3-methyl-1-(2,6,6-trimethylcyclohex-1-en-1-yl)penta-1,4-dien-3-ol

Skin sensitisation

Administrative data

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:

other: according to current guidelines, GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 22 July 2010

GLP compliance:

yes (incl. QA statement)

Remarks:

Harlan CCR GmbH

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

3-methyl-1-(2,6,6-trimethylcyclohex-1-en-1-yl)penta-1,4-dien-3-ol

EC Number:

226-006-5

EC Name:

3-methyl-1-(2,6,6-trimethylcyclohex-1-en-1-yl)penta-1,4-dien-3-ol

Cas Number:

5208-93-5

Molecular formula:

C15H24O

IUPAC Name:

(4E)-3-methyl-5-(2,6,6-trimethylcyclohex-1-en-1-yl)penta-1,4-dien-3-ol

Details on test material:

- Name of test material (as cited in study report):3-methyl-1-(2,6,6-trimethylcyclohex-1-en-1-yl)penta-1,4-dien-3-ol

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: CBA/CaOlaHsd, Harlan Laboratories, The Netherlands
- Age at study initiation: 9-11 weeks
- Weight at study initiation: 18.6 - 22.9 g
- Housing: group
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 + 2
- Humidity (%): 45-65
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

methyl ethyl ketone

Concentration:

Pretest 1: 50, 100%
Pretest 2: 10, 25%
Main test: 2, 5, 10%

No. of animals per dose:

Pretest 1/2: 2
Main test: 5

Details on study design:

RANGE FINDING TESTS:
Two mice were treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 50 and 100% once daily each on three consecutive days. Prior to the first application of the test item and before sacrifice the body weight was determined. Clinical signs were recorded at least once daily. Eventual signs of local irritation were graded concerning erythema of the ear skin. Furthermore, prior to the first application of the test item (day 1), on day 3 and before sacrifice (day 6) the ear thickness was determined using a micrometer. Additionally, for both animals, the ears were punched after sacrifice (day 6) at the apical area using a biopsy punch (Ø 8 mm corresponding to 0.5 cm2) and were immediately pooled per animal and weighed using an analytical balance. Eventual ear irritation was considered to be excessive if an erythema of the ear skin of a score value ≥3 was observed at any observation time and/or if an increase in ear thickness of ≥25% was recorded on day 3 or day 6.

At the tested concentrations both animals showed signs of systemic toxicity, such as eyelid closure and reduced spontaneous activity. Additionally, both animals showed an erythema of the ear skin (score 1 to 4). On day 3 the animal treated with 100% test item concentration was sacrificed, due to severe redness of ear skin, beginning eschar formation, and signs of pain. Furthermore, the animal treated with 50% test item concentration, showed an increase of ear weight above the threshold of 25%.

Therefore, a second pre-test was performed using test item concentrations of 10 and 25%. At the tested concentrations both animals did not show signs of toxicity. On day 3, both animals showed a scabby erythema of the ear skin (score 1 to 2). Furthermore, the animal treated with 25% test item concentration, showed an increase of ear weight above the threshold of 25%. Thus, the test item in the main study was assayed at 2, 5, and 10%. The highest concentration tested was the highest level that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed in the pre-experiment.

MAIN STUDY
Topical application
Each test group of mice was treated by (epidermal) topical application to the dorsal surface of each ear. The application volume, 25 μL/ear/day, was spread over the entire dorsal surface (diameter 8 mm) of each ear once daily for three consecutive days. A further group of mice (control animals) was treated with an equivalent volume of the relevant vehicle alone (control animals).

Administration of 3H-methyl-thymidine
Five days after the first topical application (day 6) 250 μL of phosphate-buffered saline containing 20.4 μCi of 3H-methyl thymidine (equivalent to 81.5 μCi/mL 3HTdR) were injected into each test and control mouse via the tail vein.

Determination of incorporated 3HTdR
Approximately five hours after treatment with 3HTdR all mice were euthanised by using CO2, which was, after harvesting of the lymph nodes, followed by cervical dislocation to ensure death. The draining lymph nodes were rapidly excised and pooled per animal (2 nodes per animal). Single cell suspensions (in phosphate buffered saline) of pooled lymph node cells were prepared. After washing, the lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 mL) and incubated at approximately +4 °C for at least 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 5 % trichloroacetic acid (1 mL) and transferred to scintillation vials with 10 mL of scintillation liquid and thoroughly mixed. The level of 3HTdR incorporation was then measured in a β-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1 mL-aliquots of 5 % trichloroacetic acid. The β-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute.

Determination of Lymph Node Weight and Cell Count
After excision, the lymph nodes were pooled per animal and weighed immediately using an analytical balance.
Furthermore, the lymph node cell count was determined for each animal. For this, the volume of the cell suspensions was adjusted to an equal final volume and vortexed. Subsequently, individual cell counts were determined using a cell counter.

Determination of Ear Weights
After the lymph nodes had been excised, both ears (left and right) of mice were punched at the apical area using a biopsy punch (Ø 8 mm corresponding to 0.5 cm2). For each animal both punches were immediately weighed (pooled per animal).

Positive control substance(s):

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

Statistics:

A statistical analysis was conducted on the DPM values, the ear weights, the lymph node weights and the lymph node cell count to assess whether the difference was statistically significant between the test item groups and negative control group. For all statistical calculations custom made statistical program ´R` Decision Tree was used. Statistical significance was set at the five per cent level (p < 0.05). The Dean-Dixon-Test and the Grubb’s test were used for detection of possible outliers (performed with custom made statistical program ´R` Decision Tree). An outlier animal (animal 4) was detected in both outlier tests but not excluded from any calculation, since exclusion of the outlier value would not change the overall test result.
However, both biological and statistical significance were considered together.

Results and discussion

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: 2% group: 3.83 5% group: 8.10 10% group: 8.28

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Vehicle: 702.6 +/- 295.6 2% group: 2690.6 +/-1878.4 5% group: 5691.0 +/- 1080.5 10% group: 5817.2 +/- 1338.5

Any other information on results incl. tables

Viability / Mortality

No deaths occurred during the study period.

Clinical Signs

Neither signs of systemic toxicity nor local skin effects were observed during the study period.

Body Weights

The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.

Lymph Node Weights and Cell Counts

A statistically significant and biologically relevant increase in lymph node weights and –cell counts was observed in all of the test item treated groups in comparison to the vehicle control group. The indices determined for the lymph node cell count exceeded the cut-off value for a positive response (1.55) in all dose groups (indices of 1.92, 2.48, and 2.61, respectively).

Ear Weights

A biologically relevant or statistically significant increase in ear weights was not observed. Furthermore, the cut-off value (1.1) of the ear weight index for a positive response regarding ear skin irritation was not reached or exceeded in any of the treated groups.