PEER REVIEW PANEL REPORT:The Use of In Vitro Basal Cytotoxicity
Test Methods for Estimating Starting Doses
for Acute Oral Systemic Toxicity Testing
June 2006

Peer Review Panel Report:
The Use of In Vitro Basal Cytotoxicity Test Methods
For Estimating Starting Doses For Acute Oral Systemic
Toxicity Testing

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

June 2006

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

Peer Review Panel Report: Table of Contents

June 2006

TABLE OF CONTENTS
IN VITRO ACUTE TOXICITY PEER PANEL ROSTER............................................ iv
PREFACE ........................................................................................................................ v
EXECUTIVE SUMMARY ............................................................................................ vii
1.0

2.0

3.0

4.0

Introduction and Rationale for the Use of In Vitro Neutral Red Uptake
Cytotoxicity Test Methods to Predict Starting Doses for In Vivo Acute Oral
Systemic Toxicity Testing..................................................................................... 1
1.1
Background and Rationale for the Use of In Vitro Cytotoxicity Test
Methods to Predict Starting Doses for In Vivo Acute Oral Systemic
Toxicity Tests ............................................................................................. 1
1.2
Regulatory Rationale and Applicability for the Use of In Vitro
Cytotoxicity Test Methods to Predict Starting Doses for Acute Oral
Systemic Toxicity Testing........................................................................... 2
1.3
Scientific Basis for the In Vitro NRU Test Methods .................................... 3
Test Method Protocol Components of the 3T3 and NHK In Vitro NRU Test
Methods................................................................................................................. 3
2.1
Overview of the 3T3 and NHK NRU Test Methods .................................... 3
2.2
Descriptions and Rationales of the 3T3 and NHK NRU Test
Methods ...................................................................................................... 4
2.3
Basis for Selection of the In Vitro NRU Cytotoxicity Test Methods ............ 6
2.4
Proprietary Components of the 3T3 and NHK NRU Test Method
Protocols..................................................................................................... 7
2.5
Basis for Number of Replicate and Repeat Experiments for the 3T3
and NHK NRU Test Methods ..................................................................... 7
2.6
Basis for Modifications to the 3T3 and NHK NRU Test Method
Protocols..................................................................................................... 7
2.7
Differences in 3T3 and NHK NRU Test Method Protocols and the
Guidance Document Standard Protocols...................................................... 8
2.8
Overview of the Solubility Protocol ............................................................ 8
2.9
Components of the Solubility Protocol ........................................................ 8
2.10 Basis of the Solubility Protocol ................................................................... 9
2.11 Summary .................................................................................................... 9
Reference Substances Used for Validation of the 3T3 and NHK NRU Test
Methods................................................................................................................. 9
3.1
Rationale for the Reference Substances Selected for Testing ....................... 9
3.2
Rationale for the Number of Reference Substances Selected ..................... 10
3.3
Characteristics of the Selected Reference Substances ................................ 11
3.4
Reference Substance Procurement, Coding, and Distribution .................... 11
3.5
Reference Substances Recommended by the Guidance Document(ICCVAM 2001b)..................................................................................... 12
3.6
Summary .................................................................................................. 12
In Vivo Rodent Toxicity Reference Values Used to Assess the Accuracy ofthe 3T3 and NHK NRU Test Methods............................................................... 12
4.1
Methods Used to Determine In Vivo Rodent Toxicity Reference Values.... 12i

Peer Review Panel Report: Table of Contents

June 2006

4.2
4.3
4.4
5.0

6.0

7.0

8.0

9.0

Final In Vivo Rodent Toxicity Reference Values....................................... 12
Relevant Toxicity Information for Humans............................................... 13
Accuracy and Reliability of the In Vivo Rodent Toxicity Reference
Values ...................................................................................................... 13
4.5
Summary .................................................................................................. 13
3T3 and NHK NRU Test Method Data and Results ......................................... 13
5.1
3T3 and NHK NRU Test Method Protocols .............................................. 13
5.2
Data Obtained to Evaluate Accuracy and Reliability ................................. 13
5.3
Statistical Approaches to the Evaluation of 3T3 and NHK NRU Data ....... 14
5.4
Summary of Results.................................................................................. 14
5.5
Coded Reference Substances and GLP Guidelines .................................... 15
5.6
Study Timeline and NICEATM/ECVAM Study Participatory
Laboratories.............................................................................................. 15
5.7
Availability of Data .................................................................................. 15
5.8
Solubility Test Results .............................................................................. 15
5.9
Summary .................................................................................................. 15
Accuracy of the 3T3 and NHK NRU Test Methods .......................................... 15
6.1
Accuracy of the 3T3 and NHK NRU Test Methods for Predicting Acute
Oral Systemic Toxicity ............................................................................. 16
6.2
Improving the Prediction of In Vivo Rodent LD50 Values from In Vitro
NRU IC50 Data.......................................................................................... 16
6.3
Accuracy of the 3T3 and NHK NRU Test Methods for Toxicity
Category Predictions................................................................................. 17
6.4
Strengths and Limitations of the 3T3 and NHK NRU Test Methods
for In Vivo Toxicity Prediction.................................................................. 18
6.5
Salient Issues of Data Interpretation.......................................................... 19
6.6
Comparison to Established Performance Standards................................... 19
6.7
Summary .................................................................................................. 19
Reliability of the 3T3 and NHK NRU Test Methods......................................... 19
7.1
Substances Used to Determine the Reliability of the 3T3 and NHK
NRU Test Methods ................................................................................... 20
7.2
Reproducibility Analyses for the 3T3 and NHK NRU Test Methods......... 20
7.3
Historical Positive Control Data................................................................ 22
7.4
Laboratory Concordance for Solvent Selection ......................................... 22
7.5
Summary .................................................................................................. 22
3T3 and NHK NRU Test Method Data Quality ................................................ 22
8.1
Adherence to Good Laboratory Practice Guidelines .................................. 22
8.2
Results of Data Quality Audits.................................................................. 23
8.3
Impact of Deviations from GLPs/Non-compliance.................................... 23
8.4
Availability of Laboratory Notebooks....................................................... 23
8.5
Summary .................................................................................................. 23
Other Scientific Reports and Reviews of In Vitro Cytotoxicity Test Methods
and the Ability of These Test Methods to Predict Acute Systemic Toxicity..... 23
9.1
Relevant Studies ....................................................................................... 24
9.2
Independent Scientific Reviews ................................................................ 24ii

Peer Review Panel Report: Table of Contents

June 2006

9.3
10.0

11.0

Studies Using In Vitro Cytotoxicity Test Methods with Established
Performance Standards.............................................................................. 24
9.4
Summary .................................................................................................. 25
Animal Welfare Considerations (Refinement, Reduction, andReplacement) ...................................................................................................... 25
10.1 Use of 3T3 and NHK NRU Test Methods to Predict Starting Doses for
Acute Systemic Toxicity Assays ............................................................... 26
10.2 Reduction and Refinement of Animal Use for the UDP............................. 26
10.3 Reduction and Refinement of Animal Use for the ATC............................. 26
10.4 Summary .................................................................................................. 26
Practical Considerations .................................................................................... 26
11.1 Transferability of the 3T3 and NHK NRU Test Methods........................... 27
11.2 3T3 and NHK NRU Test Method Training Considerations........................ 27
11.3 Test Method Cost Considerations.............................................................. 27
11.4 Time Considerations for the 3T3 and NHK NRU Test Methods ................ 28
11.5 Summary .................................................................................................. 28

VALIDATION STATUS OF THE NRU TEST METHODS ....................................... 29
DRAFT ICCVAM RECOMMENDATIONS FOR IN VITRO ACUTETOXICITY TEST METHODS...................................................................................... 31
APPENDIX A: DRAFT PERFORMANCE STANDARDS FOR IN VITRO
ACUTE TOXICITY METHODS ................................................................................. 39
APPENDIX B: DRAFT RECOMMENDED TEST METHOD PROTOCOLS .......... 43
REFERENCES............................................................................................................... 47

iii

Peer Review Panel Report: Panel Roster

June 2006

IN VITRO ACUTE TOXICITY PEER PANEL ROSTER
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•

David H. Blakey, D.Phil., Health Canada, Ottawa, Ontario, Canada
June Bradlaw, Ph.D., International Foundation for Ethical Research (IFER),
Rockville, Maryland
Robert Copeland, Ph.D., Howard University College of Medicine, Washington, DC
Gianni Dal Negro, D.V.M., Ph.D., GlaxoSmithKline Medicine Research Centre,
Verona, Italy
Marion Ehrich, Ph.D., RPh., D.A.B.T. Virginia-Maryland Regional College of
Veterinary Medicine, Blacksburg, Virginia
Eugene Elmore, Ph.D., University of California, Irvine, Irvine, California
Benjamin Gerson, M.D., Thomas Jefferson University School of Medicine,Philadelphia, Pennsylvania
Michael Greene, Ph.D., U.S. Consumer Product Safety Commission, Bethesda, MD
Janice Kuhn, Ph.D., D.A.B.T., Stillmeadow Inc., Sugar Land, Texas
Daniel Marsman, D.V.M., Ph.D., D.A.B.T., Procter & Gamble Company,
Cincinnati, Ohio
Andrew Rowan, Ph.D., Humane Society of the United States, Washington, DC
Hasso Seibert, Ph.D., University Medical School Schleswig-Holstein, Kiel, Germany
Nigel Stallard, Ph.D., The University of Warwick, Coventry, United Kingdom
Katherine Stitzel, D.V.M., (Panel Chair), Consultant, West Chester, Ohio
Shinobu Wakuri, MSc., Hatano Research Institute, Japan
Daniel Wilson, Ph.D., D.A.B.T., The Dow Chemical Company, Midland, Michigan

iv

Peer Review Panel Report: Preface

June 2006

PREFACEThis is an independent report of the In Vitro Acute Toxicity Peer Review Panel (“Panel”)
organized by the Interagency Coordinating Committee on the Validation of Alternative
Methods (ICCVAM) and the National Toxicology Program (NTP) Interagency Center for
the Evaluation of Alternative Toxicological Methods (NICEATM). The reportsummarizes discussions, conclusions, and recommendations of the public meeting of the
Panel that was held at the National Institutes of Health in Bethesda, MD, on May 23, 2006.
The ICCVAM and the Acute Toxicity Working Group (ATWG) will consider the Panelreport, along with public comments, to prepare final test method recommendations for U.S.
Federal agencies. ICCVAM test method recommendations will be forwarded to U.S.
Federal agencies for consideration and action, in accordance with the ICCVAM
Authorization Act of 2000 (P.L. 106-545).
NICEATM and the European Centre for the Validation of Alternative Methods (ECVAM)
organized and conducted the NICEATM/ECVAM In Vitro Basal Cytotoxicity Validation
Study. NICEATM, in coordination with the ATWG and ICCVAM, prepared acomprehensive draft background review document (BRD) reviewing the study. The draft
BRD documents the procedures and results generated from the multi-phase study using the
BALB/c 3T3 murine fibroblast (3T3) and normal human epidermal keratinocyte (NHK)neutral red uptake (NRU) test methods for the prediction of starting doses for acute oral
toxicity test methods. The draft BRD was made publicly available on the
ICCVAM/NICEATM website (http://iccvam.niehs.nih.gov) or from NICEATM on
request.
NICEATM, in collaboration with the ATWG and ICCVAM, announced the independentPeer Panel review of the test methods in March 2005. Comments from the public andscientific community were solicited and provided to the Panel for their consideration (FRNotice Vol. 71, No. 54, pp. 14229-30, 3/21/06).
The Panel was charged with:• Developing conclusions and recommendations regarding the usefulness andlimitations of in vitro NRU basal cytotoxicity test methods using the 3T3 andNHK cells to estimate the rat oral acute LD50 for the purpose of determining
the starting dose for in vivo acute oral toxicity test methods and thereby
reducing animal use• ‘Peer reviewing’ the NICEATM/ECVAM In Vitro Acute Toxicity Test
Methods Draft BRD for completeness and for any errors or omissions• Evaluating the information in the Draft BRD to determine the extent to which
each of the applicable criteria for validation and acceptance of toxicologicaltest methods (ICCVAM 20031) have been appropriately addressed

1

ICCVAM. 2003. ICCVAM Guidelines for the Nomination and Submission of New, Revised, andAlternative Test Methods. NIH Publication No. 03-4508. Research Triangle Park, NC:NIEHS. The
guidelines can be obtained at: http://iccvam.niehs.nih.gov/docs/guidelines/subguide.htmvPeer Review Panel Report: Preface•

June 2006(validation2 of a new test method is a prerequisite for it to be considered forregulatory decision-making)Considering the ICCVAM draft test method recommendations for these test
methods (i.e., the proposed test method uses, the proposed recommended
standardized protocols, and the proposed test method performance standards)
and comment on whether the recommendations are supported by the
information provided in the Draft BRD

During the public meeting on May 23, 2006, the Panel discussed the current validation
status of the in vitro test methods. The Panel also provided formal comment on the Draft
BRD and made recommendations for revisions to the Draft BRD. The Panel also provided
formal comment on the ICCVAM recommendations for test method use, future studies,
test method performance standards, and the cytotoxicity protocols. In addition, the public
were provided time at the public meeting to comment on the Draft BRD. The Panel thenprovided final endorsement regarding the validation status of the test methods.
The Panel gratefully acknowledges the efforts of the NICEATM staff in coordinating thepeer review logistics and accommodations and in the preparation of the Draft BRD andvarious other materials for the review.

