# Presentation: 003371

## NICEATM			ICCVAM
National Toxicology Program  Interagency 	Interagency Coordinating Committee Center for the Evaluation Of Alternative 	on the Validation of Alternative 	 Toxicological Methods 			Methods

**NICEATM****	****	****	****ICCVAM**
**National Toxicology Program  Interagency ****	****Interagency Coordinating Committee Center for the Evaluation Of Alternative ****	****on the Validation of Alternative ****	**** Toxicological Methods ****	****	****	****Methods**
**	****	****	****	**

**The Isolated Chicken Eye (ICE) Test Method**
**BRD Summary**

**Expert Panel Meeting**
**January 11-12, 2005**
**Bethesda, Maryland **

## Current U.S. Regulatory Status of ICE

**ICCVAM agencies were surveyed and, to the best of their knowledge, ICE test method data have not been submitted to U.S. Regulatory Agencies.**

## Primary ICE Data Sources

| Study
 |  | Accuracy |  |  | Intralab |  | Interlab |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  |  | GHS | EPA | EU | CVs | GHS classific. | CVs | GHS classific. |
| Prinsen and Koëter (1993) | S | 3 | 3 | 8 | - | - | - | - |
|  | NS  | 7 | 7 | 13 | - | - | - | - |
|  | Total  | 10 | 10 | 21 | - | - | - | - |
| Balls et al. (1995) | S | 22 | 20 | 21 | - | - | 59 | 22 |
|  | NS  | 34 | 34 | 38 | - | - |  | 34 |
|  | Total | 56 | 54 | 59 | - | - |  | 56 |
| Prinsen (1996) | S | 0 | 0 | 6 | - | - | - | - |
|  | NS  | 29 | 29 | 38 | - | - | - | - |
|  | Total | 29 | 29 | 44 | - | - | - | - |

**S: severe or corrosive irritants; NS: nonsevere irritants or nonirritants;    classific.: classification; CV: coefficient of variation**

## Other ICE Reports Considered

**Two other reports were identified that could not be used for an evaluation of accuracy or reliability due to the lack of:**
**comparative *****in vivo *****rabbit test***** *****data**
**incomplete substance identification **
**quantitative *****in vitro *****data**
**These reports discussed in Section 9**
**No additional data were obtained****   **

## ICE Database

**121 Different substances evaluated in three tests**
**15 Chemical classes tested***
**Most frequent classes: **
**alcohols **
**acids**
**surfactants**
**14 Product classes tested***
**Most frequent classes: **
**chemical/pharmaceutical intermediates**
**herbicides/pesticides**
**industrial chemicals**
**soaps/surfactants/detergents**

*** Classes with at least 3 entries**

## Major ICE Protocol Variations

| Study | n | # Eyes |  |  | Exp. Dur. | CO | CS | FR | Morph. Eval.  | Histopath. |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  |  | Neg  | Treat | Pos |  |  |  |  |  |  |
| Prinsen and Koëter (1993) | 21 | 1 | 5 | - | 10 sec | X | X | X | X | Case-by-case basis |
| Balls et al. (1995) | 59 | 1 | 3 | - | 10 sec | X | X | X | X | Not specified |
| Prinsen (1996) | 44 | 1 | 3 | - | 10 sec | X | X | X | X | Not specified |

**CO: Corneal opacity; CS: Corneal swelling; Exp. Dur.: Exposure duration; FR: Fluorescein retention; Histopath.: Histopathology; Morph. Eval.: Morphological evaluation;  n: Number of substances tested; Neg: negative control; Pos: positive control;  Treat: test substance treated**** **

## ICE Accuracy

**Ability to correctly identify ocular corrosives and severe irritants determined for**
**GHS classification system (Category 1)**
**EPA classification system (Category I)**
**EU classification system (R41)**
**Accuracy statistics calculated:**
**for each ICE test method protocol by report and where appropriate **
**classifications were pooled into one classification per substance (i.e., majority call among studies used)**
**using individual studies, where a balanced design existed (multiple substances in multiple labs)**

## Recommended ICE Version Accuracy

| Statistic | GHS (n=92)* |  | EPA (n=90)* |  | EU (n=121)* |  |
| --- | --- | --- | --- | --- | --- | --- |
|  | % | n | % | n | % | n |
| Accuracy | 82 | 75/92 | 82 | 74/90 | 85 | 103/121 |
| Sensitivity | 60 | 15/25 | 61 | 14/23 | 70 | 26/37 |
| Specificity | 90 | 60/67 | 90 | 60/67 | 92 | 77/84 |
| False Positive Rate | 10 | 7/67 | 10 | 7/67 | 8 | 7/84 |
| False Negative Rate | 40 | 10/25 | 39 | 9/23 | 30 | 11/37 |

***from Prinsen and Koëter (1993), Balls et al. (1995), and Prinsen (1996); Additional chemicals available for EU analysis only (individual animal data not available for GHS or EU classification)**

## ICE GHS Accuracy By Chemical/Physical  Class (1)

| Class | # of Substances |  |  | False Positive Rate |  | False Negative Rate |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
|  | Total | Cat 1 | Cat 2A, 2B, NI | % | n | % | n |
| OVERALL | 92 | 25 | 67 | 10 | 7/67 | 40 | 10/25 |
| Surfactant | 13 | 7 | 6 | 0 | 0/6 | 57 | 4/7 |
| Alcohol | 10 | 0 | 10 | 50 | 5/10 | - | - |
| Acid | 7 | 5 | 2 | 0 | 0/2 | 20 | 1/5 |
| Acetate | 6 | 0 | 6 | 17 | 1/6 | - | - |
| Heterocyclic | 6 | 5 | 1 | 0 | 0/1 | 40 | 2/5 |
| Hydrocarbon | 6 | 2 | 4 | 0 | 0/4 | 50 | 1/2 |

