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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:
2016-06-10 to 2016-12-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)
Version / remarks:
2010
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA): BrdU-ELISA

Test material

Reference
Name:
Unnamed
Type:
Constituent

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Remarks:
CBA/N, SPF
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS

- Age at study initiation: 9 weeks
- Weight at study initiation: 19.2–23.2 g (Dose range finding study), 18.2-21.8 g (Main study)
- Housing: 2–5 animals/cage (during the quarantine-acclimation period) / 2–3 animals/cage (during the study)
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 4-5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.6–22.9°C
- Humidity (%): 51.4–61.0%
- Air changes (per hr): 10–15 clean, fresh, filtered air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hour light/ dark cycle (7 AM to 7 PM via automated timer) 150–300 Lux

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Remarks:
The test substance was dissolved in AOO in a preliminary solubility test. Therefore, AOO was utilized as vehicle for this study.
Concentration:
10, 25, 100 %
No. of animals per dose:
5
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: The test substance was dissolved in AOO in a preliminary solubility test. Therefore, AOO was utilized as vehicle for this study.
Concentrations: 100, 50, 25, 10 and 5%
Two animals were observed per dose group.

MAIN STUDY
The positive and negative control groups were included in the main study. Used method for Ear thickness measurement is the same like in the pre-screen test.

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Group assignment
- Criteria used to consider a positive response:
SI < 1.6: Negative
SI ≥ 1.6: Positive

TREATMENT PREPARATION AND ADMINISTRATION:
Route: Application to the dorsum of each ear
Method of administration: A volume of 25 μL was applied to the dorsum of both ears of all animals daily for three consecutive days. The dose level of the positive substance was selected at 25%, which is the dose recommended in the guideline.
Negative control animals were dosed with the vehicle, AOO solution.

Observations:
- Clinical signs and mortality: All mice were observed daily for any clinical signs of systemic toxicity or local irritation at the application site
- Body weight: Body weights were recorded prior to dosing (Day 1) and on the day of necropsy, Day 6
- Ear thickness measurements: Ear thickness measurement was taken using a thickness gauge on Day 1 (pre-dose), Day 3 (approximately 48 hours after the first dose) and Day 6 (the day of necropsy).
- Erythema scores: Both ears of each mouse were observed for erythema and scored for 6 days.
No erythema: 0
Very slight erythema (barely perceptible): 1
Well-defined erythema: 2
Moderate to severe erythema: 3
Severe erythema (beet redness) to eschar formation preventing grading of erythema: 4

Necropsy
Approximately 24 hours (24 h) after BrdU injection, the animals were euthanized under CO2 gas. The draining auricular lymph nodes from each mouse ear were excised and processed separately in phosphate buffered saline (PBS) for each animal.

Determination of cellular proliferation
A volume of 0.5 mL (5 mg/mouse) of 5-Bromo-2’-deoxyuridine (BrdU) (10 mg/mL) solution was injected inter-peritoneally.
BrdU was measured by ELISA using a commercial kit.
Absorbance at 370 nm (Emission wavelength, em) with a reference wavelength of 492 nm (Reference wavelength, ref) was measured.

EVALUATION CRITERIA
- When the SI < 1.6, the result is negative.
- When the SI >= 1.6, the result is positive.
The EC1.6 value is used to classify the test substance as follows.
EC1.6 Value (%) ≥10 to ≤100: ECETOC Potency Classification Weak
EC1.6 Value (%) ≥1 to ≤10: ECETOC Potency Classification Moderate
EC1.6 Value (%) ≥0.1 to ≤1: ECETOC Potency Classification Strong
EC1.6 Value (%) <0.1: ECETOC Potency Classification Extreme
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Statistical analysis was conducted using a statistical program (version 9.3, SAS Institute Inc., U.S.A.) for the data including body weight, erythema score, ear thickness, ear weight and stimulation index.
Bartlett’s test was employed on homogeneity of variance (significance level: 0.05) for body weights, ear thickness, ear weight and stimulation index data. One-way analysis of variance (ANOVA) was employed on homogeneous data.
Dunnett’s test was applied for multiple comparisons.
Kruskal-wallis test for the erythema score was employed on heterogeneous data, and Steel’s test was applied for multiple comparisons.

Results and discussion

Positive control results:
Body Weights
In the positive control group at 25%, the mean body weight was 19.8–20.5 g. There were no significant differences when compared to the negative control group.

