# Presentation: 066391

## 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**

**Current Ocular Toxicity Regulatory Testing Procedures**
**Debbie McCall, U.S. EPA**
**January 11, 2005**

**ICCVAM**

**NICEATM**

## Statutes and Regulations Requiring Ocular Corrosivity / Irritation Testing

**Statutes and Regulations Requiring Ocular Corrosivity / Irritation Testing**

| Agency | Authority | Regulation | Guideline |
| --- | --- | --- | --- |
| EPA | FIFRA (1947) TSCA (1977) | 40CFR | OPPTS 870.2400 |
| CPSC | FHSA (1964) | 16CFR1500 | 16CFR1500.42 |
| FDA | FDCA (1938) | 21CFR | 16CFR1500.42 |
| OSHA | OHSA (1970) | ?? | 16CFR1500.42 |
| EU | Council Directive 67/548/EEC | Commission Directive 2004/73/EC | Annex V B.5 |
| OECD | - | - | Test Guideline 405 |

**CFR: Code of the Federal Register; CPSC: Consumer Product Safety Commission; EPA: Environmental Protection Agency; EU: European Union; FDA: Food and Drug Administration FDCA: Food, Drug and Cosmetic Act; FHSA: Federal Hazardous Substances Act; FIFRA: Federal Insecticide, Fungicide, and Rodenticide Act; OECD: Organisation for Economic Co-ordination and Development; OPPTS: EPA, Office of Prevention, Pesticides, and Toxic Substances;  OSHA: Occupational Safety and Health Administration; TSCA: Toxic Substances Control Act  **

## The In Vivo Rabbit Test

**The *****In Vivo***** Rabbit Test**

**Healthy adult albino rabbits (e.g., White New Zealand) **
**0.1 mL or 0.1 g  instilled into the conjunctival sac of 1 eye**
**The untreated eye serves as a control**
**Observation for at least 3 days, and may extend up to 21 days to evaluate for reversibility/irreversibility of effects**
**Some regulatory authorities permit the use of a single animal to screen for corrosive**** ****effects.  **
**If severe effect is seen, no further testing.**
**If no severe effects, up to 2 additional animals tested to confirm results.**
**Additional animals may be necessary to confirm weak or equivocal responses. **

## Relevant Testing Guidelines 

| Test Method Component | EPA* | EU* | FHSA* | OECD* |
| --- | --- | --- | --- | --- |
| Number of Animals | n = 1 to screen for corrosive, then n ≥ 2 | n = 1 to screen for corrosive, then n ≥ 2 | ≥ 6 | n = 1 to screen for corrosive, then n ≥ 2 |
| Quantity | 0.1 mL or 0.1 g | 0.1 mL or 0.1 g | 0.1 mL or 0.1 g | 0.1 mL or 0.1 g |
| Observation Times | 1-72 hr (up to 21 days) | 1-72 hr (up to 21 days) | 1-3 days (up to 7 days) | 1-72 hr (up to 21 days) |
| Post-dosing Irrigation | 24 hr** | Liquids: 24 hr
Solids: 1 hr | 24 hr | Liquids: 24 hr
Solids: 1 hr |
| Use of Anesthetics | May be used prior to dosing if pain anticipated | May be used prior to dosing if pain anticipated | May be used prior to dosing if pain anticipated | May be used prior to dosing if pain anticipated |

***EPA: TG OPPTS 870.2400 (1998); EU: Annex V B.05 (2004); FHSA: 16CFR1500.42 (2003);      OECD: TG 405 (2002)**
****For substances shown to be irritating by this test, additional testing using animals with eyes washed 30 seconds after instillation may be indicated**

## Rabbit Eye Test Method Scoring (I)

**Eyes are subjectively evaluated using the Draize method for three endpoints:**
**Corneal opacity**
**Iris effects**
**Conjunctival effects**

