N-ethyl-N-methylcarbamoyl chloride

Endpoint summary

Description of key information

Skin sensitisation: Key study. Test method according to OECD TG 429, GLP study. The test item is a skin sensitizer.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From November 21st to December 10th, 2007.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

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:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River France, L' Arbresle Cedex, France.
- Age at study initiation: 9 weeks old.
- Weight at study initiation: 19 - 24 g.
- Housing: Individually housed in labeled Macrolon cages containing sterilized sawdust as bedding material. Paper and Surrey was supplied as cage-enrichment. The paper was removed on day 1 prior to dosing and was supplied again after scoring of the ears on day 3.
- Diet (e.g. ad libitum): Pelleted rodent diet ad libitum.
- Water (e.g. ad libitum): Tap water ad libitum.
- Acclimation period: Five days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.6 - 23.6 ºC.
- Humidity (%): 39-69 %
- Air changes (per hr): 15 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hours artificial fluorescent light and 12 hours darkness per day.

Vehicle:

unchanged (no vehicle)

Concentration:

100, 50, 25 and 10% in methyl ethyl ketone. 25 µL/ear.

No. of animals per dose:

5 females per group.

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: The tests were conducted with methyl ethyl ketone as vehicle which was selected based on trial formulations.
- Irritation: The test system, procedures and techniques, were identical to those used during Days 1-3 of the main study unless otherwise specified. Two young adult animals sere selected (8-14 weeks old). Each animal was treated with one concentration on 3 consecutive days. Approximately 3-4h after the last exposure, the ear was cleaned of residual test substance with tap water and the irritation was assessed. The animals were sacrificed after the final observation and no necropsy was performed. A maximum score of 3 for erythema was recorded. Based on the results, the highest test substance concentration selected for the main study was 100% concentration.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: A stimulation index = or > to 3 indicate that the test subtance can be regarded as sensitizer.

TREATMENT PREPARATION AND ADMINISTRATION:
Inductions: day 1, 2 and 3
The dorsal surface of both ears was epidermally treated (25µg/ear) at approximately same time of day. The control were treated the same as experimental animals, except that, instead of the test subtance, the vehicle alone was administered.
Treatment day 6
Each animal was injected via tail vein with 0.25 mL of sterile phosphate buffered saline containing µCi of 3H-methyl thymidine.
After approximately 5 hours, all animals were killed by intraperitoneal injection with phernobarbital. The draining (auricular) lymph node of each ear was excised. The relative size of the nodes (as compared to the normal) was estimated by visual examination and abnormalities of the nodes and surrounding area were recorded. The nodes were pooled for each animal in approximately 3mL PBS.
Tissue processing for radioactivity. Day 6
A single cell supension of lymph node cells was prepared in PBS by gentle separation through stainless steel gauze (diamter 125µm). LNC were washed twice with an excess of PBS by centrifugation at 200g for 10 minutes at 4ºC. To precipitate the DNA, the LNC were exposed to 5 % trichloroacetic at 4ºC during night.
Radioactivity measurements, day 7:
Precipitates were recovered by centrifugation, resuspended in 1mL TCA and transferred to 10 mL of a scintillation fluid. Counting time was to a statistical precision of ± 0.2 or a maximum of 5 minutes whichever comes first. The scntillation counter was programmed to automatically substract background and convert Counts Per Minute (CPM) to Desintigrations Per Minute (DPM).

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Positive control results:

The SI values calculated for alpha-hexyl cinnamic aldehyde concentrations 5 %, 10% and 25 % were 1.0, 2.0 and 5.7 respecitvely. An EC3 value of 14.1 % was calculated using linear interpolation.
The EC3 value was found to be in the acceptable range of 2 and 20 %.

Key result

Parameter:

SI

Value:

26.1

Test group / Remarks:

Group at 25%

Key result

Parameter:

SI

Value:

56.3

Test group / Remarks:

Group at 50%.

Key result

Parameter:

EC3

Value:

> 0 - <= 25

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA: see 'Any other information on results'

EC3 CALCULATION: It was possible to strengthen the outcome of the study by adding lower concentrations, nevertheless it was considered not appropriate for ethical reasons since SI values clearly exceeded 3 and the extention of the study would not alter the classification.

CLINICAL OBSERVATIONS: Severe erythema was noted among the animals at the 100% dose. Slight to well-defined erythema of the ears was observed in the animals at 25 and 50% and slight erythema of one ear was noted in one animal of the control group. No oedema was observed in any of the animals examined. The irritation of the ears as shown by the animals was considered not to have a toxicologically significant effect on the activity of the nodes.
All nodes of the groups at 25 and 50% were considered enlarged in size and all nodes of the control group were considered in normal size. No macroscopic abnormalities of the surrounding area were noted.

BODY WEIGHTS: Marked body weight loss, a sign of systemic toxicity, was noted among the animals at the 100% dose. Body weights and body weight gain of animals at 25 and 50% groups remained in the same range as controls over the study period.

Based on the sign of systemic toxicity and severe erythema, the results of the group at 100% could not be used for interpretation.

- Radioactivity measurements: Mean DPM/animal for the experimental groups treated with the test subtance concentrations 25 and 50 % were 6166 and 13295 respectively. Based on signs of systemic toxicity (marked body weight loss) and severe erythema among the animals at 100 %, the results of the group at 100 % could not be used for interperetation.

Table 1. Preliminary Study. Skin reactions after exposure and body weights.

Animal number	% test substance	Day 1	Day 3
		Body weight (g)	Skin reactions dorsal surface ear (left)		Skin reactions dorsal surface ear (right)		Body weight (g)
			Erythema	oedema	Erythema	oedema
1^2	10	19	0	0	0	0	20
2^2	25	20	1	0	2	0	21
1	50	23	3	0	3	0	23
2	100	24	2	0	3	0	22
3^2	100	21	1	0	2	0	20

¹Vehicle: Methyl ethyl ketone.

²Additional animal in preliminary irritation study.

Table 2. Main Study. Skin reactions, body weights and relative size auricular lymph nodes.

Group	% test item^1	Animal No.	Day 1	Day 3				Day 6
			Body weight (g)	Skin reactions dorsal surface ear (left)		Skin reactions dorsal surface ear (right)		Size nodes^2		Body weight (g)
				Erythema	oedema	Erythema	oedema	Erythema	oedema
1	0%	1	22	0	0	0	0	N	N	21
		2	23	0	0	0	0	N	N	22
		3	20	0	0	1	0	N	N	19
		4	23	0	0	0	0	N	N	23
		5	24	0	0	0	0	N	N	22
2	25%	6	21	0	0	1	0	+	+	19
		7	21	1	0	1	0	+	+	18
		8	24	1	0	2	0	+	+	23
		9	23	1	0	2	0	+	+	21
		10	20	2	0	2	0	+	+	20
3	50%	11	22	1	0	2	0	++	++	21
		12	20	2	0	2	0	++	++	19
		13	21	2	0	2	0	++	++	22
		14	22	1	0	2	0	++	++	22
		15	23	2	0	2	0	++	++	22
4^3	100%	16	21	2	0	2	0	++	N	18
		17	21	3	0	2	0	++	++	20
		18	20	1	0	3	0	++	+	15
		19	21	2	0	3	0	++	++	16
		20	21	2	0	2	0	++	++	18

 

¹Vehicle: Methyl ethyl ketone.

²Relative size auricular lymph nodes (-, -- or ---: degree of reduction; +, ++, or +++: degree of enlargement, N: normal).

³Due to marked body weight loss (sign of systemic toxicity) and severe erythema among the animals at 100%, data of the group at 100% could not be used for interpretation.

 

Table 3. Main Study. Radioactivity measurements (individual animals).

Group	% test item^1	Animal No.	DPM / animal
1	0%	1	249
		2	266
		3	208
		4	78
		5	279
2	25%	6	8257
		7	5080
		8	5534
		9	4368
		10	7592
3	50%	11	17013
		12	9455
		13	9063
		14	15369
		15	15573
4^3	100%	16	12682
		17	15449
		18	13834
		19	10840
		20	353

¹Vehicle: Methyl ethyl ketone.

²Due to marked body weight loss (sign of systemic toxicity) and severe erythema among the animals at 100%, data of the group at 100% could not be used for interpretation.

Interpretation of results:

sensitising

Remarks:

EU

Conclusions:

The test subtance elicited a stimulation index of 26.1 and 56.3 in concentrations of 25 and 50 % respectively. The test subtance is concluded to be as skin sensitizer.

Executive summary:

A local lymph node assay was conducted according to the OECD guideline 429 under GLP conditions with the test item. In the preliminary studies 100 % of the test item was selected as the top dose for the main experiments and methyl ethyl ketone was selected as the vehicle.Two animals were used for each concentration in the preliminary studies. On day 1, 2 and 3, 25 µL of each of the test item solutions (100, 50, 25%) and positive and negative controls were applied to the dorsal surface of each ear of the mice. There was no treatment on day 4, 5 or 6. Clinical signs and signs of irritancy were monitored during the 6 days, there were some signs of irritancy, but no signs of systemic toxicity. Five mice were used per concentration and for each of the controls in the main experiment. On day 6 the weight was determined, the preliminary test finished at this point. In the main tests 250 µL of sterile phosphate buffered saline (PBS) containing approximately 20 µCi of 3HTdR was injected via tail vein. Five hours after injection the mice were euthanized . The draining auricular lymph nodes were prepared and pooled. Single cell suspensions (SCS) of pooled lymph node cells (LNCs) were prepared and collected in disposable tubes for each group of pooled lymph nodes, and then the determination of incorporated³HTdR was determined as DPM for each pooled group. The stimulation index was calculated (SI = DPN value of a treated group/ DPN value of the negative control group) for each treatment group. A stimulation index of 3 or greater was considered a positive result.

Based on signs of systemic toxicity (marked body weight loss) and severe erythema among the animals at 100 %, the results of the group at 100 % could not be used for interperetation. The stimulation indexes of the test item at concentrations of 50, 25 % were 26.1 and 56.3 respectively, a dose-related response was observed. The SI of the positive and negative controls were within the acceptable range. The test item was regarded as skin sensitizer.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

Key study: A local lymph node assay was conducted according to the OECD guideline 429 under GLP conditions with the test item. A stimulation index of 3 or greater was considered a positive result. Based on signs of systemic toxicity (marked body weight loss) and severe erythema among the animals at 100 %, the results of this group could not be used for interperetation. The stimulation indexes of the test item at concentrations of 50, 25 % were 26.1 and 56.3 respectively, a dose-related response was observed. The SI of the positive and negative controls were within the acceptable range. The test item was regarded as a skin sensitizer.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available experimental results, the test substance is classified as Skin sensitiser Category 1, H317, in accordance with CLP Regulation (EC) No. 1272/2008.