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Description of key information

Skin sensitisation (OECD TG 429): sensitising (read across from Cedrol, Cedarwood Texas oil distilled)

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:
14 Mar 2017 - 30 May 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
- 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.
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)
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)
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.
Key result
Parameter:
EC3
Value:
13.4
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
Parameter:
SI
Value:
5.4
Variability:
1.3
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 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.
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).

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:
SEE ALSO ATTACHED READ-ACROSS JUSTIFICATION DOCUMENT
29 November 2017 READ-ACROSS / CW-TX-CRUDE / 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 as appropriated to predict the endpoint “Skin sensitisation” for the target substance “Cedarwood Texas oil, Crude” (CW-TX-Crude) because:

 An OECD Guideline 429. Skin Sensitisation: Local Lymph Node Assay is available for the source substance “Cedarwood Texas oil, Cedrol” (CW-TX-Cedrol), which composition is very similar to CW-TX-Crude, notably regarding the constituents that may drive the skin sensitisation effects;
 The read-across is based on the comparison of the composition of two UVCBs that are very close one another, the raw material being the same, the process the same except an additional distillation step for the source substance, and the constituents the same with variations in their concentrations;
 This read-across prediction intends to propose a classification, according to CLP Regulation EC/1272/2008 and its amendments, as a skin sensitiser (Category 1B) and labelled as H317.
 Conclusions on CLP Classification and Risk Assessment comply with the REACh EC/1907/2006 and amendments regulatory requirements.

This report follows the RAAF method and so presents:
1) The hypothesis: analogue read-across approach based on the constituents, notably the ones related to the skin sensitisation/corrosion effects;
2) The scientific justifications (“Assessment Elements”) and their evaluation (“Assessment Options”), which underline the coherence of the relationship between the variations of the sensitising constituents and the skin sensitising properties of the whole UVCB;
3) The conclusions, which are summarised hereafter.
Reason / purpose:
read-across source
No. of animals per dose:
5
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.
Key result
Parameter:
EC3
Value:
13.4
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
Parameter:
SI
Value:
5.4
Variability:
1.3
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 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.
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 obtained using Cedrol, Cedarwood Texas oil distilled skin sensitisation information for read across (the calculated EC3 value is 13.4%), Cedarwood Texas crude oil needs to be classified as category 1B skin sensitiser according to the classification criteria outlined in Annex I of 1272/2008/EC (CLP). (1272/2008/EC).
Executive summary:

Cedarwood Texas crude oil is considered a skin sensitiser category 1B, based on the results from the (OECDTG429) source study performed with Cedrol, Cedarwood Texas oil distilled. The justification for this read across is provided in the attached justification for type of information.

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).

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:

The skin sensitisation for Cedarwood Texas crude oil is assessed by using read across from Cedrol, Cedarwood Texas oil distilled. The experimental skin sensitisation information on Cedrol, Cedarwood Texas oil distilled is summarised and the read-across justification is attached to the respective target records.

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 0, 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 category 1B skin sensitiser according to the classification criteria outlined in Annex I of 1272/2008/EC (CLP).

Justification for classification or non-classification

Based on the available data, it can be concluded that Cedarwood Texas oil - Crude needs to be classified as category 1B skin sensitiser according to the classification criteria outlined in Annex I of 1272/2008/EC (CLP).