## Rabbit Eye Test Method Scoring (II)

**Cornea**
**Degree of opacity**
**1 = Scattered or diffuse area – details of iris visible**
**2 = Easily discernible translucent areas – details of iris slightly obscured**
**3 = Opalescent areas, no details of iris visible, size of pupil barely discernable**
**4 = Opaque – iris not visible**
**Area of cornea involved***
**1 = One quarter (or less) but not zero**
**2 = Greater than one quarter, but less than one half**
**3 = Greater than one half, but less than three quarters**
**4 = Greater than three quarters, up to whole area**

***Not used for regulatory hazard classification.**

## Rabbit Eye Test Method Scoring (III)

**Iris**
**1 = Folds above normal, congestion, swelling, circumcorneal injection (any one or all of there, or combination of any thereof), iris still reacting to light**
**2 = No reaction to light, hemorrhage, gross destruction (any one or all of these)**

## Rabbit Eye Test Method Scoring (IV)

**Conjunctiva**
**Redness**
**1 = Vessels definitely injected above normal**
**2 = More diffuse, deeper crimson red, individual vessels not easily discernable**
**3 = Diffuse, beefy red**
**Chemosis**
**1 = Any swelling above normal (includes nictitating membrane)**
**2 = Obvious swelling with partial eversion of the lids**
**3 = Swelling with lids about half closed**
**4 = Swelling with lids half to completely closed**
**Discharge***
**1 = Any amount different from normal **
**2 = Discharge with moistening of lids and hairs adjacent to the lids**
**3 = Discharge with moistening of lids and considerable area around the eye**

***Not used for regulatory hazard classification.**

## Test Guidelines: Summary

**All four test guidelines are based on the original method of Draize et al. (1944)**
**FSHA requires the greatest number of animals in an initial test (n = 6)**
**EPA, EU, and OECD recommend up to 3 animals in an initial test (with the possibility of only one animal classifying a corrosive substance).**
**All four test guidelines permit the use of anesthetics.**
**Not recommended for routine use, only when pain is anticipated**
**EPA, EU, and OECD requires studies to be carried out to 21 days to evaluate reversible/irreversible effects, while FHSA only requires observations out to 3 days.**
**All four test guidelines allow irrigation of eyes after 24 hr**
**EU and OECD allows for irrigation at 1 hr for solid substances**

## 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**

**Overview of Ocular Hazard Regulatory Testing Requirements and Classification Schemes**
**Debbie McCall, U.S. EPA**
**January 11, 2005**

**ICCVAM**

**NICEATM**

## Background

**Background**

**Ocular toxicity classification definitions and criteria vary among regulatory hazard classification systems (EPA, EU, GHS, FHSA)**
**All current ocular toxicity classification systems are based on the Draize rabbit eye test method (Draize et al. 1944) and scoring system**

## Classification Systems

## EPA Classification System (1996)

**At least 3 animals per test (one-animal screen permitted)**
**Maximum score in any animal used for classification**
**Positive: CO or IR ≥ 1 or CC or CR ≥ 2**
**Most severe response used for classification of substance**

| EPA Category | In Vivo Effect |
| --- | --- |
| I | Corrosive; (irreversible) 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 |
| IV | Minimal effects clearing within 24 hr. |

**CC: Conjunctival Chemosis; CO: Corneal Opacity; CR: Conjunctival Redness; IR: Iritis**

## EPA Labeling

| Cat. | Signal Word | Statements | Protective Equipment/Actions |
| --- | --- | --- | --- |
| I | DANGER | Corrosive.  Causes irreversible eye damage.  Do not get in eyes or on clothing.   | Wear protective [eyewear goggles, face shield, or safety glasses]. Wash thoroughly with soap and water after handling and before eating, drinking, chewing gum, or using tobacco.  Remove and wash contaminated clothing prior to reuse. |
| II | WARNING | Causes substantial but temporary eye injury.  Do not get in eyes or on clothing | Same as Category I  above |
| III | CAUTION | Causes moderate eye irritation.  Avoid contact with eyes or clothing. | Wear protective [eyewear goggles, face shield, or safety glasses]. Wash thoroughly with soap and water after handling and before eating, drinking, chewing gum, or using tobacco. |
| IV | (CAUTION optional) | None required | None required, but may chose cat. III |

