Appendix B – Summary Review Document

B-1

Appendix B

ICCVAM Summary Review Document:

The Low Volume Eye Test
ICCVAM LVET Test Method Evaluation Report

B-2

This page intentionally left blank
Appendix B – Summary Review Document

B-3

ICCVAM Summary Review Document:

The Low Volume Eye Test

Interagency Coordinating Committee on the

Validation of Alternative Methods

National Toxicology Program Interagency Center for the

Evaluation of Alternative Toxicological Methods

National Institute of Environmental Health Sciences

National Institutes of Health

U.S. Public Health Service

Department of Health and Human Services

2010
ICCVAM LVET Test Method Evaluation Report

B-4

This page intentionally left blank
Appendix B – Summary Review Document

B-5

Table of Contents 

List of Tables.....................................................................................................................................B-6

List of Abbreviations and Acronyms..............................................................................................B-7

Interagency Coordinating Committee on the Validation of Alternative Methods: Agency 

Representatives ......................................................................................................................B-9

Acknowledgements......................................................................................................................... B-10

Preface ............................................................................................................................................. B-13

Executive Summary ....................................................................................................................... B-15

1.0 Background on Ocular Safety Testing ................................................................................ B-17

2.0 Regulatory Testing Requirements for Ocular Hazards..................................................... B-18

3.0 Principle of the Low Volume Eye Test................................................................................ B-22

4.0 Performance of the Low Volume Eye Test vs. the Draize Rabbit Eye Test..................... B-24

5.0 Performance of the Low Volume Eye Test vs. the Draize Rabbit Eye Test 

Considering Human Study Data and Experience .............................................................. B-30

5.1 Ghassemi et al. (1993).................................................................................................. B-30

5.2 Roggeband et al. (2000)................................................................................................ B-31

5.3 Freeberg et al. (1984).................................................................................................... B-32

5.4 Freeberg et al. (1986a).................................................................................................. B-33

5.5 Freeberg et al. (1986b).................................................................................................. B-33

6.0 Summary................................................................................................................................ B-35

7.0 References.............................................................................................................................. B-37

8.0 Glossary.................................................................................................................................. B-39
ICCVAM LVET Test Method Evaluation Report

B-6

List of Tables

Table 1-1 Summary of Current U.S. Legislation Related to Ocular Health............................B-17

Table 2-1 Ocular Toxicity Classification Systems..................................................................B-19

Table 3-1 Comparison of LVET and Draize Rabbit Eye Test Protocols................................B-22

Table 3-2 Scale of Weighted Scores for Grading the Severity of Ocular Lesions..................B-23

Table 4-1 Performance of the LVET in Identifying Ocular Hazard Classification 

According to the EPA Classification System When Compared to Draize Rabbit 

Eye Test Results......................................................................................................B-24

Table 4-2 Performance of the LVET in Identifying Ocular Hazard Classification 

According to the GHS Classification System When Compared to Draize Rabbit 

Eye Test Results......................................................................................................B-24

Table 4-3 Extent of Underprediction of LVET vs. Draize Rabbit Eye Test Results 

According to the EPA Classification System1 ........................................................B-25

Table 4-4 Extent of Underprediction of LVET vs. Draize Rabbit Eye Test Results 

According to the GHS Classification System1

........................................................B-25

Table 4-5 Summary of Available Rabbit LVET and Draize Data from Gettings et al. 

(1996b)....................................................................................................................B-26

Table 4-6 Summary of MAS Categorization Data..................................................................B-27

Table 4-7 Summary of Available Rabbit LVET Data.............................................................B-27

Table 4-8 Summary of Available Rabbit LVET Data.............................................................B-28

Table 5-1 Summary of Rabbit and Human Responses to an Undiluted Liquid Household 

Cleaner....................................................................................................................B-31

Table 5-2 Human and Rabbit Eye Responses to a Liquid Laundry Detergent (1 μL)............B-32

Table 5-3 Summary of Rabbit and Human Accidental Exposure Data from Freeberg et al. 

(1984)......................................................................................................................B-32

Table 5-4 Human Clinical Study and Rabbit Data..................................................................B-33

Table 5-5 Human Accidental Exposure and Rabbit Data .......................................................B-34

Table 6-1 Summary of Ocular Hazard Classifications for EPA Registered Antimicrobial 

Cleaning Products: Consideration of LVET Data and EPA Determinations1 ........B-35
Appendix B – Summary Review Document

B-7

List of Abbreviations and Acronyms 

A.I.S.E. International Association for Soaps, Detergents and Maintenance Products

ATSDR Agency for Toxic Substances and Disease Registry

BRD Background review document

CASRN Chemical Abstracts Service Registry Number

Conj Conjunctiva

CPSC (U.S.) Consumer Product Safety Commission

CR Conjunctival redness

DER Data Evaluation Report

EC European Commission

ECETOC European Centre for Ecotoxicology and Toxicology of Chemicals

EC/HO European Commission/British Home Office

ECVAM European Centre for the Validation of Alternative Methods

EEC European Economic Community

EI Extremely irritating

EPA (U.S.) Environmental Protection Agency

EU European Union

FDA (U.S.) Food and Drug Administration

FR Federal Register

FHSA U.S. Federal Hazardous Substances Act

FIFRA Federal Insecticide, Fungicide, and Rodenticide Act

GHS Globally Harmonized System of Classification and Labelling of Chemicals

GLP Good Laboratory Practices

hr Hour

ICCVAM Interagency Coordinating Committee on the Validation of Alternative Methods

JaCVAM Japanese Center for the Validation of Alternative Methods 

LVET Low volume eye test

μL Microliter

MAS Maximum average score

MeSH (National Library of Medicine) Medical Subject Headings

MI Maximally irritating

Minim Minimally irritating

MMAS Mean maximum average score

MSDS Material Safety Data Sheet

NA Not applicable

NI Nonirritating

NICEATM National Toxicology Program Interagency Center for the Evaluation of Alternative 

Toxicological Methods
ICCVAM LVET Test Method Evaluation Report

B-8

NIEHS National Institute of Environmental Health Sciences

NIOSH National Institute of Occupational Safety and Health

NP Not provided

NT Not tested

NTP (U.S.) National Toxicology Program 

OECD Organisation for Economic Co-operation and Development

OPPTS Office of Prevention, Pesticides and Toxic Substances

OSHA Occupational Safety and Health Administration

OTWG Ocular Toxicity Working Group

PNI Practically nonirritating

PSB Product Safety Branch

scs Test substance dosed on the superior conjunctival sac

SD Standard deviation

TSCA Toxic Substances Control Act

TG Test Guideline

UN United Nations
Appendix B – Summary Review Document

B-9

Interagency Coordinating Committee on the Validation of

Alternative Methods: Agency Representatives

Agency for Toxic Substances and Disease 

Registry

*Moiz Mumtaz, Ph.D.

Bruce Fowler, Ph.D.

Edward Murray, Ph.D.

Eric Sampson, Ph.D.

Consumer Product Safety Commission

*Marilyn L. Wind, Ph.D. (Chair)

+Kristina Hatlelid, Ph.D.

Joanna Matheson, Ph.D.

Department of Agriculture

*Jodie Kulpa-Eddy, D.V.M. (Vice-Chair)

+Elizabeth Goldentyer, D.V.M.

Department of Defense

*Robert E. Foster, Ph.D.

+Patty Decot

Harry Salem, Ph.D.

Peter J. Schultheiss, D.V.M., DACLAM

Department of Energy

*Michael Kuperberg, Ph.D.

+Marvin Stodolsky, Ph.D.

Department of the Interior

*Barnett A. Rattner, Ph.D.

+Sarah Gerould, Ph.D. (to Feb. 2009)

Department of Transportation

*George Cushmac, Ph.D.

+Steve Hwang, Ph.D.

Environmental Protection Agency

Office of Pesticide Programs

*John R. “Jack” Fowle III, Ph.D., DABT

+Vicki Dellarco, Ph.D.

+Tina Levine, Ph.D.

Deborah McCall

Christine Augustyniak, Ph.D. (U.S. Coordinator, 

OECD Test Guidelines Program)

Office of Pollution Prevention and Toxics

Jerry Smrchek, Ph.D. (U.S. Coordinator, OECD 

Test Guidelines Program, to July 2009)

Office of Research and Development

Suzanne McMaster, Ph.D. (to Dec. 2008)

Julian Preston, Ph.D. (to July 2009)

Stephanie Padilla, Ph.D. (to July 2009)

Office of Science Coordination and Policy

Karen Hamernik, Ph.D. (to July 2009)

Food and Drug Administration

Office of the Commissioner

* Suzanne Fitzpatrick, Ph.D., DABT

Center for Biologics Evaluation and Research

Richard McFarland, Ph.D., M.D.

Ying Huang, Ph.D.

Center for Devices and Radiological Health

Melvin E. Stratmeyer, Ph.D.

Vasant G. Malshet, Ph.D., DABT

Center for Drug Evaluation and Research

+Abigail C. Jacobs, Ph.D.

Paul C. Brown, Ph.D.

Center for Food Safety and Applied Nutrition

David G. Hattan, Ph.D.

Robert L. Bronaugh, Ph.D.

Center for Veterinary Medicine

Devaraya Jagannath, Ph.D.

M. Cecilia Aguila, D.V.M.

National Center for Toxicological Research

Paul Howard, Ph.D.

Donna Mendrick, Ph.D. 

William T. Allaben, Ph.D. (to Jan. 2009)

Office of Regulatory Affairs

Lawrence D'Hoostelaere, Ph.D.

National Cancer Institute

* T. Kevin Howcroft, Ph.D.

Chand Khanna, D.V.M., Ph.D.

Alan Poland, M.D. (to Oct. 2008)

National Institute of Environmental Health 

Sciences

* William S. Stokes, D.V.M., DACLAM

+Raymond R. Tice, Ph.D.

Rajendra S. Chhabra, Ph.D., DABT

Jerrold J. Heindel, Ph.D.

National Institute for Occupational Safety and 

Health

* Paul Nicolaysen, V.M.D.

+K. Murali Rao, M.D., Ph.D.

