Reaction mass of cis-1-methyl-1-(4-methylcyclohexyl) ethyl acetate and trans-1-methyl-1-(4-methylcyclohexyl) ethyl acetate and cis- 4-isopropyl-1-methylcyclohexyl acetate and trans-4-isopropyl-1-methylcyclohexyl acetate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 March - 04 April 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was performed according to the OECD guideline 429 and in compliance with GLP without any deviations.

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:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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:

(1r,4r)-1-methyl-4-(propan-2-yl)cyclohexyl acetate; (1s,4s)-1-methyl-4-(propan-2-yl)cyclohexyl acetate; 2-[(1r,4r)-4-methylcyclohexyl]propan-2-yl acetate; 2-[(1s,4s)-4-methylcyclohexyl]propan-2-yl acetate

EC Number:

939-728-7

Molecular formula:

C12H22O2

IUPAC Name:

(1r,4r)-1-methyl-4-(propan-2-yl)cyclohexyl acetate; (1s,4s)-1-methyl-4-(propan-2-yl)cyclohexyl acetate; 2-[(1r,4r)-4-methylcyclohexyl]propan-2-yl acetate; 2-[(1s,4s)-4-methylcyclohexyl]propan-2-yl acetate

Details on test material:

- Name of test material (as cited in study report): Menthanyl acetate multiconstituent
- Physical state: Colorless, slightly amber liquid
- Analytical purity: 98.3 %
- Composition of test material (%):Trans para menthanyl 1 (17.4 %), trans para menthanyl 8 (27.3 %), cis para menthanyl 8 (33.9 %), cis para menthanyl 1 (19.7 %) and others (1.7 %)
- Lot/batch No.: 110996
- Purity test date: 10 September 2010
- Expiration date of the lot/batch: 09 September 2012
- Storage condition of test material: Stored between 2-8 °C, protected from light, humidity and under nitrogen environment

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France.
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 18-25 g
- Housing: Animals were housed by group of two (preliminary test) or four (main test) in polycarbonate cages (Techniplast 1145T, 435 cm2, 36.9 x 15.6 x 13.2 cm) containing autoclaved sawdust (SICSA, Alfortville, France). In the main test, on Day 6 before the [3H] methyl-thymidine (3H-TdR) injections, the animals were individually housed in disposable crystal polystyrene cages (22.0 cm x 8.5 cm x 8.0 cm).
- Diet: SSNIFF R/M-H pelleted diet (SSNIFF Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: Tap water (filtered using a 0.22 µm filter), ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2 °C
- Humidity: 30-70 %
- Air changes: Approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod: 12 h dark / 12 h light

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

other: Not applicable

Vehicle:

other: Not applicable

Induction 2

Concentration / amount:

Not applicable

Challenge

Challenge 1

Route:

other: Not applicable

Vehicle:

other: Not applicable

Challenge 2

Concentration / amount:

Not applicable

No. of animals per dose:

Not applicable

Details on study design:

Not applicable

Challenge controls:

Not applicable

Study design: in vivo (LLNA)

Vehicle:

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

Remarks:

The vehicle used for the test item and positive control was a mixture Acetone/Olive Oil (4/1, v/v) (AOO): - acetone: batch No. K41254613, supplied by Merck, - olive oil: batch No. 0001428703, supplied by Sigma

Concentration:

Preliminary test: 10, 25, 50 and 100 %
Main test: 5, 10, 25, 50 and 100 %

No. of animals per dose:

Preliminary screening test: 2 females/dose: right and left ear were treated with different concentrations
Main test: 4 females/dose

Details on study design:

RANGE FINDING TEST:
- Compound solubility: The test item was soluble in the first recommended vehicle, acetone/olive oil (4/1, v/v). A solution was obtained at all tested dilutions (10-50%).
- Irritation: for 3 consecutive days, the animals received applications of 25 µL of the dosage form preparations to the external surface of both ears (one concentration per ear). Measurement of the ear thickness (using a micrometer) was performed each day before treatment and 72 hours after the last application. Dryness of skin was noted on day 6 in the right ear of females 315 and 316 treated at 100%. No notable increase in ear thickness was observed at any tested concentrations. The highest concentration retained for the main test was therefore 100%.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: The results were expressed as disintegration per minute (dpm) per group. Stimulation indices (SI) were calculated according to the following formula: SI = dpm of treated group / dpm of control group. The test item was considered as a skin sensitizer when the SI for a dose group is higher than or equal to 3. Other relevant criteria such as radioactivity levels and ear thickness were also taken into account to evaluate the data.

