α,α,α-trifluoro-m-toluidine

Eye irritation

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:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

This study has been undertaken in line with the ICCVAM - Recommended Test Method Protocol isolated Rabbit Eye Test method with some deviations identified. The study does not include key information such as the sex and strain of the rabbit used, there is no use of a positive or negative control, additionally the cut off value for maximum corneal opacity (cloudiness x area) is greater than that outlined in the ICCVAM protocol. ICCVAM has indicated that this IRE test method protocol is for non-regulatory use it does allow for a first stage assessment of the ocular irritancy potential of the test substance and to substantiate the overall irritant potential of the test chemical. The study is performed under GLP conditions in accordance with directive 2004/9/EC.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

other: ICCVAM - Test method protocol Isolated Rabbit Eye Test method

Version / remarks:

ICCVAM-Recommended Test Method Protocol Isolated Rabbit Eye Test Method
Originally published as Appendix B5 of “ICCVAM Test Method Evaluation Report: Current Validation Status of In Vitro Test Methods Proposed for Identifying Eye Injury Hazard Potential of Chemicals and Products”
NIH Publication No. 10-7553 – Published 2010
Available at: http://iccvam.niehs.nih.gov/methods/ocutox/MildMod-TMER.htm

Deviations:

yes

Remarks:

no use of a positive/negative control, source of enucleated eyes differ, cut off value for maximum corneal opacity (cloudiness x area) is greater than that outlined.

Principles of method if other than guideline:

- Principle of test: The purpose of the protocol is to provide details of the essential procedures required to (1) insure induction of corneal irritancy in the enucleated eye of the rabbit by a potentially irritating test substance, (2) evaluate the degree of irritancy, and (3) enable assignment of an appropriate regulatory classification on the potential ocular irritancy of a test substance. ocular corrosion or severe irritation.
- Short description of test conditions: the donor rabbits were sacrificed by intravenous administration of an overdose of sodium pentobarbitone. Immediately afterwards, two/three doses of saline solution was applied to the cornea to prevent desiccation during excision. The eye was 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 to allow for irrigation of the surface of the cornea. The eyes were allowed to equilibrate for approximately 30 minutes where they were re-examined to ensure they had not been damaged during excision. Corneal thickness was measured with an ultrasonic pachymeter, with any eyes where the corneal swelling was greater than 10% relative to the pre-enucleation measurement were rejected. Three eyes were treated with the test item, with two additional eyes remaining untreated for control purposes. The test item was used undiluted as supplied. A volume of 0.1 ml of the test item was applied evenly over the surface of the cornea , after 10 seconds the test item was washed off the cornea using a minimum of 20ml saline solution. Immediately after washing of the corneal surface, the treated eye was returned to the superfusion chamber and the saline drop repositioned to irrigate the eye. The untreated eyes were similarly washed and used for control purposes.
- Parameters analysed / observed: Toxic effects in the isolated rabbit eye are measured by (1) subjective assessment of changes in corneal opacity, (2) uptake of fluorescein dye within the cornea (permeability), (3) increased corneal thickness (swelling), and (4) corneal epithelial changes (pitting, sloughing, mottling, etc.) which are evaluated macroscopically or by slit- lamp. The opacity, swelling, and permeability assessments following exposure to a test substance are assessed individually and are used to determine if the test substance has the potential to induce

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

3-trifluoromethylaniline

IUPAC Name:

3-trifluoromethylaniline

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

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Batch number: 101009
- Purity, including information on contaminants, isomers: Not stated


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored at room temperature in the dark
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage:N/A
- Stability in the medium, i.e. sensitivity of the test material to hydrolysis and/or photolysis: Not stated
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: Not stated
- Reactivity of the test material with the incubation material used (e.g. plastic ware): Not stated

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): NA
- Preliminary purification step (if any): N/A
- Final concentration of a dissolved solid, stock liquid or gel: N/A
- Final preparation of a solid (e.g. stock crystals ground to fine powder using a mortar and pestle): N/A

Test animals / tissue source

Species:

rabbit

Strain:

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

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Not stated
- Number of animals: Three
- Characteristics of donor animals (e.g. age, sex, weight):Not stated
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): N/A
- Time interval prior to initiating testing: Not stated, the eyes were allowed to equilibrate for approximately 30 minutes following enucleation
- Indication of any existing defects or lesions in ocular tissue samples: No defects were noted
- Indication of any antibiotics used: Not stated
- Selection and preparation of corneas: Prior to enucleation, the eyes of provisionally selected rabbits were examined for evidence of occular irritation or defects following the 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 recored 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
- Quality check of the isolated corneas: Corneal thickness was measured using the ultrasonic pachymeter, with any eyes with swelling greater than 10% relative to the pre-enucleation measurement rejected.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

other: Control eyes were used in the study

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 0.1 ml
- Concentration (if solution): test material is applied neat

VEHICLE
- Amount(s) applied (volume or weight with unit): NA
- Concentration (if solution): NA
- Lot/batch no. (if required): NA
- Purity: NA

Duration of treatment / exposure:

10 seconds

Duration of post- treatment incubation (in vitro):

60, 120, 180 and 240 minutes

Number of animals or in vitro replicates:

Three replicate enucleated eyes were used

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 vitro

Results

Results 1

Irritation parameter:

cornea opacity score

Run / experiment:

Maximum score obtained in 3 eyes

Value:

8

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

not examined

Remarks on result:

positive indication of irritation

Results 2

Irritation parameter:

other: fluoroscein uptake

Run / experiment:

Maximum score obtained in 3 eyes

Value:

8

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

not examined

Remarks on result:

positive indication of irritation

Results 3

Irritation parameter:

percent corneal swelling

Run / experiment:

Mean swelling calculated for 3 eyes - 4 hours following treatment

Value:

115.1

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

not examined

Remarks on result:

positive indication of irritation

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:

other: The test item was considered to have the potential to cause severe ocular irritancy in vivo

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.

Executive summary:

The study was performed under GLP conditions in accordance with the ICCVAM - Test method protocol isolated rabbit eye (IRE) test method, with deviations noted.

The deviations noted inlcude key information such as the sex and strain of the rabbit used, there is no use of a positive or negative control and additionally the cut of value for maximum corneal opacity (cloudiness x area) is greater than that outlined in the ICCVAM protocol.

ICCVAM have indicated that the use of IRE test method protocol is not for use in regulatory testing straregies but is used as a first stage assessment of the ocular irritancy potential of a test substance and to substantiate the overall irritant potential of a test chemical. The EURL ECVAM have indicated that the rabbit eye enuclation test is not a validated test method to determine the endpoint of eye irritation but the results can be used as a weight of evidence in the classification of the substance. 

Application of the test substance to the enucleated eye resulted in all parameters; Corneal Opacity, Fluorscein and Corneal Swelling (%) exceeding the REET cut off values. It was therefore concluded that the test substance has the potential to cause severe ocular irritancy in vivo.