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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:
From January 31 to March 19, 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted in compliance with OECD Guideline No. 429 without any deviation.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014
Reference Type:
other: Report Amendment 1
Title:
Unnamed
Year:
2014
Report Date:
2014

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
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP Compliance Monitoring Programme (inspection date: 10 July 2012/ signed on 30 November 2012)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
liquid
Details on test material:
Description: clear colourless liquid
Specific details on test material used for the study:
- Storage conditions: room temperature in the dark
- Expiration date of the lot/batch: 2014-06-30

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/Ca (CBA/CaOlaHsd)
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories UK Limited, Oxon, UK.
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 15-23 g
- Housing: Animals were individually housed in suspended solid-floor polypropylene cages furnished with softwood wood flakes.
- Diet: Food (2014C Teklad Global Rodent diet supplied by Harlan Laboratories UK Ltd., Oxon, UK), ad libitum
- Water: Mains tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19-25 °C
- Humidity: 30-70 %
- Air changes: Approximately 15 changes/h
- Photoperiod: 12 h dark / 12 h light


IN-LIFE DATES: From: 2014-01-31 To: 2014-03-19

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
100 % and 50 % v/v
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: For the purpose of the study, the test item was used undiluted and freshly prepared as a solution in acetone/olive oil 4:1. This vehicle was chosen as it produced the most suitable formulation at the required concentration.
-The mouse treated with undiluted test material showed a 10% body weight loss. Very slight erythema (score 1) was noted on both ears on Day 4. A maximum mean ear thickness increase of 23%, i.e. close to the threshold of 25%, was measured during the test, indicating that some irritation has occurred.
Based on this information the undiluted test item was selected for the main test.

MAIN STUDY - INITIAL TEST (100 %) & ADDITIONAL TEST (50 % v/v in 4/1 acetone/olive oil)
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Step-by-step individual approach, using tritiated (3H)-methyl thymidine, according to the OECD 429 test guideline. Due to mortality in the initial main test (100 %), an additional group of animals was treated with 50 μL (25 μL per ear) of the test item as a solution in acetone/olive oil 4:1 at a concentration of 50% v/v.
- Additional investigation: Measurement of ear thickness - The ear thickness of each ear was measured using a Mitutoyo 547-300S gauge (Mitutoyo Corporation) pre-dose and 1 hour post dose on Day 1, post dose on Days 2 and 3 and on Days 4 to 6. Any changes in the ear thickness were noted. Mean ear thickness changes were calculated between time period and every Day after. A mean ear thickness increase of equal to or greater than 25% was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitisation.
- Criteria used to consider a positive response: The test item will be regarded as a sensitizer if at least one concentration of the test item results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test item failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a "non-sensitizer."

TREATMENT PREPARATION AND ADMINISTRATION:
All formulations were used within two hours of preparation and assumed to be stable for this period. The concentration and homogeneity of the formulations were not determined by analysis.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Not applicable

Results and discussion

Positive control results:
A group of five animals was treated with 50 µl (25 µl per ear) of α-Hexylcinnamaldehyde, tech., 85% as a solution in acetone/olive oil 4:1 at a concentration of 25 % v/v. A further control group of five animals was treated with acetone/olive oil 4:1 alone. With a SI = 8.42, α-Hexylcinnamaldehyde, tech., 85% was considered to be a sensitizer under the conditions of the test.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
12.39
Test group / Remarks:
100% in acetone/olive oil 4:1
Key result
Parameter:
SI
Value:
6.04
Test group / Remarks:
50% in acetone/olive oil 4:1
Key result
Parameter:
SI
Value:
8.42
Test group / Remarks:
Positive control
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
Initial test: Mean DPM / animal for vehicle and 100% were 2236.38 and 27706.26, respectively.
Additional test: Mean DPM / animal for vehicle and 50% v/v were 4654.48 and 28121.18, respectively.

DETAILS ON STIMULATION INDEX CALCULATION
The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows: 12.39 and 6.04 at 100 and 50% in acetone/olive oil 4:1, respectively.

EC3 CALCULATION : Not applicable.

CLINICAL OBSERVATIONS:
Initial test:
One animal treated with the undiluted test item (Animal Number 2-1) was humanely killed, on Day 4, due to the occurrence of clinical signs of toxicity that exceeded the moderate severity limit set forth in the UK Home Office Project Licence. Signs of systemic toxicity noted in this animal were hunched posture, lethargy and ptosis. This animal showed an 11% body weight loss. Very slight erythema (score 1) was noted on both ears of this animal on Days 1 to 3 and well-defined erythema (score 2) was noted on Day 4.
No signs of systemic toxicity were noted in the surviving test animals or control animals during the test. Very slight erythema (score 1) was noted in all surviving test animals on Days 1 to 4. Mean ear thickness increases up to 22% (Day 4), i.e. close to the threshold of 25%, were measured during the initial test, indicating that irritation has occurred.
Additional test:
There were no deaths. No signs of systemic toxicity or skin irritation were noted in the test or control animals during the test.

BODY WEIGHTS
Initial test:
Body weight change of the surviving test animals between Day 1 and Day 6 was comparable to that observed in the corresponding control group animals over the same period.
A slight body weight loss was noted in one surviving test group animal and one control group animal during the observation period but this was considered to be within an acceptable range for this Species/Strain and considered not to be of toxicological relevance.

Additional test:
Body weight change of the test animals between Day 1 and Day 6 was comparable to that observed in the corresponding control group animals over the same period.
A slight body weight loss was noted in one surviving test group animals and one control group animals during the observation period but this was considered to be within an acceptable range for this Species/Strain and considered not to be of toxicological relevance.

Any other information on results incl. tables

See the attached document for information on tables of results

Applicant's summary and conclusion

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
Under the test conditions, test material is classified as a skin sensitizer according to the Regulation EC No. 1272/2008 (CLP) and to the GHS.
Executive summary:

A study was performed to assess the skin sensitisation potential of test material in the CBA/Ca (CBA/CaOlaHsd) strain mouse following topical application to the dorsal surface of the ear. The method was conducted according to the OECD test guideline No 429 and in compliance with GLP. Following a preliminary screening test in a single animal in which no clinical signs of systemic toxicity or excessive local skin irritation were noted at a concentration of 100%, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. A group of five animals was treated with 50 μL (25 μL per ear) of the undiluted test item. A further group of five control animals were untreated. Due to mortality in the initial main test, an additional group of animals was treated with 50 μL (25 μL per ear) of the test item as a solution in acetone/olive oil 4:1 at a concentration of 50% v/v. A further group of five control animals was treated with acetone/olive oil 4:1 alone. The irritant potential of the test item was assessed in parallel by measurement of ear thickness on days 1 to 6. The Stimulation Index values expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:

Test Item Treatment Group

Stimulation Index

Result

Initial Test 100%

12.39

Positive

Additional Test 50% v/v in acetone/olive oil 4:1

6.04

Positive

The historical positive control, α-Hexylcinnamaldehyde, gave a SI of 8.42, when tested at 25 % v/v. The test system was therefore considered to be valid.

No systemic toxicity or excessive local skin irritation were noted at a concentration of 50%, therefore this dose level was considered to be of biological relevance to the endpoint of skin sensitization.

Under the test conditions, test material is classified as a skin sensitizer in the Local Lymph Node Assay according to the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

This study is considered as acceptable and satisfies the requirement for sensitisation endpoint.