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Description of key information

The test item was found to be a skin sensitiser and an EC3 value of 24.7 % (w/w) was derived.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Jan 15 to May 26, 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2010
Qualifier:
equivalent or similar to
Guideline:
EU Method B.42 (Skin Sensitisation: 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 lot/batch No.of test material: Batch No. 6F11027000
- Expiration date of the lot/batch: November 17, 2016
- Purity: 96%
Species:
mouse
Strain:
CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories B.V. (the Netherlands)
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: 1st pre-test: 9 - 10 weeks (beginning of treatment); 2nd pre-test: 10 - 11 weeks (beginning of treatment); Main study: 9 - 10 weeks (beginning of treatment)
- Weight at study initiation: 18.9 – 21.8 g
- Housing: By group In cages (Makrolon Type II for pre-test/III for main study), with wire mesh top; all animals belonging to the same experimental group were kept in one cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: At least 5 days prior to the start of dosing under test conditions after health examination

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 45-65%
- Photoperiod (hrs dark / hrs light): artificial light 6.00 a.m. - 6.00 p.m.
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
undiluted and 5%, 10%, 25% (w/w); The highest concentration tested was the highest concentration that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed by two pre-experiments.
No. of animals per dose:
4 animals/group
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: highest test item concentration which could be technically used was 100% of the undiluted test item
- Irritation: Eventual signs of local irritation documented, possible erythema of the ear skin was scored
- Systemic toxicity: At the tested concentrations the animals did not show any signs of systemic toxicity
- Ear thickness measurements: Yes, measured with a micrometer
- Erythema scores: The animal treated with 50% test item concentration showed an erythema of the ear skin (Score 1 on days 4 and 5, and Score 4 on day 6). The animal treated with 100% test item concentration showed an erythema of the ear skin (Score 2 on days 3 and 4, Score 1 on day 5, and Score 4 on day 6). Furthermore, ear swelling was observed for the animal treated with 100% test item concentration. In addition, the ear weight of both animals exceeded the threshold recommended by OECD 429 and ear thickness was exceeded in the animal treated with 100%. A second pre-test was performed using test item concentrations of 10 and 25%. The animal treated with 10% test item concentration showed an erythema of the ear skin between day 2 and 5 (Score 1 on days 2, 3 and 4 and Score 2 on day 5). The animal treated with 25% test item concentration showed an erythema of the ear skin (Score 1 on day 2 and 5, and score 2 on days 3 and 4).


MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: A test item is regarded as a sensitiser in the LLNA if the exposure to one or more test concentration resulted in a 3-fold or greater increase in incorporation of 3HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.).

TREATMENT PREPARATION AND ADMINISTRATION:
- Each test group of mice was treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 5, 10, and 25% (w/w) in acetone/olive oil (4+1, v/v). The application volume, 25 µL/ear/day, was spread over the entire dorsal surface (~8 mm) of each ear once daily for 3 consecutive days. A further group of mice (control animals) was treated with an equivalent volume of the relevant vehicle alone (control animals).
- 5 days after the first topical application (day 6) 250 µL of phosphate-buffered saline containing 19.6 µCi of 3H-methyl thymidine (equivalent to 78.3 µCi/mL 3HTdR) were injected into each test and control mouse via the tail vein.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The mean values and standard deviations were calculated in the body weight tables and for the DPM values (group mean DPM ± standard deviation).
Positive control results:
The S.I. Index results for the positive control during the course of the study ranged from 1.00 (0% test item concentration in vehicle) to 5.14 (25% test item concentration in vehicle). Calculation of the EC3 Value from Test Group2 (10% test item concentration) and Test Group 3 (25% test item concentration) was 13.8% (w/v)
Key result
Parameter:
EC3
Value:
ca. 24.7
Test group / Remarks:
Test item found to be a skin sensitiser
Parameter:
SI
Value:
ca. 1.83
Test group / Remarks:
5% (w/w) in acetone/olive oil (4+1, v/v)
Parameter:
SI
Value:
ca. 2.03
Test group / Remarks:
10% (w/w) in acetone/olive oil (4+1, v/v)
Parameter:
SI
Value:
ca. 3.02
Test group / Remarks:
25% (w/w) in acetone/olive oil (4+1, v/v)
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
- DPM/lymph node values. Mean scintillation-background DPM was subtracted from test and control raw data

DETAILS ON STIMULATION INDEX CALCULATION
- expressed as the number of radioactive disintegrations per minute per lymph node (DPM/lymph node) and as the ratio of 3HTdR incorporated into lymph node cells of test animals relative to that recorded for lymph nodes of control animals

EC3 CALCULATION
EC3 = (a-c) [(3-d)/(b-d)] + c

Where EC3 is the estimated concentration of the test item required to produce a 3-fold increase in draining lymph node cell proliferative activity; (a, b) and (c, d) are respectively the co-ordinates of the two pair of data lying immediately above and below the S.I. value of 3 on the local lymph node assay dose response plot

CLINICAL OBSERVATIONS:
- All treated animals survived the scheduled study period
- No symptoms of local skin irritation at the ears of the animals and no signs of systemic toxicity were observed during the study period
- On days 2 to 6, the animals treated with test item concentrations of 10 and 25% showed an erythema of the ear skin (Score 1 and 2). Animals treated with 5% test item concentration showed an erythema of the ear skin (Score 1) on days 4 and 5

BODY WEIGHTS
- The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age
Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Remarks:
according to EC 1272/2008 as amended
Conclusions:
The test item 1,3,5-Triisopropylbenzene was found to be a skin sensitiser and an EC3 value of 24.7 % (w/w) was derived.
Executive summary:

In the study the test item 1,3,5-Triisopropylbenzene formulated in acetone/olive oil (4+1, v/v) was assessed for its possible skin sensitising potential following the OECD 429 local lymph node assay. The highest concentration tested was the highest concentration that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed by two pre-experiments.

In this study Stimulation Indices (S.I.) of 1.83, 2.03 and 3.02 were determined with the test item at concentrations of 5, 10, and 25% (w/w) in acetone/olive oil (4+1, v/v), respectively. A clear dose response was observed.

The test item was found to be a skin sensitiser and an EC3 value of 24.7 % (w/w) was derived.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The test item was found to be a skin sensitizer and an EC3 value of 24.7 % (w/w) was derived. Therefore, according to EC 1272/2008 as amended, the substances meets the criteria for classification as a Skin Sensitizer Category 1B.