N-ethyl-N-methylcarbamoyl chloride

Endpoint summary

Description of key information

Skin irritation. Key study: Test method according to OECD 404, GLP study. The test item was irritating to the skin under test conditions (Category 2).

Eye irritation. Key study. Test method according to OECD 438, GLP study. The test item causes serious eye damage (Category 1).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: C0014
- Purity: 99.3%
- Expiration date of the lot/batch: 31 July 3008

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In refrigerator (2-8ºC) in the dark
- Stability under test conditions: stable

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan, Horst, The Netherlands
- Age at study initiation: At least 6 weeks old.
- Weight at study initiation: At least 1.0 kg.
- Housing:Individually housed in labelled cages with perforated floors.
- Diet (e.g. ad libitum): Pelleted diet for rabbits ad libitum.
- Water (e.g. ad libitum): Tap water ad libitum.
- Acclimation period: At least 5 days.
- Other: 3 male rabbits were used.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.9-21.7 ºC
- Humidity (%): 42-61 %
- Air changes (per hr): 15 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hours artificial fluorescent light/12 hours dark

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 0.5 mL.

Duration of treatment / exposure:

Four hours

Observation period:

14 days.

Number of animals:

Three

Details on study design:

TEST SITE
- Area of exposure: The test subtance was applied to the skin of one flank using a metalline path of 2x3cm.
- Type of wrap if used: The patch was mounted with Micropore tape and wrapped around the abdomen and secured with Coban elastic bandage.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): After removing the dressing, the skin was cleaned of residual test item with tap water.
- Time after start of exposure: 4 hours.

SCORING SYSTEM:
Erythema and Eschar formation
No erythema: 0
Very slight erythema (barely perceptible): 1
Well defined erythema: 2
Moderate to severe erythema: 3
Severe erythema (beef redness) to slight eschar formation (injuries in depth) preventing grading of erythema: 4

Oedema formation
No oedema: 0
Very slight oedema (barely perceptible): 1
Slight oedema (edges of area well-defined by definite raising): 2
Moderate oedema (raised approximately 1 mm): 3
Severe oedema (raised more than 1 mm and extending beyond area of exposure: 4

Irritation parameter:

erythema score

Basis:

mean

Time point:

other: 24, 48 and 72 hours

Score:

2.3

Max. score:

3

Reversibility:

not fully reversible within: At termination (14 days after treatment) very slight erythema, very slight oedema and bald skin was observed in one animal. In the other two animals scaliness and bald skin were observed at termination.

Remarks on result:

other: The individual score for each animal was the same to the mean score.

Irritation parameter:

edema score

Basis:

mean

Time point:

other: 24, 48 and 72 hours

Score:

3.3

Max. score:

4

Reversibility:

not fully reversible within: At termination (14 days after treatment) very slight erythema, very slight oedema and bald skin was observed in one animal. In the other two animals scaliness and bald skin were observed at termination.

Remarks on result:

other: The individual score for each animal was the same to the mean score.

Irritant / corrosive response data:

There was no evidence of corrosion on the skin.
The skin reactions persisted until day 14 after exposure.

Other effects:

Dry remnants of the test substance were present on day 1.
No staining of the treated skin by the test item was observed.
No signs of systemic toxicity were observed in the animals during the test period and no mortality occured.

Table 1. Individual skin irritation scores

Animal	610 (1)			626			632
Time after exposure	Erythema	Oedema	Comments	Erythema	Oedema	Comments	Erythema	Oedema	Comments
1 hour	2	3	B	2	4	B	2	3	B
24 hours	2	3	-	2	3		2	3	-
48 hours	2	3	-	2	3	F	2	3	F
72 hours	3	4	F	3	4	F	3	4	F
7 days	1	2	H, L	1	2	H, L	1	2	H, L
14 days	1	1	H	0	0	H, L	0	0	H, L

(1)= Sentinel

Comments:

B: Dry remnants of the subtance present

F: Reduced Flexibility of the skin

H: Bald skin

L: Scaliness

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Remarks:

EU

Conclusions:

The test item caused skin reactions which persisted until the end of the observation period (14 days). No evidence of corrosion was found.

Executive summary:

A study was performed to assess the skin irritation potential of the test item to the rabbit in accordance to OECD Guideline 404 (GLP study).The study was performed in a stepwise manner and was started by the treatment of a single rabbit (sentinel). The other two animals were treated in a similar manner 4 weeks later, after considering the degree of skin irritation observed in the first animal. Rabbits received a single four hour, semi-occlusive, dermal administration of approximately 0.5 mL of the test substance as supplied and were observed for 14 days. No signs of corrosion were noted. At termination very slight erythema, very slight oedema and bald skin was observed in one animal. In the other two animals scaliness and bald skin were observed at termination. The observed mean scores (24, 48 and 72h) for each animal were equal to the mean for all animals and were 2.3 for erythema and 3.3 for oedema. The test item was determined to be irritating to the skin (Category 2 according to CLP Regulation).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

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

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)

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.

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

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.

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.

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

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

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.

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

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

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation. Key study: A study was performed to assess the skin irritation potential of the test item to the rabbit in accordance with OECD Guideline 404 (GLP study). No signs of corrosion were noted. At termination very slight erythema, very slight oedema and bald skin was observed in one animal. In the other two animals scaliness and bald skin were observed at termination.The observed mean scores (24, 48 and 72h) for each animal were equal to the mean for all animals and were 2.3 for erythema and 3.3 for oedema. The test item was determined to be irritating to the skin Category II according to CLP Regulation.

Key study. An in vitro skin irritation test of the test item was performed according to OECD TG 439. Three EPISKIN disks were treated with the test item and incubated for 15 minutes at room temperature. Exposure of the test item was terminated by rinsing with Phosphate Buffered Saline (PBS). The epidermis units were then incubated at 37ºC for 42h in an incubator with 5% CO2. The viability of each disk was assessed by incubating the tissues for 3h with MTT solution at 37ºC in an incubator with 5% CO2 protected from light. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically. Concurrent negative (PBS) and positive (5% (w/v) SDS solution) controls were run in triplicate; the acceptance criteria were met. The results indicate that the test item is non-irritant to skin (No Category).

Based on the available information (in vivo study), the in vitro result might be considered as a false negative.

Eye irritation. Key study. 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, and 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.

Justification for classification or non-classification

Skin irritation: Based on the available data (24 -72h mean erythema score of 2.3 and 24 -72h mean oedema score of 3.3 in all animals), the substance is classified for Skin Irritation Category 2, H315, acccording to CLP Regulation (EC). No. 1272/2008.

Eye irritation: Based on the available data (2xIV and 1xIII combination in an OECD 438 study), the substance is classified as Eye Damage Category 1, H318, according to CLP Regulation (EC) No. 1272/2008.