# Presentation: 030715

## Proposed Panel Conclusions and Recommendations for the Hen’s Egg Test - Chorioallantoic Membrane (HET-CAM) Test Method 

**Expert Panel Meeting**
**National Institutes of Health**
**Natcher Conference Center **
**Bethesda, Maryland**
**January 11-12, 2005**

## HET-CAM Test Method Primary Reviewers
Shayne Gad, Ph.D., D.A.B.T, A.T.S, Gad Consulting Services
Donald A. Fox, Ph.D., University of Houston
Martin Stephens, Ph.D., Humane Society of the United States
Frederick Guerriero, M.S., GlaxoSmithKline
Sidney Green, Ph.D., A.T.S., Howard University
Philippe Vanparys, Ph.D., Johnson & Johnson
Nancy Flournoy, Ph.D., University of Missouri (Biostatistician)

**Shayne Gad, Ph.D., D.A.B.T, A.T.S, *****Gad Consulting Services***
**Donald ****A.**** Fox, Ph.D., *****University of Houston***
**Martin Stephens, Ph.D., *****Humane Society of the United States***
**Frederick Guerriero, M.S., *****GlaxoSmithKline***
**Sidney Green, Ph.D., A.T.S., *****Howard University***
**Philippe Vanparys, Ph.D., *****Johnson & Johnson***
**Nancy Flournoy, Ph.D., *****University of Missouri***** (Biostatistician)**

## BRD Section 1.0:  HET-CAM Test Method Rationale 

## 1.1	Scientific Basis for the HET-CAM Test Method
The CAM is most similar to the conjunctiva.
Deficiency of the method is that:
The CAM tissue structure is not similar to the cornea.
It cannot assess substances that induce delayed effects in rabbits, nor can it assess lesion reversibility.
Add discussion of cellular mechanisms of corrosion and severe irritation (e.g., necrosis, apoptosis) and relevance to in vitro testing. (ICE, HET-CAM)
Discuss the role of responsive inflammatory cells in isolated rabbit eyes.  (ICE, CAM in HET-CAM)

1.2 	Regulatory Rationale and Applicability
The ability of HET-CAM to provide an improved prediction of adverse health effects in humans may not be achieved.
Any analysis requires comparative human data.  
HET-CAM may have the potential to complement other methods in a battery or tiered testing approach.

**The CAM is most similar to the conjunctiva.**
**Deficiency of the method is that:**
**The **_**CAM tissue structure is not similar**_** to the cornea.**
**It cannot assess substances that induce delayed effects in rabbits, nor can it assess lesion reversibility.**
_**Add discussion of cellular mechanisms of corrosion and severe irritation (e.g., necrosis, apoptosis) and relevance to **__***in vitro***__** testing. (ICE, HET-CAM)**_
_**Discuss the role of responsive inflammatory cells in isolated rabbit eyes.  (ICE, CAM in HET-CAM)**_
**1.2 ****	****Regulatory Rationale and Applicability**
**The ability of HET-CAM to provide an improved prediction of adverse health effects in humans may not be achieved.**
**Any analysis requires comparative human data.  **
**HET-CAM may have the potential to complement other methods in a battery or tiered testing approach.**

## BRD Section 2.0:  HET-CAM Test Method Protocol Components

## 2.1	Test Method Components for the Recommended Version of the Protocol
Recommendations in the draft BRD appear to appropriately integrate protocol components from various protocols.  
BRD appropriately identifies the need to develop consistent methods for scoring and calculating irritation indices. 
Identification of reference substances that are part of the performance standards developed for the validated test method
The BRD should identify the decision criteria (Prediction Model) for identifying ocular corrosives and severe irritants

2.2	Basis for Selection of the Test Method System
Strain, stock, and age of recommended eggs are fairly easy to obtain.
Standardization would increase consistency in HET-CAM test results. 

**Recommendations in the draft BRD appear to appropriately integrate protocol components from various protocols.  **
**BRD appropriately identifies the need to develop consistent methods for scoring and calculating irritation indices. **
_**Identification of reference substances that are part of the performance standards developed for the validated test method**_
_**The BRD should identify the decision criteria (Prediction Model)**__** **__**for identifying ocular corrosives and severe irritants**_
**2.2****	****Basis for Selection of the Test Method System**
**Strain, stock, and age of recommended eggs are fairly easy to obtain.**
**Standardization would increase consistency in HET-CAM test results. **

## 2.3	Proprietary Components 
Panel agrees with BRD, no proprietary components are in the test method.

2.4	Number of Replicate and/or Repeat Experiments for Each Test
BRD recommendations would increase consistency of test results.

