Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline Study OECD 429 with GLP compliance

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Identity: N-Vinylcaprolactam

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River UK Manston Road Margate, Kent CT9 4LT United Kingdom
- Age (beginning of treatment): 8 - 10 weeks
- Weight at study initiation: 15.1 - 20.0 g
- Housing: Group
- Diet : Pelleted standard diet, ad libitum
- Water: Tap water, ad libitum
- Acclimatisation: At least 5 days prior to the start of dosing under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 +/- 2°C
- Relative humidity: 45 – 100% (acclimation period / pre-test) 43 – 65% (main study)
- Artificial light: 6.00 a.m. - 6.00 p.m.

IN-LIFE DATES:
From: 2011-09-07 To: 2011-11-21

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
1) 2.5 %
2) 5 %
3) 10 %
No. of animals per dose:
5 (main study)
Details on study design:
RANGE-FINDING TEST
A solubility experiment was performed. The highest test item concentration, which can be technically used, was 100% (undiluted test item). Test
item solutions at different concentrations up to 50 % were prepared using acetone:olive oil (4+1 v/v) as vehicle.

Two mice were treated by topical application to the dorsal surface of each ear with test item concentrations of 50% (w/w) and 100% once daily each on three consecutive days. On day 2, the animal treated with the undiluted test item was found dead and the animal treated with 50% test item concentration showed reduced spontaneous activity. On day 3, the animal treated with 50% test item concentration was sacrificed due to severe signs of systemic toxicity (severely reduced spontaneous activity, associated with hunchback posture, ruffled fur).

Thus, a second pre-test was performed on two mice using test item concentrations of 5 and 10 % (w/w). No signs of excessive local skin irritation or systemic toxicity were observed over the whole period of the second pre-test.

In a third pre-test, the test item concentration of 25 % (w/w) was evaluated in one mouse. On day 2 prior to the second application, the animal showed reduced spontaneous activity and an erythema of the ear skin (Score 1). On days 3 and 4, reduced spontaneous activity was again noted and on day 3-5, an erythema of the ear skin (Score 2) was observed. On day 6, reduced spontaneous activity was again observed, also, the animal showed eyelid closure. 25 % was thus not suitable for use as high dose group in the main experiment.


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
-Name of the test method: Local Lymph Node Assay

- Criteria used to consider a positive response: A test item is regarded as a sensitiser in the LLNA if exposure to one or more test item concentration results in a 3-fold or greater increase in incorporation of 3HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.). Theestimated test item concentration required to produce a S.I. of 3 is referred to as the EC3 value.

TREATMENT PREPARATIONS AND ADMINISTRATION
The test item was placed into an appropriate container on a tared balance and acetone:olive oil (4+1 v/v) was added to achieve the required test item concentration. The different test item concentrations were prepared individually. The preparations were made freshly before each dosing occasion. Concentrations were in terms of material as supplied. A concentration control analysis of all three doses was performed as a separate study under
the responsibility of the sponsor and the results were reported in a separate report.

Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear with test item concentrations of 2.5 %, 5 % and 10 % (w/w) in acetone:olive oil (4+1 v/v). The application volume, 25 μL/ear/day, was spread over the entire dorsal surface 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.

Five days after the first topical application (day 6) 250 μL of phosphate-buffered saline (PBS) containing 20.2 μCi of 3HTdR (equivalent to
approximately 80.9 μCi/mL 3HTdR) were injected into each test and control mouse via the tail vein.

Approximately five hours after treatment with 3HTdR all mice were euthanised by intraperitoneal injection of Pentobarbital-sodium. The draining
lymph nodes were rapidly excised and pooled for each animal (2 nodes per animal) and weighted immediately using an analytical balance. Single cell
suspensions of pooled lymph node cells were prepared and after several washing steps the level of 3HTdR incorporation was measured by mean of
liquid scintillation counting .

After the lymph nodes were excised, both ears of mice were punched at the apical area using a biopsy punch. For each animal both punches were
immediately weighed (pooled per animal) using an analytical balance.
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 as well as for the DPM values (group mean DPM ± standard deviation).

The EC3 value was calculated according to the equation:
EC3 = (a-c) [(3-d)/(b-d)] + c
where EC3 is the estimated concentration of the test item required to produce a 3-fold increase in draining lymph node cell proliferative activity; (a,
b) and (c, d) are respectively the co-ordinates of the two pair of data lying immediately above and below the S.I. value of 3 on the local lymph node assay dose response plot.

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 test item groups and negative control group. For all statistical calculations SigmaStat for Windows (Version 2.0) was used. A One-Way-Analysis-of-Variance was used as statistical method. In case of significant results of the One-Way-ANOVA,
multiple comparisons were performed with the Dunnett test. Statistical significance was set at the five per cent level (p < 0.05).

The Dean-Dixon-Test and Grubb’s test were used for identification of possible outliers (performed with Microsoft Excel 2003).

However, both biological and statistical significance were considered together.

Results and discussion

Positive control results:
In the periodic positive control experiment with alpha-hexylcinnamaldehyde performed in August 2011 the EC3 (= estimated concentration for a
S.I. of 3) was calculated to be 12.1 %.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
S.I.: control (vehicle) : 1.00 2.5 % N-Vinylcaprolactam: 1.53 5 % N-Vinylcaprolactam : 1.75 10 % N-Vinylcaprolactam : 3.33 EC3 = (a-c) [(3-d)/(b-d)] + c = 9.0% (w/w) In this study S.I. of 1.53, 1.75, and 3.33 were determined with the test item at concentrations of 2.5, 5, and 10% (w/w) in acetone:olive oil (4+1 v/v), respectively. Based on the S.I.s obtained with 5 and 10% test item concentration, an EC3 value of 9.0% (w/w) was calculated.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
Mean DPM per animal (was determined by dividing the sum of the measured values from lymph nodes of all animals within a group by the number of animals in that group): control (vehicle) : 866.4 2.5 % N-Vinylcaprolactam: 1323.2 5 % N-Vinylcaprolactam : 1512.6 10 % N-Vinylcaprolactam : 2881.4

Any other information on results incl. tables

Viability/Mortality

No death occured during the study

Clinical Signs

The animals did not show any signs of systemic toxicity during the study period. On day 3, the animals treated with a test item concentration of 10% showed an erythema of the ear skin (Score 1). Animals treated with 2.5 and 5% test item concentration did not show any signs of local skin irritation.

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.

Lymph Nodes Weights and Cell Count

The measured lymph node weights and –cell counts of all animals treated were recorded after sacrifice. A statistically significant increase in lymph node weights was not observed in any of the test item treated groups in comparison to the vehicle control group. A statistically significant increase in lymph node cell count was observed in all tested dose groups in comparison to the vehicle control group ( p<0.05), and furthermore, the cutoff-value for a positive response regarding the lymph node cell count index of 1.55 reported for BALB/c mice was exceeded in the high dose group (index of 1.7).

Ear Weights

The measured ear weights of all animals treated were recorded after sacrifice. A statistically significant or biologically relevant increase in ear weights was not observed in any test item treated group in comparison to the vehicle control group. For BALB/c mice, a threshold for the ear weight index of 1.1 was reported for a positive response regarding ear skin irritation. This threshold was not exceeded in any test item treated group.

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU