4-(cyclohex-1-en-1-yl)-3-methylbutan-1-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:

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)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Test guideline 3

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

Test material information

Constituent 1

Reference substance name:

4-(cyclohex-1-en-1-yl)-3-methylbutan-1-ol

EC Number:

806-826-5

Cas Number:

1359994-45-8

Molecular formula:

C11H20O

IUPAC Name:

4-(cyclohex-1-en-1-yl)-3-methylbutan-1-ol

Test material form:

liquid

Specific details on test material used for the study:

- Test substance storage In refrigerator (2-8°C) in the dark
- Description: Clear colourless liquid
- Expiry date: 19 March 2015

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Females nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: SPF-Quality
- Age at study initiation: approx. 10 weeks old
- Weight at study initiation: 19 - 24 g
- Housing: Animals were group housed in labeled Makrolon cages (MIII type; height 18 cm) containing sterilised sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany). Paper (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) and shelters (disposable paper corner home, MCORN 404, Datesand Ltd, USA) were supplied as cage-enrichment.
- Diet: pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany, ad libitum)
- Water: tap water, ad libitum
- Acclimation period: at least 5 days
- Indication of any skin lesions: At least prior to dosing. It was ensured that the animals were healthy and that the ears were intact and free from any abnormality.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24
- Humidity (%): 40-70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

0, 5, 10, and 25% w/w

No. of animals per dose:

5

Details on study design:

PRE-SCREEN TESTS:
Initially, two test substance concentrations were tested; a 50% and 100% concentration. The highest concentration was the maximum concentration as required in the test guidelines (undiluted for liquids). The test system, procedures and techniques were identical to those used in the main study except that the assessment of lymph node proliferation and necropsy were not performed. Two young adult animals per concentration were selected. Each animal was treated with one concentration on three consecutive days. Animals were group housed in labeled Makrolon cages (MII type, height 14 cm). Ear thickness measurements were conducted using a digital thickness gauge (Kroeplin C110T-K) prior to dosing on Days 1 and 3, and on Day 6.
Animals were sacrificed after the final observation. Based on the results of the initially treated animals, two additional animals per concentration were treated in a similar manner with two lower concentrations (10% and 25%) at a later stage.

MAIN STUDY
- Induction - Days 1, 2 and 3: The dorsal surface of both ears was topically treated (25 μL/ear) with the test substance concentration, at approximately the same time on each day. The concentrations were stirred with a magnetic stirrer immediately prior to dosing. The control animals were treated in the same way as the experimental animals, except that the vehicle was administered instead of the test substance.
- Excision of the nodes - Day 6: Each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline (PBS) (Merck, Darmstadt, Germany) containing 20 μCi of 3H-methyl thymidine (PerkinElmer Life and Analytical Sciences, Boston, MA, US). After approximately five hours, all animals were killed by intraperitoneal injection (0.2 mL/animal) with Euthasol® 20% (AST Farma BV, Oudewater, The Netherlands). The draining (auricular) lymph node of each ear was excised. The relative size of the nodes (as compared to normal) was estimated by visual examination and abnormalities of the nodes and surrounding area were recorded. The nodes were pooled for each animal in approximately 3 mL PBS.
- Tissue processing for radioactivity - Day 6: Following excision of the nodes, a single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel gauze (diameter 125 μm). LNC were washed twice with an excess of PBS by centrifugation at 200g for 10 minutes at 4oC. To precipitate the DNA, the LNC were exposed to 5% trichloroacetic acid (TCA) (Merck, Darmstadt, Germany) and stored in the refrigerator until the next day.
- Radioactivity measurements - Day 7: Precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL of Ultima Gold cocktail (PerkinElmer Life and Analytical Sciences, Boston, MA, US) as the scintillation fluid. Radioactive measurements were performed using a Packard scintillation counter (2800TR). Counting time was to a statistical precision of ± 0.2% or a maximum of 5 minutes whichever came first. The scintillation counter was programmed to automatically subtract background and convert Counts Per Minute (CPM) to Disintegrations Per Minute (DPM).
- Observations: Mortality/Viability Twice daily; Body weights On Day 1 (pre-dose) and Day 6 (prior to necropsy); Clinical signs Once daily on Days 1-6 (on Days 1-3 between 3 and 4 hours after dosing); Irritation Once daily on Days 1-6 (on Days 1-3 within 1 hour after dosing) according to the following numerical scoring system. Furthermore, a description of all other (local) effects was recorded.

Positive control substance(s):

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

Results and discussion

Positive control results:

The SI values of a reliability test with three concentrations of Hexylcinnamaldehyde in Acetone/Olive oil (4:1 v/v), performed not more than 6 months previously, calculated for the substance concentrations 5, 10 and 25%, were 1.2, 1.4 and 4.7 respectively. An EC3 value of 17.3% was calculated using linear interpolation. The calculated EC3 value was found to be in the acceptable range of 4.8 and 19.5%. The results of the 6 monthly HCA reliability checks of the recent years were 16.9, 14.4, 16.5, 14.5, 13.4 and 14.1%.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1.3

Test group / Remarks:

5%

Results 2

Key result

Parameter:

SI

Value:

1.1

Test group / Remarks:

10%

Results 3

Key result

Parameter:

SI

Value:

1.7

Test group / Remarks:

25%

Cellular proliferation data / Observations:

Pre-screen test:
- At 100%, it was noted on Day 1 that the eyes of both animals were partly closed. On Day two, before dosing, one animal was found dead and one animal was sacrificed for humane reasons. At 50%, lethargy and partly closed eyes were noted for both animals on Days 2 and 3. At a 10% or 25% test substance concentration no signs of systemic toxicity were noted. No irritation was observed in any of the animals examined. Variations in ear thickness during the observation period were less than 25% from Day 1 pre-dose values. Based on these results, the highest test substance concentration selected for the main study was a 25% concentration.
Main study
- Skin reactions / Irritation: No irritation of the ears was observed in any of the animals examined.
- Systemic toxicity (Body weights): No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. Body weights and body weight gain of the experimental animals remained in the same range as controls over the study period.
- Macroscopy of the auricular lymph nodes and surrounding area: 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.
- Radioactivity measurements and SI values: Mean DPM/animal values for the experimental groups treated with test substance concentrations 5, 10 and 25% were 364, 298 and 455 DPM, respectively. The mean DPM/animal value for the vehicle control group was 270 DPM. The SI values calculated for the substance concentrations 5, 10 and 25% were 1.3, 1.1 and 1.7, respectively.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met