National Institutes of Health

* Margaret D. Snyder, Ph.D.

National Library of Medicine

* Pertti (Bert) Hakkinen, Ph.D.

+ Jeanne Goshorn, M.S.

Occupational Safety and Health Administration

* Surender Ahir, Ph.D.

* Principal agency representative

+ Alternate principal agency representative
ICCVAM LVET Test Method Evaluation Report

B-10

Acknowledgements

Interagency Coordinating Committee on the Validation of 

Alternative Methods (ICCVAM) Ocular Toxicity Working Group (OTWG)

U.S. Consumer Product Safety Commission

Marilyn L. Wind, Ph.D. 

Adrienne Layton, Ph.D.

U.S. Department of Defense

Harry Salem, Ph.D.

U.S. Department of Transportation

Steve Hwang, Ph.D.

U.S. Environmental Protection Agency

Office of Pesticide Programs

Meta Bonner, Ph.D.

Jonathan Chen, Ph.D.

John R. “Jack” Fowle III, Ph.D., DABT

Masih Hashim, D.V.M., Ph.D.

Karen Hicks

Marianne Lewis

Debbie McCall

Timothy McMahon, Ph.D.

Mark Perry

John Redden

Jenny Tao, Ph.D.

Office of Research and Development

Andrew Geller, Ph.D.

Office of Science Coordination and Policy

Karen Hamernik, Ph.D.

U.S. Food and Drug Administration

Center for Drug Evaluation and Research

Paul Brown, Ph.D.

Wiley Chambers, M.D.

Abigail (Abby) Jacobs, Ph.D.

Jill Merrill, Ph.D., DABT (OTWG Chair)

Center for Food Safety and Applied Nutrition 

Robert Bronaugh, Ph.D.

Donnie Lowther

Office of the Commissioner

Suzanne Fitzpatrick, Ph.D., DABT

National Institute of Environmental Health 

Sciences

Warren Casey, Ph.D., DABT

Mark F. Cesta, D.V.M, DACVP

Raymond (Buck) Grissom, Ph.D.

William Stokes, D.V.M., DACLAM

Occupational Safety and Health Administration

Surender Ahir, Ph.D.

European Centre for the Validation of 

Alternative Methods – Liaison

João Barroso, Ph.D.

Thomas Cole, Ph.D.

Valerie Zuang, Ph.D.

Japanese Center for the Validation of 

Alternative Methods – Liaison

Hajime Kojima, Ph.D.
Appendix B – Summary Review Document

B-11

National Toxicology Program Interagency Center for the 

Evaluation of Alternative Toxicological Methods (NICEATM)

National Institute of Environmental Health Sciences

William Stokes, D.V.M., DACLAM

Director; Project Officer

Warren Casey, Ph.D., DABT

Deputy Director

Deborah McCarley

Special Assistant; Assistant Project Officer

NICEATM Support Contract Staff (Integrated Laboratory Systems [ILS], Inc.)

David Allen, Ph.D.

Jonathan Hamm, Ph.D.

Nelson Johnson

Brett Jones, Ph.D.

Elizabeth Lipscomb, Ph.D.

Linda Litchfield

Steven Morefield, M.D.

Catherine Sprankle

James Truax, M.A.

Linda Wilson

Statistical Consultant for ILS, Inc.

Joseph Haseman, Ph.D.
ICCVAM LVET Test Method Evaluation Report

B-12

This page intentionally left blank
Appendix B – Summary Review Document

B-13

Preface

Accidental contact with hazardous chemicals frequently causes eye injury and visual impairment. 

United States and international regulatory agencies currently use the Draize rabbit eye test (Draize et 

al. 1944) to identify potential ocular hazards associated with chemicals. The U.S. Consumer Product 

Safety Commission, U.S. Environmental Protection Agency, U.S. Food and Drug Administration, and 

U.S. Occupational Health and Safety Administration have testing regulations and/or guidelines and 

recommendations for assessing the ocular irritation potential of substances such as pesticides, 

household products, pharmaceuticals, cosmetics, and agricultural and industrial chemicals. 

Although ocular safety assessment has clearly helped to protect consumers and workers, concerns 

have been raised about the humane aspects of the Draize rabbit eye test. Regulatory authorities have 

adopted various modifications that reduce the number of animals used and the potential pain and 

distress associated with the procedure. Significant progress has been made during the last decade. 

Now only one to three rabbits are required per test, compared to six rabbits in the original protocol. 

Provisions have been added that allow for animals with severe lesions or discomfort to be humanely 

euthanized. 

The low volume eye test (LVET) was developed by Griffith et al. (1980) with the intent of refining 

the Draize rabbit eye test to reduce overlabeling of commercial products and more closely predict the 

human accidental response to ocular hazard. The Draize test was refined by applying the test 

substance to the corneal surface rather than to the conjunctival sac and by reducing the volume of 

exposure from 100 μL to 10 μL. However, the hypothesis that the LVET more closely predicts the 

human response than the Draize test for a wide applicability domain of test substances has not been

clearly demonstrated yet. Thus the LVET has yet to be adopted as a reference test method by any 

regulatory agency. 

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

reviewed the validity of the LVET because LVET data was used to support the validity of a test 

method described in the ICCVAM Test Method Evaluation Report: Current Validation Status of a 

Proposed In Vitro Testing Strategy for U.S. Environmental Protection Agency Ocular Hazard 

Classification and Labeling of Antimicrobial Cleaning Products (ICCVAM 2010). The ICCVAM 

Ocular Toxicity Working Group and the National Toxicology Program Interagency Center for the 

Evaluation of Alternative Toxicological Methods (NICEATM) have prepared this draft summary 

review document to summarize the current validation status of the LVET based on available 

information and data obtained by NICEATM. This draft summary review document forms the basis 

for draft ICCVAM test method recommendations, which are provided in a separate document. 

An independent international scientific peer review panel met in public forum on May 19–21, 2009, 

to develop conclusions and recommendations for the LVET. The Panel included expert scientists 

nominated by the European Centre for the Validation of Alternative Methods and the Japanese Center 

for the Validation of Alternative Methods. We anticipate that these organizations will be able to use 

the Panel’s independent report for their deliberations and development of test method 

recommendations. The Panel considered this summary review document and evaluated the extent to

which the available information supported the draft ICCVAM test method recommendations. 

ICCVAM considered the conclusions and recommendations of the Panel, along with comments 

received from the public and the Scientific Advisory Committee on Alternative Toxicological 

Methods, before finalizing the summary review document and test method recommendations. These 

will be forwarded to Federal agencies for their consideration and acceptance decisions where 

appropriate. 

We gratefully acknowledge the organizations and scientists who provided data and information for 

this document. We also acknowledge the efforts of those individuals who helped prepare this 
ICCVAM LVET Test Method Evaluation Report

B-14

summary review document, including the following staff from the NICEATM support contractor, 

Integrated Laboratory Systems, Inc.: David Allen, Jon Hamm, Nelson Johnson, Elizabeth Lipscomb, 

Brett Jones, Linda Litchfield, Gregory Moyer, Catherine Sprankle, and James Truax. We also thank 

the members of the ICCVAM Ocular Toxicity Working Group, chaired by Karen Hamernik, Ph.D. 

(EPA), and Jill Merrill, Ph.D. (U.S. Food and Drug Administration), and ICCVAM representatives 

who reviewed and provided comments throughout the process leading to this draft version. We also 

want to thank Valerie Zuang, Ph.D., and Dr. Hajime Kojima, Ph.D., the Ocular Toxicity Working 

Group liaisons from the European Centre for the Validation of Alternative Methods and the Japanese 

Center for the Validation of Alternative Methods, respectively, for their participation.

Marilyn Wind, Ph.D.

Deputy Associate Executive Director

Directorate for Health Sciences

U.S. Consumer Product Safety Commission 

Chair, ICCVAM

William S. Stokes, D.V.M., DACLAM

Rear Admiral/Assistant Surgeon General, U.S. Public Health Service

Director, NICEATM

Executive Director, ICCVAM
Appendix B – Summary Review Document

B-15

Executive Summary

Accidental eye injury due to contact with hazardous chemicals is a major cause of visual impairment. 

United States and international regulatory agencies currently use the Draize rabbit eye test (Draize et 

al. 1944) to identify potential ocular hazards associated with chemicals. In the Draize rabbit eye test, 

100 μL of the test substance is introduced into the conjunctival sac of each animal’s eye. Alternatives 

to the Draize test have been explored to reduce the possibility of pain and distress during the test 

procedure. 

Griffith et al. (1980) developed the low volume eye test (LVET) to both refine the rabbit eye test and 

more closely predict the human response to ocular hazard. In the LVET, the test substance is applied 

to the corneal surface rather than the conjunctival sac. The volume of exposure is decreased from 

100 μL to 10 μL. However, the LVET has not been shown to predict the human response more 

closely than the Draize test for a wide array of test substances. Thus, the LVET has not yet been 

adopted as a reference test method by any regulatory agency. This report reviews available scientific 

literature and summarizes the usefulness and limitations of the LVET as an acceptable in vivo

reference test method.

Most available LVET data were generated with surfactant-based mixtures or products, which produce 

only a mild ocular irritant response or no response. Gettings et al. (1996a) evaluated 25 surfactant 

formulations and their hazard classifications by the Environmental Protection Agency and Globally 

Harmonized System of Classification and Labelling of Chemicals. The authors reported several 

instances in which the LVET underpredicted an ocular corrosive or severe irritant response identified 

in the Draize test. While some claim that these data show the Draize test to be excessively 

overpredictive, there is limited information on the performance of known human corrosives in the 

LVET. 

Freeberg et al. (1984) conducted both the LVET and the Draize test on 29 household cleaning 

products for which human accidental exposure data are available. The authors concluded that the 

LVET more accurately predicts the human accidental response to such substances. Similarly, 

Freeberg et al. (1986b) tested 14 cleaning products with both the LVET and Draize tests and 

compared the responses to human accidental eye exposures. They concluded that the LVET response 

corresponds more closely to the human experience than does the Draize rabbit eye test. 

