T-A06

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 August 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Deviations:

yes

Remarks:

2 hrs 20 minutes start of enucleation-selection instead of 2 hours. As the positive and negative control conformed within expectation, this deviation is considered to not impact the result.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

ethyl (2E)-3-(cyclopropylamino)-2-[(2,4-dichloro-3-methylphenyl)carbonyl]prop-2-enoate

Molecular formula:

C16H17Cl2N1O3

IUPAC Name:

ethyl (2E)-3-(cyclopropylamino)-2-[(2,4-dichloro-3-methylphenyl)carbonyl]prop-2-enoate

Test material form:

solid: particulate/powder

Test animals / tissue source

Species:

chicken

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: slaughterhouse (Etablissement Brun, 33820
Etauliers, France) where they are killed for human consumption
- Number of animals: not specified
- Characteristics of donor animals (e.g. age, sex, weight): approx 7 weeks old, 1.5 - 2.5 kg
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Heads have been removed immediately after sedation of the chickens by electric shock, and incision of the neck for bleeding. The heads have been collected on 31 August 2020 at 8:15 am. Because eyes were dissected in the laboratory, the intact heads were transported from the slaughterhouse at ambient temperature in plastic boxes humidified with towels moistened with physiological saline. The eyes were enucleated at Laboratoire ICARE –Site de Martillac on 31 August 2020 at 9:50 am.
- Time interval prior to initiating testing: 2 hours 20 minutes
- Indication of any existing defects or lesions in ocular tissue samples: no
- Indication of any antibiotics used: not reported
- Selection and preparation of corneas:The eyelids were carefully excised, taking care not to damage the cornea. Then, the eye was further dissected from the skull, taking care not to damage the cornea. The eyeball was pulled from the orbit by holding the nictitating membrane firmly with surgical forceps, and the eye muscles were cut with a bent, blunt-tipped scissor. When the eye is removed from the orbit, a visible portion of the optic nerve should be left attached. Once removed from the orbit, the eye was placed on an absorbent pad and the nictitating membrane and other connective tissue were cut away. The enucleated eye was mounted in a stainless-steel clamp with the cornea positioned vertically. The clamp was then transferred to a chamber of the superfusion apparatus. The clamps were positioned in the superfusion apparatus such that the entire cornea was supplied with the physiological saline drip (in the range 0.1 to 0.15 mL/min). The chambers of the superfusion apparatus was temperature controlled at 32.0°C.
- Quality check of the isolated corneas: the eyes were examined with a slit-lamp microscope
to ensure that they have not been damaged during the dissection procedure. Corneal thickness was also measured at this time at the corneal apex using the depth measuring device on the slit-lamp microscope. Eyes with; (i), a fluorescein retention score of > 0.5; (ii) corneal opacity > 0.5; or, (iii), any additional signs of damage were replaced. For eyes that are not rejected based on any of these criteria, individual eyes with a corneal thickness deviating more than 10% from the mean value for all eyes are to be rejected

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

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

Duration of treatment / exposure:

10 seconds

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
The eyelids were carefully excised, taking care not to damage the cornea. Then, the eye was further
dissected from the skull, taking care not to damage the cornea. The eyeball was pulled from the orbit
by holding the nictitating membrane firmly with surgical forceps, and the eye muscles were cut with a
bent, blunt-tipped scissor. When the eye is removed from the orbit, a visible portion of the optic nerve
should be left attached. Once removed from the orbit, the eye was placed on an absorbent pad and the
nictitating membrane and other connective tissue were cut away.
The enucleated eye was mounted in a stainless-steel clamp with the cornea positioned vertically. The
clamp was then transferred to a chamber of the superfusion apparatus. The clamps were positioned in the superfusion apparatus such that the entire cornea was supplied with the physiological saline drip (in the range 0.1 to 0.15 mL/min). The chambers of the superfusion apparatus was temperature controlled at 32.0°C. After being placed in the superfusion apparatus, the eyes were examined with a slit-lamp microscope to ensure that they have not been damaged during the dissection procedure. Corneal thickness was also measured at this time at the corneal apex using the depth measuring device on the slit-lamp microscope. Eyes with; (i), a fluorescein retention score of > 0.5; (ii) corneal opacity > 0.5; or, (iii), any additional signs of damage were replaced. For eyes that are not rejected based on any of these criteria, individual eyes with a corneal thickness deviating more than 10% from the mean value for all eyes are to be rejected.


