Cyclohexane-1,2,4-triyltris(ethylene)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

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:

Cyclohexane-1,2,4-triyltris(ethylene)

EC Number:

220-668-9

EC Name:

Cyclohexane-1,2,4-triyltris(ethylene)

Cas Number:

2855-27-8

Molecular formula:

C12H18

IUPAC Name:

1,2,4-triethenylcyclohexane

Constituent 2

Reference substance name:

Trivinylcyclohexane

IUPAC Name:

Trivinylcyclohexane

Details on test material:

- Name of test material (as cited in study report):
- Physical state:liquid
- Analytical purity: 99.7 area-%
- Lot/batch No.: 8101/06/196

In vivo test system

Test animals

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, 97633 Sulzfeld
- Age at study initiation: 6 – 12 weeks
- Weight at study initiation: 18.9 g – 22.5 g
- Housing: single housing
- Diet (e.g. ad libitum): Kliba-Labordiät (Maus / Ratte Haltung “GLP”), Provimi Kliba SA, Kaiseraugst, Basel, Switzerland, ad libitum
- Water (e.g. ad libitum): Tap water, ad libitum
- Acclimation period: 7 days befor the first test-substance application

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 – 24°C
- Humidity (%): 30 - 70 %
- Air changes (per hr): fully air-conditioned rooms
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

other: acetone

Concentration:

Main test 1, 3 and 10%

No. of animals per dose:

5

Details on study design:

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 part of both ears
Frequency of application: 3 consecutive applications (day 0 – day 2) to the same application site
Mortality: Twice each workday (beginning and end) and once on Saturdays, Sundays and on public holidays.
The animals of control group 1 and test groups 2-4 were treated with vehicle or testsubstance preparation.
On study day five (about 66 to 72 hours after the last application of test substance to the ears) the mice were injected intravenously with 20 μCi of 3H-thymidine in 250 μl of sterile saline into a tail vein.
The animals were sacrificed on study day 5 about 5 hours after 3H-thymidine injection by cervical dislocation. Immediately after the death of each animal a circular piece of tissue (diameter 0.8 cm) was punched out of the apical part of each ear of all animals. The weight of the pooled punches was determined for each test group. These measurements serve for detecting a potential inflammatory ear swelling.

Results and discussion

Positive control results:

A concurrent positive control (reliability check) with a known sensitizer was not included into this study. Studies using the positive control substance Alpha-Hexylcinnamaldehyde, techn. 85% are performed twice a year in the laboratory in order to show that the test system is able to detect sensitizing compounds under the test conditions chosen.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

EC3

Value:

4.5

Results 2

Parameter:

SI

Remarks:

³H-thymidine incorporation

Value:

0.82

Test group / Remarks:

1% in acetone

Results 3

Parameter:

SI

Remarks:

³H-thymidine incorporation

Value:

2.44

Test group / Remarks:

3% in acetone

Results 4

Parameter:

SI

Remarks:

³H-thymidine incorporation

Value:

5

Test group / Remarks:

10% in acetone

Any other information on results incl. tables

Cell count, 3H-thymidine incorporation and lymph node weight:

When applied as 10% preparation in acetone, the test substance induced a biologically relevant response (increase to 1.5 fold or above of control value = stimulation index (SI) ≥1.5) in the auricular lymph node cell counts. Concomitantly, the increase of 3H-thymidine incorporation into the cells was biologically relevant (increase above the cut off stimulation index of 3) at this concentration. The 3% test substance preparation caused some increase in cellularity and 3H-thymidine incorporation into the lymph node cells, which failed to reach the cut off respective stimulation index and thus lie below the threshold of immunologic relevance. In addition there was an increase in lymph node weights induced by both concentrations. No clear cut change in lymph node parameters was observed in the test group treated with the 1% preparation.

Ear weight:

The 1%, 3% and 10% test-substance preparations caused minimal to slight increase in ear weights, without clear concentration-response.

Test group	Treatment	Cell count Stimulation Index	³H-thymidine incorporation Stimulation Index	Lymph Node Weight Stimulation Index	Ear Weight Stimulation Index
1	vehicle acetone	1.00	1.00	1.00	1.00
2	1% in acetone	0.99	0.82	1.19	1.06
3	3% in acetone	1.45	2.44	1.45	1.05
4	10% in acetone	2.72	5.00	2.10	1.10

The expected body weight gain was generally observed in the course of the study. No abnormalities were observed during general observation.

The threshold concentration for sensitization induction was >3% <10%. The estimated concentration that leads to the SI of 1.5 for cell count (EC 1.5) and the estimated concentration that leads to the SI of 3.0 for 3H-thymidine incorporation (EC 3) was calculated

by linear regression from the results of the 3% and 10% concentrations to be 3.3% and 4.5%, respectively.

Applicant's summary and conclusion

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

Thus it is concluded that Trivinylcyclohexane shows a skin sensitizing effect in the Murine Local Lymph Node Assay under the test conditions chosen.