2,4,6-trichloroaniline

Eye irritation

Administrative data

Endpoint:

eye irritation

Remarks:

in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-12-11 until 2013-12-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2400 (Acute Eye Irritation)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: INVITTOX (1994) Protocol 80: Chicken Enucleated Eye Test

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2,4,6-trichloroaniline

EC Number:

211-219-8

EC Name:

2,4,6-trichloroaniline

Cas Number:

634-93-5

Molecular formula:

C6H4Cl3N

IUPAC Name:

2,4,6-trichloroaniline

Test material form:

solid: crystalline

Details on test material:

- Physical state: Off white crystals
- Expiration date of the lot/batch: Recertification date end November 2015
- Storage condition of test material: Room temperature (<30°C)

Test animals / tissue source

Species:

other: Isolated chicken eyes

Strain:

other: Not applicable

Details on test animals or tissues and environmental conditions:

ISOLATED CHICKEN'S EYES
- Source: Heads from chickens used for human consumption supplied by a commercial abattoir.
- Preparation: Eyelids were carefully cut away with scissors, avoiding damaging the cornea. One small drop of fluorescein solution 2 % (w/v) was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL isotonic saline. The fluorescein-treated cornea was examined with a hand-held slit lamp or slit lamp microscope, with the eye still in the head, to ensure that the cornea was not damaged. If the cornea was in good condition, the eyeball was carefully removed from the orbit by holding the nictitating membrane with surgical forceps, while cutting the eye muscles with bent scissors. Care was taken to remove the eyeball from the orbit without cutting off the optical nerve too short. The procedure avoided pressure on the eye while removing the eyeball from the orbit, in order to prevent distortion of the cornea and subsequent corneal opacity. Once removed from the orbit, the eye was placed onto damp paper and the nictitating membrane was cut away with other connective tissue. The prepared eyes were kept on the wet papers in a closed box so that the appropriate humidity was maintained.

The prepared eye was placed in a steel retainer with the cornea in the correct relative position. The clamp with the eyeball was transferred to a chamber of the superfusion apparatus. The retainer holding the eye was positioned in such a way that the entire cornea was supplied with isotonic saline dripping from a stainless steel tube, at a rate of approximately 3-5 drops/minute.

The appropriate number of eyes (9-10) was selected, after being placed in the superfusion apparatus they were examined again with the slit lamp microscope to ensure that they were in good condition. The focus was adjusted to clearly see the isotonic saline which was flowing on the cornea surface. Eyes with a high baseline fluorescein staining (i.e. > 0.5) or corneal opacity score (i.e. > 0.5) were rejected. The cornea thickness was measured with an optical pachymeter on a slit-lamp microscope. Any eye with cornea thickness deviating by more than 10 % from the mean value for all eyes, or eyes that showed any other signs of damage were rejected and replaced. If the selected eyes were appropriate for the test, acclimatization was started and conducted for approximately 45 to 60 minutes. The chambers of the superfusion apparatus were at controlled temperature (32±1.5°C) during the acclimatization and treatment periods.

EXPERIMENTAL DATES: 11 to 18 December 2013:

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 mg

Duration of treatment / exposure:

10 seconds

Observation period (in vivo):

Pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse.

Number of animals or in vitro replicates:

3 test, 3 positive control, 1 negative control isolated eyes

Details on study design:

- BASE LINE ASSESSMENT: A zero reference measurement was recorded for cornea thickness and opacity to serve as a base line (t=0) for each individual eye.
- TREATMENT: The eye in its retainer was taken out of the chamber and placed on a layer of tissue with the cornea facing upwards. The eye was held in a horizontal position, while the test item was applied onto the centre of the cornea. The test item was applied in an amount of 30 mg onto the entire surface of the cornea. The positive control eyes were treated in a similar way with 30 mg powdered imidazole and the negative control was treated with 30 µL sodium chloride solution.
- TEST ITEM REMOVAL: 10 seconds after application, the cornea surface was rinsed thoroughly with 20 mL isotonic saline at ambient temperature. The eye was returned to the chamber after rinsing. (The imidazole (in all eyes) was stuck on the corneas’ surface after the post-treatment rinse. Gentle rinsing with 20 mL saline was performed at each observation time point. The cornea surfaces treated were not cleared 240 minutes after the post-treatment rinse)..
- OBSERVATION / ASSESSMENT: Corneal thickness and corneal opacity were measured at all time points. Fluorescein retention was measured on two occasions, at base line (t=0) and approximately 30 minutes after the post-treatment rinse using a slit-lamp microscope.

Endpoints evaluated were corneal opacity, swelling and fluorescein retention. Other observations which indicate damage, such as loss of epithelium were taken into account in making a classification.

