Ethyl ethyl(phenyl)carbamate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

November 7, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

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:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Ethyl ethyl(phenyl)carbamate

EC Number:

213-796-1

EC Name:

Ethyl ethyl(phenyl)carbamate

Cas Number:

1013-75-8

Molecular formula:

C11H15NO2

IUPAC Name:

ethyl ethyl(phenyl)carbamate

Test material form:

liquid

Details on test material:

Colorless

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:room temperature
- Stability under test conditions:yes

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)
- Characteristics of donor animals (e.g. age, sex, weight): 7 weeks old, 1.5-2.5 kg.
Animals were killed for human consumption.

- Storage, temperature and transport conditions of ocular tissue:
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 07 November 2016 at 8:20 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 Phycher on 07 November 2016 at 9:45 am.
- Time interval prior to initiating testing: November 7,2016 (8:20-9:45)
- indication of any existing defects or lesions in ocular tissue samples: NO
- Indication of any antibiotics used: NO

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): 30 μL of the test item was applied, as supplied.

Duration of treatment / exposure:

10 seconds

Duration of post- treatment incubation (in vitro):

240 minutes (± 5 minutes)

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 between 32.3°C and 32.6°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 using sodium fluorescein. 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 the eyes were incubated between 45 and 65 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 30 μL (one eye)

SOLVENT CONTROL USED: not applicable

POSITIVE CONTROL USED: 5% Benzalkonium chloride 30 μL (three eyes)

APPLICATION DOSE AND EXPOSURE TIME: The test item was applied for 10 seconds

OBSERVATION PERIOD: Pretreatment and starting at 30, 75, 120, 180, and 240 minutes (± 5 minutes) after the post-treatment rinse.


REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period:20 mL of physiological saline at ambient temperature
- Indicate any deviation from test procedure in the Guideline: No

METHODS FOR MEASURED ENDPOINTS:
- CORNEAL OPACITY: It 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

- SWELLING: measured with Haag-Streit BP900 slit-lamp microscope with depth-measuring device no. I and slit-width setting at 9½, equaling 0.095 mm: 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 gtime=0 )/ corneal thicnkness at time=0 )*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.

- 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.

- Macroscopic morphological damage to the surface: pitting of corneal epithelial cells, loosening of epithelium, rougheningof the corneal surface and sticking of the test item to the cornea. This findings can vary in severity and may occur simultaneously.

SCORING SYSTEM:
- Mean maximum opacity score:
0 - No opacity,
0.5 -Very faint opacity
1- Scattered or diffuse areas; details of the iris clearly visible
2- Easily discernible translucent area; details of the ris are slightly obscured,
3-Severe corneal opacity; no specific details of the iris are visible; size of the pupil is barely discernible
4-Complete corneal opacity; iris invisible

- Mean fluorescein retention score at 30 minutes post-treatment :
0-No fluorescein retention,
0.5-Very minor single cell staining,
1-Single cell staining scattered throughout the treated area of the cornea,
2-Focal or confluent dense single cell staining,
3-Confluent large areas of the cornea retaining fluorescein

DECISION CRITERIA:YES as indicated in the TG was used.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

cornea opacity score

Run / experiment:

MEAN

Value:

ca. 0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE class I

Results 2

Irritation parameter:

fluorescein retention score

Run / experiment:

MEAN

Value:

ca. 1.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE class III

Results 3

Irritation parameter:

percent corneal swelling

Run / experiment:

MEAN

Value:

ca. 0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE class I

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No, no morphological effects were noted, whatever the examination time.

Three separated tests were conducted with this substance (performed on 05 January 2015, 02 March 2015 and 15 Apri l 2015), and all tests lead to the conclusion "No prediction can be made". This result is considered as not critical since all test items that come out "No prediction can be made" would be subsequently tested with other adequately validated in vitro test(s), or as a last option in rabbits, depending on regulatory requirements, using a sequential testing strategy.

DEMONSTRATION OF TECHNICAL PROFICIENCY: YES

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, 3xI. The negative control is classified as “No Category”.
- Acceptance criteria met for positive control: Yes, 3xIV,The positive control is classified as “Corrosive/Severe Irritant.

In vivo

Other effects:

- Lesions and clinical observations: NO
- Ophthalmoscopic findings:NO
- Effects of rinsing or washing: NO
- Other observations: NO

Any other information on results incl. tables

Table No.1: Selected eyes for the performance of the ICE test

Acceptability criteria ensuring eyes quality:

Eyes with (i) a fluorescein retention score > 0.5; (ii) corneal opacity > 0.5; or, (iii) any additional signs

of damage were replaced.

For eyes that were 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 were rejected.

