Registration Dossier

Diss Factsheets

Administrative data

Description of key information

Skin sensitisation (OECD 442B): sensitising

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:
21 Apr - 16 Nov 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)
Version / remarks:
(2010)
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA): BrdU-ELISA
Species:
mouse
Strain:
other:
Remarks:
CBA/N
Details on test animals and environmental conditions:
TEST ANIMALS
- Females nulliparous and non-pregnant: not specified
- Microbiological status of animals, when known: SPF
- Age at study initiation: 8 weeks (Range-finding study); 9 weeks (Main study Set 1 and 2)
- Weight at study initiation: 16.1 - 19.8 g (Range-finding study); 14.9 - 19.0 g (Main study Set 1); 16.0 - 20.3 g (Main study Set 2)
- Housing: in groups of 2 - 3 animals per cage in polysulfone cages (200W x 320D x 140H mm)
- Diet: pelleted rodent chow (Teklad Certified Irradiated Global 18% Protein Rodent Diet 2918C), ad libitum
- Water: tap water (filtered and irratiated by uv-light), ad libitum
- Acclimation period: 4 days (Range-finding study, Main study Set 2); 11 days (Main study Set 1)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.3 - 22.4 (Range-finding study); 21.3 - 22.8 (Main study Set 1); 21.1 - 22.3 (Main study Set 2)
- Humidity (%): 47.0 - 55.8 (Range-finding study) 45.7–59.1 (Main study Set 1); 49.5 - 55.4% (Main study Set 2)
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12 / 12
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Range-finding study: 5, 10, 25, 50 and 100% (w/v)
Main study Set 1: 25, 50 and 100% (w/v)
Main study Set 2: 1, 5 and 10% (w/v)
No. of animals per dose:
2 (range-finding study), 5 (main study)
Details on study design:
RANGE-FINDING STUDY: Dose selection was based on consecutive doses and dose levels were selected from a series of appropriate concentrations such as 100, 50, 25, 10 and 5%.
- Compound solubility: The test substance was dissolved in aceton/olive oil in a preliminary solubility test. Therefore, aceton/olive oil was utilized as vehicle for this study.
Two animals were observed per dose group. All mice were observed daily for any clinical signs of systemic toxicity or local irritation at the application site. Body weights were recorded prior to dosing (Day 1) and on the day of necropsy, Day 6. Both ears of each mouse were observed for erythema and scored using erythema scores. Ear thickness measurement was taken using a thickness gauge on Day 1 (pre-dose), Day 3 and Day 6. Additionally, on Day 6, ear weight was determined by balance.
- Irritation: not specified
- Systemic toxicity: toxicity was not observed in the high dose group (100%)
- Ear thickness measurements: not specified
- Erythema scores: not specified

MAIN STUDY: Based on the result of the dose range finding study, the high dose for the main study was selected at 100%. Two additional low dose levels (50 and 25%) were produced by applying a geometric ratio of 2. In addition, positive and negative control groups were included in the main study. Additional doses of the test substance were included at 1, 5, 10 % (Main Study, Set 2) after discussion with the sponsor.

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: ELISA BrdU
- Criteria used to consider a positive response: A stimulation index (SI) was calculated for each group using the BrdU labelling index of each test group divided by the BrdU labelling index of the vehicle control group. SI < 1.6 is considered as negative result. SI ≥ 1.6 is considered as positive result.

The EC1.6 value was used to classify the test substance according to ECETOC Potency classification as follows:
EC1.6 Value(%) ≥ 10 to ≤ 100 -> Weak
EC1.6 Value(%) ≥ 1 to ≤ 10 -> Moderate
EC1.6 Value(%) ≥ 0.1 to ≤ 1 -> Strong
EC1.6 Value(%) < 0.1 -> Extreme
Cindy A et al. Extrapolating local lymph node assay EC3 values to estimate relative sensitizing potency. Cutaneous and Ocular Toxicology, 2007, 26: 135-145.