TREATMENT PREPARATION AND ADMINISTRATION:
The test item was administered as a solution in the vehicle and was mixed with the required quantity of vehicle. Dose formulations were prepared by the CIT Pharmacy extemporaneously on the day of each administration. They were stored at between 2 and 8°C and delivered to the study room in brown flasks.
On days 1, 2 and 3, a dose-volume of 25 μL of the appropriate dose formulations was applied to the dorsal surface of both ears (one concentration per ear), using an adjustable pipette fitted with a plastic tip. In order to avoid licking, to ensure an optimized application of the test material and to facilitate ear thickness measurement, the animals were placed under light isoflurane anesthesia during the administration.
No massage was performed but the tip was used to spread the preparation over the application site. No rinsing was performed.

25 µL of test material at concentrations of 0 (vehicle control), 5, 10, 25, 50 and 100 % were applied to the dorsal surface of each ear on Days 1, 2 and 3. On Day 6, 250 µL NaCl 0.9 % containing 20 μCi of 3H-TdR (specific activity of 20 Ci/mmol) was injected into the tail vein of each experimental mouse. Five hours later, all mice were killed by a lethal intraperitoneal injection of pentobarbital sodium followed by a cervical dislocation and the auricular lymph node of each ear was excised. The lymph nodes were pooled for each experimental group. A single cell suspension of auricular lymph node cell (ALNC) was prepared by mechanical disaggregation in Petri dishes using the plunger of a syringe. LNC were washed with 0.9 % NaCl and precipitated with 5 % (w/v) trichloroacetic acid (TCA) at 4 °C. Pellets were re-suspended in 1 mL TCA and 3 mL of Ultima GoldxR scintillation fluid (Packard) was added in order to measure incorporation of 3H-TdR using β-scintillation counting.

Positive control substance(s):

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

Statistics:

No data

Results and discussion

Positive control results:

The threshold positive value of 3 for the SI was reached in the positive control group (SI = 7.90). The experiment was therefore considered valid.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: See table 7.4.1/1

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: See table 7.4.1/1

Any other information on results incl. tables

Table 7.4.1/1: Results of skin sensitization

Treatment and concentrations	No. of nodes per group	dpm per group	dpm per node	Stimulation index (SI)	Increase in ear thickness (% between Day 1 and Day 6)	Irritation level	EC3 value
Vehicle	8	2431	303.88	-	-2.86	-	-
5 %	8	3245	405.63	1.33	-3.88	I	42.12 %
10 %	8	2888	361.00	1.19	-5.50	I
25 %	8	4240	530.00	1.74	0.97	I
50 %	7	7611	1087.29	3.58	0.00	I
100 %	8	14907	1863.38	6.13	23.15	II
α-hexylcinnamaldehyde 25 %	8	19213	2401.63	7.90	-	-	-

                            

dpm = disintegrations per minute

I = non-irritant (increase in ear thickness < 10 %)

II = slightly irritant (increase in ear thickness = 10 to 25 %)

EC3 value = theoretical concentration resulting in a SI value of

stimulation index = dpm of treated group / dpm of control group

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

A significant lymphoproliferation (SI > 3) was noted at the concentration ≥ 50%. In the absence of local irritation, the significant lymphoproliferative responses observed were attributed to delayed contact hypersensitivity.
Under these test conditions, Menthanyl acetate multiconstituent is classified "R43: May cause sensitisation by skin contact" according to the Annex VI of the Directive 67/548/EEC and skin sensitiser Category 1B according to the Regulation (EC) N° 1272-2008 (CLP).

Executive summary:

In a local lymph node assay performed according to OECD Guideline 429 and in compliance with GLP, groups of CBA/J mice (4 females/dose) were exposed to 25 µL of Menthanyl acetate multiconstituent in Acetone/Olive oil (4/1, v/v) at concentrations of 0 (vehicle control), 5, 10, 25, 50 and 100 % (v/v) to the dorsal surface of both ears for three consecutive days. On Day 6, all animals were injected with 3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised and measured for radioactivity expressed as number of disintegrations per minute (DPM) and stimulation index. The test concentrations for the main study were determined from a preliminary study where dryness of skin was noted on day 6 in the right ear of females 315 and 316 treated at 100%. No notable increase in ear thickness was observed at any tested concentrations. The highest concentration retained for the main test was therefore 100%.

No clinical signs and no mortality were observed during the main test. Dryness of ear skin was observed on day 6 in all females at the concentration of 100%, associated with erythema in 3/4 females. Stimulation index for 5, 10, 25, 50 and 100 % were 1.33, 1.19, 1.74, 3.58 and 6.13, respectively. The calculated effective concentration inducing an SI of 3 (EC3) was 42 %. Positive control (α-hexylcinnamaldehyde) exhibited evidence of sensitisation (SI = 7.90).

A significant lymphoproliferation (SI > 3) was noted at the concentration 25%. In the absence of local irritation, the significant lymphoproliferative responses observed were attributed to delayed contact hypersensitivity.

Under these test conditions, Menthanyl acetate multiconstituent is classified "R43: May cause sensitisation by skin contact" according to the Annex VI of the Directive 67/548/EEC and skin sensitiser Category 1B according to the Regulation (EC) N° 1272-2008 (CLP).