Registration Dossier

Administrative data

Description of key information

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:
2010-08-02 to 2010-12-09
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and Guideline Study
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
July 22, 2010
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
30 May 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sulzfeld, Germany
- Age at study initiation: 8 – 12 weeks
- Weight at study initiation: 18.8 g – 24.1 g
- Housing: The animals were housed in fully air-conditioned rooms.Type of cage: Makrolon cage, type II Enrichment: PLEXX mouse tunnel (red, transparent) and nest building material Nestlets NES 3600 (PLEXX b.v.; AB Elst, Netherlands). No. of animals per cage: 1
- Diet: Kliba-Labordiät (Maus / Ratte Haltung “GLP”), Provimi Kliba SA, Kaiseraugst, Basel, Switzerland, ad libitum
- Water: Tap water ad libitum
- Acclimation period: at least 5 days before the first test substance application

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 – 24
- Humidity (%): 30 – 70
- Air changes (per hr): Central air-conditioning
- Photoperiod (hrs dark / hrs light): 12 h / 12 h (6.00 a.m. – 6.00 p.m. / 6.00 p.m. – 6.00 a.m.)

IN-LIFE DATES: From: 2010-07-21 To: 2010-08-09
Vehicle:
other: Methyl ethyl ketone
Concentration:
0, 0.75, 2.5, 7.5 % (w/w)
No. of animals per dose:
5 animals per dose
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: MEK was used as the vehicle because good solubility of the preparation was achieved.
- Irritation: At the tested concentration of 50% the animals showed severely increased lymph node weights and slightly increased ear weights.
- Lymph node proliferation response: After application of a 5% test-substance preparation the animals did not show any signs of local irritation as confirmed by the ear weight measurements, but considerable increase in lymph node weights was observed. Signs of systemic toxicity were not observed during the pre-tests.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: In order to reveal a possible induction of sensitization, the response in the draining lymph node after epicutaneous application of several concentrations of the test substance to the skin of the dorsal surface of both ears is determined by measuring 3H-thymidine incorporation into the lymph node cells. Additional parameters used to characterize the response are, lymph node cell count and, to a certain extent, lymph node weight. Because irritation by the test substance may also induce lymph node responses, the weights of ear punches taken from the area of test-substance application are determined as an indicator for inflammatory ear swelling due to any irritant action of the test substance.

TREATMENT PREPARATION AND ADMINISTRATION:
The study comprised three treatment groups and a vehicle control group. Each group consisted of 5 mice.
Randomization: Prior to first application, the animals were distributed to the individual groups, received their animal numbers and were allocated to the respective cages according to the randomization instructions of „Nijenhuis, A. and Wilf, H.S.: Combinatorial Algorithms, Academic Press, New York, San Francisco, London, 1978, pp. 62 – 64“.
Body weight determination: Individual body weights on day 0 prior to the first application and on day 5 prior to the sacrifice of the animals.
Signs and symptoms: No detailed clinical examination of the individual animals was performed but any obvious signs of systemic toxicity and/or local inflammation at the application sites were noted.
Mortality: A check for moribund and dead animals was made twice each workday (beginning and end) and once on Saturdays, Sundays and on public holidays.
Form of application: Epicutaneous application is simulating dermal contact with the compound which is possible to occur under practical use conditions.
Application volume: 25 μL per ear
Site of application: Dorsal surface of both ears
Frequency of application: 3 consecutive applications (day 0 – day 2) to the same application site
The animals of control group 1 and test groups 2-4 were treated with vehicle or test substance preparation
³H-thymidine injection: On study day five (about 66 to 72 hours after the last application of test substance to the ears) the mice were injected into a tail vein with 20 μCi of 3H-thymidine in 250 μL of sterile saline.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Stimulation Index (SI): A value calculated to assess the skin sensitization potential of a test substance that is the ratio of the proliferation in treated groups to that in the concurrent vehicle control group.
EC3 = estimated concentration needed to produce a stimulation index of 3;
EC1.5 = estimated concentration needed to produce a stimulation index of 1.5
Positive control results:
The sensitivity of mice (CBA/J, Charles River Laboratories, Research Models and Services, Germany GmbH, Sulzfeld, Germany) and the reliability of experimental techniques were assessed regularly using a known sensitizer as recommended by the test guidelines.
Positive results were consistently obtained over the years using several variations of the methods and different vehicles.
A list of tests including stimulation indices (fold of change as compared to the vehicle control) for 3H-thymidine incorporation and cell count is presented.
Parameter:
SI
Remarks on result:
other: Test Group / Treatment / Stimulation Index 1 * / vehicle MEK / 1.00 2 / 0.75% in MEK / 1.66 3 / 2.5% in MEK / 2.20 4 / 7.5% in MEK / 5.52 * Calculation on basis of 4 animals, as one animal died during 3H-thymidine injection
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
Test Group / Treatment / [DPM/Lymph Node Pair] / Stimulation Index 1 * / vehicle MEK / 580.0 / 1.00 2 / 0.75% in MEK / 962.1 / 1.66 3 / 2.5% in MEK / 1,273.6 / 2.20 4 / 7.5% in MEK / 3,202.2 / 5.52 - test group x / test group 1 (vehicle control) * Calculation on basis of 4 animals, as one animal died during 3H-thymidine injection.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