Ghassemi et al. (1993) and Roggeband et al. (2000) concluded that the smaller volume used in the 

LVET (10 μL) is more appropriate when compared directly with human clinical data. However, the 

lack of available Draize test data in these studies precludes any direct comparison with the LVET.

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

reviewed the validity of the LVET because LVET data was used to support the validity of a test 

method described in the ICCVAM Test Method Evaluation Report: Current Validation Status of a 

Proposed In Vitro Testing Strategy for U.S. Environmental Protection Agency Ocular Hazard 

Classification and Labeling of Antimicrobial Cleaning Products (ICCVAM 2010). LVET data are 

available for only limited types and numbers of substances (i.e., surfactant-containing personal and 

household cleaning products), precluding comprehensive evaluation of LVET performance. 

Comparative human data from clinical studies and accidental exposures have been proposed to 

support the accuracy of the LVET. However, these data are primarily for mild or nonirritating 

substances. Ethical considerations have limited the severity of substances that can be tested in human 

clinical studies. As a result, LVET comparisons to human clinical study data are based on tests with 

mild irritants or substances not labeled as irritants. Regulatory agencies charged with protecting 

public health cannot be assured that the LVET can adequately protect against substances that may 

cause moderate or severe ocular injuries in humans.
ICCVAM LVET Test Method Evaluation Report

B-16

The LVET may approximate experimentally the volume of a substance that could enter the human 

eye accidentally, but there are limited data to indicate whether it can accurately identify the ocular 

hazard of substances known to cause moderate, severe, or permanent human ocular injuries. In 

contrast, there are no documented instances in which a substance that produced a severe 

irritant/corrosive response in humans was not also classified as a severe irritant/corrosive in the 

Draize rabbit eye test.
Appendix B – Summary Review Document

B-17

1.0 Background on Ocular Safety Testing

Accidental eye injury is a leading cause of visual impairment in the United States. Many of these 

injuries occur due to contact with workplace or household chemicals. According to the National 

Institute of Occupational Safety and Health (NIOSH), each day about 2,000 U.S. workers have a jobrelated eye injury that requires medical treatment. Additional eye injuries occur in the home, with 

about 125,000 eye injuries a year caused by accidents involving common household products such as 

oven cleaner and bleach (source, American Academy of Ophthalmology). U.S. regulatory agencies 

such as the Consumer Product Safety Commission (CPSC), Environmental Protection Agency (EPA), 

Food and Drug Administration (FDA), and Occupational Safety and Health Administration (OSHA) 

have testing regulations and/or guidelines and recommendations to assess the hazard potential of 

substances that may come in contact with human eyes. 

These testing requirements have effectively protected consumers and workers from potential eye 

injury (Wagoner 1997; Chiou 1999; McGwin et al. 2006). The primary method currently accepted by 

U.S. and international regulatory agencies for assessing ocular safety hazards is the Draize rabbit eye 

test (Draize et al. 1944). Testing guidelines describing the procedure have been published (EPA 

OPPTS 870.2400 [EPA 1998]), Organisation for Economic Co-operation and Development Test 

Guideline 405 [OECD 2002]) and several legislative statutes have been enacted that enable 

government agencies to regulate a variety of substances with the potential to pose a risk to ocular 

health and safety (see Table 1-1).

Table 1-1 Summary of Current U.S. Legislation Related to Ocular Health

Legislation

(Year of Initial Enactment) Agency Substance

Food, Drug, and Cosmetic Act (1938) Food and Drug Administration Pharmaceuticals and 

cosmetics

Federal Insecticide, Fungicide, and 

Rodenticide Act (1947) and Federal 

Environmental Pesticide Control Act (1972)

Environmental Protection 

Agency

Pesticides

Federal Hazardous Substances Act (1964) Consumer Product Safety 

Commission

Household products

Federal Hazardous Substances Act (1964) 

and Toxic Substances Control Act (1976)

Department of Agriculture and 

Environmental Protection 

Agency

Agricultural and 

industrial chemicals

Occupational Safety and Health Act (1970) Occupational Safety and Health 

Administration

Occupational materials

Clean Air Act Amendments (1990) Chemical Safety and Hazard 

Investigation Board and 

Environmental Protection 

Agency

Accidentally released 

chemicals and air 

pollutants

Adapted from Wilhelmus (2001).
ICCVAM LVET Test Method Evaluation Report

B-18

2.0 Regulatory Testing Requirements for Ocular Hazards

The classification of irritant responses evaluated by each regulatory agency varies depending on their 

legislative mandate and specific goals for protecting human health (Table 2-1). The EPA ocular 

irritation classification regulation and testing guidelines (EPA 1998, 2003) are based on the most 

severe response in one animal in a group of three or more animals. This classification system takes 

into consideration the kinds of ocular effects produced, as well as the reversibility and severity of the 

effects. The EPA classifies substances in ocular irritant Categories I through IV (EPA 2003). 

Category I substances are defined as corrosive or severe irritants, while classification from II to IV is 

based on decreasing severity of irritation and time required for irritation to clear. Irritation that clears 

in 8 to 21 days is classified as Category II, while irritation that clears within 7 days is classified as 

Category III. For Category IV substances, irritation clears within 24 hours. 

The U.S. Federal Hazardous Substances Act (FHSA) guideline for ocular irritation classification 

(CPSC 1995) categorizes a test substance as corrosive, irritant, or substance not labeled as irritant. A 

corrosive, according to the FHSA, is a substance that causes visible destruction or irreversible 

alterations in the tissue at the site of contact (CPSC 1995). FHSA classification depends on the 

number of test animals that exhibit a positive ocular response within 72 hours after application of the 

test substance in the conjunctival sac. 

For the purpose of harmonizing the classification of ocular irritants internationally, the United 

Nations Globally Harmonized System of Classification and Labelling of Chemicals (GHS; UN 2007) 

includes two harmonized categories. One designates irreversible effects on the eye/serious damage to 

the eye (Category 1), and one designates reversible effects on the eye (Category 2). Reversible effects

are further classified based on the duration of persistence. Category 2A (irritating to eyes) reverses 

within 21 days, and Category 2B (mildly irritating to eyes) reverses within 7 days. The GHS 

categories are based on severity of the lesions and/or the duration of persistence.

Hazard classification of ocular irritants in the European Union is characterized by two risk phrases: 

(1) R36 denotes “irritating to eyes”; (2) R41 denotes “risk of serious damage to the eyes” (EU 2001). 

These risk phrases are based on whether the levels of damage, averaged across the 24-, 48- and 

72-hour observation times for each ocular lesion, fall within or above certain ranges of scores. 
Appendix B – Summary Review Document

B-19

Table 2-1 Ocular Toxicity Classification Systems

Regulatory 

Agency 

(Authorizing Act)

Number 

of 

Animals

Observation 

Days (after 

treatment)

Mean 

score 

taken?

Positive 

Response

Classification Criteria

U.S. CPSC 

(Federal 

Hazardous 

Substances Act)

OSHA 

(Occupational 

Safety and 

Health Act)

6 (12, 18 

possible)

1, 2, 3 No Opacity or Iritis 

≥1 or Redness

or Chemosis ≥2 

for any animal 

on any day

4 or more positive animals = 

Irritant

1st Tier:

2–3 positive animals = Go to 

2nd Tier

3 or more positive animals = 

Irritant

2nd Tier

1–2 positive animals = Go to 

3rd Tier

1 positive animal = Irritant

3rd Tier :

U.S. EPA 

(FIFRA, Federal 

Environmental 

Pesticide 

Control Act, and

TSCA)

At least 3 1 hr, 1, 2, 3, 7, 

21

No –Maximum 

score in an 

animal used for 

classification

–Opacity or 

Iritis ≥1 or 

Redness or 

Chemosis ≥2

– One or more positive animals 

needed for classification in 

categories below.

Category:

I = Corrosive, corneal 

involvement, or irritation 

persisting more than 

21 days

II = Corneal involvement or 

irritation clearing in 8–

21 days

III = Corneal involvement or 

irritation clearing in 7 days 

or less

IV = Minimal effects clearing 

in less than 24 hours

Definition of Full Reversal:

Opacity and Iritis scores = 0

and

Redness and Chemosis 

scores ≤1

continued
ICCVAM LVET Test Method Evaluation Report

B-20

Table 2-1 Ocular Toxicity Classification Systems (continued)

Regulatory 

Agency 

(Authorizing Act)

Number 

of 

Animals

Observation 

Days (after 

treatment)

Mean 

score 

taken?

Positive 

Response Classification Criteria

European Union 1 if severe 

effects are 

suspected 

or 3 if no 

severe 

effects are 

suspected

1, 2, 3 

(observation 

until Day 21)

Yes Mean study 

values (scores 

of all animals in 

study averaged 

over Days 1, 2, 

and 3) of:

Opacity or 

Chemosis ≥2,

Redness ≥2.5, 

or

Iritis ≥ 1

OR

Individual 

animal mean 

values (scores 

for each 

endpoint are 

averaged for 

each animal 

over Days 1, 2, 

and 3) of:

Opacity or 

Chemosis ≥2,

Redness ≥ 2.5, 

or

Iritis ≥ 1

R36 Classification

(3) Mean study value where:

2 ≤ Opacity < 3 or

1 ≤ Iritis < 1.5 or

Redness ≥2.5 or

Chemosis ≥2

(2) If 2/3 tested animals have 

individual animal mean 

values that falls into one of 

the following categories:

2 ≤ Opacity <3

1 ≤ Iritis <2

Redness ≥2.5

Chemosis ≥2

R41 Classification

(3)Mean study value where:

Opacity ≥3 or

Iritis >1.5

(2) If 2/3 tested animals have 

individual animal mean 

values that fall into one of 

the following categories:

Opacity ≥3

Iritis = 2

(3) At least one animal (at the 

end of the observation 

period, typically Day 21) 

where Opacity or 

Chemosis ≥2, 

Redness ≥2.5 or Iritis ≥1

GHS: 

Irreversible Eye 

Effects

3 1, 2, 3 

(observation 

until Day 21)

Yes Mean animal 

values (over 

Days 1, 2, and 

3) of:

Opacity ≥3 

and/or 

Iritis ≥1.5

–At least 2 positive response 

animals = Eye Irritant 

Category 1

–At least 1 animal with an 

Opacity, Iritis, Redness, or 

Chemosis score >0 on Day 21 

= Eye Irritant Category 1

Definition of Full Reversal:

Opacity, Iritis, Redness, and 

Chemosis scores = 0

continued
Appendix B – Summary Review Document

B-21

Table 2-1 Ocular Toxicity Classification Systems (continued)

Regulatory 

Agency 

(Authorizing Act)

Number 

of 

Animals

Observation 

Days (after 

treatment)

Mean 

score 

taken?

