1,3-divinylimidazolidin-2-one

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014-10-15 to 2015-07-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and 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)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

1,3-divinylimidazolidin-2-one

EC Number:

237-457-2

EC Name:

1,3-divinylimidazolidin-2-one

Cas Number:

13811-50-2

Molecular formula:

C7H10N2O

IUPAC Name:

1,3-diethenylimidazolidin-2-one

Details on test material:

- Name of test material (as cited in study report): 1,3-divinylimidazolidin-2-one
- Test item No.: 14/0450-1
- Physical state: Colourless to yellow solid
- Homogeneity: The test item appeared to be homogenous
- Analytical purity: 99.8 corr. area-%
- Lot/batch No.: P53/13
- Expiration date of the lot/batch: 11 August 2015
- Storage condition of test material: At room temperature

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS B.V. Inc. Postbus 6174 5960 AD Horst / The Netherlands
- Age at study initiation: Pre-test: 9 - 10 weeks: Main study: 10 - 11 weeks
- Weight at study initiation: Pre-test: 20.8 - 21.6 g; Main study: 20.1 - 20.4g
- Housing: grouped by test groups, Makrolon Type II (pre-test) / III (main study), with wire mesh top
- Diet: ad libitum 2018C Teklad Global 18 % protein rodent diet
- Water: ad libitum tap water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 (except for several hours)
- Humidity (%): 45-65 (except for several hours)
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

10, 25, 50 %

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: experiment was performed according to the recommendations given by OECD 429
- Irritation: 2 animals treated with 25 and 50 % on 3 consecutive days
- Lymph node proliferation response: no

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response:
a) exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at
least 3-fold or greater than that recorded in control mice, as indicated by the Stimulation Index
b) data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either
local toxicity or immunological suppression.

TREATMENT PREPARATION AND ADMINISTRATION:
- Animals were treated topically on the dorsal surface of each ear (25 μL/ear/day) on 3 consecutive days
- 5 days after first application 78.2 μCi/mL 3HTdR were injected
- 5 h after injection animals were sacrificed

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 validated statistical
program R Script DecisionTree_2.Rnw 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 validated statistical program R Script Outlier.Rnw). No outlier was detected.
However, both biological and statistical significance were considered together.

Results and discussion

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: - Vehicle control: 1.0 - 10 %: 1.16 - 25 %: 1.24 - 50 %: 1.41

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Mean DPM per animal (2 lymph nodes) - Vehicle control: 1264.1 ± 171.1 - 10 %: 1470.1 ± 273.0 - 25 %: 1568.5 ± 864.8 - 50 %: 1786.7 ± 737.6

Any other information on results incl. tables

Viability / Mortality

No deaths occurred during the study period.

 

Clinical Signs

No cases of mortality were observed. On day 3, 24 h after the second application, the animals treated with 50% of the test item showed reduced spontaneous activity, ruffled fur and an erythema of the ear skin (score: 1). One hour after the third application, three animals treated with

25% of the test item and all animals treated with 50% of the test item showed an erythema of the ear skin (score 1), and ruffled fur. Except for animal 12, all animals treated with 25 and 50% showed reduced spontaneous activity. Furthermore, the animals treated with 50% of the test item transiently showed partially closed eyes, tippy-toe walk and sunken flanks. On day 4, 24 h after the third application, all animals treated with 50% of the test item showed an erythema of the ear skin (score 1), as well as slightly reduced spontaneous activity, tippy-toe walk and hunched posture. The animals treated with 25% and 10% of the test item did not show any clinical symptoms. On day 5, the animals treated with 50% of the test item showed a very slight erythema of the ear skin (score 1).

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. No relevant body weight loss was observed in any dose group over the course of the study.

Lymph Node Weights and Cell Counts

The measured lymph node weights and –cell counts of all animals treated were recorded after sacrifice. A statistically significant or biologically relevant increase in lymph node weights or – cell counts was not observed in any of the test item treated groups in comparison to the vehicle

control group. For BALB/c mice, a cut-off value for the lymph node cell count index of 1.55 was reported for a positive response. The indices determined for the lymph node cell count did not reach or exceed this threshold.

