N
P1
(mM)
Test for differences between phases within each laboratory
Study Phase/
Laboratory

0.031

FAL

IIVS

Log Mean IC50
(mM)

SD

N

-0.811

0.046

9

P

Log Mean IC50
(mM)

SD

N

P1

0.015

-0.850

0.034

7

0.854

1

Phase Ia

-0.8760.042

6Phase Ib

-0.8640.066

6-0.8460.065

8-0.8380.025

5Phase II

-0.8480.027

16-0.7960.057

19-0.8540.025

8Phase III-0.8420.036

36-0.8510.066

27-0.8440.041

23-0.8260.062

63-0.8470.035

44

Test for differences between laboratories (phases combined)
All Phases-0.849

0.039

64

0.006

Test for differences between phases (laboratories combined)
Phase Ia

-0.8390.049

22Phase Ib

-0.8500.056

19Phase II

-0.8310.047

34Phase III

0.845

0.045

86

0.304

Abbreviations: ANOVA=Analysis of variance; SLS=Sodium lauryl sulfate; 3T3=BALB/c 3T3 fibroblasts; NRU=Neutral
red uptake; N=Number of values; SD=Standard deviation; ECBC=Edgewood Chemical Biological Center; FAL=Fund for
the Replacement of Animals in Medical Experiments Alternatives Laboratory; IIVS=Institute for In Vitro Sciences.
1
Statistically significant at p <0.01.

7-47

In Vitro Cytotoxicity Test Methods BRD Section 7

Table 7-12
Laboratory
ECBC
FAL
IIVS
ECBC
FAL
IIVS

November 2006

Linear Regression Analysis of SLS IC50 Values Over Time1
Slope
P-value (Slope)2
3T3 NRU Test Method
0.000204
0.001-0.000324
0.012
0.0000304
0.651
NHK NRU Test Method-0.000559
0.002-0.00112<0.001-0.000445
0.002Intercept-0.874-0.796-0.850-1.901-1.737-1.885Abbreviations: SLS=Sodium lauryl sulfate; 3T3=BALB/c 3T3 fibroblasts;
NHK=Normal human epidermal keratinocytes; NRU=Neutral red uptake; N=Numberof values; ECBC=Edgewood Chemical Biological Center; FAL=Fund for the
Replacement of Animals in Medical Experiments Alternatives Laboratory;
IIVS=Institute for In Vitro Sciences.
1
Time was expressed as index values. The index value of each test reflected the orderof testing without respect to the time lapsing between tests.
2
Statistically significant from zero at p <0.05.

7.3.2

ANOVA and Linear Regression Results for the NHK NRU Test Method

7.3.2.1 Variation of SLS IC50 Values with Time
Table 7-13 shows the ANOVA results for the NHK NRU test method. When the IC50 values
within each laboratory were compared by study phase, the values were statistically different(p <0.01) at each laboratory. The IC50 values from the various study phases were alsosignificantly different from one another when the laboratory data were combined (p <0.001).
The change in cell culture methods at FAL after Phase Ib (see Section 5.3.3.1) contributed tothis difference. Table 7-13 shows that FAL had clearly the lowest log mean SLS IC50 for
Phases Ia and Ib. Linear regression analyses showed that the IC50 slopes over time (expressedas an index values) were statistically significantly less than zero for each laboratory (seeTable 7-12). Because the slopes were so small (-0.000559, -0.00112, and -0.000445), andnegative, their statistical significance was considered to be irrelevant.
7.3.2.2 Comparison of SLS IC50 Values Among the Laboratories
The ANOVA results, with laboratory as a factor (Table 7-13), showed that the SLS IC50 was
statistically significantly different among the laboratories when the data from the study
phases were pooled (p <0.001). Figure 7-5b shows that the SLS data from ECBC and IIVS
were rather similar to one another for Phases Ia, Ib, and III. The SLS IC50 data from FAL are
different from the other two laboratories for Phases Ia, Ib, and II, but the SDs for Phase III
show that the data from all laboratories produced similar values.

7-48

In Vitro Cytotoxicity Test Methods BRD Section 7

Table 7-13

November 2006

ANOVA Results for the SLS IC50 Values in the NHK NRU Test Method

ECBC
Log MeanSD
N
P1
IC50 (mM)
Test for differences between phases within each laboratory
Study Phase/
Laboratory

0.001

FAL

IIVS

Log Mean
IC50 (mM)SD

N-1.656

0.125

5

P

Log Mean
IC50 (mM)

SD

N

P1

<0.001-1.9040.060

12<0.0011

Phase Ia

-1.8670.135

5Phase Ib

-1.9360.092

6-1.8290.141

10-1.9650.046

5Phase II

-2.0070.109

11-1.9820.173

15-1.8630.058

12Phase III-1.9900.098

31-1.9410.113

34-1.9720.070

19-1.8790.175

64-1.9240.073

48

Test for differences between laboratories (phases combined)
All Phases-1.9710.113

53<0.001

Test for differences between phases (laboratories combined)
Phase Ia

-1.8330.143

22

Phase Ib

-1.8910.125

21Phase II

-1.9640.139

38

Phase III-1.9710.100

84<0.001Abbreviations: ANOVA=Analysis of variance; SLS=Sodium lauryl sulfate; NHK=Normal human epidermal keratinocytes; NRU=Neutral red uptake; N=Number of values;
SD=Standard deviation; ECBC=Edgewood Chemical Biological Center; FAL=Fund for the Replacement of Animals in Medical Experiments Alternatives Laboratory;
IIVS=Institute for In Vitro Sciences.
1
Statistically significant at p <0.01.

7-49

In Vitro Cytotoxicity Test Methods BRD Section 7

7.4

November 2006

Laboratory Concordance for Solvent Selection

The solvents used for the reference substances are shown in Table 7-14. For Phases Ib and II,
the SMT based their selection of solvents on the results provided by BioReliance (see Table
5-9) using the solubility protocol in Appendix G2. Despite the fact that the solubility of anindividual substance might be different in 3T3 and NHK growth media, the SMT selected thesame solvent (i.e., medium or DMSO) for both test methods, rather than having different
solvents for each method.
BioReliance occasionally achieved higher solubility values for the Phase I and II substances
than the three cytotoxicity laboratories (e.g., see the results for arsenic trioxide, aminopterin,
and chloramphenicol in Table 5-10). The laboratories were using the solubility protocols inAppendices C3 through C6 (for Phases Ib and II), which were somewhat different from theprotocol used by BioReliance. Although all the laboratories used the same protocols, they did
not always obtain similar results with respect to the solvent to be used (e.g., see the results
for aminopterin, cadmium chloride, and chloramphenicol in Table 5-10). In an attempt to
avoid the selection of a solvent for which one or more laboratories could not achieve thedesired solubility, the SMT used the solubility data from all the laboratories to determine thesolvents to be used for each chemical tested in Phase III. Table 7-14 shows that cell culture
medium was used as the solvent for 38 substances and DMSO was used for 34 substances.
Five of the substances were insoluble in medium and DMSO in at least one testinglaboratory. Arsenic trioxide was insoluble at all laboratories. IIVS also found sodium oxalate,strychnine, and triethylenemelamine insoluble in media and DMSO, and FAL found thallium
sulfate insoluble in both solvents. Therefore, the SMT used the results from the laboratories
that did achieve solubility to select the solvents to be used for testing these substances.
The testing laboratories selected the same solvent for 55 of the 72 reference substances(76%). Excluding the five substances that were found to be insoluble in both solvents by atleast one laboratory, there were 12 substances on which the laboratories disagreed:
acetaminophen, acetylsalicylic acid, carbamazepine, carbon tetrachloride, chloramphenicol,
dichlorvos, meprobamate, methanol, phenobarbital, phenylthiourea, physostigmine, andvalproic acid. Each laboratory reported relatively low solubility, ≤2 mg/mL, in medium forthese substances. Because 2 mg/mL in medium is the departure point for the selection of
medium or DMSO, small variations in solubility lead the laboratories to select different
solvents. The solubility of acetaminophen, for example was reported as 2 mg/mL in culture
media by ECBC and FAL, but <2 mg/mL by IIVS. IIVS found it soluble in 200 mg/mL
DMSO and selected DMSO as the solvent. ECBC and FAL selected culture media as thesolvent. The SMT selected DMSO as the solvent for acetaminophen to be used by alllaboratories so that they would all be assured of obtaining usable test results.

7-50

In Vitro Cytotoxicity Test Methods BRD Section 7

Table 7-14

November 2006

Solvent Determinations by Laboratory

Reference SubstanceAcetaminophen
Acetonitrile
Acetylsalicylic acid
Aminopterin
5-Aminosalicylic acid
Amitriptyline HCl
Arsenic III trioxide
Atropine sulfate
Boric aid
BusulfanCadmium II chloride
Caffeine
Carbamazepine
Carbon tetrachloride
Chloral hydrate
Chloramphenicol
Citric acidColchicine
Cupric sulfate pentahydrate
Cycloheximide
Dibutyl phthalate
Dichlorvos
Diethyl phthalate
DigoxinDimethylformamide
Diquat dibromide
monohydrate
Disulfoton
Endosulfan
Epinephrine bitartrate
Ethanol
Ethylene glycol
Fenpropathrin
Gibberellic acid
Glutethimide
GlycerolHaloperidol
Hexachlorophene
Lactic acid
Lindane
Lithium I carbonate
Meprobamate
Mercury II chloride
MethanolNicotine
Paraquat
Parathion
Phenobarbital
PhenolPhenylthiourea

Solvent Used for
Testing1
DMSO
MediumDMSO
DMSO
MediumDMSO
MediumMediumMediumDMSO
DMSO
MediumDMSO
DMSO
MediumDMSO
MediumMediumMediumMedium
DMSO
DMSO
DMSO
DMSO
Medium

ECBC

FAL

IIVS

MediumMediumMediumDMSO
MediumDMSO
ID
MediumMediumDMSO
DMSO
MediumMediumMediumMediumDMSO
MediumMediumMediumMediumDMSO
MediumDMSO
DMSO
MediumMediumMediumDMSO
DMSO
MediumDMSO
ID
MediumMediumDMSO
DMSO
MediumDMSO
DMSO
MediumDMSO
MediumMediumMediumMedium
DMSO
DMSO
DMSO
DMSO
MediumDMSO
MediumMediumDMSO
MediumDMSO
ID
MediumMediumDMSO
DMSO
MediumDMSO
MediumMediumMediumMediumMediumMediumMediumDMSO
MediumDMSO
DMSO
MediumMediumMediumMediumMediumDMSO
DMSO
MediumMediumMediumDMSO
MediumDMSO
MediumDMSO
DMSO
MediumDMSO
Medium
DMSO
DMSO
DMSO
MediumMediumDMSO
DMSO
Medium
DMSO

DMSO
DMSO
MediumMediumMediumDMSO
MediumDMSO
MediumDMSO
DMSO
MediumDMSO
MediumMediumDMSO
MediumMediumMediumDMSO
MediumMedium
DMSO

DMSO
DMSO
MediumMediumMediumDMSO
MediumDMSO
MediumDMSO
DMSO
MediumDMSO
MediumMediumDMSO
MediumMediumMediumDMSO
DMSO
MediumMediumDMSO
DMSO
MediumMediumMediumDMSO
MediumDMSO
MediumDMSO
DMSO
MediumDMSO
Medium
DMSO
DMSO
DMSO
MediumMediumDMSO
DMSO
MediumDMSO

7-51

In Vitro Cytotoxicity Test Methods BRD Section 7

Table 7-14

November 2006

Solvent Determinations by Laboratory

Reference SubstancePhysostigmine
Potassium I chloride
Potassium cyanide
Procainamide HCl
2-Propanol
Propranolol HCl
Propylparaben
Sodium arsenite
Sodium chloride
Sodium dichromate dihydrate
Sodium fluoride
Sodium hypochlorite
Sodium oxalate
Sodium selenate
Strychnine
Thallium I sulfate
Trichloroacetic acid
1,1,1-Trichloroethane
Triethylenemelamine
Triphenyltin hydroxide
Valproic acid
Verapamil HCl
XyleneDMSO Total
Medium Total

Solvent Used for
Testing1
DMSO
MediumMediumMediumMediumDMSO
DMSO
MediumMediumMediumMediumMediumMediumMediumMediumMediumMediumMedium
DMSO
DMSO
DMSO
DMSO
DMSO
34
38

ECBC

FAL

IIVS

MediumMediumMediumMediumMediumMediumDMSO
MediumMediumMediumMediumMediumMediumMediumMediumMediumMediumMediumMediumDMSO
Medium
DMSO
DMSO
22
49

DMSO
MediumMediumMediumMediumMediumDMSO
MediumMediumMediumMediumMediumMediumMediumMediumID
MediumMedium
DMSO
DMSO
DMSO
DMSO
DMSO
29
41

DMSO
MediumMediumMediumMediumMediumDMSO
MediumMediumMediumMediumMediumID
MediumID
MediumMediumMedium
ID
DMSO
DMSO
DMSO
DMSO
28
40

Abbreviations: DMSO=Dimethyl sulfoxide; ECBC=Edgewood Chemical Biological Center; FAL= Fund for the
Replacement of Animals in Medical Experiments Alternatives Laboratory; IIVS=Institute for In Vitro Sciences;
ID=Insufficient data to select solvent; Medium=Cell culture medium.
1
Solvents selected by the SMT for use by all laboratories.

7.5

Summary

Intra- and inter-laboratory reproducibility were assessed by comparing the laboratoryspecific IC50-LD50 regressions to the mean, across-laboratory regression for each method,
ANOVA, CV analysis, and comparison of maximum:minimum mean laboratory IC50 values.
ANOVA permitted statistical comparisons of laboratories and experimental averages, while
controlling for other factors. CV analysis compared the relative magnitudes of variability ona standardized scale. Reproducibility was evaluated using the results from the reference
substances that yielded IC50 values from all three laboratories: 64 and 68 reference
substances in the 3T3 and the NHK NRU test methods, respectively. The analysis of
intralaboratory reproducibility, by evaluating the similarity of the laboratory specific IC50LD50 regressions, showed that the laboratory regressions for both NRU test methods were
within the 95% confidence limits of the laboratory mean regressions.
The ANOVA showed significant interlaboratory differences for 23 substances in the 3T3
NRU test method and six in the NHK NRU test method. Intralaboratory CV values ranged
from 1-122% in the 3T3 test method and 1-129% in the NHK NRU test method. Meaninterlaboratory CV values were 26% for both NRU test methods, but NHK had a lower mean
7-52

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November 2006

interlaboratory CV (28% vs 47% for 3T3 NRU). Interlaboratory CV values ranged from 3135% in the 3T3 NRU test method and 1-91% in the NHK NRU test method. FAL had thehighest mean intralaboratory CV in both NRU test methods (33% in 3T3, 43% in NHK).
An analysis to determine the relationship between the chemical attributes and interlaboratory
CV indicated that chemical structure, physical form, solubility, and volatility had little effecton CV. The CV seemed to be related, however, to GHS acute toxicity category, IC50, andboiling point. Mean interlaboratory CV values were larger for substances in the most toxic
GHS categories than for substances in the other toxicity categories, especially with the 3T3
NRU test method. The mean interlaboratory CV for substances in the LD50 ≤5 mg/kg (72%)
and 5< LD50 ≤50 mg/kg (78%) classes were larger than the mean overall interlaboratory CV(47%) with the 3T3 NRU test method. The mean interlaboratory NHK CV was 37% forsubstances with LD50 ≤5 mg/kg, and 41% for substances with 5< LD50 ≤50 mg/kg, while themean overall interlaboratory CV was 28%. A Spearman correlation analysis showed that theIC50 was inversely correlated to interlaboratory CV for both the 3T3 (p=0.015) and NHK(p=0.014) test methods, and that boiling point was positively correlated to interlaboratory CV(p=0.007) (i.e., higher boiling points were associated with higher CV values) for the 3T3 but
not the NHK NRU test method (p=0.809).
The ANOVA results for the PC IC50 in the 3T3 NRU test method showed that there weresignificant differences among laboratories (p=0.006) but not among study phases withinlaboratories (p >0.01). However, interlaboratory CV values, which ranged from 2% to 10%
for the different study phases, were small and the intralaboratory CV values ranged from 5%
to 24%. The SLS IC50 values from the NHK NRU test method were more variable than thosefrom the 3T3 NRU test method. The ANOVA results for SLS in the NHK NRU test method
indicated that there were significant differences among laboratories (p <0.001) and among
study phases within laboratories (p ≤0.001). A change in cell culture methods at FAL after
Phase Ib decreased the SLS IC50 in subsequent phases, but FAL’s CV values still tended tobe higher than in the other laboratories. Intralaboratory CV values for the NHK SLS IC50
during the various study phases ranged from 11% to 51% and interlaboratory CV values for
SLS in the NHK NRU test method ranged from 8% in Phase III to 39% in Phase Ia.
Cell culture medium was used as the solvent for 38 substances and DMSO was used for 34
substances. Concordance among all three laboratories in selecting the solvent for thereference substances was 76% (55/72).

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8.0

3T3 AND NHK NRU TEST METHOD DATA QUALITY ................................. 8-3

8.1

Compliance with Good Laboratory Practice Regulations ................................... 8-3
8.1.1 Guidelines Followed for Cytotoxicity Testing .............................................. 8-3
8.1.2 Quality Assurance (QA) for NRU Cytotoxicity Test Data............................ 8-4
8.1.3 Guidelines Followed for Rodent Acute Oral LD50 Data Collection .............. 8-6

8.2

Results of Data Quality Audits ............................................................................... 8-6
8.2.1 QA Statements ............................................................................................... 8-6
8.2.2 QA Statements from the Laboratories ........................................................... 8-7

8.3

Effect of Deviations or Non-compliance with GLPs ............................................. 8-9
8.3.1 Laboratory Error Rates .................................................................................. 8-9
8.3.2 Failure Rates for Definitive and PC Tests ................................................... 8-10
8.3.3 Intralaboratory Reproducibility ................................................................... 8-11
8.3.4 Prediction of GHS Acute Oral Toxicity Categories .................................... 8-12

8.4

Availability of Laboratory Notebooks ................................................................. 8-12

8.5

Summary................................................................................................................. 8-13

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In Vitro Cytotoxicity Methods BRD Section 8

8.0

November 2006

3T3 AND NHK NRU TEST METHOD DATA QUALITY

This section of the BRD presents the extent of adherence to GLP regulations for generationof the validation study data. Data quality is described, along with deviations from theregulations and their effect (if any) on the quality of the data. Statistical analyses areprovided to compare the data generation, collection, and reporting by the two GLP compliant
laboratories and the one non-GLP compliant laboratory, as well as for the GLP-compliant
laboratory that distributed the reference substances and performed solubility studies.
Discussions of various quality assurance aspects of the study are included.
8.1

Compliance With Good Laboratory Practice Regulations

8.1.1

Guidelines Followed for Cytotoxicity Testing

8.1.1.1 Good Laboratory Practices
The SOW provided the following definition of U.S. Regulatory agency GLPs to eachlaboratory:“Regulations governing the conduct, procedures, and operations of toxicology
laboratories; regulations to assure the quality and integrity of the data and to address suchmatters as organization and personnel, facilities, equipment, facility operations, test andcontrol articles, and validation study protocol, and conduct (U.S. Food and Drug
Administration, Title 21 CFR Part 58; U.S. Environmental Protection Agency, Title 40
CFR Part 160).”
IIVS, ECBC, and BioReliance performed testing under GLP guidelines. The details of GLP
compliance and training are addressed in Section 11.2.
8.1.1.2 Spirit of GLPThe SMT determined a definition for “spirit of GLP” and provided the following to thelaboratories:“Laboratories that are non GLP-compliant shall adhere to GLP principles and othermethod parameters as put forth in this Statement of Work and the Test Method Protocols(provided by NIEHS/NICEATM); documentation and accountability shall be equal to
GLP requirements; laboratories must make assurances that they are equal in performance
criteria and that there is parity amongst the laboratories.”
FAL performed testing in the “spirit of GLP” (see Section 11.2.2.1) by following theinternational GLP standards referenced in the ECVAM Workshop 37 Report (CooperHannan 1999) and the OECD Principles of GLP (OECD 1998). The laboratory did not havetheir data and test procedures reviewed by an independent, quality assurance (QA) auditor.
The SOW directed FAL to, at a minimum, routinely document their equipment monitoring
and record keeping (see Table 8-1), and to archive all documents. The FAL already had mostof the requested procedures and guidelines in place for routine laboratory procedures before
initiation of this study. The various general laboratory-related activities were documented inworkbooks and logbooks, and the information was made available to the SMT.

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Table 8-1

November 2006

SMT-Recommended Documentation for FAL
Daily

Per UseTemperatures
Laboratory (ambient), incubators,
water baths, refrigerators, freezers

Cryogenic Storage Unit
Liquid N2 volume

Humidity/CO2
Cell culture incubators

Equipment Calibration
Balances, pH meters, cell counters

Visual Observations
Cell Culture Growth

Reagents
Lot numbers and expiration datesof medium/supplements

Periodic
Laboratory Supplies1
Lot numbers and expiration dates
for stock media formulations andcomponents, NRU reagents, tissue
culture plasticware
Cells
Quantity, and cryogenic storage
conditions, for 3T3 and NHK cells
Equipment Calibration
Incubators, laminar flow hoods,
autoclaves, micropipettors,
spectrophotometer plate readers,
computers (software)

Abbreviations: SMT=Study Management Team; FAL=Fund for the Replacement of Animals in Medical Experiments
Alternatives Laboratory; NRU=Neutral red uptake; 3T3=BALB/c 3T3 fibroblasts; NHK=Normal human epidermal
keratinocytes.
1
Documentation for laboratory supplies begins when supplies are purchased and received by the laboratory

8.1.1.3 Good Cell Culture Practices (GCCP)The SMT provided guidance in the SOW for implementing GLPs in a cell culture laboratoryenvironment. The initial assumption by the SMT was that each laboratory had the basic cell
culture skills and knowledge (e.g., as described in Freshney 2000) to reliably perform the invitro NRU cytotoxicity test methods. Reviews of historical laboratory documents, andscientific and professional exchanges with the laboratory personnel, assured the SMT thateach laboratory had demonstrated, through previous validation studies and other experience,that the personnel were capable of providing quality scientific data through the use of goodcell culture practices. A comparison of the SOW and the in vitro NRU cytotoxicity protocols
showed that the guidelines developed for the NICEATM/ECVAM study were harmonious
with the guidelines in the ECVAM Good Cell Culture Practices Reports (Hartung 2002;
Coecke et al. 2005), and the OECD document on GLPs and in vitro studies (OECD 2004a).
8.1.2

Quality Assurance (QA) for NRU Cytotoxicity Test Data

8.1.2.1 Coded Reference Substances
BioReliance acquired 73 high purity chemicals (72 reference substances and one positive
control substance) from reputable commercial sources. Sixty-four of the reference substanceswere ≥99% pure, and seven were between 90 and 99% pure. Lactic acid had the lowest
purity, 89% (See Appendix F1). The substances were coded with unique identification
numbers and provided to the testing laboratories in a blinded fashion. Procurement ofchemicals and their preparation for distribution was performed under GLP guidelines and the
SOW provided by the SMT (see Appendix G). Section 3.4 provides detailed information on
the acquisition and distribution of reference substances.

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8.1.2.2 Solubility Testing and Data Review
All laboratories performed solubility tests on all reference substances using the solvents andprocedures specified in the protocols provided by the SMT, and submitted solubility data to
the SMT in the form of hard copy printouts and electronic worksheets. The Study Directors
reviewed all laboratory procedures and all data produced at their respective laboratories, andthe QA designee in each GLP-compliant laboratory reviewed all data in their laboratory. The
SMT Project Coordinators served as informal QA reviewers for FAL (i.e., reviewed all theraw data sheets). The errors and omissions detected were reported to FAL, and corrections
were requested. The SMT reviewed all solubility data and NRU assay data produced by all of
the laboratories.
The SMT reviews of the submitted data in Phases Ia and Ib revealed that, even after data
review by the Study Directors, the data files contained an unacceptably high frequency oferrors (see Section 2.6.2.5). The laboratories were alerted to the problem and personnel fromall laboratories attended a weeklong training session at the IIVS laboratories in Gaithersburg,
Maryland to enhance harmonization among the laboratories. Errors continued to be found indata files submitted for Phase III after the training, albeit less frequently; however, such
errors generally resulted from the rush to rapidly complete the data files for submission to the
SMT shortly after the conclusion of each test. The formal QA reviews of the files occurred
later in each phase of the study.
The most common errors included typographical mistakes, transcriptional and data entry
errors in the Microsoft® EXCEL® and the GraphPad PRISM® 3.0 templates, and incorrectlabeling of files. The SMT reviewed every electronic file and hard copy printout throughout
the study and alerted the Study Directors of the affected laboratories when errors were found.
All data files were checked for consistency within the documents, and for compliance withthe protocols. The SMT also documented errors on the hard copy printouts in the form ofhandwritten notations to the files (at NICEATM) and added these notations to the electronic
data summary files compiled for data management. Files that were revised and/or correctedby the Study Director were resubmitted to the SMT and identified as corrected files.
8.1.2.3 NRU Cytotoxicity Test Tallies
The Study Directors periodically received individualized test tallies specific to theirlaboratories from NICEATM that detailed:• The number of range finder tests performed by the laboratory• The number of definitive tests performed, and the pass/fail status of each
• The number of PC tests performed, and the pass/fail status of each
• The number of acceptable tests completed• The test completion status for each chemical (i.e., whether one range finder
test had been completed, and the number of acceptable definitive tests had
been completed)
The laboratories compared the NICEATM tallies to their own records to verify theirconsistency and accuracy. Discrepancies were resolved through direct communication
between the Study Director and the SMT.

