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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Remarks:
(LLNA: BrdU)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 May 2016 - 22 December 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
This information is used for Geranyl Isobutyrate MCS.

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:
22 July 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
- Source: Japan SLC, Inc., Japan (producer); Central Lab. Animal Inc., Republic of Korea (Supplier)
- Females (if applicable) nulliparous and non-pregnant: not specified]
- Age at study initiation: 9 - 12 weeks
- Weight at study initiation: 18.5 - 21.0g (dose range finding); 17.6 - 21.7g (main set1); 19.1 - 25.7g (main set2)
- Housing: Group housing 2–3 animals/Polysulfone cage
- Diet: free access to Pelleted rodent chow (Teklad Certified Irradiated Global 18% Protein Rodent Diet 2918C)
- Water: free access to public tap water filtered and irradiated by ultraviolet light
- Acclimation period: 4 days
- Indication of any skin lesions: no

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.1 - 24.7
- Humidity (%): 44.5 - 65
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12
- IN-LIFE DATES: From: 31 May 2016 To: 21 December 2016

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0, 25, 50 and 100% (set 1); 0, 1, 5 and 10% (set 2)
No. of animals per dose:
5
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: the test substance was dissolved in the vehicle in a preliminary solubility test.
- 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 (predose), Day 3 and Day 6. Additionally, on Day 6, ear weight was determined by balance.

MAIN STUDY : the main study was conducted in two sets, with lower treatment doses used in the second set.

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Following the quarantine-acclimation period (group assignment), 25 healthy females were selected and randomly distributed into 5 groups (5 females per group).
- Criteria used to consider a positive response: SI ≥ 1.6

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. The dose level of the positive substance was selected at 25%. Negative control animals were dosed with the vehicle, acetone/olive oil solution.

OBSERVATIONS:
- Clinical signs: All animals were observed for mortality, general condition and clinical signs for 6 days.
- Body weights: Body weights were recorded prior to dosing (day 1) and on the day of necropsy (day 6).
- Erythema: Both ears of each mouse were observed for erythema and scored for 6 days.
- Ear thickness: Ear thickness measurement was taken using a thickness gauge (543-681B, Mitutoyo Co., Japan) on day 1 (pre-dose), day 3 (approximately 48 hours after the first dose) and day 6 (the day of necropsy).

On day 5, a volume of 0.5 mL (5 mg/mouse) of BrdU (10 mg/mL) solution was injected interperitoneally. 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 for each animal.

Preparation of cell suspension:
For each mouse, a single-cell suspension of lymph node cells (LNC) excised bilaterally was prepared by #70 nylon mesh to generate a single cell suspension. In each case, the target volume of the LNC suspension was adjusted to the determined optimized volume. The optimized volume was based on the mean absorbance within 0.1-0.2 in the NC group.

Determination of cellular proliferation: BrdU was measured by ELISA: 100 μL of the LNC suspension was added to the wells of a flat-bottom microplate in triplicate. After fixation and denaturation of the LNC suspension, anti-BrdU antibody was added to each well and allowed to react. Subsequently, anti-BrdU antibody was removed by washing and the substrate solution was then added and allowed to produce chromogen. Absorbance at 370 nm with a reference wavelength of 492 nm was measured.

BrdU labelling index = (ABSem - ABSblank,em) - (ABSref-ABSblank,ref)
with ABS = Absorption; em = emission wavelength; ref= reference substance

SI= mean of BrdU labelling index in the test substance / mean of BrdU labelling index in the negative control

Acceptability criterium: positive control should have a SI ≥ 1.6.

Evaluation criteria:
SI Result
SI < 1.6 Negative
SI ≥ 1.6 Positive
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 (G1, G6) and each of the test substance groups (G2–G4, G7–G9) or positive substance group (G5, G10). 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 (G1, G6) and each of the test substance groups (G2–G4, G7–G9) or positive substance group (G5, G10).

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 (G1, G6) and each of the test substance groups (G2–G4, G7–G9) or positive substance group (G5, G10).

Results and discussion

Positive control results:
Set 1: In the positive control group at 25%, the mean value of stimulation index was 3.10. 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 value of stimulation index was 4.32. There was a significant increase when compared to the negative control group (p<0.05).

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
other: EC1.6 (%)
Value:
26.4
Key result
Parameter:
SI
Value:
1.22
Remarks on result:
other: Test group: 1%
Key result
Parameter:
SI
Value:
1.1
Remarks on result:
other: Test group: 5%
Key result
Parameter:
SI
Value:
1.4
Remarks on result:
other: Test group: 10%
Key result
Parameter:
SI
Value:
1.78
Remarks on result:
other: Test group: 25%
Key result
Parameter:
SI
Value:
2.15
Remarks on result:
other: Test group: 50%
Key result
Parameter:
SI
Value:
1.7
Remarks on result:
other: Test group: 100%
Cellular proliferation data / Observations:
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 1.78, 2.15 and 1.70, respectively. There were significant increases 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 test substance groups at 1, 5, 10%, the mean values of stimulation index were 1.22, 1.10 and 1.40, respectively. There were no significant differences when compared to the negative control group.

EC1.6 CALCULATION
Three concentration showed SI of <1.6, and EC1.6 was calculated to be 26.4%.

