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

Endpoint summary

Description of key information

Skin sensitisation (OECDTG429)

Cedarwood Texas oil distilled - Terpenes (Thujopsene): not sensitising

Cedarwood Texas oil distilled - Terpenes (Cedrene): sensitising (read-across from Cedarwood Virginia oil)

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2010

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

2003

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)

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.

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

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)

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

Parameter:

SI

Value:

1

Variability:

0.1

Test group / Remarks:

0% dose

Parameter:

SI

Value:

1.2

Variability:

0.2

Test group / Remarks:

5% dose

Parameter:

SI

Value:

1.3

Variability:

0.1

Test group / Remarks:

10% dose

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.

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).

Reference 2

Endpoint:

skin sensitisation: in vitro

Data waiving:

study technically not feasible

Justification for data waiving:

other:

Reference 3

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 Jun 2017 - 20 Jul 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2010

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

2003

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)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: Batch: 1002960562, provided by sponsor
- Expiration date of the lot/batch: 31 August 2017
- Purity test date: 22 Aug 2016

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.

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, when known: SPF-quality
- Age at study initiation: approx. 8-10 weeks old
- Weight at study initiation: 18.7 to 22.7 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: 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 (%): 47 to 72
- Air changes (per hr): >10 (no recirculation)
- Photoperiod (hrs dark / hrs light): 12 / 12
- IN-LIFE DATES: Study start:13 Jun 2017 To: 17 Jul 2017

Vehicle:

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

Concentration:

0% (w/w), 25% (w/w), 50% (w/w), 100% (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 25, 50 or 100% 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)

Positive control results:

The six-month reliability check with Alpha-hexylcinnamaldehyde indicates that the Local Lymph Node Assay as performed at Charles River Den Bosch is an appropriate model for testing for contact hypersensitivity

Key result

Parameter:

EC3

Remarks:

%

Value:

22

Test group / Remarks:

All groups

Parameter:

SI

Value:

1

Variability:

0.3

Test group / Remarks:

0% Virginia Oil

Parameter:

SI

Value:

3.6

Variability:

1.1

Test group / Remarks:

25% Virginia Oil

Parameter:

SI

Value:

6.9

Variability:

1.4

Test group / Remarks:

50% Virginia Oil

Parameter:

SI

Value:

11.8

Variability:

5

Test group / Remarks:

100% Virginia Oil

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
The majority of auricular lymph nodes were considered normal in size, except for the nodes in the animals at 50% and 100%, of which two animals per group showed enlarged auricular lymph nodes.
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 25, 50 and 100% were 1095, 2077 and 3547 DPM, respectively. The mean DPM/animal value for the vehicle control group was 302 DPM. The individual SI is the ratio of the DPM/animal compared to the DPM/vehicle control group mean. The SI values calculated for the test item concentrations 25, 50 and 100% were 3.6, 6.9 and 11.8, respectively.

CLINICAL OBSERVATIONS:
Ptosis was noted for all animals treated at 100% between Days 3 and 5 and three animals treated at 50% on Day 3. These clinical signs were considered not relevant for the outcome of the study, since the 25% group was for classification of the test item. No mortality occurred.

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

Interpretation of results:

other: Category 1B Skin sensitiser

Remarks:

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

Conclusions:

Based on the results of this LLNA, the Cedarwood Oil Virginia needs to be classified as category 1B skin sensitiser according to the classification criteria outlined in Annex I of 1272/2008/EC (CLP).

Executive summary:

An LLNA, skin sensitization study was performed according to OECD TG 429 and in compliance with GLP. three experimental groups of five female CBA/J mice were treated with Cedarwood Virginia Oil concentrations of 25, 50 or 100% 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)). Positive control tests were performed regularly with α-Hexylcinnamaldehyde.

Ptosis was noted for all animals treated at 100% between Days 3 and 5 and three animals treated at 50% on Day 3. These clinical signs were considered not relevant for the outcome of the study, since the classification of the test item is based on the results of the 25% group. No mortality occurred. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. 