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8.1.3
Guidelines Followed for Rodent Acute Oral LD50 Data Collection
For the purposes of this validation study, the in vitro NRU test methods were proposed forpredicting starting doses for rodent acute oral toxicity test methods, rather than as
replacement tests for the in vivo test method. No in vivo tests were performed for thisvalidation study. All in vivo data (i.e., rat and mouse LD50 values) were collected by
NICEATM through reviews of the literature and from publicly available databases. All
relevant data and pertinent information were gathered and stored in an Excel® spreadsheet.
8.1.3.1 Rodent Acute Oral LD50 Values Used in the Registry of Cytotoxicity (RC)The RC is a database of acute oral LD50 values for rats and mice obtained primarily from the
1983/84 RTECS database compiled by NIOSH, and IC50 values from in vitro cytotoxicity
assays using multiple cell lines and cytotoxicity endpoints for chemicals with known
molecular weights (Halle 1998, 2003). Collection and reporting methods used for generating
the data in RTECS® were not a part of the data collection hierarchy employed by NIOSH,
and the data in this database were not evaluated for quality and accuracy. Many of the values
come from secondary sources with no citation to the original report. GLP guidelines were notused to determine acceptable data for the database. The only criterion used by NIOSH forreporting acute oral toxicity data in RTECS® was that the LD50 value was the most toxic
LD50 value for a chemical that could be found in the literature, regardless of the number ofother values available, or their distribution.
8.1.3.2 Rodent Acute Oral LD50 Values Collected by NICEATM from Other Sources
One critical aspect of the validation study design was the establishment of a rat acute oral
LD50 reference value for each of the 72 reference substances (see Section 4). These reference
values were used to evaluate the extent to which the two in vitro NRU test methods could
predict rat acute oral LD50 values. Primary rat acute oral LD50 studies were located throughsearching electronic databases, published articles, and secondary references. Rat data were
not available for three of the reference substances and mouse acute oral LD50 values wereused. Only seven of the 455 LD50 values collected from the literature were produced under
GLP guidelines.
8.2

Results of Data Quality Audits

The QA unit or designee in each GLP laboratory provided a systematic and criticalcomparison of the data provided in the laboratory’s study reports to the raw data in thelaboratory records. The SOW provided to each laboratory contained the following guidance
regarding QA statements:“The Final Reports for all phases of the Validation Study shall be audited by the Quality
Assurance unit of the Testing Facility for GLP compliance and a QA Statement shall be
provided by the Testing Facility. Each Final Report shall identify: 1) the phases and datainspected, 2) dates of inspection, and 3) dates findings were reported to the Study
Director and Testing Facility management. The QA Statement shall identify whether the
methods and results described in the Final Report accurately reflect the raw data
produced during the Validation Study.”
8.2.1
QA Statements
The QA statements from the GLP-compliant laboratories addressed the reviews of:• Protocols• Laboratory standard operating procedures (SOPs)
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November 2006

Laboratory operations, in general
3T3 and NHK NRU experiment data
The report submitted to the SMT

The QA statements from the GLP laboratories affirm that the methods described in the
protocols are the methods that the laboratory personnel used, and that the data reported to the
SMT accurately reflect the raw data obtained by the laboratory. See Section 8.2.2 for
information about the QA statements for the non-GLP laboratory.
8.2.2

QA Statements from the Laboratories

8.2.2.1 BioReliance QA Statements
The Study Director/Laboratory Director provided the following statement in all of the final
reports:“The solubility studies, acquisition, preparation, and distribution of the test chemicals
were conducted in compliance with GLP. Although not audited (per SOW), the work
described in this report for Phase X (i.e., Ia, Ib, and II) fully and accurately reflects to thebest of my knowledge the raw data generated in the study.”
8.2.2.2 FAL QA Statements
The Study Director for FAL performed the final review of all data and reports before sending
them to the SMT, and provided the following two statements in the final reports provided to
the SMT.
• “The laboratory worked under the principles of GLP whilst not being a GLPcompliant laboratory.”• “The report accurately reflects the work undertaken and the results obtained at
the FRAME Alternatives Laboratory.”
Formal QA statements were not provided to FAL because the SMT performed informal QA
reviews.
8.2.2.3 ECBC QA Statements
The QA statements reported the particular study phase and laboratory procedures that were
examined for GLP compliance. In addition, the laboratory’s statement noted that the scope ofwork, associated protocols, and quality control (QC) acceptance criteria were updated orchanged during the study, which made the assessment of the procedures and data for
conformance to the SOPs more difficult. However, compliance with the requirements andintent of GLP guidelines was continually assessed during the review of the SOPs and theobservance of operations. The QA reviews found the ECBC protocols to be in compliancewith the NICEATM/ECVAM study protocols. The aspects of the studies inspected by the
QA designee were:• Review of protocols and laboratory SOPs
• Review of waste handling procedures• Review of laboratory operations• Certification of new personnel• Review of data
• Review of the final report for each testing phase

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In Vitro Cytotoxicity Methods BRD Section 8

November 2006

The QA designee also observed the preparation of reference substances, 96-well plate
configuration, application of reference substance, annotation to the workbook, andappropriate sterile technique while performing the testing. The number of inspections oflaboratory operations was reduced in the latter phases of the study because the same
personnel conducted the testing throughout the entire study.
ECBC Review Dates of the Study Phases• Phase Ia: July 2002 through May 2003
• Phase Ib: July 2002 through January 2003
• Phase II: May 2003 through February 2004
• Phase III: November 2003 through March 2005
8.2.2.4 IIVS QA Statements
Because the IIVS QA unit is small, it carried out reviews of different aspects of theprocedures at different times. The IIVS QA Statement reads:“This study has been divided into a series of in-process phases. Using a random sampling
approach, Quality Assurance monitors each of these phases over a series of studies.
Procedures, documentation, equipment records, etc., are examined to assure that the study
is performed in accordance with the U.S. FDA Good Laboratory Practice regulations (21CFR 58), the U.S. EPA GLP Standards (40 CFR 792 and 40 CFR 160) and the OECD
Principles of Good Laboratory Practice and to assure that the study is conductedaccording to the protocol and relevant Standard Operating Procedures.”
The aspects of the studies inspected by the QA designee were as follows:• Protocol and initial paperwork• Reading of the plates (definitive test)• Dilution of the test articles (definitive test)• Treatment of the cells• Termination of treatment and addition of the NR dye (definitive test)• Cell concentration determination and seeding of the plates (third definitivetest)• Termination of treatment and addition of the NR dye• Washing the cells• Draft report and data
• Final report
IIVS Review Dates of Various Aspects of the Test Phases• Phase Ia: August 2002
Final Report Review: October 2005
• Phase Ib: January 2003
Final Report Review: October 2005
• Phase II: July-August 2003
Final Report Review: October 2005
• Phase III: January-November 2004 Final Report Review: October 2005
8.2.2.5 Other QA Information
Data generated by the laboratories and reviewed by their respective Study Directors were
submitted to the SMT. Often, the data were provided electronically within days of the end of
testing. The SMT was active as a secondary QA reviewer of all information provided by the

8-8In Vitro Cytotoxicity Methods BRD Section 8

November 2006

Study Directors. If the SMT found discrepancies, the Project Coordinators corresponded withthe appropriate Study Director to identify and rectify the error. The Study Director madecorrections/adjustments to the discrepancies in data reporting and presented the changes to
the SMT. The SMT did not initiate any external data quality audits.
The quality of the reference substances was assured in the form of certificates of analysis
provided by the chemical manufacturer to BioReliance at the time of purchase. The SMT andthe laboratories obtained certificates of analysis from CAMBREX for Clonetics® NHK
culture medium and supplements. In addition, the SMT obtained QC data directly fromCAMBREX technical departments concerning the NHK medium’s ability to support
keratinocyte growth.
8.3

Effect of Deviations or Non-compliance with GLPs

Rates for several types of errors (i.e., documentation, testing methods, and data management)
were determined by the SMT. Many of the errors (particularly in Phases Ia and Ib) were theresult of minor mistakes (e.g., typographical, mislabeling) and did not affect the quality ofthe data.
8.3.1
Laboratory Error Rates
The SMT was concerned about the number of errors that were seen in documentation andtesting methods during Phases Ia and Ib, and compiled the detected errors from eachlaboratory. The types of errors found included errors in documentation (e.g., reference
substance identification did not match on all associated data sheets; IC20 and IC80 values weretransposed in the EXCEL® template; a test acceptance criterion flag in a data sheet wasincorrect) and in testing (e.g., wrong dilution scheme was used for the PC; wrong SLS IC50
was used as the PC IC50). Error rates were compiled as the number of tests with errors per
total number of tests. As shown in Table 2-3, FAL had the highest error rates: 93% for the
3T3 NRU test method and 41% for the NHK NRU test method. The highest error rates in theother laboratories were 10% for the 3T3 NRU test method and 23% for the NHK NRU test
method (both ECBC).
There were relatively few errors detected in the Phase III data files. The SMT did notcompile the typographical and transcriptional errors found, but reported them directly to theappropriate Study Director so that the data sheets could be immediately corrected. The SMT
did not detect errors in the raw optical density data from the 96-well plates provided in eachdata file. The laboratories and the SMT corrected typographical and transcriptional errors(e.g., incorrect logIC50 value entered) in the EXCEL® templates. The EXCEL® template
formulas were used for the statistical analyses.
An assessment of error rates was performed specifically for Phase III for one particularclerical error – the transfer of the final results (e.g., ICx values) from the GraphPad PRISM®
3.0 template to the Microsoft® EXCEL® template. It was often necessary for the SMT torevise the EXCEL® data files provided by the laboratories because the incorrect values had
been transferred to EXCEL®. Table 8-2 summarizes the Phase III error rates resulting fromthe transfer of data from PRISM® to EXCEL®.

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In Vitro Cytotoxicity Methods BRD Section 8

November 2006

Phase III Error Rates in the Transfer of Data to the EXCEL® Template

Table 8-2

Laboratory

Number of Errors
Detected

Number of Definitive
Tests

Percentage of Tests
with Detected Errors

ECBC

49

402

12

FAL

171

513

33

IIVS

25

419

6

Abbreviations: ECBC=Edgewood Chemical Biological Center; FAL=Fund for the Replacement of Animals in
Medical Experiments Alternatives Laboratory; IIVS=Institute for In Vitro Sciences.

8.3.2
Failure Rates for Definitive and PC Tests
Table 8-3 presents the test failure (i.e., did not meet test acceptance criteria) rates
experienced in Phase III. Approximately 25% of all 3T3 definitive tests and 18% of all NHK
definitive tests failed. If a definitive test (see Section 2.3.2.2 for the definition of a definitivetest) failed, the laboratory repeated the test and attempted to obtain three acceptabledefinitive tests for each reference substance in each cell type (see Section 2.5 for criteria for
repeating tests). The PC tests failed 0 to 18% of the time with a combined average failure rateof 8% for both cell types. FAL had the highest individual laboratory test failure rates for 3T3
definitive tests (30%), NHK definitive tests (32%), and NHK PC tests (18%). ECBC had thehighest failure rate for 3T3 PC tests (11%). IIVS had no PC test failures.
Table 8-3

Definitive Test and Positive Control (PC) Test Failure Rates in Phase III
Test Type

3T3 NRU Test Method

NHK NRU Test Method

ECBC FAL IIVS Total ECBC FAL IIVS Total

Total

Definitive Tests - Acceptable

169

177

176

522

173

175

174

522

1044

Definitive Tests - Total

215

257

225

697

187

256

194

637

1334

% Failed Definitive Tests

21

30

22

25

8

32

10

18

22

PC Tests - Acceptable

66

40

16

122

58

37

20

115

237

PC Tests - Total

74

42

17

133

59

45

20

124

257

% Failed PC Tests

11

5

6

8

2

18

0

7

8

14

6

14

34

0

22

0

22

56

7

2

6

5

0

9

0

4

4

Definitive Tests Failed Only
Because PC Tests Failed% Definitive Tests Failed Only
Because PC Tests Failed

Abbreviations: ECBC=Edgewood Chemical Biological Center; FAL=Fund for the Replacement of Animals in
Medical Experiments Alternatives Laboratory; IIVS=Institute for In Vitro Sciences; NRU=Neutral red uptake;
3T3=BALB/c 3T3 fibroblasts; NHK=Normal human epidermal keratinocytes.
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In Vitro Cytotoxicity Methods BRD Section 8

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The Phase III guidelines required each laboratory to provide three acceptable definitive tests
for each substance for both cell types (3 x 60 x 2 = 360 definitive tests). PC tests were runconcurrently with the definitive tests, and more than one reference substance was usuallytested in conjunction with each PC test. Because of test failures, each laboratory performed
additional testing to obtain the three acceptable definitive tests required for each substance.
Table 8-4 presents the success rates for each laboratory for Phase III testing and a total for
all the laboratories combined.
Table 8-4

Combined Definitive and Positive Control (PC) Test Success Rates for the
3T3 and NHK Methods in Phase III

Test TypeAcceptable Definitive Tests/
Total Definitive Tests% Acceptable Definitive
Tests
Acceptable PC Tests/Total
PC Tests% Acceptable PC Tests

ECBC

FAL

IIVS

Total

342/402352/513350/4191044/1334

85%

69%

84%

78%

124/133

77/87

36/37

237/257

93%

89%

97%

92%

Abbreviations: ECBC=Edgewood Chemical Biological Center; FAL=Fund for the Replacement of Animals in
Medical Experiments Alternatives Laboratory; IIVS=Institute for In Vitro Sciences; NRU=Neutral red uptake;
3T3=BALB/c 3T3 fibroblasts; NHK=Normal human epidermal keratinocytes.

8.3.3
Intralaboratory Reproducibility
CV values for each method were determined for each reference substance in each laboratory
using the IC50 values from the acceptable definitive tests, as described in Section 5.5.2.
Table 8-5 presents the average CV values for the substances tested in each of the study
phases, and for the entire study.
Table 8-5

CV Values for Definitive Tests

Cell
Type

Labs

3T3

ECBC
FAL
IIVS

NHK

ECBC
FAL
IIVS

Phases I & II
Number of
Average
Reference% CV
Substances
12
17
11
28
11
20
12
12
12

Phase IIIAll Phases
Number of
Number of
Average
Average
Reference
Reference% CV% CV
Substances
Substances
57
24
69
23
55
33
66
33
56
22
68
21

24
31
14

57
57
58

22
45
14

69
69
70

23
42
14

Abbreviations: ECBC=Edgewood Chemical Biological Center; FAL=Fund for the Replacement of Animals in
Medical Experiments Alternatives Laboratory; IIVS=Institute for In Vitro Sciences; 3T3=BALB/c 3T3
fibroblasts; NHK=Normal human epidermal keratinocytes; CV=Coefficient of variation.
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In Vitro Cytotoxicity Methods BRD Section 8

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8.3.4
Prediction of GHS Acute Oral Toxicity Categories
Predicted LD50 values were determined using the in vitro NRU IC50 values in the IC50-LD50
regressions presented in Table 6-5. The predicted LD50 values were used to assign each
substance to a predicted GHS acute oral toxicity category (UN 2005). The accuracy of the
3T3 and NHK NRU test methods for predicting GHS categories was determined bycomparison with categorization based on in vivo rat oral LD50 values (in mg/kg) in Table 42. Using the RC rat-only millimole regression, the accuracy of the predictions and the extent
of underprediction or overprediction are shown for each laboratory in Table 8-6. The
laboratories generally agreed with each other in their predictions. Although FAL had thehighest error rates and CV values, their predictions of GHS categories were consistent withthe other laboratories. The laboratories determined category matches for 25 to 30% of thereference substances for the 3T3 NRU test method and 29 to 31% of the reference substances
for the NHK NRU test method. For the 3T3 NRU test method, toxicity was overpredicted for
38% of the reference substances and underpredicted for 33 to 38% of them. For the NHK
NRU test method, toxicity was overpredicted for 35 to 38% of the reference substances andunderpredicted for 32 to 34% of them. (See Appendix J for additional laboratorycomparisons for the other in vitro – in vivo regressions evaluated in Section 6.)
8.4

Availability of Laboratory Notebooks

All laboratories maintained laboratory notebooks using a template provided by IIVS, andprovided copies of the notebooks to the SMT (archived at NICEATM) after completion ofeach testing phase. The notebooks contained information from all aspects of testing
including, but not limited to:• Environmental conditions• Reagent identification• Preparation of 96-well plates• Preparation of reference substances• Treatment of cell cultures• Visual observations of cell cultures• NRU assays• Data analysis

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In Vitro Cytotoxicity Methods BRD Section 8

GHS Acute Oral Toxicity Category Predictions by Laboratory1

Table 8-6

ECBC

Total
Reference
Substances
64

FAL

Labs

3T3

NHK

November 2006

Category
Match

Toxicity
Toxicity
Overpredicted Underpredicted

30%

38%

33%

64

25%

38%

38%

IIVS

64

27%

38%

36%

ECBC

68

31%

35%

34%

FAL

68

29%

38%

32%

IIVS

68

31%

37%

32%

Abbreviations: ECBC=Edgewood Chemical Biological Center; FAL=Fund for the Replacement of Animals in
Medical Experiments Alternatives Laboratory; IIVS=Institute for In Vitro Sciences; 3T3=BALB/c 3T3
fibroblasts; NHK=Normal human epidermal keratinocytes; GHS=Globally Harmonized System forClassification and Labelling of Chemicals (UN 2005).
1
3T3 and NHK NRU test method IC50 data (geometric mean of within laboratory replicates) used with the RC
rat-only millimole regression, log LD50 (mmol/kg) = 0.439 x log IC50 (mM) + 0.621, to assign GHS category. In
vivo category was based on reference rodent oral LD50 values (mg/kg) in Table 4-2. For each method, thereference substances evaluated were those for which all three laboratories obtained IC50 values.

8.5

Summary••••

The determinations of test method and data collection errors showed that FAL
consistently had the highest error levels; however, the laboratory’s GHS acuteoral toxicity category predictions were comparable to the other laboratories’
results.
The laboratories reported no significant deviations from the protocols, anddeviations that did occur during the testing phases were generally quickly
acknowledged and addressed by the Study Directors. If a deviation occurredthat would affect the data (e.g., improper concentration of DMSO solvent), theStudy Director would reject the test, notify the SMT, and perform an
additional test. Improper transfer of data to either the EXCEL® or PRISM®
templates, which would affect the data summaries and analyses, wererecognized, documented, and rectified by the Study Director and/or the SMT.
The SMT reviewed all data sheets to ensure that data were not inadvertently
attributed to the incorrect data summary files, and that the correct data wereused in all statistical analyses.
An electronic copy of all data for this validation study can be obtained fromNICEATM upon request by mail, fax, or e-mail to Dr. William S. Stokes,
NICEATM, NIEHS, P. O. Box 12233, MD EC-17, Research Triangle Park,NC, 27709, (phone) 919-541-2384, (fax) 919-541-0947, (e-mail)niceatm@niehs.nih.gov.

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9.0

OTHER SCIENTIFIC REPORTS AND REVIEWS OF IN VITROCYTOTOXICITY TEST METHODS AND THEIR ABILITY TO
PREDICT IN VIVO ACUTE TOXICITY AND OTHER TOXICEFFECTS.................................................................................................................. 9-3

9.1

Relevant Studies ....................................................................................................... 9-3
9.1.1 Correlation of NRU Cytotoxicity Values with Rodent Lethality .................. 9-3
9.1.2 Use of In Vitro Cytotoxicity Data to Reduce the Use of Animals in
Acute Oral Toxicity Testing .......................................................................... 9-7
9.1.3 Other Evaluations of 3T3 or NHK NRU Methods ...................................... 9-10

9.2

Independent Scientific Reviews ............................................................................ 9-13
9.2.1 In Vitro Acute Toxicity Testing for the Classification and Labeling ofChemicals..................................................................................................... 9-13
9.2.2 Use of In Vitro Cytotoxicity Data for Estimation of Starting Doses for
Acute Oral Toxicity Testing ........................................................................ 9-14
9.2.3 Validation of 3T3 NRU Assay for Phototoxicity ........................................ 9-17

9.3

Studies Using In Vitro Cytotoxicity Methods with Established
Performance Standards......................................................................................... 9-18
9.3.1 Guidance Document (ICCVAM 2001b)...................................................... 9-18
9.3.2 King and Jones (2003) ................................................................................. 9-19
9.3.3 A-Cute-Tox Project: Optimization and Pre-Validation of an In Vitro
Test Strategy for Predicting Human Acute Toxicity (Clemedson 2005)..... 9-19

9.4

Summary................................................................................................................. 9-20

9-1

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In Vitro Cytotoxicity Test Methods BRD Section 9

9.0

November 2006

OTHER SCIENTIFIC REPORTS AND REVIEWS OF IN VITROCYTOTOXICITY TEST METHODS AND THEIR ABILITY TO PREDICTIN VIVO ACUTE TOXICITY AND OTHER TOXIC EFFECTS

In vitro cytotoxicity methods based on NRU have been evaluated for a number of uses. Thissection reviews studies that used in vitro NRU cytotoxicity methods to:• Predict acute rodent oral toxicity• Predict starting doses for acute systemic toxicity tests• Predict other in vivo toxicity endpoints, including phototoxicity and eye
irritation.
Section 9.1 describes studies that evaluated in vitro cytotoxicity test methods that measured
NRU for its ability to predict acute systemic toxicity in rodents, and other in vivo endpoints.
Also reviewed are studies that evaluated the use of in vitro cytotoxicity results to reduceanimal use in acute toxicity testing. Section 9.2 reviews independent evaluations of the useof in vitro cytotoxicity methods to predict acute oral toxicity, and to determine starting doses
for acute systemic toxicity assays. Also discussed is a 3T3 NRU test method that has beenvalidated and accepted for regulatory use for detecting phototoxic potential using a protocol
similar to that used in the NICEATM/ECVAM validation study. The conclusions of thesereports will be compared to the conclusions reached in this study, wherever possible. Section
9.3 reviews published studies that used the Guidance Document approach (ICCVAM 2001b),
which established the current test method performance standard.
9.1

Relevant Studies

9.1.1
Correlation of NRU Cytotoxicity Values with Rodent Lethality
This section reviews five published in vitro cytotoxicity studies that correlated cytotoxicity
values (i.e., IC20 or IC50) from NRU cytotoxicity test methods that used various cell types, torat and/or mouse acute LD50 values from various exposure routes. In these sections, italics
are used to identify reference substances tested in the reviewed studies that were also tested
in the NICEATM/ECVAM validation study. Table 9-1 characterizes the substances tested in
the reviewed studies by providing the ranges of their rat oral LD50 values. Also shown forcomparison are the mouse and/or rat oral LD50 ranges for the NICEATM/ECVAM validation
study and the RC. The table shows that the substances tested by Peloux et al. (1992), Fautrel
et al. (1993), and Rasmussen (1999), covered a wide range of rat acute LD50 values. The
substances used by Roguet et al. (1993) and Creppy et al. (2004) covered a much smaller
range. Table 9-2 characterizes the test substances by chemical class based on NLM Medical
Subject Heading (MeSH®) descriptors.

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In Vitro Cytotoxicity Test Methods BRD Section 9

Table 9-1

November 2006

Rat Acute Oral LD50 Ranges for Test Substances Used in Previous In
Vitro NRU Cytotoxicity Studies and the NICEATM/ECVAM Study1

Study/Database
Peloux et al. (1992)
Fautrel et al. (1993)
Roguet et al. (1993)
Rasmussen (1999)
Creppy et al. (2004)
NICEATM/ECVAM Validation3
RC4

Rat Acute Oral LD50 Range (mg/kg)2
2 – 14500
2 – 14500
0.04 – 176
1 – 10298
48 – 9245
2 – 19770
1 – 31015

N
30
31
28
20
2
72
347

Abbreviations: N=Number of substances in the study/database; RC=Registry of Cytotoxicity.
1
Studies reviewed in Section 9.1.1.
2
Values cited in the studies or from references provided by the studies.
3
Current study summarized in this BRD.
4The RC includes both rat and mouse LD50 values.
5Upper limit of range is an LD50 calculated from the in vitro NRU IC50 because there was no in vivo value
available for that substance.

Table 9-2

Chemical Classes Represented by the Substances Used in Published
Studies for Correlation of In Vitro NRU Cytotoxicity with Rodent Acute
Lethality

Chemical Class1

Study2

Chemical Class1

Alcohols
AmidesAminesArsenicals
Carboxylic Acids
Chlorine
CopperEthers

1, 2, 3, 4
1, 2, 3
1, 2
3, 4
1, 2, 3, 4
3, 4
3, 4
1, 2

Fluorine
Heterocyclics
Hydrocarbons
Iron
Lactones
Lithium
Mercury
MetalsStudy2

Chemical Class1

Study2

3, 4
1, 2, 3, 4, 5
1, 2, 3, 4, 5
3
1, 2
1, 2, 3, 4
3, 4
3, 4

NitrilesNitrogen
Organophosphates
Phenols
Polycyclics
Potassium
SodiumSulfur

1, 2
3, 4
3, 4
3, 4
3
3, 4
3, 4
1, 2, 3, 4

Study references: 1=Peloux et al. (1992) (24/25 substances were organic compounds); 2=Fautrel et al. (1993)(30/31 substances were organic compounds); 3=Roguet et al. (1993) (22/30 substances were organic
compounds); 4=Rasmussen (1993) (13/20 substances were organic compounds); 5=Creppy et al. (2004) (2/2
substances were organic compounds).
1
Classification by NLM Medical Subject Heading (MeSH®) descriptors.
2
Studies reviewed in Section 9.1.1.

9.1.1.1 Peloux et al. (1992)
The authors used several different in vitro cytotoxicity methods with primary rat hepatocytes
to determine the correlation with rat/mouse intraperitoneal (i.p.) or intravenous (i.v.) LD50
values for the 25 substances tested. The in vitro cytotoxicity methods, which used 20-hour
test substance exposure durations, assessed the following endpoints: NRU, total protein
content, LDH release, MTT reduction. MTT is metabolized by mitochondrial succinate
dehydrogenase of viable cells to yield a purple formazan reaction product. The IC50 values

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In Vitro Cytotoxicity Test Methods BRD Section 9

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obtained using the four endpoints were highly correlated (r = 0.973 to 0.999) to each other.
When performing the IC50-LD50 regressions, Peloux et al. (1992) used the lowest reported
published LD50 value for acute rat or mouse studies that administered the test substances
using the i.p. or i.v. routes. The IC50 values obtained using NRU as the endpoint had thehighest correlation coefficient, r = 0.877, to the rat/mouse i.p./i.v. LD50 values. The total
protein assay yielded r = 0.872, the MTT reduction assay yielded r = 0.808, and the LDH
release assay yielded r = 0.789.
Peloux et al. (1992) followed the recommendations of Fry et al. (1988, 1990) and usedparenteral LD50 values rather than oral LD50 values for comparison with in vitro values. Fry
et al. (1988, 1990) recommended the use of the i.p./i.v. LD50 values for comparisons because
they proposed that cells in vivo receive a more direct test substance exposure via these routes
than through the oral route. They had posited that in vitro cell cultures would mirror this(direct) toxicity because they also receive direct exposure to test substances via the cell
culture medium. The authors also noted that the oral route of exposure presents confounding
variables such as, 1) only a fraction of a test substance would be available in the systemic
circulation due to limited absorption or pre-systemic metabolism, and 2), the level of thesubstance in the systemic circulation decreases due to elimination mechanisms (e.g.,
metabolism, excretion). Fry et al. (1990) had reported a correlation of only r = 0.49 for invivo/in vitro comparisons of oral LD50 and IC50 values (from a total protein assay) and acorrelation of r = 0.68 for i.p. LD50 and ID50 values1.
9.1.1.2 Fautrel et al. (1993)
Six laboratories tested the cytotoxicity of 31 substances in primary rat hepatocyte culturesusing a 24-hour exposure followed by measurement of NRU. The investigators performed
linear regression analyses for the prediction of rat i.v., i.p., and oral LD50 values from the
NRU IC50 values. The regressions for the various in vivo administration routes did not use thesame substances because LD50 values were not available for all of the tested substances in allof the routes. Oral, i.v., and i.p. LD50 values were available for 27, 24, and 18 substances,respectively, and IC50 values were obtained for 15, 14, and 11 of these substances,respectively. The regression for the i.v. data was statistically significant (r = 0.88, n = 11),
but the i.p. (r = 0.48, n = 14) and oral regressions (r = 0.17, n = 15) were not. The finding thatthe i.v. LD50 values corresponded more closely with the in vitro cytotoxicity data than did theoral LD50 was thought to be the result of having fewer pharmacokinetic variables (i.e.,
absorption, distribution, etc.) to consider following i.v. administration.
9.1.1.3 Roguet et al. (1993)
Roguet et al. (1993) tested the cytotoxicity of 28 MEIC substances in primary rat hepatocytes
exposed for 21 hours, followed by the measurement of NRU. A correlation of the NRU IC50
values to oral LD50 values obtained from the unpublished data of B. Ekwall et al. (personalcommunication) yielded a statistically significant linear correlation (p <0.001) with r = 0.80
when the in vivo and in vitro data were in molar units. [NOTE: The LD50 values subsequently
published by Ekwall et al. (1998) were from the 1997 edition of RTECS®.] The authors
reported that the toxicities of thioridazine, malathion, and copper sulfate were overestimated,
and the toxicity of potassium cyanide was underestimated by the correlation, but their criteria
for over- and under- estimation were not provided.
1

ID50: index of cytotoxicity; concentrations (µg/mL) producing a 50% reduction in protein value.
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In Vitro Cytotoxicity Test Methods BRD Section 9

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The in vivo toxicity of potassium cyanide was also underpredicted in the
NICEATM/ECVAM validation study. Table 6-3 shows that potassium cyanide was an
outlier for which toxicity was underpredicted when using the IC50 values from both the 3T3
and NHK NRU test methods in the RC millimole regression (log LD50 mmol/kg = 0.435 log
IC50 mM + 0.625). The GHS category predictions using both NRU test methods and the RC
rat-only millimole regression (log LD50 mmol/kg = 0.439 log IC50 mM + 0.621), and the RC
rat-only weight regression (i.e., log LD50 =0.372 log IC50 + 2.024), were also higher (i.e., lesstoxic) than the in vivo category (see Appendix L2).
9.1.1.4 Rasmussen (1999)
Twenty MEIC substances were tested for cytotoxicity using NRU release from 3T3 cells
following 24-hr exposure, with and without the addition of a Aroclor-induced rat liver
microsomal preparation (S9 mix). Similar to the present validation study, Rasmussen (1999)
observed that xylene was non-toxic to the cells, even though it was dissolved in ethanol
instead of DMSO. In the presence of S9, the cytotoxicities of malathion, 2,4dichlorophenoxyacetic acid, propranolol, thioridazine, lithium sulfate, copper sulfate, andthallium sulfate, were significantly decreased (p <0.05), while the cytotoxicities of 1,1,1trichloroethane, phenol, nicotine, and paraquat were significantly increased (p <0.05).
Because the NICEATM/ECVAM validation study used cells with little or no xenobiotic
metabolizing capability, it could be expected that these systems would overpredict the
toxicity of substances that would be inactivated by the addition of a metabolizing system, or
to underpredict the toxicity of substances that are metabolized to more toxic substances.
None of the four substances in common for which toxicity was decreased by the addition of
S9 were overpredicted in the NICEATM/ECVAM study. However, the toxicities of two of
the four substances in common for which toxicity was increased by the addition of S9, were
underpredicted in the NICEATM/ECVAM study. Table 6-3 shows that nicotine was an
outlier whose toxicity was underpredicted when using the 3T3 and NHK IC50 values in the
RC millimole regression (log LD50 mmol/kg = 0.435 log IC50 mM + 0.625). Paraquat was an
outlier whose toxicity was underpredicted when using the NHK IC50 value in the RC
millimole regression. The GHS category predictions for both substances using both NRU test
methods with the RC rat-only millimole regression (log LD50 mmol/kg = 0.439 log IC50 mM+ 0.621) and the RC rat-only weight regression (log LD50 mg/kg = 0.357 log IC50 µg/mL +2.194) were also higher than the in vivo category (see Appendix L2).
Although both the IC20 and IC50 values were determined in the Rasmussen (1999) study, only
the IC20 values were used for correlations with the rat acute oral LD50 values from RTECS®.
The units of the LD50 values were not reported, but the correlations were assumed to be inmolar units because the IC20 and IC50 values were reported in µM units. Significant
correlations (p <0.001) between IC20 and LD50 values were obtained with and without rat
liver microsomes. The correlation of IC20 with LD50 was slightly higher with the S9 mix (r =0.72 vs. 0.68 for oral LD50 values, and 0.82 vs. 0.78 for i.p. LD50 values).
Although the presence of S9 increased the cytotoxicity of some substances to the 3T3 cells, it
decreased the toxicity of others, and yielded only a small improvement in the correlation to invivo data. Rasmussen (1999) concluded that the toxicity of the S9 mix (0.32 mg protein/mL),
itself, was insignificant because it reduced cell survival by less than 10% compared with cells

9-6In Vitro Cytotoxicity Test Methods BRD Section 9

November 2006

without S9. However, others have shown that S9 microsomal mixes could produce
significant cytotoxic effects. Kohn (1993) showed that an S9 mix containing 0.07 mg
protein/mL was cytotoxic to all types of murine neurons in culture when the cells were
exposed for four days or longer. Non-neuronal cells tolerated higher concentration exposuresof S9, but exhibited cytoplasmic inclusions when exposed to S9 at 0.35 mg protein/mL. Dal
Negro et al. (2006) reported 100% cell death of human monocyte-derived U-937 cells when
the S9 fraction (1 mg protein/mL) and co-factors were applied to the cells for a 72-hour
incubation. Both of these studies used longer exposure durations, and/or higher protein
concentrations, than the Rasmussen (1999) study.
9.1.1.5 Creppy et al. (2004)
Creppy et al. (2004) used a 48-hour NRU assay to determine the cytotoxicity of ochratoxin A(OTA) and fumonisin B1 (FB1) on cultured C6 glioma (rat brain), Caco-2 (human intestinal),
and Vero (green monkey kidney) cells. The IC50 determined in the NRU assay was used in
the RC millimole regression to predict rodent acute oral LD50 values. The predicted LD50 for
OTA using the C6 glioma cells was similar to mouse LD50 values generated from four in vivo
mouse studies, but the LD50 values predicted by the other cell lines were about 50 times
greater. The authors found the relative insensitivity of the Vero cells surprising because OTA
is a kidney toxin. There were no available in vivo rodent oral LD50 values with which tocompare the predicted LD50 of FB1, which ranged from 671 to 924 mg/kg for the three cell
types tested.
9.1.2

Use of In Vitro Cytotoxicity Data to Reduce the Use of Animals in Acute Oral
Toxicity Testing

9.1.2.1 Halle et al. (1997): Animal Savings with the ATC Method Using Cytotoxicity Data
This study assessed the animal savings that would be produced by using IC50 data in an IC50LD50 regression to determine a starting dose for ATC testing. No cytotoxicity testing was
performed for this study. Instead, the authors used the IC50 values from the RC database andthe RC millimole regression to predict the LD50 for 347 RC substances. The predicted LD50
values were then used to determine the starting doses for simulated ATC testing.
At the time of the Halle et al. (1997) study, the ATC method (1996 version from OECD) was
designed to classify substances using three classes of acute oral toxicity and an unclassified
group, as defined by the acute oral toxicity classification system of the EU (see Table 9-3).
As a result, the fixed doses for the ATC testing were 25, 200, and 2000 mg/kg. The authors
used the LD50 predicted by the RC IC50 and the RC millimole regression for the 347 RC
substances as a starting point to estimate the number of ATC dose steps, and number ofanimals, that would be needed to classify the substances in the EU category associated withthe rodent oral LD50 (i.e., rat or mouse values from RTECS®). The method required thesimulated ATC testing for each substance to start at the fixed ATC dose nearest to thepredicted LD50. The outcome of the simulated testing of three animals per fixed dose wasdetermined by the in vivo LD50. If the test dose was lower than the in vivo LD50, animals
were assumed to live and, if the test dose was higher than the LD50, the animals wereassumed to die. Testing of the substance would proceed with higher (when the animals lived)or lower fixed doses (when the animals died) until the substance was placed into the EU
toxicity category indicated by the in vivo rodent oral LD50.

