Essential oil of Cedarwood Texas obtained from the wood of Juniperus mexicana (Cupressaceae) by distillation - Terpenes

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 Mar 2017 - 19 Apr 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:

2017

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:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

2003

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: EC, No 440/2008, part B "Skin Sensitization: Local Lymph Node Assay"

Version / remarks:

2008

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:

2,4a,8,8-tetramethyl-1H,1aH,4H,4aH,5H,6H,7H,8H-cyclopropa[e]naphthalene; 2,6,6,8-tetramethyltricyclo[5.3.1.0¹,⁵]undec-8-ene

EC Number:

946-670-6

Molecular formula:

Not applicable due to UVCB nature of the substance

IUPAC Name:

2,4a,8,8-tetramethyl-1H,1aH,4H,4aH,5H,6H,7H,8H-cyclopropa[e]naphthalene; 2,6,6,8-tetramethyltricyclo[5.3.1.0¹,⁵]undec-8-ene

Test material form:

liquid

Remarks:

Almost colourless, very pale yellow liquid

Details on test material:

Name of test material as cited in study report: Cedarwood Texas oil Terpenes 2 (Thujopsene)
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Obtained from sponsor; batch: B-64543
- Expiration date of the lot/batch: 25 January 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was tested neat.

OTHER SPECIFICS: UVCB

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: Batch: B-64543, provided by sponsor
- Expiration date of the lot/batch: 25 January 2019
- Purity test date: 26 January 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
Test item dosing formulations (w/w) were homogenized 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 dosing formulations were stirred until and during dosing.

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Remarks:

/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
- Microbiological status of animals: SPF-quality
- Age at study initiation: approx. 11 weeks old
- Weight at study initiation: 20.4 to 25.0 g
- Housing: animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Makrolon MIII type; height 18 cm.) containing sterilized sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles.
- Diet: ad libitum, pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany)
- Water: ad libitum, municipal tap-water (periodically analysed)
- 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
- Humidity (%): 42 to 47
- Air changes (per hr): >10 (no recirculation)
- Photoperiod (hrs dark / hrs light): 12 / 12
- IN-LIFE DATES: Study start:13 Mar 2017 To: 19 Apr 2017

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

0% (w/w), 5% (w/w), 10% (w/w), 25% (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 treated with test item concentrations of 0% (w/w), 5% (w/w), 10% (w/w), 25% (w/w) 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:
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. 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.
- Consideration was given to the EC3 value (the estimated test item concentration that will give a SI =3)

Results and discussion

Positive control results:

The SI values calculated for the item concentrations 5, 10 and 25% were 1.4, 2.4 and 4.3 respectively. An EC3 value of 14.7% 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 13.4, 14.1, 17.3, 9.8, 17.8% and 18.0%.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

Parameter:

SI

Value:

1

Variability:

0.1

Test group / Remarks:

0% dose

Results 2

Parameter:

SI

Value:

1.2

Variability:

0.2

Test group / Remarks:

5% dose

Results 3

Parameter:

SI

Value:

1.3

Variability:

0.1

Test group / Remarks:

10% dose

Results 4

Key result

Parameter:

SI

Value:

2.1

Variability:

0.3

Test group / Remarks:

25% dose

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
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.

DETAILS ON STIMULATION INDEX CALCULATION
Mean DPM/animal values for the experimental groups treated with test item concentrations 5, 10 and 25% were 937, 1021 and 1656 DPM, respectively. The mean DPM/animal value for the vehicle control group was 784 DPM. The SI values calculated for the test item concentrations 5, 10 and 25% were 1.2, 1.3 and 2.1, respectively.

EC3 CALCULATION
Since there was no indication that the test item elicits a SI ≥ 3 when tested up to 25%, Terpenes 2 (Thujopsene), Cedarwood Texas oil distilled was not considered to be a skin sensitizer. It was established that the EC3 value (the estimated test item concentration that will give a SI =3) (if any) exceeds 25%.

CLINICAL OBSERVATIONS:
No erythema was noted for any of the animals, scaliness was noted for the majority of animals treated at a 10% and 25% test item concentration between Days 4 and 6.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:

other: not sensitising

Remarks:

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

Conclusions:

Based on the results of this LLNA, the Cedarwood Texas oil distilled - Terpenes (Thujopsene) does not need to be classified as skin sensitiser according to the classification criteria outlined in Annex I of 1272/2008/EC (CLP).

Executive summary:

The skin sensitisation potential of  Cedarwood Texas oil distilled - Terpenes (Thujopsene) was tested according to  OECD, Section 4, Health Effects, No.429 (2010). Three experimental groups of five female CBA/J mice were treated with test item concentrations of 5, 10 or 25% 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 pre-screen test. Five vehicle control animals were similarly treated, but with the vehicle alone (AcOO).  No erythema was noted for any of the animals, scaliness was noted for the majority of animals treated at a 10% and 25% test item concentration between Days 4 and 6. 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. The mean body weight gain shown by the animals over the study period was considered to be normal. Mean DPM/animal values for the experimental groups treated with test item concentrations 5, 10 and 25% were 937, 1021 and 1656 DPM, respectively. The mean DPM/animal value for the vehicle control group was 784 DPM. The SI values calculated for the test item concentrations 5, 10 and 25% were 1.2, 1.3 and 2.1, respectively. Based on the results of this LLNA, the Cedarwood Texas oil distilled - Terpenes (Thujopsene) does not need to be classified as skin sensitiser according to the classification criteria outlined in Annex I of 1272/2008/EC (CLP).