The majority of auricular lymph nodes were considered normal in size, except for the nodes in the animals at 50% and 100%, of which two animals per group showed enlarged auricular lymph nodes. No macroscopic abnormalities of the surrounding area were noted for any of the animals. Mean DPM/animal values for the experimental groups treated with test item concentrations 25, 50 and 100% were 1095, 2077 and 3547 DPM, respectively. The mean DPM/animal value for the vehicle control group was 302 DPM. The SI values calculated for the test item concentrations 25, 50 and 100% were 3.6, 6.9 and 11.8, respectively. These results indicate that the test item could elicit a SI ≥ 3. An EC3 value (the estimated test item concentration that will give a SI =3) of 22.0% was calculated. The EC3 value of 22.0% was calculated. Based on the results of this study the test substance should therefore be classified as a skin sensitizer (Category 1B) in accordance with the CLP Regulation (1272/2008/EC).

Reference 4

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

The full read across justification report is attached under "Attached justification".

20 December 2017 READ-ACROSS STUDY / CW TX OIL TERPENES 1 (CEDRENE) / SKIN SENSITISATION I&B9W8768R001F2.0

According to Annex VII, 8.3 of the REACh Regulation (EC) No 1907/2006, Skin Sensitisation is standard information required for the registration of substances manufactured or imported in quantities of one tonne per year or more. However, according to Annex XI, 1.5 of the REACH Regulation, Read-across and grouping approaches can be used to adapt the standard testing regime. This read-across study report follows notably the recommendations made by the European Chemicals Agency in its “Guidance on information requirements and chemical safety assessment Chapter R.6 – QSARs and grouping of chemicals” (ECHA, 2008) and in its document “Read-Across Assessment Framework (RAAF)” (ECHA, 2017).

A read-across approach appears appropriate to predict the endpoint “Skin Sensitisation” for the substance Cedarwood Texas oil distilled - Terpenes 1 (Cedrene) [CW TX T1] because:

A Skin Sensitisation study, according to OECD test guideline 429, is available for the substance Cedarwood Virginia oil [CW Virginia], which composition is very similar, including the constituent Cedrol and beta-caryophyllene, which are foreseen as leading the skin sensitisation effects.

Analysis of literature and data available for the constituent thujopsene tends to confirm that variations in this constituent, which is the main difference with the source substance, do not significantly influence the skin sensitisation;

This report follows the RAAF method and so presents:
1) The hypothesis: analogue read-across approach, based on the similarity of the structures and the potential Skin Sensitisation effects for these types of structures;
2) The scientific justifications (“Assessment Elements”) and their evaluation (“Assessment Options”); which demonstrate the confidence that can be put in this prediction.
3) The conclusions, usable for classification assessment or risk assessment, which are summarised hereafter.

Reason / purpose for cross-reference:

read-across source

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: Batch: 1002960562, provided by sponsor
- Expiration date of the lot/batch: 31 August 2017
- Purity test date: 22 Aug 2016

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.

Positive control results:

The six-month reliability check with Alpha-hexylcinnamaldehyde indicates that the Local Lymph Node Assay as performed at Charles River Den Bosch is an appropriate model for testing for contact hypersensitivity

Parameter:

SI

Value:

1

Variability:

0.3

Test group / Remarks:

0% Virginia Oil

Parameter:

SI

Value:

3.6

Variability:

1.1

Test group / Remarks:

25% Virginia Oil

Parameter:

SI

Value:

6.9

Variability:

1.4

Test group / Remarks:

50% Virginia Oil

Parameter:

SI

Value:

11.8

Variability:

5

Test group / Remarks:

100% Virginia Oil

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
The majority of auricular lymph nodes were considered normal in size, except for the nodes in the animals at 50% and 100%, of which two animals per group showed enlarged auricular lymph nodes.
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 25, 50 and 100% were 1095, 2077 and 3547 DPM, respectively. The mean DPM/animal value for the vehicle control group was 302 DPM. The individual SI is the ratio of the DPM/animal compared to the DPM/vehicle control group mean. The SI values calculated for the test item concentrations 25, 50 and 100% were 3.6, 6.9 and 11.8, respectively.

CLINICAL OBSERVATIONS:
Ptosis was noted for all animals treated at 100% between Days 3 and 5 and three animals treated at 50% on Day 3. These clinical signs were considered not relevant for the outcome of the study, since the 25% group was for classification of the test item. No mortality occurred.

