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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:
18-12-2015 to 25-03-2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
in accordance with GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
- Name of test material (as cited in study report): Guaiacwood oil
- Physical state: viscous liquid
- Storage condition of test material: at room temperature protected from light

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: approximately 10 weeks old
- Weight at study initiation: body weight variation was within +/- 20% of the sex mean
- Housing: labeled Makrolon cages containing sterilised sawdust as bedding material. Paper and shelters were supplied as cage-enrichment
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days before the start of treatment under laboratory conditions

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C
- Humidity (%): 40 to 70%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 hrs dark/ 12 hrs light

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Pre-screen test: 50 - 100%Main study: 0 - 25 - 50 -100 % (v/v)
No. of animals per dose:
5
Details on study design:
MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: Increased T-lymphocyte proliferation in the test groups as compared to the concurrent vehicle control group.

EVALUATION
The disintegrations per minute (dpm) value was determined for each test group. This information was used to calculate the Stimulation Index (SI) for each of the test groups (disintegrations per minute of treatment group / disintegrations per minute of control group). A positive response is indicated when one or more test groups shows a SI of 3. The EC3 value is the concentration at which a 3-fold increase of lymph node proliferation is observed. Interpolation is used to determine the EC3 value between two test concentrations (based on the following reference: Basketter DA, Lea LJ, Dickens A, Briggs, D, Pate I, Dearman RJ and Kimber I.A comparison of statistical approaches to the derivation of EC3 values from local lymph node assay dose responses. J Appl Toxicol 1999; 19:261-266
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
The six monthly reliability check with Hexylcinnamaldehyde, indicates that the Local Lymph Node Assay as performed at WIL Research Europe is an appropriate model for testing for contact hypersensitivity.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
EC3
Value:
42.6
Parameter:
SI
Value:
1.8
Test group / Remarks:
25%
Parameter:
SI
Value:
3.5
Test group / Remarks:
50%
Parameter:
SI
Value:
4.4
Test group / Remarks:
100%
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
Mean DPM/animal values for the experimental groups treated with test substance concentrations 25, 50 and 100% were 1116, 2158 and 2746 DPM respectively.
The mean DPM/animal value for the vehicle control group was 623 DPM.

DETAILS ON STIMULATION INDEX CALCULATION
The Stimulation Index was calculated as a ratio of the DPM from test groups and the DPM vehicle control group (DPM test group / DPM vehicle control group).

EC3 CALCULATION
The data showed a dose-response relationship and an EC3 value (the estimated test substance concentration that will give a SI =3) of 42.6% was calculated.

CLINICAL OBSERVATIONS:
The results of the LLNA study showed that no irritation was observed in any of the animals. Scaliness was noted for three animals treated at 50% and all animals treated at 100% on Day 6. The relative size of the auricular lymph nodes of all animals of the experimental and control groups were considered normal in size. There was no mortality, neither where there any clinical signs of systemic toxicity.

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

Any other information on results incl. tables

Pre-screen test: The results of the epidermal exposures for the selection of highest test substance concentration show that there was no clear irritation detected, apart from very slight erythema (barely perceptible). Based on these results, the highest test substance concentration selected for the main study was a 100% concentration.

Applicant's summary and conclusion

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
Under the conditions of this test, the test substance was found to elicit a positive response (SI ≥ 3) and a clear dose-response relationship was found. The EC3 value was calculated to be 42.6%. Based on these results, Guaiacwood oil needs to be classified for skin sensitization (H317, Skin Sens. 1B) in accordance with the criteria outlined in Annex I of CLP (1272/2008/EC).
Executive summary:

The Local Lymph Node Assay (according to OECD 429) was conducted to determine the sensitising potential of Guaiacwood oil in mice. Lymph node proliferation was determined after exposure to 0%, 25%, 50% or 100% Guaiacwood oil in vehicle (Acetone/Olive oil (4:1 v/v)), using radioactivity counts (DPM). A pooled approach (per test group) was used. Stimulation indices were calculated.

 

Measured disintegrations per node (pooled) were 623, 1116, 2158 and 2746 for the 0%, 25%, 50% and 100% dosing groups, respectively. Corresponding stimulation indices (SI) were calculated to be 1.8, 3.5 and 4.4 for the 25%, 50% and 100% concentration, respectively. The EC3 was calculated to be 42.6%. Based on these results, Guaiacwood oil needs to be classified for skin sensitization (H317, Skin Sens. 1B) in accordance with the criteria outlined in Annex I of CLP (1272/2008/EC).