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

BODY WEIGHTS (mean)
- Set 1:
In the negative control group, the mean values of body weights were 19.3–20.2 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 were 19.1–19.5, 19.5–19.6 and 19.3–19.3 g, respectively. There were no significant differences when compared to the negative control group.
In the positive control group at 25%, the mean values of body weights were 19.6–19.9 g. There were no significant differences when compared to the negative control group.
- Set 2:
In the negative control group, the mean values of body weights were 21.1–21.3 g prior to dosing until Day 6 after dosing.
In the test substance groups at 1, 5, 10%, the mean values of body weights were 21.7–21.4, 21.6–20.6 and 21.9–20.8 g, respectively. There were no significant differences when compared to the negative control group.
In the positive control group at 25%, the mean values of body weight were 21.4–20.9 g. There were no significant differences when compared to the negative control group.

ERYTHEMA SCORE:
- Set 1:
In the negative control group, the mean values of erythema score at 0.0 from prior to dosing until Day 6 after dosing.
In the test substance groups at 25, 50 and 100%, the mean values of erythema score was 0.0–0.7, 0.0–0.9 and 0.0–1.2, respectively. There were significant increases when compared to the negative control group (p<0.05: Day 4 (25%), p<0.01: Days 4 (50 and 100%), 5 and 6 (25, 50 and 100%)).
In the positive control group at 25%, the mean values of erythema score was 0.0–1.9. There were significant increases when compared to the negative control group (p<0.01: Days 3, 4, 5 and 6).
- Set 2:
In the negative control group, the mean values of erythema score at 0.0 prior to dosing until Day 6 after dosing.
In the test substance groups at 1, 5, 10%, the mean values of erythema was 0.0, 0.0–1.1 and 0.0–1.0, respectively. There were significant increases when compared to the negative control group (p<0.01: Days 5 and 6 (5 and 10%)).
In the positive control group at 25%, the mean values of erythema score was 0.0–1.7. There was a significant increase when compared to the negative control group (p<0.01: Days 3, 4, 5 and 6).

EAR THICKNESS (mean):
- Set 1:
In the negative control group, the mean values of ear thickness at 0.19–0.20 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 was 0.19–0.21, 0.20–0.21 and 0.19–0.21 mm, respectively. There were significant increases when compared to the negative control group (p<0.05: Day 6 (25%)).
In the positive control group at 25%, the mean value of ear thickness was 0.19–0.22 mm. There were significant increases when compared to the negative control group (p<0.01: Day 6).
- Set 2:
In the negative control group, the mean values of ear thickness at 0.19–0.19 mm prior to dosing until Day 6 after dosing.
In the test substance groups at 1, 5, 10%, the mean values of ear thickness were 0.20–0.19, 0.20–0.20, 0.20–0.20 mm, respectively. There was a significant increase when compared to the negative control group (p<0.05: Day 1 (1%), Day 3 (5%), p<0.01: Days 1 (5 and 10%), 3 (10%) and 6 (5 and 10%)).
In the positive control group at 25%, the mean value of ear thickness was 0.20–0.21 mm. There was a significant increase when compared to the negative control group (p<0.01: Days 1, 3 and 6).

EAR WEIGHTS (mean):
- Set 1:
In the negative control group, the mean value of ear weight was 12.6 mg.
In the test substance groups at 25, 50 and 100%, the mean values of ear weight were 13.5, 13.5 and 13.7 mg, respectively. There were significant increases when compared to the negative control group (p<0.05: 100%).
In the positive control group at 25%, the mean value of ear weight was 14.2 mg. There was a significant increase when compared to the negative control group (p<0.01).
- Set 2:
In the negative control group, the mean value of ear tissue was 12.1 mg.
In the test substance groups at 1, 5, 10%, the mean values of ear tissue were 12.9, 13.3 and 12.6 mg, respectively. There were significant increases when compared to the negative control group (p<0.05: 5%).
In the positive control group at 25%, the mean value of ear tissue was 13.9 mg. There was a significant increase when compared to the negative control group (p<0.01).

Applicant's summary and conclusion

Interpretation of results:
other: Skin sensitiser Category 1B
Remarks:
according to EU CLP (EC No. 1272/2008 and its amendments).
Conclusions:
The substance produced a SI ≥ 1.6 and an EC1.6 value of 26.4% was calculated. Based on these results, the substance is considered to be a skin sensitiser.
Executive summary:

In a Local Lymph Node Assay (BrdU-ELISA), the skin sensitisation potential of the substance was tested according to OECD TG 442B under GLP. Per concentration, 5 female mice were used. Skin sensitization potential was evaluated by ear thickness measurements. Cellular proliferations and SI were determined. In the first set, at 25, 50 and 100%, SI values were 1.78, 2.15 and 1.70, respectively. In the second set, at 1, 5 and 10%, SI values were 1.22, 1.10 and 1.40, respectively. Negative and positive controls were included and all acceptability criteria were met (SI values positive controls: 3.10 and 4.32). These results show that the substance could elicit a SI ≥ 1.6. An EC1.6 value of 26.4% was calculated. Based on the results, the substance is considered a skin sensitiser 1B.