2

Validation is the process by which the reliability and accuracy of a test method are established for a specificpurpose (ICCVAM 2003).
vi

Peer Review Panel Report: Executive Summary

June 2006

EXECUTIVE SUMMARY
Introduction
This report describes the conclusions and recommendations of the In Vitro Acute Toxicity
Peer Panel (“Panel”) regarding the validation status of the BALB/c 3T3 murine fibroblast(3T3) and normal human epidermal keratinocyte (NHK) in vitro neutral red uptake (NRU)
basal cytotoxicity test methods (hereafter designated as NRU test methods) and the ability to
use these test methods to estimate starting doses for acute oral systemic toxicity tests. The
Panel accepts the sections of the Draft Background Review Document for In Vitro Acute
Toxicity Test Methods (BRD) for which it had no comments and recommendations as
adequate and acceptably accurate.
Panel Recommendations for the BRD
The Panel stated that, in general, the information presented in the Draft BRD was sufficientfor its purpose. Exceptions are noted within the body of the Panel report. The Panelconcluded that the objectives of the validation study were appropriate, and agreed that theapplicable validation criteria were adequately addressed in the Draft BRD for using these invitro test methods to determine starting doses for acute oral systemic toxicity tests.
The Panel made numerous recommendations for additional explanations (e.g., provide therationale for using serum that is not heat-inactivated) and clarifications (e.g., provide
additional details for using the GraphPad PRISM® software to calculate IC50 values) to theDraft BRD that will not require additional statistical analyses. Some recommendations
included presentation of the existing data in other formats (e.g., using the relative IC50 ratios
between the reference substances and the positive control [at the level of the individuallaboratory] to compare similar substances across test methods), or additional analyses (e.g.,determine the usefulness of the test methods to estimate starting doses for the Fixed Dose
Procedure [FDP] acute toxicity test method).
The Panel concluded that several confounding factors were not addressed in the selection or
evaluation of test substances but should be. They recommended that the octanol:water
coefficients and the surface-active potential (to the extent possible) for the 72 reference
substances should be characterized and incorporated into the assessment of accuracy. The
Panel also recommended that protein binding should also be taken into account in the data
analyses (i.e., to the extent possible, the free fraction in serum corresponding to the LD50
should be considered). Another potential confounder was the attempt to select chemicals toprevent the entire set of reference substances from having proportionally more outlier
substances than the Registry of Cytotoxicity (RC) linear regression.
In the evaluation of test method accuracy, substances with neurotoxic and cardiotoxic
mechanisms, and those that interfere with energy utilization or that alkylate cellular
macromolecules were excluded. Such substances were excluded because it was expected thatthese mechanisms of action could not be detected by the NRU test methods. The Paneldisagreed with their exclusion because interference with energy metabolism and alkylation ofproteins and deoxyribonucleic acid (DNA) represent important mechanisms of cytotoxicityvii

Peer Review Panel Report: Executive Summary

June 2006

that should be detected by these two test methods. Additionally, there was consensus among
the Panel members that the available data on the mechanism of acute in vivo toxicity were
not sufficient to justify the exclusion of substances based on mechanism and/or possible
involvement of biotransformation reactions. However, the Panel recommended that theproperties (e.g., metabolism, receptors, transporters) of the cell types that are important forbasal cytotoxicity be better characterized. Despite the fact that there was no significant
difference between rat and mouse LD50 data from the RC, the Panel indicated that the
separation of such data (in developing in vitro-in vivo regressions) is useful because it
decreases the biological variability associated with species differences.
Although the Panel recommended additional analyses for the evaluation of intra- and interlaboratory reproducibility (i.e., the comparison of ratios of the maxima and minima mean
laboratory IC50 values), the Panel agreed that these would not change the conclusion that the
NHK NRU test method was more reproducible than the 3T3 version. The Panel suggested
that an explanation for the difference in interlaboratory reproducibility be provided.
The Panel recommended that the analyses to determine the reduction of animal use consider
prevalence (i.e., the distribution of the universe of substances that are likely to be tested
within each hazard classification). The Panel also recommended that animal
reduction/refinement be evaluated for the use of the NRU test methods to determine thestarting dose for the FDP.
The Panel suggested that costs for equipment and working time needed to perform the NRU
test methods and a cost-benefit analysis, including information on the reduction of thenumber of animals used, should be included in the Draft BRD. The time needed to prescreen
NHK culture medium should also be included.
Validation Status of the NRU Test Methods
The Panel agreed that the applicable validation criteria have been adequately addressed forusing these in vitro test methods in a weight-of-evidence approach to determine the starting
dose for acute oral in vivo toxicity protocols. However, the Panel was aware that validationof the two NRU test methods was carried out not only to determine if they could be used to
set starting doses for in vivo acute toxicity studies, but also to determine the extent to which
the tests could be useful step in an in vitro tiered testing strategy for acute toxicity. The Panelagreed the validation study showed that neither of the two NRU test methods evaluated could
be used as a stand-alone replacement for the in vivo tests even considering the variability of
the latter. The Panel encouraged future work to develop a tiered testing strategy that includes
basal cytotoxicity as part of the overall strategy.
Review of the Draft Interagency Coordinating Committee on Validation of Alternative
Methods (ICCVAM) Recommendations for Test Method Use
The Panel agreed that although neither of the NRU test methods can be used as alternatives
for the in vivo acute oral toxicity test for the purposes of hazard classification, the testviii

Peer Review Panel Report: Executive Summary

June 2006

methods may be useful in a weight-of-evidence approach to determine the starting dose for
acute oral in vivo toxicity protocols. The Panel agreed that the NRU test methods be
considered before animals are used if there was no other stronger weight-of-evidence
information on which to base a starting dose.
The Panel disagreed that the NRU test methods were not appropriate for substances thatinterfere with energy utilization or alkylation of proteins and other macromolecules and withusing the revised RC regression that excluded chemicals based on mechanism of action.
However, the Panel agreed with using the RC rat-only regression to estimate the LD50 from
IC50 data and agreed that a regression based on weight rather than molar units would be
useful for situations where the molar weight of the test substance is unknown. In situations
where the molecular weight of a test substance is known, the molar regression should be
used.
The Panel agreed that other in vitro basal cytotoxicity test methods are based on similarscientific principles and that measure or predict the same biological response (i.e., basal
cytotoxicity and the rat acute oral LD50 value, respectively) should be demonstrated to meetor exceed the accuracy and reliability of the 3T3 and NHK NRU test methods.
Some Panel members agreed that the 3T3 NRU, based on relative ease of performance andcost, should be recommended for general use, but cautioned that one test method should not
be preferred over the other. One Panel member noted that it is important to remember thathazard assessment relates to the safety of humans, not rats. The NHK NRU IC50 data had ahigher correlation with human LC50 values (R2=0.62) than did rodent 3T3 NRU IC50 data
(R2=0.51) and a higher correlation than did rodent LD50 data with human LC50 values(R2=0.56) (Casati et al. 2005).
Review of the Draft ICCVAM Recommendations for Future Studies
The Panel indicated that high quality comparative in vitro basal cytotoxicity data should becollected in tandem with in vivo rat acute oral toxicity test results to further evaluate the useof the these test methods for predicting the starting dose for acute oral toxicity tests.
However, no Panel member recommended that in vivo testing be conducted solely to collectdata to further assess the usefulness of the NRU test.
The Panel agreed that additional in vitro tests and other methods necessary to achieve
accurate acute oral hazard classification should be investigated. The Panel also agreed thatthe in vivo database of reference substances used in the validation study be used to evaluate
the utility of other non-animal approaches to estimate starting doses for rat acute oral toxicity
tests.
The Panel agreed that standardized procedures to collect information pertinent to anunderstanding of the mechanisms of lethality should be included, to the extent possible, in
future rat acute oral toxicity studies. Such information will likely be necessary to support thefurther development of predictive mechanism-based in vitro test methods. The Panelrecommended that ICCVAM consider convening a working group to explore mechanisms ofix

Peer Review Panel Report: Executive Summary

June 2006

action of acute toxicity and approaches for acquiring additional information on acute toxic
mechanisms during acute toxicity testing.
The Panel agreed that an expanded list of reference substances with estimated rat LD50 values
substantiated by high quality in vivo data should be developed for use in future in vitro test
method development and validation studies and that there should be a concerted effort toobtain higher quality proprietary data from regulated industries.
Review of the Draft Performance Standards for In Vitro Acute Toxicity Test Methods
and Draft Recommended Test Method Protocols
The Panel agreed that the available data from the validation study appeared to support thevalidity of the recommended performance standards for the two NRU test methods. The
usefulness and limitations were well covered. Although the two NRU test methods may be
useful, there would be cause for concern if use of the test methods were made compulsory for
regulatory purposes as other information such as structure-property relationships, whenavailable, could provide better estimates of starting doses for acute toxicity studies.
The Panel identified several aspects of the performance standards that should be clarified.
Specifically, the Panel recommended that more thorough explanations and more detail fortest method procedures should be added to the recommended test method protocols but thatan effort should be made to streamline them, where possible, to assure easy use andtransferability. Clarification of solubility procedures for the determination of test substances
should be provided since the variability between laboratories in the selection of solvent
indicates a possible flaw in the solvent determination procedure. The Panel also suggested
including other methods for calculating the IC50 values and a recommendation for taskspecific training for laboratory technicians.

xPeer Review Panel Report

1.0

June 2006

Introduction And Rationale for the Use of In Vitro Neutral Red Uptake (NRU)
Cytotoxicity Test Methods to Predict Starting Doses for In Vivo Acute Oral
Systemic Toxicity TestingThis section of the Draft In Vitro Acute Toxicity Test Methods Background Review
Document (BRD) provided valuable historical background on the use of in vitro NRU test
methods to predict starting doses for in vivo acute oral systemic toxicity. The objectives ofthe validation study were valid. The introduction acknowledged that in vitro cytotoxicity
could not replace the Up-and-Down Procedure (UDP) or the Acute Toxic Class method(ATC) acute oral toxicity tests in animals. Furthermore, these in vitro tests would not beappropriate substitutes for any of the other standard acute toxicity tests. The Draft BRD
recommended that in vitro cytotoxicity testing be part of a weight-of-evidence approach todetermining the starting dose for in vivo acute oral systemic toxicity testing.
1.1

Background and Rationale for the Use of In Vitro Cytotoxicity Test Methods to
Predict Starting Doses for In Vivo Acute Oral Systemic Toxicity Tests

This section briefly mentioned the concept of using the predicted LD50 value as a starting
dose for acute oral toxicity to reduce the number of animals. This was first discussed at a
European Centre for the Validation of Alternative Methods (ECVAM) workshop in 1996
(Seibert et al. 1996). The Panel suggested that this section also include the other major
conclusions and recommendations of that workshop. The 1996 ECVAM workshop arrived ata general consensus, that• Testing for basal cytotoxicity is not sufficient for prediction of acute systemic
toxicity.
• Biokinetic factors must be considered before performing in vitro/in vivo
comparisons, in order to make the in vivo and in vitro data more comparable andthe resulting comparison more meaningful.
The Panel also recommended including information from an international project supportedby the Commission of the European Communities. The project was performed in 1992 and1993 by the Fund for Replacement of Animals in Medical Experiments (FRAME); Instituteof Toxicology, Kiel, Germany; University of Nottingham, United Kingdom (UK); andGesellschaft für Strahlen- und Umweltforschung (Society for Radiological andEnvironmental Research, for which the name changed to Forschungszentrum für Umwelt undGesundheit [Center for Environmental and Health Research]), Neuherberg, Germany. The
report, An International Evaluation of Selected in Vitro Toxicity Test Systems for Predicting
Acute Systemic Toxicity (Fentem et al. 1993), contains results on the in vitro cytotoxicity of
42 substances determined with a 3T3 NRU test method and several other in vitro systems.
Many of the substances tested are identical to those tested in the National Toxicology
Program Interagency Center for the Evaluation of Alternative Toxicological Methods(NICEATM)/ECVAM validation study. Furthermore, the report contains statistical analysesof correlations between rodent LD50 values and in vitro IC50 values, and evaluations of theaccuracy of the in vitro methods for predicting LD50 values and acute toxicity categories,
respectively.

