# Presentation: 523868

## 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 Rabbit Eye (IRE) Test Method**
**BRD Summary**

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

## Current U.S. Regulatory Status of IRE

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

## Primary IRE Data Sources

| Study
 |  | Accuracy |  |  | Intralab |  | Interlab |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  |  | GHS | EPA | EU | CVs | GHS classific. | CVs | GHS classific. |
| CEC (1991) | S | - | - | 8 | - | - | 21 | - |
|  | NS  | - | - | 13 | - | - |  | - |
|  | Total  | - | - | 21 | - | - |  | - |
| Balls et al. (1995) | S | 22 | 18 | 21 | - | - | 59 | 22 |
|  | NS  | 34 | 34 | 38 | - | - |  | 34 |
|  | Total | 56 | 52 | 59 | - | - |  | 56 |
| Gettings et al. (1996) | S | 16 | 17 | 7 | - | - | - | - |
|  | NS  | 9 | 8 | 18 | - | - | - | - |
|  | Total | 25 | 25 | 25 | - | - | - | - |

**S = severe or corrosive irritants; NS = nonsevere irritants or nonirritants; classific. = classification**** **

## Primary IRE Data Sources

| Study
 |  | Accuracy |  |  | Intralab |  | Interlab |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  |  | GHS | EPA | EU | CVs | GHS classific. | CVs | GHS classific. |
| Guerriero et al. (2004) | S | 12 | 12 | 15 | - | - | - | - |
|  | NS  | 24 | 24 | 29 | - | - | - | - |
|  | Total | 36 | 36 | 44 | - | - | - | - |
| Guerriero et al. (2004)* | S | 12 | 12 | 12 | - | - | - | - |
|  | NS  | 24 | 24 | 24 | - | - | - | - |
|  | Total | 36 | 36 | 36 | - | - | - | - |

***Performance calculated excluding test substances with pH extreme or skin corrosivity data as was done for EPA and GHS analyses **
**S = severe or corrosive irritants; NS = nonsevere irritants or nonirritants; classific. = classification**** **

## Other IRE Reports Considered

**23 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 **
**quantitative *****in vitro *****data**
**These reports discussed in Section 9**
**No additional data obtained****   **

## IRE Database

**149 different substances evaluated in 4 tests**
**124 Chemicals  **
**  ****25 Products or formulations**
**15 Chemical classes tested***
**Most frequent classes**
**heterocyclics/aromatics**
**acetates/esters**
**formulations **
**14 Product classes tested***
**Most frequent classes: **
**Industrial chemicals**
**chemical intermediates**
**soaps/surfactants**

*** Classes with at least 3 entries**

## Major IRE Protocol Variations

| Study1 | n | # Eyes |  |  | Exp. Dur. | CO | CS | FP | Epithel.
Integ.  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  |  | Neg  | Treat | Pos |  |  |  |  |  |
| 1991 | 21 | 1 | 3 | - | 10 s | X | X | X | - |
| 1995 | 59 | 1 | 3 | - | 10  s | X | X | - | - |
| 1996 | 25 | 1 | 3 | - | 10 s | X | X | - | - |
| 2004 | 44 | 1 | 3 | - | 10 s | X2 | X | X2 | X |

**1**** CEC (1991), Balls et al. (1995), Gettings et al. (1966), Guerriero et al. (2004)**
** ****2 ****Area and Intensity**

## IRE 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 IRE 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 IRE Version Accuracy

| Statistic | GHS (n=36)* |  | EPA (n=36)* |  | EU (n=44)1 |  |
| --- | --- | --- | --- | --- | --- | --- |
|  | % | n | % | n | % | n |
| Accuracy | 78 | 28/36 | 78 | 28/36 | 77 | 34/44 |
| Sensitivity | 100 | 12/12 | 100 | 12/12 | 100 | 15/15 |
| Specificity | 67 | 16/24 | 67 | 16/24 | 66 | 19/29 |
| False Positive Rate | 33 | 8/24 | 33 | 8/24 | 34 | 10/29 |
| False Negative Rate | 0 | 0/12 | 0 | 0/12 | 0 | 0/15 |

***Guerriero et al. (2004)**
**1****Additional 8 chemicals available for EU analysis only**

## IRE GHS Accuracy By Chemical/Physical Class*

| Class | # of Substances |  |  | False Negative Rate |  | False Positive Rate |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
|  | Total | Cat 1 | Cat 2A, 2B, NI | % | n | % | n |
| OVERALL | 36 | 12 | 24 | 0 | 0/12 | 33 | 8/24 |
| Amine | 5 | 2 | 3 | 0 | 0/2 | 33 | 1/3 |
| Heterocyclic | 7 | 1 | 6 | 0 | 0/1 | 33 | 2/6 |
| Amide | 6 | 2 | 4 | 0 | 0/2 | 25 | 1/4 |
| Sulfur-containing | 4 | 1 | 3 | 0 | 0/1 | 0 | 0/3 |
| Acetate | 2 | 0 | 2 | - | - | 100 | 2/2 |
| Ketone | 2 | 0 | 2 | - | - | 100 | 2/2 |
| Acid; Alcohol | 2 | 0 | 2 | - | - | 100 | 2/2 |
| Aldehyde; Inorganic; Hydrocarbon;  surfactant | 4 | 1 | 3 | 0 | 0/1 | 0 | 0/3 |
| Solids | 28 | 12 | 16 | 0 | 0/12 | 13 | 2/16 |
| Liquids | 12 | 4 | 8 | 0 | 0/4 | 75 | 6/8 |