Key study:

The skin sensitizing potential of cyclohexylvinylether was assessed using the radioactive Murine Local Lymph Node Assay. The assay simulates the induction phase for skin sensitization in mice. It determines the response of the auricular lymph nodes on repeated application of the test substance to the dorsal skin of the ears.

Groups of 5 female CBA/J mice each were treated with 0.75%, 2.5% and 7.5% w/w preparations of the test substance in MEK (methyl ethyl ketone) or with the vehicle alone.

The study used 3 test groups and 1 control group. Each test animal was treated with 25 μL per ear of the appropriate test-substance preparation, applied to the dorsal surfaces of both ears for three consecutive days. The control group was treated with 25 μL per ear of the vehicle alone.

When applied as 7.5% preparation in MEK, the test substance induced a biologically relevant increase of 3H-thymidine incorporation into the cells from the auricular lymph nodes. There was a relevant increase in lymph node weights, as well.

Concomitantly, the increase of cell counts was biologically relevant (increase to 1.5 fold or above of control value = stimulation index (SI) ≥ 1.5) at this concentration.

The test-substance preparations did not cause any relevant increase in ear weights demonstrating the absence of ear skin irritation.

Thus it is concluded that cyclohexylvinylether exhibits a skin sensitizing potential in the Murine Local Lymph Node Assay under the test conditions chosen. The estimated concentration that leads to the SI of 3.0 for 3H-thymidine incorporation (EC 3) and the estimated concentration that leads to the SI of 1.5 for cell count (EC 1.5) was calculated by linear regression from the results of these concentrations to be 3.6% and 3.7%, respectively. (BASF, 2010).

Supporting study:

The potential of cyclohexylvinylether to cause delayed contact hypersensitivity in guinea-pigs was assessed by the Magnusson-Kligman Maximisation Test according to OECD Guideline 406.

The closely-clipped dorsa of ten male and ten female Dunkin-Hartley guinea-pigs was subject to intradermal injections of Freunds Complete Adiuvant, 30% v/v test substance in paraffin oil an 30% v/v test substance in the adjuvant on Day 1.

Seven days later the same area of skin was treated by topical application of test substance as supplied and the test site was covered by an occlusive dressing for 48 hours. The same induction procedures were carried out on a contemporaneous control group, except that the test material was replaced by vehicle in all doses.

On Day 22, all animals were challenged by occluded application of paraffin oil to the left flank and test substance as supplied and 30% v/v test substance in paraffin oil to two sites on the right flank. The occlusive dressings were removed on the following day and the condition of the test sites was assessed approximately 24 and 48 hours later.

Challenge application of test substance as supplied caused a significant response (slight erythema or a more marked reaction) in nineteen test and one control animals.

Challenge application of 30% v/v test substance in paraffin oil caused a significant response in twelve test and no control animal. Challenge application of paraffin oil alone caused a significant response in one test and one control animals.

It was concluded that, under the conditions of this study, repeated administration of cyclohexylvinylether caused delayed contact hypersensitivity in guinea-pigs (LSR, 1990).


Migrated from Short description of key information:
Cyclohexylvinylether was tested for skin sensitizing effects in a mouse LLNA test. The test item was concluded to be sensitizing in this test.
In addition, cyclohexylvinylether was tested for sensitizing effects in a skin maximisation test in guinea pigs. The test item also caused sensitisation in this test.

Justification for selection of skin sensitisation endpoint:
Only one guideline and GLP study available.

Justification for classification or non-classification

According to the results in the performed tests, cyclohexylvinylether was classified as skin sensitizer - Xi,R43 - May cause sensitisation by skin contact - according to Directive 67/548/EEC (DSD) and cat 1b - H317: May cause an allergic skin reaction - according to Regulation (EC) No 1272/2008 (CLP, GHS).