Registration Dossier

Toxicological information

Skin sensitisation

Currently viewing:

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 Mar 2017 - 30 May 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 guidelineopen allclose all
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)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Remarks:
LIGHT YELLOW, SOLID MASS
Details on test material:
Name of test material as cited in study report: Cedrol - Cedarwood Texas oil distilled
Mfg Date 01/26/2016
Exp Date 01/25/2019

- Identification: Cedrol, Cedarwood Texas oil distilled
- Appearance: Light yellow, solid mass
- Batch: B-64530
- Purity/Composition: UVCB
- Test item storage: At room temperature
- Stable under storage conditions until: 25 January 2019 (expiry date)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature protected from light

ODOUR DESCRIPTION: CONFORMS TO STANDARD
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and Batch No.of test material: Provided by sponsor, B-64530
- Expiration date of the lot/batch: 25 January 2019
- Purity test date: 26 January 2017
- Test Facility test item number: 207804/A

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

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
-Test item was equilibrated to 100C for several minutes until completely liquefied to obtain a homogeneous sample. 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. 10 weeks old
- Weight at study initiation: 18.7 to 23.6 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 66%
- Air changes (per hr): >10 (no recirculation)
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES:
-Experimental study start date 09 March 2017 - 30 May 2017 (completion of In-life)

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Pre-screen: 10, 25, 50, 100 % w/w
Main study: 0, 5, 10, 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 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

EVALUATION CRITERIA
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)

Resultsopen allclose all
Parameter:
SI
Value:
1
Variability:
0.3
Test group / Remarks:
0% dose
Parameter:
SI
Value:
2
Variability:
0.6
Test group / Remarks:
5% dose
Parameter:
SI
Value:
2.3
Variability:
0.6
Test group / Remarks:
10% dose
Key result
Parameter:
SI
Value:
5.4
Variability:
1.3
Test group / Remarks:
25% dose
Key result
Parameter:
EC3
Value:
13.4
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 561, 647 and 1524 DPM, respectively. The mean DPM/animal value for the vehicle control group was 285 DPM. The SI values calculated for the test item concentrations 5, 10 and 25% were 2.0, 2.3 and 5.4, 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 25%. The data showed a dose response and an EC3 value of 13.4% was calculated.

CLINICAL OBSERVATIONS:
-No erythema was noted for any of the animals. Scaliness was noted on the ears of four animals treated at 25% between Days 3 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 main study period.

Applicant's summary and conclusion

Interpretation of results:
Category 1B (indication of 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 LLNA, the calculated EC3 value is 13.4%. Therefore, Cedrol, Cedarwood Texas oil distilled 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 sensitisation potential of Cedrol, Cedarwood Texas oil distilled 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 on the ears of four animals treated at 25% between days 3 and 6. 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 5, 10 and 25% were 561, 647 and 1524 DPM, respectively. The mean DPM/animal value for the vehicle control group was 285 DPM. The SI values calculated for the test item concentrations 5, 10 and 25% were 2.0, 2.3 and 5.4, respectively. The data showed a dose-response and an EC3 value (the estimated test item concentration that will give a SI =3) of 13.4% was calculated. Based on the results of this LLNA, Cedrol, Cedarwood Texas oil distilled needs to be classified as Skin Sensitiser Category 1B according to the classifcation critria outlined in Annex I of 1272/2008/EC (CLP).