N-ethyl-N-methylcarbamoyl chloride

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From July 12th to August 11th, 2016.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

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:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

479-390-3

EC Name:

-

Cas Number:

42252-34-6

Molecular formula:

C4H8ClNO

IUPAC Name:

N-ethyl-N-methylcarbamoyl chloride

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: M15564C
- Expiration date of the lot/batch: May 2020
- Purity: 99.1%
- Purity test date: June 19, 2015

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: stored at room temperature.
- Stability under test conditions: stable.

Test animals / tissue source

Species:

chicken

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: the eyeballs were collected from chickens obtain a licensed slaughterhouse, i.e. Zakład Przemysłu Drobiarskiego JAS-DROP in Krzyżowice where they were killed for human consumption.
- Characteristics of donor animals (e.g. age, sex, weight): age approximately 7 weeks, weight 1.5 - 2.5 kg. All eyeballs used in the tests came from the same group of eyes collected on a specific day.
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): After electric shock and incision of the neck for bleeding, the chicken heads were transported to the laboratory in a plastic container at ambient temperature. During the transport, the heads were humidified with a physiological salt solution by placing moistened paper towels inside the container.Till the moment of their transfer, the eyeballs were kept in special containers at temperature of -18°C.
- Time interval prior to initiating testing: The time interval between the collection of the chickens’ heads and the use of their eyeballs in the ICE test was 30 minutes.
- 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): 0.03 mL

Duration of treatment / exposure:

10 seconds

Observation period (in vivo):

240 ± 5 minutes

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES: In order to control the quality of the procedure, the eyeballs used for the purpose of the experiment were assessed for potential damage by corneal opacity profusion and fluorescein retention determination. Result of corneal opacity for all examined eyeballs was less than 0.5. Deviation of corneal thickness for all examined eyeballs was less than 10%.
After a careful excision of the eyelids so as not to damage the cornea, the eyeball surface was treated with a 2% fluorescein solution (w/v) for a few seconds in order to evaluate the corneal integrity. After the removal of the dye by rinsing the corneal surface with a physiological salt solution, fluorescein retention and corneal opacity scores were determined using the slit-lamp microscope BP 900 LED (HAAG STREIT) to ensure that the cornea was undamaged. Only eyeballs without damage were analyzed (fluorescein retention and opacity profusion scores fell below 0.5). The enucleated eyeball was mounted in a stainless steel clamp with the cornea positioned vertically. The clamp was then transferred to the superfusion apparatus so that the entire cornea was supplied with the physiological salt drip.
After the insertion of the eyeballs to the apparatus, another evaluation of corneal opacity and swelling was performed. Result of corneal opacity for all examined eyeballs was less than 0.5. Deviation of corneal thickness for all examined eyeballs was less than 10%. The corneal thickness was determinated using the depth measuring device no. 1 on the slit-lamp microscope and an SP-100 pachymeter (TOMEY).

EQUILIBRATION AND BASELINE RECORDINGS: Before the application of the test item and the control items, all examined and approved eyeballs were incubated at temperature of 32 ± 1.5°C for 45-60 minutes in order to equilibrate them to the test system. After that, a zero reference measurement for corneal thickness and opacity was recorded. The fluorescein score determined at dissection served as the baseline measurement for that endpoint.

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: physiological saline, 0.03 mL

POSITIVE CONTROL USED: 10% acetic acid, 0.03 mL

APPLICATION DOSE AND EXPOSURE TIME: 0.03 mL, exposure 10s

OBSERVATION PERIOD: The corneas treated with the test item and the control items 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: the test item and the control items were rinsed from the eye with 20 mL of physiological salt solution at ambient temperature. Next, the eyeballs in their holders were placed in the superfusion apparatus in the original upright position.
- Indicate any deviation from test procedure in the Guideline: no.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: qualitative assessment by assigning appropiate values to opaque areas, the mean corneal opacity value was calculated for all test eyeballs for all observation time points. Based on the highest mean score corneal opacity the final score was given.
- Damage to epithelium based on fluorescein retention: yes. Qualitative assessment of damage to epithelium based on application of fluorescein to the eye (fluorescein retention) 30 min after end of exposure.
- Swelling: measured with optical pachymeter on a slit-lamp microscope; quantitative measurement, using a slit-lamp microscope and an SP-100 pachymeter.The mean percentage of corneal swelling for all test eyeballs was calculated for all observation time points. Based on the highest mean score for corneal swelling, at any time point, the final category score was given.
- Macroscopic morphological damage to the surface: qualitative assessment.
- Others (e.g, histopathology): histopathological evaluations of morphological damage to the surface.