Positive 

Response Classification Criteria

GHS: 

Reversible Eye 

Effects

3 1, 2, 3 

(observation 

until Day 21)

Yes Mean animal 

values (over 

Days 1, 2, and 

3) of:

Opacity or Iritis 

≥1 or

Redness or 

Chemosis ≥2 

and the effect 

fully reverses in 

7 or 21 days

–At least 2 positive response 

animals and the effect fully 

reverses in 21 days = Eye 

Irritant Category 2A

–At least 2 positive response 

animals and effect fully 

reverses in 7 days = Eye 

Irritant Category 2B

Definition of Full Reversal:

Opacity, Iritis, Redness, and 

Chemosis scores = 0

Abbreviations: CPSC = U.S. Consumer Product Safety Commission; EPA = U.S. Environmental Protection 

Agency; FDA = U.S. Food and Drug Administration; FIFRA = Federal Insecticide, Fungicide, and 

Rodenticide Act; GHS = United Nations (UN) Globally Harmonized System of Classification and Labelling 

of Chemicals; OSHA = U.S. Occupational Safety and Health Administration; TSCA = Toxic Substances 

Control Act.
ICCVAM LVET Test Method Evaluation Report

B-22

3.0 Principle of the Low Volume Eye Test

The low volume eye test (LVET) is an in vivo rabbit eye test that, like the Draize test, was designed to 

determine the extent of potential ocular hazard of a test substance. The tests evaluate the ocular 

irritation response when a test substance is administered as a single dose to the eye of a rabbit. 

Developed by Griffith et al. (1980), the LVET differs from the Draize rabbit eye test primarily by 

applying 10 μL (instead of 100 μL) of a test substance directly on the cornea (instead of the 

conjunctival sac) (Table 3-1). Scoring of corneal, iridal, and conjunctival lesions in the LVET is 

identical to that of the Draize rabbit eye test (Table 3-2). 

Table 3-1 Comparison of LVET and Draize Rabbit Eye Test Protocols

LVET Draize

Dose volume 10 μL 100 μL

Dose location Applied directly onto the cornea Applied into the lower 

conjunctival sac

Eyelid closure No forced eyelid closure Eyelids held closed for one second

Scale for scoring ocular 

lesions

Draize Draize

Abbreviation: LVET = low volume eye test

To date, the LVET has not been demonstrated as an adequately valid in vivo reference test method. It 

has not been formally adopted by any regulatory agency. For this reason, the Interagency 

Coordinating Committee on the Validation of Alternative Methods (ICCVAM) is reviewing the 

validity of the LVET as an acceptable in vivo reference test method. In February 2007, the 

International Association for Soaps, Detergents and Maintenance Products (A.I.S.E.) submitted a 

background review document to the European Centre for the Validation of Alternative Methods 

(ECVAM) for an independent peer review by their Scientific Advisory Committee. The A.I.S.E. 

background review document provides a comprehensive summary of available data and information 

with which to evaluate the usefulness and limitations of the LVET. 

Since its original development, proponents of the LVET have suggested that it is a more appropriate 

in vivo reference test method for comparisons to in vitro data than is the Draize rabbit eye test. This is 

based primarily on the assertion that the LVET is more representative of the human response to a 

potential ocular hazard than the Draize rabbit eye test, given that the site (corneal surface) and volume 

of exposure used in the LVET more closely resemble that of accidental human exposure. As a result, 

a reported advantage of the LVET is that it underpredicts the Draize test and is thereby less 

overpredictive of the human response than the Draize test. However, definitive data to support this 

claim are not available.
Appendix B – Summary Review Document

B-23

Table 3-2 Scale of Weighted Scores for Grading the Severity of Ocular Lesions

Cornea

Lesion Score1

A. Opacity – Degree of density (area which is most dense is taken for reading)

Scattered or diffuse area – details of iris clearly visible 1

Easily discernible translucent areas, details of iris slightly obscured 2

Opalescent areas, no details of iris visible, size of pupil barely discernible 3

Opaque, iris invisible 4

B. Area of cornea involved

One quarter (or less) but not zero 1

Greater than one quarter but less than one half 2

Greater than one half but less than three quarters 3

Greater than three quarters up to whole area 4

Score equals A x B x 5 Total maximum = 80

Iris

Lesion Score1

A. Values

Folds above normal, congestion, swelling, circumcorneal injection (any one or all of these or 

combination of any thereof), iris still reacting to light (sluggish reaction is positive) 1

No reaction to light, hemorrhage; gross destruction (any one or all of these) 2

Score equals A x 5 Total possible maximum = 10

Conjunctiva

Lesion Score1

A. Redness (refers to palpebral conjunctiva only)

Vessels definitely injected above normal 1

More diffuse, deeper crimson red, individual vessels not easily discernible 2

Diffuse beefy red 3

B. Chemosis

Any swelling above normal (includes nictitating membrane) 1

Obvious swelling with partial eversion of the lids 2

Swelling with lids about half closed 3

Swelling with lids about half closed to completely closed 4

C. Discharge

Any amount different from normal (does not include small amount observed in inner canthus 

of normal animals 1

Discharge with moistening of the lids and hairs just adjacent to the lids 2

Discharge with moistening of the lids and considerable area around the eye 3

Score equals (A + B + C) x 2 Total maximum = 20

From Draize et al. (1944).

1The maximum total score is the sum of all scores obtained for the cornea, iris and conjunctiva. Scores of 0 are 

assigned for each parameter if the cornea, iris, or conjunctiva is normal.
ICCVAM LVET Test Method Evaluation Report

B-24

4.0 Performance of the Low Volume Eye Test vs. the Draize Rabbit Eye 

Test

In general, most of the original data generated with the LVET were from surfactant-based mixtures or 

surfactant-based products (Freeberg et al. 1984; Gettings et al. 1996a, 1998). A comparison of the 

substances that have been classified by the Draize rabbit eye test as ocular corrosives or severe 

irritants that have also been tested in the LVET indicates that the LVET routinely underpredicts the 

ocular corrosive or severe irritant response in the Draize, in many cases by more than one hazard 

category. Gettings et al. (1996a, 1998) illustrate this in their evaluation of 25 surfactant-containing 

formulations and the resulting hazard classifications according to the EPA and GHS classification 

systems (EPA 2003; UN 2007) (Tables 4-1 and 4-2). 

Table 4-1 Performance of the LVET in Identifying Ocular Hazard Classification 

According to the EPA Classification System When Compared to Draize Rabbit 

Eye Test Results

EPA Category1 LVET Classification

I II III IV Totals

Draize

I 3 1 6 0 10

II 0 0 0 0 0

III 0 0 9 2 11

IV 0 0 0 4 4

Totals 3 1 15 6 25

From Gettings et. al. 1996a and 1998.

Abbreviations: EPA = Environmental Protection Agency; LVET = low volume eye test 1 EPA classification system (EPA 2003).

Table 4-2 Performance of the LVET in Identifying Ocular Hazard Classification 

According to the GHS Classification System When Compared to Draize Rabbit 

Eye Test Results

GHS Category1 LVET Classification

1 2A 2B Not Labeled Totals

Draize

1 0 0 4 4 8

2A 0 0 0 0 0

2B 0 0 0 1 1

Not Labeled 0 0 0 16 16

Totals 0 0 4 21 25

From Gettings et. al. 1996a and 1998.

Abbreviations: GHS = Globally Harmonized System; LVET = low volume eye test 1 GHS classification system (UN 2007).

Tables 4-1 and 4-2 show multiple instances of underprediction of an ocular corrosive or severe 

irritant response in the Draize rabbit eye test by the LVET. When the EPA hazard classification 

system (EPA 2003) was used, the LVET underpredicted 60% (6/10) of Draize Category I substances 

as Category III (mild irritant) (Table 4-3). When the GHS hazard classification system (UN 2007) 

was used, the LVET underpredicted all eight of the Draize Category 1 substances: 50% (4/8) as 
Appendix B – Summary Review Document

B-25

Category 2B (mild irritant) and 50% (4/8) as Not Labeled (not labeled as an irritant) (Table 4-4).

These data raise concern about the ability of the LVET to reliably detect ocular corrosives or severe 

irritants (i.e., EPA Category I, EU Category R41, GHS Category 1). 

Table 4-3 Extent of Underprediction of LVET vs. Draize Rabbit Eye Test Results 

According to the EPA Classification System1

EPA Category LVET Category Product

Category I Category II HZY (Antidandruff shampoo)

Category I Category III HZA (Shampoo #7)

Category I Category III HZE (Gel cleanser)

Category I Category III HZF (Baby shampoo #2)

Category I Category III HZL (Foam bath)

Category I Category III HZR (Facial cleaning foam)

Category I Category III HZX (Shampoo #2)

Abbreviations: EPA = Environmental Protection Agency; LVET = low volume eye test 1 EPA classification system (EPA 2003).

Table 4-4 Extent of Underprediction of LVET vs. Draize Rabbit Eye Test Results 

According to the GHS Classification System1

GHS Category LVET Category Product

Category 1 Category 2B HZI (Skin cleanser)

Category 1 Category 2B HZK (Bubble bath)

Category 1 Category 2B HZS (Shower gel)

Category 1 Category 2B HZY (Antidandruff shampoo)

Category 1 Not Classified HZL (Foam bath)

Category 1 Not Classified HZF (Baby shampoo #2)

Category 1 Not Classified HZX (Shampoo #2)

Category 1 Not Classified HZA (Shampoo #7)

Abbreviations: GHS = United Nations Globally Harmonized System; LVET = low volume eye test 1

GHS classification system (UN 2007).