**Prinsen and Koëter (1993), Balls et al. (1995), and Prinsen (1996)**

## ICE GHS Accuracy By Chemical/Physical Class (2)

| Class | # of Substances |  |  | False Positive Rate |  | False Negative Rate |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
|  | Total | Cat 1 | Cat 2A, 2B, NI | % | n | % | n |
| Inorganic | 3 | 1 | 2 | 0 | 0/2 | 100 | 1/1 |
| Ketone | 3 | 0 | 3 | 33 | 1/3 | - | - |
| Amine | 2 | 1 | 1 | 0 | 0/1 | 0 | 0/1 |
| Acyl halide; Lactone; Aldehyde; Amide; Organometallic; Organophoshpate | 1 | 0 | 1 | 0 | 0/1 | - | - |
| Alkali | 1 | 1 | 0 | - | - | 0 | 0/1 |
| Diol | 1 | 1 | 0 | - | - | 100 | 1/1 |
| Solids | 23 | 11 | 12 | 0 | 0/12 | 55 | 6/11 |
| Liquids | 69 | 14 | 55 | 13 | 7/55 | 29 | 4/14 |

**Prinsen and Koëter (1993), Balls et al. (1995), and Prinsen (1996****)**

## Limitations of ICE Accuracy Analysis

**Lack of individual rabbit data for all substances prevents an accuracy evaluation using the GHS and EPA classification schemes.**
**The small number of substances in each chemical class allow for limited conclusions with respect to the accuracy of ICE by chemical class or physicochemical property.  However, it appears that:**
**Alcohols tend to be overpredicted **
**Surfactants and solids tend to be underpredicted**

## ICE Reliability

**Intralaboratory Repeatability and Reproducibility **
**Not conducted due to the lack of published intralaboratory ICE data**
**Interlaboratory Reproducibility**
**Qualitative analysis: Extent of agreement between testing laboratories when identifying ocular corrosives and severe irritants**
**Quantitative analysis: Coefficient of variation **

## ICE Classification Agreement Among Four Laboratories

| % Interlaboratory Agreement | GHS (59 substances)* |  | EPA (59 substances)* |  | EU (59 substances)* |  |
| --- | --- | --- | --- | --- | --- | --- |
|  | % | n | % | n | % | n |
| 100% (all) | 75 | 44/59 | 75 | 44/59 | 76 | 45/59 |
| 75% (all) | 90 | 53/59 | 90 | 53/59 | 90 | 53/59 |
| 100% (severes)** | 72 | 16/22 | 75 | 15/20 | 71 | 15/21 |
| 75% (severes)** | 95 | 21/22 | 100 | 20/20 | 95 | 20/21 |

***Balls et al. (1995)**
****Scores for fluorescein retention and corneal swelling were not provided**
**    ****for one severe irritant/corrosive (30% trichloroacetic acid), which was **
**    ****therefore classified based on results from only 3 laboratories**

## ICE Interlaboratory %CV Values*

|  |  | %CV |  |  |
| --- | --- | --- | --- | --- |
|  |  | FR | CO | CS |
| Total 
(59 Substances) | Mean | 38.8 | 46.8 | 77.2 |
|  | Median | 35.6 | 37.1 | 74.5 |
|  | Range  | 0-158.7 | 0-158.7 | 30.8-159.4 |
| GHS Category 1
(22 Substances) | Mean | 29.9 | 34.2 | 72.4 |
|  | Median | 23.0 | 25.0 | 69.5 |
|  | Range  | 0-158.7 | 0-118.6 | 32.2-132.2 |

***Balls et al. (1995)**
**CO: Corneal opacity; CS: Corneal swelling; CV: standard deviation/mean; %CV: Coefficient of variation, expressed as a percentage; FR: Fluorescein retention**
**Interlaboratory %CV values based on results from four laboratories**

## Limitations of ICE Reliability Analysis

**Intralaboratory reliability unknown due to lack of published data**
**Interlaboratory reproducibility based on only one study (4 laboratories, 59 substances)**

## Draft ICE BRD Proposals (1) 

**A proposed ICE version, which evaluates corneal opacity, corneal swelling, fluorescein retention, and morphological effects**
**A proposed standardized protocol**
**Protocol based on method of TNO Nutrition and Food Research (INVITTOX 2004)**
**Only significant difference is inclusion of additional eyes for negative controls, a concurrent positive control, and, when appropriate, benchmark controls **** **

## Draft ICE BRD Proposals (2) 

**Proposed additional optimization studies, including:**
**Retrospective analysis of decision criteria used to identify corrosives and severe irritants**
**An evaluation of the potential causes of the greater level of interlaboratory variability for the corneal swelling endpoint**
**Additional evaluation of possible increased interlaboratory variability for specific chemical classes appearing more variable, based on the small numbers of representative substances in this evaluation (i.e., alcohols, acetates/esters, cationic surfactants)**
**Determining the feasibility of introducing quantitative measurement for corneal opacity**
**Determining the utility of histopathology and when it should be included.**
**Once optimized, additional validation studies to further characterize accuracy and reliability of the optimized method **