TREATMENT PREPARATION AND ADMINISTRATION: A volume of 25 μL was applied to the dorsum of both ears of all animals daily for three consecutive days. Two days after the third application on Day 5, an intraperitoneal injection of 0.5 mL (5 mg/mouse) of BrdU solution (10 mg/mL) was made. Approximately 24 h after BrdU injection, the mice were sacrificed and draining auricular lymph nodes from each mouse ear were excised and processed separately in phosphate buffered saline for each animal. A single cell suspension was prepared by separation through a nylon mesh. In each case, the target volume of the cell suspension was adjusted to the determined optimized volume. The optimized volume was based on the mean absorbance within 0.1 - 0.2 in the negative control group. BrdU was measured by ELISA using a commercial kit. Briefly, 100 µL of the lymph node cell suspension was added to the wells of a microplate in triplicate. After fixation and denaturation of the cell suspension, anti-BrdU antibody was added to each well. Subsequently, anti-BrdU antibody was removed by washing and the substrate solution was added. Absorbance was measured at 370 nm with a reference wavelength of 492 nm.

Negative control animals were dosed with the vehicle, acetone/olive oil solution.
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 t-test was applied for multiple comparisons (significance levels: 0.05 and 0.01, one-tailed) between the negative control group and each of the test substance groups or positive substance group. Since it was not significant, Kruskal-Wallis test was employed on heterogeneous data and Steel’s test was applied for multiple comparisons (significance levels: 0.05 and 0.01, one-tailed) between the negative control group and each of the test substance groups or positive substance group.
Kruskal-Wallis test for the erythema score was employed on heterogeneous data, and Steel’s test was applied for multiple comparisons (significance levels: 0.05 and 0.01, one-tailed) between the negative control group and each of the test substance groups or positive substance group.
Positive control results:
Body weights: Set 1 : The mean values of body weight ranged from 18.5 to 19.6 g. There were no significant differences when compared to the negative control group. Set 2: The mean values of body weight ranged from 18.6 to 19.2 g. There were no significant differences when compared to the negative control group.
Irritation: Set 1: The mean value of erythema scores ranged from 0.0 to 0.7. There was a significant increase when compared to the negative control group (p < 0.01: Day 4, 5 and 6). Set 2: The mean value of erythema score range from 0.0 to 1.8. There was a significant increase when compared to the negative control group (p < 0.01: Day 2, 3, 4, 5 and 6).
Ear thickness: Set 1 and 2: The mean value of ear thickness ranged from 0.19 to 0.21 mm. There was a significant increase when compared to the negative control group (Set 1: p < 0.01: Day 3, p < 0.05: Day 6; Set 2: p < 0.01: Day 6).
Ear weights: Set 1: The mean value of ear tissue weight was 14.9 mg. There was a significant increase when compared to the negative control group (p < 0.01). Set 2: The mean value of ear tissue weight was 14.0 mg. There was a significant increase when compared to the negative control group (p < 0.01).
Stimulation Index: Set 1: The mean value of stimulation index was 2.41. There was a significant increase when compared to the negative control group (p < 0.05). Set 2: The mean value of stimulation index was 3.16. There was a significant increase when compared to the negative control group (p < 0.01).
Key result
Parameter:
SI
Value:
1.33
Test group / Remarks:
1% (w/v)
Key result
Parameter:
SI
Value:
1.2
Test group / Remarks:
5% (w/v)
Key result
Parameter:
SI
Value:
1.27
Test group / Remarks:
10% (w/v)
Key result
Parameter:
SI
Value:
1.91
Test group / Remarks:
25% (w/v)
Key result
Parameter:
SI
Value:
3.53
Test group / Remarks:
50% (w/v)
Key result
Parameter:
SI
Value:
2.35
Test group / Remarks:
100% (w/v)
Key result
Parameter:
other: EC1.6
Value:
12.73
Cellular proliferation data / Observations:
IRRITATION, EAR THICKNESS and EAR WEIGHTS:
Erythema Score: Set 1: In the negative control group, the mean value of erythema score ranged from 0.0 to 0.0 prior to dosing until Day 6 after dosing. In the test substance groups at 25, 50 and 100%, the mean values of erythema score ranged from 0.0 to 0.3, 0.0 to 0.6 and 0.0 to 0.5, respectively. There was a significant increase when compared to the negative control group (p < 0.05: Days 4, 5 and 6: 50 and 100%).
Set 2: In the negative control group, the mean value of erythema score ranged from 0.0 to 0.0 prior to dosing until Day 6 after dosing. In the test substance groups at 1, 5 and 10%, the mean values of erythema score ranged from 0.0 to 0.0, 0.0 to 0.1 and 0.0 to 1.0, respectively. There was a significant increase when compared to the negative control group (p < 0.01: Days 4, 5 and 6: 10%).