SCORING SYSTEM:
- Mean corneal swelling (%): The degree of corneal swelling was determined by measuring corneal thickness using a slit-lamp microscope and an SP-100 pachymeter.The mean percentage of corneal swelling for all test eyeballs was calculated for all observation time points. Based on the highest mean score for corneal swelling, at any time point, the final category score was given.
- Mean maximum opacity score:
0 No opacity
0.5 Very faint opacity
1 Scattered or diffuse areas; details of the iris are clearly visible
2 Easily discernible translucent areas; 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
Corneal swelling was calculated as % as follows:corneal swelling = [corneal thickness at time t – corneal
thickness at time t = 0/ corneal thickness at time t = 0] x100

- 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: decision criteria as indicated in the TG was used.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

fluorescein retention score

Run / experiment:

mean

Value:

2

Vehicle controls validity:

not valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE class III.

Results 2

Irritation parameter:

cornea opacity score

Run / experiment:

mean

Value:

3.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE class IV

Results 3

Irritation parameter:

percent corneal swelling

Run / experiment:

mean

Value:

>= 11.8 - <= 38.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE class II to ICE class IV

Results 4

Irritation parameter:

morphological effects

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Remarks:

roughening of the corneal surface.

Results 5

Irritation parameter:

histopathological observations

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Remarks:

severe vacuolation and necrosis of the anterior corneal epithelium (eyeballs no. 2, no. 3), very severe vacuolation and necrosis of the single cells of the anterior corneal epithelium (eyeball no. 1), vacuolation of the corneal stroma (eyeball no. 3).

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: yes. Gross examinations of the eyeballs treated with the test item showed roughening of the corneal surface.
- Histopathological examinations of the corneas treated with the test item revealed: severe vacuolation and necrosis of the anterior corneal epithelium (eyeballs no. 2, no. 3), very severe vacuolation and necrosis of the single cells of the anterior corneal epithelium (eyeball no. 1), vacuolation of the corneal stroma (eyeball no. 3)

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

Any other information on results incl. tables

Table 1. Fluorescein retention.

Observation after time t (minutes)	Test item			Positive control 10% acetic acid			Negative control physiological saline
	Eyeball no.			Eyeball no.			Eyeball no.
	1	2	3	4	5	6	7	8	9
0	0	0	0	0	0	0	0	0	0
30	2	2	2	3	3	3	0	0	0

 

Table 2. Corneal opacity.

Observation after time t (minutes)	Test item			Positive control 10% acetic acid			Negative control physiological saline
	Eyeball no.			Eyeball no.			Eyeball no.
	1	2	3	4	5	6	7	8	9
0	0	0	0	0	0	0	0	0	0
30	2	2	2	3	3	3	0	0	0
75	2	3	3	4	4	4	0	0	0
120	3	3	3	4	4	4	0	0	0
180	3	3	4	4	4	4	0	0	0
240	3	4	4	4	4	4	0	0	0

 

Table 3. Corneal swelling (%).

Observation after time t (minutes)	Test item			Positive control 10% acetic acid			Negative control physiological saline
	Eyeball no.			Eyeball no.			Eyeball no.
	1	2	3	4	5	6	7	8	9
0	-	-	-	-	-	-	-	-	-
30	3.43	15.78	16.04	20.93	17.40	19.81	-3.02	-6.50	-3.57
75	10.45	16.72	21.96	24.96	24.85	27.92	-4.38	-6.50	-3.26
120	13.73	30.94	24.45	37.21	27.63	39.12	-1.21	-9.89	-7.13
180	23.87	34.06	31.62	50.85	31.73	41.23	-2.27	-11.30	-9.46
240	33.39	41.25	40.97	53.02	37.72	52.11	0	-12.01	-12.71

The negative values are regarded as no swelling [ICE Class I]

 

Table 4. Gross evaluation of the treated corneas.

Observation after time t (minutes)	Test item			Positive control 10% acetic acid			Negative control physiological saline
	Eyeball no.			Eyeball no.			Eyeball no.
	1	2	3	4	5	6	7	8	9
0	NC	NC	SIGNS	SIGNS	SIGNS	SIGNS	NC	NC	NC
30	NC	NC	SIGNS	SIGNS	SIGNS	SIGNS	NC	NC	NC
75	NC	SIGNS	SIGNS	SIGNS	SIGNS	SIGNS	NC	NC	NC
120	SIGNS	SIGNS	SIGNS	SIGNS	SIGNS	SIGNS	NC	NC	NC
180	SIGNS	SIGNS	SIGNS	SIGNS	SIGNS	SIGNS	NC	NC	NC
240	SIGNS	SIGNS	SIGNS	SIGNS	SIGNS	SIGNS	NC	NC	NC

NC = no changes; SIGNS = roughening of the corneal surface

Applicant's summary and conclusion

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Remarks:

EU criteria.

Conclusions:

The test item causes serious damage to the eye, and therefore it is classified as Eye Damage Category 1.

Executive summary:

An in vitro (ex vivo) study was conducted in order to determine the potential severe eye damaging effects of 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 the test item, 10% acetic acid (positive control) or physiological saline (negative control). Three eyeballs were used for the test item and for each control. 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 recomendations, histopathological evaluation of the corneal layers was conducted. The combination of the three endpoints was 2 x IV and 1 x III. According to the overall in vitro classification, the test item may cause serious eye damage, and therefore, it is classified as Eye Damage Category 1.