Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 MAY 2011
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Although there is no guideline for this type of in-vitro eye irritation study, the rabbit enucleated eye test is used as a first stage in the assessment of ocular irritancy potential. The preferred species of choice is the rabbit. The assay has undergone inter-laboratory validation and has been shown to reliably detect test items that are negligible, or moderate to severe ocular irritants. This study was conducted in in compliance with UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by SI 2004/0994)).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011

Materials and methods

Principles of method if other than guideline:
The rabbit enucleated eye test is used (in-house), as a first stage in the assessment of ocular irritancy potential. The preferred species of choice is the rabbit. The assay has undergone inter-laboratory validation and has been shown to reliably detect test items that are negligible, or moderate to severe ocular irritants.
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Identification : 3-trifluoromethylaniline
Description : extremely pale yellow liquid
Batch number : 101009
Purity : not supplied
Date received : 25 October 2010
Storage conditions : room temperature in the dark

Test animals / tissue source

Species:
rabbit
Strain:
other: The strain of rabbit eye used in this in vitro study was not reported

Test system

Vehicle:
unchanged (no vehicle)
Controls:
other: Control eyes were used in the study
Details on study design:
METHODS
Pre-Test Procedures
Superfusion Chamber
The water heating circulator (Julabo MP5, Jencons (Scientific) Ltd., Leighton Buzzard,
Beds, UK), was adjusted so that the temperature of the water flowing through the water
jacket of the superfusion apparatus, gave a stable temperature, of 32 ±1.5°C, within the
chambers of the apparatus. A peristaltic pump (205S/BA, Watson Marlow Ltd, Falmouth,
Cornwall; UK) was used to supply saline solution at a flow rate of 0.15 to 0.4 ml/minute
(at approximately 30°C) into the rear of each chamb er of the apparatus in order to
irrigate the surface of the cornea.

Selection of Eyes
Prior to enucleation, the eyes of the provisionally selected rabbits were examined for
evidence of ocular irritation or defect, following application of Fluorescein Sodium drops
BP (1% w/v). Examination was aided with the Kowa SL-5 slit-lamp biomicroscope
(Keeler Ltd, Windsor, Berks; UK). Corneal thickness values were also recorded using
the DGH-55 Ultrasonic pachymeter (DGH Technology Inc, Solana Beach, CA). Only
animals whose eyes showed no evidence of ocular irritation or defect were used for
testing purposes (Appendix 1).

Enucleation of Eyes
The donor rabbits were sacrificed by intravenous administration of an overdose of
sodium pentobarbitone. Immediately afterwards, two to three drops of saline solution
(approximately 32°C) were applied to the cornea to prevent desiccation during excision.
The eye was then carefully removed, positioned in a perspex clamp and placed within
the chamber of the superfusion apparatus, with the saline drip at the rear of the chamber
adjusted so that saline solution was allowed to irrigate the surface of the cornea. The
eyes were then allowed to equilibrate for approximately thirty minutes. Following the
equilibration period, the eyes were re-examined to ensure they had not been damaged
during excision. Corneal thickness was also measured using the ultrasonic pachymeter.
Any eyes in which the corneal swelling was greater than 10% relative to the preenucleation
measurement, or in which the cornea was stained with fluorescein, were
rejected. The post equilibration corneal thickness values for each eye were recorded
(Appendix 1).

PROCEDURE
Test Item Administration
Three eyes were treated with test item, two additional eyes remained untreated for
control purposes. The treatment eye was removed from the superfusion apparatus
whilst still being held in the perspex clamp. The clamp/eye was then placed horizontally
into a petri dish.
The test item was used undiluted as supplied. A volume of 0.1 ml of the test item was
applied as evenly as possible to the surface of the cornea. After ten seconds the test
item was washed off the cornea using a minimum of 20 ml of saline solution
(approximately 32°C).
Immediately following washing of the corneal surface, the treated eye was returned to
the superfusion chamber and the saline drip repositioned to irrigate the eye.
The untreated eyes were similarly washed and used for control purposes.

Observations
Assessment of corneal cloudiness was made pre-enucleation, post equilibration and
approximately 60, 120, 180 and 240 minutes following treatment, according to the
numerical evaluation given in Appendix 2 adopted from Advances in Modern Toxicology:
Dermatoxicology, 4th Ed, (F Marzulli and H Maibach, eds) Hemisphere Publishing
Corporation, Washington DC, 1991, pp 749-815.
Examination of the eye was facilitated by use of a slit-lamp biomicroscope. The
thickness of the cornea was measured using an ultrasonic pachymeter. For each
enucleated eye a measurement was made at the optical centre, and at a further four
locations at the apex of the cornea. A mean value for corneal thickness was then
calculated. Measurements for corneal thickness were carried out pre-enucleation, post
equilibration and approximately 60, 120, 180 and 240 minutes following treatment.
The condition of the corneal epithelium was assessed approximately 60, 120, 180 and
240 minutes following treatment. Assessment was facilitated by the use of the slit-lamp
biomicroscope.
The uptake of fluorescein by the corneal epithelium was assessed pre-enucleation, post
equilibration and approximately 240 minutes following treatment, according to the
numerical evaluation given in Appendix 2. This was carried out using the cobalt blue
filter of the slit-lamp biomicroscope, following application of Fluorescein Sodium drops.

Results and discussion

In vivo

Irritant / corrosive response data:
Raw data provided in attached background material.

Any other information on results incl. tables

Corneal Opacity

Individual scores for corneal opacity are given in Table 1 of the raw data provided in the attached background material.

Some loss of transparency was noted in one test eye and moderate loss of transparency

was noted in two test eyes at the 60-Minute observation with moderate loss of

transparency noted in all test eyes at the 120, 180 and 240 minute observations. No

corneal effects were noted in the control eyes during the study period.

Corneal Thickness

Individual and mean corneal thickness measurements and corneal swelling calculations

are given in Table 2 and Table 3 of the raw data provided in the attached background

material.

Corneal swelling of the test eyes during the study period was considerably greater than

that observed in the control eyes over the same period.

Corneal Condition

The condition of the corneal epithelium following treatment is given in Table 4 of

the raw data provided in the attached background material.

Sloughing of the corneal epithelium was noted in all test eyes. The condition of the

corneal epithelium of the control eyes appeared normal during the study period.

Fluorescein Uptake

Individual scores for fluorescein uptake are given in Table 5 of the raw data provided

in the attached background material.

Fluorescein uptake was noted in the test eyes 240 minutes following test item

application. No fluorescein uptake was noted in the control eyes 240 minutes following

treatment.

Applicant's summary and conclusion

Interpretation of results:
Category 1 (irreversible effects on the eye)
Remarks:
Migrated information Based on itro screening data available. Criteria used for interpretation of results: EU
Conclusions:
Following assessment of the data for all endpoints, the test item was considered to have the potential to cause severe ocular irritancy in vivo.