Chamber	Fluorescein retention	Corneal opacity	Morphological effects	Corneal thickness (e)
1	0.5	0	N.t.R.	0.54
2	0.5	0	N.t.R.	0.60
3	0.5	0	N.t.R.	0.56
4	0.5	0	N.t.R.	0.57
5	0.5	0	N.t.R.	0.54
6	0.5	0	N.t.R.	0.57
7	0.5	0	N.t.R.	0.54
8	0.5	0	N.t.R.	0.62
9	0.5	0	N.t.R.	0.60
10	0.5	0	N.t.R.	0.61
11	0.5	0	N.t.R.	0.62
12	0.5	0	N.t.R.	0.60
13	0.5	0	N.t.R.	0.62
14	0.5	0	N.t.R.	0.59
15	0.5	0	N.t.R.	0.60
16	0.5	0	N.t.R.	0.54
Mean corneal thickness value = Range of accepted thickness :				0.58 0.52≤e≤0.64

N.t.R.: Nothing to Report

Table No.2: Results for negative control, 30 μL of negative control, 07 November 2016

Endpoint measured	Eye No.	0	30	75	120	180	240
Corneal opacity	16	0.0	0.0	0.0	0.0	0.0	0.0
ICE class					I
Fluorescein retention	16	0.5	0.5	-	-	-	-
ICE class			I	-	-	-	-
Corneal thickness	16	0.54	0.54	0.54	0.55	0.55	0.55
Corneal swelling (%)	16	-	0	0	2	2	2
ICE class					I
Combination of the 3 Endpoints	3 x I
CLASSIFICATION	No Category

No morphological effects were noted, whatever the examination time.

Table No.3:Results of positive control, 30 μL of positive control, 07 November 2016

Endpoint measured	Eye No.	0	30	75	120	180	240
Corneal opacity	1 2 3	0 0 0	3 3 3	3 3 3	3 3 3	3 3 3	3 3 3
Mean		0.0	3.0	3.0	3.0	3.0	3.0
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	0.54 0.6 0.56	0.63 0.78 0.72	0.98 0.89 0.72	1.06 0.99 0.88	1.20 0.99 0.88	1.20 0.99 0.90
Corneal swelling (%)	1 2 3	- - -	17 30 29	81 48 29	96 65 57	122 65 57	122 65 61
Mean		-	25	53	73	81	83
ICE class					IV
Combination of the 3 Endpoints	3 x IV
CLASSIFICATION	Category 1 : Corrosive/Severe irritant

Severe loosening of the corneal epithelium noted from 30 minutes post-dose in eyes No. 1, 2 & 3.

Table No.4:Results of test item, 30 μL of pure test item, 07 November 2016

Endpoint measured	Eye No.	0	30	75	120	180	240
Corneal opacity	10 11 12	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	10 11 12	0.5 0.5 0.5	1 2 2	- - -	- - -	- - -	- - -
Mean		0.5	1.7	-	-	-	-
ICE class			III
Corneal thickness	10 11 12	0.6 0.62 0.60	0.61 0.62 0.60	0.61 0.62 0.60	0.61 0.62 0.60	0.61 0.62 0.60	0.61 0.62 0.60
Corneal swelling (%)	10 11 12	0 0 0	0 0 0	0 0 0	0 0 0	0 0 0	0 0 0
Mean		-	0	0	0	0	0
ICE class					I
Combination of the 3 Endpoints	1 x III, 2 x I
CLASSIFICATION	No prediction can be made

No morphological effects were noted, whatever the examination time.

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

The combination of the three endpoints for the test item was 1 x III, 2 x I, therefore the test item is not predicted as causing serious eye damage or not classified for eye irritation/serious eye damage.

Executive summary:

In accordance with OECD TG 438 and GLP study, the test item was tested for evaluation the possible ocular corrosive or severe irritating effects. Eyes were collected from the chicken killed for human consumption and transported in plastic boxes humified with towels moistened with physiological saline at ambient temperature in 1 hour 25 minutes to the laboratory at the same day. Thes test item was applied as supplied in amount 30 μL to 3 enucleated chicken eyes during 10 seconds. Then the eyes were rinsed twice with 20 mL of physiological saline at ambient temperature. Three eyes were treated in the same manner with a positive control and one eye with a negative control. Damages by the test item were assessed by determination of corneal swelling, opacity, and fluorescein retention at 30, 75, 120, 180 and 240 minutes post-dose. Then the results of each endpoint were assigned to ICE classes according the OECD guideline 438. Their combination - 1xIII and 2xI leads to the category : no prediction can be made.Therefore, the test item is not predicted as causing serious eye damage (Category 1) or as not classified for eye irritation/serious eye damage (No category) with ICE method.