Gettings et al. (1996b) published another study investigating the relationship between the LVET and 

Draize eye irritation test data for 10 representative hydroalcoholic personal-care formulations. 

Table 4-5 provides the eye irritation profile for each of the 10 substances tested. A range of irritancy 

classification was demonstrated for the LVET; however, only one of the test substances was 

considered moderately irritating and none severely irritating according to the criteria developed by 

Kay and Calandra (1962). A further comparison of the LVET using the classification scheme of 

Bruner et al. (1992) revealed a range of responses from nonirritating to moderately irritating. The 

Bruner et al. (1992) LVET classification appeared to be more consistent with the Kay and Calandra 

irritancy classification as determined by the Draize rabbit eye test (Table 4-5). 
ICCVAM LVET Test Method Evaluation Report

B-26

Table 4-5 Summary of Available Rabbit LVET and Draize Data from Gettings et al.

(1996b)

Ethanol (%)

Rabbit LVET Rabbit Draize

MAS Category1 Category2 MAS Category1

5 2.2 PNI I 7.7 Mild

10 1.3 PNI I 3.0 Minim

15 0.7 PNI I 0.7 PNI

20 0.7 PNI I 0.7 PNI

33 4.3 Minim I 14.3 Mild

40 15.5 Mild III 38.7 Moderate

55 14.3 Mild II 36.7 Moderate

65 22.5 Mild III 28.3 Moderate

83 22.5 Mild III 36.0 Moderate

90 26.0 Moderate III 45.7 Moderate

Modified from Gettings et al. (1996b).

Abbreviations: LVET = low volume eye test; MAS = maximum average score. 1 Kay and Calandra (1962): PNI = practically nonirritating; Minim = minimally irritating; Mild = mildly 

irritating; Moderate = moderately irritating. 2 Bruner et al. (1992): I = none to inconsequential irritation (LVET-MAS = 0–5); II = slight irritation 

(LVET-MAS > 5–15); III = moderate to severe irritation (LVET-MAS > 15–50); IV = severe irritation 

(LVET-MAS > 50–65); V = extremely irritating to corrosive (LVET-MAS > 65–110).

The authors noted a similarity between the irritant responses observed in the Draize rabbit eye test 

and the LVET, with both tests ranking the substances in a similar order. In addition, the observed 

irritation for both tests significantly increased when ethanol levels exceeded 33%. Indeed, the LVET 

consistently underpredicted ethanol solutions above this range when compared to the Draize rabbit 

eye test data (Table 4-5). 

Maurer et al. (2001a, 2001b) used pathology to evaluate the relationship of the ocular irritation 

response to the extent of initial injury for several nonsurfactant materials using the LVET. In these 

studies, they reported maximum average score (MAS) data for the LVET and irritation classifications 

based on Kay and Calandra (1962) as shown in Table 4-6. These LVET data are compared to 

available Draize data obtained from the database of the European Centre for Ecotoxicology and 

Toxicology of Chemicals (ECETOC 1998) in Table 4-7. Maurer et al. (2001a, 2001b) applied test 

substances directly to the cornea and performed macroscopic assessments for irritation 3 hours after 

dosing and periodically thereafter up to 35 days. The alcohols, cyclohexanol and parafluoroaniline, 

were moderate to severe irritants in the LVET. Only cyclohexanol was tested in the Draize test, and it 

was a severe irritant/corrosive. Of the aldehydes, acetone was a mild irritant in the LVET and a 

moderate irritant in the Draize test. Formaldehyde (37%; w/v) was a severe irritant in the LVET but 

was not tested in the Draize test. 

Four bleaches, sodium perborate monohydrate (NaBO3), sodium hypochlorite (NaOCl), 10% 

hydrogen peroxide (H2O2), and 15% H2O2, were evaluated in the LVET, but no corresponding Draize 

data were available. NaBO3 and NaOCl were classified as mild and minimal irritants in LVET 

respectively, with corneal injuries being limited to the epithelium and superficial stroma, as 

determined using in vivo confocal microscopy. It should be noted that some Material Safety Data 

Sheets (MSDS) from various manufacturers label NaOCl as moderately irritating or a severe 

irritant/corrosive in humans at or above 5.25%, while label it corrosive in humans above 14%. The 
Appendix B – Summary Review Document

B-27

15% H2O2 solution would be classified as a severe irritant based on LVET data. Both concentrations 

affected the epithelium and deep stroma, as determined using in vivo confocal microscopy. In 

undiluted form, H2O2 is a known human ocular corrosive/severe irritant.

Table 4-6 Summary of MAS Categorization Data

MAS Score Ocular Irritation Rating

0–0.5 Nonirritating— NI

0.5–2.5 Practically nonirritating— PNI

2.5–15 Minimally irritating— Minim

15–25 Mildly irritating— Mild

25–50 Moderately irritating— Moderate

50–80 Severely irritating— Severe

80–100 Extremely irritating— EI

100–110 Maximally irritating— MI

From Kay and Calandra (1962).

Abbreviation: MAS = maximum average score.

Table 4-7 Summary of Available Rabbit LVET Data 

Chemical Class

Eye Data

Rabbit LVET Rabbit Draize

MAS Category1 MMAS Category2

Alcohols - - - -

Cyclohexanol 50.8 Moderate/Severe 79.8 1/I/R41

Parafluoroaniline 55.0 Moderate/Severe 69.8

Aldehydes - - - -

Acetone 19.1 Mild 65.8 2A/II/R36

Formaldehyde, 37% (w/v) 80.0 Severe

Bleaching Agents - - - -

Sodium Perborate Monohydrate 23 ± 31.2 Mild - -

Sodium Hypochlorite (2.4%) 11 ± 3.6 Minim - -

10% Hydrogen Peroxide 16 ± 7.5 Mild - -

15% Hydrogen Peroxide 58.3 ± 26.1 Severe - -

Data from Maurer et al. (2001a, 2001b).

Abbreviations: ECETOC = European Centre for Ecotoxicology and Toxicology of Chemicals; LVET = low 

volume eye test; MAS = maximum average score; MMAS = mean maximum average score; 1 MAS categorization data compiled from classification table of Kay and Calandra (1962): PNI = practically 

nonirritating; Minim = minimally irritating; Mild = mildly irritating; Moderate = moderately irritating; 

Severe = severely irritating. Eye irritancy classification scores based on in vivo confocal microscopy and light 

microscopy also available in Jester (2006). 2 Data obtained from ECETOC database (ECETOC 1998). Hazard classifications based on the Globally 

Harmonized System (UN 2007)/EPA (EPA 2003)/European Union (EU 2001) were determined by 

NICEATM based on available ECETOC Draize data.
ICCVAM LVET Test Method Evaluation Report

B-28

Maurer et al. (2001a, 2001b) concluded that results obtained on these nonsurfactant materials support 

their hypothesis that ocular irritation is principally defined by the extent of initial injury, despite clear 

differences in the means by which irritants cause tissue damage.

Jester (2006) used the LVET to investigate the ocular irritancy of 22 substances varying in type (i.e., 

surfactant, acid, alkali, bleach, alcohol, aldehyde, and acetone) and severity (Table 4-8). Jester 

evaluated the extent of ocular irritation using light microscopy, in vivo confocal microscopy, and laser 

scanning confocal microscopy. Of the 22 substances, five produced slight irritation, nine produced 

mild irritation, three produced moderate/severe irritation, and five produced severe irritation. 

However, of the three substances for which Draize data were identified (i.e., 10% acetic acid, 

cyclohexanol, and acetone), the LVET underpredicted Draize results. 

Table 4-8 Summary of Available Rabbit LVET Data 

Chemical Class

Eye Data

Human2 Rabbit LVET Rabbit Draize1

MAS Category3 MMAS Category4

Surfactant - - - - - -

Nonionic - - - - - -

Polyoxyethylene glycol 

monoalkylether - - 0.0 NI - -

Polyoxyethelenesorbitan - - 0.0 NI - -

Alkyl E ethoxylate - - 33.0 Moderate - -

Anionic - - - - - -

Sodium lauryl sulfate, 5% - - 4.8 Minim - -

Sodium linear alkylbenzene 

sulfonate - - 49.3 Moderate - -

Sodium alkyl ethoxylate sulfate - - 31.2 Moderate - -

Cationic - - - - - -

Cetyltrimethylammonium 

chloride, 50% - - 76.3 Severe - -

3-Isotridecyloxypropylbis(polyoxyethylene) ammonium 

chloride

- - 7.7 Minim - -

3-Decyloxypropylbis(polyoxyethylene amine, 5% - - 40.0 Moderate - -

Alkylbenyldimethylammonium 

chloride, 10% - 70.6 Severe - -

Acid - - - - - -

3% Acetic Acid - - 5.0 Minim - -

10% Acetic Acid - - 9.5 Minim 68 1/I/R41

Base - - - - - -

2% Sodium Hydroxide - - 5.0 Minim - -

8% Sodium Hydroxide - - 50.8 Severe - -

continued
Appendix B – Summary Review Document

B-29

Table 4-8 Summary of Available Rabbit LVET Data (continued)

Chemical Class

Eye Data

Human2 Rabbit LVET Rabbit Draize1

MAS Category3 MMAS Category4

Aldehyde - - - - - -

Acetone - - 3.8 Minim 65.8 2A/II/R36

Formaldehyde, 37% - - 79.7 Severe - -

Alcohol - - - - - -

Parafluoroaniline - - 43.3 Moderate - -

Cyclohexanol - - 45.8 Moderate 79.8 1/I/R41

Bleach - - - - - -

Sodium Perborate Monohydrate - - 8.3 Minim - -

Sodium Hypochlorite (2.4%) Severe5 - 11.8 Minim - -

10% Hydrogen Peroxide - - 30.3 Moderate - -

15% Hydrogen Peroxide - - 68.3 Severe - -

Data from Jester (2006).

