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Administrative data

Description of key information

Sensitising in the LLNA, EC3 = 12.6%.

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:
2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Guideline study under GLP. This in vivo study was conducted prior to passage of Commission Regulation (EU) 863/2016.
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Principles of method if other than guideline:
This in vivo study was conducted prior to passage of Commission Regulation (EU) 863/2016.
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories S.r.l.
- Females (if applicable) nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: SPF
- Age at study initiation: 9 weeks
- Weight at study initiation:19.8-21.9 g
- Housing:
- Diet (e.g. ad libitum): ad libitum, ssniff SM
- Water (e.g. ad libitum): ad libitum, municipal water supply
- Acclimation period: 14
- Indication of any skin lesions: no

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.0-25.8
- Humidity (%): 24-86
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12
- IN-LIFE DATES: From: To:
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
25, 10 and 5% (w/v) for LLNA
No. of animals per dose:
4
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: Yes
- Irritation: Yes
- Systemic toxicity:
- Ear thickness measurements: Yes
- Erythema scores: Yes

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: standard LLNA
- Criteria used to consider a positive response: SI of 3 or greater is an indication of a positive response. SI is the mean DPN of treated group divided by mean DPN of control group.

TREATMENT PREPARATION AND ADMINISTRATION:
Test item was administered to the dorsal side of ears on days 1, 2 and 3. No treatments occurred on days 4, 5 and 6. Mice were then injected with 25 microCi of 3HtdR and left for 5 hours. The mice were euthanised by asphyxiation with CO2 and draining auricular lymph nodes were excised and processed on an individual basis. A single cell suspension of lymph node cells was prepared and collected, washed thrice with PBS and pelleted. After overnight incubation at 2-8 degrees C, pellets were resuspended in 5% TCA solution, mixed with 10 ml of scintillation liquid in vials, counted in a scintillation counter and expressed as DPM.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
means of duplicate DPM counts indiviidual animals within the dose groups
Positive control results:
HCA dissolved in the same vehicle demonstrated an appropriate lymphoproliferative response in the study, with a SI of 5.5.
Key result
Parameter:
EC3
Value:
12.6
Parameter:
SI
Value:
4.4
Test group / Remarks:
25% (high) dose group
Parameter:
SI
Value:
2.7
Test group / Remarks:
10% (mid) dose group
Parameter:
SI
Value:
1.6
Test group / Remarks:
5% (low) dose group

As the stimulation index of two of the three dose groups was over 3, the substance is considered a dermal sensitiser. The EC3 is calculated at 12.6%. No systemic toxicity was observed in the mice.

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Remarks:
Stimulation index (SI) over 3
Conclusions:
The test substance, solubilized in acetone:olive oil (4:1 v/v), was shown to be a sensitiser in the Local Lymph Node Assay in CBA/Ca mice. Irritation and thickening of the skin of the ear was found in a preliminary skin irritation test. The stimulation index values were 4.4, 2.7 and 1.6 at concentrations of 25, 10 and 5% (w/v) respectively. Under the conditions of this study, the calculated EC3 value of the substance is 12.6% (w/v).
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

The test substance, solubilized in acetone:olive oil (4:1 v/v), was shown to be a sensitiser in the Local Lymph Node Assay (OECD 429) in CBA/Ca mice.  Irritation and thickening of the skin of the ear was found in a preliminary skin irritation test, and the maximum concentration of the test material was set at 25%.  The stimulation index values were 4.4, 2.7 and 1.6 at concentrations of 25, 10 and 5% (w/v) respectively.  Under the conditions of this study, the calculated EC3 value of the substance was 12.6% (w/v).

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

According to the criteria of Regulation EC No. 1272/2008, the test substance is a sensitiser (Category 1B) in the LLNA assay, with a stimulation index over 3 at a 25% concentration (AOO vehicle).