3,7-dimethylocta-1,3,6-triene

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 May 2010 to 03 Jun 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

24th April 2002

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

CIT, BP 563, 27005 Evreux, France

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

7-methyl-3-methyleneocta-1,6-diene

EC Number:

204-622-5

EC Name:

7-methyl-3-methyleneocta-1,6-diene

Cas Number:

123-35-3

Molecular formula:

C10H16

IUPAC Name:

7-methyl-3-methyleneocta-1,6-diene

Test material form:

liquid

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
- Age at study initiation: 9 weeks
- Weight at study initiation: 19.4 ± 0.8 g
- Housing: The animals were housed individually in disposable crystal polystyrene cages (22.00 cm x 8.50 cm x 8.00 cm). Each cage contained (except for the 5 hours following the 3H-TdR injections) autoclaved sawdust (SICSA, Alfortville, France).
- Diet: SSNIFF R/M-H pelleted maintenance diet (SSNIFF Spezialdiäten GmbH, Soest, Germany) ad libitum
- Water: tap water (filtered using a 0.22 micron filter) ad libitum
- Acclimation period: at least 5 days before the beginning of the study
- No contaminants are known to be present in the diet, drinking water or sawdust at levels which may be expected to interfere with or prejudice the outcome of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 30 to 70
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

0, 2.5, 5, 10, 25 and 50% / 25 µL

No. of animals per dose:

4

Details on study design:

PRE-SCREEN TESTS:
- The concentrations selected for the preliminary test were 10, 25, 50 and 100%.
- 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.
- Since the test item was irritant at the concentration of 100% in the preliminary test, the highest tested concentration retained for the main test was 50%.

MAIN STUDY
- During the induction phase, the test item, vehicle or reference item was applied over the ears for 3 consecutive days.
- On day 6, all animals of all groups received a single intravenous injection of 250 μL of 0.9% NaCl containing 20 μCi of 3H-TdR (specific activity of 20 Ci/mmol) via the tail vein.
- Approximately 5 hours later, the animals were killed by cervical dislocation and the auricular lymph nodes were excised. The lymph nodes were pooled for each experimental group. For each experimental group, a single cell suspension of Auricular Lymph Node Cells (ALNC) was prepared by mechanical disaggregation in Petri dishes with the plunger of a syringe.
- The irritant potential of the test item was assessed in parallel by measurement of ear thickness on days 1, 2, 3 and 6.

TREATMENT PREPARATION AND ADMINISTRATION:
- On days 1, 2 and 3, a dose-volume of 25 μL of the control or dosage form preparations was applied to the dorsal surface of both ears, using an adjustable pipette fitted with a plastic tip.
- In order to avoid licking and to ensure an optimized application of the test materials, the animals were placed under light isoflurane anesthezia during the administration.
- No massage was performed but the tip was used to spread the preparation over the application sites. No rinsing was performed between each application.

CLINICAL EXAMINATIONS
- The animals were observed at least once a day during the study for clinical signs, signs of morbidity or mortality.
- In the main test, the animals were weighed individually on the first day of the study (day 1) and on the day of sacrifice (day 6).
- On days 1, 2 and 3 (before each cutaneous application) and on day 6 (after sacrifice), the thickness of the left ear of each animal of the vehicle control and treated groups was measured using a micrometer.
- Any irritation reaction (erythema and edema) was recorded in parallel.
- Any other observation (coloration, presence of residual test item, …) was noted.

Positive control substance(s):

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

Results and discussion

Positive control results:

The threshold positive value of 3 for the SI was reached in the positive control group. The study was therefore considered valid.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

0.66

Test group / Remarks:

2.5%

Results 2

Parameter:

SI

Value:

0.96

Test group / Remarks:

5%

Results 3

Parameter:

SI

Value:

0.79

Test group / Remarks:

10%

Results 4

Parameter:

SI

Value:

2.59

Test group / Remarks:

25%

Results 5

Parameter:

SI

Value:

1.78

Test group / Remarks:

50%

Results 6

Key result

Parameter:

EC3

Value:

> 50

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
The stimulation index rose from 0.66 (concentration 2.5%) to 2.59 (concentration 25%). Therefore, the stimulation index was lower than 3 for the test item at any tested concentration.

CLINICAL OBSERVATIONS:
- Neither mortality nor clinical signs were observed during the study.
- No cutaneous reactions and no notable increase in ear thickness were observed at any of the tested concentrations.

BODY WEIGHTS
- The body weight change of treated animals was similar to that of control animals.

Applicant's summary and conclusion

Interpretation of results:

other: Not sensitizing

Remarks:

in accordance with CLP (1272/2008 and its updates)

Conclusions:

Under the experimental conditions of this study, the test item did not induce delayed contact hypersensitivity in the murine Local Lymph Node Assay.

Executive summary:

The skin sensitisation potential of the test substance has been tested using the Local Lymph Node Assay (LLNA) according to OECD TG 429 and GLP principles. Test concentrations were determined in a preliminary test ( including 10, 25, 50 and 100%). Since the test item was irritant at the concentration of 100%, the highest tested concentration retained for the main test was 50%. The application of the test substance at concentrations of 2.5, 5, 10, 25 and 50% in a vehicle of acetone and olive oil (4:1 v/v) for three consecutive days did not result in an increase in isotope incorporation which was greater than 3-fold. No clinical effects or mortality were observed. Based on the results, the substance was not considered to be a skin sensitizer.