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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:
01 Feb - 02 Aug 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
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

Test material

Reference
Name:
Unnamed
Type:
Constituent

In vivo test system

Test animals

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; not older than 12 weeks
- Weight at study initiation: dose range finding study: 17.7 - 22.7 g; main study (Set 1): 17.8 - 21.4 g; main study (Set 2): 17.7 - 25.0 g
- Housing: 2 - 3 animals per cage in polysulfone cages (200Wx320Dx140H mm)
- Diet: Teklad Certified Irradiated Global 18% Protein Rodent Diet 2918C
- 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): 20.4 - 23.9
- Humidity (%): 39.9 - 66.4
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Dose range finding study: 5, 10, 25, 50% (w/v) and 100%
Set 1: 5, 10 and 25% (w/v)
Set 2: 25, 50% (w/v) and 100%
No. of animals per dose:
2 (range-finding study), 5 (main study)
Details on study design:
RANGE-FINDING STUDY:
- 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.
Dose selection was based on the consecutive doses and dose levels were selected from a series of appropriate concentrations such as 5, 10, 25, 50 and 100%. 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: toxicity was observed (decreased body weight between 1-5% in all treatment groups
- 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 25%. Two additional lower dose levels (5 and 10%) were established. In addition, the positive and negative control groups were included in the main study. Test substance concentrations of 25, 50 and 100% were added (Set 2) at the request of the sponsor after systemic toxicity (body weight loss) could not be verified 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.

Results and discussion

Positive control results:
Body weights: Set 1: In the positive control group at 25%, the mean body weight was 19.0–19.4 g. There were no significant differences when compared to the negative control group. Set 2: In the positive control group at 25%, the mean body weight was 20.5–20.8 g. There were no significant differences when compared to the negative control group.
Erythema Score: Set 1: In the positive control group at 25%, the mean erythema score was 0–3. There were significant increases when compared to the negative control group (p<0.01: Days 3, 4, 5 and 6). Set 2: In the positive control group at 25%, the mean erythema score was 0–1. There were significant increases when compared to the negative control group (p<0.05: Day 5 and 6, p<0.01: Day 4).
Ear thickness: Set 1: In the positive control group at 25%, the mean ear thickness was 0.19–0.23 mm. There were significant increases when compared to the negative control group (p<0.05: Days 1 and 3, p<0.01: Day 6). Set 2: In the positive control group at 25%, the mean ear thickness was 0.19–0.20 mm. There were significant increases when compared to the negative control group (p<0.05: Day 6).
Ear weights: Set 1: In the positive control group at 25%, the mean ear weight was 15.0 mg. There were significant increases when compared to the negative control group (p<0.01). Set 2: In the positive control group at 25%, the mean ear weight was 13.5 mg. There were significant increases when compared to the negative control group (p<0.05).
Stimulation index: Set 1: In the positive control group at 25%, the mean stimulation index was 2.98. There was a significant increase when compared to the negative control group (p<0.05). Set 2: In the positive control group at 25%, the mean stimulation index was 1.65. There was a significant increase when compared to the negative control group (p<0.01).

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
1.01
Test group / Remarks:
5% (w/v)
Key result
Parameter:
SI
Value:
1.02
Test group / Remarks:
10% (w/v)
Key result
Parameter:
SI
Value:
1.34
Test group / Remarks:
Set 1: 25% (w/v)
Key result
Parameter:
SI
Value:
1.21
Test group / Remarks:
Set 2: 25% (w/v)
Key result
Parameter:
SI
Value:
1.61
Test group / Remarks:
50% (w/v)
Key result
Parameter:
SI
Value:
1.88
Test group / Remarks:
100% (w/v)
Key result
Parameter:
other: EC1.6
Value:
49.4

Any other information on results incl. tables

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 body weight was 19.2–19.5 g from Day 1 to Day 6 after dosing. In the test substance groups at 5, 10 and 25%, the mean body weights were 19.2–19.5, 19.0–19.4 and 19.6–19.7 g, respectively. There were no significant differences when compared to the negative control group.

Set 2: In the negative control group, the mean body weight was 20.5–20.8 g from Day 1 to Day 6 after dosing. In the test substance groups at 25, 50 and 100%, the mean body weights were 19.8–20.2, 19.1–20.3 and 20.2–20.5 g, respectively. There were no significant differences when compared to the negative control group.