*** Guerriero et al. (2004)**

## Limitations of IRE Accuracy Analysis*

**The small number of substances in each chemical class allow for limited conclusions with respect to the accuracy of IRE by chemical class or physicochemical property.  However, it appears that:**
**Liquids tend to be overpredicted**
**Acetates, alcohols, and ketones tend to be overpredicted across all studies, and these limited data appear to support this trend. **
***Guerriero et al. (2004)**

## IRE Reliability

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

## IRE Classification Agreement Among “n” Laboratories

| % Interlaboratory Agreement | GHS (4 labs, 59 substances)* |  | EPA (4 labs, 59 substances)* |  | EU (4 labs, 59 substances)* |  | EU (3 labs, 21 substances)** |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  | % | n | % | n | % | n | % | n |
| 100% (all) | 59 | 35/59 | 59 | 35/59 | 61 | 36/59 | 81 | 17/21 |
| 75% (all) | 85 | 50/59 | 85 | 50/59 | 85 | 50/59 | 95 | 20/21 |
| 100% (severes) | 100 | 14/14 | 100 | 13/13 | 100 | 14/14 | 83 | 5/6 |
| 75% (severes) | 100 | 14/14 | 100 | 13/13 | 100 | 14/14 | 100 | 6/6 |

***Balls et al. (1995)**
****CEC (1991)**

## IRE Interlaboratory %CV Values (Balls et al. 1995)

|  | %CV | CO (1 HR) | CO (4 HR) | CS (1 HR) | CS (4 HR) |
| --- | --- | --- | --- | --- | --- |
| Total 
(59 Substances) | Mean | 84.1 | 63.8 | 56.2 | 53.5 |
|  | Median | 74.6 | 43.4 | 50.8 | 49.7 |
|  | Range  | 0-200 | 0-200 | 11-129 | 10-118 |
| GHS Category 1
(22 Substances) | Mean | 46.6 | 40.5 | 37.6 | 36.9 |
|  | Median | 40.6 | 33.6 | 36.0 | 35.5 |
|  | Range  | 0-200 | 0-200 | 11-118 | 11-118 |

**CO: Corneal opacity; CS: Corneal swelling; %CV: Coefficient of variation, expressed as a percentage; CV: standard deviation/mean**
**Interlaboratory %CV values based on results from four laboratories**

## IRE Interlaboratory %CV Values (CEC 1991)

|  | %CV | CS 
(1.25 HR) | CS 
(4 HR) | FR
(4 HR) | CO
(4 HR) |
| --- | --- | --- | --- | --- | --- |
| Total 
(21 Substances) | Mean | 53.3 | 57.3 | 58.9 | 37.7 |
|  | Median | 43.0 | 40.0 | 28.0 | 24.0 |
|  | Range  | 6.7-148 | 7.2-173 | 0-175 | 0-141 |
| GHS Category 1
(8 Substances) | Mean | 36.6 | 35.4 | 22.1 | 15.5 |
|  | Median | 30.5 | 35.5 | 21.0 | 15.4 |
|  | Range  | 19-63 | 20-61 | 0-78 | 0-40 |

***CEC (1991)**
**CO: Corneal opacity; CS: Corneal swelling; %CV: Coefficient of variation, expressed as a percentage;  CV = standard deviation/mean; FR: Fluorescein retention**
**Interlaboratory %CV values based on results from four laboratories**

## Limitations of IRE Reliability Analysis

**Intralaboratory reliability unknown due to lack of published data**
**Interlaboratory reproducibility based on a small number of substances (n=21)**

## Draft IRE BRD Proposals

**A recommended IRE version identified, which evaluates**
**corneal opacity and area, corneal swelling, fluorescein penetration, and epithelial integrity**
**A standardized protocol proposed for the recommended version of the IRE test method**
**Protocol based on the method used by SafePharm Laboratories (UK)**
**Only significant difference is inclusion of additional concurrent,controls (negative, solvent, positive, and, when appropriate, benchmark) from validated reference list**
**Additional optimization studies recommended, including:**
**Retrospective analysis of decision criteria used to identify corrosives and severe irritants**
**Once optimized, additional validation studies recommended to further characterize accuracy and reliability of the optimized method **