Erythema Scores
In the positive control group at 25%, the mean erythema score was 0.0–1.0. There were significant increases when compared to the negative control group (p<0.05: Days 3 and 4, p<0.01: Days 5 and 6).

Ear Thickness
In the positive control group at 25%, the mean ear thickness was 0.18–0.21 mm. There were significant increases when compared to the negative control group (p<0.05: Day 3, p<0.01: Day 6).

Stimulation Index
In the positive control group at 25%, the mean stimulation index was 3.20. There was a significant increase when compared to the negative control group (p<0.05).

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
1.48
Test group / Remarks:
test substance group 10%
Key result
Parameter:
SI
Value:
2.86
Test group / Remarks:
test substance group 25%
Key result
Parameter:
SI
Value:
3.68
Test group / Remarks:
test substance group 100%
Cellular proliferation data / Observations:
Dose Range Finding Study
Ear weight did not show toxicity but it was increased when compared to the negative control group.

CELLULAR PROLIFERATION DATA (mean values of 5 animals):
BrdU labelling index:
1)Negative control: 0.12
2)Test substance 10%: 0.18
3)Test substance 25%: 0.34
4)Test substance 100%: 0.44

DETAILS ON STIMULATION INDEX CALCULATION
The average BrdU labelling index of each group was substituted into the following equation of stimulation index (Stimulation index, SI) to calculate SI.
SI =Mean of BrdU labeling index in the test substance/ Mean of BrdU labeling index in the negative control

CLINICAL OBSERVATIONS:
There were no abnormal clinical signs or deaths in any dosing group during the observation period.

BODY WEIGHTS
In the negative control group, the mean body weight was 20.0–20.1 g from Day 1 to Day 6 after dosing.
In the test substance groups at 10, 25 and 100%, the mean body weights were 19.8–19.9, 19.5–20.2 and 19.8–20.6 g, respectively. There were no significant differences when compared to the negative control group.

Erythema Scores
In the negative control group, the mean erythema score was 0.0–0.0 from Day 1 to Day 6 after dosing.
In the test substance groups at 10, 25 and 100%, the mean erythema scores were 0.0–0.0, 0.0–0.0 and 0.0–1.0, respectively. There were significant increases when compared to the negative control group (p<0.01: Days 5 and 6 (100%)).

Ear Thickness
In the negative control group, the mean ear thickness was 0.19–0.18 mm from Day 1 to Day 6 after dosing.
In the test substance groups at 10, 25 and 100%, the mean ear thickness were 0.19–0.19, 0.19–0.19 and 0.18–0.21 mm, respectively. There were significant increases when compared to the negative control group (p<0.05: Day 1 (10%), Day 3 (10 and 25%), p<0.01: Day 3 (100%), Day 6 (100%)).

Stimulation Index EC 1.6
One concentration (test group at 10%) showed SI of <1.6, and EC1.6 was calculated to be 15 %

Applicant's summary and conclusion

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
Based on the calculated EC1.6 value of 15 %, the test substance is considered to be a weak sensitizer.
Executive summary:

The purpose of this study was to assess the skin sensitization potential of the test substance, after application to the dorsum of each ear of female CBA/N mice.

The dose range finding study was conducted at dose levels of 5, 10, 25, 50 and 100% to determine the high dose level for the main study. In the dose range finding study, the clinical signs, body weights, erythema score, ear thickness and ear weights by the toxicity of the test substance were evaluated.

Based on the result of the dose range finding study, the ear weight did not show toxicity, but it was increased when compared to the negative control group. According to the dose range finding study, the EC1.6 concentration was estimated. The high dose level for the main study was selected at 100% with two additional lower dose levels at 25 and 10 % (in consultation with the sponsor). In addition, the positive and negative control groups were included in the main study.

In clinical signs, no abnormalities were observed in any animal. In the test substance groups, the body weight was not significantly different when compared to the negative control group. But the erythema score, ear thickness, ear weight and stimulation index were significantly increased when compared to the negative control group. In the positive control group, the body weight was not significantly different when compared to the negative control group. The erythema score, ear thickness, ear weight and stimulation index were significantly increased when compared to the negative control group.

The test substance produced a stimulation index of ≥1.6 in all groups except the 10 % group of CBA/N mice, and it is therefore considered to be a sensitizer (defined as producing a positive response).

Based on the calculated EC1.6 value of 15 %, the test substance is considered to be a weak sensitizer.