Registration Dossier

Administrative data

Description of key information

Skin sensitisation (OECD 442B): skin 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:
06 Jul - 26 Jul 2017
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 Jul 2010
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA): BrdU-ELISA
Species:
mouse
Strain:
other: CBA/N
Sex:
female
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: 9 weeks (dose range finding study and main study)
- Weight at study initiation: 19.0 - 21.6 g (dose range finding study); 17.5 - 22.3 g (main study)
- Housing: 2 - 3 animals per cage in polysulfone cages (200W x 320D x 140H mm)
- Diet: Teklad Certified Irradiated Global 18% Protein Rodent Diet 2918C), ad libitum
- Water: tap water filtered and irradiated by ultraviolet light, ad libitum
- Acclimation period: 4 days
- Indication of any skin lesions: not specified

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.3 - 23.6 (dose range finding study); 21.6 - 23.2 (main study)
- Humidity (%): 44.7 - 58.4 (dose range finding study); 47.6 - 57.2 (main study)
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Dose range finding study: 5, 10, 25, 50% (w/v) and 100%
Main study: 2.5, 25% (w/v) and 100%
No. of animals per dose:
2 (dose range finding study); 5 (main study)
Details on study design:
RANGE-FINDING STUDY: Dose levels are selected from a series of appropriate concentrations such as 5, 10, 25, 50 and 100%.
- 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 score. 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 ear punch weight measurement.
- Irritation: not specified
- Systemic toxicity: no toxicity was 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 levels (2.5 and 25%) were established. In addition, the positive and negative control groups were included in the main study.

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
Ryan CA 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. Negative control animals were dosed with the vehicle, acetone/olive oil solution. 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.

OBSERVATIONS:
Animals were observed daily for mortality, general condition, clinical signs of toxicity and signs of local irritation (erythema) at the test site for 6 days. Individual body weights were recorded on Day 1 prior to dosing and on Day 6. Ear thickness was taken using a thickness gauge on Day 1 prior to dosing, Day 3 (approximately 48 h after the first dose) and Day 6.

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 weight: The mean body weights on Day 1 and Day 6 were 20.4 and 20.8 g. There were no significant differences when compared to the negative control group.
Erythema: The mean erythema score was in the range of 0–1 from Day 1 to Day 6. There were significant increases when compared to the negative control group (p<0.05: Day 3, p<0.01: Days 4–6)
Ear thickness: The mean ear thickness values were in the range 0.19–0.20 mm from Day 1 to Day 6, respectively. There were significant increases when compared to the negative control group (p<0.05: Days 3 and 6).
Ear weight: The mean ear weight was 13.5 mg. The observed effects were significantly increased when compared to the negative control group (p<0.05).
Stimulation index: The mean stimulation index was 2.06. There was a significant increase when compared to the negative control group (p<0.01).
Parameter:
SI
Value:
1.09
Test group / Remarks:
2.5% (w/v)
Parameter:
SI
Value:
2.02
Test group / Remarks:
25% (w/v)
Parameter:
SI
Value:
2.84
Test group / Remarks:
100%
Key result
Parameter:
other: EC1.6
Value:
14.8
Cellular proliferation data / Observations:
IRRITATION, EAR THICKNESS and EAR WEIGHTS:
Erythema Score: In the negative control group, the mean erythema score was 0 from Day 1 to Day 6 after dosing.
In the test substance groups at 2.5, 25 and 100%, the mean erythema scores were 0, 0–1 and 0–2 from Day 1 to Day 6, respectively.

Ear thickness: In the negative control group, the mean ear thickness was 0.19 mm from Day 1 to Day 6 after dosing.
In the test substance groups at 2.5, 25 and 100%, the mean ear thickness values (Day 1 to Day 6) were in the ranges of 0.19–0.19, 0.19–0.20 and 0.19–0.21 mm.

Ear weights: In the negative control group, the mean ear weight was 12.4 mg.
In the test substance groups at 2.5, 25 and 100%, the mean ear weights were 13.0, 13.9 and 16.5 mg, respectively.

DETAILS ON STIMULATION INDEX CALCULATION:

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 = 14.8%

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 weights were 19.9 and 20.3 g on Day 1 and Day 6 after dosing, respectively.
In the test substance groups at 2.5, 25 and 100%, the mean body weights were 20.1, 20.2 and 20.2 g on Day 1, and 20.9, 20.6 and 20.4 g on Day 6, respectively.
Interpretation of results:
other: Skin Sens Cat 1B 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 and 100%. The calculated EC1.6 value was 14.8%. Therefore, the test substance is considered to be a weak skin sensitiser.
Executive summary:

Under the conditions of the local lymph node assay, the test substance revealed a SI ≥ 1.6 at concentrations of 25 and 100%. The calculated EC1.6 value was 14.8%. Therefore, the test substance is considered to be a weak skin sensitiser.

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

The skin sensitisation potential of the test substance was assessed by a LLNA test using the BrdU ELISA method according to OECD Guideline 442B in compliance with GLP (2017). Treatment of CBA/N mice with the test substance revealed stimulation indices of 1.09, 2.02 and 2.84 at test substance concentrations of 2.5, 25, 100% (w/v) in acetone/olive oil (4:1 v/v), respectively. The EC1.6 value was calculated to be 14.8%.

In conclusion, based on the available data the test substace is considered to be a weak skin sensitiser.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The available data on skin sensitisation meets the criteria for classification as Skin Sens 1B (H317) according to Regulation (EC) No. 1272/2008.