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Description of key information

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:
From May 12 to September 9, 2015.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test method according to OECD Guideline 429 and GLP study.
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymphnode assay (LLNA)
Test material information:
Composition 1
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories S.r.l. San Pietro al Natisone (UD). Zona Industriale Azzida, 57, 33049 Italy.
- Age at study initiation: 9 weeks old (age-matched, within one week)
- Weight at study initiation: 19.3 – 20.3 g (the weight variation in animals in the study did not exceed 20% of the mean weight).
- Housing: SPF at arrival; standard housing conditions during the study. Type II. polypropylene / polycarbonate cage type. Group caging / mice were provided with glass tunnel-tubes. Bedding was available to animals during the study.
- Diet (e.g. ad libitum): ssniff® SM Rat/Mouse – “Breeding & Maintenance, 15 mm, autoclavable Complete diet for rats/mice” (Batch number: 814 3108, Expiry date: August 2015) produced by ssniff Spezialdiäten GmbH (Ferdinand-Gabriel-Weg 16, D-59494 Soest, Germany), ad libitum.
- Water (e.g. ad libitum): Animals received tap water from the municipal supply from 500 mL bottle, ad libitum.
- Acclimation period: at least 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.1-25.8°C
- Humidity (%): 31-73 %
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.

IN-LIFE DATES: From: June 24th 2015; To: June 30th 2015.
Vehicle:
dimethyl sulphoxide
Concentration:
Preliminary study: 25 and 50% (w/v) in DMSO.
Main study: 10, 5 and 2.5% (w/v) in DMSO.
No. of animals per dose:
Preliminary study: 2 animals/dose.
Preliminary study II: 2 animals/dose
Main study: 4 animals/group.
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: The 50% (w/v) formulation using DMSO was achievable and the most homogenous solution tested.
- Irritation: No erythema was observed in any case. Increased ears thickness values were detected in some cases on Days 3 and 6 but all were within the acceptable range, as well as the ear punch weights.
- Lymph node proliferation response: The lymph nodes were visually examined: they were larger and normal in both dose groups. The radioactive proliferation assay was not performed in the range finding test.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Proliferation assay.
- Criteria used to consider a positive response: A Stimulation Index of 3 or greater is an indication of a postive response.

TREATMENT PREPARATION AND ADMINISTRATION:
The test item was freshly diluted with the selected vehicle to obtain appropriate concentrations. The applicable dose levels were based on the results of the Preliminary Irritation / Toxicity Test. Formulations were prepared on weight: volume basis as % (w/v), and were considered to be stable for this short period (no correction for purity of the test item was applied). The formulations were checked for visible homogeneity and physical stability.


Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Parameter:
SI
Remarks on result:
other: 10% (w/v) = 3.6 5% (w/v) = 2.2 2.5% (w/v) = 1.3
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: 10% (w/v) = 18895 5% (w/v) = 11452.5 2.5% (w/v) = 6534.5

In the preliminary study no mortality was observed. Marked body weight loss (>5%) was detected for each animal, the mean body weight loss in both dose groups was above the acceptable limit indicating systemic toxicity. Minimal alopecia was observed in the 50% (w/v) dose group on Days 5 -6. Increased ear thickness values were detected in some cases on Days 3 and 6 but all were below the limit of positivity. The ear punch weights were within the acceptable range. No erythema was observed. The draining auricular lymph nodes were visually examined: they were larger than normal in both dose groups. Based on these results, 50 and 25% (w/v) doses showed signs of systemic toxicity, and excluded from the examined concentration series of the main test. An additional Preliminary experiment was performed to provide data for dose selection. This experiment was using 3 doses (2 mice/dose) at test item concentrations of 10, 5 and 2.5 % (w/v) in DMSO. During this test no mortality, signs of systemic toxicity, marked body weight loss (>5%) nor erythema were detected. Slightly increased ear thickness was detected on Days 3 and 6 in some cases, but the values were below the positivity. The observed punch weights were also in the acceptable range. Therefore, 10% (w/v) was selected as the top dose for the main test.

Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item was shown to have sensitisation potential (sensitizer) in the Local Lymph Node Assay under the test conditions.
Executive summary:

The aim of this study was to determine the skin sensitisation potential of the test item following dermal exposure, according to OECD Guideline 429, EU Method B.42 and following GLP. It was conducted with CBA/CaOlaHsd female mice.

The Preliminary Irritation / Toxicity Tests (I and II) were performed in CBA/CaOlaHsd mice using 5 doses (50, 25, 10, 5 and 2.5% (w/v) in DMSO). Based on the observations of systemic toxicity signs in the 50 and 25 % (w/v) dose groups, 10% (w/v) was chosen as the top dose for the main test.

In the Main Test, 20 animals were allocated in 5 groups: 3 dose, 1 positive control (25% (w/v) HCA in DMSO) and 1 solvent control group. The test item solutions were applied on the dorsal surface of ears of experimental animals (25 μL/ear) for 3 consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6. On Day 6, the cell proliferation in the local lymph nodes was measured by incorporation of tritiated methyl thymidine (3HTdR) and the values obtained were used to calculate Stimulation Indices (SI).

No mortality or signs of systemic toxicity were observed during the study. No treatment related body weight loss was observed and there was no indication of any irritancy at the site of application.

The Stimulation Index (SI) values were 3.6, 2.2 and 1.3 at concentrations of 10, 5 and 2.5% (w/v), respectively (surpassing the threshold of 3). The extrapolated EC3 value was 7.9% (w/v). The performance of the assay is considered valid as the SI of the positive control was 3.1 (above de threshold).

In conclusion, under the conditions of the present assay, the test item was shown to have sensitisation potential (sensitizer) in the Local Lymph Node Assay.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

Key study: The skin sensitization test was performed according to OECD Guideline 429, EU Method B.42 and following GLP.

No mortality or signs of systemic toxicity were observed during the study.

The Stimulation Index (SI) values were 3.6, 2.2 and 1.3 at concentrations of 10, 5 and 2.5% (w/v), respectively (surpassing the threshold of 3). The extrapolated EC3 value was 7.9% (w/v). The performance of the assay is considered valid as the SI of the positive control was 3.1 (above de threshold).

In conclusion, under the conditions of the present assay, the test item was shown to have sensitisation potential (sensitizer) in the Local Lymph Node Assay.


Migrated from Short description of key information:
Key study: OECD guideline 429, GLP study. The substance has sensitisation potential (sensitizer) in the Local Lymph Node Assay.

Justification for selection of skin sensitisation endpoint:
One study available (according to OECD 429, with GLP).

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the available results, the substance is classified as Skin Sensitizer Category 1B, H317 according to CLP Regulation (EC) no. 1272/2008.