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In Vitro Cytotoxicity Test Methods BRD Section 9

Table 9-3

November 2006

EU1 Classes of Acute Oral Toxicity
Category

LD50 (mg/kg)

1

LD50 ≤252

25 < LD50 ≤200

3

200 < LD50 ≤2000

Unclassified

LD50 >2000

Abbreviations: EU=European Union
1
Anon (1993)

The method of Halle et al. (1997) can be illustrated with digoxin, which has an in vivo mouse
LD50 of 18 mg/kg (from RTECS®). The predicted LD50 of 414 mg/kg was calculated using
the RC IC50 in the RC millimole regression (log LD50 [mmol/kg] = 0.435 x log IC50 (mM) +0.625). Simulated ATC testing would start at the nearest fixed dose, 200 mg/kg. The three
animals tested were assumed to die, and then three more animals would be tested at 25
mg/kg. The animals tested at 25 mg/kg were assumed to die and digoxin would be classifiedin category 1 for LD50 ≤25 mg/kg. Thus, the classification of digoxin using the 4-categorysystem required six animals.
Using such simulations of ATC testing, Halle et al. (1997) estimated that 2139 animals
would be used to test the 347 substances:• Three hundred twenty-eight would require testing with two doses using threetest animals each.
• Nineteen would require testing with three doses using three animals each.
Halle et al. (1997) cited Schlede et al. (1995) in reporting that the average number of animals
required to classify substances using the ATC method was 9.11 animals per test. Using thisaverage, ATC testing of the 347 RC substances would require 3161 animals. Thus, Halle etal. (1997) estimated that there would be a 32% reduction ([3161-2139]/3161) in the numberof test animals used when the LD50 prediction from the RC millimole regression was usedwith the 1996 version of the ATC method, in lieu of the standardanimal classification
procedure (Halle et al. 1997).
The simulated average animal savings for the ATC in the NICEATM/ECVAM validation
study at dose-response slopes of 2.0 and 8.3 was 4.8% to 10.2% (0.51 to 1.09 animals) for
the 3T3 (67 reference substances) and NHK (68 reference substances) NRU test methods(see Section 10.3.3.2), depending on the regression evaluated. This is considerably lower
than the average savings of 32% estimated by Halle et al. (1997). However, there are anumber of differences between the evaluation performed by Halle et al. (1997) and the
NICEATM/ECVAM study that contribute to the difference in calculated animal savings:• The NICEATM/ECVAM study used six GHS acute toxicity categories forclassification whereas Halle et al. (1997) used the EU toxicity classification
scheme, which had only four toxicity categories. The accuracy of category
prediction by any method would be higher with fewer categories.

9-8

In Vitro Cytotoxicity Test Methods BRD Section 9

•

•

•

•

November 2006

The NICEATM/ECVAM study used experimentally derived in vitro
cytotoxicity data from a standardized protocol to estimate starting doses(using two regressions based on the RC substances with rat LD50 data),
whereas Halle et al. (1997) used IC50 data from the RC database.
The reference substances tested in the NICEATM/ECVAM study poorly fit
the RC millimole regression. Nearly half of the reference substances evaluatedwere outliers (28/70 [40%] in the 3T3 NRU test method, and 31/71 [44%] in
the NHK NRU test method) (see Table 6-3). The RC database had 95/347(27.4%) substances outside of the prediction intervals.
The NICEATM/ECVAM study used computer simulations of ATC testing,which incorporated assumptions about mortality distributions, to determine
animals used, whereas Halle et al. (1997) used simplified assumptions (i.e., allanimals lived when test dose was less than the in vivo LD50 and all animals
died when test dose was greater than the in vivo LD50).
The NICEATM/ECVAM study determined animal savings by comparing
animal use with starting doses determined by the in vitro data, to animals usedat the default starting dose of 300 mg/kg. Halle et al. (1997) used the averageanimal use for the ATC for comparison to animal use with simulated testing.

9.1.2.2 Spielmann et al. (1999): Animal Savings Using Cytotoxicity Data with the UDP
Spielmann et al. (1999) recommended an in vitro cytotoxicity procedure as a range finding
test for the in vivo toxicity test to reduce the number of animals used in acute toxicity tests.
The authors identified nine substances in both the RC database and an evaluation of acutetoxicity methods by Lipnick et al. (1995). They then compared the LD50 values from Lipnick
et al. (1995) to LD50 predictions calculated when using the RC IC50 values in the RC
millimole regression formula (log LD50 [mmol/kg] = 0.435 x log IC50 [mM] + 0.625). For
seven of the nine substances, the LD50 prediction was within an order of magnitude of theexperimental LD50 reported by Lipnick et al. (1995). Spielmann et al. (1999) concluded that
the RC millimole regression provided an adequate prediction of LD50, and that in vitro
cytotoxicity data could be used to predict starting doses for the UDP. The authors
recommended using the IC50, with the RC millimole regression, to calculate a starting dose
(i.e., an estimated LD50) for the UDP, FDP, or ATC method whenever an IC50 was available.
If no IC50 was available, Spielmann et al. (1997) recommended determining cytotoxicity
using a standard cell line and specific cytotoxic endpoint (e.g., NRU, total protein, MTT
reduction). They recommended testing 10 to 20 RC substances to demonstrate that the invitro cytotoxicity test methods provide results that are consistent with the RC millimole
regression. The resulting IC50 values would then be used to calculate a new regression (using
the LD50 values reported in the RC), which would be compared to the RC millimole
regression. If the new regression fit into the acceptance interval (± log 5 of the fitted
regression line) of the RC millimole regression, the RC millimole regression would be used
to predict starting doses for the UDP. If the new regression is parallel to the RC millimole
regression, but outside the ± log 5 acceptance interval, then the new regression would be
used for the prediction of the starting dose.
Spielmann et al. (1999) contended that the RC millimole regression provides a sufficient
prediction of LD50 values from IC50 values for substances that do not require metabolic
9-9In Vitro Cytotoxicity Test Methods BRD Section 9

November 2006

activation and are not very toxic (i.e., LD50 > 200 mg/kg). The authors acknowledged that thefit of substances with LD50 <200 mg/kg to the RC millimole regression is not good, andattributed the poor fit of these substances to the need for metabolic activation to a more toxic
substance. They suggested that the prediction of starting doses using cytotoxicity data can beused with the UDP and ATC methods, but not with the FDP because dosing is not sequential(which contradicted a claim made earlier in the paper that the approach could be used with
the FDP). They did not estimate the number of animals that might be saved with thisapproach, but did recommend that the approach be validated experimentally using several
established cell lines with a limited number of representative substances from the RC.
9.1.2.3 EPA (2004): U.S. EPA HPV Challenge Program Submission
In response to the EPA HPV Chemical Challenge Program, PPG Industries, Inc., themanufacturer of Propanoic acid, 2-hydroxy-, compound with 3-[2-(dimethylamino)ethyl] 1(2-ethylhexyl) (4-methyl-1,3-phenylene)bis[carbamate] (1:1) [CASRN 68227-46-3], and thesponsor of this compound, submitted data to the EPA. This is an isolated intermediate used to
produce a resin component of paint products. PPG provided the following types of data intheir submission to the EPA: physical-chemical, environmental fate and pathway,
ecotoxicity, and toxicology. The acute mammalian toxicology data were generated using
both in vitro and in vivo methods.
An in vitro NRU cytotoxicity test was conducted with 3T3 cells to estimate a starting dose
for the in vivo acute UDP oral toxicity test (OECD 2001a) (see Appendix M1 for the OECD
UDP test guideline). The use of this in vitro NRU test method was intended to minimize thenumber of animals used for in vivo testing. The estimated LD50 of the compound as
determined by the NRU assay was 489 mg/kg. Therefore, the starting dose for the UDP study
was set at 175 mg/kg, which is the first default dose below the estimated LD50 value; this isalso the default starting dose for the UDP, and is used when no information on which to basea starting dose is available. A total of fifteen female rats received the compound at 175, 550,
or 2000 mg/kg. Five of nine rats treated at 2000 mg/kg died prematurely on Days 2 and 3,
and by Day 15, 2/4 surviving animals at this dose had lost up to 25% of their Day 1 bodyweights. The LD50 was estimated to be 2000 mg/kg, with a 95% confidence interval of 11235700 mg/kg. Thus, the in vitro NRU test method overpredicted the toxicity of the compoundby estimating an LD50 value that was lower than that determined in the UDP test. The reportauthors reported that a greater than predicted number of animals was used for the UDP
testing because the estimated LD50, 489 mg/kg and, consequently, the starting dose, wasmuch lower than the in vivo LD50 of 2000 mg/kg. However, because the UDP started with thedefault starting dose of 175 mg/kg, the claim that more animals were used is incorrect,
because animal use with the default starting dose is the baseline against which other animal
use should be compared.
9.1.3
Other Evaluations of 3T3 or NHK NRU Test Methods
This section briefly reviews five studies that evaluated NRU test methods for purposes otherthan the prediction of starting doses for acute oral toxicity assays. NRU test methods using
either 3T3 or NHK cells have been evaluated for use as alternatives to the Draize eyeirritation test, to measure phototoxicity, and to predict acute lethality in humans. Except forthe 3T3 NRU phototoxicity assay, NRU methods have not been scientifically validated by anindependent review for any of these purposes or accepted for regulatory use. The use of the

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November 2006validated 3T3 NRU test method to determine phototoxic potential is addressed in Section
9.2.
The in vitro NRU protocols evaluated in the five reviewed studies are similar to those used in
the NICEATM/ECVAM validation study, all of which were based on the method ofBorenfreund and Puerner (1985). The major difference is that most studies used a 24-hour
test substance exposure duration for the 3T3 NRU test method, while the
NICEATM/ECVAM 3T3 study used a 48-hour exposure duration. The major difference
between the NHK protocols used in the reviewed studies and the protocol used in the
NICEATM/ECVAM study is that the cell culture medium was changed at the time of test
substance application in the NICEATM/ECVAM study.
9.1.3.1 Draize Eye Irritation
Triglia et al. (1989)
Four laboratories collaborated in an interlaboratory validation study to test the NHK NRU
assay marketed by Clonetics® Corporation2 for its intra- and inter-laboratory reproducibility
and ability to predict in vivo ocular irritancy. Each laboratory tested 11 blind-codedsurfactant-based substances and compared the IC50 values to in vivo Draize ocular irritancy
scores.
The test exhibited the following performance characteristics for the comparison of in vitro
and in vivo data:• Specificity (percentage of non-irritants correctly detected) = 93%• Sensitivity (percentage of true irritants correctly detected) = 80%• Predictive values (probability that an unknown agent will be properlyclassified)o Positive predictive value = 90%
o Negative predictive value = 87%
The authors reported that there was excellent correlation among the laboratories, and goodcorrelation between the in vitro IC50 values and in vivo Draize scores (Spearman Rankcorrelation coefficients between in vivo and in vitro data for the laboratories ranged from
0.67-0.76). The authors also concluded that the NRU test could not replace the Draize test,
but may be an effective screening tool for use in a battery of in vitro alternatives
Sina et al. (1995)
Sina et al (1995) evaluated the NHK NRU test method along with six other in vitro methods
to determine whether they could be used as complimentary tests in a battery approach toestimate ocular irritation. The NRU data correlated poorly with Draize ocular scores for the
33 pharmaceutical intermediates tested. The Spearman correlation coefficient for the IC50 andmaximum average Draize score (MAS) was -0.10, and the Pearson correlation coefficient
was -0.04.

2

Clonetics® Corporation sponsored this study. It was not clear in the publication if Clonetics® Corporation
participated as one of the testing laboratories.
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Brantom et al. (1997)
This study examined the potential of 10 alternative methods to predict the eye irritation
potential of cosmetic ingredients. Four laboratories tested 55 coded substances (23 single
ingredients and 32 formulations) using the 3T3 NRU test method, and used the resulting IC50
values to predict modified maximum average scores (MMAS) for the Draize test.
An endpoint was generated for each test by interpolation from a plot of percent cell survival
versus test substance concentration. A prediction model was developed from data of 30
single ingredients (29 surfactants and one substance not classified by the authors) to equate
the IC50 value to an MMAS.
The interlaboratory CV for the IC50 values was 37.3 ± 29.8% (7.5 ± 6.8, log transformed).
Most of the mean IC50 values from a single laboratory differed by plus or minus an order ofmagnitude from the means of all the laboratories for each substance, which the authorsinterpreted as “no significant outliers”. Correlations of NRU-predicted MMAS scores with invivo MMAS scores yielded Pearson’s r values ranging from 0.25 to 0.32 for the four
laboratories.
Although the authors concluded the interlaboratory reproducibility was good, the IC50 values
did not predict the MMAS. The r values for the in vitro/in vivo correlations were low (0.246
to 0.316) and the tests all underpredicted irritants and overpredicted non-irritants. Four
substances were outside of the 95% confidence intervals and the authors concluded that the
3T3 NRU test method had wide applicability to test the remaining 51 coded substances
according to the limitations in the prediction model, but that it was not effective as a standalone replacement for the Draize test across the entire irritation scale. The authors did not
identify the test substances.
Harbell et al. (1997)
This publication reported the results of the evaluation of 12 in vitro cytotoxicity assays topredict ocular irritation. Data were voluntarily submitted to the U.S. Interagency Regulatory
Alternatives Group (IRAG), composed of members from CPSC, EPA, and FDA. The NHK
NRU test method was one of the tests evaluated by six laboratories testing surfactants andsurfactant-containing formulations (the 3T3 NRU test method was not tested). Twolaboratories submitted results for the same test substances, but the other four submitted datafor various sets of substances and formulations.
The correlation of results from the two laboratories that independently tested the same
substances was r=0.99. Correlations between the IC50 data and in vivo maximum average
Draize score (MAS) ranged from -0.92 to -0.54. The IRAG concluded that the assays weresuitable as a screening and adjunct assay to assess eye irritation over the range of toxicities
found in personal care and household products, and recommended that its use be limited towater-soluble materials. Although the method was also evaluated for surfactants, IRAG
recommended that the evaluation continue for its performance in predicting eye irritation forvarious product classes (e.g., fabric softeners, shampoos). In addition, the substance’s
physical form should be considered because the in vitro toxicity of a solution of the test
substance will not necessarily predict toxicity of the parent, solid substance in vivo.

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9.1.3.2 Predicting Human Lethal Blood Concentrations (LC)Seibert et al. (1992)
This single laboratory study was designed to evaluate various aspects of cellular toxicity infour in vitro test systems for their relevance and reliability with respect to acute systemic
toxicity, in particular, human LC. The 3T3 NRU test method was one of four methods
evaluated with 10 MEIC substances.
The authors stated that final conclusions on the relevance of the in vitro systems for in vivo
data could not be determined because the variations in LC were unknown so that limits forover or underprediction of human in vivo toxicity using experimental models could not be
defined. In addition, the ability of in vitro toxicity to predict in vivo toxicity may depend on
toxicokinetic factors that were not considered in the in vitro systems.
9.2

Independent Scientific Reviews

This section summarizes independent scientific reviews of the use of in vitro cytotoxicity
methods for the prediction of rodent acute oral toxicity, and for the reduction of animal use inacute toxicity testing. The conclusions of these reviews are compared to the conclusions of
the current study. Also discussed is the 3T3 NRU phototoxicity method, because it is similar
to the 3T3 NRU test method used in the current validation study and has been validated by
ECVAM and is the subject of OECD Test Guideline 432 (OECD 2004).
9.2.1

In Vitro Acute Toxicity Testing for the Classification and Labelling of Chemicals

9.2.1.1 Seibert et al. (1996): ECVAM Workshop 16
ECVAM sponsored a workshop in 1994 to review the current status of various in vitro
methods and to determine their potential uses for reducing, refining, and/or replacing the useof laboratory animals for acute systemic toxicity testing. The workshop participants reviewed
various types of toxicity, in vitro cytotoxicity testing schemes and strategies, inclusion of
biokinetic parameters, biotransformation, biodistribution in vitro and in vivo, and a proposed
acute toxicity testing scheme for the classification of substances.
The workshop participants agreed that some studies showed good correlations between invitro cytotoxicity data and LD50 values. They also acknowledged that in vitro basal
cytotoxicity tests could not address all the different of mechanisms of acute systemic toxicity.
Additional approaches to replacing animals would have to incorporate the three main typesof cellular level toxic effects that can lead to in acute systemic toxicity (i.e., basal
cytotoxicity, selective toxicity, and cell-specific function toxicity). The participants
determined that it is also important that any alternative method take into account the active
concentration and meaningful dose of a test substance in an in vitro cell culture system.
Quantitative comparisons of test substance concentrations must be made to evaluate theeffects of the test substances regarding the three types of cytotoxicity.
The biokinetics of a test substance (determined by its absorption, distribution, metabolism,and elimination) must be considered when making predictions of in vivo toxicity using invitro toxicity data. Various methods can be used to convert in vitro effective concentrationsof a test substance to equivalent body doses. Test substance factors, such as physicochemical
characteristics (e.g., pKa, lipophilicity, volatility), estimates of protein binding, and in vitro

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characteristics (e.g., cell concentration, cell protein concentration, ratio of cell/medium
volumes, medium albumin concentration), are needed for such conversions.
An in vitro tiered testing scheme was proposed by the workshop participants for using invitro methods to determine the acute oral toxicity of a substance:• Stage 1: Basal cytotoxicity test
• Stage 2: Hepatocyte-specific cytotoxicity test to assess the role ofbiotransformation in producing toxicity• Stage 3: Test system that evaluates non-hepatocyte-specific selective
cytotoxicity (i.e., effects on cell-specific functions)This testing scheme was proposed as an approach to classify substances by their in vitro
toxicity. The lowest IC50 value determined at any of the testing stages would be used toclassify a substance (i.e., very toxic, toxic, harmful, and no label). The workshop participants
recommended that a feasibility study be conducted to determine the practicability, relevance,and reliability of this tiered testing scheme. As noted in the NICEATM/ECVAM study (seeSection 6.4), the in vitro basal cytotoxicity tests are not suitable as replacements for rodent
acute oral toxicity tests and could only be used as an adjunct test, and not a stand-alone test,
for classifying substances for acute oral toxicity. However, in vitro tests could be used to
identify starting doses for acute toxicity testing to reduce the number of animals used.
9.2.2

Use of In Vitro Cytotoxicity Data for Estimation of Starting Doses for Acute Oral
Toxicity Testing

9.2.2.1

ICCVAM (2001a): Estimation of Animal Savings Using Cytotoxicity Data with the
ATC Method
Participants at Workshop 2000 examined the influence of starting dose on animal use in the
ATC method (ICCVAM 2001a; Section 2.2.3, pp.12-14; no testing was performed at theWorkshop). The participants made inferences from the 1996 version of the ATC method thatwas based on the EU toxicity classification system (Table 9-1). The fixed doses for testing
were 25, 200, and 2000 mg/kg. Normally, classification of a substance requires testing three
animals in two to four dosing steps (i.e., six to 12 animals). The number of dosing steps
increases with increasing distance between the true toxicity class and the starting dose. They
estimated that one to three dosing steps could be avoided (i.e., three to nine animals saved) if
the optimum starting dose could be predicted by in vitro cytotoxicity testing.
The predicted savings of one to three dosing steps was made under ideal conditions. The
Workshop 2000 report (ICCVAM 2001a) provides a biometric analysis at a dose-mortality
slope of 2.0 that shows that the greatest animal savings would occur for substances with very
high and very low toxicity. Three animals are needed to classify a substance in the <25
mg/kg class if the true LD50 is 1 mg/kg and 25 mg/kg is the starting dose, but six animals areneeded if the test starts from the default starting dose of 200 mg/kg (i.e., an animal savings of50%). For a substance with a true LD50 of 10000 mg/kg, 11.3 animals on average are needed
when the default starting dose is used, but only 7.7 animals would be needed at the 2000
mg/kg starting dose (i.e., an animal savings of 31%). For substances with a true LD50 of 2000
mg/kg, no animals would be saved by starting at the 2000 mg/kg dose (compared to startingat the default starting dose of 200 mg/kg).

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Although these analyses were performed assuming the 1996 ATC method used starting dosesof 25, 200, 2000 mg/kg, the Workshop 2000 participants noted that the animal savings thatwould be produced by improving the starting dose would not be significantly different for thecurrent ATC method that uses GHS doses of 5, 50, 300, and 2000 mg/kg (or up to 5000
mg/kg) (OECD 2001c; see Appendix M for the current ATC test guideline). The Workshop
2000 participants did not predict the animal savings when in vitro cytotoxicity methods areused to estimate starting doses for the ATC, other than the biometric analysis describedabove.
The NICEATM/ECVAM study yielded patterns of animal savings with the ATC that weresimilar to those discussed at the 2000 Workshop (i.e., animal savings were greater for
substances with a lower or higher LD50 than the default starting dose; see Section 10.3.3.3).
Depending on the NRU test method and regression evaluated, the average animal savings per
test (for the 67 or 68 reference substances evaluated) predicted by the NICEATM/ECVAM
7validation study at a dose-mortality slope of 2.0 were:• 22.6 to 30.4 % (2.21 to 2.96 animals) for substances in the LD50 ≤5 mg/kg
category• 10.2 to 13.0 % (1.17 to 1.51 animals) for substances in the 5< LD50 ≤50
mg/kg category• 3.8 to 4.3 % (0.42 to 0.47 animals) for substances in the 50< LD50 ≤300
mg/kg category• -9.5 to -6.1% (-0.93 to -0.60 animals) for substances in the 300< LD50 ≤2000
mg/kg category• -0.03 to 12.7% (-0.30 to 1.43 animals) for substances in the 2000< LD50
≤5000 mg/kg category• 17.1 to 25.5% (2.03 to 3.02 animals) for substances with LD50 >5000 mg/kg
The major differences between the evaluation reviewed by the Workshop 2000 participants
and the NICEATM/ECVAM study were:• The NICEATM/ECVAM study used the GHS toxicity categories forclassification whereas the Workshop participants used the EU classification
scheme, which has fewer toxicity categories. The accuracy of category
prediction is higher with fewer categories.
• The NICEATM/ECVAM study used in vitro cytotoxicity data to estimate
starting doses using two regressions based on the RC substances with rat LD50
data, whereas the Workshop 2000 participants used the fixed ATC doses as
starting doses.
• The NICEATM/ECVAM study used computer simulations of ATC testing forindividual substances whereas Workshop 2000 participants used an evaluationthat estimated animal use based on fixed in vivo LD50 values and the fixed
ATC doses.

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9.2.2.2

ICCVAM (2001a): Estimation of Animal Savings Using Cytotoxicity Data with the
UDP
Workshop 2000 participants examined the effect of starting dose on animal usage in the UDP
assay by making inferences from the computer simulations of animal use shown in the peerreview BRD for the UDP (ICCVAM 2000). When the rule that requires testing to stop whenfour animals have been tested after the first reversal is used, and no other stopping rules are
considered, the animal use is relatively insensitive to the slope of the dose-mortality curve.
The number of animals required when the starting dose equals the true LD50 is approximately
six. However, approximately nine animals are required when the starting dose is 1% of thetrue LD50. Thus, animal use is 30% less when the starting dose is the true LD50 compared to astarting dose that is 1% of the true LD50 (ICCVAM 2001a, section 2.2.4, pg. 16). When UDP
testing stops based on the likelihood-ratio stopping rule, the animal use depends principallyon the slope of the dose-mortality curve. The Workshop 2000 participants estimated that 25
to 40% of the animals would be saved when the starting dose is equal to the true LD50,
compared to the savings at a starting dose 1% of the true LD50.
According to the UDP BRD (ICCVAM 2000) used by the Workshop participants, UDP
simulations at a mortality-response slope of 2.0 showed that an average of 12.4 animals per
test were used when the starting dose was 1% of the true LD50, but an average of 8.7 animals
was used when the starting dose was the true LD50 (i.e., a 30% reduction). At a slope of 8.3,
an average of 11 animals per test were used when the starting dose was 1% of the true LD50,
but an average of only six animals were used when the starting dose was the true LD50 (i.e., a
46% reduction). The animal savings predicted by Workshop 2000 participants was 25 to 40%
based on starting at the true LD50 in comparison to starting at a dose that is 1% of the true
LD50.
Depending on the regression evaluated, the average animal savings predicted in the
NICEATM/ECVAM validation study at dose-response slopes of 2.0 and 8.3 were 5.8 to
7.8% (0.49 to 0.66 animals) using the 3T3 (67 reference substances) and NHK (68 reference
substances) NRU test methods (see Section 10.2.3). When averaged for the reference
substances in each GHS category, the highest mean animal savings at a mortality-response
slope of 2.0 was obtained for reference substances in the 2000 < LD50 ≤5000 mg/kg and LD50
>5000 mg/kg categories. Animal savings were 11.3 to 16.7% (1.28 to 1.65 animals) using the
3T3 and NHK NRU test methods for the two regressions evaluated. The average animalsavings for the substances in these categories at a dose-mortality slope of 8.3 were 12.1 to
21.0% (1.11 to 1.63 animals) for both methods and regressions. The major differences
between the evaluation performed by the Workshop 2000 participants and the
NICEATM/ECVAM study were that:• The default starting dose used for the NICEATM/ECVAM simulations was
175 mg/kg (see Section 10.2.2), rather than 1% of the true LD50 assumed by
the Workshop 2000 participants.
• The NRU IC50 was used in two regressions of in vitro data against in vivo data
to estimate starting doses. This estimation was not always close to the true
LD50, which was the value used by the Workshop 2000 participants. For
example, LD50 values predicted by the NICEATM/ECVAM study forphenylthiourea were approximately 540 mg/kg by the 3T3 IC50 and

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approximately 904 mg/kg by the NHK IC50 using the RC rat-only millimole
regression. The true in vivo LD50 for phenylthiourea is 3 mg/kg. Workshop
2000 participants used a best-case scenario when they assumed that in vitro
cytotoxicity precisely predicted the true LD50.
9.2.3
Validation of the 3T3 NRU Assay for Phototoxicity
An NRU assay using 3T3 cells was validated by ECVAM, and accepted for regulatory use,
to detect the phototoxic potential of test substances. The 3T3 NRU test for phototoxicity
requires a 60-minute exposure to the test substance, a 50-minute exposure to ultraviolet(UVA, 315-400 nm) light, followed by removal of test substance and incubation for another24 hours in fresh medium (Spielmann et al. 1998). NR medium is then added, and NRU is
measured after a 3-hour incubation. Phototoxic potential is assessed by comparing thedifferences in cytotoxicity between test plates containing the test substance that have notbeen exposed to UVA and comparable test plates exposed to UVA.
Two different models, employing the Photoinhibition Factor (PIF) and the Mean Photo
Effect (MPE), were validated for the prediction of in vivo phototoxic potential. The accuracyof the models for classifying the phototoxic potential of the 30 substances tested in ninelaboratories was 88% for the PIF, and 92% for the MPE, when compared with in vivo
classifications. Interlaboratory variability for classification (i.e., phototoxic vs. nonphototoxic) was assessed using a bootstrapping approach. For each substance, theclassification based on a single experiment was compared to the classification based on themean PIF or mean MPE. The interlaboratory variability for classification was 0 to 18.8%
using PIF and 0 to 20% using MPE.
The ECVAM Scientific Advisory Committee confirmed the scientific validity of the method
in 1997 (ECVAM 1997) and its regulatory acceptance was noted in Annex V of Council
Directive 67/548/EEC part B.41 on phototoxicity, in 2000. An OECD Test Guideline, 432,
was finalized in 2004 (OECD 2004). The 3T3 NRU phototoxicity test is used in a tiered
testing approach to determine the phototoxic potential of test substances.
The performance of the 3T3 NRU phototoxicity assay could not be compared with theperformance of the 3T3 NRU test method used in this validation study because different
classification schemes were used (i.e., a two-category classification for the phototoxicity vs.
a six-class scheme for acute oral toxicity). The ECVAM measurements of interlaboratory
variability also used different techniques and were not comparable to those used for the
NICEATM/ECVAM study.
9.2.3.1 NHK NRU Phototoxicity Assay
FAL participated in the European Union/European Cosmetic, Toiletry and Perfumery
Association (EU/COLIPA) study (30 substances tested using NHK and 3T3 cells) and the
ECVAM/COLIPA study (20 substances tested using NHK cells) (Clothier et al. 1999). The
studies showed that the NHK NRU test method could be used to predict phototoxic potential.
The accuracy for predicting in vivo results was similar to that of the 3T3 NRU phototoxicity
test (see Table 9-4). The NHK NRU phototoxicity test uses the same test substance exposure
duration (approximately 2 hours) as the 3T3 NRU test method, but the duration of culture
after UV exposure is 72 hours rather than 24 hours. NRU was measured after a 45-minuteincubation with NR.
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Although the NHK NRU phototoxicity method achieved good concordance with in vivo
phototoxicity, it has not yet been validated for regulatory use.
Table 9-4

Correct Identification of In Vivo Phototoxicants by the NHK NRU
Phototoxicity Assay
Study

EU/COLIPA(Spielmann et al. 1998)
ECVAM/COLIPA
Combined Study Data

3T3 NRU Phototoxicity
MethodNHK NRU Phototoxicity
Method29/30 (97%)1

28/30 (93%)1

NA
45/45 (100%)2

18/20 (90%)1
19/20 (95%)2
44/45 (98%)2

Abbreviations: 3T3=BALB/c 3T3 fibroblasts; NHK=Normal human epidermal keratinocytes; NRU=Neutral red
uptake; EU=European Union; ECVAM=European Centre for the Validation of Alternative Methods;
COLIPA=The European Cosmetic Toiletry and Perfumery Association; NA=not available.
1
Mean Photo Effect (MPF) prediction model.
2
Photoinhibition Factor (PIF) prediction model.