1

Peer Review Panel Report

June 2006

The Registry of Cytotoxicity (RC) is a registry of lethality and IC50 values. The Panel agreed
that this database is important and that increasing the numbers of chemicals in this databasewould be of value. However, IC50 values do not indicate the steepness of slope for thecytotoxicity concentration response relationship nor the number of points the value is basedon. Furthermore, the RC used many endpoints for cytotoxicity, some of which may bereversible (e.g., cell detachment, effects on cell proliferation). These deficiencies must be
mentioned.
The stepwise approach for the validation study was a good approach because it allowed forthe review of intermediate progress.
1.2

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

1.2.1
Current Regulatory Testing Requirements for Acute Systemic Toxicity
This section provided a great deal of detail regarding the context of the regulatory
requirements for acute oral toxicity assays.
1.2.2
Intended Regulatory Uses for the In Vitro Cytotoxicity Test Methods
This section should clarify that the NRU cytotoxicity test methods are to be used in a weightof-evidence approach to determining the starting dose for acute oral systemic toxicity assays.
The default starting dose is usually used when there is no information upon which to base astarting dose (e.g., no toxicity information from chemicals with similar structure, etc.).
The Draft BRD indicated that the NRU cytotoxicity test methods could not be used todetermine the starting dose for the Fixed Dose Procedure (FDP) because it is not possible topredict a dose that leads to non-fatal toxicity (the TD50). The Panel suggested the TD50 and
IC50 are highly correlated, so that, given TD50 data, a regression model for prediction of TD50
from IC50 could be constructed. Even in the absence of TD50 data, a simple procedure such asassuming that the FDP starting dose is two doses below the estimated LD50 would be worth
investigating. The studies of one Panel member, who has compared IC50 values for growth
inhibition and mitochondrial function of various epithelial cell lines from normal humantissues, showed that adverse events in clinical studies were observed only after plasma levels
exceeded the in vitro IC50 levels by about one log or more.
1.2.3

Similarities and Differences in the Endpoints of the In Vitro Cytotoxicity Test
Methods and In Vivo Acute Oral Toxicity Test Methods
Animal death and death of cells in culture may or may not have similarities at the cellularlevel. As noted in the Draft BRD, extrapolation to the whole organism may involve morethan just cellular death.
The Draft BRD recognized the ability of normal human epidermal keratinocytes (NHK) tometabolize some xenobiotic substances. The fact that BALB/c mouse fibroblast 3T3 (3T3)cells and NHK cells responded differently to several of the reference substances tested could
result from differences in doubling times between the two cell lines. It also could result fromdetoxification mechanisms or metabolites generated in the NHK cells. The use of serum can2

Peer Review Panel Report

June 2006

complicate the issue of determining and/or identifying mechanism of toxicity. The 3T3 cell
culture system included serum, while the NHK cell culture system did not. Mechanistic
differences in cell type are recognized for toxicants that act at particular receptors.
Toxin should be used to refer to a biological product. Since the NICEATM/ECVAM
validation study tested pure chemicals, the term toxicant should be used.
1.2.4

Use of In Vitro Cytotoxicity Test Methods in the Overall Strategy of Hazard
Assessment
The Draft BRD indicated that the RC millimole regression cannot be used with mixtures andunknown substances because the equation requires molecular weight information for themole units. The new regression formula (developed in Section 6) based on gram units should
be described in this section, too. The new regression formula would be applicable to mixturesand unknown substances.
1.3

Scientific Basis for the In Vitro NRU Test Methods

1.3.1
Purpose and Mechanistic Basis of the In Vitro NRU Test Methods
The Draft BRD should clarify the extent to which Borenfreund and Puerner (1985) relied onmorphology to determine the maximal tolerated dose.
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
This section well delineated the differences between the cell types.
1.3.3
Range of Substances Amenable to the In Vitro NRU Test Methods
This section of the Draft BRD appropriately identified problems concerning substances withspecific toxicity mechanisms, those that were insoluble or volatile, the presence of serum,
lysosomal sequestration, and red color. It should be noted that other colored compounds may
present a problem as well.
2.0

Test Method Protocol Components of the 3T3 and NHK In Vitro NRU Test
Methods

The information presented in Section 2 of the Draft BRD appeared to be sufficient. There
was a great deal of detail regarding the equipment, methods, and procedures required for
implementation of the proposed 3T3 and NHK NRU test methods.
The Guidance Document (ICCVAM 2001b) recommendations were good. This section
should explain why it is important to have an exposure period of at least the duration of one
cell cycle.
2.1

Overview of the 3T3 and NHK NRU Test Methods

This section of the Draft BRD noted the similarities and differences of the 3T3 and NHK
NRU cytotoxicity test methods. The similarities included preparation of reference substances3

Peer Review Panel Report

June 2006

and the positive control, cell culture environmental conditions, determination of test
substance solubility, 96-well plate configuration, 48 hour exposures, microscopic evaluation,
NRU measurement as % of control with concentration in µg/mL, and data analysis. The 3T3
and NHK NRU differed in conditions for cell propagation, cell growth media, andapplication of reference substances (volume). The Panel noted that the IC50 values obtained
during the study are only valid under the conditions used in the conduct of the test methods.
2.1.1
The 3T3 NRU Test Method
The Panel noted that the serum for the 3T3 NRU test method was not heat-inactivated. Serumthat is not heat-inactivated can contain enzymes (i.e., esterases) that transform certain
chemicals. The Draft BRD should explain the rationale for using serum that is not heatinactivated. Of the 21 substances deleted from the accuracy analyses (Table 6.3 of the Draft
BRD), one Panel member noted that eight substances (atropine, carbamazepine, dichlorvos,
disulfoton, fenpropathrin, parathion, physostigmine, procainamide) had structures that could
have been biotransformed by serum enzymes.
The Draft BRD should also discuss the rationale for the restriction of the use of the 3T3 cells
to less than 18 passages after thawing.
2.1.2
The NHK NRU Test Method
Keratinocytes were not subcultured beyond the second passage, which is not unusual forprimary cells. The Draft BRD should acknowledge that the use of different lots of NHK cellsby an investigator might increase variability.
2.1.3
Measurement of NRU for both 3T3 and NHK Test Methods
The Panel found that the Draft BRD discussion and evaluation in this section wasappropriate.
2.2

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

The Draft BRD mentioned that there were problems concerning the growth of both the 3T3
and NHK cells. Since the growth rate can be very important for the results of the cytotoxicity
test methods, the Draft BRD should report the doubling times after seeding the cells in 96well plates and during exposure.
2.2.1
Materials, Equipment, and Supplies
Materials and equipment were listed in this section. There was no information regarding the
maximum absorbance required of the plate reader; this must be provided as many
spectrophotometers following Beer’s Law can only read a maximum optical density (OD) of
~ 3.
2.2.2
Reference Substance Concentrations/Dose Selection
A commercial medium (keratinocyte basal medium [KBM] supplied by Clonetics) wasused for culturing the NHK cells. There was no specific information on the composition ofthis medium. The exact composition of the medium should be specified, especially, whether
sera are included, and, if so, the types and concentrations. Without this information, it is4Peer Review Panel Report

June 2006

impossible to judge whether differences in medium composition may contribute to thediffering results of the test methods for several of the test substances.
2.2.3
NRU Endpoints Measured
The Panel found that the Draft BRD discussion and evaluation in this section wasappropriate.
2.2.4
Duration of Reference Substance Exposure
The 48-hour duration of exposure was justified in this section. The differences between invitro cytotoxicity at 24- and 72-hour exposures were noted. As part of future research, it
might be of interest to extend the duration of exposure to 96 hours to parallel the 4-day
exposure used in animal test protocols. On the other hand, a time course may be important.
Recovery and cell growth would suggest that an agent’s IC50 could change at 72 or 96 hours
relative to that at 48 hours. If recovery occurs, then lethality would require a higher dose.
Perhaps two time points as used by Elmore (2001, 2002) would be useful. These studies used
three days and five days for exposure and noted differences in the IC50 values. These time
points were chosen to facilitate detection of growth inhibition. Increasing toxicity at five days
suggested the agent was more toxic while decreasing toxicity suggested recovery of the cells.
2.2.5
Known Limits of Use
This section of the Draft BRD contained caveats on solubility, volatility, andpharmacokinetics, noting that the latter was not addressed. The organ-specific section
contained a 5-step in vitro test method. The value of including this organ-specific section was
unclear since it did not refer to the use of organ-specific cells. The organ-specific section was
more concerned with metabolism, energy production, and disruption of epithelial barriers.
Another limitation of use of the in vitro test methods is for substances that etch plastics andthose that film out (i.e., form a film on the medium surface or plastic well wall). Substancesthat etch plastics can be detected by looking for the presence of etched rings in the 96-wellplates after exposure. Some substances that film out in medium may etch plastic.
Additionally, substances that film out decrease the concentration delivered to the cells. Such
substances can be identified by the changes produced in the meniscus of the medium or by
the presence of a film where the surface of the medium was in the well.
2.2.6
Nature of Response Assessed
The Panel found that the Draft BRD discussion and evaluation in this section wasappropriate.
2.2.7
Appropriate Vehicle, Positive, and Negative Controls
The Panel found that the Draft BRD discussion and evaluation in this section wasappropriate.

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June 2006

2.2.8 Acceptable Ranges of Control Responses
The Draft BRD should explain why vehicle control (VC) ODs were lower during Phase II
and Phase III testing. Higher viability appeared to correlate with high absorbance. The VC
OD ranges of each laboratory should be described so that the stability of cell growth
conditions in each laboratory can be evaluated.
The doubling time of each cell type (for each laboratory) should be described in this section.
2.2.9
Nature of Experimental Data Collected
Since the Study Director decided whether to remove outliers at 99% level, the Study Director
must be an expert in theory and practice of cell culture.
2.2.10 Type of Media for Data Storage
The Panel found that the Draft BRD discussion and evaluation in this section wasappropriate.
2.2.11 Measures of Variability
The Panel found that the Draft BRD discussion and evaluation in this section wasappropriate.
2.2.12 Methods for Analyzing NRU Data
The Panel found that the Draft BRD discussion and evaluation in this section wasappropriate.
2.2.13 Decision Criteria for Classification of Reference Substances
The Panel found that the Draft BRD discussion and evaluation in this section wasappropriate.
2.2.14 Information and Data Included in the Test Report
The Panel found that the Draft BRD discussion and evaluation in this section wasappropriate.
2.3

Basis for Selection of the In Vitro NRU Cytotoxicity Test Methods

The selection of NRU cytotoxicity test methods was derived from the Report of theInternational Workshop on In Vitro Methods for Assessing Acute Systemic Toxicity(ICCVAM 2001a). Workshop participants evaluated several in vitro initiatives to evaluate
the prediction of systemic toxicity from in vitro toxicity. Workshop participants concluded
that there were no differences between species sources or between continuous cell lines andprimary cells.
2.3.1

Guidance Document Rationale for Selection of In Vitro NRU Cytotoxicity Test
Methods
The Panel found that the Draft BRD discussion and evaluation in this section wasappropriate.