Results from corneal swelling, opacity and fluorescein retention were evaluated separately to generate an Isolated Chicken Eye (ICE) class for each endpoint. The ICE classes for each endpoint were then combined to generate an Irritancy Classification for each test substance

Effects were divided into 4 categories (I = none, II = slight, III = moderate, IV = severe).

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

other: Mean maximum corneal swelling (Run 1)

Basis:

mean

Time point:

other: 75 min

Score:

0

Remarks on result:

other: ICE Class I

Results 2

Irritation parameter:

other: Mean maximum corneal swelling (Run 1)

Basis:

mean

Time point:

other: 240 min

Score:

0

Remarks on result:

other: ICE Class I

Results 3

Irritation parameter:

other: Mean maximum corneal opacity change (Run 1)

Score:

0

Remarks on result:

other: ICE Class I

Results 4

Irritation parameter:

other: Mean fluorescein retention change (Run 1)

Basis:

mean

Score:

0.17

Remarks on result:

other: ICE Class I

Results 5

Irritation parameter:

other: Mean maximum corneal swelling (Run 2)

Basis:

mean

Time point:

other: 75 min

Score:

0

Remarks on result:

other: ICE Class I

Results 6

Irritation parameter:

other: Mean maximum corneal swelling (Run 2)

Basis:

mean

Time point:

other: 240 min

Score:

0

Remarks on result:

other: ICE Class I

Results 7

Irritation parameter:

other: Mean maximum corneal opacity change (Run 2)

Basis:

mean

Score:

0

Remarks on result:

other: ICE Class I

Results 8

Irritation parameter:

other: Mean fluorescein retention change (Run 2)

Basis:

mean

Score:

0

Remarks on result:

other: ICE Class I

Irritant / corrosive response data:

No corneal swelling or corneal opacity was observed during the four hour observation period. Fluorescein retention (severity 0.5) was noted only in one eye in the first run. No other corneal effect was observed. The negative and positive control group results demonstrate that the study was valid.

Any other information on results incl. tables

Table 1: Eye irritation scores in the in vitro eye irritation test in isolated chicken eyes – Run 1

Observation	Test item		Positive control		Negative control
	Value	ICE class	Value	ICE class	Value	ICE class
Mean maximum corneal swelling at up to 75 min	0%	I	4%	I	0%	I
Mean maximum corneal swelling at up to 240 min	0%	I	7%	II	0%	I
Mean maximum corneal opacity change	0	I	3.83	IV	0	I
Mean fluorescein retention change	0.17	I	2.83	IV	0	I
Other Observations	None		The imidazole was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces were not cleared 240 minutes after the post-treatment rinse.		None
Overall ICE Class	3 x I		1 x II 2 x IV		3 x I

 

Table 2: Eye irritation scores in the in vitro eye irritation test in isolated chicken eyes – Run 2

Observation	Test item		Positive control		Negative control
	Value	ICE class	Value	ICE class	Value	ICE class
Mean maximum corneal swelling at up to 75 min	0%	I	2%	I	0%	I
Mean maximum corneal swelling at up to 240 min	0%	I	5.3%	II	0%	I
Mean maximum corneal opacity change	0	I	3.83	IV	0	I
Mean fluorescein retention change	0	I	2.83	IV	0	I
Other Observations	None		The imidazole was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces were not cleared 240 minutes after the post-treatment rinse.		None
Overall ICE Class	3 x I		1 x II 2 x IV		3 x I

 

Applicant's summary and conclusion

Interpretation of results:

not classified

Remarks:

Migrated information Criteria used for interpretation of results: other: UN GHS

Conclusions:

Based on this in vitro eye irritation study in isolated chicken eyes, the test item is non-irritating, GHS Classification: Non-classified.

Executive summary:

An in vitro eye irritation study was performed in isolated chicken’s eyes. After the zero reference measurements, the eye was held in a horizontal position and 30 mg of the test item was applied onto the centre of the cornea such that the entire surface of the cornea was covered. After 10 seconds, the surface was rinsed with saline. The positive control eyes were treated in a similar way with 30 mg imidazole. The negative control eye was treated with 30 μL of 0.9% sodium chloride. Corneal thickness, corneal opacity and fluorescein retention were measured and any morphological effects (pitting or loosening of the epithelium) evaluated.

In line with the most recent OECD guideline, the negative effect was confirmed in a repeat run of 3 eyes, such that the guideline allows the test item to be classified as negative, without the need for an in vivo confirmatory study.

No corneal swelling or corneal opacity was observed during the four hour observation period. Fluorescein retention (severity 0.5) was noted only in one eye in the first run. No other corneal effect was observed. The negative and positive control group results demonstrate that the study was valid.

Based on this in vitro eye irritation study in isolated chicken eyes, the test item is non-irritating, GHS Classification: Non-classified.