reaction mass of 3-methyl-4-(2,6,6-trimethylcyclohex-2-en-1-yl)but-3-en-2-one and 1-(2,6,6-trimethylcyclohex-2-en-1-yl)pent-1-en-3-one

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From July 29 to August 11, 2004

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP study conducted similarly to OECD 429 Guideline with deviations: no ear thickness measurement; no range-finding test was performed. However, no sign or irritation or systemic toxicity were observed at the concentration giving a SI above 3. The supporting substance was only tested up to 50% without jutification, however it could be explained by the classification of the substance as skin irritant. The supporting substance is considered adequate for read-across purpose as data relates to one of the two isomers of the registered substance (see Iuclid section 13 for additional justification).

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

not tested up to 100% but at up to 50% with no justification, no data reported on clinical observations and local skin irritations and no preliminary study performed, no ear thickness measurement unless the absence of preliminary study

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

yes

Remarks:

same as above

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

3-methyl-4-(2,6,6-trimethyl-2-cyclohexen-1-yl)-3-buten-2-one

EC Number:

204-846-3

EC Name:

3-methyl-4-(2,6,6-trimethyl-2-cyclohexen-1-yl)-3-buten-2-one

Cas Number:

127-51-5

Molecular formula:

C14H22O

IUPAC Name:

3-methyl-4-(2,6,6-trimethylcyclohex-2-en-1-yl)but-3-en-2-one

Test material form:

other: Liquid

Details on test material:

- Physical state: Colourless liquid
- Storage condition of test material: Stored at ambient temperature in the dark.

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/Ca/Ola/Hsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Interfauna UK Limited, Blackthorne, Bicester, Oxon, UK
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 16.8-20.6 g
- Housing: Four animals were housed per cage.
- Diet: RM1 (supplied by Special Diet Services Limited, Witham, Essex, UK), ad libitum
- Water: Mains water (supplied by an automatic system), ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 30-70 %
- Air changes: A minimum of 15 changes/h
- Photoperiod: 12 h dark/ 12 h light

IN-LIFE DATES: From: August 04, 2004 To: August 11, 2004

Study design: in vivo (LLNA)

Vehicle:

other: ethanol:diethyl phthalate (1:3)

Concentration:

2.5, 5, 10, 25 and 50 % w/v in 1:3 ethanol:diethyl phthalate

No. of animals per dose:

4 females/dose

Details on study design:

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: The criterion for a positive response is that one or more concentrations of the test material should elicit a 3-fold or greater increase in isotope incorporation relative to the vehicle control group. A test material which does not fulfil the above criterion is designated as unlikely to be a skin sensitiser.

TREATMENT PREPARATION AND ADMINISTRATION:
- All dose preparations were used within 24 h of preparation. Approximately 25 µL of control or test material were applied to the dorsal surface of both ears daily for 3 consecutive days. Three days after the third application, all animals were injected via the tail vein with 250 µL of phosphate buffered saline (PBS) containing 20 µCi of a 2 Ci/mmol specific activity 3H-methyl thymidine. Approximately 5 h later, all animals were killed and the draining auricular lymph nodes were removed from each animal, together with the nodes from the other animals in the group in PBS. A single cell suspension was prepared by mechanical disaggregation of lymph nodes through 200-mesh stainless steel gauze. The cell suspensions were then washed three times by centrifugation with approximately 10 mL of PBS. Approximately 3 mL of 5 % w/v trichloroacetic acid (TCA) was added and, after overnight precipitation at 4°C, the samples were pelleted by centrifugation and the supernatant was discarded. The cells were then resuspended in approximately 1 mL of TCA. The lymph node suspensions were transferred to scintillation vials and 10 mL of scintillant (Optiphase) was added prior to β-scintillation counting using a Packard Tri-Carb Liquid Scintillation Counter. Disintegrations per minute (DPM) values were presented for each dose group. EC3 value was calculated as percentage dose and µg/cm2.

Positive control substance(s):

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

Statistics:

None

Results and discussion

Positive control results:

Stimulation index for positive control group treated with 10 and 25 % w/v of hexylcinnamicaldehyde in acetone: olive oil (4:1) was found to be 3.3 and 10.9 respectively; classified as skin sensitiser.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: Stimulation index for test material at 2.5, 5, 10, 25 and 50 % w/v were 0.6, 0.6, 1.5, 3.4 and 4.6, respectively.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: DPM/group for 0 (vehicle), 2.5, 5, 10, 25 and 50 % w/v were 3254, 1997, 2012, 4848, 11008 and 14974, respectively.

Any other information on results incl. tables

- Body weight: The mean body weight of positive control and treatment group animals was comparable to that of the vehicle control group.

- EC3 value: The EC3 value calculated for the test material was found to be 21.8 % w/v (5450 µg/cm2).

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Under the test conditions, test material is classified as “Category 1” skin sensitiser according to the annex VI of the Regulation EC No. 1272/2008 (CLP).

Executive summary:

In a Local Lymph Node Assay (LLNA) performed similarly to OECD Guideline 429 and in compliance with GLP, groups of female CBA/Ca/Ola/Hsd mice (4 females/group) were topically applied with test material at the dose concentrations of 2.5, 5, 10, 25 and 50 % w/v in 1:3 ethanol:diethyl phthalate on the dorsal surface of both ears (25 µL/ear) daily for three consecutive days. A vehicle control group was treated using 1:3 ethanol:diethyl phthalate alone and a positive control group was treated with α-hexylcinnamaldehyde at the dose concentration of 5, 10 and 25 % w/v in acetone:olive oil (4:1) in same manner to confirm the sensitivity and reliability of the test method. On Day 6, the proliferation of lymph node cells in the lymph node draining the application site was measured by incorporation of 3H-methyl thymidine and stimulation index (SI) was calculated. Body weight of individual animal was recorded prior to dosing on Day 1 and prior to injection of 3H-methyl thymidine on Day 6.

Body weight of positive control and treatment group animals was comparable to that of the vehicle control group. Mean DPM / animal for 0 (vehicle), 2.5, 5, 10, 25 and 50 % w/v were 3254, 1997, 2012, 4848, 11008 and 14974, respectively. Stimulation Index (SI Value) calculated for test material treated groups was found to be 0.6, 0.6, 1.5, 3.4 and 4.6 for the dose concentrations of 2.5, 5, 10, 25 and 50 % w/v, respectively.

The EC3 value calculated for the test material was found to be 21.8 % w/v (5450 µg/cm2).

Stimulation index for positive control group treated with 10 and 25 % w/v of hexylcinnamicaldehyde in acetone: olive oil (4:1) was found to be 3.3 and 10.9, respectively. Therefore, hexylcinnamaldehyde was shown to be a skin sensitiser and confirming the validity of the study.

Under the test conditions,test material is classified as “Category 1” skin sensitiser according to the annex VI of the Regulation EC No. 1272/2008 (CLP).

This study is considered as acceptable and satisfies the requirement for sensitisation endpoint. The supporting substance is considered adequate for read-across purpose as data relates to one of the two isomers of the registered substance (see Iuclid section 13 for additional justification).