Ethyl diphenylcarbamate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

November 16, 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 diphenylcarbamate

EC Number:

210-047-0

EC Name:

Ethyl diphenylcarbamate

Cas Number:

603-52-1

Molecular formula:

C15H15NO2

IUPAC Name:

ethyl N,N-diphenylcarbamate

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

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

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: spring chickens traditionally processed by a poultry slaughterhouse (approximately 7 weeks old, 1.5-2.5 kg).
- Storage, temperature and transport conditions of ocular tissue: Eyes were dissected in the laboratory. The heads have been collected and transported intact from slaughterhouse at ambient temperature in plastic boxes humidified with towels moistened with physiological saline. The eyes were enucleated at laboratory on 16 November 2016 at 9:50 am.
- Time interval prior to initiating testing: Heads were collected on 16 November 2016 at 8:15 am. The eyes were enucleated at laboratory on 16 November 2016 at 9:50 am. The eyes were enucleated at laboratory on 16 November 2016 at 9:50 am. The examined and approved eyes were incubated between 45 and 58 minutes to equilibrate them to the test system prior to dosing.
- 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 applied: 30 mg

Duration of treatment / exposure:

10 seconds

Duration of post- treatment incubation (in vitro):

4 hours

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
Dissection of the 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. One removed from the orbit, the eye was placed on an absorbent pad and the nictitating membrane and other connective tissue were cut away.
Selection of the eyes: 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 32.3ºC and 32.6ºC.
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 deph 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
The examined and approved eyes were incubated between 45 and 58 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: 30 μL of Phisiological saline - Dutscher, Batch No. 3012316 (1 replicate)

POSITIVE CONTROL USED: 3. 30 mg of Sodium hydroxide - Sigma, Batch No. MKBP7805V (3 replicates)

APPLICATION DOSE AND EXPOSURE TIME: 30 mg for 10 seconds

OBSERVATION PERIOD: Treated corneas were evaluated 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: Test item rinsed from the eye with 20 mL of physiological saline at ambient temperature.
- Indicate any deviation from test procedure in the Guideline: no deviations

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: the corneal opacity was calculated using the area of the cornea that was most densely opacied 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, and 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 HaagStreit BP900 slit-lamp microscope with deph-measuring device no. I; the slit-width was set at 9 1/2 equlling 0.095 mm: corneal swelling was determined from corneal thickness measurements made with an optical pachymeter on a slit-lamp microscope. 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, and overall category score was the 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.

SCORING SYSTEM:
- Mean corneal swelling (%): calculated according to the following formula and expressed as a percentage

[(Corneal thickness at time 1 - Corneal thickness at time 0) / Corneal thickness at time 0] x 100

- 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 iris 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: the decision criteria as indicated in the TG was used. Results from corneal opacity, swelling and fluorescein retention were evaluated separately to generate an ICE class for each endpoint. The ICE classes for each endpoint were then combined to generate an Irritancy Classification for the test item. Once each endpoint was evaluated, ICE classes were assigned based on a predetermined range.
The test will considered acceptable if the concurrent negative control and the concurrent positive control are identified as GHS Non-classified and GHS Category 1, respectively

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

cornea opacity score

Run / experiment:

mean

Value:

ca. 0

Vehicle controls validity:

not valid

Negative controls validity:

valid

Remarks:

ICE class I

Positive controls validity:

valid

Remarks:

ICE class IV

Remarks on result:

no indication of irritation

Remarks:

ICE class I

Results 2

Irritation parameter:

fluorescein retention score

Run / experiment:

mean

Value:

ca. 0.5

Vehicle controls validity:

not valid

Negative controls validity:

valid

Remarks:

ICE class II

Positive controls validity:

valid

Remarks:

ICE class IV

Remarks on result:

no indication of irritation

Remarks:

ICE class I

Results 3

Irritation parameter:

percent corneal swelling

Run / experiment:

mean

Value:

ca. 1

Vehicle controls validity:

not valid

Negative controls validity:

valid

Remarks:

ICE class I

Positive controls validity:

valid

Remarks:

ICE class IV

Remarks on result:

no indication of irritation

Remarks:

ICE class I

Other effects / acceptance of results:

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

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes, 12 of the 13 chemicals tested obtained the same category as OECD 438. There was a borderline chemical (DMSO) over-classified ("No prediction can be made" vs. "No Category") in three separated tests. It should be tested with other adequately validated in vitro test.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, the combination of the three endpoints for the negative contro, physiological saline, was 2 x I, 1 x II. 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

Results: Negative Control (Physiological Saline)

Endpoint measured	Eye No.	Time (min)
		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	1	-	-	-	-
ICE class		II
Corneal thickness	13	0.58	0.58	0.58	0.58	0.58	0.58
Corneal swelling (%)	13	-	0	0	0	0	0
ICE class		I
Combination of the 3 Endpoints		2 x I, 1 x II
CLASSIFICATION		No Category

Note:

No morphological effects were noted, whatever the examination time.

Results: Positive control (Sodium hydroxide)

Endpoint measured	Eye No.	Time (min)
		0	30	75	120	180	240
Corneal opacity	1	0	4	4	4	4	4
	2	0	4	4	4	4	4
	3	0	4	4	4	4	4
Mean		0.0	4.0	4.0	4.0	4.0	4.0
ICE class		IV
Fluorescein retention	1	0.5	3	-	-	-	-
	2	0.5	3	-	-	-	-
	3	0.5	3	-	-	-	-
Mean		0.5	3.0	-	-	-	-
ICE class		IV
Corneal thickness	1	0.58	-	-	-	-	-
	2	0.58	-	-	-	-	-
	3	0.58	-	-	-	-	-
Corneal swelling (%)	1	-	(-)	(-)	(-)	(-)	(-)
	2	-	(-)	(-)	(-)	(-)	(-)
	3	-	(-)	(-)	(-)	(-)	(-)
Mean		-	-	-	-	-	-
ICE class		IV
Combination of the 3 Endpoints		3 x IV
CLASSIFICATION		Category I

Note:

(-): evaluation of corneal swelling not possible (Corneal opacity = 4 at each examination time, leading to a marked refraction of the light preventing from the evaluation of the corneal swelling with the biomicroscope)

Severe loosening of the corneal epithelium noted from 30 minutes post dose in eyes nº2 and nº3

Results: Test item

Endpoint measured	Eye No.	Time (min)
		0	30	75	120	180	240
Corneal opacity	4	0	0	0	0	0	0
	5	0	0	0	0	0	0
	6	0	0	0	0	0	0
Mean		0.0	0.0	0.0	0.0	0.0	0.0
ICE class		I
Fluorescein retention	4	0.5	0.5	-	-	-	-
	5	0.5	0.5	-	-	-	-
	6	0.5	0.5	-	-	-	-
Mean		0.5	0.5	-	-	-	-
ICE class		I
Corneal thickness	4	0.59	0.60	0.61	0.61	0.61	0.61
	5	0.59	0.59	0.59	0.59	0.59	0.59
	6	0.58	0.58	0.58	0.58	0.58	0.58
Corneal swelling (%)	4	-	2	3	3	3	3
	5	-	0	0	0	0	0
	6	-	0	0	0	0	0
Mean		-	1	1	1	1	1
ICE class		I
Combination of the 3 Endpoints		3 x I
CLASSIFICATION		No Category

Note:

No morphological effects were noted, whatever the examination time.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

The combination of the three endpoint (corneal swelling, opacity and fluorescein retention) evaluated for the test item was 3 x I. The test item is classified as "No Category"

Conclusions:

The combination of the tree endpoints for the test item was 3x1. Therefore, the test item is classified as "No Category".

Executive summary:

An 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 was performed according OECD guideline 438 and test method B.48, to evaluate the possible ocular corrosive or severe irritating effects of the test item after administration on enucleated chicken eyes. The test item was applied, as supplied, at the dose of 30 mg, to 3 enucleated chicken eyes, during 10 seconds. Then the eyes were rinsed twice with 10 mL of physiological saline. 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. The ocular reactions observed in eyes treated with the test item were: maximal mean score of corneal opacity: 0.0, corresponding to ICE class I; mean score of fluorescein retention: 0.5, corresponding to ICE class I; maximal mean corneal swelling: 1%, corresponding to ICE class I. The combination of the three endpoints for the test item was 3 x I. Positive and negative control were classified as expected. In accordance with the Regulation (EC) No. 1272/2008, the results obtained under these experimental conditions enable to conclude that the test item does not require classification for eye irritation and serious eye damage as defined by UN GHS (No category).