EQUILIBRATION AND BASELINE RECORDINGS
Once all eyes had been examined and approved (see table in appendix 4), the eyes were incubated between 45 and 61 minutes to equilibrate them to the test system prior to dosing. Following the equilibration period, a zero-reference measurement was recorded for corneal thickness and opacity to serve as a baseline (i.e., time = 0). The fluorescein score determined at dissection was used as the baseline measurement for that endpoint.

NUMBER OF REPLICATES
3

NEGATIVE CONTROL USED
physiological saline – Dutscher Batch No. 3014011

POSITIVE CONTROL USED
sodium hydroxide – Fisher Scientific, Batch No. 1550248

APPLICATION DOSE AND EXPOSURE TIME
30mg for 10 seconds

OBSERVATION PERIOD
30, 75, 120, 180 and 240 minutes

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: rinsed twice with 10ml of physiological saline at ambient temperature
- Indicate any deviation from test procedure in the Guideline: The start of the enucleation-selection was carried out 2 hours and 20 minutes after the collection of the heads instead of 2 hours at the maximum, as initially scheduled.As the results obtained with the eyes treated with the negative control and the positive control were conformed to what was expected, this deviation is considered as without impact on the results of the study.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Corneal opacity was calculated by using the area of the cornea that was most densely opacified for scoring. The mean corneal opacity value for all test eyes was calculated for all observation time points.Based on the highest mean score for corneal opacity, as observed at any time point, an overall category score was then given for each test or control item.
- Damage to epithelium based on fluorescein retention: The mean fluorescein retention value for all test eyes was calculated for the 30-minute observation time point only, which was used for the overall category score given for each test or control item
- Swelling: measured with optical pachymeter on a slit-lamp microscope; slit-width setting: Corneal swelling was determined from corneal thickness measurements made with an optical
pachymeter on a slit-lamp microscope. It was expressed as a percentage and was calculated from corneal thickness measurements according to the following formula: [(corneal thickness at time t - corneal thickness at time = 0)/corneal thickness at time = 0] x 100
The mean percentage of corneal swelling for all test eyes was calculated for all observation time
points. Based on the highest mean score for corneal swelling, as observed at any time point, an overall category score was then given for each test item.
- Macroscopic morphological damage to the surface: Morphological effects include “pitting” of corneal epithelial cells, “loosening” of epithelium, “roughening” of the corneal surface and “sticking” of the test item to the cornea. These findings can vary in severity and may occur simultaneously. The classification of these findings is subjective according to the interpretation of the investigator.
- Others (e.g, histopathology):

SCORING SYSTEM:
- Mean corneal swelling [(corneal thickness at time t - corneal thickness at time = 0)/corneal thickness at time = 0] x 100
The mean percentage of corneal swelling for all test eyes was calculated for all observation time
points. Based on the highest mean score for corneal swelling, as observed at any time point, an overall category score was then given for each test item.

- Mean maximum opacity score
See table 1
- Mean fluorescein retention score at 30 minutes post-treatment
See table 2


DECISION CRITERIA: Decision criteria was used as indicated in the TG

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

cornea opacity score

Run / experiment:

Maximal mean

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class 1

Results 2

Irritation parameter:

fluorescein retention score

Run / experiment:

mean score

Value:

0.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE class I

Results 3

Irritation parameter:

percent corneal swelling

Run / experiment:

maximal mean

Value:

3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE class I

Results 4

Irritation parameter:

morphological effects

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system:no

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes, historical lab data records

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, the combination of the three endpoints for the negative control, physiological saline, was 3 x I. Therefore, the negative control is classified as “No Category”, as expected.
- Acceptance criteria met for positive control: Yes, the combination of the three endpoints for the positive control, Sodium hydroxide, was 3 x IV. Therefore, the positive control is classified as “Corrosive/Severe Irritant”, as expected.