IRRITATION, EAR THICKNESS and EAR WEIGHTS:

Erythema score: Set 1: 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 5, 10 and 25%, the mean erythema scores were 0–0, 0–1 and 0–2, respectively. There were significant increases when compared to the negative control group (p<0.05: Days 5 and 6 (10%), Day 3 (25%), p<0.01: Days 4, 5 and 6 (25%)).

Set 2: 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 25, 50 and 100%, the mean erythema scores were 0–1, 0 and 0–1, respectively. There were significant increases when compared to the negative control group (p<0.05: Days 5 and 6 (25%), Days 4, 5 and 6 (100%)).

Ear thickness: Set 1: In the negative control group, the mean ear thickness was 0.19–0.20 mm from Day 1 to Day 6 after dosing. In the test substance groups at 5, 10 and 25%, the mean ear thickness was 0.19–0.21, 0.19–0.21 and 0.19–0.22 mm, respectively. There were significant increases when compared to the negative control group (p<0.05: Day 6 (5%), p<0.01: Day 6 (10%), Days 1 and 6 (25%)).

Set 2: 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 25, 50 and 100%, the mean ear thickness was 0.19, 0.19–0.20 and 0.19–0.21 mm, respectively. There were significant increases when compared to the negative control group (p<0.05: Day 6 (25%), p<0.01: Day 6 (50%)).

Ear weights: Set 1: In the negative control group, the mean ear weight was 12.7 mg. In the test substance groups at 5, 10 and 25%, the mean ear weights were 13.5, 13.7 and 14.2 mg, respectively. There were significant increases when compared to the negative control group (p<0.05: 10%, p<0.01: 25%).

Set 2: In the negative control group, the mean ear weight was 12.3 mg. In the test substance groups at 25, 50, 100% the mean ear weights were 13.2, 13.5 and 15.0 mg, respectively. There were significant increases when compared to the negative control group (p<0.05: 50%, p<0.01: 100%).

DETAILS ON STIMULATION INDEX CALCULATION: Set 1: In the negative control group, the mean stimulation index was 1.00. In the test substance groups at 5, 10 and 25%, the mean stimulation index was 1.01, 1.02 and 1.34, respectively. There were significant increases when compared to the negative control group (p<0.05: 25%).

Set 2: In the negative control group, the mean stimulation index was 1.00. In the test substance groups at 25, 50 and 100%, the mean stimulation index was 1.21, 1.61 and 1.88, respectively. There were significant increases when compared to the negative control group (p<0.01: 50 and 100%).

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 = 49.4%

Applicant's summary and conclusion

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
Under the conditions of the local lymph node assay, the test substance revealed a SI ≥ 1.6 at concentrations of 50 and 100% (w/v). The calculated EC1.6 value was 49.4%. Therefore, the test substance is considered as weak sensitiser.
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. Based on the results of the dose range finding study, the high dose level of the main study was selected at 25%, and two additional lower dose levels (10 and 5%) were established. In addition, positive and negative control groups were included in the main study (Main Study, SET1). As no clinical signs or effects on body weights were observed, the concentrations 25%, 50 %, and 100% were added (Main Study, SET2). In the main study, no clinical signs and abnormalities were observed in any animal.

<SET1>

In the test substance groups, body weights were not significantly different when compared to the negative control group. Erythema score, ear thickness, ear weights and stimulation index (SI) were significantly different when compared to the negative control group. In the positive control group, body weights were not significantly different when compared to the negative control group. Erythema score, ear thickness, ear weights, and SI values were significantly different when compared to the negative control group.

<SET2>

In the test substance groups, body weights were not significantly different when compared to the negative control group. Erythema score, ear thickness, ear weights, and SI values were significantly different when compared to the negative control group.

In the positive control group, body weights were not significantly different when compared to the negative control group. Erythema score, ear thickness, ear weights, and SI values were significantly different when compared to the negative control group.

Based on the results of this study, the test substance produced the following SI values: 1.01, 1.02, 1.34, 1.61, and 1.88 at the concentrations of 5, 10, 25, 50 and 100%, respectively. The EC1.6 value was 49.4%, thus the test substance was considered to be a weak sensitizer.