Essential oil of Dipterocarpus (Dipterocarpaceae) obtained from the resin tapped from Dipterocarpus trees by steam distillation (gurjunene quality)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14-11-2017 to 13-12-2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2010

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 (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Essential oil of Gurjun obtained from the resin tapped from Dipterocarpus trees (Dipterocarpaceae) by steam distillation (Gurjunene quality)

EC Number:

947-839-7

IUPAC Name:

Essential oil of Gurjun obtained from the resin tapped from Dipterocarpus trees (Dipterocarpaceae) by steam distillation (Gurjunene quality)

Test material form:

liquid

Details on test material:

Name in used in report: Gurrjun Balsam oil (Gurjunene)
Test facility number 209144/A


SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: obtained from sponsor, Batch no. 2642704.
- Expiration date of the lot/batch: 28-02-2018 and extended to 28-12-2018 (30-03-2018)
- Purity test date: 20-09-2017


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room tempersture.

OTHER SPECIFICS: UVCB

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: obtained from sponsor, Batch no. 2642704.
- Test facility number 209144/A
- Expiration date of the lot/batch: 28-02-2018 and extended to 28-12-2018 (30-03-2018)
- Purity test date: 20-09-2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room tempersture.

OTHER SPECIFICS: UVCB

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 (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Young adult animals approximately 11 weeks old
- Weight at study initiation: 20.4 to 24.2 g
- Housing: up to 5 animals /polycarbonate cage, Makrolon MIII type; height 18 cm. Bedding: sterilized sawdust (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany)
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days
- Indication of any skin lesions: before the initiation of dosing, a health inspection was performed and any assigned animal considered unsuitable for use in the study were replaced by alternate animals obtained from the same shipment and maintained under the same environmental conditions.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C
- Humidity (%): 40-70%
- Air changes (per hr): At least 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12-hours light and 12-hours dark

IN-LIFE DATES: not specified

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

Pre-screen test: 50 and 100% (w/w)
Second pre-screen test: 2, 5, 10 and 25% (w/w)
Main test: 0, 1, 2 and 5% (w/w)

No. of animals per dose:

5

Details on study design:

PRE-SCREEN TESTS:
- Irritation: erythema and eschar formation observations were performed once daily on days 1-6 (on days 1-3 within 1 hour after dosing).
- Systemic toxicity: observations were performed once daily on days 1-6 (on days 1-3 between 3 and 4 hours after dosing).
- Ear thickness measurements: 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.
- Erythema scores:
0 No erythema
1 Very slight erythema (barely perceptible)
2 Well-defined erythema
3 Moderate to severe erythema (beet redness) to slight eschar formation (injuries in depth)
4 Severe erythema (beet redness) to eschar formation preventing grading of erythema

MAIN STUDY
In the main study, three experimental groups of five female CBA/J mice were topically treated with 25µL/ear test item concentrations on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with the vehicle alone (Aceton/Olive Oil (4:1 v/v)). Three days after the last exposure, all animals were injected with 3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised and pooled for each animal. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of disintegrations per minute (DPM) and a stimulation index (SI) was subsequently calculated for each group.

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response: A Stimulation Index (SI) is calculated for each group using the individual SI values. The individual SI is the ratio of the DPM/animal compared to the DPM/vehicle control group mean. If the results indicate a SI ≥ 3, the test item may be regarded as a skin sensitizer. Consideration was given to the EC3 value (the estimated test item concentration that will give a SI =3), EC3 value ≤ 2%: sub-category 1A, EC3 value > 2%: sub-category 1B.

TREATMENT PREPARATION AND ADMINISTRATION:
Test item dosing formulations (w/w) were homogenized in the vehicle (acetone/olive oil (4:1 v/v)) to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily and dosed within 4 hours after adding the vehicle to the test item. The dosing formulations were kept at room temperature until dosing. The dorsal surface of both ears was topically treated (25 μL/ear) with the test item, 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 item.