2.5	Study Acceptance Criteria
Due to variations in study acceptance criteria, a common definition of a positive result is needed.

**Panel agrees with BRD, no proprietary components are in the test method.**
**2.4****	****Number of Replicate and/or Repeat Experiments for Each Test**
**BRD recommendations would increase consistency of test results.**
**2.5****	****Study Acceptance Criteria**
**Due to variations in study acceptance criteria, a common definition of a positive result is needed.**
**	**

## 2.6	Basis for any Modifications to the Original Test Method Protocol
No concerns with regard to this section of the BRD.

2.7	Adequacy of the Recommended Standardized Protocol
Panel agrees with BRD recommendation that a standardized protocol is needed.
Critical recommendation is the inclusion of concurrent negative and positive controls.

**No concerns with regard to this section of the BRD.**
**2.7****	****Adequacy of the Recommended Standardized Protocol**
**Panel agrees with BRD recommendation that a standardized protocol is needed.**
**Critical recommendation is the inclusion of concurrent negative and positive controls.**
**	**

## BRD Section 3.0:  Substances Used for Previous Validation Studies of the HET-CAM Test Method 

## 3.1	Types & Numbers of Substances/Products Used for Prior Validation Studies
Type and number of substances tested in prior studies were adequate.

3.2 	Coding Procedures for Test Substances and Quality of HET-CAM Test Method Data
Difficult to determine if coding procedures were appropriate; not enough information provided in all studies.
As long as the quality and multiplicity of data sources are sufficient to draw conclusions, coding does not matter.

**Type and number of substances tested in prior studies were adequate.**
**3.2 ****	****Coding Procedures for Test Substances and Quality of HET-CAM Test Method Data**
**Difficult to determine if coding procedures were appropriate; not enough information provided in all studies.**
**As long as the quality and multiplicity of data sources are sufficient to draw conclusions, coding does not matter.**

## BRD Section 4.0:  In Vivo Reference Data Used for an Assessment of Test Method Accuracy 

## 4.1	In Vivo Rabbit Eye Test Method Protocols Used to Generate Reference Data
In vivo test method protocols used to generate data used in the BRD were appropriate.

4.2 	Interpretation of In Vivo Test Method Results for Cited Studies
Interpretation of in vivo rabbit eye test results were correct.
The concern can reasonably be raised that the regulatory classification methods may not be adequate for use in evaluating in vitro methods for their suitability in identifying corrosives and severe irritants.

***In vivo *****test method protocols used to generate data used in the BRD were appropriate.**
**4.2 ****	****Interpretation of *****In Vivo***** Test Method Results for Cited Studies**
**Interpretation of *****in vivo***** rabbit eye test results were correct.**
**The concern can reasonably be raised that the regulatory classification methods may not be adequate for use in evaluating *****in vitro***** methods for their suitability in identifying corrosives and severe irritants.**

## 4.3	Data Quality for Test Substances When Original Study Records Are/Are Not Available
Original study records were not available for any reports. 
However, study quality otherwise appears to be adequate.

4.4 	Data Quality With Respect to Extent of GLP Compliance
Not all studies evaluated referenced GLP compliance.
However, a distinction in the weight given to GLP-compliant v. non-GLP-compliant studies in the BRD may not be necessary.
When the basic requirements of the GLP procedure (the “spirit” of GLPs) are implemented in a study, lack of complete/formal GLP compliance is not an adequate criterion to exclude data.

**Original study records were not available for any reports. **
**However, study quality otherwise appears to be adequate.**
**4.4 ****	****Data Quality With Respect to Extent of GLP Compliance**
**Not all studies evaluated referenced GLP compliance.**
**However, a distinction in the weight given to GLP-compliant v. non-GLP-compliant studies in the BRD may not be necessary.**
**When the basic requirements of the GLP procedure (the “spirit” of GLPs) are implemented in a study, lack of complete/formal GLP compliance is not an adequate criterion to exclude data.**

## 4.5	Availability of Relevant Human Ocular Toxicity Information
There needs to be greater effort to obtain and consider information on human topical ocular chemical injury. 
Most of the available information originates from accidental exposure 
COLIPA and ILSI should be consulted for information on human eye irritation databases to assess current ocular hazard classification system adequacy in protecting human health.