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June 2006

2.3.2 Guidance Document Rationale for Selection of Cell Types
ICCVAM wanted rodent cells used in a cytotoxicity test method because LD50 data is
obtained with rodents. Cell lines rather than primary cultures would hasten generation of anin vitro database. Highly differentiated cells were not used and neither were metabolically
active cells such as liver.
2.4

Proprietary Components of the In Vitro NRU Cytotoxicity Test Methods

Proprietary cells and media were used for the NHK NRU method (Clonetics).
2.5

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

The Panel found that the Draft BRD discussion and evaluation in this section wasappropriate.
2.6

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

The Panel recommended that the OD of the positive control be included in Table 2-2 of the
Draft BRD. The VC OD range was eventually deleted as a test acceptance criterion.
The Panel asked whether something other than mechanism of action contributed to theunusual concentration-response curves for aminopterin and colchicine. The Draft BRD
should identify those substances for which the IC50 was calculated using only one point
between 0 and 100% when a substance had a steep concentration-response curve. The Panel
preferred that there be three points between 10 and 90% viability
2.6.1
Phase Ia: Laboratory Evaluation Phase
The ring of dead NHK cells was produced by the use of the plate inversion technique forremoving the cell culture medium prior to refeeding the cells. Such a technique leaves
residual media around the edges of each well. The ring of dead cells can be avoided byaspirating the medium from the wells prior to refeeding. Aspiration also obviates the need toprepare chemicals as a 2X dilution. A 1X chemical solution (or vehicle control) can be added
to the cells immediately after aspiration to avoid drying of the cells.
2.6.2
Phase Ib: Laboratory Evaluation Phase
The Panel found that the Draft BRD discussion and evaluation in this section wasappropriate.
2.6.3
Phase II: Laboratory Qualification Phase
The approach for handling of volatile agents by covering the 96-well plates with plastic film
was appropriate. The Panel recommended that oil not be used to cover the culture media
surface because agents that bind to lipids can bind to the oil, which reduces their effective
concentration.

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Prism® software calculations for IC50 using Hillslope and midpoints may under- or
overestimate the IC50 depending on the inclusion of nontoxic concentrations for which
viability is >100%, highest test concentrations that produce less than complete toxicity (i.e.,
viability >0%), or concentration-response curves for which the lowest nontoxic concentration
produced <100% viability. The Panel was not satisfied with the current explanation for the
IC50 calculation.
2.6.4
Phase III: Laboratory Testing Phase
The Panel found that the Draft BRD discussion and evaluation in this section wasappropriate.
2.7

Differences in 3T3 and NHK NRU Test Method Protocols and the GuidanceDocument Standard Protocols

2.7.1
Optimization of the Guidance Document Protocols Prior to Initiation of the Study
The Panel found that the Draft BRD discussion and evaluation in this section wasappropriate.
2.7.2
Optimization of the Guidance Document Protocols During the Study
The Panel found that the Draft BRD discussion and evaluation in this section wasappropriate.
2.8

Overview of the Solubility Protocol

A complex flow chart for the solvent selection for each test substance was provided.
2.9

Components of the Solubility Protocol

The Panel found that the Draft BRD discussion and evaluation in this section wasappropriate.
2.9.1
Medium, Supplies, and Equipment Required
The Panel suggested that the visual solubility determination be performed using a
microscope.
2.9.2
Data Collection
The Panel found that the Draft BRD discussion and evaluation in this section was appropriate
2.9.3
Variability in Solubility Measurement
The Panel found that the Draft BRD discussion and evaluation in this section wasappropriate.
2.9.4
Solubility and the 3T3 and NHK NRU Test Methods
The Panel found that the Draft BRD discussion and evaluation in this section was appropriate

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June 2006

2.9.5
Methods for Analyzing Solubility Data
The Panel found that the Draft BRD discussion and evaluation in this section was appropriate
2.10

Basis of the Solubility Protocol

The Panel had no comments on this section, although the comments on the protocol itself areaddressed below.
2.10.1 Initial Solubility Protocol Development
The Draft BRD noted that sometimes BioReliance and the cytotoxicity testing laboratories
did not get the same solubility results and additional explanation as to why this occurred
would be useful. However, as a whole, solubility was not a major issue.
2.10.2 Basis for Modification of the Phase II Protocol
The Panel found that the Draft BRD discussion and evaluation in this section wasappropriate.
2.11

Summary

The Panel found that the Draft BRD discussion and evaluation in this section wasappropriate.
3.0

Reference Substances Used for Validation of the 3T3 And NHK NRU Test
Methods

3.1

Rationale for the Reference Substances Selected for Testing

The selection of test chemicals, the determination of reference in vivo data, as well as test
method standardization and validation appeared to be well described, and generally of highquality. A wide range of substances, belonging to many chemical classes, with varying
physical properties, and different mechanisms of toxicity were included. The list included
pharmaceuticals, pesticides, solvents and a number of metal-containing molecules; however,there were no polycyclic aromatic hydrocarbons, catalysts, simple aldehydes, ketones,
biocides, cosmetic ingredients, mixtures/formulations, plant toxins, or other naturalcompounds. The molecular structures were not provided and should be.
The adequacy of the range of reference substances and their mechanisms of oral toxicity wasdifficult to judge because there is often very limited knowledge about their mechanisms ofaction. The overall poor characterization of modes or mechanisms of action of acute oral
toxicity in vivo makes it difficult to strategically select reference substances for broad acutetoxicity validation of in vitro methods. However, since the NRU methods are expected todetect basal cytotoxicity, the selected substances should be sufficient to evaluate reliability
and accuracy. Specifically, the Draft BRD provided little information about the 72 reference
substances to indicate that specific modes of action of acute systemic toxicity had been
robustly explored.

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The standardized methodology for acute toxicity protocols (i.e., the traditional LD50 or the
reduced UDP procedure), which include only the most rudimentary collection of endpoints,
makes no attempt to characterize even the simplest modes of action of a test substance. As
such, the overall poor characterization of these reference substances for modes ormechanisms of action of acute oral toxicity in vivo made it difficult to strategically selectreference substances for broad acute toxicity validation of in vitro methods.
Within this context, there may be some limited value in adding data from additional
substances to improve precision. Inclusion of substances at the extremes of the GHS toxicity
categories may be helpful.
3.1.1
Reference Substance Selection Criteria
The Panel found that the Draft BRD discussion and evaluation in this section wasappropriate.
3.1.2
Candidate Reference Substances
The Panel found that the Draft BRD discussion and evaluation in this section wasappropriate.
3.1.3
Selection of Reference Substances for Testing
The selection of reference substances for evaluating the reliability and the accuracy of the
NRU cytotoxicity test methods was well planned and executed, arriving at a broad and fairly
complete selection of model chemicals. However, many test substances in the regulatory
testing realm are mixtures. It would have been useful to include some mixtures similar tocommon pesticide or household product formulations.
Also regarding the selection of reference substances used to determine the accuracy of the
3T3 and NHK test methods, there was an attempt to maintain the same proportion of“outliers” as was present in the RC. However, the total percentage of RC outliers in the set ofreference substances (38%) was greater than the total percentage of outliers in the RC (27%).
This should be highlighted and addressed as a potential confounder. Conversely, there wassome concern that the potential for bias may exist if chemicals were pre-selected based onbest fit to a regression line plotting cytotoxicity versus in vivo LD50 to evaluate in vitro test
methods to estimate the acute oral LD50. This bias likely predisposed the results to
overprediction of the value of the NRU test methods for predicting random source chemicals.
This potential bias needs to be discussed.
3.2

Rationale for the Number of Reference Substances Selected

The Panel found that the Draft BRD discussion and evaluation in this section wasappropriate.

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3.3

June 2006

Characteristics of the Selected Reference Substances

3.3.1
Source Databases Represented by the Selected Reference Substances
The Panel found that the Draft BRD discussion and evaluation in this section wasappropriate.
3.3.2
Chemical Classes Represented by the Selected Reference Substances
The Panel found that the Draft BRD discussion and evaluation in this section wasappropriate.
3.3.3
Product/Use Classes Represented by the Selected Reference Substances
The Panel found that the Draft BRD discussion and evaluation in this section wasappropriate.
3.3.4
Toxicological Characteristics of the Selected Reference Substances
Several confounding factors were addressed in the selection or evaluation of the reference
substances (e.g., the octanol:water partition coefficient and the surface-active potential).
These should be characterized and this information should be incorporated into theassessment.
Surface active molecules, in particular those that can partition at the oil-water interface, cansignificantly influence absorption, toxicity, and interactions with other molecules, and may
enhance or diminish the predictive capacity of an in vitro test method. Test substanceconcentration and inherent toxic potential may be heavily influenced by molecular charge
and surface activity.
Another example of a physical-chemical feature that can represent a confounding factor isgiven by the cationic amphiphilic molecules that contain a hydrophobic ring structure and ahydrophilic side chain with a charged cationic amine group. This chemical structure enables
the substance to penetrate the cell membranes very rapidly and to interfere with phospholipid
metabolism, causing phospholipidosis. This issue needs to be addressed.
3.3.5
Selection of Reference Substances for Testing in Validation Study Phases Ib and II
The Panel found that the Draft BRD discussion and evaluation in this section wasappropriate.
3.3.6
Unsuitable and Challenging Reference Substances
The cytotoxicity endpoint for the test method is based on uptake of neutral red intolysosomes. The Draft BRD did not mention whether any of the reference substances causelysosomal swelling, which could cause artifacts.
3.4

Reference Substance Procurement, Coding, and Distribution

The Panel found that the Draft BRD discussion and evaluation in this section wasappropriate.

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3.5

June 2006

Reference Substances Recommended by the Guidance Document (ICCVAM
2001b)The Panel found that the Draft BRD discussion and evaluation in this section wasappropriate.
3.6

Summary

To the extent possible, characterization of the metabolic profiles of the reference substances
should be added.
4.0

In Vivo Rodent Toxicity Reference Values Used to Assess the Accuracy of the
3T3 and NHK NRU Test Methods

This section described the problems that arise in finding and using rodent LD50 values takenfrom the published literature. These problems have been well known for decades (e.g., areview by Morrison et al. 1968) and little has improved since then as indicated by the lack ofdata collected under Good Laboratory Practice (GLP) guidelines. Given the shortcomings ofthe existing data, the information provided was adequate and revisions are unlikely to lead toany significant improvement.
The mechanisms of oral toxicity of the reference substances were difficult to determine
because LD50 values are so rarely accompanied by more detailed information concerning theactual lesions observed and the reason for the animals’ deaths. The overall poor
characterization of modes or mechanisms of acute toxicity resulted in some difficulty indeveloping more sophisticated comparisons of in vitro and in vivo data.
4.1

Methods Used to Determine In Vivo Rodent Toxicity Reference Values

4.1.1
Identification of Candidate In Vivo Rodent Toxicity Reference Data
The selection of reference in vivo data was well described. A wide range of databases was
searched and a comprehensive set of in vivo LD50 identified. In general, the actual data did
not conform to modern standards of toxicity testing, hence their quality would be difficult todetermine (99% - 452 of 459 LD50 values would have to be eliminated if a GLP requirement
were to be mandated).
4.1.2

Criteria Used to Select Candidate In Vivo Rodent Toxicity Data for Determinationof Reference Values
The Panel found that the Draft BRD discussion and evaluation in this section wasappropriate.
4.2

Final In Vivo Rodent Toxicity Reference Values

The Panel found that the Draft BRD discussion and evaluation in this section wasappropriate.

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4.3

June 2006

Relevant Toxicity Information for Humans

The Panel found that the Draft BRD discussion and evaluation in this section wasappropriate.
4.4

Accuracy and Reliability of the In Vivo Rodent Toxicity Reference Values

Because many of the 72 reference substances had multiple LD50 values in the literature, thesevalues had to be transformed to a single reference value for each chemical. The mean
maximum:minimum values for those chemicals that had multiple LD50 values showed atendency to decline as the toxicity decreased (See Table 4.4 of the Draft BRD). This may
simply reflect the fact that inherent biological variability has a greater impact at low LD50
values than at high.
4.5

Summary

There was a general consensus that adequate data have been generated to draw conclusions
about the accuracy and validity of the methods. The majority of the most relevant in vivo datafrom the available literature were collected to compare the two in vitro tests with in vivo
acute toxicity in rodents.
5.0

3T3 and NHK NRU Test Method Data and Results

In general, the results section adequately presented the data and results. The statisticalmethods adopted provide a good quality analysis. However, several outcomes (indicated in
the following subsections) were not adequately addressed.
5.1

3T3 and NHK NRU Test Method Protocols

The Panel found that the Draft BRD discussion and evaluation in this section wasappropriate.
5.2

Data Obtained to Evaluate Accuracy and Reliability

5.2.1
Positive Control (PC) Data
The Draft BRD should explain the considerably higher sensitivity of NHK cells to thepositive control (sodium lauryl sulfate [SLS]).
5.2.2
Reference Substance Data
Consistently, carbon tetrachloride could not be tested in the 3T3 and NHK NRU test
methods. The reason that this chemical could not be tested should be addressed. Several
additional reference substances could not be adequately tested by one or two of the threelaboratories, although they had used the same cell types and harmonized protocols. The
reason(s) for these differences between the laboratories should be discussed.

