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

Endpoint summary

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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)

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

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.

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.

Key result

Parameter:

EC3

Value:

4.5

Parameter:

SI

Remarks:

³H-thymidine incorporation

Value:

0.82

Test group / Remarks:

1% in acetone

Parameter:

SI

Remarks:

³H-thymidine incorporation

Value:

2.44

Test group / Remarks:

3% in acetone

Parameter:

SI

Remarks:

³H-thymidine incorporation

Value:

5

Test group / Remarks:

10% in acetone

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.

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

The skin sensitizing potential of Trivinylcyclohexane 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 1%, 3% and 10% w/w preparations of the test substance in acetone or with the vehicle alone. The study used 3 test groups and 1 control group. Each test animal was applied with 25 μL per ear of the respective test-substance preparation to the dorsum of both ears for three consecutive days. The control group was treated with 25 μL per ear of the vehicle alone. Three days after the last application the mice were injected intravenously with 20 μCi of 3H-thymidine in 250 μL of sterile saline into a tail vein. About 5 hours after the 3H-thymidine injection, the mice were sacrificed and the auricular lymph nodes were removed. The weights of each animal’s pooled lymph nodes were determined. Thereafter lymph nodes were pooled group wise and further evaluated by measuring their cellular content and 3H-thymidine incorporation into the lymph node cells (indicators of cell proliferation). Moreover, a defined area with a diameter of 0.8 cm was punched out of the apical part of each ear and for each test group the weight of the pooled punches was determined in order to obtain an indication of possible skin irritation. No signs of systemic toxicity were noticed. 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. The 1%, 3% and 10% test-substance preparations caused minimal to slight increase in ear weights as indication of ear skin irritation without a clear concentration-response, which cannot fully explain the concentration dependent increase in lymph node parameters. Thus it is concluded that Trivinylcyclohexane shows a skin sensitizing effect in the Murine Local Lymph Node Assay under the test conditions chosen. 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.

Justification for classification or non-classification

Based on the results of the skin sensitization testing, the test item is classified as skin sensitization cat. 1B (H317) according to Regulation (EC) No 1272/2008 (CLP).