_**There needs to be greater effort to obtain and consider information on human topical ocular chemical injury.**__** **_
**Most of the available information originates from accidental exposure **
**COLIPA and ILSI should be consulted for information on human eye irritation databases to assess current ocular hazard classification system**** adequacy in protecting human health.**

## 4.6 	Accuracy and Reliability of the In Vivo Rabbit Eye Test
The potential variability of the in vivo rabbit data has not been adequately discussed.
Evaluation of an alternative method is unavoidably biased by the selection of the in vivo data used in that evaluation
Any optimization and validation studies should use existing animal data; if available.
Additional animal studies should only be conducted if important data gaps are identified and such studies should be carefully designed to maximize the amount of pathophysiological information obtained (e.g., wound healing).
Minority opinion – no animal testing for this purpose.

**The potential variability of the *****in vivo***** rabbit data has not been adequately discussed.**
**Evaluation of an alternative method is unavoidably biased by the selection of the *****in vivo***** data used in that evaluation**
_**Any optimization and validation studies should use existing animal data; if available.**_
_**Additional animal studies should only be conducted if important data gaps are identified and such studies should be carefully designed to maximize the amount of pathophysiological information obtained (e.g., wound healing).**_
_**Minority opinion – no animal testing for this purpose.**_

## BRD Section 5.0:  HET-CAM Test Method Data and Results

## 5.1	HET-CAM Test Method Protocols Used to Generate Each Set of Data 
The test method protocols used to generate data were adequately described in the BRD.
Panel recommends including a description of which type of irritation score (IS(A) or IS(B)) was evaluated by each study in BRD Section 5.4.	

5.2	Other Comparative HET-CAM - In Vivo Rabbit Eye Test Data Not Considered in the BRD
Recently submitted data of Gautheron et al. (1994) may be useful and should be included in the evaluation in the BRD.

**The test method protocols used to generate data were adequately described in the BRD.**
**Panel recommends including a description of which type of irritation score (IS(A) or IS(B)) was evaluated by each study in BRD Section 5.4.****	**
**5.2****	****Other Comparative HET-CAM - *****In Vivo***** Rabbit Eye Test Data Not Considered in the BRD**
**Recently submitted data of Gautheron et al. (1994) may be useful and should be included in the evaluation in the BRD.**

## 5.3	Statistical and Nonstatistical Approaches Used to Evaluate the Resulting HET-CAM Data
The approaches used to evaluate the HET-CAM data appear to adequately describe the accuracy and reliability of the test method.  However, given the unavailability of original HET-CAM data, a definitive  statement regarding the adequacy of these approaches is not feasible.      
Lack of consistent evaluation methods complicated BRD evaluations.
IS(B) appears to be the optimal approach.

5.4 	Use of Coded Substances, Blind Studies, and GLP Guidelines for Cited Studies
BRD documents the use of coding and GLP guidelines.
Lack of GLP compliance should not exclude data from evaluation.
If study data can be interpreted and no serious deficiencies are identified, it should be accepted for use.

_**The approaches used to evaluate the HET-CAM data appear to adequately describe the accuracy and reliability of the test method.  However, given the unavailability of original HET-CAM data, a definitive  statement regarding the adequacy of these approaches is not feasible.**_**      **
**Lack of consistent evaluation methods complicated BRD evaluations.**
**IS(B) appears to be the optimal approach.**
**5.4 ****	****Use of Coded Substances, Blind Studies, and GLP Guidelines for Cited Studies**
**BRD documents the use of coding and GLP guidelines.**
**Lack of GLP compliance should not exclude data from evaluation.**
**If study data can be interpreted and no serious deficiencies are identified, it should be accepted for use.**

## 5.5	“Lot-to-Lot” Consistency of Test Substances and Timeframe of Studies
Insufficient information on lot-to-lot consistency in studies reviewed.
Panel expects substances from different batch could result in different results.
Major qualitative differences should not occur except when irritancy grade is borderline.