9.3

Studies Using In Vitro Cytotoxicity Methods with Established PerformanceStandards

The procedure provided in the Guidance Document for evaluating basal cytotoxicity assays
for use in predicting starting doses for acute oral toxicity assays provides the existing
performance standards for the 3T3 and NHK NRU test methods (ICCVAM 2001b).
9.3.1
Guidance Document (ICCVAM 2001b)
In addition to guidance for evaluating in vitro basal cytotoxicity methods for use inpredicting starting doses for rodent acute oral toxicity assays, the Guidance Document
provided results from testing 11 reference substances using the recommended 3T3 and NHK
NRU protocols (ICCVAM 2001b). The 11 substances were chosen from the RC database so
as to have a close fit to the RC millimole regression and to cover a wide range ofcytotoxicity. The major differences between the Guidance Document protocols and theprotocols used in this validation study are the reduced NR concentrations (from 50 µg/mL to
25 µg/mL in the 3T3 NRU test method, and from 50 µg/mL to 33 µg/mL in the NHK NRU
test method), the increased duration of test substance exposure in the 3T3 NRU test method,from 24 to 48 hours, and the lack of a refeeding step in the NHK NRU test method just prior
to substance application (see Sections 2.6 and 2.7 for further detail). Despite thesedifferences, the Guidance Document shows that the test results for the 11 substances in boththe 3T3 and NHK NRU test methods were similar to the results in the RC database. The
calculated regressions for the 11 Guidance Document substances were:• log LD50 = 0.506 log IC50 + 0.475 (R2=0.985) for the 3T3 NRU test method• log LD50 = 0.498 log IC50 + 0.551 (R2=0.936) for the NHK NRU test method• log LD50 = 0.435 log IC50 + 0.625 for the RC millimole regression
The 3T3 and NHK NRU regressions were compared with the RC millimole regression (347substances) to show that the regression lines, as well as all 11 substance data points, were

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within the acceptance interval (± 0.5 log around the regression) of the RC millimole
regression (see Guidance Document Figures 3 and 4, p.13 [ICCVAM 2001b]).
9.3.2
King and Jones (2003)
This study also tested the 11 substances recommend in the Guidance Document using therecommended 3T3 NRU protocol. The IC50 - LD50 regression obtained was comparable to the
RC millimole regression and to the 11 substance regression provided in the GuidanceDocument (ICCVAM 2001b). The regression was log LD50 = 0.552 log IC50 + 0.503(R2=0.929) and the RC millimole regression was log LD50 = 0.435 log IC50 + 0.625. The 11substance regression fit within the acceptance interval (± 0.5 log) of the RC millimole
regression.
King and Jones (2003) also showed that a 3T3 NRU test method that was adapted for highthroughput testing by using three test sample concentrations yielded approximately the same
IC50 as an eight concentration-response. A regression used to compare the IC50 values using
the two different concentration-response approaches yielded R2=0.945.
9.3.3

A-Cute-Tox Project: Optimization and Pre-Validation of an In Vitro Test Strategy
for Predicting Human Acute Toxicity (Clemedson 2005)
The A-Cute-Tox Project is an Integrated Project under the EU 6th framework program thatstarted in January 2005, with a termination date of January 2010. It was initiated in responseto the REACH (Registration, Evaluation, Authorisation and Restriction of Chemicals)
Directive and the 7th amendment of the Cosmetics Directive, which calls for the broad
replacement of animal experiments for finished products by 2003, and for ingredients by
2009. The project is an extension of the NICEATM/ECVAM validation study and the EDIT
program, which is the continuation of the MEIC program. The partnership is made up of the
EDIT Consortium, ECVAM, and 35 other European toxicity research group partners.
The aim of the project is to develop a simple and robust in vitro testing strategy forprediction of human acute oral toxicity, which could replace the animal acute oral toxicity
tests currently used for regulatory purposes. The objectives of A-Cute-Tox are:• Compilation, critical evaluation, and generation of high quality in vitro and invivo data for comparative analysis.
• Identifying factors (e.g., kinetics, metabolism, and organ specificity) that
influence the correlation between in vitro toxicity (concentration) and in vivo
toxicity (dosage), and to define an algorithm that accounts for these effects.
• Explore innovative tools and cellular systems to identify new toxicity endpoints and strategies to better anticipate animal and human toxicity.
• To design a simple, robust and reliable in vitro test strategy associated withthe prediction model for acute toxicity that is amenable to high-throughput
testing.
The project has been divided into the following workpackages that will be implemented byvarious configurations of research partners:• WP1: Generation of a “high quality” in vivo database (through literaturesearches and historical data) and establishment of a depository list ofreference substances

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•
•
•
•
•
•
•
•

November 2006

WP2: Generation of a “high quality” in vitro database (including data from the
NICEATM/ECVAM study, EDIT studies, and MEIC studies)
WP3: Iterative amendment of the testing strategy
WP4: New end-points and new cell systems
WP5: Alerts and correctors in toxicity screening (I): Role of absorption,
distribution, and excretion
WP6: Alerts and correctors in toxicity screening (II): Role of metabolism
WP7: Alerts and correctors in toxicity screening (III): Role of target organ
toxicity (i.e., neuro-, nephro-, hepato-toxicity)
WP8: Technical optimisation of the amended test strategy
WP9: Pre-validation of the test strategy

A-Cute-Tox aims to extend the NICEATM/ECVAM and MEIC/EDIT approaches toward afull replacement test strategy by improving the prediction of acute oral toxicity using invitro methods, and then validating the testing procedure.
9.4

Summary••••

In vitro NRU cytotoxicity test methods using various cell types have been
evaluated for their correlation with rodent lethality endpoints (e.g., rat/mousei.v., i.p., and oral toxicity). Peloux et al. (1992) and Fautrel et al. (1993)
showed good correlations (r=0.877 and 0.88, respectively) of in vitro
cytotoxicity with rodent i.p./i.v. and i.v. toxicity data, respectively.
3T3 and NHK NRU test methods have been evaluated for purposes other thanthe prediction of starting doses for acute toxicity studies (e.g., ocular irritancy;
human LC values, in vivo phototoxicity).
A 3T3 NRU test method has been validated by ECVAM for the identificationof in vivo phototoxic potential.
No in vitro test methods have been validated for the prediction of acute oral
toxicity. Estimations of animal savings using in vitro cytotoxicity data toestimate starting doses for the UDP did not use actual in vitro cytotoxicity
data. Instead, animal savings were estimated by assuming that the in vivo
starting dose equals the true LD50, which is an approach that assumes thatcytotoxicity data can perfectly predict in vivo lethality. These theoreticalpredictions of animal savings in the UDP ranged from 25 to 40% (ICCVAM
2001a), as compared with the average animal savings of 5.3 to 7.8% predictedusing computer simulation modeling of the UDP for the reference substancestested in the NICEATM/ECVAM study. Halle et al. (1997) used the in vitro
cytotoxicity data in the RC to determine that an animal savings of 32% can beattained for the ATC method by using the LD50 predicted by the RC
regression as the starting dose. For the reference substances tested in the
NICEATM/ECVAM validation study, most of which were a poor fit to the
RC millimole regression, the average animal savings for the ATC, as
determined by computer simulation modeling, was 4.8 to 10.2%.

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10.0

ANIMAL WELFARE CONSIDERATIONS (REFINEMENT,REDUCTION, AND REPLACEMENT)............................................................. 10-3

10.1

Use of 3T3 and NHK NRU Test Methods to Predict Starting Doses forRodent Acute Oral Toxicity Assays ..................................................................... 10-4

10.2

Reduction and Refinement of Animal Use for the UDP..................................... 10-4
10.2.1 In Vivo Testing Using the UDP ................................................................... 10-4
10.2.2 Computer Simulation Modeling of the UDP ............................................... 10-6
10.2.3 Animal Savings in the UDP When Using 3T3- and NHK-Based
Starting Doses .............................................................................................. 10-7
10.2.4 Refinement of Animal Use for the UDP When Using 3T3- and NHKBased Starting Doses ................................................................................. 10-15
10.2.5 Accuracy of UDP Outcomes Using the IC50-Based Starting Doses .......... 10-16

10.3

Reduction and Refinement of Animal Use in the ATC Method ...................... 10-16
10.3.1 In Vivo Testing Using the ATC Method .................................................... 10-16
10.3.2 Computer Simulation Modeling of the ATC Method................................ 10-17
10.3.3 Animal Savings for the ATC Method When Using 3T3- and NHKBased Starting Doses ................................................................................. 10-18
10.3.4 Refinement of Animal Use in the ATC Method When Using 3T3- andNHK-Based Starting Doses ....................................................................... 10-26
10.3.5 Accuracy of the ATC Method Outcomes Using the IC50-Based
Starting Doses ............................................................................................ 10-27

10.4

The Impact of Accuracy on Animal Savings ..................................................... 10-28

10.5

The Impact of Prevalence on Animal Savings................................................... 10-30

10.6

Summary............................................................................................................... 10-31

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10.0

November 2006

ANIMAL WELFARE CONSIDERATIONS (REFINEMENT, REDUCTION,
AND REPLACEMENT)As demonstrated in Section 6, in vitro basal cytotoxicity methods cannot be used as
replacement assays1 for rodent acute oral toxicity test methods for hazard classification.
However, as described in this section, these methods can be used to reduce2 and refine3
animal use in the UDP or ATC acute oral toxicity assays, as shown by the computer
simulations of such testing. Although the use of in vitro cytotoxicity data to determine
starting doses for the FDP may reduce the use of animals for the FDP, even though death is
not the primary endpoint, such an evaluation will not be provided in this document.
The test guidelines recommend using information on structurally-related substances and theresults of any other toxicity tests (EPA 2002b) for the test substance, including in vitro
cytotoxicity results, to approximate the LD50 and the slope of the dose-mortality curve(OECD 2001a; OECD 2001d; EPA 2002a). However, for the purposes of the reduction andrefinement evaluation conducted in this section, it was assumed that no information otherthan 3T3 and NHK NRU IC50 data would be available. To determine the extent of animal
reduction or refinement that would occur in the UDP and the ATC method when using astarting dose based on 3T3 or NHK NRU IC50 values rather than the default starting dose,computer models were used to simulate the in vivo testing of the reference substances used in
the validation study.
Section 10.1 lists the regressions that were used with IC50 data from the 3T3 and NHK NRU
test methods to determine starting doses for the UDP and the ATC. Sections 10.2.1 and10.3.1 summarize the animal testing procedures in the current test guidelines for the UDP
and the ATC, respectively. The procedures for using computer simulation of the animal
testing of the reference substances are described in Sections 10.2.2 and 10.3.2. The computer
simulations were used to determine the numbers of animals used and the numbers of animals
that “died” for each test. The modeling was performed using five different dose-mortality
slopes4 (i.e., 8.3, 4.0, 2.0, 0.8, and 0.5) because such slope information was not available forall of the reference substances used. To simplify the presentation of results, the animal use
figures provided in Sections 10.2.3, 10.2.4, 10.3.3, and 10.3.4 include the data for only twoof the slopes, 8.3 and 2.0. The slope of 2.0 is the default used for the calculation of LD50 by
the UDP method (OECD 2001a; EPA 2002a) and the slope of 8.3 is shown to represent
substances, such as pesticides, with higher slopes. The results for the other three slopes were
calculated, and are provided in Appendices N and Q. The numbers of animals used aresummarized to show the mean number of animals tested when the default starting dose isused and the mean number of animals used when the starting dose was determined from the
3T3 or NHK NRU IC50 values. The difference in animal use between the default starting
doses and the IC50-based starting doses is referred to as the animal savings. Differences were1

Replacement alternative: a new or modified test method that replaces animals with nonanimal systems or one
animal species with a phylogenetically lower one (e.g., a mammal with an invertebrate).
2
Reduction alternative: a new or modified test method that reduces the number of animals required.
3
Refinement alternative: a new or modified test method that refines procedures to lessen or eliminate pain or
distress in animals or enhances animal well-being.
4
The dose-mortality slope is the slope of the dose-response curve for mortality.
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tested for statistical significance (at p <0.05) using a one-sided Wilcoxon signed ranked test
based on the number of substances evaluated. Sections 10.2 and 10.3 summarize mean
animal use by the total number of substances tested and by the number of substances in each
GHS category. Sections 10.2.4 and 10.3.4 provide the mean number of animal deaths
compared to the mean number of animals used for each default and IC50-based starting doseto determine whether the IC50-based starting doses lead to a reduction in the number ofanimals used and the number that die (i.e., refinement). Sections 10.2.5 and 10.3.5 discussconcordance for the reference substance outcomes of simulated testing using the IC50-based
starting doses, with the outcomes of the default starting doses. Sections 10.4 and 10.5 discuss
the impact of accuracy and the impact of prevalence (i.e., the number of substances to betested in each GHS category) on animal savings.
10.1

Use of the 3T3 and NHK NRU Test Methods to Predict Starting Doses forRodent Acute Oral Toxicity Assays

The IC50 values developed from the 3T3 and NHK NRU test methods were used to predict
starting doses for rodent acute oral toxicity tests using the following linear regressions of
IC50-LD50 values (from Section 6.3):• The RC rat-only millimole regression: log LD50 (mmol/kg) = 0.439 log IC50(mM) + 0.621• The RC rat-only weight regression: log LD50 (mg/kg) = 0.372 log IC50(µg/mL) + 2.024
The IC50 values from each in vitro NRU test method were evaluated with each regression andsimulated acute oral toxicity test method,. The criteria for the use of a reference substance forthis evaluation were that it must have:• Replicate IC50 values from at least one laboratory• A rat acute oral LD50 reference value (from Table 4-2)
Sixty-seven and 68 reference substances were evaluated for the 3T3 and the NHK NRU test
methods, respectively. Of the 72 reference substances tested, epinephrine bitartrate,colchicine, and propylparaben were excluded because they did not have associated rat oral
LD50 data. Carbon tetrachloride and methanol were excluded from the 3T3 evaluations, andcarbon tetrachloride was excluded from the NHK evaluations, because none of thelaboratories achieved sufficient toxicity in any test for the calculation of an IC50.
10.2

Reduction and Refinement of Animal Use for the UDP

10.2.1In Vivo Testing Using the UDPThis section describes the general dosing procedure for the UDP (OECD 2001a; EPA 2002a).
Although doses, interval between doses, and dose progression, may be adjusted as necessary,
the procedures described reflect the default guidance. Guidance on the types of animals thatcan be used, animal housing, clinical observations, etc., are outside the scope of the current
discussion and are provided in the test guidelines (see Appendices M1 and M2).
10.2.1.1 Main Test
The UDP is based on a staircase design in which single animals are dosed, in sequence, at
48-hour intervals. The effect on the first animal determines the dose of the next animal. If thefirst animal dies or is in a moribund state within 48 hours after dosing, the dose administered

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to the next animal is lowered by dividing the original dose by one-half log (i.e., 3.2, which isthe default dose progression). If the first animal survives, the dose administered to the next
animal is increased by one-half log times the original dose. A dose progression of one-half
log unit corresponds to a dose-mortality slope of 2.0. The default dose progression can be
adjusted if the analyst has prior information upon which to estimate a slope.
The starting dose recommended by the guideline is one dose progression step below theanalyst’s best estimate of the LD50, because, in the UDP test method, the LD50 estimate tends
to move toward the starting dose. A default starting dose of 175 mg/kg is used if there is no
information on which to base a starting dose. The default dosing scheme, using the dose
progression of 3.2, is 1.75, 5.5, 17.5, 55, 175, 550, 1750, and 5000 mg/kg (EPA 2002a) or
1.75, 5.5, 17.5, 55, 175, 550, and 2000 mg/kg (OECD 2001a). The difference between thetwo reflects the different maximum doses emphasized in the different guidelines. Dosing
single animals, upward or downward, in sequence proceeds until the first of three conditions,referred to as stopping rules, is met:
• Three consecutive animals survive at the upper dose limit (2000 or 5000
mg/kg)• Five reversals5 occur in any six consecutive animals tested• Four or more animals have followed the first reversal, and the likelihoodratios specified by the guideline exceed the critical value. For a wide varietyof LD50 values and dose-mortality slopes, this rule is satisfied with four to six
animals after the first reversal. Three likelihood values are calculated: alikelihood at an LD50 point estimate (called the rough estimate or doseaveraging estimate); a likelihood at a value below the point estimate (the point
estimate divided by 2.5); and a likelihood at a value above the point estimate(the point estimate multiplied by 2.5). The ratios of the likelihoods are
examined to determine whether they exceed a critical value.
If none of these conditions is met, the dosing stops after 15 animals have been used.
10.2.1.2 Limit Test
The UDP guidelines include a limit test using three to five animals dosed sequentially at2000 mg/kg (OECD 2001a) or 5000 mg/kg (EPA 2002a). The EPA guideline for testing at alimit dose calls for proceeding to the main test if the first animal dosed at 5000 mg/kg dies
(EPA 2002a). If the first animal lives, two more animals are dosed, in sequence, with 5000
mg/kg. If both animals live, then testing is terminated, and the substance is designated ashaving an LD50 >5000 mg/kg. If one or both animals die, then two more animals are dosed insequence. As soon as a total of three animals survive, the test is terminated, with theconclusion that LD50 >5000 mg/kg. However, the main test is conducted if three animals die.

5

Reversal: a situation where a nonresponse (i.e., animal lives) is observed at some dose, and a response isobserved at the next dose tested (i.e. animal dies), or vice versa. Reversal is created by a pair of responses. (SeeAppendices M1 and M2)

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The OECD guideline for testing at a limit dose calls for proceeding to the main test if the firstanimal dosed at 2000 mg/kg dies (OECD 2001a). If the animal lives, four more animals aresequentially dosed. The main test is performed if three animals die. If three or more animals
survive, testing is terminated with the conclusion that the LD50 >2000 mg/kg.
10.2.2Computer Simulation Modeling of the UDP
Ten thousand UDP testing simulations were run for each substance, in vitro NRU test
method, and dose-mortality slope. Because the analysis assumed there was no information
upon which to estimate a dose-mortality slope, the modeling used the default dose
progression factor of 3.2, and 5000 mg/kg as the upper limit dose because this upper limit isemphasized in the EPA guideline (EPA 2002a)6. If the starting dose estimated from the invitro IC50 value was ≥4000 mg/kg, then the limit test, rather than the main test, wasperformed. If, during the dose progression, the next highest dose to be administered wasapproximately 4000 mg/kg or greater, then the limit dose of 5000 mg/kg was administered. If
a dose one step below the IC50-estimated LD50 was used as the starting dose, the other doses
administered corresponded to the default doses specified in the guidelines (OECD 2001a;
EPA 2002a). The simulation modeling procedures also used a lower limit of 1 mg/kg. Thus, adose of 1 mg/kg was administered if the dose progression fell below 1 mg/kg. To estimate
animal use by the default method, a starting dose of 175 mg/kg was used; the other doses
administered after the default starting dose corresponded to the doses specified in theguidelines (OECD 2001a; EPA 2002a).
The simulation was performed using SAS version 8 (SAS 1999) and implemented the
distributional assumptions underlying the dose-mortality relationship. The lowest dose atwhich an animal dies in response to the administration of a toxic substance varies fromanimal to animal. For an entire population of animals, mortality is assumed to have a lognormal distribution, with the mean equal to the log of the true LD50. Sigma (σ), thevariability of the simulated population, is the inverse of the slope of the dose-mortality curve.
Because of a lack of information concerning the actual dose-mortality curves, the simulations
assumed several different values of the slope, but no corresponding changes were made in
the dose progression. Dose-mortality slopes of 0.5, 0.8, 2.0, 4.0, and 8.3 were used because
these were used in the simulation modeling used to evaluate the current version of the UDP
guidelines (ICCVAM 2001c).
To model the variability of the IC50 values within and among laboratories, the values for eachreference substance were log-transformed to normalize their distribution. The mean andvariance of these log-transformed values were used to generate a log-normal distribution
from which an IC50 value was randomly selected. This IC50 value was used with theregressions to determine starting doses using two different methods. One method used theLD50 estimated from the IC50 and the regression as the starting dose, while the other used theclosest default dose that was lower than the estimated LD50. The latter method isrecommended by the EPA and OECD test guidelines (EPA 2002a; OECD 2001a), and theresults from that simulation are presented in Section 10.2. The UDP is only usable forregulatory purposes if the starting dose is set below the expected LD50. Appendix Q contains
6

The results from UDP simulations for a limit dose of 2000 mg/kg will be presented in a future addendum tothis document.
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In Vitro Cytotoxicity Test Methods BRD Section 10

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the results obtained when the LD50 that was estimated by the IC50 and the regression wasused as the starting dose.
The simulation procedure used the following steps for each reference substance:
1. The LD50 value (in mg/kg) from Table 4-2 was entered as the true LD50 value
and the choices of assumed slope were entered as the true slopes for the dosemortality curve.
2. An IC50 value was selected from a distribution identified by the mean andvariance of the IC50 values for each chemical to reflect the variation in IC50
values produced by the different laboratories (see Tables 5-4 and 5-5 formean IC50 values and standard deviations for the 3T3 and NHK NRU test
methods, respectively).
3. The IC50 value from Step 2 was used in the regression model being evaluatedto predict a LD50 value, which was used to determine the starting dose.
4. The dosing simulation was run three times: once with the default starting doseof 175 mg/kg, once at the next default dose below the LD50 estimated by the
regression being evaluated, and once at a dose equal to that of the LD50
estimated by the regression being evaluated.
5. For each simulated trial, the animals are dosed sequentially; therefore for eachanimal (i) there is a corresponding dose (i) that is administered to the animal.
For the first animal in each trial, it is the starting dose for that trial. For eachsubsequent animal, the dose is dependent on the previous dose and theprevious animal’s response, as described in Section 10.2.1. For animal (i), theprobability of a response is computed with the cumulative log-normal
distribution at the dose administered. That is,
P ( response ) = P ( x < log[ dose (i )]) where x ~ N ( µ , σ ) ,where µ is the log of the true LD50 value, and σ is the inverse of theassumed slope of the dose-mortality curve. One observation is then sampledfrom a binomial distribution with this calculated probability of success todetermine whether the animal lives or dies.
6. Dosing simulation is stopped as soon as one of the stopping rules is satisfied.
Steps 2-6 were repeated 10,000 times in order to compute an average animal use for eachmethod evaluated.
10.2.3

Animal Savings in the UDP When Using 3T3- and NHK-Based Starting Doses

10.2.3.1 The Effect of the Dose-Mortality Slope on Animal Use
As described in Section 10.2.2, the simulation modeling of animal use for the UDP assumed
five different dose-mortality slopes in order to assess animal use under various conditions of
population variability. Table 10-1 shows that the number of animals used for the UDP
decreases with increasing slope for both the default starting dose and the IC50-determined
starting dose when based on the RC rat-only millimole regression. The IC50-determined
starting dose was the next default dose lower than the regression-estimated LD50. For
example, because the LD50 predicted for cadmium chloride by the 3T3 NRU IC50 with the
RC rat-only millimole regression was 16 mg/kg, the starting dose was 1.75 mg/kg (i.e., thenext default dose below the predicted LD50). This approach is consistent with the UDP

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guidelines (OECD 2001a; EPA 2002a) as a means of reducing the number of animals thatmight experience pain and suffering from a treatment. This approach also overcomes thenonconservative bias of the UDP, which tends to yield an LD50 close to the starting dose.
Table 10-1 shows that, for each dose-mortality slope, the mean number of animals saved wasstatistically significant (p<0.05) when compared to mean number of animals needed when
the default starting dose was used. When expressed as a percentage of the number of animals
used when the default starting dose is used, animal savings also generally increased withincreasing slope of the dose-response. The animal savings is the same at all slopes tested, but
fewer animals are used at the steeper slopes, which increases the relative percentages ofanimals saved.
Table 10-1

Change in Animal Use1 with Dose-Mortality Slope for the UDP2

Dose-Mortality Slope

With Default Starting
Dose1,3With IC50-Based Starting
Dose1,4

Animals Saved5

0.5

3T3 NRU Test Method
10.01 ±0.10
9.48 ±0.11

0.53* (5.3%)

0.8

9.95 ±0.139.34 ±0.14

0.61* (6.1%)

2.0

9.35 ±0.168.80 ±0.17

0.54* (5.8%)

4.0

8.68±0.18

8.15 ±0.19

0.52* (6.0%)

8.3

7.95 ±0.18

7.42 ±0.20

0.53* (6.6%)

NHK NRU Test Method
0.5

10.01 ±0.09

9.53 ±0.12

0.49* (4.9%)

0.8

9.96 ±0.139.41 ±0.15

0.55* (5.5%)

2.0

9.36 ±0.168.86 ±0.18

0.50* (5.3%)

4.0

8.66 ±0.178.18 ±0.20

0.48* (5.6%)

8.3

7.92 ±0.187.43 ±0.20

0.49* (6.2%)

Abbreviations: UDP=Up-and-Down Procedure; 3T3=BALB/c 3T3 fibroblasts; NHK=Normal human epidermal
keratinocytes; RC=Registry of Cytotoxicity.
*Statistically significant (p<0.05) by a one-sided Wilcoxon signed rank test. Percentage difference is shown in parentheses.
1
Mean numbers of animals ±standard errors for 10,000 simulations for each of the 67 substances in the 3T3 NRU test
method and 68 substances in the NHK NRU test method. Although the simulations used whole animals, averaging theresults over a large number of simulations produced fractional numbers. Upper limit dose =5000 mg/kg.
2
OECD (2001a); EPA (2002a).
3
Default starting dose = 175 mg/kg.
4
The starting dose = next lower default dose to the predicted LD50, which was calculated from the IC50 value in the RC ratonly millimole regression: log LD50 (mmol/kg) = 0.439 log IC50 (mM) + 0.621. The IC50 value for each reference substance
was randomly selected from the distribution of values obtained during the testing with each method.
5
Difference between mean animal use with the default starting dose and mean animal use with the IC50-based starting dose.

To simplify the presentation of animal savings and the comparison of the various regressions
and starting doses, the results of subsequent analyses presented in Section 10.2.3 are limited
to the dose-mortality slopes of 2.0 and 8.3. The slope of 2.0 is the default used for the
calculation of LD50 by the UDP method (OECD 2001a; EPA 2002a) and the slope of 8.3 isshown to represent substances, such as pesticides, with higher slopes. Animal savings results
for the other dose-mortality slopes were calculated, and are presented in Appendices N1-N3.
Although using the next lower default dose to the in vitro-determined LD50 value overcomes

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the bias of the UDP toward the starting dose (OECD 2001a, EPA 2002a) and is theappropriate approach for regulatory use, animal savings results using the estimated LD50 as
the starting dose were also calculated (see Appendix Q).
10.2.3.2 Mean Animal Use for UDP Simulations – Comparison of Regressions andPredictions from the 3T3 and NHK NRU Test Methods
Table 10-2 shows the mean animal use for the simulated UDP testing of the reference
substances described in Section 10.1. Mean animal use is shown for the default starting doseand for starting doses that were one default dose lower than the LD50 predicted from the invitro NRU methods and the regressions evaluated in Section 6.4 for the prediction of GHS
category. The difference in animal use between the two starting doses is the mean animalsavings produced by using the starting dose based on the in vitro NRU methods. All
differences (i.e., mean animal savings) were statistically significant (p <0.05) by a one-sided
Wilcoxon signed rank test. Mean animal savings ranged from 0.49 to 0.66 (6.2% to 7.0%)
animals per test depending upon the in vitro NRU test method, regression, and dose-mortality
slope. The lowest mean animal savings were obtained for the RC rat-only millimole
regression (0.49 [6.2%] to 0.54 [5.8%] animals for the different test methods and dosemortality slopes), and the greatest mean animal savings were obtained with the RC rat-only
weight regression (0.54 [6.8%] to 0.66 [7.0%] animals per test).
The animal savings using the in vitro NRU test methods with the RC rat-only regressions
apply only to the reference substances evaluated in this validation study, and are based on
substances pre-selected for their known in vivo toxicities and may not be broadly applicableto other substances. Table 3-4 shows that 22 (38%) of the 58 RC substances selected fortesting were known to have a poor fit to the RC millimole regression (i.e., the in vivo LD50
was outside the RC acceptance interval for the predicted LD50). Table 6-3 shows that 40%(28/70 for the 3T3) and 44% (31/71 for the NHK) of the reference substances that produced
IC50 values were outliers. The RC rat-only millimole regression evaluated here is very similar
to the RC millimole regression (see Table 6-5). Substances with better fits to the regression
are more likely to yield greater animal savings.
10.2.3.3 Animal Savings in the UDP by GHS Acute Oral Toxicity Category Using 3T3- andNHK-Based Starting Doses
Tables 10-3 and 10-4 show mean animal use and mean animal savings for the UDP when thedefault starting dose and the IC50-predicted starting doses were used, and when the reference
substances are grouped by GHS category (UN 2005). The data come from the same analyses
as the data provided in Table 10-2. The IC50-predicted starting doses were based on the:• RC rat-only millimole regression (Table 10-3)• RC rat-only weight regression (Table 10-4)

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Table 10-2

November 2006

Mean Animal Use1 in the UDP2 Using Starting Doses Based on the 3T3 and NHK NRU Test
Methods with the Different Regressions
With
With IC50Animals
Default
Based
Starting
Starting
Saved5
3
4
Dose
Dose
Dose-mortality Slope = 2.0

With
With IC50Animals
Default
Based
Starting
Starting
Saved5
3
5
Dose
Dose
Dose-mortality Slope = 8.3

RC rat-only millimole6

9.35 ±0.168.80 ±0.17

0.54* (5.8%)

7.95 ±0.18

7.42 ±0.20

0.53* (6.6%)

RC rat-only weight7

9.36 ±0.168.70 ±0.16

0.66* (7.0%)

7.94 ±0.18

7.32 ±0.19

0.62* (7.8%)

Assay/Regression
3T3 NRU Test Method

NHK NRU Test Method
6

RC rat-only millimole
7

RC rat-only weight

Dose-mortality Slope = 2.0

Dose-mortality Slope = 8.3

9.36 ±0.168.86 ±0.18

0.50* (5.3%)

7.92 ±0.187.43 ±0.20

0.49* (6.2%)

9.36 ±0.168.80 ±0.17

0.56* (6.0%)

7.92 ±0.18

7.38 ±0.20

0.54* (6.8%)

Abbreviations: 3T3=BALB/c 3T3 fibroblasts; NHK=Normal human epidermal keratinocytes; RC=Registry of Cytotoxicity; UDP=Up-and-Down Procedure.
*Statistically significant (p <0.05) by a one-sided Wilcoxon signed rank test. Percentage difference is shown in parentheses.
1
Mean numbers of animals ±standard errors for 10,000 simulations for each of the 67 substances in the 3T3 NRU test method and 68 substances in the NHK
NRU test method. Although the simulations used whole animals, averaging the results over a large number of simulations produced fractional numbers. Upper
limit dose =5000 mg/kg.
2
OECD (2001a); EPA (2002a).
3
Default starting dose =175 mg/kg.
4
The starting dose = one default dose lower than the predicted acute oral LD50 calculated using the IC50 value in the specified regression. The IC50 value for eachreference substance was randomly selected from the distribution of values obtained during the in vitro testing with each test method.
5
Difference between mean animal use with default starting dose and mean animal use with the IC50-based starting dose.
6
log LD50 (mmol/kg) = 0.439 log IC50 (mM) + 0.621.
7
log LD50 (mg/kg) = 0.372 log IC50 (µg/mL) + 2.024.