## EU Classification System (2001)

**At least 3 animals per test (one-animal screen for corrosive effect permitted)**
**Two Possibilities for Classification**
**If > 3 animals, mean study values (each endpoint averaged over days 1-3 for all animals) used**
**If 3 animals, individual animal mean values (each endpoint averaged over days 1-3) used**

| EU Category | # Animals | In Vivo Effect |
| --- | --- | --- |
| R41 | > 3 | Mean* study value where CO ≥ 3 or IR ≥ 1.5  |
| R41 | 3 | ≥ 2 animals with individual means* of CO ≥ 3 or IR ≥ 2 OR ≥ 1 animal with CO or CC ≥ 2, CR ≥ 2.5, or IR ≥ 1 at end of observation (usually Day 21) |
| R36 | > 3 | Mean* study value where 2 ≤ CO < 3 or 1 ≤ IR < 1.5 or CR ≥ 2.5 or CC ≥ 2 |
| R36 | 3 | ≥ 2animals with individual means* of 2 ≤ CO < 3 or 1 ≤ IR < 2 or CR ≥ 2.5 or CC ≥ 2 |

***Mean calculated over days 1-3                                                                                           CC: Conjunctival Chemosis; CO: Corneal Opacity; CR: Conjunctival Redness; IR: Iritis**

## GHS Classification System (2003)

**Is test method neutral**
**Classification based on severity of effect and reversibility of the effect**

| Category | In Vivo Effect |
| --- | --- |
| 1 | ≥ 1 animal with CO = 4 at any time OR ≥ 2 animals with mean* CO ≥ 3 or IR ≥ 1.5 OR ≥1 animal at day 21 with CO or IR ≥ 1 or CC or CR≥ 2 |
| 2A | ≥ 2 animals with mean* CO or IR ≥ 1 or CC or CR≥ 2 which reverses within 21 days. |
| 2B | ≥ 2 animals with mean* CO or IR ≥ 1 or CC or CR≥ 2 which reverses within 7 days. |

***Mean values calculated over days 1-3                                                                              CC: Conjunctival Chemosis; CO: Corneal Opacity; CR: Conjunctival Redness; IR: Iritis **

## GHS Labeling

| Category  | Symbol | Signal Word | Label Statement |
| --- | --- | --- | --- |
| 1 | Corrosive symbol | Danger | Causes severe eye damage |
| 2A | Exclamation mark | Warning | Causes severe eye irritation |
| 2B | No symbol used | Warning | Causes eye irritation |

**!**

## FHSA Classification System (1995)

**FHSA Classification System (1995)**

**At least 6 animals per test**
**Corrosive: ≥ 1 animal with destruction or irreversible alterations at the site of contact**
**For irritants, maximum score in any animal on any day used for classification**
**Positive: CO or IR ≥ 1 or CC or CR ≥ 2**
**Testing may be carried out in multiple tiers (6 animals/tier)**
**Tier 1:**
**≥ ****4 positive animals = Irritant**
**2-3 positive animals = Go to Tier 2**
**1 positive animal = negative**
**Tier 2:**
**≥ ****3 positive animals = Irritant**
**1-2 positive animals = Go to Tier 3**
**0 = negative**
**Tier 3:**
**1 positive animal = Irritant**

## Classification Systems: Summary

**Classification Systems: Summary**

**EPA, EU, and GHS allow for classification of corrosive based on a one-animal screen.  If the initial animal indicates corrosivity, no additional testing is required.  ****	**
**Classification according to EPA and FHSA is based on the most severe lesion in any animal and on any day.**
**Classification according to EU and GHS takes into account the most severe mean scores over days 1-3, in addition to persistent lesions. **
**All four systems have only 1 classification for ocular**** ****corrosives/severe irritants.****  **
**However, different numbers of classifications for nonsevere irritants**
**EPA: n = 3 (Category II, III, or IV)**
**EU: n = 1 (R36)**
**FHSA: n = 1 (Irritant)**
**GHS: n = 2 (Category 2A or 2B)**