Reaction mass of 4-Vinylcyclohexene, Cyclododeca-1,5,9-triene (1Z,5E,9E) and Cycloocta-1,5-diene

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 June 2014 - 24 June 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

(1E,5E,9E)-cyclododeca-1,5,9-triene; (1Z,5Z)-cycloocta-1,5-diene; 4-ethenylcyclohex-1-ene

EC Number:

940-766-1

Molecular formula:

unspecified

IUPAC Name:

(1E,5E,9E)-cyclododeca-1,5,9-triene; (1Z,5Z)-cycloocta-1,5-diene; 4-ethenylcyclohex-1-ene

Details on test material:

- Name of test material (as cited in study report): 4-Vinyl-1-cyclohexen (constituent of registered reaction mass)
- Physical state: Liquid, clear colorless
- Analytical purity: 99.8 area-%
- Lot/batch No.: 20140404

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories, Horst / The Netherlands
- Age at study initiation: Pre-test: 8 - 9 weeks (beginning of treatment); Main study: 9 - 10 weeks (beginning of treatment)
- Housing: group (Makrolon Type II (pr-test9/ III (main study with wire mesh top
- Diet (e.g. ad libitum): ad libitum (2018C Teklad Global 18% protein rodent diet (certified))
- Water (e.g. ad libitum): ad libitum (tap water)
- Acclimation period: at least 5 days prior

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

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

10, 25 and 50% (w/w)

No. of animals per dose:

5

Details on study design:

Each test group of mice was treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 10, 25 and 50% in acetone/olive oil (4+1, v/v). 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 relevant vehicle alone (control animals).
Five days after the first topical application (day 6) 250 μL of phosphate-buffered saline containing 20 μCi of 3H-methyl thymidine (equivalent to 80 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 using CO2, which was, after harvesting of the lymph nodes, followed by cervical dislocation to ensure death.
After excision, the lymph nodes were pooled per animal and weighed immediately using an analytical balance.

Positive control substance(s):

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

Statistics:

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 SigmaStat for Windows (Version 2.0) was used. A One-Way-Analysis-of-Variance was used as a 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 was used for detection of possible outliers (performed with Microsoft Excel 2007). However, both biological and statistical significance were considered together.

Results and discussion

Positive control results:

The sensitivity and reliability of the experimental technique employed was assessed by use of α-hexyl cinnamaldehyde dissolved in acetone/olive oil (4+1, v/v) (compound listed in OECD 429 Guideline) which is known to have skin sensitisation properties in mice. The periodic positive control experiment was performed using CBA/CaOlaHsd mice in April 2014.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1

Test group / Remarks:

for the control (vehicle) the S.I. was 1.00

Results 2

Parameter:

SI

Value:

0.5

Test group / Remarks:

10% (w/w) in acetone/olive oil (4+1, v/v)

Results 3

Parameter:

SI

Value:

1.16

Test group / Remarks:

25% (w/w) in acetone/olive oil (4+1, v/v)

Results 4

Parameter:

SI

Value:

1.74

Test group / Remarks:

50% (w/w) in acetone/olive oil (4+1, v/v)

Any other information on results incl. tables

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 and biologically relevant increase in DPM values and lymph node cell count was not observed in any dose groups in comparison to the vehicle control group. A statistically significant increase in lymph node weights was observed in the high dose group in comparison to the vehicle control group. This was considered to be not biologically relevant, as the corresponding DPM value was not statistically significant increased in comparison to the vehicle control. 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 exceed this threshold. Moreover, the cut-off value for a positive response regarding the lymph node cell count index reported for BALB/c mice was not exceeded in any of the test item groups.

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 exceeded in any of the treated groups.

 

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

 

Calculation of Stimulation Indices per Dose Group

Test item concentration	Group Calculation
	Mean DPM per animal (2 lymph nodes)^a)	SD	S.I.
Vehicle Control Group (acetone:olive oil (4+1, v/v))	1877.6	710.5	1.00
10% 4-Vinyl-1-cyclohexen	944.4	273.5	0.50
25% 4-Vinyl-1-cyclohexen	2182.0	1617.9	1.16
50% 4-Vinyl-1-cyclohexen	3270.4	405.6	1.74

a) Mean DPM/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 (5 animals)

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item 4-Vinyl-1-cyclohexen was thus not a skin sensitiser under the test conditions of this study.

Executive summary:

In this study the test item 4-Vinyl-1-cyclohexen was assessed for its skin sensitising potential using the Local Lymph Node Assay (LLNA) in mice. Test item solution at different concentrations was prepared in the vehicle acetone/olive oil (4+1, v/v). The local lymph node assay is recommended by international test guidelines (e.g., OECD) as an animal test for predicting skin sensitisation in humans and provides a rational basis for risk assessment. The basic principle underlying the LLNA is that sensitisers induce a primary proliferation of lymphocytes in the lymph node draining the application site. The ratio of proliferation in test item treated groups compared to that in vehicle controls is termed the Stimulation Index (S.I.). Radioactive labeling is used to measure cell proliferations. For this purpose a local lymph node assay was performed using test item concentrations of 10, 25 and 50% (w/w). The highest concentration tested was the highest concentration that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation (as determined by a pre-experiment). The animals did not show any signs of systemic toxicity during the course of the study and no cases of mortality were observed. On day 3, the animals treated with a test item concentration of 25% showed an erythema of the ear skin (score 1). The animals treated with a test item concentration of 50% showed an erythema of the ear skin (score 1) on day 3 and 4. Animals treated with 10% test item concentration did not show any signs of local skin irritation. A statistically significant or biologically relevant increase in ear weights was not observed in any treated group in comparison to the vehicle control group. 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 3 HTdR 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 0.50, 1.16 and 1.74 were determined with the test item at concentrations of 10, 25 and 50% (w/w) in acetone/olive oil (4+1, v/v), respectively. A statistically significant and biologically relevant increase in DPM values and lymph node cell count was not observed in any dose groups in comparison to the vehicle control group. A statistically significant increase in lymph node weights was observed in the high dose group incomparison to the vehicle control group. This was considered to be not biologically relevant, as the corresponding DPM values were not statistically significant increased in comparison to the vehicle control and as the S.I. determined for the high dose group did not reach the value of 3. 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 exceed this threshold at all tested concentrations. The test item 4-Vinyl-1-cyclohexen was thus not a skin sensitiser under the test conditions of this study.