# Presentation: 610738

## Expert Panel Meeting to Assess the Current Validation Status of In Vitro Testing Methods For Identifying Ocular Corrosives and Severe Irritants: The Isolated Rabbit Eye (IRE)Test Method 

**Robert Guest **
**Head of Alternative and Acute Toxicology**
**SafePharm Laboratories Ltd, UK**

***Natcher Conference Center******National Institutes of Health******Bethesda, Maryland USA***
***January 12, 2005***

**Notes:**
Introduction
Name
Position
Company
Background

## Scope of the Presentation

- Test Facility
- Eye Irritancy Testing Strategy
- Scientific basis for the IRE
- Apparatus
- Source of Tissues
- Pre-test Procedures
- Test substance application
- Observations
- Prediction Model
- Conclusion

## SafePharm Laboratories Limited

- UK based CRO
- Established 30 Years
- Derby/Nottingham
- GLP Accredited
- www.safepharm.co.uk

**Notes:**
Safepharm Laboratories Ltd 

## Eye Irritation Testing

- 300 - 400 eye irritation studies per year
- Wide range of test substances
- To satisfy regulatory requirements worldwide
- Occupational safety assessment
- In accordance with official test guidelines (e.g. OECD 405, 2002)
- Tiered Testing Strategy

## Sequential Testing Strategy (OECD, 2002)

**Existing data**

**SAR**

**Physico-chemical**

**Use of valid *****in vitro***
** ****or *****ex vivo***** tests**

**Test in single animal**

**Full Study**

- IRE

- Classify

- Positive

- Negative

## Isolated Rabbit Eye Test (IRE)

- Used ‘in-house’ since 1999
- Screening test for severe eye irritants & corrosives
- ‘Pre-validated’ in-house (14 chemicals)
- Validated on behalf of GSK for worker safety assessment
- (30 chemicals)
- Has minimised exposure of animals to severe irritants

## Scientific Basis for the IRE

- Severely irritant materials have the potential to:
-  - damage the cells and structure of the cornea
-  - cause swelling of the cornea
-  - cause corneal opacity (cloudiness)
- A whole cornea model is therefore relevant
- The IRE involves use of rabbit eyes without causing pain or distress

## Why IRE?

- Utilises target tissue (intact cornea)
- Corneal damage is ‘weighted’in the Draize Test
- Easily accessible source of fresh tissues
- Donor animals previously used for other purposes
- No major health and safety concerns
- Techniques transferable from *in vivo* testing
- Quantifiable endpoint (corneal thickness) supplements qualitative assessments
- Rapid (1 working day)

## IRE Outline of Procedures

- 3 Test and 2 control eyes

- Maintain in superfusion chamber at 32oC

- Examine eyes pre-enucleation and post equilibration

- Dose: 0.1 ml or 100mg

- Exposure: 10 seconds

- Multi-endpoint evaluation of effects

## Multi-Endpoint Evaluation

- Macroscopic & microscopic evaluation of condition of the cornea
- Assessment of corneal opacity (cloudiness)
- Measurement of corneal thickness (swelling)
- Uptake of sodium fluorescein by the cornea
- Histopathology may be performed if required

## Apparatus

- Superfusion apparatus
-  - 11 temperature controlled chambers
-  - 4 temperature probes

- Material: Perspex or polypropylene
- Cost:  ~ £ 1200

## Apparatus

- Portable slit-lamp
- Model SL-5

- Supplier: Kowa
- Cost:  ~ £3000

## Apparatus

- Ultrasonic pachymeter
- Model DGH-1000

- Supplier: DGH Technology, Inc.
- Cost (Model DGH-550):  ~ £4000

## Apparatus

- Temperature-controlled circulating water bath
- Multi-channel Peristaltic Pump
- Diaphragm pump
- Temperature monitors

## Source of Tissues

- Rabbits from local accredited supplier
- Strain: New Zealand White (albino)
- No specified weight or age range. Typically 2.5 – 4.0 kg animals
- May previously have been used for skin irritation tests at Safepharm
- Control eyes from animals that have undergone an eye irritation test

## Pre-test Procedures: Apparatus Set-Up

- Temperature of water bath adjusted to achieve a stable temperature of 32 ± 1.5oC
-  Flow-rate of peristaltic pump adjusted to 0.15 – 0.4 ml/minute

## Pre-test Procedures: In vivo Examination 

- Application of Sodium Fluorescein (1% w/v) to the rabbit eye + rinse
- Slit-lamp examination of the cornea
- Measurement of corneal thickness (t = -1)

- Animals with corneal defects are rejected

## Pre-test Procedures: Corneal Thickness

- Using Ultrasonic pachymeter
- No pain, no distress
- Five positions
- Optical centre
-  3, 6, 9 & 12 o’ clock positions
- Mean corneal thickness calculated
-                                              (n = 5)

- Iris

- Pupil

## Pre-test Procedures: Enucleation of the Eye

- Animals humanely sacrificed by i.v. overdose of sodium pentobarbitol (marginal ear vein)
- 2-3 drops physiological saline (approximately 32oC) applied to the eye to prevent dessication
- Eye removed by careful dissection

## Pre-test Procedures: Mounting of Eye

-  Eye mounted vertically in perspex clamp (Burton, York & Lawrence, 1980)
-  Held in place by adjustable jaws with stainless steel ‘pins’
- Saline drip adjusted if necessary

## Pre-test Procedures:Equilibration

-  30 minute equilibration
-  Eyes re-examined
-  - corneal thickness (t = 0)
-  - slit-lamp examination
-  - Sodium fluorescein 1%

- Eyes showing an increase in corneal thickness of  _>_10%, from t = -1 are rejected

## Method of Test Substance Application

- Eye removed from chamber & placed in petri dish
- Test material applied from a disposable syringe
- Liquids -  0.1 ml
- Solids – volume occupying 0.1 ml
- (or maximum of 100 mg)

## Method of Test Substance Application

- After 10 seconds cornea rinsed with 20 ml of saline (32oC)

## Chemical Range

- Broad range of chemicals and formulations
-  - solids
-  - liquids
-  - pastes
-  - various chemical classes
- Same classes of chemicals as the Draize Test
- If material runs off or adheres to the cornea, this is recorded

## Observations (1, 2, 3 and 4 hours after treatment)

- Macroscopic (visual) assessment of cornea (diffuse illumination)
- Mottling
- Pitting
- Sloughing of epithelium
- Slit-lamp biomicroscopic examination of the condition of the cornea

- Epithelial & more in depth injuries (stroma & endothelium)

## Observations (1, 2, 3 and 4 hours after treatment)

- Corneal opacity (cloudiness)
- Severity & Area
- 0 - 4 scale (Hackett & McDonald, 1991)

## Observations (1, 2, 3 and 4 hours after treatment)

- Severity of opacity (cloudiness)
- Abridged version of the 0-4 scale
- 0 = Normal
- 1 = Some loss of transparency
- 2 = Moderate loss of transparency
- 3 = Involvement of entire thickness of stroma underlying structures barely visible)
- 4 = Involvement of entire thickness of stroma (underlying structures cannot be seen)

## Observations (1, 2, 3 and 4 hours after treatment)

- Area of opacity
- 0  = Normal cornea with no area of cloudiness
- 1  = 1 to 25% area of stromal cloudiness
- 2  = 26 to 50% area of stromal cloudiness
- 3  = 51 to 75% area of stromal cloudiness
- 4  = 76 to 100% area of stromal cloudiness

## Observations (1, 2, 3 and 4 hours after treatment)

- Measurement of corneal thickness (ultrasonic pachymeter)
- Five positions
- Optical centre
- 3, 6, 9 & 12 o’ clock positions
- Mean corneal thickness calculated for each eye at each observation

## Observation (4 hours after treatment)

- Evaluation of sodium fluorescein uptake by the cornea
- Conducted after application of  one drop of sodium fluorescein (1% w/v) to the rabbit eye (with rinse)
- Degree & Area
- 0 - 4 scale (Hackett & McDonald, 1991)

