N-(1,3-dimethylbutylidene)-5-[(1,3-dimethylbutylidene)amino]-1,3,3-trimethylcyclohexanemethylamine

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 September 2014 - 25 September 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)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Reference

Name:

Unnamed

Type:

Constituent

Test material form:

liquid

Specific details on test material used for the study:

Identification: CAS#71077-09-3
Batch: 76200
Purity: 98,70%
Physical state: Pale yellow liquid
Expiry date 04 March 2015
Storage conditions: room temperature in dark

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan laboratories UK Ltd, Oxon, UK
- Age at study initiation: 8-12 weeks old
- Weight at study initiation: 15-23g
- Diet (e.g. ad libitum): Free access to tap water and certified laboratory food (2014C Teklad Global Rodent Diet)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25 °C
- Humidity (%): 30-70 %
- Air changes (per hr): 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

A preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25ul of the test item at a concentration of 50% v/v in acetone/olive oil 4:1, to the dorsal surface of each ear for three consecutive days (D 1, 2, 3). Clinical signs and irritation were recorded daily until day 6. The thickness of ear was measured on D1, D3 and D6.

In the main test the substance was tested at three concentrations (10 %, 25 % and 50 %) in acetone/olive oil 4:1. The mice were treated by daily application og 25ul of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (D 1, 2, 3).

No. of animals per dose:

5 animals / dose

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: The test item was soluble in acetone/olive oil based on trial preparations performed at Charles River Den Bosch and on information provided by the Sponsor.
- Irritation: No signs of visual local skin irritation or irritation indicated by an equal to or greater than 25% increase in mean ear thickness were noted.
- Systemic toxicity: No signs of systemic toxicity

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
Animals were assigned to the study at the discretion of the coordinating biotechnician according to body weights, with all animals within ± 20% of the sex mean. Animals in poor health or at extremes of body weight range were not assigned to the study.

- Criteria used to consider a positive response: If the results indicate a SI ≥ 3, the test item may be regarded as a skin sensitizer.

TREATMENT PREPARATION AND ADMINISTRATION:
The dosing formulations were prepared daily and dosed within 2 hours after adding the vehicle to the test item. The dorsal surface of both ears was topically treated (25 μL/ear) with the test item, at approximately the same time on each day.
The control animals were treated in the same way as the experimental animals, except that the vehicle was administered instead of the test item.

Positive control substance(s):

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

Statistics:

DPM values are presented for each animal and for each dose group. A Stimulation Index (SI) is calculated for each group using the individual SI values. The individual SI is the ratio of the DPM/animal compared to the DPM/vehicle control group mean.
If the results indicate a SI ≥ 3, the test item may be regarded as a skin sensitizer.

Results and discussion

Positive control results:

A reliability check is carried out at regular intervals to check the sensitivity of the test system and the reliability of the experimental techniques as used by Charles River Den Bosch

Based on the results, it was concluded that the Local Lymph Node Assay as performed at Charles River Den Bosch is an appropriate model for testing for contact hypersensitivity.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

21.3

Test group / Remarks:

10% test item

Results 2

Key result

Parameter:

SI

Value:

22.81

Test group / Remarks:

25% test item

Results 3

Key result

Parameter:

SI

Value:

30.98

Test group / Remarks:

50% test item

Results 4

Key result

Parameter:

EC3

Value:

< 10

Cellular proliferation data / Observations:

DETAILS ON STIMULATION INDEX CALCULATION
Mean DPM/animal values for the experimental groups treated with test item concentrations 10, 25 and 50% were 50 862, 54 461 and 73 980 DPM, respectively. The mean DPM/animal value for the vehicle control group was 2 388 DPM.

EC3 CALCULATION
It was established that the EC3 value (the estimated test item concentration that will give a SI =3) is lower than 10 %.

CLINICAL OBSERVATIONS:
No mortality occurred and no clinical signs of systemic toxicity were observed in the animals.

BODY WEIGHTS
Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

The skin sensitising potential of the test item was assessed using murine local lymph node assay (LNNA). The substance was considered sensitiser in the LLNA study under the experimental conditions described in this report.

Executive summary:

The objective of this study was to evaluate whether the substance induces skin sensitization in mice after three epidermal exposures of the animals.

Test item concentrations selected for the main study were based on the results of a pre-screen test. The preliminary screening test suggested that the test item would not produce systemic toxicity or excessive local irritation at the highest suitable concentration.

 

In the main study, three experimental groups of five female mice were treated with test item concentrations of 10, 25% or 50 v/v on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with the vehicle alone (Acetone/Olive oil (4:1 v/v)). Three days after the last exposure, all animals were injected with³H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised and pooled for each animal. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of disintegrations per minute (DPM) and a stimulation index (SI) was subsequently calculated for each group.

All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

The SI values calculated for the test item concentrations 10, 25 and 50% were 21.3, 22.8 and 31.0, respectively.

Based on these results, the test item is regarded as a skin sensitizer according to the recommendations made in the test guidelines. The test item have to be classified for sensitization by skin contact according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments) and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).