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5.3

June 2006

Statistical Approaches to the Evaluation of 3T3 and NHK NRU Data

The Panel found that the Draft BRD discussion and evaluation in this section wasappropriate.
5.4

Summary of Results

Further discussion exploring the biological significance of and possible reasons for thedifferences in sensitivity and selectivity between the two cell lines is needed; this may be
useful for selecting the appropriate cell line(s) for future use.
The significance of the steepness of the concentration-response curve was unclear from thedata. The IC50 alone does not address this issue. While IC20 and IC80 (or at least a dose below
and above the IC50) were collected for most of the reference substances, they were not usedin the analysis. The slope of the concentration-response curve should be included along with
the IC50 data as additional information about the concentration-response characteristics.
The Draft BRD should include an explanation as to why 3T3 IC50 values for numerous
reference substances were orders of magnitude different from those determined in the NHK
test method. Was this due to cell-specific cytotoxicity? Or was it a consequence ofdifferences in cell culture medium (i.e., presence or absence of serum)?
Table 5-4 in the Draft BRD was highly confusing. The column labeled “Difference (Ordersof Magnitude)” contained the calculated ratios of the 3T3/NHK mean IC50 values. However,the column contained several mistakes. For example, potassium cyanide, with IC50 values of
34.6 vs. 29.0 µg/mL (ratio=1.2), has a difference of 1 order of magnitude while parathion,
37.4 vs. 30.3 (ratio=1.2), has a difference of 0. There were several more such cases (e.g.,phenol, carbamazepine, nicotine). A more useful column to compare materials across the two
NRU test methods would show the relative difference from the positive control. Since Table
5-5 uses some of the same data as Table 5-4, it must also be revised.
Noted in the summary but not discussed in Section 5.4 were the results in Table 5-4 showingthat the IC50 values for aminopterin and digoxin differed by five orders of magnitude whentested in 3T3 versus NHK cells. Aminopterin and digoxin are established substrates fororganic anionic transporters (OATs). Such transporters are very important for in vivo toxicity
responses in terms of the ability of molecules to be absorbed, reach target tissues,
accumulate, be excreted or secreted. Expression, induction, interference and binding to OATs
can strongly influence the in vivo effects of a compound. Single nucleotide polymorphisms,
which can strongly affect normal function, have been identified in human OATs. The
differential susceptibility of the two studied cell lines could be explained by differentialfunctioning of OATs between the cell types, but that was not examined or discussed. At least
one publication indicated that NHK cells have at least five different OAT subclass members,
with one shown to bind digoxin but not be constitutively expressed in the NHK, which could
explain their sensitivity to this chemical. This issue needs to be addressed.
The summary indicated that the IC50 values were commonly (92%) within one order ofmagnitude of each other. A more descriptive and helpful summary would include the fraction

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that was within specific IC50 ranges. For example, “for nine substances ratios between 3T3
IC50 values and NHK IC50 values were ≥10 or 0.1, respectively.”
5.5

Coded Reference Substances and GLP Guidelines

The Panel found that the Draft BRD discussion and evaluation in this section wasappropriate.
5.6

Study Timeline and NICEATM/ECVAM Study Participatory Laboratories

The Panel found that the Draft BRD discussion and evaluation in this section wasappropriate.
5.7

Availability of Data

The Panel found that the Draft BRD discussion and evaluation in this section wasappropriate.
5.8

Solubility Test Results

The Panel found that the Draft BRD discussion and evaluation in this section wasappropriate.
5.9

Summary

One approach for comparing data generated on the same substance in different laboratories
would be to normalize the data using the relative IC50 ratios between the reference substances
and the positive control (at the level of the individual laboratory). This approach should beconsidered.
6.0

Accuracy of the 3T3 and NHK NRU Test Methods

This section adequately summarized the accuracy of the studies. The performance andlimitations of the two NRU basal cytotoxicity tests were well defined. The overall accuracy
for the prediction of Globally Harmonized System (GHS; UN 2005) acute oral hazard
category was modest, and enhancement of accuracy through material selection (modular
approach), model refinement, or tiered testing strategy should be pursued. Further
performance at the extremes of LD50 should be considered. Although some analysis of
accuracy was conducted related to physical-chemical properties (e.g., solubility) andabsorption, distribution, metabolism, and excretion (ADME) (e.g., biotransformation), andother factors (e.g., surface active properties, protein binding, receptor mediation) should beassessed to refine the test methods or draw greater precision by using a modular approach todefine the types of materials suitable for the test methods.

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Although there was not a significant difference between rat and mouse LD50 data (because of
the variability of the data), separation was useful because it decreased the biologicalvariability associated with species differences.
6.1

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

Graphs should be added to compare the responses of the 58 RC substances to the same
substances when tested using the 3T3 and NHK NRU test methods.
6.2

Improving the Prediction of In Vivo Rodent LD50 Values from In Vitro NRU IC50
Data

6.2.1
The RC Rat-Only Regression in Millimolar Units
The Panel found that the Draft BRD discussion and evaluation in this section wasappropriate.
6.2.2
The RC Rat-Only Regression in Weight Units
Optimization of the IC50-LD50 regression to allow for testing of mixtures was undertaken, yet
no mixtures were used in fitting the regression curve. Since the test methods have limitationsin accurately predicting the toxicity of materials with known or uncertain mechanisms, thetesting of mixtures seems highly controversial.
6.2.3

The RC Rat-Only Regression in Weight Units Excluding Substances with SpecificMechanisms of Toxicity
It is true that many of the reference substances with underpredicted toxicity had mechanismsof toxicity that could not be expected to be detected in the 3T3 and NHK cell cultures;
however, the Draft BRD incorrectly identified the mechanisms inactive in the cell cultures.
The Draft BRD indicated that neurotoxic and cardiotoxic mechanisms, interference withenergy utilization, and alkylation of macromolecules would not be active in the cell cultures.
Interference with energy metabolism and alkylation of proteins and deoxyribonucleic acid
(DNA) actually represent important mechanisms of cytotoxic action, which, in principle,
should be detected by cytotoxicity assays such as the 3T3 and NHK NRU test methods. The
rationale for excluding the 50 substances with specific mechanisms of action appears veryquestionable. Indeed, Table 6-2 of the Draft BRD shows that the linear regression between
rodent LD50 values and IC50 values was not improved by the exclusion of these substances(R2=0.353).
In addition, errors were made in the exclusion process based on the rules cited in the Draft
BRD. For example, triethylene melamine and busulfan are both alkylating agents, but were
not excluded. Paraquat and potassium cyanide were excluded based on interference withenergy utilization. However, arsenic trioxide, which can uncouple oxidative phosphorylation,
should have been excluded, but was not. Paraquat and potassium cyanide exert their acutesystemic toxicity by means of cytotoxic action and should not have been excluded. If using a
modular approach based upon proposed mechanisms (e.g., all substances interfering withenergy metabolism), then hexachlorophene (a potent uncoupler of mitochondrial

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phosphorylation), digoxin (a cardiac glycoside), or propanolol (a β-blocker) should havebeen included.
The Panel recommended against excluding reference substances based on mechanism given
the numerous mechanisms of induction of cytotoxicity, the poor mechanistic understandingof the acute toxicity of many of these materials, and the incomplete knowledge of theappropriateness of the models for the individual modes/mechanisms of action.
6.3

Accuracy of the 3T3 and NHK NRU Test Methods for Toxicity Category
Predictions

There was general consensus that adequate data were generated to draw conclusions about
the accuracy and validity of the methods. The statistical approaches adopted to analyze data
enable accurate and scientifically robust analyses of the two methods with regards to all theiraspects.
The evaluation of the accuracy of the NRU basal cytotoxicity test methods for estimating
GHS acute oral toxicity category was very extensive and detailed, and it identified areas ofconcern relative to specific chemical classes, chemicals with known mechanisms of toxicity
and particular properties such as solubility, volatility, and so on. The evaluation of
concordance of the observed and predicted GHS toxicity categories for each substance was
performed correctly. Although a modular approach for using the model may be more reliable,
the database was probably too small for most mechanisms of action to draw soundconclusions regarding strengths and limitations of the test methods with respect to chemicalclasses, mechanisms of toxicity, or physico-chemical properties. Since a mode of action isunlikely to be known about a random source material, it is also unlikely that a modular
approach based upon mechanism will often be a viable option. A better approach would be amodular approach to validation based on chemical class, implying similar mode of action.
Thus, the justification for the exclusion of 21 substances with specific modes of toxicity was
not appropriate. The 26% accuracy for prediction of GHS class without removal of the 21
substances was poor, but better than a random selection using the 72 chemicals (1/6
accuracy).
Corrosivity was an exclusionary criterion intended to be applied to the selection of reference
substances (see Section 3 of the Draft BRD). However, corrosive materials as a class were
not subsequently deleted from the data when the regression curves were made. Corrosive
chemicals are excluded from testing in in vivo acute toxicity tests because testing suchchemicals in vivo is not appropriate, but using data for such chemicals in these analyses is
acceptable.
For those classes of substances found to be appropriate for the assay, the NRU-based test
methods may also be useful in a development context. During industry screening of newmaterials, a tool such as this may be useful to rank compounds belonging to the samechemical class (e.g., early lead optimization phase of drug development).

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6.3.1

Prediction of Toxicity Category by the 3T3 and NHK NRU Test Methods Using the
RC Millimole Regression
The Panel found that the Draft BRD discussion and evaluation in this section wasappropriate.
6.3.2

Prediction of Toxicity Category by the 3T3 and NHK NRU Test Methods Using the
RC Rat-Only Weight Regression
The Panel found that the Draft BRD discussion and evaluation in this section wasappropriate.

6.3.3

Prediction of Toxicity Category by the 3T3 and NHK NRU Test Methods with the
RC Rat-Only Weight Regression Excluding Substances with Specific Mechanisms of
Toxicity
The Panel found that the Draft BRD discussion and evaluation in this section wasappropriate.

6.3.4
Summary of the Regressions Evaluated
The Panel found that the Draft BRD discussion and evaluation in this section wasappropriate.
6.4

Strengths and Limitations of the In Vitro NRU Test Methods for In Vivo Toxicity
Prediction

Use of metabolically competent systems was recommended as one approach to improve theaccuracy of in vitro predictions of acute toxicity; this should be explored in the future. The
use of metabolizing systems is a general requirement for all in vitro tests for the prediction ofgenetic and carcinogenic potential and is considered necessary and scientifically justified.
However, the contribution of metabolism of the reference substances was likely misstated,
given the incomplete understanding of the acute toxicity of many of them. The substances
listed in Table 3-7 of the Draft BRD, which were noted in the analysis of discordant
substances, were highly variable in structure and purported mechanism. Of this set of
substances, several (e.g., phthalates, valproic acid) may have active metabolites that
contribute to their chronic toxicologic effects but which play little or no role in their acutetoxicologic effects. Conversely, one may speculate that there may be substances not includedin Table 3-7 of the Draft BRD for which active metabolism was an important component of
its acute effects. Therefore, a more robust analysis of the contribution of metabolism to theaccuracy of the models is recommended by incorporating a metabolic system into the in vitro
assays.
As a future task, the properties of the cell lines (e.g., metabolism, receptors, transporters) thatare important for basal cytotoxicity should be better characterized. Identified important
properties could be used as performance standards.