**Insufficient information on lot-to-lot consistency in studies reviewed.**
**Panel expects substances from different batch could result in different results.**
**Major qualitative differences should not occur except when irritancy grade is borderline.**

## BRD Section 6.0:  HET-CAM Test Method Accuracy

**The closeness of agreement between a test method result and an accepted reference value.  **** **
**the proportion of correct outcomes of a test method**

## 6.1	Accuracy Evaluation of the HET-CAM Test Method for Identifying Ocular Corrosives and Severe Irritants as Defined by the EPA (1996), the EU (2001), and the GHS (2003)
Accuracy evaluation adequately described; overall false positive rates ranged from 20% to 33% and false negative rates ranged from 0% to 7%.
It is essential that chemical classes the test method works well for and poorly for are identified.
Panel recommends replacing the term “accuracy” with “concordance” or “agreement”. Minority opinion – Theran, Stephens
HET-CAM accuracy not being evaluated directly against human data.
Accuracy being evaluated indirectly, by comparison to the in vivo test.
More suitable to use one of the proposed alternative terms to describe the evaluation being conducted.
In addition to the analyses conducted, the Panel suggests an assessment based on ranking of experimental data for severity for both the reference method and the in vitro test

**Accuracy evaluation adequately described; overall false positive rates ranged from 20% to 33% and false negative rates ranged from 0% to 7%.**
**It is essential that chemical classes the test method works well for and poorly for are identified.**
_**Panel recommends replacing the term “accuracy” with “concordance” or “agreement”. Minority opinion – Theran, Stephens**_
**HET-CAM accuracy not being evaluated directly against human data.**
**Accuracy being evaluated indirectly, by comparison to the *****in vivo *****test.**
_**More suitable to use one of the proposed alternative terms to describe the evaluation being conducted.**_
_**In addition to the analyses conducted, the Panel suggests an assessment based on ranking of experimental data for severity for both the reference method and the in vitro test**_

## 6.2	Strengths & Limitations of the Test Method, Including Those Applicable to Specific Chemical Classes or to Certain Physicochemical Properties
Limitations of the analysis are discussed.
Lack of sufficient data for many chemical and by physicochemical properties. 
Differences between methods and analysis used in different studies.
Categorization method differences.
Studies not designed to meet regulatory agency requirements.
Panel notes that there is in vivo data variability.
In cases where false positives and negatives are noted, variability of the in vivo responses should be reviewed.

**Limitations of the analysis are discussed.**
**Lack of sufficient data for many chemical and by physicochemical properties. **
**Differences between methods and analysis used in different studies.**
**Categorization method differences.**
**Studies not designed to meet regulatory agency requirements.**
**Panel notes that there is *****in vivo *****data variability.**
**In cases where false positives and negatives are noted, variability of the *****in vivo *****responses should be reviewed.**

## 6.3	Issues of Data Interpretation
Data interpretation are sufficiently described in the BRD.
BRD highlights IS(B) method as being the best in identifying ocular corrosives and severe irritants.
Panel notes that a standardized test method protocol is needed to produce more consistent data.
Panel recommends development of a table with non-accepted studies (Section 9.0) to evaluate outcomes of these studies using IS(B) analysis.

**Data interpretation are sufficiently described in the BRD.**
**BRD highlights IS(B) method as being the best in identifying ocular corrosives and severe irritants.**
**Panel notes that a standardized test method protocol is needed to produce more consistent data.**
**Panel recommends development of a table with non-accepted studies (Section 9.0) to evaluate outcomes of these studies using IS(B) analysis.**

## BRD Section 7.0:  HET-CAM Test Method Reliability(Repeatability/Reproducibility)A measure of the degree to which a test method can be performed reproducibly within and among laboratories over time.

## 7.1	Selection Rationale for the Substances Used to Evaluate Test Method Reliability
Rationale for substance selection was primarily based on data availability.
Quality of in vivo data is a limitation of all studies used.
Selection of compounds in which in vivo irritancy grades were confirmed by ≥ 2 studies would give more power to the studies.
Limitations noted are of the studies used in the BRD and thus are limitations of the BRD evaluation.

**Rationale for substance selection was primarily based on data availability.**
**Quality of *****in vivo *****data is a limitation of all studies used.**
**Selection of compounds in which *****in vivo *****irritancy grades were confirmed by ≥ 2 studies would give more power to the studies.**
**Limitations noted are of the studies used in the BRD and thus are limitations of the BRD evaluation.**

## 7.2	Analyses & Conclusions Regarding Intralaboratory Repeatability and Intra- & Inter-laboratory Reproducibility
No intralaboratory repeatability and reproducibility evaluation due to lack of data.
Weakness, but should not be a roadblock for use.
Qualitative and quantitative interlaboratory variability was well addressed.
Analysis clearly shows classification agreement occurred frequently.
Use of %CV has limitation when evaluating a narrow range of scores (i.e., 0-21).  Alternative approaches for measuring agreement could be used for evaluating reproducibility.
Panel noted several items for revision in this section.