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These analyses showed that:• For each in vitro NRU test method and regression, animal savings were
statistically significant for substances in the 2000 < LD50 ≤5000 mg/kg and
LD50 >5000 mg/kg toxicity categories.
• For substances with 5 < LD50 ≤50 mg/kg and 50 < LD50 ≤300 mg/kg, both invitro NRU test methods with each regression used slightly more animals thanthe default-starting dose, but the differences were not statistically significant.
Animal Savings for the UDP by GHS Acute Oral Toxicity Category Using 3T3- and NHKBased Starting Doses with the RC Rat-Only Millimole Regression
Table 10-3 shows the animal savings by GHS category when the IC50 values are used with
the RC rat-only millimole regression. Mean animal savings were statistically significant (p<0.05) by a one-tailed Wilcoxon signed rank test for the following GHS toxicity categories,
test methods, and dose-mortality slopes:• The use of the NHK NRU test method at both dose-mortality slopes for
substances with 300 < LD50 ≤2000 mg/kg that produced savings of 0.49
(6.5%) to 0.52 (6.1%) animals per test.
• The use of the 3T3 NRU test method at the 8.3 dose-mortality slope for
substances with 300 < LD50 ≤2000 mg/kg that produced a saving of 0.31
(4.1%) animals per test.
• The use of both in vitro NRU test methods at both dose-mortality slopes for
substances with 2000 < LD50 ≤5000 mg/kg that produced savings of 1.11
(12.1%) to 1.28 (11.9%) animals per test.
• The use of both in vitro NRU test methods and both dose-mortality slopes for
substances with an LD50 >5000 mg/kg that produced savings of 1.47 (14.8%)
to 1.58 (20.3%) animals per test.
The mean animal savings for the 3T3 and NHK NRU test methods were similar for most
toxicity categories at both dose-mortality slopes, with the mean savings with the 3T3 slightly
higher than with the NHK. For the dose-mortality slope of 2.0, the mean animal savings withthe 3T3 NRU test method ranged from -0.42 (-5.5%) to 1.58 (16.0%) animals per test for thevarious toxicity categories, and savings for the NHK NRU test method ranged from -0.34 (3.5%) to 1.47 (14.8%) animals per test. For the dose-mortality slope of 8.3, animal savingsfor the 3T3 NRU test method ranged from -0.29 (-4.3%) to 1.58 (20.3%) animals per test andsavings for the NHK NRU test method ranged from -0.33 (-3.9%) to 1.47 (19.2%) animals
per test. Animal savings were also obtained for highly toxic substances (LD50 ≤5 mg/kg) withboth the 3T3 (0.96 [9.9%] to 1.14 [10.0%] animals per test) and NHK (0.71 [7.3%] to 0.75
[6.7%] animals per test) NRU test methods, but the savings were not statistically significant.
No mean animal savings (≤-0.28 animal per test) were observed for substances with 50<
LD50 ≤300 mg/kg by either the 3T3 or the NHK NRU test method. This category includes thedefault starting dose of 175 mg/kg. Animal savings were not expected for this category
because savings were determined by comparing animal use with the IC50-based starting dosewith animal use at the default starting dose. No animal savings (-0.07 to -0.34 animals pertest) were observed for substances with 5< LD50 ≤50 mg/kg for either NRU test method.
None of these differences in animal use was statistically significant.

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In Vitro Cytotoxicity Test Methods BRD Section 10

Table 10-3

November 2006

Animal Use1 for the UDP2 by GHS Acute Oral Toxicity Category3 Using Starting Doses Based on the 3T3
and NHK NRU Test Methods with the RC Rat-Only Millimole Regression4
Dose-mortality Slope = 2.0

GHS Acute Oral Toxicity
Category3

Number ofReference
Substances

With Default
Starting
Dose5

With IC50Based
Starting
Dose6

LD50 ≤5 mg/kg
5 < LD50 ≤50 mg/kg
50 < LD50 ≤300 mg/kg
300 < LD50 ≤2000 mg/kg
2000 < LD50 ≤5000 mg/kg
LD50 >5000 mg/kg

6
11
12
16
10
12

11.32 ±0.20
9.68 ±0.23
7.76 ±0.108.53 ±0.2110.73 ±0.109.87 ±0.34

10.19 ±0.70
9.74 ±0.458.18 ±0.218.14 ±0.219.46 ±0.158.29 ±0.49

LD50 ≤5 mg/kg
5 < LD50 ≤50 mg/kg
50 < LD50 ≤300 mg/kg
300 < LD50 ≤2000 mg/kg
2000 < LD50 ≤5000 mg/kg
LD50 >5000 mg/kg

6
11
12
16
10
13

11.21 ±0.24
9.65 ±0.16
7.78 ±0.118.55 ±0.2210.75 ±0.08
9.87 ±0.32

10.47 ±0.71
9.99 ±0. 45
8.12 ±0.218.03 ±0.239.54 ±0.20
8.41 ±0.44

Animals
Saved7

Dose-mortality Slope = 8.3
With Default
Starting
Dose5

3T3 NRU Test Method
1.14 (10.0%)
9.70 ±0.28-0.07 (-0.7%)
8.46 ±0.28-0.42 (-5.5%)
6.61 ±0.19
0.38 (4.5%)
7.46 ±0.24
1.28* (11.9%)9.17 ±0.23
1.58* (16.0%)
7.76 ±0.59
NHK NRU Test Method0.75 (6.7%)
9.66 ±0.27-0.34 (-3.5%)
8.43 ±0.26-0.34 (-4.4%)
6.57 ±0.19
0.52* (6.1%)
7.49 ±0.25
1.21* (11.3%)
9.17 ±0.23
1.47* (14.8%)
7.66 ±0.59

With IC50Based
Starting
Dose6

Animals
Saved78.74 ±0.438.54 ±0.476.90 ±0.197.15 ±0.19
7.96 ±0.31
6.18 ±0.69

0.96 (9.9%)-0.08 (-1.0%)-0.29 (-4.3%)
0.31* (4.1%)
1.21* (13.2%)
1.58* (20.3%)

8.95 ±0.528.77 ±0.496.85 ±0.19
7.00 ±0.20
8.06 ±0.29
6.18 ±0.69

0.71 (7.3%)-0.33 (-3.9%)-0.28 (-4.2%)
0.49* (6.5%)
1.11* (12.1%)
1.47* (19.2%)

Abbreviations: 3T3=BALB/c 3T3 fibroblasts; GHS=Globally Harmonized System of Classification and Labelling of Chemicals (UN 2005); NHK=Normal humanepidermal keratinocytes; RC=Registry of Cytotoxicity; UDP=Up-and-Down Procedure.
*Statistically significant (p<0.05) by a one-sided Wilcoxon signed rank test. Percentage difference shown in parentheses.
1
Mean numbers of animals used ±standard errors for 10,000 simulations for each substance with an upper limit dose of 5000 mg/kg. Although the simulations used wholeanimals, averaging the results over a large number of simulations produced fractional numbers. Results are provided for 67 substances in the 3T3 NRU test method and 68
substances in the NHK NRU test method. Substances were categorized using the rat acute oral LD50 reference values in mg/kg from Table 4-2.
2
OECD (2001a); EPA (2002a).
3
UN (2005).
4
The RC rat-only millimole regression is log LD50 (mmol/kg) = 0.439 log IC50 (mM) + 0.621.
5
Default starting dose = 175 mg/kg.
6
The starting dose was one default dose lower than the predicted LD50 calculated using the IC50 value for each reference substance in the RC rat-only millimole regression.
The IC50 value for each reference substance was randomly selected from the distribution of values obtained during the testing with each method.
7
Difference between mean animal use with the default starting dose and mean animal use with the predicted starting dose.

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November 2006

The animal savings from the future use of these in vitro NRU test methods with the RC ratonly millimole regression will depend on the proportion of test substances that will fall into
each of the GHS categories.
Animal Savings for the UDP by GHS Category Using 3T3- and NHK-Based Starting Doses
with the RC Rat-Only Weight Regression
Table 10-4 shows the mean animal savings by GHS acute oral toxicity category when the
IC50 values are used with the RC rat-only weight regression. A comparison of mean animalsavings, by category, with the RC rat-only millimole regression, indicates that, in most cases,
animal savings were slightly higher for the RC rat-only weight regression than for the
millimole regression. In the RC rat-only weight regression, the mean differences between
animal use for the default starting dose and mean animal use with the IC50-determined
starting dose were statistically significant (p <0.05) by a one-sided Wilcoxon signed rank test
for the following GHS categories, NRU test methods, and dose-mortality slopes:• The use of the 3T3 NRU test method at the 8.3 mortality-slope for substanceswith 300< LD50 ≤2000 mg/kg that produced a savings of 0.28 (3.8%) animals
per test.
• The use of both in vitro NRU test methods at both dose mortality slopes for
substances with 2000< LD50 ≤5000 mg/kg that produced savings of 1.28
(14.0%) to 1.64 (15.2%) animals per test.
• The use of both in vitro NRU test methods at both dose-mortality slopes for
substances with LD50 >5000 mg/kg that produced savings of 1.53 (20.0%) to
1.65 (16.7%) animals per test.
For the dose-mortality slope of 2.0, the mean animal savings (for the various GHS
categories) with the 3T3 NRU test method ranged from -0.25 (-3.3%) to 1.65 (16.7%)
animals per test, and from -0.24 (-3.1%) to 1.54 (15.6%) animals per test using the NHK
NRU test method. At the dose-mortality slope of 8.3, animal savings with the 3T3 NRU test
method ranged from -0.18 (-2.7%) to 1.63 (21.0%) animals per test, and savings for the NHK
NRU test method ranged from -0.18 (-2.7%) to 1.53 (20.0%) animals per test. Animalsavings were also obtained for highly toxic substances (LD50 ≤5 mg/kg) with both the 3T3(0.78 [8.0%] to 0.90 [8.0%] animals per test) and NHK (0.69 [7.1%] to 0.72 [6.4%] animals
per test) NRU test methods, but these savings were not statistically significant.
There were no mean animal savings (≤-0.18 animals per test) for substances with 50 < LD50
≤300 mg/kg with either in vitro NRU test method. This category includes the default starting
dose of 175 mg/kg. Animal savings were not expected for this category because savings were
determined by comparing animal use at the IC50-based starting dose with animal use at thedefault starting dose. For the NHK NRU test method, there were no animal savings (-0.07 to-0.13 animals per test) when used for substances with 5 < LD50 ≤50 mg/kg. None of thesesmall changes in animal use were statistically significant.
The animal savings from testing new substances with these in vitro NRU test methods using
the RC rat-only weight regression will depend on the proportion of test substances that fall
into each of the GHS categories.

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In Vitro Cytotoxicity Test Methods BRD Section 10

Table 10-4

November 2006

Animal Use1 for the UDP2 by GHS Acute Oral Toxicity Category3 Using Starting Doses Based on the 3T3
and NHK NRU Test Methods with the RC Rat-Only Weight Regression4

GHS Acute Oral Toxicity
Category3

Number ofReference
Substances

LD50 ≤5 mg/kg
5 < LD50 ≤50 mg/kg
50 < LD50 ≤300 mg/kg
300 < LD50 ≤2000 mg/kg
2000 < LD50 ≤5000 mg/kg
LD50 >5000 mg/kg

6
11
12
16
10
12

LD50 ≤5 mg/kg
5 < LD50 ≤50 mg/kg
50 < LD50 ≤300 mg/kg
300 < LD50 ≤2000 mg/kg
2000 < LD50 ≤5000 mg/kg
LD50 >5000 mg/kg

6
11
12
16
10
13

Dose-mortality Slope = 2.0
Dose-mortality Slope = 8.3
With
With IC50With Default
With IC50Animals
Animals
Default
Based
Starting
Based
7Starting
Saved
Saved7
Starting
5
Dose
Starting Dose
5
Dose
Dose
3T3 NRU Test Method
11.29 ±0. 20
10.38 ±0.62
0.90 (8.0%)
9.70 ±0.28
8.92 ±0.37
0.78 (8.0%)
9.71 ±0.229.58 ±0.42
0.13 (1.3%)
8.47 ±0.28
8.41 ±0.44
0.06 (0.8%)
7.74 ±0.10
7.99 ±0.18-0.25 (-3.3%)
6.58 ±0.19
6.76 ±0.18-0.18 (-2.7%)
8.52 ±0.218.16 ±0.19
0.35 (4.1%)
7.46 ±0.24
7.17 ±0.16
0.28* (3.8%)
10.78 ±0.11
9.14 ±0.24
1.64* (15.2%)
9.20 ±0.24
7.61 ±0.37
1.59* (17.3%)
9.87 ±0.348.23 ±0.48
1.65* (16.7%)
7.76 ±0.59
6.14 ±0.69
1.63* (21.0%)
NHK NRU Test Method
11.21 ±0.24
10.49 ±0.71
0.72 (6.4%)
9.66 ±0.27
8.97 ±0.52
0.69 (7.1%)
9.70 ±0.189.78 ±0.41-0.07 (-0.8%)
8.45 ±0.27
8.59 ±0.44-0.13 (-1.6%)
7.75 ±0.11
7.99 ±0.21-0.24 (-3.1%)
6.58 ±0.19
6.76 ±0.18-0.18 (-2.7%)
8.54 ±0.21
8.20 ±0.22
0.34 (3.9%)
7.48 ±0.23
7.17 ±0.16
0.31 (4.1%)
10.77 ±0.08
9.40 ±0.25
1.38*(12.8%)
9.18 ±0.237.90 ±0.33
1.28* (14.0%)
9.88 ±0.32
8.34 ±0.44
1.54*(15.6%)
7.66 ±0.566.12 ±0.63
1.53* (20.0%)

Abbreviations: 3T3=BALB/c 3T3 fibroblasts; GHS=Globally Harmonized System of Classification and Labelling of Chemicals (UN 2005); NHK=Normal humanepidermal keratinocytes; RC=Registry of Cytotoxicity; UDP=Up-and-Down Procedure.
*Statistically significant (p <0.05) by a one-sided Wilcoxon signed rank test. Percent difference is shown in parentheses.
1
Mean number of animals used ±standard errors for 10,000 simulations for each substance with a limit dose of 5000 mg/kg. Although the simulations used whole animals,
averaging the results over a large number of simulations produced fractional numbers. Results are provided for 67 substances for the 3T3 NRU test method and 68
substances for the NHK NRU test method categorized using the rat acute oral LD50 reference values in mg/kg from Table 4-2.
2
OECD (2001a); EPA (2002a).
3
UN (2005).
4
The RC rat-only weight regression is log LD50 (mg/kg) = 0.372 log IC50 (µg/mL) + 2.024
5Default starting dose = 175 mg/kg.
6
The starting dose was one default dose lower than the predicted LD50 calculated using the IC50 values for each reference substance in the RC rat-only weight regression.
The IC50 value for each reference substance was randomly selected from the distribution of values obtained during the testing with each method.
7
Difference between mean animal use with the default starting dose and mean animal use with the predicted starting dose.

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November 2006

10.2.4

Refinement of Animal Use for the UDP When Using 3T3- and NHK-Based
Starting Doses
A procedure refines animal use when it lessens or eliminates pain or distress in animals or
enhances animal well-being (ICCVAM 2003). This section evaluates whether the use of 3T3and NHK-based starting doses refines animal use by reducing the number of animals that die
and experience accompanying pain and distress during UDP testing, compared to the numberof animals that die when the default starting dose of 175 mg/kg is used. Table 10-5 reports
the results for the UDP simulation modeling using the 5000 mg/kg limit dose. For every
regression evaluated, the mean number of deaths when using the IC50-based starting doses
were essentially equal to the mean number of deaths when using the default starting dose.
The percentage of deaths, however, was slightly higher for the IC50-based starting doses thanfor the default starting dose because the total number of animals used was lower for the IC50based starting doses. Thus, fewer animals were used when using an IC50-based starting dosecompared with use of the default starting dose, but the same numbers of animals died.
Table 10-5

Animal Deaths1 in the UDP2 Using Starting Doses Based on the 3T3 andNHK NRU Test Methods

Assay/Regression

With Default Starting Dose3

With IC50-Based Starting Dose4

Used

Used

Dead

3T3 NRU Test Method
RC rat-only millimole5
6

RC rat-only weight% DeathsDead% Deaths

Dose-Mortality Slope = 2.0
9.35

4.11

44.0%

8.80

4.09

46.5%

9.36

4.11

43.9%

8.70

4.05

46.6%

Dose-Mortality Slope = 8.3
5

RC rat-only millimole
6

RC rat-only weight

7.95

3.44

43.3%

7.42

3.43

46.2%

7.94

3.43

43.2%

7.32

3.39

46.3%

NHK NRU Test Method5

RC rat-only millimole

Dose-Mortality Slope = 2.0
9.36

4.08

43.6%

8.86

4.07

45.9%

RC rat-only weight

9.36

4.08

43.6%

8.80

4.02

45.7%

RC rat-only millimole5

7.92

3.39

42.8%

7.43

3.39

45.6%

7.92

3.39

42.8%

7.38

3.35

45.4%

6

Dose-Mortality Slope = 8.3
6

RC rat-only weight

Abbreviations: 3T3=BALB/c 3T3 fibroblasts; NHK=Normal human epidermal keratinocytes; RC=Registry ofCytotoxicity; UDP=Up-and-Down Procedure.
1
Numbers are mean numbers of animals used for 10,000 simulations for each substance. Although the simulations usedwhole animals, averaging the results over a large number of simulations produced fractional numbers. Upper limit dose =5000 mg/kg. Results are provided for 67 substances in the 3T3 NRU test method and 68 substances in the NHK NRU test
methods.
2
OECD (2001a); EPA (2002a).
3
Default starting dose = 175 mg/kg.
4
The starting dose was one default dose lower than the predicted LD50 calculated using the IC50 value in the regression
specified. The IC50 value for each reference substance was randomly selected from the distribution of values obtained during
the testing with each method.
5log LD50 (mmol/kg) = 0.439 log IC50 (mM) + 0.621.
6
log LD50 (mg/kg) = 0.372 log IC50 (µg/mL) + 2.024.

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November 2006

10.2.5
Accuracy of UDP Outcomes Using the IC50-Based Starting Doses
For each of the reference substances, the outcome of the simulated UDP testing, thesimulated LD50 was used to classify the substance into a GHS acute oral toxicity category.
The accuracy of GHS toxicity category assignments using the IC50-based starting doses wasdetermined by calculating the proportion of reference substances for which the GHS acuteoral toxicity category obtained using the IC50-based starting dose matched the categories
obtained using the default starting dose.
The concordance between the GHS categories determined using the 3T3 and NHK NRU test
methods with the RC rat-only millimole regression, and those determined using the UDP
default starting dose, was 96% for 3T3 and 97% for NHK (see Appendix N1). The
discordant reference substances were acetaminophen and sodium dichromate dihydrate in the
3T3 NRU test method, and acetaminophen, caffeine, and sodium dichromate dihydrate in the
NHK NRU test method. The use of the IC50-based starting dose from both in vitro NRU test
methods resulted in a higher GHS category (i.e., higher simulated LD50) for acetaminophen(simulated LD50 = 2047 vs. 1765 mg/kg for 3T3, and LD50 = 2174 vs. 1755 mg/kg for NHK),and a lower GHS category for sodium dichromate dihydrate (simulated LD50 = 44 vs. 52
mg/kg for 3T3 and LD50 = 45 vs. 52 mg/kg for NHK) than when using the default starting
dose. The NHK-based starting dose resulted in a lower GHS category for caffeine (simulated
LD50 = 280 vs. 357 mg/kg).
The concordance of GHS acute toxicity category predictions with those determined using thedefault starting dose was 97% for the 3T3 and NHK NRU test methods when the RC rat-only
weight regression was used (see Appendix N2). The discordant reference substances were
caffeine and sodium dichromate dihydrate. The simulated LD50 outcome for caffeine waslowered from 338 mg/kg for the default starting dose to 272 mg/kg for the 3T3-based starting
dose, and from 339 mg/kg to 270 mg/kg for the NHK-based starting dose. The simulated
LD50 outcome for sodium dichromate dihydrate was lowered from 51 mg/kg for the default
starting dose to 48 mg/kg for the 3T3-based starting dose, and from 51 mg/kg to 49 mg/kg
for the NHK-based starting dose.
Thus, the use of the IC50-based starting doses did not significantly alter the outcome of thesimulated UDP tests compared with the outcome obtained using the default starting doses.
10.3

Reduction and Refinement of Animal Use in the ATC Method

10.3.1In Vivo Testing Using the ATC Method
This section describes the general dosing procedure for the conduct of the ATC procedure(OECD 2001d). The ATC is used to assign a test substance to the appropriate GHS category
for classification and labeling. This is done by estimating the range of the LD50 values for thetest substance, rather than calculating a point estimate of the LD50. The time between
administration of test substance doses is determined by the onset, duration, and severity of
toxic signs. Guidance on the types of animals to use, animal housing, clinical observations,etc., which are outside the scope of the current discussion, are provided in the test guideline(See Appendix M3).

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November 2006

10.3.1.1 Main Test
The ATC method uses a stepwise administration of test substances to three animals at a time,at one of a number of fixed doses: 5, 50, 300, and 2000 mg/kg (and 5000 mg/kg, if
necessary). The starting dose is selected so that at least some of the animals die at that dose.
If no information on which to base a starting dose is available, a default starting dose of 300
mg/kg is used. The next step is determined by the starting dose and the outcome of the three
animals tested at the starting dose and may be a decision to stop testing, test additional
animals at the same dose, test at the next higher dose, or test at the next lower dose. For
example, if two to three animals die or are in a moribund state after receiving the 300 mg/kg
starting dose, the next step is to administer 50 mg/kg to three more animals. However, if no,or one, animal dies at 300 mg/kg, three additional animals are tested at that dose. Most
substances require two to four dosing steps before they can be classified, and testing can be
stopped. See Appendix M3 for the outcome-based testing sequence for each starting dose.
10.3.1.2 Limit Test
For test substances that are likely to be nontoxic, the ATC guideline includes a limit test inwhich six animals (three animals per step [see Appendix M3]) are tested at the limit dose of2000 mg/kg or three animals are tested at a limit dose of 5000 mg/kg (OECD 2001d).
10.3.2Computer Simulation Modeling of the ATC Method
The simulation for the ATC method was performed using MATLAB® (The MathWorks, Inc.
1996-2004) computational software, which is functionally comparable with SAS® version 8.
Two thousand simulations of ATC testing were run for each substance, in vitro NRU test
method, and dose-mortality slope, using an upper limit dose of 2000 mg/kg7. The simulation
implements the distributional assumptions underlying the dose-mortality response. The
lowest dose at which an animal dies in response to the administration of a toxic substancevaries from animal to animal. For an entire population of animals, mortality is assumed tohave a log-normal distribution with the mean equal to the log of the true LD50. Sigma (σ), thevariability of the simulated population, is the inverse of the slope of the dose-mortality curve.
For any given dose, the probability that an animal will die is computed by the cumulative
log-normal distribution:

1 log dose $( t$log trueLD )Probability (death) =dt& e 2"" 2# $%
50

2

2

Because of a lack of information regarding the real dose-mortality curves, the simulations
assumed several different values of the slope (i.e., the inverse of σ). Dose-mortality slopes of! chosen, so as to be comparable to the slopes chosen for
0.5, 0.8, 2.0, 4.0, and 8.3 were
simulation modeling of the UDP (see Section 10.2.2).
To model the variability of the IC50 values within and among laboratories, the values for each
substance were log-transformed to normalize their distribution. The mean and variance of

7

The results from ATC simulations for a limit dose of 5000 mg/kg will be presented in a future addendum tothis document.

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In Vitro Cytotoxicity Test Methods BRD Section 10

November 2006

these log-transformed values were used to generate a log-normal distribution from which torandomly select an IC50 value.
The simulation procedure used the following steps for each substance:
1. The rodent acute oral LD50 value (in mg/kg) from Table 4-2 was entered as
the true LD50 value and the choices of assumed slope were entered as the true
slope for the dose-mortality curve.
2. An IC50 value was selected from a distribution identified by the mean andvariance of the IC50 values computed from the data to reflect that different
laboratories produce different IC50 values in different situations (see Tables 54 and 5-5 for mean IC50 values and standard deviations for the 3T3 and NHK
NRU test methods, respectively).
3. The IC50 value from Step 2 was used in the regression model being evaluatedto compute a predicted LD50 value for determining the starting dose.
4. The dosing simulation (of 2000 iterations) was run twice: once with thedefault starting dose of 300 mg/kg and once with a starting dose equal to thenext fixed dose below the predicted LD50, which was estimated by the
regression being evaluated (i.e., the IC50-based starting dose). If the IC50based starting dose was greater than the 2000 mg/kg limit dose, then testingproceeded using the 2000 mg/kg limit test rather than the main test.
5. For every dose group of three animals, one observation was sampled from abinomial distribution with the probability of death calculated by theprobability equation for a population of three. The sampled value, referred toas N1, indicates the number of animals, 0, 1, 2, or 3, in the dosing group thatdie.
6. If N1 ≤1, step 4 is repeated with the same dose. The resulting sampled value
from the binomial distribution is referred to as N2.
7. If N2 ≤1 and the dose is the highest dose tested, or the dose has already been
decreased, a toxicity category is assigned and testing is terminated. If the doseis not the highest dose tested, or if the dose has not been decreased, the next
higher fixed dose is administered and step 4 is repeated.
8. If N1 >1 or N2 >2, and the dose is the lowest dose tested, or if the dose has
already been increased, a toxicity category is assigned and testing isterminated. If the dose is not the lowest dose tested, or if the dose has notalready been increased, the next lower fixed dose is administered and step 4 isrepeated.
10.3.3

Animal Savings for the ATC Method When Using 3T3- and NHK-Based Starting
Doses

10.3.3.1 The Effect of the Dose-Mortality Slope on Animal Use
As described in Section 10.3.2, the simulation modeling of animal use for the ATC used fivedifferent dose-mortality slopes to assess animal use under various conditions of population
variability. Table 10-6 shows how mean animal use for the simulated ATC changes withdose-mortality slope for both the default starting dose of 300 mg/kg and a starting dose thatwas one fixed dose lower than that predicted by the 3T3 and NHK NRU IC50 values with the
RC rat-only millimole regression. The mean number of animals used for the ATC method

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In Vitro Cytotoxicity Test Methods BRD Section 10

November 2006

decreased slightly with increasing slope for both the default starting dose and the IC50-based
starting dose.
The mean numbers of animals saved at all dose-mortality slopes were statistically significant(p <0.05 by one-sided Wilcoxon signed rank tests) when compared with mean animal use
with the default dose, and tended to decrease with increasing slope. To simplify thepresentation of animal savings and comparisons of the various regressions and starting doses,
subsequent results in Section 10.3.3 are shown only for dose-mortality slopes of 2.0 and 8.3.
As stated earlier, these slopes are shown here because the slope of 2.0 is the default used forthe calculation of LD50 by the UDP method (OECD 2001a; EPA 2002a) and the slope of 8.3
is shown to represent substances, such as pesticides, with higher slopes. Results for the otherdose-mortality slopes were computed, and are presented in Appendices N3 and N4.
Table 10-6

Change in Animal Use1 with Dose-Mortality Slope in the ATC Method2

Dose-Mortality Slope

With Default Starting
With IC50- Based
Dose1,3Starting Dose1,4
3T3 NRU Test Method

Animals Saved5

0.5

11.25 ±0.05

10.56 ±0.17

0.69* (6.1%)

0.8

11.10 ±0.07

10.46 ±0.19

0.64* (5.8%)

2.0

10.89 ±0.1210.27 ±0.24

0.62* (5.7%)

4.0

10.73 ±0.15

10.15 ±0.26

0.58* (5.4%)

8.3

10.64 ±0.1710.13 ±0.27

0.51* (4.8%)

NHK NRU Test Method
0.5

11.25 ±0.05

10.43 ±0.16

0.82* (7.3%)

0.8

11.10 ±0.07

10.31 ±0.18

0.79* (7.1%)

2.0

10.91 ±0.11

10.11 ±0.24

0.80* (7.3%)

4.0

10.75 ±0.15

9.98 ±0.27

0.77* (7.1%)

8.3

10.67 ±0.179.96 ±0.29

0.70* (6.6%)

Abbreviations: 3T3=BALB/c 3T3 fibroblasts; ATC=Acute Toxic Class method; NHK=Normal human epidermal
keratinocytes; RC=Registry of Cytotoxicity.
*Statistically significant (p <0.05) by a one-sided Wilcoxon rank test. Percent difference is shown in parentheses.
1
Mean numbers of animals used ± standard errors for 2000 simulations each for 67 substances in the 3T3 NRU test method
and 68 substances in the NHK NRU test method. Although the simulations used whole animals, averaging the results over alarge number of simulations produced fractional numbers. Upper limit dose = 2000 mg/kg.
2
OECD (2001d).
3
Default starting dose = 300 mg/kg.
4
Next fixed dose lower than the predicted LD50 calculated using the IC50 value for each reference substance in the RC ratonly millimole regression: log LD50 (mmol/kg) = 0.439 log IC50 (mM) + 0.621. The IC50 value for each reference substance
was randomly selected from the distribution of values obtained during the testing with each method.
5
Difference between mean animal use with the default starting dose and mean animal use with the IC50-based starting dose.