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6.5

June 2006

Salient Issues of Data Interpretation

The Panel found that the Draft BRD discussion and evaluation in this section wasappropriate.
6.6

Comparison to Established Performance Standards

It would be informative to show comparisons of the RC LD50 values for the selectedreference substances used in this study versus the individual laboratory responses for eachtest instead of the data shown in Figures 6-6 to 6-8 of the Draft BRD, which compares the invitro responses to the overall RC millimole regression data.
While the mean IC50 values from one laboratory were generally higher than the rest,
comparison to regressions with animal data (Appendix J) suggest there are no majordifferences between the laboratories in their ability to predict LD50 values. In fact the
responses in Figures 6-6 to 6-8 look similar. When the in vitro response data from alllaboratories with the agents selected from the RC are compared to the same agents for theRC, they provide a better correlation with the LD50 than did the overall RC data. Given thisobservation coupled with the variability in the data from animal studies, the data from the invitro test methods would suggest that, as long as the appropriate controls (VC and PC) areused, the data from valid assays should be fairly predictive of animal response. It would be
informative to show comparisons of the regression lines using the RC data for the 11 agentsshown versus the individual laboratory responses for each test method instead of the data
shown in Figures 6-6 to 6-8, which compares the in vitro responses to the overall RC
millimole regression.
6.7

Summary

Protein binding should be taken into account in the data analyses. This parameter could beeventually taken into account in an additional data analysis (i.e., to the extent possible,
consider the free fraction in serum corresponding to the LD50 dose). The Hill function slope
data and LD50 slope data should be compared.
7.0

Reliability of the 3T3 and NHK NRU Test Methods

In general, the analyses in Section 7 adequately addressed the issues regarding both intraand inter-laboratory reproducibility for the 3T3 and NHK NRU test methods. It was a little
bit surprising, however, that some laboratories failed to obtain IC50 results for some of thereference substances. The Draft BRD should include an explanation or at least a discussionof these discrepancies, which may relate to the solvent protocol (discussed later). The
compounds failing to yield IC50 values were mostly solvents (carbon tetrachloride, methanol,
xylene, and 1,1,1-trichloroethane). Solvents are an important class of industrial substances
for which Toxic Substances Control Act (TSCA) applies. The Draft BRD should offer an
explanation if possible. Additional IC50 data are available for three of these substances:methanol (1000 mM), 1,1,1-trichloroethane (5.6 mM), and carbon tetrachloride (4.8 mM)
using 3T3 cells after 24 hours of exposure (Gülden et al. 2005).

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7.1

June 2006

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

The Panel found that the Draft BRD discussion and evaluation in this section wasappropriate.
7.2

Reproducibility Analyses for the 3T3 and NHK NRU Test Methods

Additional consideration as to the underlying reasons for the variability between thelaboratories would be helpful. The issue of intra- and inter-laboratory reproducibility due tovariations in laboratory practices was addressed during the study and the findings indicatedthat the data from the two laboratories with GLP compliant procedures were in closer
agreement and tended to show less variability and lower error rates than the other laboratory(which had an error rate of 93% for Phases 1a and 1b). Following a common training session
for all laboratories, the interlaboratory variability decreased. This indicates the need fortraining in basic methodology and emphasis on protocol compliance. Everyone participatingin such studies should be adequately trained in the basics of cell and tissue culture and soundscientific methods.
In order to increase the transparency of the comparison of the results from the different
laboratories, an additional analysis of the IC50 data could be added: for each substance and
NRU test method, the ratio between the highest and the lowest mean IC50 from thelaboratories should be calculated. Those reference substances having ratios ≥ 3.0 should bepresented in a separate table together with their calculated ratios and the names of thelaboratories that delivered the corresponding IC50 values. From the Panel’s analysis, itappeared that 17 substances for the 3T3 NRU test method and 11 substances for the NHK
NRU test method had ratios ≥3.0. Extreme cases were cupric sulfate with a ratio of 22 (3T3
NRU test method) and digoxin with a ratio of 107 (NHK NRU test method). Furthermore, it
became apparent that even for a simple compound such as sodium chloride, the results fromdifferent laboratories deviated by a factor of more than 3.0 for the NHK NRU test method.
It would be helpful to include a figure in the Draft BRD depicting all IC50 values for each test
substance from all laboratories. Graphing of IC50 values plus-or-minus (±) the standard
deviation (SD) and rat LD50 values ± SD should provide a better comparison of variation in
the two sets of values.
It might also be helpful to look at ratios of the maximum IC50 values to the minimum IC50
values to see how they compare vs. rodent LD50 values. Given the variability in animal data
where LD50 values (when more than one LD50 was available) could differ from 4 to 14 fold,
the determination of a precise IC50 in each of the test methods to facilitate the selection of astarting dose does not seem necessary. Although the comparison of intra- and interlaboratory
reproducibility for the purpose of validating the initial performance was appropriate, the useof multiple, costly test methods to identify precise IC50 values to establish initial doses fordetermining LD50 values seems counterproductive on the basis of cost and would limit
acceptance of such methods.

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For some of the reference substances, there was only one point and possibly even no points
between 0 and 100% viability. These substances should be identified in the BRD.
NHK NRU IC50 data had a better correlation with human LC50 values (R2=0.62) than didrodent 3T3 NRU IC50 data (R2=0.51), as reported by Casati et al. (2005) at the 5th World
Congress in Berlin in 2005. The correlation of NHK NRU IC50 data with human LC50 values(R2=0.62) was also better than the correlation of rodent LD50 data with human LC50 values(R2=0.56) (Casati et al. 2005). Discussion of this relationship should be considered forinclusion in the BRD.
7.2.1
ANOVA Results for the 3T3 and NHK NRU Test Methods
The Panel questioned the utility of the ANOVA for addressing the issue of intra- and interlaboratory reproducibility. Depending upon the sample size and intralaboratory variation, asignificant difference could correspond to a very small variation between laboratories or a
non-significant difference could correspond to a very large difference between laboratories.
Examples include parathion and procainamide. Parathion had reported IC50 values of 22.7,
141, and 22 µg/mL (p=0.014, not significant), and procainamide had reported IC50 values of400, 431, and 497 µg/mL (p=0.007, significant). As a consequence, procainamide withsatisfying, low interlaboratory reproducibility was included in Table 7-4 (because the
ANOVA indicated significant laboratory differences) while parathion was not. There weremore such examples that make the utility of the ANOVA questionable.
Based on the ANOVA analysis performed, FAL reported significantly different results fromthe two other laboratories for 20 substances (3T3 NRU test method). For 18 of thesesubstances FAL reported the highest values. This phenomenon should be explained.
The statistically significant differences among the laboratories for 26 of the reference
substances in the 3T3 NRU was worth noting, especially since it was greater than 1/3 of theagents tested. Volatility and/or presence of a precipitate were only noted for nine agents.
7.2.2
CV Results for the 3T3 and NHK NRU Test Methods
This section adequately elucidated associations between intra- or interlaboratory
reproducibility and chemical classes, chemical properties, and potency categories. The result
was that there were no clear associations between any of these parameters and CV values.
What was evident, however, was that the reproducibility of both methods depends on thelaboratory performing the measurements. A discussion of the possible reasons for thislaboratory-specific reproducibility would be helpful.
7.2.3

Comparison of Laboratory-Specific Linear Regression Analyses for the Predictionof In Vivo Rodent LD50 Values from In Vitro NRU IC50 Values
The Panel found that the Draft BRD discussion and evaluation in this section wasappropriate.

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June 2006

7.2.4
Laboratory Concordance for the Prediction of GHS Acute Oral Toxicity Category
The most important information given here was how often the data generated by the different
laboratories would produce different starting doses for the ATC or UDP.
7.3

Historical Positive Control Data

The Panel found that the Draft BRD discussion and evaluation in this section wasappropriate.
7.4

Laboratory Concordance for Solvent Selection

Concern was raised about the differences in solvent selection between laboratories ascompared to the BioReliance solvent information. For whatever reason, the variability
between laboratories in the selection of solvent pointed out a possible flaw in the solvent
determination protocol. This should be evaluated for future studies.
7.5

Summary

Irrespective of the statistical method used (ANOVA or calculation of the ratio between
maximum and minimum IC50), there were many more reference substances with deviating
results between laboratories in the 3T3 NRU test method than in the NHK NRU test method.
This should be explained.
8.0

3T3 and NHK NRU Test Method Data Quality

Section 8 adequately addressed the purpose of this section. No additional data are needed.
8.1

Adherence to Good Laboratory Practice Guidelines

8.1.1
Guidelines Followed for In Vitro NRU Cytotoxicity Testing
The Panel found that the Draft BRD discussion and evaluation in this section wasappropriate.
8.1.2
Quality Assurance (QA) for In Vitro NRU Cytotoxicity Test Data
The Panel found that the Draft BRD discussion and evaluation in this section wasappropriate.
8.1.3
Guidelines Followed for In Vivo Rodent Oral LD50 Data Collection
The use of the NRU test relied on the relationship between rat LD50 data and the observed
IC50. This relationship required reliable LD50 measurements for the RC substances used toconstruct the regression line. Since the LD50 values reported by the Registry of Toxic Effects
for Chemicals Substances (RTECS®) were the most toxic found in the literature, one isunsure to what extent these LD50 estimates can be considered the gold-standard. Theseestimates may be appropriate for risk assessment but these extreme values can be unreliable
and could lead to a misleading model of the desired linear relationship.

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For comparative purposes with the IC50 values, LD50 values should reflect the variation
observed. In most cases, a range of values should be shown. Such a range should reflectreasonable data with outliers omitted. If no range is shown, then a mean value (whenavailable) plus-or-minus (±) SD should be used for the LD50. The variability in animal data is
usually much greater than that found in vitro. Therefore, comparing IC50 ± SD and Rat LD50
± SD or data range should provide a better comparison. The Panel recommended that thesedata be shown in the report possibly in a bar graph similar to those in Figure 5-1. Based on
the current data, it was not anticipated to have a major effect of the predictive potential of thetwo in vitro test methods. However, it could be important for future studies with othersubstances. The positive control response limits for a definitive test in Phase III was IC50 ±2.5 SD. If the positive control showed this amount of variation, then why should thereference substances be expected to show any less? The test methods were not designed to
predict hazard class but to predict starting animal dose in the acute LD50 tests.
8.2

Results of Data Quality Audits

The Panel found that the Draft BRD discussion and evaluation in this section wasappropriate.
8.3

Impact of Deviations from GLPs/Non-compliance

The Panel found that the Draft BRD discussion and evaluation in this section wasappropriate.
8.4

Availability of Laboratory Notebooks

The Panel found that the Draft BRD discussion and evaluation in this section wasappropriate.
8.5

Summary

The Panel found that the Draft BRD discussion and evaluation in this section wasappropriate.
9.0

Other Scientific Reports and Reviews of In Vitro Cytotoxicity Test Methods
and the Ability of These Test Methods to Predict Acute Systemic Toxicity

In general, reports on other in vitro test methods using NRU were useful in providing insights
into the correlation as well as the disparities between in vitro IC50 and in vivo LD50. This wasparticularly true for the previously reported attempts to compare in vitro toxicity to in vivo
lethality. However, it was less clear that the comparisons between eye irritation and NRU in
vitro test methods were of use in interpreting the data used to compare in vitro IC50 to in vivo
LD50. While the mode of exposure is much more comparable between the in vitro test
methods and the eye irritation (i.e., the test substance is applied directly to the target cell
population), the endpoint is dissimilar. Furthermore, direct exposure of the target cells often

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cannot adequately predict systemic effects, especially for some classes of substances that act
through a known mechanism that does not relate to basal cytotoxicity.
Care was taken in the NICEATM/ECVAM study to cover a range of potencies and mode ofaction was also considered. It would be useful to compare the range of in vivo toxicities andmodes of action represented in the other studies reported in Section 9 with the present
NICEATM/ECVAM study.
9.1

Relevant Studies

9.1.1
Correlation of In Vitro NRU Cytotoxicity Results with Rodent Lethality
Additional discussion from the published literature about the advantages and limitations ofusing various supplemental metabolizing systems in cell culture for cytotoxicity testing could
be included. For the Peloux et al. (1992) study, it may be worth including a discussion about
the high correlation and whether the relatively good predictive value was a result of the routeof exposure (i.e., intravenous [iv] and intraperitoneal [ip]). It should be clarified that the
goodness of correlation for the in vivo/in vitro values for the different routes of exposure wasiv>ip>oral and reflected different kinetic variables.
The results of the workshop presented in Seibert et al. 1996 should be included.
9.1.2
Use of Cytotoxicity Data to Reduce the Use of Animals in Acute Toxicity Testing
The Panel found that the Draft BRD discussion and evaluation in this section wasappropriate.
9.1.3
Other Evaluations of 3T3 or NHK NRU Test Methods
The Panel found that the Draft BRD discussion and evaluation in this section wasappropriate.
9.2

Independent Scientific Reviews

9.2.1

Use of In Vitro Cytotoxicity Data for Estimation of Starting Doses for Acute Oral
Toxicity Testing
Clarification about the percentage reduction of animal use as referenced in the ICCVAM
2001a report should be included in Section 9 with the present ICCVAM study (i.e., what is
the likely basis for the difference between then and now).
9.2.2
Validation of 3T3 NRU for Phototoxicity
The Panel found that the Draft BRD discussion and evaluation in this section wasappropriate.
9.3

Studies Using In Vitro Cytotoxicity Test Methods with Established PerformanceStandards

The Panel found that the Draft BRD discussion and evaluation in this section wasappropriate.

