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Description of key information

Skin sensitisation (OECD TG 429): sensitising

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

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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:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2010
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
2003
Qualifier:
according to
Guideline:
other: EC, No 440/2008, part B "Skin Sensitization: Local Lymph Node Assay"
Version / remarks:
2008
GLP compliance:
yes (incl. certificate)
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, extended expery date until: 28 February 2018 (21 Oct 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.

EC3 CALCULATION

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:

A 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:
skin sensitisation: in vitro
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

A 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.

Since the lowest dose could elicit a SI ≥ 3, the EC3 value was calculated by log-linear extrapolation according to the method described by Ryan et al (2007). An EC3 value of 22.0% was calculated.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the available data, Cedarwood Virginia oil should be classified as a skin sensitizer (Skin Sens. 1B / H317) in accordance with the criteria outlined in Annex 1 of the CLP Regulation (1272/2008/EC).