Any other information on results incl. tables

Table 7

				Time (min)
Endpoint measured	Eye No.	0	30	75	120	180	240
Corneal opacity	13	0.0	0.0	0.0                       0.0		0.0	0.0
ICE class				I
Fluorescein retention	13	0.5	0.5	-                            -		-	-
ICE class			I	-                            -		-	-
Corneal thickness	13	0.50	0.50	0.50                    0.50		0.50	0.50
Corneal swelling (%)	13	-	0	0                           0		0	0
ICE class				I
Combination of the 3 Endpoints				3 x I
CLASSIFICATION				No category

Table 8

			Time (min)
Endpoint measured	Eye No.	0	30                   75		120                 180	240
Corneal opacity	1 2 3	0 0 0	4                     4                      4                     4            4                     4                      4                     4            4                     4                      4                     4			4 4 4
Mean		0.0	4.0                  4.0                  4.0                  4.0			4.0
ICE class			IV
Fluorescein retention	1 2 3	0.5 0.5 0.5	3                      -                      -                      -            3                      -                      -                      -            3                      -                      -                      -			- - -
Mean		0.5	3.0	-                       -                      -		-
ICE class			IV                     -                      -                      -			-
Corneal thickness	1 2 3	0.47 0.47 0.46	-                                  -           -              - -                                  -           -              - -                                  -           -              -			- - -
Corneal swelling (%)	1 2 3	( - ) ( - ) ( - )	( - )                 ( - )                 ( - )                 ( - )          ( - )                 ( - )                 ( - )                 ( - )          ( - )                 ( - )                 ( - )                 ( - )			( - ) ( - ) ( - )
Mean		-	-                       -                      -                      -			-
ICE class			IV
Combination of the 3 Endpoints			3 x IV
CLASSIFICATION			category 1 : "Corrosive/severe irritant"

Table 9

				Time (min)
Endpoint measured	Eye No.	0	30	75	120	180	240
Corneal opacity	7 8 9	0 0 0	0 0 0	0                        0             0                        0             0                        0		0 0 0	0 0 0
Mean		0.0	0.0	0.0                     0.0		0.0	0.0
ICE class				I
Fluorescein retention	7 8 9	0.5 0.5 0.5	0.5 0.5 0.5	-                                      - -                                      - -                                      -		- - -	- - -
Mean		0.5	0.5	-                         -		-	-
ICE class			I	-                         -		-	-
Corneal thickness	7 8 9	0.50 0.46 0.49	0.50 0.48 0.50	0.50               0.50 0.48      0.49           0.50                   0.50		0.50 0.49 0.50	0.50 0.49 0.50
Corneal swelling (%)	7 8 9	- - -	0 4 2	0                        0             4                        7             2                        2		0 7 2	0 7 2
Mean		-	2	2                        3		3	3
ICE class				I
Combination of the 3 Endpoints				3 x I
CLASSIFICATION				No category

 

Applicant's summary and conclusion

Interpretation of results:

other: Not classified (CLP Regulation EC no. 1272/2008)

Conclusions:

The test item was determined to not cause severe damage or irrritation for eyes in the ICE test.

Executive summary:

An in vitro (ex vivo) study was conducted in order to determine the potential severe eye damaging effectsof the test item according to the OECD guideline 438 under GLP conditions. Eyeballs were isolated from chickens killed for human consumption and after the appropriate preparation were exposed to either 30 mg of the test item, 30 mg of sodium hidroxide (positive control) or 30μL of physiological saline (negative control). Three eyeballs were used in test item and positive groups, and one for the negative control group. Fluorescein retention, corneal opacity and corneal swelling were evaluated, then the results of each endpoint were assigned to ICE classes according to OECD guideline 438. According to CLP Regulation EC no. 1272/2008 the test item does not require classification for eye irritation and serious eye damage as defined by the UN GHS (No category), since the combination of the 3 endpoints for the test item was 3x I. The test item was determined to not cause severe damage or irrritation for eyes in the ICE test.