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November 2006

10.3.3.2 Mean Animal Use for ATC Simulations – Comparison of Regressions andPredictions from the 3T3 and NHK NRU Test Methods
Table 10-7 shows the mean animal use for testing the reference substances using thesimulated ATC method, when the starting dose was the default starting dose and when thestarting dose was one fixed dose lower than that determined by the 3T3 and NHK-predicted
LD50, and the regressions evaluated in Section 6.4 for prediction of GHS category. The meandifference in animal use between the two starting doses is the mean animal savings. All mean
animal savings were statistically significant (p <0.05 using one-sided Wilcoxon signed rank
tests), and ranged from 0.51 (4.8%) to 1.09 (10.2%) animals per test depending upon the
NRU test method, regression, and dose-mortality slope. The lowest mean animal savings
were obtained for the RC rat-only millimole regression (0.51 [4.8%] to 0.80 [7.3%] animals
per test), and the highest were obtained with the RC rat-only weight regression (0.91 [8.6%]
to 1.09 [10.2%] animals per test).
The animal savings obtained using the in vitro NRU test methods with the RC rat-onlyregressions apply only to the reference substances evaluated in this validation study, and are
based on substances pre-selected for their known in vivo toxicities and may not be broadly
applicable to other substances. Table 3-4 shows that 22 (38%) of the 58 RC substances
selected for testing were known to have a poor fit to the RC millimole regression (i.e., thepredicted LD50 was outside the RC acceptance interval). Table 6-3 shows that 40% (28/70 in
the 3T3) and 44% (31/71 in the NHK) of the reference substances that yielded IC50 values
were outliers. Substances that better fit the regression are likely to yield greater animalsavings.

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Table 10-7

November 2006

Animal Use1 for the ATC2 Method Using Starting Doses Based on the 3T3 and NHK NRU Test Methods with
the Different Regressions

Method/Regression

With Default
Starting Dose3

3T3 NRU Test Method
RC rat-only millimole6
7

RC rat-only weight7

RC rat-only weight

Animals
Saved5Dose-Mortality Slope = 2.0

With Default
Starting Dose3

With IC50- Based
Starting Dose5

Animals
Saved5Dose-Mortality Slope = 8.3

10.89 ±0.1210.27 ±240.62* (5.7%)

10.64 ±0.1710.13 ±0.27

0.51* (4.8%)

10.89 ±0.129.85 ±0.24

1.04* (9.6%)

10.64 ±0.179.55 ±0.29

1.09* (10.2%)

NHK NRU Test Method
RC rat-only millimole6

With IC50- Based
Starting Dose4

Dose-Mortality Slope = 2.0

Dose-Mortality Slope = 8.3

10.91 ±0.11

10.11 ±0.24

0.80* (7.3%)

10.67 ±0.179.96 ±0.29

0.70* (6.6%)

10.91 ±0.119.95 ±0.24

0.96* (8.8%)

10.67 ±0.179.75 ±0.30

0.91* (8.6%)

Abbreviations: 3T3=BALB/c 3T3 fibroblasts; ATC=Acute Toxic Class method; NHK=Normal human epidermal keratinocytes; RC=Registry of Cytotoxicity.
*Statistically significant (p<0.05) using a one-sided Wilcoxon signed rank test. Percentage difference is shown in parentheses.
1
Mean numbers of animals used ±standard errors for 2000 simulations each for 67 substances in the 3T3 NRU test method and 68 substances in the NHK NRU
test method. Upper limit dose =2000 mg/kg.
2
OECD (2001d).
3
Default starting dose =300 mg/kg.
4
Starting dose was one fixed dose lower than the predicted LD50 calculated using the IC50 value for each reference substance in the regression specified. The IC50
value for each reference substance was randomly selected from the distribution of values obtained during the testing with each test method.
5
Difference between mean animal use with the default starting dose and mean animal use with the IC50-based starting dose.
6
log LD50 (mmol/kg) = 0.439 log IC50 (mM) + 0.621.
7
log LD50 (mg/kg) = 0.372 log IC50 (µg/mL) + 2.024.

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November 2006

10.3.3.3 Animal Savings in the ATC Method by GHS Acute Oral Toxicity Category Using
the 3T3- and NHK -Based Starting Doses
Tables 10-8 and 10-9 show mean animal use and mean animal savings for the ATC whenused with the in vitro NRU test methods, organized by GHS category (UN 2005), and whenbased on the:• RC rat-only millimole regression (Table 10-8)• RC rat-only weight regression (Table 10-9)
The following data come from the same analyses as the data provided in Table 10-7.
The analyses showed that:• For each in vitro NRU test method and regression, the highest mean animalsavings were generally in the LD50 ≤5 mg/kg and LD50 >5000 mg/kg toxicity
categories.
• For each NRU test method and regression, the lowest mean animal savings
were in the 300 < LD50 ≤2000 mg/kg toxicity category.
Animal Savings in the ATC Method by GHS Category Using the 3T3- and NHK-Based
Starting Doses with the RC Rat-Only Millimole Regression
Table 10-8 shows the mean animal savings in the ATC method by GHS category for the invitro NRU test methods used with the RC rat-only millimole regression. Mean differences
between animal use for the default starting dose and with the IC50-determined starting dosewere statistically significant (p <0.05) by a one-sided Wilcoxon signed rank test for thefollowing GHS toxicity categories, NRU test methods, and dose-mortality slopes:• The use of both in vitro NRU test methods at both dose-mortality slopes for
substances with 5 < LD50 ≤50 mg/kg produced savings of 1.15 (9.8%) to 1.33
(11.4%) animals per test
• The use of the 3T3 NRU test method at both dose-mortality slopes for
substances with 300 < LD50 ≤2000 mg/kg used more animals per test (i.e.,
produced savings of -0.92 [-9.5%] to -1.30 [-14.0%] animals per test)• The use of both in vitro NRU test methods at both dose-mortality slopes for
substances with LD50 >5000 mg/kg produced savings of 2.03 (17.1%) to 2.66
(22.2%) animals per test
At the dose-mortality slope of 2.0, the mean animal savings with the 3T3 NRU test method
ranged from -0.92 (-9.5%) to 2.68 (27.4%) animals per test, and the animal savings with the
NHK NRU test method ranged from -0.60 (-6.1%) to 2.96 (30.4%) animals per test. At thedose-mortality slope of 8.3, the mean animal savings with the 3T3 NRU test method ranged
from -1.30 (-14.0%) to 2.70 (29.7%) animals per test, and the animal savings with the NHK
NRU test method ranged from -0.85 (-9.2%) to 2.99 (33.0%) animals per test.

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Table 10-8

November 2006

Animal Savings1 for the ATC2 Method by GHS Acute Oral Toxicity Category3 Using Starting Doses Basedon the 3T3 and NHK NRU Test Methods with the RC Rat-Only Millimole Regression4
Dose-Mortality Slope = 2.0

GHS Acute Oral Toxicity
Category3

Number ofReference
Substances

With Default
Starting
Dose5

With IC50Based
Starting
Dose6

LD50 ≤5 mg/kg
5 < LD50 ≤50 mg/kg
50 < LD50 ≤300 mg/kg
300 < LD50 ≤2000 mg/kg
2000 < LD50 ≤5000 mg/kg
LD50 >5000 mg/kg

6
11
12
16
10
12

9.77 ±0.1711.56 ±0.2110.81 ±0.20
9.75 ±0.07
11.22 ±0.08
11.85 ±0.04

7.09 ±1.09
10.39 ±0.52
10.39 ±0.1710.67 ±0.48
11.14 ±0.08
9.82 ±0.78

LD50 ≤5 mg/kg
5 < LD50 ≤50 mg/kg
50 < LD50 ≤300 mg/kg
300 < LD50 ≤2000 mg/kg
2000 < LD50 ≤5000 mg/kg
LD50 >5000 mg/kg

6
11
12
16
10
13

9.74 ±0.16
11.56 ±0.21
10.83 ±0.219.77 ±0.06
11.22 ±0.08
11.86 ±0.03

6.78 ±1.31
10.38 ±0.35
10.39 ±0.29
10.37 ±0.49
11.25 ±0.129.43 ±0.73

Animals Saved

Dose-Mortality Slope = 8.3
7With Default
Starting Dose5

3T3 NRU Test Method
2.68 (27.4%)
9.08 ±0.08
1.17* (10.2%)
11.75 ±0.16
0.42 (3.9%)9.42 ±0.26-0.92* (-9.5%)
9.26 ±0.10
0.08 (0.7%)
11.88 ±0.10
2.03* (17.1%)
12.00 ±0.000
NHK NRU Test Method
2.96 (30.4%)
9.09 ±0.08
1.18* (10.2%)
11.76 ±0.17
0.44 (4.0%)
9.44 ±0.26-0.60 (-6.1%)
9.26 ±0.10-0.03 (-0.3%)
11.87 ±0.102.43* (20.5%)
12.00 ±0.000

WithIC50Based
Starting
Dose6
6.38 ±1.09
10.60 ±0.439.27 ±0.1110.56 ±0.62
11.77 ±0.109.81 ±0.84
6.09 ±1.2310.42 ±0.45
9.63 ±0.49
10.11 ±0.63
11.89 ±0.15
9.34 ±0.80

Animals
Saved72.70 (29.7%)
1.15* (9.8%)
0.15 (1.6%)-1.30* (-14.0%)

0.11 (0.9%)
2.19* (18.3%)
2.99 (33.0%)
1.33* (11.4%)-0.20 (-2.1%)-0.85 (-9.2%)-0.02 (-0.2%)
2.66* (22.2%)

Abbreviations: 3T3=BALB/c 3T3 fibroblasts; ATC=Acute Toxic Class method; GHS=Globally Harmonized System of Classification and Labelling of Chemicals (UN2005); NHK=Normal human epidermal keratinocytes; RC=Registry of Cytotoxicity.
*Statistically significant (p <0.05) by a one-sided Wilcoxon signed rank test. Percentage difference is shown in parentheses.
1
Mean number of animals used ±standard errors for 2000 simulations for each substance with an upper limit dose of 2000 mg/kg. Results are provided for 67 substances inthe 3T3 NRU test method and 68 substances in the NHK NRU test method categorized using the rat acute oral LD50 reference values in mg/kg from Table 4-2. Although
the simulations used whole animals, averaging the results over a large number of simulations produced fractional numbers.
2
OECD (2001d).
3
GHS for acute oral toxicity (UN 2005).
4
The RC rat-only millimole regression is log LD50 (mmol/kg) = 0.439 log IC50 (mM) + 0.621.
5
Default starting dose =300 mg/kg.
6
The starting dose was the next fixed dose lower than the predicted LD50 using the IC50 for each reference substance in the RC rat-only millimole regression. The IC50 value
for each reference substance was randomly selected from the distribution of values obtained during the testing with each method.
7
Difference between mean animal use with the default starting dose and mean animal use with the IC50-based starting dose.

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In Vitro Cytotoxicity Test Methods BRD Section 10

November 2006

At both the 2.0 and 8.3 dose-mortality slopes, the mean animal savings using the 3T3 NRU
test method were lower than the corresponding savings using the NHK NRU test method, forsubstances in at least four of the six toxicity categories: LD50 ≤5 mg/kg; 5 < LD50 ≤50 mg/kg;
300 < LD50 ≤2000 mg/kg; and LD50 >5000 mg/kg. The mean animal savings per test werehigher with the 3T3 NRU test method than the NHK NRU test method for substances in the
2000< LD50 ≤5000 mg/kg category at both dose-mortality slopes. For substances in the 50 <LD50 ≤300 mg/kg category, the mean animal savings using the 3T3 NRU test method was
greater than the savings using the NHK NRU test method, when the dose-mortality slope
equaled 8.3. When the 3T3 NRU test method was used, the highest mean animal savings
occurred when testing substances in the LD50 ≤5 mg/kg category (2.68 [27.4%] animals per
test at dose-mortality slope = 2.0, and 2.70 [29.7%] at dose-mortality slope = 8.3). When the
NHK NRU test method was used, the highest mean animal savings occurred when testing
substances in the LD50 ≤5 mg/kg category (2.96 [30.4%] animals per test at dose-mortality
slope = 2.0, and 2.99 [33.0%] animals per dose at dose-mortality slope = 8.3). However, theanimal savings were not statistically significant with either in vitro NRU test method.
The smallest mean animal savings (≤0.44) in both in vitro NRU test methods were observed
for substances with LD50 values between 50 and 5000 mg/kg. Because the default starting
dose was 300 mg/kg, little change in mean animal use was expected for substances in the 50< LD50 ≤300 mg/kg and 300 < LD50 ≤2000 mg/kg categories. The mean animal savings fromboth in vitro NRU test methods and both dose-mortality slopes for the substances in the 50 <LD50 ≤300 mg/kg category were -0.20 to 0.44 animals per test. There were no animal savingsfor substances in the 300 < LD50 ≤2000 mg/kg category using either NRU test method or
dose-mortality slope. In fact, significantlyore animals were used when the starting doses werebased on the 3T3 NRU IC50 than using the default starting dose (-0.92 to -1.30 animals pertest). More animals were also used when the starting doses were based on the NHK NRU
IC50 (-0.85 to -0.60 animals/test), but the difference was not statistically significant.
The animal savings in the various GHS acute oral toxicity categories using the in vitro NRU
test methods with the RC rat-only millimole regression applies only to the reference
substances evaluated in this validation study, and may not be broadly applicable to othersubstances. The animal savings for future testing using the in vitro NRU test methods with
the RC rat-only millimole regression will depend on the prevalence of test substances in eachof the GHS acute oral toxicity categories.
Animal Savings with the ATC Method by GHS Category Using 3T3- and NHK-Based
Starting Doses with the RC Rat-Only Weight Regression
Table 10-9 shows the animal savings for the simulated ATC method by GHS category forthe in vitro NRU methods used with the RC rat-only weight regression. Mean animal savings
were statistically significant (p <0.05) by a one-tailed Wilcoxon signed rank test for thefollowing GHS toxicity categories, NRU test methods, and dose-mortality slopes.
• The use of both in vitro NRU test methods at both dose-mortality slopes for
substances with 5 < LD50 ≤50 mg/kg produced savings of 1.25 (10.8%) to
1.51 (13.0%) animals per test.

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Table 10-9

November 2006

Animal Savings1 for the ATC2 Method by GHS Acute Oral Toxicity Category3 Using Starting Doses
Based on the 3T3 and NHK NRU Test Methods with the RC Rat-Only Weight Regression4

GHS Acute Oral Toxicity
Category3

Number ofReference
Substances

LD50 ≤5 mg/kg
5 < LD50 ≤50 mg/kg
50 < LD50 ≤300 mg/kg
300 < LD50 ≤2000 mg/kg
2000 < LD50 ≤5000 mg/kg
LD50 >5000 mg/kg

6
11
12
16
10
12

LD50 ≤5 mg/kg
5 < LD50 ≤50 mg/kg
50 < LD50 ≤300 mg/kg
300 < LD50 ≤2000 mg/kg
2000 < LD50 ≤5000 mg/kg
LD50 >5000 mg/kg

6
11
12
16
10
13

Dose-Mortality Slope = 2.0
Dose-Mortality Slope = 8.3
With
With IC50With
With IC50Animals
Animals
Default
Based
Default
Based
Starting
Saved7
Starting
Starting
Saved7
Starting
6
5
6
5
Dose
Dose
Dose
Dose
3T3 NRU Test Method
9.77 ±0.177.56 ±1.03
2.21 (22.6%)
9.08 ±0.08
6.85 ±0.99
2.24 (24.6%)
11.56 ±0.21
10.06 ±0.38
1.51* (13.0%) 11.75 ±0.16
10.27 ±0.33
1.48* (12.6%)
10.81 ±0.20
10.35 ±0.18
0.47* (4.3%)
9.42 ±0.26
9.20 ±0.10
0.22 (2.4%)
9.75 ±0.07
10.67 ±0.50-0.93* (-9.5%)
9.26 ±0.1010.65 ±0.66-1.39 (-15.0%)
11.22 ±0.08
9.80 ±0.51
1.43* (12.7%) 11.88 ±0.109.44 ±0.88
2.43 (20.5%)
11.85 ±0.04
8.83 ±0.83
3.02* (25.5%) 12.00 ±0.00
8.67 ±0.91
3.33* (27.7%)
NHK NRU Test Method
9.74 ±0.166.87 ±1.282.87 (29.4%)
9.09 ±0.08
6.18 ±1.20
2.91 (32.0%)
11.56 ±0.2110.31 ±0.19
1.25* (10.8%) 11.76 ±0.1710.40 ±0.33
1.36* (11.5%)
10.83 ±0.21
10.41 ±0.28
0.42 (3.8%)
9.44 ±0.26
9.63 ±0.49-0.20 (-2.1%)
9.77 ±0.62
10.46 ±0.50-0.69 (-7.1%)
9.26 ±0.1010.23 ±0.65-0.97 (-10.4%)
11.22 ±0.09
10.69 ±0.37
0.53 (4.7%)
11.87 ±0.10
11.03 ±0.60
0.84 (7.1%)
11.86 ±0.03
8.91 ±0.78
2.94* (24.8%) 12.00 ±0.00
8.75 ±0.85
3.25* (27.1%)

Abbreviations: 3T3=BALB/c 3T3 fibroblasts; ATC=Acute Toxic Class method; GHS=Globally Harmonized System of Classification and Labelling of Chemicals (UN2005); NHK=Normal human epidermal keratinocytes; RC=Registry of Cytotoxicity.
*Statistically significant (p <0.05) by a one-sided Wilcoxon signed rank test. Percentage difference is shown in parentheses.
1
Mean number of animals used ±standard errors for 2000 simulations for each substance with an upper limit dose of 2000 mg/kg. Although the simulations used wholeanimals, averaging the results over a large number of simulations produced fractional numbers. Results are provided for 67 substances in the 3T3 NRU test method and68 substances in the NHK NRU test method categorized using the rat acute oral reference LD50 values in mg/kg from Table 4-2.
2
OECD (2001d).
3
GHS for acute oral toxicity (UN 2005).
4
From Table 6-2; log LD50 (mg/kg) = 0.372 log IC50 (µg/mL) + 2.024
5Default starting dose = 300 mg/kg.
6
The starting dose was one fixed dose lower than the predicted LD50 calculated using the IC50 for each reference substance in the RC rat-only weight regression. The IC50
value for each reference substance was randomly selected from the distribution of values obtained during the testing with each method.
7
Difference between mean animal use with the default starting dose and mean animal use with the IC50-based starting dose.

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In Vitro Cytotoxicity Test Methods BRD Section 10

•
•

•
•

November 2006

The use of the 3T3 NRU test method at the 2.0 dose-mortality slope for
substances with 50 < LD50 ≤300 mg/kg produced savings of 0.47 (4.3%)
animals per test.
The use of the 3T3 NRU test method at the 2.0 dose-mortality slope for
substances with 300 < LD50 ≤2000 mg/kg produced savings of -0.93 (-9.5%)
animals per test (i.e., used more animals per test than the default starting
dose).
The use of the 3T3 NRU test method at the 2.0 dose-mortality slope for
substances with 2000 < LD50 ≤5000 mg/kg produced savings of 1.43 (12.7%)
animals per test.
The use of both in vitro NRU test methods at both dose-mortality slopes for
substances with LD50 >5000 mg/kg produced savings of 2.94 (24.8%) to 3.33
(27.7%) animals per test.

The mean animal savings with the 3T3 and NHK NRU test methods were similar for mostacute oral toxicity categories at both dose-mortality slopes; the mean savings for the 3T3
NRU test method was slightly higher than for the NHK NRU test method for most toxicity
categories. At the dose-mortality slope of 2.0, the mean animal savings for the 3T3 NRU test
method (for the various toxicity categories) ranged from -0.93 (-9.5%) to 3.02 (25.5%)
animals per test, and savings for the NHK NRU test method ranged from -0.69 (-7.1%) to
2.94 (24.8%) animals per test. At the dose-mortality slope of 8.3, animal savings with the
3T3 NRU test method ranged from -1.39 (-15.0%) to 3.33 (27.7%) animals per test, andsavings with the NHK NRU test method ranged from -0.97 (-10.4%) to 3.25 (27.1%) animals
per test.
There were no mean animal savings (≤-0.69 animals) for substances with 300 < LD50 ≤2000
when either in vitro NRU test method was used. The mean animal savings for the substancesin the 50 < LD50 ≤300 mg/kg category using both in vitro NRU test methods and dosemortality slopes were also relatively small (-0.20 to 0.47 animals per test). Because thedefault starting dose was 300 mg/kg, little change in mean animal use was expected for
substances in the 50< LD50 ≤300 mg/kg and 300 < LD50 ≤2000 mg/kg categories.The highest
mean animal savings (≤-0.69 animals) occurred for substances with LD50 >5000 mg/kg
when either in vitro NRU test method was used. For both test methods and dose-mortality
slopes, the mean animal savings for substances in this category were 2.94 (24.8%) to 3.33
(27.7%) animals per test and were statistically significant. Mean animal savings were alsohigh (2.21 [22.6%] to 2.91 [32.0%] animals per test) for substances with LD50 ≤5 mg/kg, butthese savings were not statistically significant.
The animal savings in the various GHS categories using the two in vitro NRU test methods
with the RC rat-only weight regression applies only to the reference substances evaluated inthis validation study, and may not be broadly applicable to other substances.
10.3.4

Refinement of Animal Use in the ATC Method When Using 3T3- and NHK-Based
Starting Doses
A procedure refines animal use when it lessens or eliminates pain or distress in animals, orenhances animal well-being (ICCVAM 2003). This section evaluates whether the use of 3T3and NHK-based starting doses refines animal use by reducing the number of animals that die
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In Vitro Cytotoxicity Test Methods BRD Section 10

November 2006

when the IC50-predicted starting doses are used, compared to the number of animals that die
when using the default ATC starting dose of 300 mg/kg. Table 10-10 reports the results for
the ATC simulation modeling using the 2000 mg/kg limit dose. For every regression
evaluated, the mean number of deaths when using the 3T3- and NHK-based starting doses
was less than the mean number of deaths when using the default starting dose, byapproximately 0.4 to 0.5 deaths per test. For the RC rat-only millimole regression and the RC
rat-only weight regression, the percentage of deaths (compared with the numbers of animals
used) was also slightly lower with the in vitro-based starting dose compared with the default
starting dose. In general, fewer animals were used with the in vitro-based starting dose, andfewer animals died.
Table 10-10 Animal Deaths1 for the ATC2 Method Using Starting Doses Based on the
3T3 and NHK NRU Test Methods

Method/Regression

Default Starting Dose3
Used

Dead

3T3 NRU Test Method5

RC rat-only millimole
6

RC rat-only weight% Deaths

IC50- Based Starting Dose4
Used

Dead

% Deaths

Dose-Mortality Slope = 2.0
10.89

3.77

34.6%

10.27

3.31

32.2%

10.89

3.77

34.6%

9.85

3.27

33.2%

Dose-Mortality Slope = 8.3
5

RC rat-only millimole
RC rat-only weight

6

10.64

3.20

30.1%

10.13

2.77

27.3%

10.64

3.20

30.1%

9.55

2.73

28.6%

10.91

3.72

34.1%

10.11

3.19

31.6%

10.91

3.72

34.1%

9.95

3.21

32.3%

NHK NRU Test Method
RC rat-only millimole5
RC rat-only weight

6

Dose-Mortality Slope = 2.0

Dose-Mortality Slope = 8.3
5

RC rat-only millimole
RC rat-only weight

6

10.67

3.15

29.5%

9.96

2.67

26.8%

10.67

3.15

29.5%

9.75

2.67

27.4%

Abbreviations: 3T3=BALB/c 3T3 fibroblasts; ATC=Acute Toxic Class method; NHK=Normal human epidermal
keratinocytes; RC=Registry of Cytotoxicity.
1
Mean numbers of animals used for 2000 simulations for each of 67 substances in the 3T3 NRU test method and 68
substances in the NHK NRU test method. Although the simulations used whole animals, averaging the results over a large
number of simulations produced fractional numbers. Upper limit dose =2000 mg/kg.
2
OECD (2001d).
3
Default starting dose =300 mg/kg.
4
The starting dose was one fixed dose lower than the predicted LD50 calculated by using the IC50 for each reference
substance in the regression evaluated. The IC50 value for each reference substance was randomly selected from the
distribution of values obtained during the testing with each method.
5log LD50 (mmol/kg) = 0.439 log IC50 (mM) + 0.621.
6
log LD50 (mg/kg) = 0.372 log IC50 (µg/mL) + 2.024.