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9.4

June 2006

Summary

The Panel found that the Draft BRD discussion and evaluation in this section wasappropriate.
10.0

Animal Welfare Considerations (Refinement, Reduction, and Replacement)

The extent to which the NRU-based methods could contribute to a reduction in animal use
was clearly discussed. The statistical analyses were clearly presented and the conclusions areclear. However, the Panel indicated that the extent to which the NRU test methods will
reduce animal use for in vivo testing was not adequately characterized and discussed. The
calculated savings (8-21%) of animals was only valid if several assumptions were accepted.
For example, 21 of the 72 reference substances were excluded from the calculations becauseof their assumed specific modes of action. The best way to evaluate a possible reduction in
animal use by using in vitro cytotoxicity to set the starting dose of an unknown substance isto assume that nothing is known about the mechanism(s) of toxicity of that test chemical.
Therefore, all 72 reference substances should be included in the calculation of animalsavings, regardless of their mode of action.
The use of the NRU cytotoxicity test methods are warranted not only if the number ofanimals used in the studies is reduced but also if the stress resulting from chemical exposureis minimized. The decision to use the NRU test to determine the starting dose for the ATC
method or UDP is justified by the reduction in the number of animals required as indicated in
the simulation studies.
The simulation studies compared the numbers of animals used with the starting dose
indicated by the NRU basal cytotoxicity test method with the numbers of animals used withthe default starting dose. Although the reduction in animals was not that great on apercentage basis, the testing of 4000 chemicals coming on the market in a year, could save
4000 rats at a rate of one rat per chemical. The Panel indicated, however, that a requirement
to use the NRU test to determine the starting dose could lead to an increase in the number ofanimals required particularly if other data were available to provide a more accurate startingdose.
More information on the doses at which the reductions in expected animal numbers were
found should be provided in the Draft BRD. Presumably, for the most toxic substances, thesavings were at higher doses (as with the NRU test, the starting dose was below the default)
and for the least toxic substances the savings were at the lower doses. The former are more
important than the latter. For the most toxic substances, the largest savings in animal
numbers was provided by the RC millimole regression. This was in contrast to the overall
animal savings, which was smallest when this prediction is used. If the aim was to prevent
animal suffering rather than to reduce animal numbers, then it appeared that the RC
millimole regression was preferable.

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10.1

June 2006

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

This section should clarify that the NRU methods are to be used in a weight-of-evidence
approach to determining the starting dose for acute oral systemic toxicity assays. Concern
was expressed that underprediction of the toxicity by the cytotoxicity tests might lead toincreased animal suffering. Although the accuracy for predicting the exact GHS category
appears to be low, the data demonstrates that there is a reduction in animal use versus startingat the default starting dose if no other information is available (e.g., no toxicity informationfrom chemicals with similar structure, etc.).
10.2

Reduction and Refinement of Animal Use for the UDP

Based on existing data, where molecular weight information was available for a relatively
pure test substance, the millimolar regression should be used; in the absence of such data, themg/kg regression should be used.
10.3

Reduction and Refinement of Animal Use for the ATC

The Panel found the discussion and evaluation of this section to be appropriate.
10.4

Summary

The Panel found the discussion and evaluation of this section to be appropriate.
The possibility of using the NRU test methods to determine the starting dose for the fixed
dose procedure (FDP) acute toxicity test should be evaluated.
Animal savings should take into account, to the extent possible, the prevalence of chemicals
in each GHS category.
11.0

Practical Considerations

Section 11 contained evaluations of potential expense to be incurred upon approval andrequired implementation of these procedures to aid in choosing the starting dose for a UDPor other type of rat oral toxicity study. However, a cost-benefit analysis was absent. In orderto reduce the animal usage per acute oral toxicity study by approximately 1-2 rats, theestimated cost to sponsors increased by $1000-2000 for the preliminary in vitro study. This is
not cost-effective. Obviously, additional time would be required also to complete the oral
toxicity evaluation. Furthermore, although it was said that defining a starting dose to moreclosely coincide with the actual LD50 of a test substance improves the ultimate LD50 estimate,many regulatory tests are limit tests for which a preliminary in vitro test would offer nobenefit.

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11.1

June 2006

Transferability of the 3T3 and NHK NRU Test Methods

It appears that transferability was not as easy as was stated; minor protocol differences canhave profound effects. Adequate training must be conducted prior to the initiation of thestudy, and a demonstration of proficiency in running the test must be demonstrated before
testing unknowns.
11.1.1 Facilities and Major Fixed Equipment
A dedicated cell culture laboratory should be added to the list of needs.
11.1.2 Availability of Other Necessary Equipment and Supplies
A single source for NHK medium was noted to be a problem in the NICEATM/ECVAM
validation study.
Although the Draft BRD indicated that laboratories could isolate keratinocytes from donatedcultures, this could increase intralaboratory variation. The Panel agreed that therecommendation for a commercial source is better.
The Draft BRD should indicate that it is necessary to confirm that cells are free fromcontamination (e.g., bacteria, mycoplasma).
11.2

3T3 and NHK NRU Test Method Training Considerations

11.2.1 Required Training and ExpertiseThis section noted that good cell culture practices are needed. The Panel recommended
removing statements about the need for training in cloning, transfection, expression cloning,
immortalization, and virus propagation since these techniques are not necessary forcytotoxicity testing.
11.2.2 Training Requirements to Demonstrate Proficiency
The Panel found that the Draft BRD discussion and evaluation in this section wasappropriate.
11.3

Test Method Cost Considerations

11.3.1 3T3 and NHK NRU Test Methods
The Panel indicated that the costs quoted may be more than a little bit low. The Draft BRD
noted that it was possible that there wouldn’t be cost savings using NRU testing first, if only
a few rats were used. Additionally, the NHK NRU test could be almost cost-prohibitive if 5 x$380 vials are needed per 5 x 96-well plates.
The costs of performing NRU testing were charges from commercial laboratories. A roughcalculation for the cost of equipment and time need to perform each test might help
individual laboratories understand the cost and time of performing the test methods.

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11.3.2 In Vivo Rodent Acute Oral Toxicity Testing
Since the NRU test methods are to be used for reduction of animal use rather thanreplacement, it is appropriate to describe the number of animals that might be reduced in thissection.
11.4

Time Considerations for the 3T3 and NHK NRU Test Methods

Since it takes some time to screen the NHK NRU assay medium, it should be described inthis section.
11.5

Summary

The commentaries in Section 11 appeared to be appropriate. It was difficult to compare thevalue of the in vitro NRU test method ($1120-$1850) per test substance to achieve an IC50
versus an animal test ($750-$3750) to achieve an LD50. If the in vitro test can save at least asingle animal in the execution of the ATC or UDP test, this evaluation was worth the effort.

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VALIDATION STATUS OF THE NRU TEST METHODSThe Panel agreed that the applicable validation criteria have been adequately addressed forusing these in vitro test methods in a weight-of-evidence approach to determine the starting
dose for acute oral in vivo toxicity protocols. However, the Panel was aware that validationof the two NRU test methods was carried out not only to determine if they could be used to
set starting doses for in vivo acute toxicity studies, but also to determine the extent to whichthese tests could be a useful step in an in vitro tiered testing strategy for acute toxicity. The
Panel agreed the validation study showed the two NRU test methods evaluated could not be
used as a stand-alone replacement for the in vivo tests even considering the variability of thelatter. The Panel encouraged future work to develop a tiered testing strategy that includes
basal cytotoxicity as part of the overall strategy.

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Peer Review Panel Report: Draft ICCVAM Recommendations

June 2006

DRAFT ICCVAM RECOMMENDATIONS FOR IN VITRO
ACUTE TOXICITY TEST METHODS

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June 2006

Peer Review Panel Report: Draft ICCVAM Recommendations

June 2006

1.0

Draft ICCVAM Recommendations for In Vitro Acute Toxicity Test Methods

1.1

Recommended Test Method Uses1.

The 3T3 and NHK NRU test methods are not sufficiently accurate to predict
acute oral toxicity for the purpose of hazard classification (see Section 6 of the
In Vitro Acute Toxicity Test Methods BRD).

••
2.

For the purposes of acute oral toxicity testing, the 3T3 and NHK NRU test
methods may be used in a weight-of-evidence approach to determine thestarting dose for the current acute oral in vivo toxicity protocols (i.e., the Upand-Down Procedure [UDP] and Acute Toxic Class [ATC]).

•
•
•

3.

The Panel agreed with this statement in that neither of the two basal
cytotoxicity tests can be used as alternatives for the in vivo acute oral
toxicity test for the purposes of hazard classification.
In the Draft BRD, the rat in vivo data did not conform to current GLP
standards.

The Panel agreed that the in vitro test methods may be useful in a weightof-evidence approach to determine the starting dose for acute oral in vivo
toxicity protocols.
Given the test methods’ limited predictive capacity, however, it was
unclear whether they will provide substantial weight in that decision.
The overall accuracy was modest, and enhancement of accuracy throughmaterial selection (modular approach), model refinement, or tiered testing
strategy should be pursued.

Consistent with the U.S. Government Principles on the Use of Animals in
Research, Testing, and Education (National Research Council 1996), and the
U.S. Public Health Service Policy on Humane Care and Use of Laboratory
Animals (PHS 2002)3, in vitro basal cytotoxicity test methods as part of aweight-of-evidence approach to estimate the starting dose for acute oral invivo toxicity test methods should be considered and used where appropriate
before testing is conducted using animals. For some types of substances, thisapproach will reduce the number of animals needed. In some testing
situations, the approach may also reduce the numbers of animals that die orneed to be humanely killed.

•

The Panel agreed.

3

National Research Council. 1996. Guide for the Care and Use of Laboratory Animals. Washington, DC:
National Academy Press.
PHS. 2002. Public Health Service (PHS) Policy on Humane Care and Use of Laboratory Animals.
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Peer Review Panel Report: Draft ICCVAM Recommendations

4.

Substances with specific toxic mechanisms that are not expected to be active
in 3T3 or NHK cells (e.g., those that are neurotoxic, cardiotoxic, interferewith energy utilization, or alkylate proteins and other macromolecules) will
likely be underpredicted by these in vitro basal cytotoxicity test methods.
Therefore, until such time as a more predictive testing approach is developed,
the results from basal cytotoxicity testing with such substances may not beappropriate.

•

5.

•
•

The Panel did not agree with this statement.
There was consensus among the Panel that the data contained within the
Draft BRD or the open literature were not sufficient to justify theexclusion of reference substances based on mechanism.
It was not justified to (retrospectively) exclude substances because ofassumed modes of toxic action in vivo and/or possible involvement of
biotransformation reactions.

The performance of other in vitro basal cytotoxicity test methods that are
based on similar scientific principles and that measure or predict the same
biological response (i.e., basal cytotoxicity and the rat acute oral LD50 value,
respectively) should be demonstrated to meet or exceed the accuracy andreliability of the 3T3 and NHK NRU test methods.
•

7.

The Panel disagreed with elements of this statement; specific toxic
mechanisms that are not expected to be active in 3T3 and NHK cells,such as “interference with energy utilization and alkylation of proteins
and other macromolecules”, are mechanisms of cytotoxic action andshould be detectable with 3T3 and NHK cells.

The regression formula used to determine starting doses should be the revised
Registry of Cytotoxicity (RC) regression line [with IC50 values in µg/mL and
LD50 values in mg/kg] developed with the RC chemicals using rat LD50 data
only and excluding chemicals with mechanisms of action that are not expected
to be active in in vitro basal cytotoxicity test methods.

•

6.

June 2006

The Panel agreed with this statement although the reliability of the test
methods in this study was not quite satisfying (e.g., inter-laboratory
reproducibility), the reproducibility of these methods (e.g., intralaboratory reproducibility) was modest, and the accuracy of thesemethods was poor.

Compared to the NHK NRU test method, the 3T3 NRU test method appears tobe less labor intensive and less expensive to conduct; therefore, the 3T3 NRU
cytotoxicity test method is recommended for general use.

