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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07 Jun 2017 - 06 Jul 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
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2010
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
2003
Guideline:
other: EC, No 440/2008, part B: "Skin Sensitization: Local Lymph Node Assay"
Version / remarks:
2008
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Reference substance name:
Cypress, Cupressus funebris, ext.
EC Number:
285-360-9
EC Name:
Cypress, Cupressus funebris, ext.
Cas Number:
85085-29-6
Molecular formula:
Not applicable due to UVCB nature of the substance
IUPAC Name:
Essential oil of Cedarwood obtained from the wood of Cupressus funebris (Cupressaceae) by steam distillation
Test material form:
other: Pale yellow to yellow liquid
Remarks:
could crystallize
Details on test material:
- Name of test material (as cited in study report): Cedarwood Oil China
- Substance type: pure substance
- Physical state: liquid
- Analytical purity: no data
- Lot/batch No.: confidential information


Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Batch 1002515317
- Expiration date of the lot/batch: 31 August 2017
- Purity: 100.0% (UVCB)

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: homogenised in Acetone/Olive oil (4:1 v/v)
- Final dilution of a dissolved solid, stock liquid or gel: 25% w/w

In vivo test system

Test animals

Species:
mouse
Strain:
CBA:J
Remarks:
Inbred
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
- Age at study initiation: Young adult animals (approximately 9 weeks old)
- Weight at study initiation: 18.1 to 23.7 g
- Housing: polycarbonate cages (Makrolon MIII type; height 18 cm.)
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5 days before the commencement of dosing
- Indication of any skin lesions: health inspection was performed

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C
- Humidity (%): 40 to 70%
- Air changes (per hr): 10 or greater air changes per hour with 100% fresh air
- Photoperiod (hrs dark / hrs light): 12 hour light/12 hour dark
- IN-LIFE DATES: From:07 Jun 2017 (start dosing) To: 03 jul 2017

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0, 5, 10 and 25%
No. of animals per dose:
5
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: Soluble up to 100%
- Irritation: Very slight erythema, scaliness and scabs were noted for all animals between Days 2 and 6.
- Systemic toxicity: Hunched posture, piloerection and/or lethargy was noted for the animals treated at 100% on Days 3. Piloerection was noted for one animal treated at 50% on Day 3.
- Ear thickness measurements: Variations in ear thickness during the observation period were more than 25% from Day 1 pre-dose values for animals treated at 50% and 100%.

MAIN STUDY
According to OECD TG 429.

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: the test item is regarded as a sensitiser in the LLNA if the exposure to at least one concentration of the test item resulted in an incorporation of 3H-methyl thymidine at least 3-fold or greater than that recorded in control mice, as indicated by the stimulation index.

TREATMENT PREPARATION AND ADMINISTRATION:
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), at approximately the same time on each day. The formulation was 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):
other: Historical control
Statistics:
All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented. 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.

Results and discussion

Positive control results:
For both scientific and animal welfare reasons, no concurrent positive control group was included in the study. An database is available with reliability checks performed each half year during at least the recent 9 years showing reproducible and consistent positive results.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
9.1
Variability:
1.9
Test group / Remarks:
25% w/w
Key result
Parameter:
SI
Value:
3.3
Variability:
0.7
Test group / Remarks:
10% w/w
Key result
Parameter:
SI
Value:
3.1
Variability:
0.7
Test group / Remarks:
5% w/w
Key result
Parameter:
EC3
Remarks:
%
Value:
3.5
Test group / Remarks:
The EC3 value was determined using a log-linear extrapolation according to Ryan et al (2007)
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
Mean DPM per lymph node:
- Vehicle control group: 165 +/- 27
- 5%: 515 +/- 74
- 10%: 541 +/- 65
- 25%: 1502 +/- 190

DETAILS ON STIMULATION INDEX CALCULATION
The nodes were pooled for each animal. 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.

EC3 CALCULATION
The EC3 value (the estimated test item concentration that will give a SI =3) was determined, using a log-linear extrapolation as described by Ryan et al. Extrapolating local lymph node assay EC3 values to estimate relative sensitizing potency. Cutan Ocul Toxicol 2007;26:135-145.

CLINICAL OBSERVATIONS:
One animal in the vehicle group died during injection of thymidine due to a non-test item related technical error. During necropsy no abnormalities were observed. No further 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: sensitising
Remarks:
based on CLP criteria
Conclusions:
Under the conditions of the test Cedarwood Chinese Oil needs to be classified as skin sensitizer. The results indicate that the test item could elicit a SI ≥ 3. An EC3 value of 3.5% was calculated. Based on the results of this study the test substance should therefore be classified as a skin sensitizer (Skin Sens. 1B / H317) in accordance with the CLP Regulation (1272/2008/EC).
Executive summary:

An LLNA, skin sensitization study was performed according to OECD TG 429 and in compliance with GLP. 4 mice per test group were exposed to 0, 5, 10 and 25% w/w Cedarwood Chinese Oil. Historical positive control tests were performed with α-Hexylcinnamaldehyde. The animals did not show any treatment related clinical signs during the course of the study. One animal in the vehicle group died during injection of thymidine due to a non-test item related technical error. In this study Stimulation Indices (S.I.) of 3.1, 3.3 and 9.1 were determined with the test item at concentrations of 5, 10 and 25% w/w in acetone/olive oil (4:1 v/v), 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 3.5% was calculated. The test substance should therefore be classified as a skin sensitizer (Skin Sens. 1B / H317) in accordance with the CLP Regulation (1272/2008/EC).