**No intralaboratory repeatability and reproducibility evaluation due to lack of data.**
**Weakness, but should not be a roadblock for use.**
**Qualitative and quantitative interlaboratory variability was well addressed.**
**Analysis clearly shows classification agreement occurred frequently.**
**Use of %CV **_**has limitation when**__** evaluating a narrow range of scores (i.e., 0-21).  **__**Alternative approaches for measuring agreement could be used for evaluating reproducibility**__**.**_
**Panel noted several items for revision in this section.**

## 7.3	Availability of Historical Negative & Positive Control Data
Absence of historical data is a weakness.
Should not be a roadblock for use in detecting severe irritants and corrosives.
Information from non-accepted studies (Section 9.0) may be sources of control data.
e.g., Gilleron et al. 1996, 1997.
Appropriate recommendations are made in the BRD for positive and negative controls (selection and use in test method).

**Absence of historical data is a weakness.**
**Should not be a roadblock for use in detecting severe irritants and corrosives.**
**Information from non-accepted studies (Section 9.0) may be sources of control data.**
**e.g., Gilleron et al. 1996, 1997.**
**Appropriate recommendations are made in the BRD for positive and negative controls (selection and use in test method).**

## 7.4	Effect of Minor Protocol Changes to Recommended Test Method Protocol and Transferability of Test Method
Effect of protocol changes is unknown without having more standardized studies with measures of variability.

**Effect of protocol changes is unknown without having more standardized studies with measures of variability.**

## BRD Section 8.0:  HET-CAM Test Method Data Quality

## 8.1	Extent of Adherence to GLP Guidelines and Use of Coded Chemicals
Lack of GLP compliance may be overcome by use of coded substances and appropriate data handling	

8.2	Data Quality Audits
Panel agrees with BRD, no quality audits could have been conducted.

8.3	Impact of Deviations from GLP Guidelines
Panel agrees with BRD, conclusions about the impact of deviations from GLP guidelines can not be evaluated.

_**Lack of GLP compliance may be overcome by use of coded substances and appropriate data handling**__**	**_
**8.2****	****Data Quality Audits**
**Panel agrees with BRD, no quality audits could have been conducted.**
**8.3****	****Impact of Deviations from GLP Guidelines**
**Panel agrees with BRD, conclusions about the impact of deviations from GLP guidelines can not be evaluated.**

## 8.4	Availability of Laboratory Notebooks or Other Records for an Independent Audit
The BRD adequately discusses the availability of laboratory notebooks and records for audit.
Availability of notebooks for audit would increase “trust index” of conclusions.

**The BRD adequately discusses the availability of laboratory notebooks and records for audit.**
**Availability of notebooks for audit would increase “trust index” of conclusions.**

## BRD Section 9.0:  Other Scientific Reports and Reviews

## 9.1	Adequacy and Completeness of Relevant Data Identified in Other Published or Unpublished BCOP Studies
Panel agrees that a comprehensive review of available publications were made in BRD.
Panel recommends that additional efforts be made to obtain data for such reports and that criteria for accepting studies for analysis be expanded.

9.2	Adequacy and Completeness of the Conclusions Published in Independent Peer Reviewed Reports or Other Independent Scientific Reviews
Panel agrees that the conclusions of papers were adequate and complete.
Useful to have information on why studies were excluded from the evaluation.
Panel recommends that additional efforts be made to obtain data for such reports and that criteria for accepting studies for analysis be expanded.

**Panel agrees that a comprehensive review of available publications were made in BRD.**
**Panel recommends that additional efforts be made to obtain data for such reports and that criteria for accepting studies for analysis be expanded.**
**9.2****	****Adequacy and Completeness of the Conclusions Published in Independent Peer Reviewed Reports or Other Independent Scientific Reviews**
**Panel agrees that the conclusions of papers were adequate and complete.**
**Useful to have information on why studies were excluded from the evaluation.**
**Panel recommends that additional efforts be made to obtain data for such reports and that criteria for accepting studies for analysis be expanded.**

## 9.3	Approaches that can be Used to Expedite the Process for Obtaining Additional In-House Data from the Private Sector
Panel considers it critically important that additional HET-CAM and in vivo rabbit eye test results be obtained from government and private sources.
Panel recommends that a list of substances of special interest be developed and distributed to e.g., companies in order to request additional data on these substances that support or contradict conclusions made by the Panel.	

