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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:
29 June 2010 - 02 Mar 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
adopted 22 July 2010
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
No. 440/2008
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: TD-9024390, received November 1, 2010
- Expiration date of the lot/batch: November 1, 2011
- Purity: 99.63%

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

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: the test item was used as supplied.
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: TD-9024390
- Expiration date of the lot/batch: 01 November 2011
- Purity: 99.63%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature in the dark
- Solubility and stability of the test substance in the solvent/vehicle: The test item was formulated within two hours of being applied to the test system. It is
assumed that the formulation was stable for this duration. No analysis was conducted to determine the homogeneity, concentration or stability of
the test item formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: test item was used undiluted and freshly prepared as a solution in acetone/olive oil 4: 1
- Final dilution of a dissolved solid, stock liquid or gel: 50%

OTHER SPECIFICS: test material is a pale yellow liquid

In vivo test system

Test animals

Species:
mouse
Strain:
CBA/Ca
Remarks:
CBN/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories U.K. Ltd., Oxon, UK.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: eight to twelve weeks
- Weight at study initiation: 15 to 23 g
- Housing: suspended solid-floor polypropylene cages furnished with softwood woodflakes
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C):19 to 25°C
- Humidity (%): 30 to 70%,
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): twelve hours continuous light (06.00 to 18.00) and twelve hours darkness
- Other:The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
25%, 50% and 100%
No. of animals per dose:
4
Details on study design:
PRE-SCREEN TESTS:
The mouse was treated by daily application of 25 μI of the undiluted test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6.
Any signs of excessive local irritation noted during this period were recorded.
- Systemic toxicity: Any signs of toxicity noted during this period were recorded.
- Ear thickness easurements: The thickness of each ear was measured using an Oditest micrometer (Dyer, PA), pre-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. Daily mean ear thickness values were calculated and the overall mean ear thickness changes were calculated for Days 2 to 5. A mean ear thickness increase of ~25% was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitisation.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response: The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (disintegrations per minute/node) and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index). The test item will be regarded as a sensitiser 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-sensitiser".

TREATMENT PREPARATION AND ADMINISTRATION:
Groups of four mice were treated with the undiluted test item or the test item at concentrations of 50% or 25% v/v in acetone/olive oil 4: 1. The preliminary screening test suggested that the test item would not produce systemic toxicity or excessive local irritation at the highest suitable concentration. The mice were treated by daily application of 25 μI of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test item formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette. A further group of four mice received the vehicle alone in the same manner.
Five days following the first topical application of the test item or vehicle (Day 6) all mice were injected via the tail vein with 250 μI of phosphate buffered saline (PBS) containing 3H-methyl thymidine (3HTdR: 80 μCi/ml, specific activity 2.0 Ci/mmol, ARC UK Ltd) giving a total of 20 μCi to each mouse.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
a-Hexylcinnamaldehyde, tech., 85% induced a stimulation index of 7,25 and was thereforeconsidered to be a sensitiser under the conditions of the test.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
0.44
Variability:
not specified
Test group / Remarks:
Concentration 25%
Key result
Parameter:
SI
Value:
0.58
Variability:
not specified
Test group / Remarks:
Concentration 50%
Key result
Parameter:
SI
Value:
1.06
Variability:
not specified
Test group / Remarks:
Concentration 100%
Cellular proliferation data / Observations:
DETAILS ON STIMULATION INDEX CALCULATION
The Stimulation Index is expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group

CLINICAL OBSERVATIONS:
There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test.

BODY WEIGHTS
Bodyweight changes of the test animals between Day 1 and Day 6 were comparable to those observed in the corresponding control group animals over the same period.

Applicant's summary and conclusion

Interpretation of results:
other: Not classified
Remarks:
Annex I of the CLP Regulation (1272/2008/EC)
Conclusions:
Based on the results in this study the compound does not need to be classified as a skin sensitizer in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC). This result is used for read-across to Ethyl Amyl Ketone.
Executive summary:

A study was performed according to OECD Guideline 429 and Method 842 (EC) No. 440/2008 to assess the skin sensitisation potential of Methyl n-Amyl Ketone in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear. Following a preliminary screening test in which no clinical signs of toxicity 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. Three groups, each of four animals, were treated with 50μI (25μI per ear) of the undiluted test item or the test item as a solution in acetone/olive oil 4:1 at concentrations of 50% or 25% v/v. A further group of four animals was treated with acetone/olive oil 4:1 alone. The Stimulation Index expressed as the mean radioactive incorporation for each treatment group ( 25%, 50% and 100%) divided by the mean radioactive incorporation of the vehicle control group were 0.44, 0.58 and 1.06 respectively. Indicating a negative result for all 3 dose groups. Therefore, the test item was considered to be a non-sensitiser under the conditions of the test does not need to be classified as a skin sensitizer in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC). This result is used for read-across to Ethyl Amyl Ketone.