•

Some Panel members agreed in a general sense, however, cautioned that
one model be preferred over the other, based upon specific knowledge
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Peer Review Panel Report: Draft ICCVAM Recommendations

•

•
•

1.2

regarding known mechanisms of action (e.g., the rationale for the
disparate results observed with aminopterin and digoxin). Other Panelmembers agreed with this statement because the use of continuous celllines is more efficient, especially since the overall animal savings wererelatively low.
One Panel member noted that NHK NRU IC50 data have shown a better
correlation with human LC50 values (R2=0.62) than do rodent 3T3 NRU
IC50 data (R2=0.51) and better than rodent LD50 data correlates withhuman LC50 values (R2=0.56) as reported by Casati et al. (2005) at the 5thWorld Congress in Berlin. It is important to remember that hazard
assessment relates to the safety of humans, not rats.
Based on costs of commercial keratinocytes, the NHK NRU test method
may be cost-prohibitive.
The proprietary nature of the composition of the NHK culture medium
made it impossible to assess the role differences in media composition
may have had on the results.

Draft Recommended Test Method Limitations
•
•

1.3

June 2006

Colored substances (besides red substances) may absorb light in the opticaldensity range of the NRU test methods, which could affect the accuracy of theresults.
The Draft BRD indicated that optimization to allow for testing of mixtures
was being undertaken, yet no mixtures were used in fitting the regression
curve. Given the limitations of the test methods in accurately predicting
materials of known or uncertain mechanisms, the testing of mixtures seems
highly controversial.

Draft Recommended Future Studies
1.

Additional data should be collected using the 3T3 and/or the NHK NRU test
methods to evaluate their usefulness for predicting the in vivo acute oral
toxicity of chemical mixtures.

•

2.

The Panel generally agreed that this is a good recommendation, althoughcollecting data could be difficult and doing a correlation with in vivo data
would be even more difficult. It may be useful to suggest that such data
only be collected with the 3T3 NRU test method, and that it would benecessary to clarify the reasons for the interlaboratory variations for
future use of the method.

Additional high quality comparative in vitro basal cytotoxicity data should becollected in tandem with in vivo rat acute oral toxicity test results to
supplement the high quality validation database started by this study. Periodic
evaluations of the expanded database should be conducted to further

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Peer Review Panel Report: Draft ICCVAM Recommendations

June 2006

characterize the usefulness and limitations of using in vitro cytotoxicity dataas part of a weight-of-evidence approach to estimate starting doses.

•
•

3.

Additional efforts should be conducted to identify additional in vitro tests andother methods necessary to achieve accurate acute oral hazard classification;
specifically, studies should be conducted to investigate the potential use of invitro cell-based test methods that incorporate mechanisms of action andevaluations of ADME (absorption, distribution, metabolism, excretion) to
provide improved estimates of acute toxicity hazard categories.

•

•

4.

The Panel agreed with this statement and added that there should be
additional effort towards development of alternative methods toadequately predict the in vivo acute toxicity of chemicals for the purposesof hazard classification.
An additional statement to include could be, “and the development ofmethods to extrapolate from in vitro toxic concentrations to equivalentdoses in vivo.”

The in vivo database of reference substances used in this validation study
should be used to evaluate the utility of other non-animal approaches toestimate starting doses for acute oral systemic toxicity tests (e.g., widely
available software that uses quantitative structure-activity relationships[QSAR]).
•

5.

The Panel agreed this could be valuable under certain conditions,especially if NRU data were collected as acute toxicity testing is
conducted.
However, no reviewer wanted in vivo testing conducted solely to collectdata to assess the usefulness of the NRU test method, particularly giventhat the savings in animal numbers that arise from the use of the NRU test
method to determine the starting dose for the ATC method or UDP are
fairly modest.

The Panel agreed with this recommendation.

Standardized procedures to collect information pertinent to an understandingof the mechanisms of lethality should be included in future in vivo rat acuteoral toxicity studies. Such information will likely be necessary to support thefurther development of predictive mechanism-based in vitro methods.
••

The Panel agreed with this recommendation; this is really important andcould further the development of non-animal alternatives in the future.
To facilitate comparisons and model development, future studies shouldincorporate high quality animal data for required testing of new
substances, blood levels from animals (LC50) (where possible), and highquality in vitro data for the same substances.
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Peer Review Panel Report: Draft ICCVAM Recommendations

•
•

•

6.

June 2006

To aid in this process, the Panel recommended that an expert group be
convened to identify appropriate in vivo endpoints.
The Panel recommended also that ICCVAM consider convening aworking group to explore mechanisms of action of acute toxicity, andapproaches to acquiring additional information on acute toxic
mechanisms when conducting the required in vivo acute toxicity testing.
Although a modular approach may be more reliable, the database was
likely too small for most mechanisms of action to draw sound conclusions
regarding strengths and limitations of the test methods with respect tochemical classes, mechanisms of toxicity, or physico-chemical properties.
Since a mode of action is unlikely to be known about a random source
material, it is also unlikely that a modular approach based uponmechanism will be a viable option. A better approach to validation is one
based on chemical class, implying similar mode of action.

An expanded list of reference substances with estimated rat LD50 values
substantiated by high quality in vivo data should be developed for use in
future in vitro test method development and validation studies.
•

The Panel agreed with this recommendation; there should be a concerted
effort to collect proprietary data.

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38

June 2006

Peer Review Panel Report: Performance Standards

June 2006

APPENDIX A: DRAFT PERFORMANCE STANDARDS FOR
IN VITRO ACUTE TOXICITY METHODS

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Peer Review Panel Report: Performance Standards

1.0

June 2006

Purpose and Background of Performance Standards

The available data from this study appeared to support the validity of the recommended
performance standards for the test methods. The usefulness and limitations were wellcovered, and if validated, the methods may be a worthwhile option. However, there may besome cause for concern if use of the methods is made compulsory for regulatory purposes.
1.1

Introduction

The Panel found the discussion and evaluation of this section to be appropriate.
1.2

Elements of ICCVAM Performance Standards

The Panel found the discussion and evaluation of this section to be appropriate.
1.3

ICCVAM Process for the Development of Performance Standards

The Panel found the discussion and evaluation of this section to be appropriate.
1.4

ICCVAM Development of Recommended Performance Standards for In Vitro
Acute Toxicity Test Methods

The Panel found the discussion and evaluation of this section to be appropriate.
2.0

In Vitro Acute Toxicity Test Methods

The Panel found the discussion and evaluation of this section to be appropriate.
2.1

Background

The Panel found the discussion and evaluation of this section to be appropriate.

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Peer Review Panel Report: Performance Standards

2.2

June 2006

Principles of In Vitro Basal Cytotoxicity Assays to Predict Starting Doses for Acute
Oral Toxicity Tests

The Panel found the discussion and evaluation of this section to be appropriate.
2.3

Essential Test Method Components for In Vitro Basal Cytotoxicity Assays to
Predict Starting Doses for Acute Oral Toxicity (Lethality) Tests

A discussion is needed in this section regarding whether or not the NRU test methods arerecommended for use with unknown substances and mixtures. The recommendations made in
Section 2.3.2 (Application of the Test Substances), Section 2.3.3 (Control Substances), andSection 2.3.4 (Viability Measurements) were acceptable.
2.4

Reference Substances for In Vitro Basal Cytotoxicity Assays to Predict Starting
Doses for Acute Oral Toxicity Tests

The significance of the secondary chemical subset to be used for investigational purposes
should be better elucidated.
2.5

Accuracy and Reliability

The Panel found the discussion and evaluation of this section to be appropriate.

42Peer Review Panel Report: Test Method Protocols

June 2006

APPENDIX B:
DRAFT RECOMMENDED TEST METHOD PROTOCOLS

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Peer Review Panel Report: Test Method Protocols

1.0

June 2006

Draft Recommended Test Method Protocols

The protocols were generally quite detailed and laboratory technicians should be able follow
the procedures. The Panel recommended the following clarifications be added to the 3T3 andNHK NRU test method protocols:
1.1

Protocol Recommendations
•
•
•
•
•
•
•

•
•

1.2

Cell Culture Recommendations
•
•
•

1.3

The rationale for testing the positive control on separate plates rather than on
the test plates should be provided.
The number of definitive tests that should be performed for a test substanceshould be specified.
The range of linearity of the microplate reader should be confirmed (as per inhouse SOPs) for the recommended optical density (OD540) and stated.
Maximum absorbance values needed by a spectrophotometric plate reader
should be provided for application to the NRU test methods.
The test method protocols should be streamlined. (Undefined is how thisshould be accomplished.)
Guidance for using methods other than the Hill function to determine IC50
values should be provided.
The lowest acceptable test substance dilution factor (i.e., 1.21) should be
reduced rather than accepting only one cytotoxicity point between 0 and 100%
viability on a steep dose-response curve to use for determination of the IC50
value.
Study directors and quality assurance units are necessary only if testing is
performed under Good Laboratory Procedures (GLP), which is not usuallynecessary for dose-setting tests.
The protocol for the NHK cells should include a statement about the need to
avoid allowing the cell to reach confluence: under these conditions, these cells
can exhibit contact-induced differentiation. Once differentiation is induced,
cells lose their ability to proliferate.

Good cell culture practices (e.g., Hartung et al. 2002) must be followed.
Whether or not a prequalification test of new keratinocytes should be
performed by the laboratory prior to actual testing should be stated.
A recommendation that keratinocytes should be procured only throughcommercial sources and not by preparing primary cultures from donated tissue
should be included.

Solubility Recommendations•

Additional guidance to the solubility step-wise procedure should be added(i.e., ensure that test substance solution preparation procedures can be easily
understood by laboratory technicians).

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Peer Review Panel Report: Test Method Protocols

•
•
•
•
•

June 2006

Include a recommendation for training laboratory technicians so they better
understand solvent and solubility determinations.
Additional guidance as to the use of a microscope to assist in determining
solubility of a test substance should be added.
Test substances that may etch plastic or film out in medium should be
identified (the importance of detecting such compounds by the laboratory
technicians should be emphasized).
The protocols should recommend the use of a solvent (e.g., dimethylsulfoxide[DMSO], ethanol) at its lowest possible concentration.
There was concern about the differences in solvent selection between
laboratories as compared to the BioReliance solvent information. The
variability between laboratories in the selection of solvent points out apossible flaw in the solvent determination protocol. This should be evaluatedfor future studies.

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Peer Review Panel Report: References

June 2006

REFERENCES
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special tissue culture tests. In: In Vitro Toxicity Testing 1975-1976 (Berky J, Sherrod C, eds).
Philadelphia:Franklin Institute Press, 23-49.
Casati S, Hoffmann S, Strickland J, Paris M, Stokes W, Tice R, Malerba I, Hartung T. 2005.
Anaylsis of the correlation between in vitro cytotoxicity data and acute toxic effects inhumans. 5th World Congress on Alternatives and Animal Use in the Life Sciences, Berlin,
Germany.
Elmore E, Luc TT, Steele VE, Redpath JL. 2001. Comparative tissue-specific toxicities of 20
cancer preventive agents using cultured cells from 8 different normal human epithelia. In Vitr
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Elmore E, Siddiqui S, Desai N, Moyer MP, Steele VE, Redpath JL. 2002. The humanepithelial cell cytotoxicity test method for determining tissue specific toxicity: method
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Hartung T, Balls M, Bardouille C, Blanck O, Coecke S, Gstrauthaler G, Lewis D. 2002.
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ICCVAM. 2001a. Report of the International Workshop on In Vitro Methods for Assessing
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ICCVAM. 2001b. Guidance Document on Using In Vitro Data to Estimate In Vivo Starting
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http://iccvam.niehs.nih.gov/. [accessed 2 June 2005].
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June 2006

Seibert H, Balls M, Fentem JH, Bianchi V, Clothier RH, Dierickx PJ, Ekwall B, Garle MJ,
Gómez-Lechón MJ, Gribaldo L, Gülden M, Liebsch M, Rasmussen E, Roguet, Shrivastava
R, Walum E. 1996. Acute toxicity testing in vitro and the classification and labeling ofchemicals: The report and recommendations of ECVAM Workshop 16. Altern Lab Anim
24:499-510.
UN. 2005. Globally Harmonized System of Classification and Labelling of Chemicals(GHS), First Revised Edition. [ST/SG/AC.10/30/Rev.1]. United Nations, New York andGeneva. Available: http://www.unece.org/trans/danger/publi/ghs/ghs_rev01/01files_e.html[accessed 12 September 2005].

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