Abbreviations: ATSDR = Agency for Toxic Substances and Disease Registry; ECETOC = European Centre for 

Ecotoxicology and Toxicology of Chemicals; LVET = low volume eye test; MAS = maximum average score;

MMAS = mean maximum average score; MSDS = material safety data sheet. 1 Data obtained from ECETOC database (ECETOC 1998). Hazard classifications based on EPA (EPA 2003), 

Globally Harmonized System (UN 2007), and European Union (EU 2001) were determined by the National 

Toxicology Program Interagency Center for the Evaluation of Alternative Toxicological Methods based on 

ECETOC Draize data. 2 Data compiled from accidental exposures (ATSDR database). 3 MAS categorization data compiled from classification table of Kay and Calandra, (1962) (see Table 4-8). Eye 

irritancy classification scores based on in vivo confocal microscopy and light microscopy also available in 

Jester (2006). 4 Category classification– EPA/GHS/EU. 5 Labeled as moderately irritating or severe irritant/corrosive in humans at or above 5.25% based on some 

MSDS reports, while labeled as corrosive in humans above 14%.
ICCVAM LVET Test Method Evaluation Report

B-30

5.0 Performance of the Low Volume Eye Test vs. the Draize Rabbit Eye 

Test Considering Human Study Data and Experience

Human data on potential ocular hazards are available either from accidental exposures or from 

clinical studies. Accidental exposures are not generally considered to be a reliable source of the true 

ocular hazard potential because such exposures are likely immediately followed by flushing the eyes 

with large volumes of water. Thus they may not represent the most severe lesion that might be 

produced by such an exposure. Griffith et al. (1980) conducted a series of rabbit eye test studies using 

either 10 or 100 μL of substances “recognized as slightly irritating, moderately irritating, or severely 

irritating/corrosive to humans.”

The ocular corrosive or severe irritant substances included the following: 

• Acetic acid (10%), which is referenced as a severe irritant based on splashes of vinegar 

(containing 4% to 10% acetic acid) reported to cause pain, conjunctival hyperemia, and 

occasionally permanent opacity of the human cornea

• Calcium hydroxide (hydrated lime), which is referenced as one of the most common 

causes of severe chemical burns of the eye (McLaughlin 1946; Grant and Schuman 1993)

• Formaldehyde (38%), which is referenced for the range of injuries caused by splashes in 

the human eye from minor transient discomfort to severe, permanent corneal opacities 

(Grant and Schuman 1993)

Although detailed animal data are not available, the summary data provided by Griffith et al. (1980) 

indicate that the lesions induced by either 10 or 100 μL of these substances were not reversible within 

21 days. However, such accidental exposures as human reference data make definitive quantitative 

measures of amount and time of exposure impossible to obtain. Ethical considerations and results 

based largely on the Draize rabbit eye test have limited the severity of substances that can be tested in 

human clinical studies. As a result, comparisons to human data are based on clinical study tests with 

mild irritants or substances not labeled as irritants. Such data provide little assurance to the regulatory 

agencies charged with protecting public health that the LVET can provide adequate protection from 

substances that may cause moderate or severe ocular injuries. 

The fact that seemingly innocuous commercial consumer products were identified as ocular 

corrosives or severe irritants by the Draize eye test could be seen as supporting the contention that the 

Draize eye test is excessively overpredictive of the actual hazard to humans. However, because of the 

paucity of information on the performance of known human corrosives in the LVET, these data 

cannot simply be dismissed. 

Several studies have published supporting data for the demonstrated usefulness of the LVET

(Ghassemi et al. 1993; Roggeband et al. 2000; Freeberg et al. 1984, 1986a, 1986b).

5.1 Ghassemi et al. (1993)

Ghassemi et al. (1993) provides an evaluation of a single product, a liquid household cleaner (pH 3) 

reportedly containing the following qualitative formula: nonionic surfactant, amphoteric surfactant, 

hydrotrope, solvent, and water. This study directly compares LVET results to human clinical data 

(using either 10 or 100 μL doses) for the same test substance. No Draize rabbit eye test data had been 

reported; therefore, LVET results could not be compared to those of the standard eye test. The ocular 

lesions that were produced in this study and their subsequent time to clear suggest that this product is 

a mild ocular irritant (Table 5-1). The authors conclude that because the direct application to the 

human eye using either 10 or 100 μL doses produced similar results, the smaller volume for testing is 

more appropriate anatomically and physiologically based on eye volume capacity and subsequent tear 

volume.
Appendix B – Summary Review Document

B-31

Table 5-1 Summary of Rabbit and Human Responses to an Undiluted Liquid Household 

Cleaner

Species

Ocular 

Tissues 

Involved

Number of Eyes Affected Mean CR at

24 hr

Eyes Cleared/

Time to Clear

Max Time

Cornea to Clear Iris Conj

Rabbit LVET

Cornea

Iris

Conj

3/3 2/3 3/3 2 2/4 days

1/7 days 7 days

Human

(10 μL) Conj 0/10 0/10 10/10 0.1 1/1hr; 4/2hr; 6/4hr; 

10/24hr 48 hr

Human

(100 μL) Conj 0/10 0/10 10/10 0.2 1/1hr; 2/2hr; 9/24hr; 

2/46hr 70hr

Data from Ghassemi et al. (1993).

Abbreviations: Conj = conjunctiva; CR = conjunctival redness; hr = hour; LVET = low volume eye test (10 μL 

dose volume).

5.2 Roggeband et al. (2000)

Roggeband et al. (2000) evaluates two products, a dishwashing liquid (pH 8, contains anionic 

surfactant, nonionic surfactant, soap, ethanol, water) and a liquid laundry detergent (pH 7, contains 

anionic surfactant, nonionic surfactant, ethanol, water). This study directly compares modified LVET 

results to those of a human clinical study. Both rabbits and humans were dosed with either 3 μL 

(dishwashing detergent) or 1 μL (liquid laundry detergent) of the test products. There are no 

corresponding Draize rabbit eye test data. The ocular lesions that were produced in this study and 

their subsequent time to clear suggest that these products are mild ocular irritants (Table 5-2). The 

authors conclude that these data support the notion that (1) an accidental exposure would be 

approximately 10 μL or less and (2) a volume of 10 μL provides a suitable margin of safety. This is 

based on (1) knowledge of the anatomical and physiological characteristics of the eye and (2) the fact 

that study participants in Roggeband et al. (2000) could “only be exposed to 1 μL of dishwashing 

liquid and 3 μL of liquid laundry detergent before predetermined ‘cut-off’ ocular responses were 

observed above which it would have been ethically unacceptable to proceed” (Roggeband et al. 

2000).
ICCVAM LVET Test Method Evaluation Report

B-32

Table 5-2 Human and Rabbit Eye Responses to a Liquid Laundry Detergent (1 μL)

Human 

Volunteer

Human

Animal 

Number

Rabbit LVET1

1 hour 24 hours 1 hour 24 hours

Cornea Conj Cornea Conj Cornea Conj Cornea Conj

5 0 1/1 0 0/0 28 (c) 0/0 1/1/0 1/2 2/1/1

6 0 1/0 0 0/0 29 (c) 0/0 1/1/0 1/2 2/1/1

21 0 1/0 0 0/0 30 (c) 0/0 1/1/0 0/0 2/1/1

23 1/2 1/0 0 1/0 31 (scs) 0/0 1/1/0 1/4 2/1/0

25 1/1 1/0 0 0/0 32 (scs) 0/0 1/1/0 1/3 2/1/1

27 0 1/0 0 1/0 33 (scs) 0/0 1/1/0 1/4 2/1/1

28 0 1/0 0 0/0

30 0 0/0 0 0/0

32 0 1/0 0 0/0

34 0 1/0 0 0/0

Data from Roggeband et al. (2000).

Abbreviations: (c) = test substance dosed on the central cornea; Conj = conjunctiva; LVET = low volume eye 

test; (scs) = test substance dosed on the superior conjunctival sac. 1

Low volume eye test was modified to use 1 μL instead of 10 μL.

5.3 Freeberg et al. (1984)

A series of studies by Freeberg et al. (1984) compare data from LVET, Draize rabbit eye test, and 

human studies or experience. Freeberg et al. (1984) compares LVET and Draize rabbit eye test data 

for 29 cleaning products (laundry products, household cleaning products, and dishwashing products) 

to human experience data. The ocular lesions that were produced in this study and their subsequent 

time to clear suggest that these products are either mild ocular irritants or substances not labeled as 

irritants (Table 5-3). The human data were obtained from medical records of factory and consumer 

accidental eye exposures (515 reports over a 2-year period). The results indicate that both rabbit 

LVET and Draize eye tests overpredicted (based on time to clear of ocular lesions) the human 

response based on accidental eye exposure to the cleaning products. The time to clear was longer in 

the Draize eye test than in the LVET for the same product, forming the basis for the conclusion that 

the LVET more closely predicts the human response. 

Table 5-3 Summary of Rabbit and Human Accidental Exposure Data from Freeberg et al. 

(1984)

Species Test Method Number of 

Products

Average ± SD Mean 

Time to Clear

(Day Range)

Average ± SD Median 

Time to Clear)

(Day Range)

Average ± SD 

Number of 

Incidents (Range)

Rabbit LVET 17 7.3 ± 7.2

(1.3–28.8)

6.2 ± 8.8

(0.7–35) Not Applicable

Rabbit Draize 26 20.4 ± 7.2

(3.1–33.5)

20.2 ± 12.3

(1.4–35) Not Applicable

Human Experience data1 29 2.4 ± 2.1

(0.2–9.5)

1.5 ± 1.5

(0.1–1.8)

16.2 ± 8.4

(3–68)

Data from Freeberg et al. (1984).

Abbreviations: LVET = low volume eye test; SD = standard deviation. 1

Experience data = combined manufacturing and consumer accidental exposures.
Appendix B – Summary Review Document

B-33

5.4 Freeberg et al. (1986a)

Freeberg et al. (1986a) compared rabbit eye test results (both LVET and Draize) with those of human 

studies (both 10 μL and 100 μL dose volumes) for four cleaning products (a liquid fabric softener, 

liquid shampoo, liquid hand soap, and liquid laundry detergent). The results indicate that the LVET 

overpredicted the human response to 10 μL and 100 μL of the same product. The ocular lesions in the 

Draize rabbit eye test (100 μL) were more severe (both type and longevity) than in the human test 

using the same volume. While the majority of effects in humans were conjunctival, the corneal effects 

in humans were minimal and transient. The corneal effects in rabbits were more severe and recovered 

less quickly. The ocular lesions that were produced in this study and their subsequent time to clear 

suggest that these products would be classified as mild ocular irritants based on the Draize rabbit eye 

test results, the LVET, and human results (Table 5-4).