10.3.5
Accuracy of the ATC Method Outcomes Using the IC50-Based Starting Doses
The accuracy of the outcome of the simulated ATC testing (i.e., the simulated GHS acuteoral toxicity category) using the IC50-based starting dose was determined by calculating the
proportion of reference substances for which the simulated GHS category for the IC50-based
starting dose matched the simulated GHS category for the default starting dose.
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When the RC rat-only millimole regression with the 3T3 and NHK NRU test methods wasused, the concordance of simulated GHS categories for the IC50-based starting doses withthose for the default starting dose was 99% for both in vitro NRU test methods (seeAppendix N3). The discordant reference substance in the 3T3 NRU test method wascaffeine. The simulated GHS category using the 3T3-based starting dose was 50 < LD50 ≤300
mg/kg, and the simulated GHS category using the default starting dose was 300 < LD50
≤2000 mg/kg.
The discordant reference substance in the NHK NRU test method was sodium dichromate
dihydrate. The simulated GHS acute oral toxicity category using the NHK-based starting
dose was 5 < LD50 ≤50 mg/kg and the simulated GHS category using the default starting
dose was 50 < LD50 ≤300 mg/kg. Both discordant substances were predicted to have astarting dose one category below the actual category.
When the RC rat-only weight regression was used with the 3T3 and NHK NRU test methods,
the concordance of simulated GHS acute toxicity category predictions with those determinedusing the default starting dose was 99% and 97% for the 3T3 and the NHK NRU test
methods, respectively (see Appendix N4). The discordant reference substance in the 3T3
NRU test method was caffeine. The simulated GHS acute oral toxicity category for caffeine
using the 3T3-based starting dose was 50 < LD50 ≤300 mg/kg and that using the default
starting dose was 300 < LD50 ≤2000 mg/kg. The discordant reference substances in the NHK
NRU test method were caffeine and sodium dichromate dihydrate. The simulated GHS acuteoral toxicity category for caffeine using the NHK-based starting dose was 50 < LD50 ≤300
mg/kg and the simulated GHS category using the default starting dose was 300 < LD50 ≤2000
mg/kg. The simulated GHS acute oral toxicity category for sodium dichromate dihydrateusing the NHK-based starting dose was 5 < LD50 ≤50 mg/kg while that for the default
starting dose was 50 < LD50 ≤300 mg/kg. Similar to what was seen with the RC millimole
regression, the predicted starting doses for the discordant substances were one GHS category
below the actual category.
Thus, the use of the IC50-based starting doses did not significantly alter the outcomes of thesimulated ATC tests compared with the outcome based on the default starting dose.
10.4

The Impact of Accuracy on Animal Savings

Two types of accuracy analyses were performed for the NICEATM/ECVAM validation
study. The first analyses determined the accuracy of using the NRU IC50 values with an IC50LD50 regression to predict LD50 values. It calculated the concordance for GHS acute oral
toxicity category by comparing the GHS categorization yielded by the NRU-predicted LD50
values (using the in vitro NRU IC50 values in the regressions presented in Table 6-5) with the
GHS categorization based on rat acute oral LD50 data (see Section 6.4). The second analysis
determined the accuracy of the simulation outcomes using the IC50-based starting doses (seeSections 10.2.5 and 10.3.5). It calculated the concordance for the GHS acute oral toxicity
category outcomes obtained using the IC50-based starting doses with the GHS category
outcomes obtained using the default starting dose. The magnitude of animal savings did notcorrelate with either determination of accuracy and the accuracy determinations for IC50based predictions and IC50-based outcomes for GHS category did not correlate with one
another.
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Animal savings did not correlate with the accuracy of the GHS acute oral toxicity category
predictions based on the LD50 values calculated using the IC50 values in the RC rat-onlyregressions (see Sections 6.4.2 and 6.4.3). Substances in categories with the lowest accuracy
produced the highest animal savings. For example, using the RC rat-only millimole
regression with the in vitro NRU IC50 values yielded very low accuracy (0 to 17%) for GHS
acute oral toxicity category prediction for substances with LD50 >5000 mg/kg (see Table 67), but the highest animal savings of 14.8 to 20.3% occurred in this category (see Table 103). Animal savings were small, 4.5 to 6.5%, for substances with 300 ≤ LD50 ≤2000 mg/kg,
but the accuracy of 75-81% for GHS acute oral toxicity category prediction was relatively
high. The reason that animal savings is unrelated to the accuracy of prediction of GHS acuteoral toxicity category based on the LD50 values calculated using IC50 values in the RC ratonly regressions is because two different standards are used for comparison in the twoanalyses:•
GHS acute oral toxicity category predictions using IC50 values in the RC ratonly regressions are compared with the GHS categories derived from the invivo reference LD50•
The number of animals used (to determine animal savings) was comparedwith the animal use at the default starting dose of 175 mg/kg for the UDP or
300 mg/kg for the ATC
Despite the relatively poor GHS accuracy for the low toxicity chemicals (the toxicity ofalmost all were overpredicted by one GHS category), animal savings were greatest due to thefact that testing goes to the limit dose faster.
The accuracy of the simulated GHS toxicity category assignments using the IC50-based
starting doses for UDP and ATC test simulations was determined by calculating the
proportion of reference substances for which the GHS acute oral toxicity category obtainedusing the IC50-based starting dose matched the categories obtained using the default starting
dose (see Sections 10.2.5 and 10.3.5). The accuracy of these GHS toxicity category
assignments based on the simulation outcomes does not correlate with animal savings using
the IC50 values in the RC rat-only regressions (see Sections 6.4.2 and 6.4.3). For example,
the accuracy of GHS acute oral toxicity category outcomes for the ATC test method whenusing the RC rat-only millimole regression was 100% for the 3T3 NRU test method for
substances with 300 ≤ LD50 ≤2000 mg/kg (see Appendix N3). In contrast, the animal savingsfor those substances was negative at -6.1 to -14.0% (i.e., more animals were used comparedwith the default starting dose) (see Table 10-8). The reason the outcome-based GHS acuteoral toxicity category predictions is unrelated to animal savings is that two different
parameters are being measured in the two analyses:•
The accuracy of the simulatedGHS acute oral toxicity outcomes using theIC50-based starting doses measured outcome (i.e., simulated GHS category
based on the simulated LD50 outcome for the UDP and simulated GHS
category for the ATC)•
The animal savings analysis measured the number of animals used at the IC50based starting dose and the default starting dose of 175 mg/kg for the UDP or
300 mg/kg for the ATC

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Thus, the measurements for the two analyses are different: outcome (i.e., GHS category) andnumber of animals used to achieve the outcome.
In addition, accuracy of the GHS toxicity category assignments based on the simulation
outcomes does not correlate with the accuracy of the GHS acute oral toxicity category
predictions using the IC50 values in the RC rat-only regressions (see Section 6.4.2 and 6.4.3).
For example, the overall accuracy of GHS acute oral toxicity category outcomes for the ATCtest method when using the RC rat-only millimole regression was 99% for both in vitro NRU
test methods (see Section 10.3.5 and Appendix N3). In contrast, the overall accuracy of
GHS acute oral toxicity category predictions using the using the IC50 values in the RC ratonly millimole regression was 31% for the 3T3 NRU test method and 29% for the NHK
NRU test method (see Table 6-7). The reason the simulated outcome-based GHS acute oral
toxicity category predictions differed from the accuracy of the GHS acute oral toxicity
category predictions based on the calculation of LD50 using the IC50 in the IC50-LD50
regression is because two different standards are used for comparison in the two analyses:•
Simulated GHS acute oral toxicity outcomes for the IC50-based starting doses
were compared with the simulated GHS category outcomes using the default
starting doses•
GHS acute oral toxicity category predictions using the IC50 values in the RC
rat-only regressions were compared with the GHS category derived from thein vivo reference LD50
Thus, despite that the IC50 values and IC50-LD50 regressions predicted GHS acute oral
toxicity categories poorly, the GHS acute oral toxicity category outcomes using the IC50based starting doses were practically the same as the GHS acute oral toxicity category
outcomes using the default starting dose.
10.5

The Impact of Prevalence on Animal Savings

As stated several times in this section, the animal savings for substances tested in the
futureusing the 3T3 and NHK NRU test methods to determine the staring dose for rodent
acute oral toxicity test methods will depend on the proportion of test substances that fall into
each of the GHS acute toxicity hazard categories. Although the prevalence of substancesamong the different categories will depend, to a large extent, on the mandate of a particularregulatory agency, Spielmann et al. (1999) indicated that 76% (845/1115) of the industrial
substances submitted to the Federal Institute for Health Protection of Consumers andVeterinary Medicine in Berlin, Germany, since 1982 had LD50 >2000 mg/kg. The extent towhich these substances represent the population of substances in commerce is not known.
However, if the results of the validation study are broadly applicable to substances to betested in the future, and if such substances are relatively nontoxic, the selection of starting
doses using the in vitro NRU test methods may save a considerable number of animals sinceanimal savings for the validation study were highest for the least toxic substances.

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Summary

Computer simulation modeling of UDP testing using the default dose progression shows that,for the subset of reference substances evaluated, the prediction of starting doses using the
3T3 and NHK NRU test methods with the RC rat-only millimole regression resulted in astatistically significant (p <0.05) decrease in the number of animals used by an average of
0.49 (6.2%) to 0.54 (5.8%) animals per test, depending upon the in vitro NRU test method
and the dose-mortality slope (2.0 or 8.3) used. The mean animal savings improved slightly, to0.54 (6.8%) to 0.66 (7.0%) animals per test, when the RC rat-only weight regression wasused.
When reference substances were grouped by GHS category, there were no mean animalsavings by simulated UDP testing for substances with 50< LD50 ≤300 mg/kg. The highest,
and statistically significant, animal savings were observed with both in vitro NRU test
methods when testing substances with 2000< LD50 ≤5000 mg/kg and LD50 >5000 mg/kg.
When using the RC rat-only millimole regression, animal savings for these categories ranged
from 1.28 (11.9%) to 1.58 (20.3%) animals per test. The use of the RC rat-only weight
regression improved animal savings slightly for the substances in these toxicity categories to
1.28 (14.0%) to 1.65 (16.7%) animals per test. Although the use of IC50 values to estimate
starting doses for the simulated UDP deceased the number of animals used per test, it did notchange the number of animals that would have died during the procedures.
Computer simulation modeling of ATC testing showed that, for the reference substancestested in this validation study, the prediction of starting doses using the 3T3 and NHK NRU
test methods with the RC rat-only millimole regression resulted in a statistically significant(p <0.05) decrease in the number of animals for ATC testing by an average of 0.51 (4.8%) to
0.80 (7.3%) animals per test, depending upon the in vitro NRU test method and the dosemortality slope (2.0 or 8.3) used. Animal savings improved to a mean of 0.91 (8.6%) to 1.09
(10.2%) animals per test when the RC rat-only weight regression was used.
When test substances were grouped by GHS category, the mean animal savings for ATC
testing using the RC rat-only millimole regression were statistically significant with the 3T3
NRU test method at both dose-mortality slopes for substances with 5 < LD50 ≤50 mg/kg(1.15 [9.8%] to 1.17 [10.2%] animals per test), and for substances with LD50 >5000 mg/kg(2.03 [17.1%] to 2.19 [18.3%] animals per test). Significantly more animals were needed
when the 3T3-based starting doses were used, than the default starting dose for reference
substances with 300 < LD50 ≤2000 mg/kg (i.e., the animal savings were negative: -0.92 [9.5%] to -1.30 [-14.0%] animals). The mean animal savings with the NHK NRU test method
and the RC rat-only millimole regression were statistically significant at both dose-mortality
slopes for substances with 5 < LD50 ≤50 mg/kg (1.18 [10.2%] to 1.33 [11.4%] animals pertest), and for substances with LD50 >5000 mg/kg (2.43 [20.5%] to 2.66 [22.2%] animals pertest). When the RC rat-only weight regression was used, statistically significant savings inanimals used were observed with both in vitro NRU test methods and dose-mortality slopes
for substances with 5 <LD50 ≤50 mg/kg (1.25 [10.8%] to 1.51 [13.0%] animals per test), andfor substances with LD50 >5000 mg/kg (2.94 [24.8%] to 3.33 [27.7%] animals per test). The
use of IC50 values to estimate starting doses for the ATC refined animal use by producing

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approximately 0.5 to 0.6 fewer mean animal deaths per test than when the default starting
dose of 300 mg/kg was used.
The use of the IC50-based starting doses did not significantly alter the GHS category
outcomes of the simulated UDP or ATC when compared with the outcomes based on thedefault starting dose. The concordance for GHS acute oral toxicity category for the IC50based starting dose with the default starting dose was 97 to 99% for both in vitro NRU
methods and IC50-LD50 regressions evaluated.
The magnitude of animal savings did not correlate with the accuracy of GHS categorization
yielded by the NRU-predicted LD50 values (using the in vitro NRU IC50 values in the IC50LD50 regressions) or with the accuracy of GHS category outcomes since the accuracy andanimals savings analyses used different standards for comparison.
The specific animal savings using the 3T3 and NHK NRU test methods with the RC rat-onlyregressions apply only to the reference substances evaluated in this validation study, and may
not be broadly applicable to other substances. Spielmann et al. (1999) indicated that 76%(845/1115) of the industrial substances submitted to the Federal Institute for HealthProtection of Consumers and Veterinary Medicine in Berlin, Germany, since 1982 had LD50
>2000 mg/kg. The extent to which these substances represent the population of substances incommerce is not known. However, if the results of the validation study are broadly
applicable to substances to be tested in the future, and if such substances are relatively
nontoxic, the selection of starting doses using the in vitro NRU test methods may save aconsiderable number of animals since animal savings for the validation study were highest
for the least toxic substances.

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11.0

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PRACTICAL CONSIDERATIONS .................................................................... 11-3

11.1 Transferability of the 3T3 and NHK NRU Test Methods.................................. 11-3
11.1.1 Facilities and Major Fixed Equipment..................................................................... 11-3
11.1.2 Availability of Other Necessary Equipment and Supplies ...................................... 11-4
11.1.3 Problems Specific to the NHK Test Method ........................................................... 11-5
11.2 3T3 and NHK NRU Test Method Training Considerations .............................. 11-6
11.2.1 Required Training and Expertise ............................................................................. 11-6
11.2.2 Training Requirements to Demonstrate Proficiency................................................ 11-7
11.2.3 Personnel Needed to Perform the In Vitro NRU Test Methods............................... 11-8
11.3 Cost Considerations ............................................................................................... 11-8
11.3.1 3T3 and NHK NRU Test Methods .......................................................................... 11-8
11.3.2 Rodent Acute Oral Toxicity Testing...................................................................... 11-11
11.4 Time Considerations for Performing the 3T3 and NHK NRU Tests.............. 11-12
11.4.1 The 3T3 NRU Test Method ................................................................................... 11-12
11.4.2 The NHK NRU Test Method................................................................................. 11-12
11.4.3 Prequalification of NHK Medium ......................................................................... 11-12
11.4.4 In Vivo Testing....................................................................................................... 11-12
11.4.5 The Limit Test........................................................................................................ 11-13
11.5

Summary............................................................................................................... 11-14

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PRACTICAL CONSIDERATIONS

The 3T3 and NHK NRU test methods are proposed as adjuncts, rather than replacements for,in vivo acute oral toxicity assays. Data from these in vitro basal cytotoxicity test methods areused with a linear regression model to predict the rat acute oral LD50 of the test substance,
which is then used to determine the starting dose for subsequent rat acute oral toxicity tests,
as described in Sections 10.2.2 and 10.3.2. This section discusses practical issues involved inusing these two in vitro NRU test methods for predicting starting doses for rat acute oral
toxicity tests. Practical issues that need to be considered with respect to the implementationof these cell culture methods include the need for, and availability of, specialized equipment,
personnel training and expertise requirements, cost considerations, and time expenditures.
11.1

Transferability of the 3T3 and NHK NRU Test Methods

Transferability of a test method is defined as the ability of a method or procedure to beaccurately and reliably performed in different, competent laboratories (ICCVAM 2003).
Accuracy and reliability of these NRU test methods are discussed in Sections 6 and 7,
respectively.
Protocols for the 3T3 and NHK NRU test methods, including solubility testing, andprequalification of keratinocyte growth medium, have been optimized and are available on
the ICCVAM/NICEATM website (http://iccvam.niehs.nih.gov/methods/invitro.htm). The
protocols were designed with GLP-compliance in mind and can be easily implemented or
adapted by scientists with the appropriate technical experience.
Although the in vitro and in vivo test methods require some similar, general laboratory skills(e.g., preparation of solutions and test substance doses, record keeping), in vitro testing
requires skills specific to cell culture systems (e.g., aseptic techniques, microscopic
evaluation of cell cultures, propagation of cells in medium) but not to the maintenance,
handling, or treatment of rodents.
11.1.1
Facilities and Major Fixed Equipment
The following lists of facility requirements, equipment and supplies, and training andexpertise are common to most in vitro mammalian cell culture laboratories. Required
equipment and supplies are also described in detail in the validation study 3T3 and NHK
protocols (Appendices B and C), the Guidance Document (ICCVAM 2001b), and Hartung etal. (2002).
11.1.1.1 Facility Requirements
The testing facility should be appropriate for operating a scientific laboratory (e.g.,laboratory space, air handling procedures, access to utilities, shipping/receiving department[for appropriate receipt and handling of cell culture materials], etc.). Each facility should
provide:• Adequate facilities, equipment, and supplies• Proper health and safety guidelines• Satisfactory quality assurance procedures
Each facility should conform to all appropriate statutes (i.e., local, state, provincial, federal,national, international) concerning safety guidelines (e.g., general workplace safety
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guidelines, chemical handling and disposal guidelines, biohazard guidelines). Hartung et al.
(2002) provides recommended safety guidelines for working with potentially infectious
materials (e.g., HIV, hepatitis B, hepatitis C) and human materials (e.g., cells, tissues, fluids).
11.1.1.2 Cell Culture Laboratory
The testing facility should have a designated cell culture laboratory to ensure that in vitro
cytotoxicity assays are performed under clean and proper aseptic conditions. The dedicatedlaboratory should be located such that through traffic is minimal to reduce possible
disturbances that can lead to contamination which could compromise the cell culture assays.
The room temperature of the laboratory should be regulated, monitored, and documented.
Access to the laboratory and its supplies and test chemicals should be restricted toappropriate personnel.
11.1.1.3 Major Equipment
Each testing facility should have at a minimum the following equipment:• Incubator (37ºC ± 1ºC, 90% ± 10% humidity, 5.0% ± 1% CO2/air)• Laminar flow clean bench/cabinet (standard: "biological hazard")• Inverted phase contrast microscope• 96-well plate spectrophotometric plate reader equipped with 540 nm ± 10 nm
filter (if testing in 96-well plates)• Autoclave• Refrigerator• Freezer (-70ºC)• Cryogenic (liquid nitrogen) freezer/storage unit
• Computer
Equipment maintenance and calibration should be routinely performed and documented
according to GLP guidelines and testing facility SOPs.
11.1.2Availability of Other Necessary Equipment and Supplies

11.1.2.1 General Equipment
Each testing facility should have at a minimum the following equipment:• Low speed centrifuge• Adjustable temperature waterbath• Pipettors• Balance• pH meter• Cell counting system• Water bath sonicator• Magnetic stirrer• Vortex mixer• Antistatic bar ionizer (for reduction of static on tissue culture plates)
Equipment maintenance and calibration should be routinely performed and documented as
per GLP guidelines and testing facility SOPs. The types of equipment listed in this section
are available from scientific and laboratory supply companies (e.g., Thomas Scientific http://www.thomassci.com/index.jsp; Fisher Scientific - https://www.fishersci.com/).
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11.1.2.2 Cell Culture Materials and Supplies
The following supplies are needed for the in vitro NRU test methods. Specific product andprivate company names are provided either as an identification of actual materials/brands
used in the validation study or as examples. Mention of these names does not imply
endorsement of the product or company.
• Tissue culture plasticware (flasks [e.g., 25 cm2, 75-80 cm2], 96-well plates,
disposable pipettes)• Laboratory glassware (e.g., flasks, bottles, graduated cylinders)• Adhesive film plate sealers (e.g., Excel Scientific SealPlate)• Sterile filtration systems (e.g., vacuum filtration units with 0.22 µm and 0.45
µm sterile filters)• Culture medium and supplements (e.g., DMEM; prequalified NHK medium)• NCS (bovine)• Balanced salt solutions (e.g., HBSS, D-PBS)
Cell culture supplies are generally available through the major scientific and laboratory
supply companies and through specialty companies (e.g., GIBCO, SIGMA-Aldrich,
CAMBREX/Biowhittaker, Becton Dickinson). Compositions of culture media,
supplements/additives, salt solutions, NRU assay chemicals, and the volumes of each neededfor each test method, should be defined. All tissue culture flasks and dishes needed to assure
proper cell propagation should be identified.
11.1.2.3 Cell Cultures
3T3 Mouse Fibroblasts: BALB/c 3T3 cells, clone 31, can be obtained fromnational/international cell culture repositories (e.g., American Type Culture Collection[ATCC], Manassas, VA, product # CCL-163).
NHKs: These non-transformed keratinocyte cells from cryopreserved primary or secondary
cells can be obtained from national/international cell culture repositories (e.g., CAMBREX
Bio Science, 8830 Biggs Ford Road, Walkersville, MD), or isolated from donated tissue
using proper collection, preparation, and propagation techniques. It may be difficult, at times,
to obtain adequate supplies of keratinocytes; the preparation of a pool of cells depends on theavailability of tissue donors. It is recommended that testing laboratories procure of acommercially available stock pool of cells and store them indefinitely in a cryogenic freezer.
All cell stock and cultures used for testing must be certified as free of contamination by
mycoplasma and bacteria.
11.1.3
Problems Specific to the NHK NRU Test Method
FAL had difficulty obtaining an adequate supply of NHK medium during the validation
study. Communication between the UK distributor and the laboratory was uneven and the
SMT attempted to resolve the supply issue on several occasions. The other laboratories
periodically had difficulties in obtaining NHK medium and supplements that adequately
supported keratinocyte growth. Although the purchased medium and supplements met the
manufacturer’s QA/QC standards, certain lots of the medium and supplements did notsupport the growth of NHK cells to the extent needed in the test protocol. To deal with these
problems, an NHK medium prequalification protocol was incorporated into the study to
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avoid unnecessarily repeating studies because of medium and supplements that did notadequately support cell growth. These experiences illustrate the need for multiple sources ofkeratinocyte cell culture medium. They also suggest that the NHK results could be morevariable than the 3T3 results because of the batch-to-batch differences in NHK growth
medium and supplements.
11.2

3T3 and NHK NRU Test Method Training Considerations

The ECVAM Good Cell Culture Practice Task Force Report 1 (Hartung et al. 2002)
encouraged the establishment of practices and principles that will reduce uncertainty in thedevelopment and application of in vitro test methods. Training in good cell culture practices,in conjunction with good laboratory practices, are essential for all in vitro cytotoxicity testingand should be employed to ensure that data produced from the 3T3 and NHK NRU test
methods are reproducible, credible, and acceptable.
In vitro cytotoxicity test methods require personnel trained specifically in sterile tissue/cell
culture techniques and general laboratory procedures. Personnel should have mandatory
training in good cell culture practices, in the specialized culture procedures needed for theseassays, and in safety and handling practices appropriate to the types of substances that may
be tested in the laboratory (Hartung et al. 2002).
The facility management should establish scientific guidelines and procedures, train andsupervise professional and technical staff, and evaluate results and performance within theirdiscipline area relative to the testing requirements. Performance of the tests requires amoderate degree of technical capability and a high degree of skill in monitoring andmaintaining appropriate cell growth conditions, troubleshooting the potential and real
problems in culture systems, and analyzing and interpreting in vitro cytotoxicity data. Eachindividual engaged in the conduct of a study, or responsible for its supervision, shall haveeducation, training, and experience, or combination thereof, to enable that individual toperform the assigned duties. The NRU test methods do not require that personnel be trained
to perform in vivo testing.
11.2.1Required Training and Expertise
Personnel performing in vitro testing should have training in basic cell culture aspects suchas: sterile technique, handling culture media, feeding cultures, cell counting, subculture(trypsinization), detection and elimination of contamination, cell growth and measurement ofgrowth curves, viability assays, and storage and freezing/thawing of cells. Additionally,
training is encouraged for special culture procedures such as primary cell and tissue cultures,
toxicity testing, and viability assays. Laboratory personnel should be trained in theapplication of GLP requirements (see Section 8.1.1), and in the safe storage, handling, anddisposal of toxic substances.
11.2.1.1 Specific Training and Expertise Needed
Personnel performing the in vitro cytotoxicity test methods should be well experienced ingeneral cell culture techniques and should be able to:• Work with cryogenic freezing apparatus• Pipette solutions with large volume pipettors and multi-channel pipettors

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Establish cells in culture vessels under aseptic conditions and monitor growth;recognize normal and abnormal cell growth characteristics; and document
observations of cell cultures throughout all aspects of the procedure
Perform the in vitro assays by following the protocols to grow the cells, count,transfer, and feed the cells, treat the cells with test substances, perform
application of adhesive plate sealers to culture plates for control of volatile
substances, perform the NRU assay, perform optical density measurements,
transfer data to electronic templates
Operate equipment necessary for maintaining cell culture laboratories (e.g.,
incubators, biohazard hoods, spectrophotometric microtiter plate readers)

11.2.1.2 General Laboratory Expertise Needed
Personnel should also be able to understand and perform basic laboratory techniques andlaboratory management:• Prepare cell culture solutions (e.g., culture medium, NRU solutions), measure
pH, know proper storage conditions, and maintain proper documentation• Prepare test substances for application to cell cultures, follow solubility
protocols to adequately prepare test chemicals in solution, recognize solubility
issues (e.g., insolubility nature of chemical, precipitation), and implement
procedures for dissolving the test chemicals• Monitor and control laboratory environment (e.g., temperature, humidity,
lighting, traffic), maintain equipment to support cell cultures (e.g.,temperature, humidity, gas flow, calibrations)11.2.2Training Requirements to Demonstrate Proficiency
Laboratories establish their own criteria for proficiency but, over the course of training,laboratory personnel should be able to understand the protocol, perform the protocol withguidance from an experienced supervisor/trainer and, eventually, perform the protocol withminimal or no supervision. An experienced supervisor determines when a technician isadequately trained because there are no standardized criteria or tasks that can be used toaccurately measure competence. After the technician demonstrates competence in executing
all the aspects of the test protocols(s), it is appropriate to perform routine assessments oftechnical competence using a benchmark, coded control test substance (e.g., SLS). It isessential that the laboratory staff be certified as proficient in using the test methods to test
unknowns.
The laboratories in the validation study were selected because of their experience inperforming in vitro cytotoxicity assays but were required to develop additional skills throughPhases I and II (e.g., data collection and transfer to Excel® and PRISM® templates).
Inexperienced laboratory personnel were trained by having them perform “training” assays
using SLS. In the early phases of the validation study, the laboratories continued training bytesting coded reference substances of various toxicities, and performing solubility testing on
substances of varying solubilities. These procedures helped improve proficiency among thelaboratories for the final phase of the validation study.

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11.2.2.1 Proficiency With GLP-Compliance
Results from these test methods will be submitted to regulatory agencies that will, for themost part, require GLPs. Laboratories should work toward attaining GLP compliance. GLP
compliance in each laboratory is determined by its independent QA unit. ECBC and IIVS
conducted this validation study in compliance with GLP (see Section 8.1.1). Their respective
QA units (as per GLPs) reviewed the various aspects of the study and issued QA statementsthat addressed whether the test methods and the results described in the Final Reportaccurately followed the test protocol and reflected the raw data produced during the study,
and provided assurance that all testing was done under according to GLP. FAL (which was
non-GLP-adherent) followed the GLP standards referenced in Section 8.1.1 as guidelines for
conducting this study. FAL had no QA unit to judge GLP compliance.
11.2.3
Personnel Needed to Perform the In Vitro NRU Test Methods
The facility management will be responsible for determining which qualified personel meetthe criteria (e.g., scientific knowledge, specialized training) for the following positions
needed for adequate performance of the in vitro NRU test methods and oversight of thetesting.
• Study Director: the individual with the overall responsibility for the technicalconduct of the testing (e.g., is familiar with the test procedures, provides SOPs
and ensures GLP compliance, analyzes and interprets the data, determines test
acceptance, oversees recordkeeping procedures, and produces the test reports.
• Quality Assurance Officer: monitors the testing to assure conformance with
GLP requirements; must be independent of the Study Director.
• Laboratory Technician(s): individuals trained in sterile tissue/cell culture
techniques and general laboratory procedures and who are capable ofperforming the test methods according to GLPs.
11.3

Cost Considerations

11.3.1

3T3 and NHK NRU Test Methods

11.3.1.1 Equipment Costs
Major instruments and equipment needed to implement the in vitro cytotoxicity test methods
are described in Section 11.1.1. Ranges of costs for some of the equipment were obtained
from on-line catalogues for two major scientific equipment and supplies companies (Thomas
Scientific - http://www.thomassci.com/index.jsp; Fisher Scientific https://www.fishersci.com/). These prices are for equipment that will meet the minimum
needs of the NRU test methods (see Table 11-1). These costs were researched in August
2006.
11.3.1.2 Costs for Cell Cultures and Supplies
Supplies such as cell culture chemicals, the reagents used to measure NRU, and cell culture
plasticware are available from numerous suppliers, and are not cost prohibitive.

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Table 11-1

November 2006

Costs for Major Laboratory Equipment
Range of Costs1

Equipment
Class II Biological Safety Cabinet$7,300 – $12,200

CO2 Incubator$5,100 – $16,400

Spectrophotometer Microplate Reader$5,000 – $7,500

Freezer (capable of -70°C)$8,000 – $15,300

Refrigerator$1,300 – $9,800

Centrifuge (benchtop model)$2,100 – $8,500

Microscope (inverse phase contrast)$3,000 – $14,500

Coulter Counter2, 3$3,000 – $9,000

Autoclave (benchtop model)2$3,500 – $15,400

Cryogenic (liquid nitrogen) Storage$1,000 – $3,700

1From on-line scientific equipment catalogues (Thomas Scientific - http://www.thomassci.com/index.jsp; Fisher
Scientific - https://www.fishersci.com/). [searched August 2006]
2
May be useful, but not required for performing the tests.
3
Other automatic cell counters may be used.

The 3T3 NRU test method is generally less expensive to perform than the NHK NRU test
method. One vial of the immortalized 3T3 cells (~$200 [ATCC]) can be propagatedindefinitely by passaging cells and periodically cryopreserving batches of cells. The NHK
NRU test method requires a fresh sample of primary cells for each test run (~$380 per vial
[CAMBREX]). Because primary NHK cells are passaged only once after initiating theculture, there are no cells available to cryopreserve a stock batch of cells. The DMEM
medium used for the 3T3 cells is less expensive, more “generic”, and more readily available
than keratinocyte-specific NHK medium. (See Table 11-2.)
11.3.1.3 Commercial Testing
The following price quotes are provided as examples of test costs and were acquired fromcommercial laboratories through Internet contact or through personal communication. Use of
information from these specific laboratories does not imply endorsement of them.
A representative of MB Research Laboratories (Spinnerstown, PA,
http://www.mbresearch.com/) provided a quote (personal communication, 2005) for an invitro 24-hr cytotoxicity test (but not a 48-hour test period) of $1050 (USP standards1) or$1950 (ISO standards1) for a set of three test chemicals. The lead laboratory for the
NICEATM/ECVAM study, IIVS (Gaithersburg, MD, http://www.iivs.org/) provides
1

USP=United States Pharmacopeia; ISO=International Standards Organization. These organizations provide
international standard testing requirements for products that require high quality for public use.

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commercial laboratory GLP-compliant testing using this study’s protocols (48-hour test
period) at a cost of $1120 - $1850 per chemical/sample for one cell type (personalcommunication 2005) (see Table 11-2).
Table 11-2

Costs for Cell Culture Materials and Commercial Laboratory In Vitro
Cytotoxicity Testing

Item

Cost
(approximate)

Number ofTests Possible

Other
One vial can produce an
indefinite supply of cells by
propagating the cells in culture
and periodically freezing apool of cells.
Since cells are passaged only
once beyond cryopreservation,
new vials should be thawed asneeded to maintain continuous
testing.
Establish cells in culture (~20mL/vial of cells; 60 mL/3
vials), seed cells in 96-wellplates (12 mL/plate; 180
mL/15 plates); prepare stock
solution and eight
concentration dilutions (~20mL/chemical; 300 mL/15
plates).