## Observations (4 hours after treatment)

- Severity
- 0 = Absence of fluorescein staining
- 1 = Slight fluorescein staining
- 2 = Moderate fluorescein staining
- 3 = Marked fluorescein staining
- 4 = Extreme fluorescein staining
- Area  - as for corneal opacity

## IRE: Evaluation of Results

- The percentage increase in corneal swelling is calculated for each eye, at each time point (t = 60, 120, 180 & 240  )
- _(Mean thickness, time t) – (mean thickness, time 0)_ x100
- Mean thickness, time 0
- Mean corneal swelling is calculated for the 3 test eyes and 2 control eyes

## Prediction Model

- An assessment of ocular irritancy potential is made in accordance with the following prediction model

| Parameter | Cut-off Value |
| --- | --- |
| Maximum Corneal Opacity (cloudiness x area)  |  3 |
| Maximum Fluorescein Uptake (Intensity x Area) |  4 |
| Mean Corneal Swelling (60, 120, 180, 240 mins) |  25% |
| Corneal Epithelium Observations | Any eye with pitting, mottling or sloughing |

## Prediction Model

- If any of the PM criteria are met, then the test material is regarded as a potential severe eye irritant
- Such materials do not require testing in vivo
- If any of the criteria are met by the control eyes, the test is repeated

## Conclusion (1)

- The IRE has been routinely used at Safepharm since 1999 as a screen for severe ocular  irritants and corrosives
- It is incorporated into an ethical tiered testing strategy
- It uses normal eyes that would otherwise be discarded
- It has resulted in a reduction in the number of animals exposed to severe irritants

## Conclusion (2)

- If used in conjunction with other in vitro tests (e.g. human reconstituted tissue models) it is hoped that  complete replacement of the rabbit eye irritation test may be possible
- Efforts to standardise the test protocol and formally validate the model are encouraged

## Acknowledgements

- Fred Guerriero
- Chris Seaman
- Mike Olson
- Peter Hemsley
- Candace Prusiewicz

- Andy Whittingham
- Neil Warren
- Andy Else
- Gary Henzell

## The Isolated Rabbit Eye (IRE) Test Method

***Thank you**** *

## Toxicology Testing Services

- Local Tolerance (skin, eye)
- Skin Sensitisation
- Photosafety
- Sub-Chronic Toxicity
- Reproductive Toxicity
- Genetic Toxicology
- Ecotoxicology
- Alternative Test Methods

**Toxicology Testing Services**

## Apparatus

- Temperature-controlled circulating water bath

- Supplier: Julabo
- Cost: ~ £1000

## Apparatus

- Multi-channel Peristaltic Pump

- Supplier: Watson-Marlow
- Cost:  ~ £4000

## Apparatus

- Diaphragm pump

- Supplier: KNF Neuberger
- Cost:  ~ £400

## Apparatus

- Temperature monitors

- Supplier: Widely available
- Cost:  ~ £ 100 each

## Test Area

- 20 feet x 20 feet
- (2 systems)

- Total cost of 1 system: £15000

## IRE: Observations

**1, 2, 3 and 4 hours after treatment**
- Macroscopic (visual) assessment of the cornea
- Slit-lamp biomicroscopic examination of the cornea
- Evaluation of corneal cloudiness (severity & area)
- Measurement of corneal thickness (ultrasonic pachymeter)

## IRE: Observations

**Additional Observation: 4 hours after treatment**
- Evaluation of sodium fluorescein uptake by the cornea (severity and area)
- Application of  one drop of sodium fluorescein (1% w/v) to the rabbit eye
- Rinsed with physiological saline at 32oC (10 ml)

## Observations (4 hours after treatment)

- Area of fluorescein staining
- 0  = Normal
- 1  = 1 to 25% area of cornea
- 2  = 26 to 50% area of cornea
- 3  = 51 to 75% area of cornea
- 4  = 76 to 100% area of cornea