Table 5-4 Human Clinical Study and Rabbit Data 

Test Product Concentration 

(% in water)

Time to Clear (hr)

Dosing Volume

Rabbit Human Rabbit

10 μL 10 μL 100 μL 100 μL

Liquid fabric 

Softener

60 45 18.9 24.9 45

80 66 12.6 33.6 93

100 27 13.2 12.5 84

Liquid shampoo 4 5 1.5 2.5 NT

16 19.8 1.9 2.6 36.5

20 33 7.5 7.9 63

Liquid hand soap 8 24 1.5 31.5 63

10 42 10.5 9.1 66

12 42 1.7 NT NT

Liquid laundry 

detergent

2 8.8 2 24.1 27.8

3 19.8 4.7 1.8 60

4 39.8 4.8 19.8 75

Data from Freeberg et al. (1986a).

Abbreviation: NT = not tested.

5.5 Freeberg et al. (1986b)

Freeberg et al. (1986b) compares LVET and Draize rabbit eye test data for 14 cleaning products 

(liquid and solid laundry products, liquid and solid household cleaning products, liquid and solid 

dishwashing products, and liquid shampoos) to human experience data. The ocular lesions that were 

produced in this study and their subsequent time to clear suggest that these products would be 

classified as moderate to severe ocular irritants based on the Draize rabbit eye test results. Most

would be classified as mild ocular irritants by the LVET (Table 5-5). The human data were obtained 

from medical records of factory and consumer accidental eye exposures (218 reports over an 18-

month period). Similar to Freeberg et al. (1986a), rabbit LVET and Draize tests both overpredicted 

the human response due to accidental eye exposure (based on time to clear). Because the time to clear 

was longer for substances tested in the Draize rabbit eye test than in the LVET, the authors concluded 
ICCVAM LVET Test Method Evaluation Report

B-34

that the LVET outcome more closely relates to the human experience than the Draize rabbit eye test

does. 

Table 5-5 Human Accidental Exposure and Rabbit Data 

Product

Mean Time to Clear (Days)

Human Rabbit LVET Rabbit Draize

Liquid Laundry Product #1 1.92 26.6 35.0

Liquid Dishwashing Product #1 0.77 8.2 25.7

Solid Dishwashing Product #1 0.59 4.6 18.3

Liquid Dishwashing Product #2 0.43 7.7 11.7

Liquid Household Cleaning Product #1 0.38 - 11.1

Liquid Dishwashing Product #3 0.30 3.9 22.2

Liquid Household Cleaning Product #2 0.23 4.0 15.2

Solid Household Cleaning Product #1 0.19 1.3 29.2

Solid Dishwashing Product #1 0.08 2.1 13.8

Solid Dishwashing Product #1 0.06 2.9 15.1

Data from Freeberg et al. (1986b).

Abbreviation: LVET = low volume eye test.
Appendix B – Summary Review Document

B-35

6.0 Summary

Because studies conducted with the LVET have been limited to tests of surfactant-containing personal 

and household cleaning products, the applicability domain for which the LVET can be considered is 

necessarily restricted to these product types. As summarized in Table 6-1, LVET data have 

previously been used by one personal-care product company to support submission of data to the EPA 

for the registration of at least five antimicrobial cleaning products. The results were used by EPA 

reviewers in a weight-of-evidence approach, in conjunction with either consumer incidence data (i.e., 

commercial products for which there is an opportunity for adverse events to be reported by the 

consumer) and/or Draize data for similar, structurally related substances. Each study was considered 

on a case-by-case basis and several submissions were deemed unacceptable by the EPA because 

either the LVET study was not considered an acceptable fulfillment of the eye irritation data 

requirement and/or the further confirmatory information provided by the submitter was insufficient 

(Table 6-1). Based on the data provided to NICEATM in the Data Evaluation Reports (DERs), it 

appears that a final EPA ocular hazard classification was not assigned for any product using LVET 

data alone.

As indicated in the studies summarized above, human data on potential ocular hazards are available 

either from accidental exposures or from clinical studies. Accidental exposures are not generally 

considered to be a reliable source of the true ocular hazard potential because such exposures are likely 

immediately followed by flushing the eyes with large volumes of water. Such accidents make 

definitive quantitative measures of amount and time of exposure impossible to obtain. Although the 

Draize eye test is reported to be excessively overpredictive of the human response, ethical 

considerations based largely on results from the Draize rabbit eye test are used to limit the types of 

substances that can be tested in human clinical studies. As a result, comparisons to human clinical 

study data are based on tests of mild irritants or substances not labeled as irritants. Such data provide 

little assurance to the regulatory agencies charged with protecting public health that the LVET can 

provide adequate protection from more severe ocular injuries. 

Thus, while the LVET is proposed as more likely to approximate the volume of a substance that could 

enter the human eye accidently, there are limited data to indicate whether it can accurately identify 

the ocular hazard of substances known to cause moderate, severe, or permanent human ocular 

injuries. In contrast, there are no documented instances in which a substance with a hazard category 

determined in the Draize eye test produced a more severe hazard category response in humans 

following accidental exposures or ethical human studies.

Table 6-1 Summary of Ocular Hazard Classifications for EPA Registered Antimicrobial 

Cleaning Products: Consideration of LVET Data and EPA Determinations1

EPA 

Registration 

Number or 

Submission 

Code

Submission 

Date

Animal Data 

from LVET 

Study

EPA Hazard 

Category

Based on 

LVET Data

Additional 

Submission 

Information

Final EPA 

Classification 

Provided in DER

3573-AO

Jul 20, 2000

No corneal 

opacity, iritis, or 

conjunctival 

irritation (n=6). 

Category IV Consumer 

incidence data Study unacceptable2

Jun 6, 2001 Same as for Jul 

20, 2000

Consumer incidence data; LVET and 

Draize data for similar substances Category III

continued
ICCVAM LVET Test Method Evaluation Report

B-36

Table 6-1 Summary of Ocular Hazard Classifications for EPA Registered Antimicrobial 

Cleaning Products: Consideration of LVET Data and EPA Determinations1

(continued)

EPA 

Registration 

Number or 

Submission 

Code 

Submission 

Date

Animal Data 

from LVET 

Study

EPA Hazard 

Category 

Based on 

LVET Data

Additional 

Submission 

Information

Final EPA 

Classification 

Provided in DER

3573-TE

Aug 9, 2000

No corneal 

opacity, iritis, 

redness, or 

chemosis at day 

1 (n=3).

Category IV None Study unacceptable2

Feb 7, 2001 Repeat submission from Aug 9, 2000

Animal data for 

similar 

substances

Category IV

3573-72 Jun 6, 2001 NP Category III

Consumer 

incidence data; 

LVET and 

Draize data for 

similar 

substances

Category III

3573-AI Jun 6, 2001 NP NP NP Category II

S596273 Jun 27, 2001

No corneal 

effects or iritis 

observed. 

Conjunctivitis 

resolved by 72 

hr (n=3).

Category III None Study unacceptable2

3573-TG Jul 25, 2001 NP Category III

Consumer 

incidence data; 

Animal skin 

irritation studyCategory I

(severe irritant)

Study unacceptable3

Abbreviations: DER = Data Evaluation Reports; EPA = U.S. Environmental Protection Agency; LVET = low 

volume eye test; NP = not provided (i.e., information not contained in and/or not provided to NICEATM in 

DERs). 1 Data source: Obtained from a Freedom of Information Act request submitted to EPA for LVET data used to 

support the submission of data for the registration of antimicrobial cleaning products. 2 “The EPA does not consider the LVET study to be an acceptable fulfillment of the eye irritation data 

requirement.” 3 “It is now the Product Safety Branch’s (PSB) policy to take a weight of the evidence approach to the situation 

by considering individual LVET studies for possible acceptance on a case by case basis if they are 

significantly supplemented by further, confirmatory information. In the present case, that confirmatory 

further information is not sufficient.”
Appendix B – Summary Review Document

B-37

7.0 References

Bruner LH, Parker RD, Bruce RD. 1992. Reducing the number of rabbits in the low-volume eye test. 

Fund Appl Toxicol 19:330-335.

Chiou GCY. 1999. Ophthalmic Toxicology. In: Target Organ Toxicology Series (Hayes AW, Thoams 

JA, Gardner DG, eds). Philadelphia, PA:CRC Press.

Clean Air Act Amendments of 1990. 1990. Public Law 101-549. Available:

http://www.ehso.com/caa.htm.

CPSC. 1995. Test for eye irritants. 16 CFR 1500.42. 

Draize J, Woodard G, Calvery H. 1944. Methods for the study of irritation and toxicity of substances 

applied topically to the skin and mucous membranes. J Pharm Exp Ther 82:377–390.

ECETOC. 1998. Eye Irritation Reference Chemicals Data Bank. 2d ed. Technical Report No. 48 (2). 

European Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC). Brussels, 

Belgium:ECETOC.

EPA. 1998. Health Effects Test Guideline. OPPTS 870.2400 Acute Eye Irritation. EPA 712-C-98-

195. Washington, DC:U.S. Environmental Protection Agency.

EPA. 2003. Label Review Manual. 3rd ed. EPA735-B-03-001. Office of Prevention, Pesticides and 

Toxic Substances. Washington, DC:U.S. Environmental Protection Agency.

EU. 2001. Commission Directive 2001/59/EC of 6 August 2001 adapting to technical progress for the 

28th time Council Directive 67/548/EEC on the approximation of the laws, regulations and 

administrative provisions relating to the classification, packaging and labelling of dangerous 

substances. Official Journal of the European Communities L255:1–333.

Federal Environmental Pesticide Control Act. 1972. Public Law 92-516.

Federal Hazardous Substances Act. 1964. Public Law 86-613.

Federal Insecticide, Fungicide and Rodenticide Act. 1947. Public Law 80-102.