3T3 Cells~$200/vial1

indefinite

NHK Cells~$380/vial2~5 (96-wellplates)Dulbeccos’ Minimum
Essential Medium (DMEM) with
supplements~$20/500mL3~15 (96-wellplates)~$80/500 mL2~15 (96-wellplates)Same as DMEM (above)$1050/$1950(USP/ISO) per 3 test
materials4

1 test/material

in vitro NRU cytotoxicity test
(24-hour test period)$1120 (GLP) per test
material (minimum of 5
materials tested
simultaneously)4

1 range finder, 2
definitive tests
per test material

in vitro NRU cytotoxicity test
(48-hour test period)$1850 (GLP) per single
test material (tested
individually)4

1 range finder, 2
definitive tests
per test material

in vitro NRU cytotoxicity test
(48-hour test period)

NHK Medium with
supplements
Commercial
Laboratory Testing(MB ResearchLaboratories [GLPcompliant])
Commercial
Laboratory Testing(Institute for In Vitro
Sciences [GLPcompliant])
Commercial
Laboratory Testing(Institute for In Vitro
Sciences)

Abbreviations: 3T3=BALB/c 3T3 fibroblasts; NHK=Normal human epidermal keratinocytes; NRU=Neutral red
uptake; USP/ISO= United States Pharmacopeia/International Standards Organization GLP=Good Laboratory
Practices
1
Catalogue price from American Type Culture Collection (ATCC) (http://www.atcc.org/)
2
Catalogue price from CAMBREX (http://www.cambrex.com/Welcome.asp)
3
Catalogue price from INVITROGEN (http://www.invitrogen.com/content.cfm?pageid=1)
4
Personal communication (Raabe 2005)
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11.3.2
Rodent Acute Oral Toxicity Testing
As stated in Section 11.3.1.3, presentation of price quotes from commercial laboratories
provides examples of test costs and does not imply an endorsement of that laboratory. Table
11-3 provides some commercial prices for acute oral systemic toxicity testing. MB ResearchLaboratories performs the UDP test at a cost of $750 for three rats and charges $250 for each
additional rat needed. In the best-case scenario, the UDP test needs only three rats ($750). In
the worst-case scenario, this test would need an additional 12 rats (15 maximum for the test);
the total cost of the test would be $3,750. In this costing strategy, $250 is saved for each rat
not used by an accurate prediction of the starting dose by the 3T3 or NHK NRU test method.
Because the in vitro cytotoxicity test costs from $350 to $1850 per chemical, there is no net
savings in animal costs if fewer than two to six animals are saved.
Table 11-3

Commercial Prices for Conducting In Vivo Acute Rat Toxicity Testing
GLP-Compliant

Non GLPCompliant

$1200

$1000

$800

$650

$2700

$2200

$950

NA

$1500

NA

Acute Oral Rat Toxicity: LD50

$3000

NA

Acute Oral Toxicity – UDP$730 for the first 3
animals; $250 each
additional animal

NA

Test
Acute Oral Toxicity UDP:
Limit Test - 2000 mg/kg
Acute Oral Toxicity UDP:
Limit Test - 5000 mg/kg
Acute Oral Toxicity UDP:
LD50
Acute Oral Rat Toxicity:
single dose2
Acute Oral Rat Toxicity: twodoses2Company
Product Safety
Laboratories
Product Safety
Laboratories
Product Safety
Laboratories1
Bio ResearchLaboratories
Bio ResearchLaboratories
Bio ResearchLaboratories
MB ResearchLaboratories1

Abbreviations: UDP=Up-and-Down Procedure; GLP=Good Laboratory Practices; NA=Not available.
1
Personal communication (Wnorowski 2005).
2
Washington State Biological Testing Methods #80-12 For the Designation of Dangerous Waste; Part B: Acute Oral Rat
Toxicity Test [http://www.ecy.wa.gov/pubs/80012.pdf]. This test method is an adaptation of the EPA Health Affects Test
Guidelines OPPTS 870.110 Acute Oral Toxicity and American Society for Testing and Materials (ASTM) methods E 116390 (Standard test method for estimating acute oral toxicity in rats) and E 1372-90 (Standard test method for conducting a 90day oral toxicity study in rats).

The President of Product Safety Laboratories, Gary Wnorowski, (Dayton, NJ,
http://www.productsafetylabs.com/), provided a cost quote of $2700 for determination of a
rat LD50 value using the UDP test; the cost is independent of the number of rats that areneeded. Each test dose is administered ~24-48 hours after the previous dose and each animal
test generally does not exceed four days. The time involved in providing the LD50 value isapproximately three months (initiation of the test to provision of the final report). Having theestimated LD50 value would not affect the cost of the in vivo test but could reduce the numberof animals needed.
Bio Research Laboratories performs the rat acute oral toxicity test using a test method that
determines lethality and signs of acute toxicity from a waste sample administered in a single
dose, by gavage, to a limited number of rats. The bioassay determines if the test sample
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produces an LD50 either greater than or less than a regulatory threshold corresponding to ahazardous waste designation (i.e., 5000, 500, 50 mg/kg). A minimum of 10 rats is used at thetested dose for the regulatory threshold value that is relevant to the test sponsor. In thistesting scenario, knowledge of the estimated LD50 would not reduce animal use or test costs
if a single predetermined dose is tested.
11.4

Time Considerations for Performing the 3T3 and NHK NRU Tests

11.4.1The 3T3 NRU Test Method
Approximately one week is needed to thaw cryopreserved 3T3 cells, propagate them, andpassage them at least two times before subculturing them into the 96-well test plates. After
subculture into 96-well plates, the cells are incubated another 24 hours to reach the proper
confluence, and then exposed to test chemical for 48 hours. The initial 3T3 NRU test (range
finder or definitive test) takes approximately 10 days. However, after the cells are establishedin culture, they can be passaged for approximately two months before having to go back to
the cryopreserved cells to start a new culture. A 3T3 NRU test can be completed in less thanfour consecutive days when started from an established stock culture. Multiple substances
can be tested at the same time, and different tests can overlap each other; thus, many
substances can be tested in a relatively short time.
11.4.2The NHK NRU Test Method
Approximately one week is needed to thaw cryopreserved NHK cells, propagate them, andpassage them into the 96-well test plates. After subculture into 96-well plates, the cells are
incubated another 48-72 hours to reach the proper confluence and then exposed to test
chemical for 48 hours. The entire NHK NRU test (range finder or definitive test) requires
approximately 11-12 days. Cells can be seeded at different densities from one starter vial in
the culture flasks so that passaging the cultures can take place on different days. Once thecells are established in culture, they are passaged once to the 96-well test plates and an NHK
NRU test can usually be completed in five to six consecutive days. Multiple substances canbe tested at the same time, and different tests can overlap each other; thus, many substances
can be tested in a relatively short time.
11.4.3Prequalification of NHK Medium
The protocol for the prequalification of NHK medium requires nearly identical steps, andsimilar time-line (i.e., 11-12 days), as required for the NHK rangefinder and definitive tests.
Table 11-2 provides an estimate of how many tests could be performed using one 500 mL
bottle of medium with supplements (~15 tests in 96-well plates).
11.4.4
In Vivo Testing
According to guidelines for acute oral toxicity testing, single animals or groups of animals
are dosed in sequence, usually at 2-4 day intervals, and observations are generally made forup to 14 days (for animals that are not moribund) for the main test and limit dose test (EPA
2002a; OECD 2001a; OECD 2001b, OECD 2001c). The addition of 3T3 or NHK NRU
testing to estimate a starting dose prior to the implementation of the UDP main test or limit
dose test would take 10-12 days, but could save up to 14 days of observation for every
animal not used.

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11.4.5
The Limit Test
The in vitro NRU test methods can provide a savings of time when used to determine if an invivo acute oral toxicity limit test can be employed as the initial test for a substance withunknown in vivo toxicity. If the IC50 value from an in vitro NRU test could accurately predict
an LD50 that is greater than, or equal to, the limit dose (i.e., 2000 mg/kg or 5000 mg/kg), thein vivo test could start at the limit test dose. This approach has the potential to eliminate theneed to do the main test and could result in a net savings of six days for the UDP test method
and about one day for the ATC test method. Table 11-4 illustrates the following:••

Time needed to perform the 3T3 and NHK NRU test
Time needed to reach the limit test starting dose when initiating the in vivo
main test using the default starting doses (UDP and ATC)

The times presented in Table 11-4 use the following assumptions:• 3T3 cells reach ≤50% confluence in approximately 24 hours• NHK cells reach >20% confluence in approximately 48 hours• Animals show no evident toxicity 48 hours post-dosing, and additional
animals are dosed at the next higher default dose
• Limit test dose = 5000 mg/kg for the UDP and 2000 mg/kg for the ATC
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Table 11-4

November 2006

Comparison of Time Needed for In Vitro and In Vivo TestingTime

3T3 NRU
Test Method

NHK NRU
Test Method

UDP(5000 mg/kg
upper limit)

ATC(2000 mg/kg
upper limit)

Day 1

Seed cells in 96-wellplate
Incubate for 24 ±2 hr

Seed cells in 96-well plate
Incubate forapproximately 48 to 72 hr

Dose 1 animal at
default dose
(175 mg/kg)
Observe for 48 hr

Dose 3 animals atdefault dose(300 mg/kg)
Observe for 48 hr

Day 2

Apply test substance
Incubate for 48 ±0.5 hr

Incubate

Observe

Observe0 – 1 animal dies
Dose 3 animals atdefault dose(300 mg/kg)
Observe 48 hr

Day 3

Incubate

Apply test substance
Incubate for 48 ±0.5 hr

No death
Dose 1 animal at next
default dose
(550 mg/kg)
Observe 48 hr

Day 4

NRU: 3 ±0.1 hr
Elute NR: 0.33 to 0.75 hr
OD540 measurement
Calculate IC50
Estimate LD50 andStarting Dose*

Incubate

Observe

Observe

NRU: 3 ±0.1 hr
Elute NR: 0.33 to 0.75 hr
OD540 measurement
Calculate IC50
Estimate LD50 andStarting Dose*No death
Dose 1 animal at next
default dose
(1750 mg/kg)
Observe 48 hr

0 – 1 animal dies
Dose 3 animals at next
default dose
(2000 mg/kg)
Starting Point for the
Limit Test

Day 5

Day 6

Observe

Day 7

No death
Dose 1 animal at next
default dose
(5000 mg/kg)
Starting Point for the
Limit Test

Abbreviations: 3T3=BALB/c 3T3 fibroblasts; NHK=Normal human epidermal keratinocytes; NRU=Neutral red uptake; UDP=Up-andDown Procedure; ATC=Acute Toxic Class method; hr=Hour; NR=Neutral red; OD540=Optical density at 540 nm.

11.5

Summary•

All equipment and supplies should be readily commercially available. During
the validation study, direct communication with the NHK medium supplierinsured that specific lots of medium were available to the laboratories. The
test methods are expected to be transferable to laboratories experienced withmammalian cell culture methods.

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November 2006

Much of the training and expertise needed to perform the 3T3 and NHK NRU
test methods are common to mammalian cell culture procedures. Additional
technical training would not be extensive because these test methods are
similar to other in vitro mammalian cell culture assays, and no extraordinary
techniques are necessary. GLP training should be provided to technicians toensure proper adherence to protocols and documentation procedures.
Prices for commercial testing for one chemical are $1,120 to $1,850 (Table
11-2) for in vitro cytotoxicity testing in the 3T3 and NHK test methods,
respectively, to determine the IC50 (Raabe 2005, personal communication). In
contrast, the in vivo rat acute oral testing for LD50 determination could costfrom $750 - $3,750 (Table 11-3), depending on the test method used and the
toxicity of the test substance. Comparison of costs of in vitro testing to in vivo
testing is difficult because the in vitro NRU test methods are not replacements
for the animal testing, and animal testing would be performed regardless of
the responses of the 3T3 or NHK cells. The use of these in vitro NRU test
methods may not reduce the overall cost of the in vivo rat acute oral toxicity
test, but has the potential to reduce the number of animals needed for a study.

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12.0

November 2006

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13.0

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GLOSSARY1

Accuracy2: (a) The closeness of agreement between a test method result and an acceptedreference value. (b) The proportion of correct outcomes of a test method. It is a measure oftest method performance and one aspect of “relevance”. Accuracy is highly dependent on theprevalence of positives in the population being examined.
Acute Toxic Class (ATC) method: An acute oral systemic toxicity test method based on
testing groups of animals at fixed doses in a sequential manner. The lethality outcomes areused to classify a test substance into the appropriate GHS acute oral toxicity category.
ANOVA: One-way (and two-way) analysis of variance. ANOVA compares themeasurements (continuous variables) of three or more groups when the data are categorized
in one way (one-way) or two ways (two-way). ANOVA assumes that the populations
compared are normally distributed and that the variances for the groups to be compared areapproximately equal.
Assay2: The experimental system used. Often used interchangeably with “test” and “testmethod.”Biphasic dose-response: Dose-response in which cytotoxicity increases (as dose increases),plateaus, and then increases again. See Section 2.6.3.
Category prediction: The acute oral GHS hazard category that includes the predicted LD50
value for a test chemical.
Coded substances: Substances labeled by code rather than name so that they can be tested
and evaluated without knowledge of their identity or anticipation of test results. Coded
substances are used to avoid intentional or unintentional bias when evaluating laboratory or
test method performance.
Coefficient of determination: In linear regression, it denotes the proportion of the variancein Y and X that is shared. Its value ranges between zero and one and it is commonly called
called “R2.” For example, R2 = 0.45, indicates that 45% of the variance in Y can be explainedby the variation in X and that 45% of the variance in X can be explained by the variation inY.

1

The definitions in this Glossary are restricted to their uses with respect to in vitro cytotoxicity testing and the
NRU test methods.
2
Definition used by the Interagency Coordinating Committee on the Validation of Alternative Methods(ICCVAM 2003).
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Coefficient of variation: A statistical representation of the precision of a test. It is expressed
as a percentage and is calculated as follows: standard deviation  × 100%meanConcordance2: The proportion of all substances tested that are correctly classified as
positive or negative. It is a measure of test method performance and one aspect of“relevance.” The term is often used interchangeably with “accuracy.” Concordance is highly
dependent on the prevalence of positives in the population being examined. In the
NICEATM/ECVAM study, concordance was used to describe the proportion of test
substances that were correctly classified into GHS acute oral toxicity hazard categories, or todescribe the proportion of test substances for which the laboratories obtained the sameclassification result.
Confluence: A state in which cells in culture come into contact with other cells in the same
culture to form a complete sheet of cells (monolayer). For this study, confluence isdetermined as a percentage of cell coverage of the tissue culture vessel growth surface (e.g.,cell monolayer has 80% confluency).
Cytotoxicity: The adverse effects resulting from interference with structures and/or
processes essential for cell survival, proliferation, and/or function. For most chemicals,
toxicity is a consequence of non-specific alterations in "basal cell functions" (i.e., via
mitochondria, plasma membrane integrity, etc.), which may then lead to effects on organspecific functions and/or death of the organism. These effects may involve the integrity ofmembranes and the cytoskeleton, cellular metabolism, the synthesis and degradation orrelease of cellular constituents or products, ion regulation, and cell division.
Definitive test: The main test of the cytotoxicity assay for determining the IC50. The
concentration closest to the range finder test IC50 serves as the midpoint of the concentrations
tested in a definitive test. Compared to the range finder test, the definitive test uses a smaller
dilution factor for the concentrations tested.
Discordant chemicals: Chemicals for which the LD50 is not accurately predicted by the IC50
(and the associated regression formula) or the GHS toxicity category is not accurately
predicted by the IC50 (and the associated regression formula). Also referred to as “outliers.”
EDIT: Evaluation-guided Development of New In vitro Test Batteries. An international
project initiated by Björn Ekwall in 1998 and continued by the Scandinavian Society for Cell
Toxicology to develop new in vitro tests for toxicity and toxicokinetics to be incorporated
into test batteries for predicting acute and chronic systemic toxicity.
Endpoint2: The biological process, response, or effect assessed by a test method.

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Fixed Dose Procedure (FDP): An acute oral systemic toxicity test method based on testinggroups of animals at fixed doses. Evident toxicity outcomes are used to classify a test
substance into the appropriate GHS acute oral toxicity category.
Geometric mean: The antilog of the mean of the logarithm of the values. It is less affectedby extreme values than the arithmetic mean.
Globally Harmonized System (GHS): A classification system presented by the United
Nations that provides (a) a harmonized criteria for classifying substances and mixtures
according to their health, environmental and physical hazards, and (b) a harmonized hazard
communication elements, including requirements for labeling and safety data sheets.
Good Laboratory Practices (GLP)2: Regulations promulgated by the U.S. Food and Drug
Administration and the U.S. Environmental Protection Agency, and principles andprocedures adopted by the Organization for Economic Cooperation and Development andJapanese authorities that describe record keeping and quality assurance procedures forlaboratory records that will be the basis for data submissions to national regulatory agencies.
Guidance Document: Guidance Document on Using In Vitro Data to Estimate In Vivo
Starting Doses for Acute Toxicity (ICCVAM 2001b).
Hazard2: The potential for an adverse health or ecological effect. A hazard potential results
only if an exposure occurs that leads to the possibility of an adverse effect being manifested.
Hill function: The IC50 values are determined from the concentration-response using a Hillfunction which is a four parameter logistic mathematical model relating the concentration of
the test chemical to the response (typically following a sigmoidal shape).

Y = Bottom +Top − Bottom
1+ 10 (logEC50−log X)HillSlope

where Y=response (i.e., % viability), X is the substance concentration producing the
response, Bottom is the minimum response (0% viability, maximum toxicity), Top is the
maximum response (maximum viability), EC50 is the substance concentration at the response
midway between Top and Bottom, and HillSlope describes the slope of the curve. When
Top=100% viability and Bottom=0% viability, the EC50 is the equal to the IC50.
Hill function (rearranged): Some unusual dose-responses did not fit the Hill function well.
To obtain a better model fit, the Bottom parameter was estimated without constraints (theprevious practice was to use Bottom=0). However, when Bottom≠0, the EC50 reported by the
Hill function was not the same as the IC50 since the Hill function defines EC50 as the point
midway between Top and Bottom. Thus, the Hill function calculation using the Prism®
software was rearranged to calculate the concentration corresponding to the IC50 as follows: Top − Bottom −1log Y − Bottomlog IC50 = log EC50 −HillSlope13-3

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where IC50 is the concentration producing 50% toxicity, EC50 is the concentration producing
a response midway between the Top and Bottom responses; Top is the maximum response(maximum survival), Bottom is the minimum response (0% viability, maximum toxicity),
Y=50 (i.e., 50% response), and HillSlope describes the slope of the response. The X from thestandard Hill function equation is replaced, in the rearranged Hill function equation, by the
IC50.
Hormesis: a dose-response characterized by a compound’s ability to cause an opposite effectat low doses than it causes at high doses. A stimulatory effect at low doses and an inhibitory
effect in high doses is often the observed manifestation of hormesis.
IC50: test chemical concentration producing 50% inhibition of the endpoint measured (i.e.,cell viability).
Interlaboratory reproducibility2: A measure of whether different qualified laboratories
using the same protocol and test substances can produce qualitatively and quantitatively
similar results. Interlaboratory reproducibility is determined during the prevalidation andvalidation processes and indicates the extent to which a test method can be transferred
successfully among laboratories.
Intralaboratory repeatability2: The closeness of agreement between test results obtained
within a single laboratory when the procedure is performed on the same substance underidentical conditions within a given time period.
Intralaboratory reproducibility2: The first stage of validation; a determination of whether
qualified people within the same laboratory can successfully replicate results using a specifictest protocol at different times.
In vitro: In glass. Refers to assays that are carried out in an artificial system (e.g., in a test
tube or petri dish) and typically use single-cell organisms, cultured cells, cell-free extracts, or
purified cellular components.
In vivo: In the living organism. Refers to assays performed in multicellular organisms.
Kow: Octanol:water partition coefficient.
LC50: Acute lethal serum or blood concentrations.
LD50: The calculated value of the oral dose that produces lethality in 50% of test animals(rats and mice). The LD50 values serve as reference values for the in vitro tests.
LD50 (initial): Acute oral rat and mouse LD50 values used during the chemical selection
process. For RC chemicals, LD50 values were those used in the RC database, which werelargely from the 1983/84 RTECS®. For chemicals that were not included in the RC, theinitial LD50 values came from HSDB or 2002 RTECS®.

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LD50 (reference): Acute oral rodent LD50 values from rats and mice were located throughliterature searches and references from major toxicity databases such as RTECS®. Studies
were reviewed to identify the most appropriate LD50 values for each chemical. Values
obtained using feral animals, preanesthetized animals, or animals less than 4 weeks of age
were not used. Values reported as inequalities were not used. Reference LD50 values were
determined by calculating the geometric mean of the acceptable LD50 values. Data were usedin generation of the laboratory-specific and combined-laboratory 3T3 and NHK NRU
regressions.
Maximum:minimum value: Ratio of minimum acceptable LD50 (or IC50) to maximum
acceptable LD50 (or IC50).
MEIC: Multicentre Evaluation of In Vitro Cytotoxicity. An international effort establishedby the Scandinavian Society for Cell Toxicology and initiated in 1983 to evaluate therelationship and relevance of in vitro cytotoxicity for predicting the acute toxicity ofchemicals in humans.
Millimolar regressions: Linear regressions with IC50 values in mmol/L and LD50 values in
mmol/kg.
Negative control: An untreated sample containing all components of a test system, except
the test substance solvent, which is replaced with a known non-reactive material, such as
water. This sample is processed with test substance-treated samples and other control
samples to determine whether the solvent interacts with the test system.
Neutral red (NR): A weakly cationic water-soluble dye that stains living cells by readily
diffusing through the plasma membrane and concentrating in lysosomes where it
electrostatically binds to the anionic lysosomal matrix.
Neutral red uptake (NRU): Concentration of neutral red dye in the lysosomes of living
cells. Altering the cell surface or the lysosomal membrane by a toxicological agent causes
lysosomal fragility and other adverse changes that gradually become irreversible. The NRU
test method makes it possible to distinguish between viable, damaged, or dead cells because
these changes result in decreased uptake and binding of NR measurable by optical density
absorption readings in a spectrophotometer.
NHK: Normal Human epidermal Keratinocytes (from neonatal foreskin).
Optical density (OD): The absorption (i.e., OD measurement) of the resulting coloredsolution (colorimetric endpoint) in the NRU assay measured at 540 nm ± 10 nm in a
spectrophotometric microtiter plate reader using blanks as a reference
Outlier: For any measurement, an extreme value in the NICEATM/ECVAM study wasreferred to as an “outlier” if it passes a statistical test for outliers at the 99% level. With
respect to chemicals, it refers to chemicals that do not fit (using the specified criteria) an
IC50-LD50 linear regression model. It may also refer to chemicals for which the predicted

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acute oral GHS toxicity category does not match the reference in vivo GHS acute oral
toxicity category.
Performance2: The accuracy and reliability characteristics of a test method (see “accuracy”,“reliability”).
pH: A measure of the acidity or alkalinity of a solution. pH 7.0 is neutral; higher pHs arealkaline, lower pHs are acidic.
Plate reader: A spectrophotometric device for measuring light intensity as a function ofcolor/wavelength (i.e., optical density/absorption at 540 nm ± 10 nm for NRU) in 96-well
microtiter tissue culture plates.
Positive control: A sample containing all components of a test system and treated with asubstance known to induce a positive response, which is processed with the test substancetreated and other control samples to demonstrate the sensitivity of each experiment and toallow for an assessment of variability in the conduct of the assay over time.
Predictivity2: Proportion of in vivo category matches for all substances with in vitro
predictions for a particular category. Predictivity is an indicator of test accuracy.
Protocol2: The precise, step-by-step description of a test, including the listing of allnecessary reagents, criteria and procedures for the evaluation of the test data.
Quality assurance (QA)2: A management process by which adherence to laboratory testing
standards, requirements, and record keeping procedures is assessed independently by
individuals other than those performing the testing.
Quality control (QC): A management process for ensuring products or services are designed
and produced to adhere to a defined set of quality criteria to meet or exceed customer
requirements; similar to quality assurance.
Range finder: Initial test performed to determine starting doses for the main (definitive) test.
The NRU assays test eight concentrations of the test chemical or the PC by diluting the stock
solution in log dilutions to cover a large concentration range.
RC millimole regression: log (LD50) = 0.435 x log (IC50) + 0.625; for estimating an LD50
value in mmol/kg (body weight) from an IC50 value in mM. Developed using the 347 IC50
and oral LD50 (282 rat and 65 mouse) values from the RC.
RC rat-only millimole regression: log (LD50) = 0.439 x log (IC50) + 0.621; for estimating an
LD50 value in mmol/kg (body weight) from an IC50 value in mM. Developed from the IC50
values (in mM) and acute oral LD50 values (in mmol/kg) for the 282 substances with rat LD50
values in the RC database (Halle 1998, 2003).

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RC rat-only weight regression: log (LD50) = 0.372 x log (IC50) + 2.024; for estimating an
LD50 value in mg/kg (body weight) from an IC50 value in µg/mL. Developed from the IC50
values (in µg/mL) and acute oral LD50 values (in mg/kg) for the 282 substances with rat LD50
values in the RC database (Halle 1998, 2003).
Reduction alternative2: A new or modified test method that reduces the number of animals
required.
Reference substances: Substances selected for use during the research, development,
prevalidation, and validation of a proposed test method because their response in the in vivo
reference test method or the species of interest is known (see “reference test”). Reference
substances should represent the classes of chemicals for which the proposed test method is
expected to be used and cover the range of expected responses (negative, weak to strong
positive).
Reference test method2: The accepted in vivo test method used for regulatory purposes toevaluate the potential of a test substance to be hazardous to the species of interest.
Refinement alternative2: A new or modified test method that refines procedures to lessen or
eliminate pain or distress in animals or enhances animal well-being.
Registry of Cytotoxicity (RC): Database that consists of in vivo acute oral toxicity data (i.e.,
LD50 values) from rats and mice and in vitro cytotoxicity data (i.e., IC50 values) frommultiple cell lines and cytotoxicity endpoints for 347 chemicals with known molecularweights (Halle 1998, 2003). A regression model constructed from these data was proposed
by ZEBET, as a method to reduce animal use by identifying the most appropriate starting
doses for acute oral systemic toxicity tests.
Relevance2: The extent to which a test method correctly predicts or measures the biologicaleffect of interest in humans or another species of interest. Relevance incorporates
consideration of the “accuracy” or “concordance” of a test method.
Reliability2: A measure of the degree to which a test method can be performed reproducibly
within and among laboratories over time. It is assessed by calculating intra- and interlaboratory reproducibility and intralaboratory repeatability.
Replacement alternative2: A new or modified test method that replaces animals withnonanimal systems or one animal species with a phylogenetically lower one (e.g., a mammal
with an invertebrate).
Reproducibility2: The consistency of individual test results obtained in a single laboratory(intralaboratory reproducibility) or in different laboratories (interlaboratory reproducibility)
using the same protocol and test substances (see intra- and inter-laboratory reproducibility).
RTECS®: Registry of Toxic Effects for Chemical Substances. Compendium of data
extracted from the open scientific literature. The database includes toxicity data (e.g., acute

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toxicity) and specific numeric toxicity values (e.g., LD50). Compiled by the U.S. National
Institute for Occupational Safety and Health (NIOSH) and now licensed to MDL Information
Systems, Inc.
Sensitivity2: The proportion of all positive substances that are classified correctly as positivein a test method. It is a measure of test method accuracy.
Simulation modeling: Computer simulation modeling of the acute systemic toxicity assays
to determine animal use. The simulation process uses a simulated population of animals fortesting, a reference endpoint (i.e., “true” LD50 value), and its assumed log-normal
distribution. Mortality is assumed to have a mean equal to the log of the true LD50. The SD,
which reflects the variability of the simulated population, is the inverse of the slope of thedose-mortality curve. Due to a lack of information for the real dose-mortality curve, thesimulations assumed slopes of 0.5, 0.8, 2, 4, and 8.3.
Solubility: The amount of a test substance that can be dissolved (or thoroughly mixed with)
culture medium or solvent. The solubility protocol was based on a U.S. EPA guideline (EPA
1998) that involves testing for solubility in a particular solvent, beginning at a relatively highconcentration and proceeding to successively lower concentrations by adding more solvent as
necessary for dissolution. Testing stops when, upon visual observation, the procedure
produces a clear solution with no cloudiness or precipitate.
Solvent control: An untreated sample containing all components of a test system, including
the solvent that is processed with the test substance-treated and other control samples toestablish the baseline response for the samples treated with the test substance dissolved in thesame solvent. When tested with a concurrent negative control, this sample also demonstrates
whether the solvent interacts with the test system.
Specificity2: The proportion of all negative substances that are classified correctly as
negative in a test method. It is a measure of test method accuracy.
Spirit of GLP: Guidance provided in the Statement of Work specifically for the non GLPcompliant laboratory that participated in the validation study. Based on the GLP standards
referenced in the ECVAM Workshop 37 Report (Cooper-Hannan et al. 1999) and the OECD
Principles of GLP (OECD 1998). “Laboratories that are non GLP-compliant shall adhere to
GLP principles and other method parameters. Documentation and accountability shall beequal to GLP requirements. Laboratories must make assurances that they are equal in
performance criteria and that there is parity amongst the laboratories.”
TESS: Toxic Exposure Surveillance System. A comprehensive poisoning surveillance
database maintained by the American Association of Poison Control Centers (AAPCC).
Test2: The experimental system used; used interchangeably with “test method” and “assay”.
Test method2: A process or procedure used to obtain information on the characteristics of a
substance or agent. Toxicological test methods generate information regarding the ability of a

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substance or agent to produce a specified biological effect under specified conditions. Used
interchangeably with “test” and “assay”. See also “validated test method” and “reference
test”.
Test method component: Structural, functional, and procedural elements of a test methodthat are used to develop the test method protocol. These components include uniquecharacteristics of the test method, critical procedural details, and quality control measures.
3T3: BALB/c 3T3 clone A31 mouse fibroblasts developed in 1968 from disaggregated 14- to
17-day-old BALB/c mouse embryos (American Type Culture Collection [ATCC]; # CCL163).
Tiered testing: A testing strategy where all existing information on a test substance isreviewed, in a specified order, before in vivo testing.
Toxicity underpredicted: Measured l LD50 value of a test substance is lower than thepredicted LD50 value.
Toxicity overpredicted: Measured LD50 value of a test substance is higher than thepredicted LD50 value.
Transferability2: The ability of a test method or procedure to be accurately and reliably
performed in different, competent laboratories.
Up-and-Down Procedure (UDP): An acute oral systemic toxicity test method used tominimize the number of animals required to estimate the acute oral toxicity of a chemical,
estimate the LD50 and confidence interval (CI), and observe signs of toxicity. Single animals
are tested sequentially. Subsequent doses are based on the outcome of the previous animal.
Validated test method2: An accepted test method for which validation studies have been
completed to determine the accuracy and reliability of this method for a specific proposed
use.
Validation2: The process by which the reliability and accuracy of a procedure are established
for a specific purpose.
Vehicle control (VC): The VC consists of appropriate cell culture medium for the cells in
the test (i.e., DMEM for 3T3 cells and keratinocyte growth medium for the NHK cells). For
chemicals dissolved in DMSO, the VC consists of medium with the same amount of solvent
as that used in the test chemical concentrations that are applied to the 96-well test plate. The
final DMSO concentration is ≤0.5% (v/v) in the VCs.
Volatility: Ability of a test chemical to evaporate. A general indicator of volatility issues in
the NRU test methods is the percent difference in the mean OD values for the two VC
columns on the test plate. If the difference is greater than 15%, then chemical volatility canbe suspected, especially if the VC adjacent to the highest test concentration had a

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significantly reduced OD value. Volatility may be an issue for compounds with a specificgravity of less than 1.
Weight of evidence (process): The strengths and weaknesses of a collection of information
are used as the basis for a conclusion that may not be evident from the individual data.
Weight regressions: Linear regressions with IC50 values in µg/mL and LD50 values in
mg/kg.
ZEBET: The German National Center for the Documentation and Evaluation of Alternative
Methods to Animal Experiments.

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