Registration Dossier

Administrative data

Description of key information

An in vivo LLNA study performed with Sakura Salicylate is available (Klimisch 1 study).

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:
10 September 2015 - 22 December 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)
Version / remarks:
adopted 22 July 2010
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA): BrdU-ELISA
Species:
mouse
Strain:
CBA:JN
Sex:
female
Details on test animals and environmental conditions:
Species and strain: Mice, CBA/JN
Sex: Females (nulliparous and non-pregnant)
Age: Approximately 8 weeks old, approximately 25 grams
Supplier: Charles River Italia S.p.A., Calco (Lecco), Italy
Breeder: Charles River France Laboratories, Iffa Credo, Domaine des Oncins B.P. 0109, F 69592, L’ARBRESLE CEDEX, France.
Weight range at arrival: 18 to 23 grams
Acclimatisation period: At least 5 days

Environmental condition:
Room lighting: Artificial (fluorescent tubes), daily light/dark cycle of 12/12 hours
Air changes: Approximately 15 to 20 air changes per hour
Temperature range: 22 °C +/- 2 °C
Relative humidity range: 55%+/-15%
Vehicle:
other: ethanol/diethyl phthalate 1:3 (v/v) as vehicle
Concentration:
Main Assay: 100, 50 and 25% (w/w)
Preliminary assay: 100, 50, 25, 10 and 5% (w/w)
No. of animals per dose:
5 animals for dose
Details on study design:
PRE-SCREEN TESTS:
- Irritation: The treated sites of all animals were examined daily (once before first dosing, before dosing on Days 2 and 3 and daily thereafter).
- Systemic toxicity: The animals were observed for clinical signs on: Day 1: before and 1 hour after dosing. Day 2 to 6: daily (approximately 1 hour after daily dosing, when applicable).
- Ear thickness measurements: The ear thickness was measured by a suitable micrometer on Day 1 (before dosing), on Day 3 (before dosing) and on Day 6.
- Erythema scores: Irritation to the skin was assigned a numerical value according to the the OECD guideline.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LOCAL LYMPH NODE ASSAY (LLNA: BrdU-ELISA method)
- Criteria used to consider a positive response: The test item is considered to induce sensitisation when the SI for any single treatment dose group is >/= 1.6. It is not required that an increased response is observed at increasing dose levels, but dose-related activity and/or statistical significance may be taken as further evidence of a sensitisation effect (i.e. in case of borderline results with 1.6 <= SI <= 1.9).

TREATMENT PREPARATION AND ADMINISTRATION:
The animals were treated for three consecutive days (Days 1, 2, 3) with the vehicles, test or positive control item formulations. A dose volume of 25 μL/ear/day of each selected concentration and controls was applied to the dorsal surface of each ear (50 μL/animal/day), using a micropipette.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Differences between each treated group and the concurrent negative control group (individual BrdU labelling indices) were assessed by Dunnett’s test. The homogeneity of the data was verified by Bartlett’s test before Dunnett’s test. If data were found to be inhomogeneous a Modified t test (Cochran and Cox) was applied.
Positive control results:
In the group treated with the positive control item, a Stimulation Index of 2.43 was calculated. As it was greater than 2, the study was regarded as valid.
Key result
Parameter:
SI
Value:
1.63
Test group / Remarks:
25%
Key result
Parameter:
SI
Value:
2.23
Test group / Remarks:
50%
Key result
Parameter:
SI
Value:
2.46
Test group / Remarks:
100%
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
Dose-related increases in cell proliferation of draining lymph nodes, significant at statistical analysis at the 2 higher concentrations, were observed in the three treatment groups.

DETAILS ON STIMULATION INDEX CALCULATION
The calculated Stimulation Indices (SI) were 1.63, 2.23 and 2.46, respectively at low, mid- and high dose levels (25%, 50% and 100% (w/w)).

CLINICAL OBSERVATIONS:
No mortality or clinical signs were recorded in animals treated at all dose levels investigated.

BODY WEIGHTS:
Changes in body weight observed during the study were within the expected range for this strain and age of animals.

In the preliminary study, no signs of toxicity (significant clinical signs or body weight losses) were observed at the tested concentrations. According to the results of the irritation screening, the concentration of 100% w/w was judged to be not irritant.

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
Based on the results of an in vivo LLNA assay, performed according to the OECD guideline and GLP principles, it is concluded that Sakura Salicylate has skin sensitising properties.
Executive summary:

The potential of Sakura Salicylate to cause skin sensitisation reactions following topical application to the skin of CBA/JN (CBA/J) mice, was assessed using the LLNA:BrdU-ELISA method, according to the OECD Guideline 442b and GLP principles.

In the main assay, the test item was topically administered at the concentrations of 100, 50 and 25% (w/w), in ethanol/diethyl phthalate 1:3 (v/v). No mortality or clinical signs were recorded in any animal. Changes in body weight observed during the study were within the expected range for this strain and age of animals. Statistically significant and dose-related increases in cell proliferation of draining lymph nodes were observed in the three treatment groups, being the calculated Stimulation Indices (SI) 1.63, 2.23 and 2.46, respectively at low, mid- and high dose levels (25%, 50% and 100% (w/w)). The results obtained in this study indicate that the test item may elicit a sensitisation response in mice following dermal exposure, since in all dose groups the Stimulation Index was greater than 1.6. The calculated EC1.6 (estimated concentration needed to produce a stimulation index of a positive response with SI1.6) is 22%. Based on this result, Sakura Salicylate is classified as a skin sensitiser 1B according to Regulation (EC) 1272/2008.

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

The potential of Sakura Salicylate to cause skin sensitisation reactions following topical application to the skin of CBA/JN (CBA/J) mice, was assessed using the LLNA:BrdU-ELISA method, according to the OECD Guideline 442b and GLP principles.

In the main assay, the test item was topically administered at the concentrations of 100, 50 and 25% (w/w), in ethanol/diethyl phthalate 1:3 (v/v). No mortality or clinical signs were recorded in any animal. Changes in body weight observed during the study were within the expected range for this strain and age of animals. Statistically significant and dose-related increases in cell proliferation of draining lymph nodes were observed in the three treatment groups, being the calculated Stimulation Indices (SI) 1.63, 2.23 and 2.46, respectively at low, mid- and high dose levels (25%, 50% and 100% (w/w)). The results obtained in this study indicate that the test item may elicit a sensitisation response in mice following dermal exposure, since in all dose groups the Stimulation Index was greater than 1.6.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The EC1.6 (estimated concentration needed to produce a stimulation index of a positive response with SI1.6) from a LLNA: BrdU-ELISA

with Sakura Salicylate was calculated to be 22%. Strickland et al (" Evaluation of Two Nonradiolabeled Murine Local Lymph Node Assays (LLNA) for Potency Categorization of Substances Causing Allergic Contact Dermatitis in Humans", NICEATM–ICCVAM SOT 2012 Poster) evaluated the decision criteria to classify substances based on BrdU-ELISA data. It was concluded that an effective threshold concentration ≤ 9% for the LLNA: BrdU-ELISA classified strong human sensitizers at rates comparable to that of the conventional LLNA. An effective threshold concentration of 22%, as found for Sakura Salicylate, would thus not justify classification cat 1A. Taken all data together, it is justified to classify Sakura Salicylate Cat. 1B for skin sensitising properties in accordance with Regulation (EC) 1272/2008.