**Panel considers it critically important that additional HET-CAM and *****in vivo***** rabbit eye test results be obtained from government and private sources.**
**Panel recommends that a list of substances of special interest be developed and distributed to e.g., companies in order to request additional data on these substances that support or contradict conclusions made by the Panel.****	**

## BRD Section 10.0:  Animal Welfare Considerations (Refinement, Reduction, Replacement)

## 10.1	Extent to Which the Test Method Will Refine, Reduce or Replace Animal Use 
Additional discussion on some issues needed
The Panel recognizes that HET-CAM is an in ovo assay but for purposes of consistency the term in vitro will be used. 
Section should discuss tier testing strategy and that animals would only be used to test negatives. 
HET-CAM should be considered a “partial replacement.”
Additional discussion should state:
Optimization or validation studies should rely on existing rabbit data.
Low false negative rate has the advantage of reducing the exposure of animals to severe irritants and corrosives.
Any additional optimization should focus on decreasing the false negative rate.

**Additional discussion on some issues needed**
_**The Panel recognizes that HET-CAM is an **__***in ovo***__** assay but for purposes of consistency the term in vitro will be used.**__** **_
**Section should discuss tier testing strategy and that animals would only be used to test negatives. **
**HET-CAM should be considered a “partial replacement.”**
**Additional discussion should state:**
**Optimization or validation studies should rely on existing rabbit data.**
**Low false negative rate has the advantage of reducing the exposure of animals to severe irritants and corrosives.**
**Any additional optimization should focus on decreasing the false **_**negative**_** rate.**

## BRD Section 11.0:  Practical Considerations

## 11.1	Adequacy and Completeness of Test 		Method Transferability
The Panel agrees with the BRD that:
Facilities and major equipment should be readily available
Other necessary equipment and supplies are generally available 	
The recommended protocol should be readily transferable
A training video and other visual media on the technical aspects of the assay is recommended (place in all)
Training approaches in the application of this test method should be developed/implemented (place in all)

11.2	Adequacy and Completeness of Test Method Training 
The Panel agrees with the BRD on:
The required level of training needed to conduct the test
Training requirements needed for personnel to demonstrate proficiency  	

**The Panel agrees with the BRD that:**
**Facilities and major equipment should be readily available**
**Other necessary equipment and supplies are generally available ****	**
**The recommended protocol should be readily transferable**
_**A training video and other visual media on the technical aspects of the assay is recommended (place in all)**_
_**Training approaches in the application of this test method should be developed/implemented (place in all)**_
**11.2****	****Adequacy and Completeness of Test Method Training **
**The Panel agrees with the BRD on:**
**The required level of training needed to conduct the test**
**Training requirements needed for personnel to demonstrate proficiency****  ****	**

## 11.3	Adequacy and Completeness of Test 		Method Costs
The Panel recommends that estimated cost information for HET-CAM be included in the BRD

11.4	Adequacy and Completeness of Amount of Time to Conduct Test Method
HET-CAM would significantly reduce the time needed to assess the ability of a test substance to induce ocular corrosivity or severe irritancy relative to the in vivo rabbit eye test (typically at least 3 days and may extend up to 21 days).  However, it is recognized that a corrosive or severe irritant may be detected within a few hours using a single rabbit. 	

**The Panel recommends that estimated cost information for HET-CAM be included in the BRD**
**11.4****	****Adequacy and Completeness of Amount of Time to Conduct Test Method**
**HET-CAM would significantly reduce the time needed to assess the ability of a test substance to induce ocular corrosivity or severe irritancy relative to the *****in vivo***** rabbit eye test (typically at least 3 days and may extend up to 21 days).  **_**However, it is recognized that a corrosive or severe irritant may be detected within a few hours using a single rabbit.**__** **__**	**_

## BRD Section 12.0:  Proposed HET-CAM Test Method Recommendations

## 12.1	Recommended Version of the HET-CAM  Test Method 
The most appropriate version of the HET-CAM test method for use in a tiered-testing strategy has been recommended.
Standardized protocol was adapted from the Spielmann and Liebsch (INVITTOX 1992).
Method requires use of controls and recommends use of IS(B) analysis method.
Consistent use of this version will allow future data to be collected in a standardized manner.
Positive results from HET-CAM can be used to classify a substance as an ocular corrosive or severe irritant; negative results would follow the tier-testing strategy.
Given the relatively high false positive rate, testing of positives could include a second assay (re-testing with a modified HET-CAM assay of a lower concentration, or another screening test with a low false negative rate) to possibly identify HET-CAM false positives.   HET-CAM data exists to evaluate the use of a lower concentration – this needs to be included in the BRD.