Positive control substance(s):

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

Statistics:

-DPM values are presented for each animal and for each dose group. A Stimulation Index (SI) is calculated for each group using the individual SI values.
-The individual SI is the ratio of the DPM/animal compared to the DPM/vehicle control group mean.
-The EC3 value (the estimated test item concentration that will give a SI =3) was determined, using linear interpolation.

Results and discussion

Positive control results:

The SI values calculated for the item concentrations 1, 2 and 5% were 1.9, 3.7, and 7.3 respectively.
An EC3 value of 19.2% was calculated for the positive control 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 13.2, 14.1, 17.3, 9.8, 17.8%, 18.0%,14.7% and 13.2%. The six-month reliability check with Alpha-hexylcinnamaldehyde indicates that the Local Lymph Node Assay as performed at Charles River Den Bosch was found a appropriate model for testing for contact hypersensitivity.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

EC3

Value:

1.6

Results 2

Parameter:

SI

Value:

1

Variability:

0.1

Test group / Remarks:

0%

Results 3

Parameter:

SI

Value:

1.9

Variability:

0.3

Test group / Remarks:

1% dose

Results 4

Parameter:

SI

Value:

3.7

Variability:

0.8

Test group / Remarks:

2% dose

Results 5

Parameter:

SI

Value:

7.3

Variability:

0.9

Test group / Remarks:

5% dose

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
- All auricular lymph nodes of the animals of the experimental and control groups were considered no rmal in size. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

DETAILS ON STIMULATION INDEX CALCULATION
- Mean DPM/animal values for the experimental groups treated with test item concentrations 1, 2 and 5% were 579, 1107 and 2179 DPM, respectively. The mean DPM/animal value for the vehicle control group was 298 DPM. The SI values calculated for the test item concentrations 1, 2 and 5% were 1.9, 3.7 and 7.3 respectively.

EC3 CALCULATION
- The EC3 value (the estimated item concentration that will give a SI=3) was determined based on the dose response relationship or calculated using linear interpolation. The test item elicits a SI ≥ 3 when tested at 2%. The data showed a dose response and an EC3 value of 1.6 was calculated.

CLINICAL OBSERVATIONS:
- The very slight erythema of the ears and/or scaliness for individual test item treated animals throughout the dosing groups between days 4 and 6 was considered not to have a toxicologically significant effect on the activity of the nodes.
-No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study.

BODY WEIGHTS
- Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

Applicant's summary and conclusion

Interpretation of results:

Category 1A (indication of significant skin sensitising potential) based on GHS criteria

Remarks:

based on CLP criteria ( Annex I of 1272/2008/EC)

Conclusions:

Based on the results of this study, the calculated EC3 value is 1.6%. Therefore, Gurjun balsam oil (Gurjunene) needs to be classified as category 1A skin sensitiser according to the classification criteria outlined in Annex I of 1272/2008/EC (CLP).

Executive summary:

The skin sensitisation potential of Gurjun balsam oil (Gurjunene) was tested according to OECDTG 429. Three experimental groups of five female CBA/J mice were treated with 25µL test item concentrations of 1, 2 or 5% w/w on three consecutive days, by open application on the ears. Test item concentrations selected for the main study were based on the results of a prescreen test. Five vehicle control animals were similarly treated, but with the vehicle alone (AcOO). Very slight erythema and/or scaliness was noted was considered not to have a toxicologically significant effect on the activity of the nodes. No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. 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. Body weights and body weight gain of experimental animals remained in the same range as controls over the main study period. Mean DPM/animal values for the experimental groups treated with test item concentrations 1, 2 and 5% were 579, 1107 and 2179 DPM respectively. The mean DPM/animal value for the vehicle control group was 298 DPM. The SI values calculated for the test item concentrations 1, 2 and 5% were 1.9, 3.7 and 7.3 respectively. The data showed a dose-response and an EC3 value (the stimated test item concentration that will give a SI =3) of 1.6% was calculated.Based on these results, Gurjun balsam oil (Gurjunene) needs to be classified as category 1A skin sensitiser according to the classification criteria outlined in Annex I of 1272/2008/EC (CLP).