Food, Drug and Cosmetic Act of 1938. 1938. Public Law 75-717.

Freeberg FE, Griffith JF, Bruce RD, Bay PHS. 1984. Correlation of animal test methods with human 

experience for household products. J Toxic Cut & Ocular Toxic 1:53–64.

Freeberg FE, Hooker DT, Griffith JF. 1986b. Correlation of animal test methods with human 

experience for household products: an update. J Toxic Cut & Ocular Toxic 5:115–123.

Freeberg FE, Nixon GA, Reer PJ, Weaver JE, Bruce RD, Griffith JF, et al. 1986a. Human and rabbit 

eye responses to chemical insult. Fundam Appl Toxicol 7:626–634.

Gettings S, Lordo R, Hintze K, Bagley D, Casterton P, Chudkowski M, et al. 1996a. The CTFA 

evaluation of alternatives program: an evaluation of in vitro alternatives to the Draize primary eye 

irritation test. (Phase III) surfactant-based formulations. Food Chem Toxic 34:79–117.

Gettings SD, Lordo RA, Demetrulias J, Feder PI, Hintze KL. 1996b. Comparison of low-volume, 

Draize and in vitro eye irritation test data. I. Hydroalcoholic formulations. Food Chem Toxicol 

34:737–749.

Gettings SD, Lordo RA, Feder PI, Hintze KL. 1998. A comparison of low volume, Draize and in 

vitro eye irritation test data. III. Surfactant-based formulations. Food Chem Toxicol 36:209–231.
ICCVAM LVET Test Method Evaluation Report

B-38

Ghassemi A, Sauers LH, Bruner LH, Reer PJ, Hall RH. 1993. Demonstrating the human safety of a 

new household cleaning (HSC) product using alternatives to the Draize eye irritation test. 

Presentation made at the U.S. Society of Toxicology New Orleans, Louisiana.

Grant WM, Schuman JS. 1993. Toxicology of the Eye: Effects on the Eyes and Visual System from 

Chemicals, Drugs, Metals and Minerals, Plants, Toxins, and Venoms; Also Systemic Side Effects 

from Eye Medications. 4th ed. Springfield, IL:Charles C. Thomas.

Griffith J, Nixon G, Bruce R, Reer P, Bannan E. 1980. Dose-response studies with chemical irritants 

in the albino rabbit eye as a basis for selecting optimum testing conditions for predicting hazard to the 

human eye. Toxic Appl Pharmacol 55:501–513.

ICCVAM. 2003. ICCVAM Guidelines for the Nomination and Submission of New, Revised, and 

Alternative Test Methods. NIH Publication No. 03-4508. Research Triangle Park, NC:National 

Institute of Environmental Health Sciences.

ICCVAM. 2010. ICCVAM Test Method Evaluation Report: Current Validation Status of a Proposed 

In Vitro Testing Strategy for U.S. Environmental Protection Agency Ocular Hazard Classification and 

Labeling of Antimicrobial Cleaning Products. NIH Publication No. 10-7513. Research Triangle Park, 

NC:National Institute of Environmental Health Sciences.

Jester JV. 2006. Extent of corneal injury as biomarker for hazard assessment and the development of 

alternative models to the Draize rabbit eye test. Cutan Ocul Toxicol 25:41–54.

Kay JH, Calandra JC. 1962. Interpretation of eye irritation tests. J Soc Cosmet Chem 13:281–289.

Maurer JK, Molai A, Parker RD, Li L, Carr GJ, Petroll WM, Cavanagh HD, Jester JV. 2001a. 

Pathology of ocular irritation with bleaching agents in the rabbit low-volume eye test Toxicologic 

Pathology 29:308–319.

Maurer JK, Molai A, Parker RD, Li L, Carr GJ, Petroll WM, Cavanagh HD, Jester JV. 2001b. 

Pathology of ocular irritation with acetone, cyclohexanol, parafluoroaniline, and formaldehyde in the 

rabbit low-volume eye test. Toxicologic Pathology 29:187–199.

McGwin G. Hall TA, Xie A, Owsley C. 2006. Trends in eye injury in the United States, 1992-2001. 

Inv Ophthalmol Vis Science 47:521–527.

McLaughlin R. 1946. Chemical burns of the human cornea. Am J Ophthamol 29:1355–1362.

Occupational Safety and Health Act of 1970. 1970. Public Law 91-596

Organisation for Economic Co-operation and Development. 2002. Test guideline 405, Acute eye 

irritation/corrosion, adopted February 24, 1987. In: OECD Guidelines for Testing of Chemicals. 

Paris:OECD.

Roggeband R, York M, Pericoi M, Braun W. 2000. Eye irritation responses in rabbit and man after 

single applications of equal volumes of undiluted model liquid detergent products. Food Chem Toxic

38:727–734.

Toxic Substances Control Act of 1976. 1976. Public Law 94-469.

United Nations. 2007. Globally Harmonized System of Classification and Labelling of Chemicals 

(GHS). New York, Geneva:United Nations Publications.

Wagoner MD. 1997. Chemical injuries of the eye: Current concepts in pathophysiology and therapy. 

Surv Ophthalmol 41:275–313.

Wilhelmus K. 2001. The Draize eye test. Survey of Ophthalmology 45:493–515.
Appendix B – Summary Review Document

B-39

8.0 Glossary1

Assay:

2 The experimental system used. Often used interchangeably with test and test method.

Canthus: The angle formed by the meeting of the upper and lower eyelids at either side of the eye.

Chemosis: A form of eye irritation in which the membranes that line the eyelids and surface of the 

eye (conjunctiva) become swollen.

Classification system: An arrangement of quantified results or data into groups or categories 

according to previously established criteria.

Confocal microscopy: An optical imaging technique that increases the contrast of micrographs. It 

can used to reconstruct three-dimensional images by use of a spatial pinhole to eliminate out-of-focus 

light or flare in specimens that are thicker than the focal plane.

Conjunctiva: The mucous membrane that lines the inner surfaces of the eyelids and folds back to 

cover the front surface of the eyeball, except for the central clear portion of the outer eye (the cornea). 

The conjunctiva is composed of three sections: palpebral conjunctiva, bulbar conjunctiva, and fornix.

Conjunctival sac: The space located between the eyelid and the conjunctiva-covered eyeball. 

Substances are instilled into the sac to conduct an in vivo eye test.

Cornea: The transparent part of the coat of the eyeball that covers the iris and pupil and admits light 

to the interior.

Corneal opacity: A subjective measurement of the extent of opaqueness of the cornea following 

exposure to a test substance. Increased corneal opacity is indicative of damage to the cornea. 

Corneal stroma: The substantia propia: a tough, fibrous, transparent layer consisting of plates of 

collagen fibrils (lamellae) produced by keratocytes that make up 10% of the stroma. The fibrils run 

parallel to each other, but are positioned at right angles to adjacent lamellae.

Corrosion: Destruction of tissue at the site of contact with a substance.

Corrosive: A substance that causes irreversible tissue damage at the site of contact. 

Distress: To cause pain, or stress, or suffering to.

Endpoint:

2 The biological process, response, or effect assessed by a test method. 

Globally Harmonized System (GHS): A classification system presented by the United Nations that 

provides (a) a harmonized criteria for classifying substances and mixtures according to their health, 

environmental and physical hazards, and (b) harmonized hazard communication elements, including 

requirements for labeling and safety data sheets.

Hazard:

2 The potential for an adverse health or ecological effect. A hazard potential results only if an 

exposure occurs that leads to the possibility of an adverse effect being manifested.

Hyperemia: An increase in blood flow to a tissue (e.g., cornea).

In vitro: In glass. Refers to assays that are carried out in an artificial system (e.g., in a test tube or 

petri dish) and typically use single-cell organisms, cultured cells, cell-free extracts, or purified 

cellular components. 

 1 The definitions in this Glossary are restricted to their uses with respect to the Draize rabbit eye test method 

and in the assessment or treatment of pain and distress. 2 Definition used by the Interagency Coordinating Committee on the Validation of Alternative Methods 

(ICCVAM 2003)
ICCVAM LVET Test Method Evaluation Report

B-40

In vivo: In the living organism. Refers to assays performed in multicellular organisms.

Iris: The contractile diaphragm perforated by the pupil and forming the colored portion of the eye.

Not Labeled: (a) A substance the produces no changes in the eye following application to the 

anterior surface of the eye. (b) Substances that are not classified as GHS Category 1, 2A, or 2B; or 

EU R41 or R36 ocular irritants.

Ocular: Of or relating to the eye.

Ocular corrosive: A substance that causes irreversible tissue damage in the eye following application 

to the anterior surface of the eye. 

Ocular irritant: A substance that produces a reversible change in the eye following application to the 

anterior surface of the eye.

Pain: An unpleasant sensation occurring in varying degrees of severity as a consequence of injury, 

disease, or emotional disorder; suffering or distress.

pH: A measure of the acidity or alkalinity of a solution. A pH of 7.0 is neutral; higher pHs are 

alkaline, lower pHs are acidic.

Protocol:

2 The precise, step-by-step description of a test, including the listing of all necessary 

reagents, criteria and procedures for the evaluation of the test data. 

Severe irritant: (a) A substance that causes tissue damage in the eye following application to the 

anterior surface of the eye that is not reversible within 21 days of application or causes serious 

physical decay of vision. (b) Substances that are classified as GHS Category 1, EPA Category I, or 

EU R41 ocular irritants.

Test:

2 The experimental system used; used interchangeably with test method and assay.

Test method:

2 A process or procedure used to obtain information on the characteristics of a 

substance or agent. Toxicological test methods generate information regarding the ability of a 

substance or agent to produce a specified biological effect under specified conditions. Used 

interchangeably with test and assay. See also validated test method and reference test.

Validated test method:

2 An accepted test method for which validation studies have been completed 

to determine the relevance and reliability of this method for a specific proposed use.

Validation:

2 The process by which the reliability and relevance of a procedure are established for a 

specific purpose.

Weight of evidence (process): The strengths and weaknesses of a collection of information are used 

as the basis for a conclusion that may not be evident from the individual data.