**The most appropriate version of the HET-CAM test method for use in a tiered-testing strategy has been recommended.**
**Standardized protocol was adapted from the Spielmann and Liebsch (INVITTOX 1992).**
**Method requires use of controls and recommends use of IS(B) analysis method.**
**Consistent use of this version will allow future data to be collected in a standardized manner.**
**Positive results from HET-CAM can be used to classify a substance as an ocular corrosive or severe irritant; negative results would follow the tier-testing strategy.**
**Given the relatively high false positive rate, testing of positives could include a second assay (re-testing with a modified HET-CAM assay of a lower concentration, or another screening test with a low false negative rate) to possibly identify HET-CAM false positives.   HET-CAM data exists to evaluate the use of a lower concentration – this needs to be included in the BRD.**

## 12.2	Recommended Standardized HET-CAM Test Method Protocol
Panel agrees with recommended test method protocol presented in BRD, except procedures for applying and removing solids from the CAM need to be included.  
Optimization should increase reliability and accuracy.  
Other frequently used endpoints for the assessment of in vitro toxicity might be considered to increase test method accuracy (e.g., trypan blue absorption, antibody staining, histology, membrane changes). 
Such efforts should not block a retrospective validation of the test method with the parameters previously used.	

**Panel agrees with recommended test method protocol presented in BRD, except procedures for applying and removing solids from the CAM need to be included.  **
**Optimization should increase reliability and accuracy.  **
**Other frequently used endpoints for the assessment of *****in vitro***** toxicity might be considered to increase test method accuracy (e.g., trypan blue absorption, antibody staining, histology, membrane changes). **
**Such efforts should not block a retrospective validation of the test method with the parameters previously used.****	**

## 12.3	Recommended HET-CAM Optimization and Validation Studies
Retrospective evaluation of HET-CAM test data should be conducted to optimize the test method for the identification of corrosives and severe irritants (i.e., to reduce the false positive rate).  
Optimization may involve the development of a protocol that includes re-testing of positive substances using a modified HET-CAM protocol. 
Once an optimized HET-CAM test method protocol has been developed, small scale validation studies would be needed to evaluate reliability and accuracy for e.g., solids.
An evaluation to determine the relationship or predictability between the short-term effects observed in the HET-CAM and long-term effects in the rabbits or humans (cite ref for limbal cells). 

**Retrospective evaluation of HET-CAM test data should be conducted to optimize the test method for the identification of corrosives and severe irritants (i.e., to reduce the false positive rate).  **
**Optimization may involve the development of a protocol that includes re-testing of positive substances using a modified HET-CAM protocol. **
**Once an optimized HET-CAM test method protocol has been developed, small scale validation studies would be needed to evaluate reliability and accuracy for e.g., solids.**
_**An evaluation to determine the relationship or predictability between the short-term effects observed in the HET-CAM and long-term effects in the rabbits or humans (cite ref for limbal cells). **_

## 12.3	Recommended HET-CAM Optimization and Validation Studies
Any optimization and validation studies should use existing animal data; if available.
Panel urges that companies with in-house HET-CAM IS(B) and in vivo rabbit eye data submit such data to NICEATM. 
Additional animal studies should only be conducted if important data gaps are identified and such studies should be carefully designed to maximize the amount of pathophysiological information obtained (e.g., wound healing)
Minority opinion –sufficient data should be available so no animal testing for this purpose – Dr. Stephens
Reference substances should be identified that can be used as part of performance standards 

_**Any optimization and validation studies should use existing animal data; if available.**_
**Panel urges that companies with in-house HET-CAM IS(B) and *****in vivo***** rabbit eye data submit such data to NICEATM. **
_**Additional animal studies should only be conducted if important data gaps are identified and such studies should be carefully designed to maximize the amount of pathophysiological information obtained (e.g., wound healing)**_
_**Minority opinion –sufficient data should be available so no animal testing for this purpose – Dr. Stephens**_
_**Reference substances should be identified that can be used as part of performance standards **_