(4S)-1-methyl-4-(6-methylhepta-1,5-dien-2-yl)cyclohexene

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

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)

Remarks:

Swiss GLP Monitoring Authorities

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

6-methyl-2-(4-methylcyclohex-3-enyl)hept-2,5-diene

EC Number:

241-610-9

EC Name:

6-methyl-2-(4-methylcyclohex-3-enyl)hept-2,5-diene

Cas Number:

17627-44-0

Molecular formula:

C15H24

IUPAC Name:

4-(1,5-dimethylhexa-1,4-dien-1-yl)-1-methylcyclohexene

Constituent 2

Reference substance name:

2-methyl-6-(4-methylcyclohex-3-en-1-ylidene)hept-2-ene

EC Number:

207-805-8

EC Name:

2-methyl-6-(4-methylcyclohex-3-en-1-ylidene)hept-2-ene

Cas Number:

495-62-5

Molecular formula:

C15H24

IUPAC Name:

4-(1,5-dimethylhex-4-en-1-ylidene)-1-methylcyclohexene

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Remarks:

CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Netherlands; B.V. Postbus 6174 NL - 5960 AD Horst / The Netherlands
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 7 - 12 weeks (beginning of acclimatization)
- Weight at study initiation: 16.4 g - 20.2 g (beginning of acclimatization period)
- Housing: In groups of four
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-/+3
- Humidity (%): 30-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

other: ethanol:water, 7:3 (v/v)

Concentration:

0.1%, 1%, 10%, 100% (undiluted)

No. of animals per dose:

4

Details on study design:

PRE-SCREEN TESTS:
No prescreen test reported

MAIN STUDY

TEST ITEM PREPARATION
The test item was placed into a volumetric flask on a tared Mettler balance and the vehicle was quantitatively added. The weight/volume dilutions were
prepared individually using a magnetic stirrer as homogenizer. Homogeneity of the test item in the vehicle was maintained during treatment with the magnetic stirrer. The preparations were made shortly before each dosing.

TOPICAL APPLICATION
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear lobe (left and right) with different test item concentrations. The application volume, 25 μI, was spread over the entire dorsal surface (0 - 8 mm) of each ear lobe once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals). A hair dryer was used to dry the ear's surface as quickly as possible to avoid loss of test item applied.

ADMINISTRATION OF 3H-METHYL THYMIDINE
3H-methyl thymidine (3HTdR) was purchased from Amersham International (Amersham product code no. TRA 31 O; specific activity, 2 Ci/mmol; concentration, 1 mCi/ml). Five days after the first topical application, all mice were administered with 250 μI of 76.78 μCi/ml 3HTdR (equal to 19.2 μCi 3HTdR) by intravenous injection via a tail vein.

DETERMINATION OF INCORPORATED 3HTDR
Approximately five hours after treatment with 3HTdR all mice were euthanized by intraperitoneal injection of VETANARCOL (Veterinaria AG, Zurich).
Draining lymph nodes were rapidly excised and pooled for each experimental group (8 nodes per group).
Single cell suspensions (phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 μm mesh size). After washing three times with phosphate buffered saline (approx. 10 ml) the lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 ml) and incubated at approximately +4 °C overnight for precipitation of macromolecules. The
precipitates were then resuspended in 5 % trichloroacetic acid (1 ml) and transferred to glass scintillation vials with 10 ml of 'Ultima Gold' scintillation liquid and thoroughly mixed.
The level of 3HTdR incorporation was then measured on a beta-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1 ml-aliquots of 5 % trichloroacetic acid.

INTERPRETATION OF DATA
A test item is regarded as a sensitizer in the LLNA if the following criteria are fulfilled:
- Eposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the STIMULATION INDEX.
-The data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

Positive control substance(s):

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

Statistics:

A statistical analysis was conducted for assessment of the dose-response relationship, and the EC3 value was calculated according to the equation
EC3 = (a-c)*[(3-d)/(b-d)] + c
where EC3 is the estimated concentration of the test item required to produce a 3-fold increase in draining lymph node cell proliferative activity; (a, b) and (c, d) are respectively the co-ordinates of the two pair of data lying immediately above and below the S.I. value of 3 on the local lymph node assay dose response plot.

Results and discussion

Positive control results:

STIMULATION INDICES of 2.9, 2.6 and 7.1 were determined with the test item ALPHA-HEXYLCINNAMALDEHYDE at concentrations of 5 %, 10 % and 25 % (w/v) in acetone:olive oil, 4: 1 (v/v).
An EC3 of 11.3 % (w/v) was theoretically calculated with STIMULATION INDICES of 2.6 and 7.1 at test item concentrations of 10 % and 25 % (w/v).

In vivo (LLNA)

Results

Parameter:

EC3

Value:

8.6

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
SI:
Test item 0.1%: 0.4
Test item 1%: 0.8
Test item 10%: 3.4
Test item 100%: 13.5

DETAILS ON STIMULATION INDEX CALCULATION
DPM (-background) / DPM per lymph node:
Vehicle: 2256 / 282
Test item 0.1%: 866 / 108
Test item 1%: 1789 / 224
Test item 10%: 7695 / 962
Test item 100%: 30358 / 3795

EC3 CALCULATION
SI values for EC3 calculation:
Test item 1%: 0.8
Test item 10%: 3.4
EC3 = (1-10) * [(3-3.4) / (0.8-3.4)] + 10 = 8.6

CLINICAL OBSERVATIONS:
No deaths occurred during the study period.
No test item-related clinical signs were observed in all animals except for the 100 % (undiluted) group:
About 1 hour after the first application a moderate swelling was observed at the dosing sites of both ears in all animals of the 100 % (undiluted) dose group. The swelling reduced to slight 5 days after the first dosing.
Two days after the first local application a slight general erythema was observed at the dosing sites of both ears in all animals of the 100 % (undiluted) dose group, lasting for the remaing 4 days assessed.

BODY WEIGHTS
The body weight of the animals, recorded at the start of acclimatization period and prior to necropsy, was within the range commonly recorded for animals of this strain and age.