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

Interpretation of results:

other: Category 1B Skin sensitiser

Remarks:

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

Conclusions:

The skin sensitisition potential of Cedarwood Texas Terpenes (cedrene) was evaluated by read across with the source study for Cedarwood Virginia oil. Based on the results of the test, the Cedarwood Texas Terpenes (cedrene) needs to be classified as category 1B skin sensitiser according to the classification criteria outlined in Annex I of 1272/2008/EC (CLP).

Executive summary:

The skin sensitisition potential of Cedarwood Texas Terpenes (cedrene) was evaluated by read across with the source study for Cedarwood Virginia oil. An LLNA, skin sensitization study was performed according to OECD TG 429 and in compliance with GLP. three experimental groups of five female CBA/J mice were treated with Cedarwood Virginia Oil concentrations of 25, 50 or 100% 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)). Positive control tests were performed regularly with α-Hexylcinnamaldehyde.

Ptosis was noted for all animals treated at 100% between Days 3 and 5 and three animals treated at 50% on Day 3. These clinical signs were considered not relevant for the outcome of the study, since the classification of the test item is based on the results of the 25% group. No mortality occurred. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. 

The majority of auricular lymph nodes were considered normal in size, except for the nodes in the animals at 50% and 100%, of which two animals per group showed enlarged auricular lymph nodes. No macroscopic abnormalities of the surrounding area were noted for any of the animals. Mean DPM/animal values for the experimental groups treated with test item concentrations 25, 50 and 100% were 1095, 2077 and 3547 DPM, respectively. The mean DPM/animal value for the vehicle control group was 302 DPM. The SI values calculated for the test item concentrations 25, 50 and 100% were 3.6, 6.9 and 11.8, respectively.

These results indicate that the test item could elicit a SI ≥ 3. An EC3 value (the estimated test item concentration that will give a SI =3) of 22.0% was calculated.

The EC3 value of 22.0% was calculated. Based on the results of this study the test substance should therefore be classified as a skin sensitizer (Category 1B) in accordance with the CLP Regulation (1272/2008/EC).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

The skin sensitisation of Cedarwood Texas oil distilled - Terpenes was assessed. Cedarwood Texas oil distilled - Terpenes (Thujopsene) was tested in an OECD TG 429 study, of which the summary is presented below. The skin sensitisation of Cedarwood Texas oil distilled - Terpenes (Cedrene) was assessed by using read across from Cedarwood Virginia oil. The summary of the read-across study is presented below, and the read across justification is attached to the oral toxicity target record.

Cedarwood Texas oil distilled - Terpenes (Thujopsene): Skin sensitisation (OECDTG429)

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).

Read-across source substance Cedarwood Virginia oil: Skin sensitisation (OECDTG429)

An LLNA, skin sensitization study was performed according to OECD TG 429 and in compliance with GLP. three experimental groups of five female CBA/J mice were treated with Cedarwood Virginia oil concentrations of 25, 50 or 100% 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)). Positive control tests were performed regularly with α-Hexylcinnamaldehyde.

Ptosis was noted for all animals treated at 100% between Days 3 and 5 and three animals treated at 50% on Day 3. These clinical signs were considered not relevant for the outcome of the study, since the classification of the test item is based on the results of the 25% group. No mortality occurred. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. 

The majority of auricular lymph nodes were considered normal in size, except for the nodes in the animals at 50% and 100%, of which two animals per group showed enlarged auricular lymph nodes. No macroscopic abnormalities of the surrounding area were noted for any of the animals. Mean DPM/animal values for the experimental groups treated with test item concentrations 25, 50 and 100% were 1095, 2077 and 3547 DPM, respectively. The mean DPM/animal value for the vehicle control group was 302 DPM. The SI values calculated for the test item concentrations 25, 50 and 100% were 3.6, 6.9 and 11.8, respectively. These results indicate that the test item could elicit a SI ≥ 3. An EC3 value (the estimated test item concentration that will give a SI =3) of 22.0% was calculated. The EC3 value of 22.0% was calculated.

Justification for classification or non-classification

Based on the available data it can be concluded that 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).

Based on the available read-across data Cedarwood Texas oil distilled - Terpenes (Cedrene) was determined to be a skin sensitiser. Therefore Cedarwood Texas oil distilled - Terpenes (Cedrene) needs to be classified as category 1B skin sensitiser according to the classification criteria outlined in Annex I of 1272/2008/EC (CLP).