Ear Weights

The measured ear weight of all animals treated was recorded on test day 6 (after necropsy). A biologically relevant or statistically significant increase in ear weights was not observed. Furthermore, the cut-off value (1.1) of the ear weight index for a positive response regarding ear skin irritation reported for BALB/c mice was not reached or exceeded in any of the treated groups.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

Migrated information

Conclusions:

The test item 1,3-divinylimidazolidin-2-one was not a skin sensitizer under the test conditions of this study.

Executive summary:

In order to study a possible skin sensitising potential of 1,3-divinylimidazolidin-2-one, three groups each of five female mice were treated once daily with the test item at concentrations of 10, 25, and 50% in DMF by topical application to the dorsum of each ear for three consecutive days. The test item could be dissolved in the vehicle. The appropriateness of the used concentrations

was previously assessed by a pre-experiment. A control group of five mice was treated with the vehicle DMF only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (3H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised, pooled per animal and immediately weighed. Furthermore, after excision of the lymph nodes, both ears of the mice were punched at the apical area using a biopsy punch and were immediately weighed pooled per animal using an analytical balance. Afterwards single cell suspensions of lymph node cells were prepared from pooled lymph nodes per animal. An aliquot of each cell suspension was used for determination of lymph node cell count. Subsequently the suspensions were washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in a

β-scintillation counter.

No cases of mortality were observed. On day 3, 24 h after the second application, the animals treated with 50% of the test item showed reduced spontaneous activity, ruffled fur and an erythema of the ear skin (score: 1). One hour after the third application, three animals treated with 25% of the test item and all animals treated with 50% of the test item showed an erythema of the ear skin (score 1), ruffled fur and burrowed themselves in the bedding. Except for animal 12, all animals treated with 25 and 50% showed reduced spontaneous activity. Furthermore, the animals treated with 50% of the test item transiently showed partially closed eyes, tippy-toe walk and sunken flanks. On day 4, 24 h after the third application, all animals treated with 50% of the test item showed an erythema of the ear skin (score 1), as well as slightly reduced spontaneous activity , tippy-toe walk and hunched posture. The animals treated with 25% and 10% of the test item did not show any clinical symptoms. On day 5, the animals treated with 50% of the test item showed a very slight erythema of the ear skin (score 1). However, no relevant body weight loss was observed in any dose group over the course of the study.

No statistically significant increase in ear weights was observed in any dose group in comparison to the vehicle control group. Furthermore, for BALB/c mice, a cut-off value of 1.1 for the ear weight index was reported for a positive response regarding ear skin irritation. None of the indices determined for the test item treated groups reached or exceeded this threshold.

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.). The estimated test item concentration required to produce a S.I. of 3 is referred to as the EC3 value. In this study Stimulation Indices (S.I.) of 1.16, 1.24 and 1.41 were determined with the test item at concentrations of 10, 25, and 50% in DMF, respectively.

In the mid dose group, for animal 11 and 12, only one lymph node per animal could be harvested. However, this was considered to be not relevant for the outcome of the study, since these two values were not detected as statistical outliers in two outlier tests. Furthermore, when these two DPM values were excluded to calculate the S.I. of the mid dose group, the S.I. of the mid dose

group did not change substantially (1.64 instead of 1.24) and still remained well below the threshold index of 3 for a positive response. Furthermore, the S.I. determined for the high dose group was well below 3.

No statistically significant increase in DPM value and also in lymph node cell count and lymph node weights was observed in any dose group in comparison to the vehicle control group. Furthermore, the cut-off value of 1.55 for a positive response regarding the lymph node cell count index reported for BALB/c mice was not reached or exceeded in any dose group.

The EC3 value could not be calculated, since none of the tested concentrations induced a S.I. greater than the threshold value of 3.