N-methyl-N-vinylacetamide

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-03-05 to 2015-03-31

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2010

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Harlan Cytotest Cell Research GmbH, 64380 Rossdorf, Germany

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

N-methyl-N-vinylacetamide

EC Number:

221-698-5

EC Name:

N-methyl-N-vinylacetamide

Cas Number:

3195-78-6

Molecular formula:

C5H9NO

IUPAC Name:

N-ethenyl-N-methylacetamide

Specific details on test material used for the study:

Batch: 68005536W0
Expiry Date: 01 July 2015
Storage Conditions: In the refrigerator between 2 and 8°C, avoid temperatures > 10°C

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Remarks:

CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories B.V., 5960 AD Horst / The Netherlands
- Females nulliparous and non-pregnant: yes
- Age at study initiation: Pre-test: 10 - 11 weeks (beginning of treatment); Main study: 8 - 9 weeks (beginning of treatment)
- Weight at study initiation: 17.9-21.2 g
- Housing: group, Makrolon Type II (pre-test) / III (main study), with wire mesh top
- Diet: ad libitum, 2018C Teklad Global 18 % protein rodent diet (certified)
- Water: ad libitum, tap water
- Acclimation period: at least 5 days
- Indication of any skin lesions: Only animals without any visible signs of illness were used for the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 45-65
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

propylene glycol

Concentration:

25, 50 and 100 %

No. of animals per dose:

5

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: The highest test item concentration, which could be technically used was 100% of the undiluted test item. Test item solutions at different concentrations were prepared using PG as vehicle.
- Irritation: After treatment with 50 and 100 % test substance for 3 days animals did not show any signs of local skin irritation.
- Systemic toxicity: Not detected.
- Ear thickness measurements: No increase in ear thickness of ≥ 25 % was recorded.
- Erythema scores: No erythema was detected.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
The animals were distributed into the test groups at random.
- Criteria used to consider a positive response: The cut-off values for a positive response were stimulation index of 3, for the lymph node cell count index of 1.55 and for the ear weight index of 1.1.

TREATMENT PREPARATION AND ADMINISTRATION:
Each test group of mice was treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 25 and 50 % in PG, and 100 %. The application volume, 25 μL/ear/day, was spread over the entire dorsal surface (∅ ∼ 8 mm) of each ear once daily for three consecutive days. A further group of mice (control animals) was treated with an equivalent volume of the vehicle alone (control animals). For positive control a periodic positive control experiment was used. Five days after the first topical application (day 6) 250 μL of phosphate-buffered saline containing 20.2 μCi of 3H-methyl thymidine (equivalent to 80.9 μCi/mL 3HTdR) were injected into each test and control mouse via the tail vein.

Positive control substance(s):

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

Statistics:

The mean values and standard deviations were calculated in the body weight tables, for the ear weights, the lymph node weights and lymph node cell count, and for the DPM values (group mean DPM ± standard deviation). A statistical analysis was conducted on the DPM values, the ear weights, the lymph node weights and the lymph node cell count to assess whether the difference was statistically significant between the test item groups and negative control group. For all statistical calculations custom made statistical program ´R` Decision Tree was used. Statistical significance was set at the five per cent level (p < 0.05). The Dean-Dixon-Test and the Grubb’s test were used for detection of possible outliers (performed with custom made statistical program ´R` Decision Tree).

Results and discussion

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1.13

Test group / Remarks:

25 % treatment group

Results 2

Key result

Parameter:

SI

Value:

1.04

Test group / Remarks:

50 % treatment group

Results 3

Key result

Parameter:

SI

Value:

0.57

Test group / Remarks:

100 % treatment group

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
For the 25 % test substance group the mean DPM count was 1296.3 (±567.1). For the 50 % test substance group the mean DPM count was 1184.7 (±534.2). For the 100 % test substance group the mean DPM count was 646.3 (±253.9).

DETAILS ON STIMULATION INDEX CALCULATION
The ratio of 3HTdR incorporated into lymph node cells of test animals relative to that recorded for lymph nodes of control animals was calculated.

EC3 CALCULATION
The EC3 value could not be calculated, since all S.I.´s are below the threshold value of 3.

CLINICAL OBSERVATIONS:
No signs of systemic toxicity or local skin irritation were observed.

BODY WEIGHTS
The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age. The animals treated with the undiluted test item lost 6.3 % of their average body weight.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met