Ear Thickness: Set 1: In the negative control group, the mean value of ear thickness ranged from 0.20 to 0.20 mm prior to dosing until Day 6 after dosing. In the test substance groups at 25, 50 and 100%, the mean values of ear thickness ranged from 0.19 to 0.20, 0.20 to 0.21 and 0.19 to 0.21 mm, respectively. There was a significant increase when compared to the negative control group (p < 0.01: Day 3: 25, 50 and 100%, Day 6: 50 and 100%).
Set 2: In the negative control group, the mean value of ear thickness ranged from 0.19 to 0.19 mm prior to dosing until Day 6 after dosing. In the test substance groups at 1, 5 and 10%, the mean values of ear thickness ranged from 0.19 to 0.19, 0.19 to 0.19 and 0.19 to 0.20 mm, respectively. There was a significant increase when compared to the negative control group (p < 0.05: Day 6: 10%, p < 0.01: Day 3: 10%).

Ear weights: Set 1: In the negative control group, the mean value of ear tissue weight was 12.9 mg. In the test substance groups at 25, 50 and 100%, the mean values of ear tissue weight were 15.3, 16.0 and 18.9 mg, respectively. There was a significant increase when compared to the negative control group (p < 0.01: 25, 50 and 100%).
Set 2: In the negative control group, the mean value of ear tissue weight was 12.5 mg. In the test substance groups at 1, 5 and 10%, the mean values of ear tissue weights were 12.8, 13.4 and 14.3 mg, respectively. There was a significant increase when compared to the negative control group (p < 0.01: 10%).

DETAILS ON STIMULATION INDEX CALCULATION: Set 1: In the negative control group, the mean value of stimulation index was 1.00. In the test substance groups at 25, 50 and 100%, the mean values of stimulation index were 1.91, 3.53 and 2.35, respectively. There was a significant increase when compared to the negative control group (p<0.05: 25, 50 and 100%).
Set 2: In the negative control group, the mean value of stimulation index was 1.00. In the negative control group, the mean value of stimulation index was 1.00. In the test substance groups at 1, 5 and 10%, the mean values of stimulation index were 1.33, 1.20 and 1.27, respectively. There were no significant differences when compared to the negative control group.

EC1.6 CALCULATION: EC1.6 = c+[(1.6-d)/(b-d)] x (a-c)
a = The dose concentration with higher SI; b = The higher SI value, c = The dose concentration with lower SI; d = the lower SI value
EC1.6 = 12.73%

CLINICAL OBSERVATIONS: Set 1 and Set 2: There were no abnormal clinical signs or deaths in any dosing group during the observation period.

BODY WEIGHTS: Set 1: In the negative control group, the mean value of body weight ranged from 19.1 to 19.5 g prior to dosing until Day 6 after dosing. In the test substance groups at 25, 50 and 100%, the mean values of body weights ranged from 19.3 to 18.9, 18.9 to 19.0 and 18.9 to 19.0 g, respectively. There were no significant differences when compared to the negative control group. Set 2: In the negative control group, the mean value of body weight ranged from 18.7to 18.9 g prior to dosing until Day 6 after dosing. In the test substance groups at 1, 5 and 10%, the mean values of body weights ranged from18.6 to 18.5, 19.4 to 19.6 and 18.7 to 19.1 g, respectively. There were no significant differences when compared to the negative control group.
Interpretation of results:
other: Skin Sens. 1B (H317) required according to Regulation (EC) No 1272/2008
Conclusions:
Under the conditions of the local lymph node assay, the test substance revealed a SI ≥ 1.6 at concentrations of 25, 50 and 100%. The calculated EC1.6 value was 12.73%. Therefore, the test substance is considered to be weak sensitiser.
Executive summary:

The skin sensitising potential of the test substance was investigated in a Local Lymph Node Assay (LLNA) in female CBA/N mice using BrdU-ELISA method according to OECD Guideline 442B and in compliance with GLP (2017). A preliminary range-finding study was performed at dose levels of 5, 10, 25, 50 and 100% to determine the appropriate dose levels of the test substance in acetone/olive oil 4:1 (v/v). Based on the result of the dose range finding study, the high dose for the main study was selected at 100%. Two additional low dose levels (25 and 50%) were added for the first main experiment (Set 1). In a second experiment (Set 2) doses of 1, 5 and 10% were used. In addition, positive and negative controls were included. 5 animals per test group, negative and positive control group were used. In the main study, the test substance was applied to the dorsum of each ear for 3 consecutive days. All animals were observed for mortality, clinical signs and erythema for 6 days. Body weights were recorded on Day 1 prior to dosing and on Day 6. Ear thickness was measured on Day 1, 3 and 6. After necropsy on day 6 ear weights and stimulation indices were measured. In the test substance groups at dose levels of 25, 50 and 100% (Set 1) the body weight was not significantly increased when compared to the negative control group. The erythema score, ear weights, ear thickness and stimulation index were significantly increased when compared to the negative control group. In the test substance groups at 1, 5 and 10% the body weight and the stimulation indices were not significantly increased when compared to the negative control. Erythema score, ear weights and ear thickness were significantly increased when compared to the negative control. No abnormalities in clinical signs or death were observed in any animal. The test substance produced a stimulation index of ≥ 1.6 in three concentrations of 25, 50 and 100% (w/v) and an EC1.6 value of 12.73% was calculated. Based on the results of this LLNA, the test substance was classified as Skin Sens. 1B.

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

The skin sensitising potential of the test substance was investigated in a Local Lymph Node Assay (LLNA) in female CBA/N mice using BrdU-ELISA method according to OECD Guideline 442B and in compliance with GLP (2017). A preliminary range-finding study was performed at dose levels of 5, 10, 25, 50 and 100% to determine the appropriate dose levels of the test substance in acetone/olive oil 4:1 (v/v). Based on the result of the dose range finding study, the high dose for the main study was selected at 100%. Two additional low dose levels (25 and 50%) were added for the first main experiment (Set 1). In a second experiment (Set 2) doses of 1, 5 and 10% were used. In addition, positive and negative controls were included. 5 animals per test group, negative and positive control group were used. In the main study, the test substance was applied to the dorsum of each ear for 3 consecutive days. All animals were observed for mortality, clinical signs and erythema for 6 days. Body weights were recorded on Day 1 prior to dosing and on Day 6. Ear thickness was measured on Day 1, 3 and 6. After necropsy on day 6 ear weights and stimulation indices were measured. In the test substance groups at dose levels of 25, 50 and 100% (Set 1) the body weight was not significantly increased when compared to the negative control group. The erythema score, ear weights, ear thickness and stimulation index were significantly increased when compared to the negative control group. In the test substance groups at 1, 5 and 10% the body weight and the stimulation indices were not significantly increased when compared to the negative control. Erythema score, ear weights and ear thickness were significantly increased when compared to the negative control. No abnormalities in clinical signs or death were observed in any animal. The test substance produced a stimulation index of ≥ 1.6 in three concentrations of 25, 50 and 100% (w/v) and an EC1.6 value of 12.73% was calculated. Based on the results of this LLNA, the test substance was classified as Skin Sens. 1B.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The available data on skin sensitisation meet the criteria for classification as Skin Sens. 1B (H317) according to Regulation (EC) 1272/2008.