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Description of key information

The test substance did not show evidence of skin sensitization under the conditions of this study.

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:
12.12.2016 - 20.02.2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA): BrdU-ELISA
Test material information:
Composition 1
Species:
mouse
Strain:
CBA
Remarks:
CBA/N
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Microbiological status of animals, when known: SPF
- Age at study initiation: 9 weeks
- Weight at study initiation: 19.1 - 22.5 g (main study), 19.0-23.6 g (dose range finder)
- Housing: Polysulfone cage, 200Wx320Dx140H (mm), 2–5 animals/cage (during the quarantine-acclimation period) / 2–3 animals/cage (during the study)
- Diet: ad libitum: Pelleted rodent chow (Teklad Certified Irradiated Global 18% Protein Rodent Diet 2918C)
- Water: ad libitum: filtered and irradiated public tap water
- Acclimation period: animals were quarantined for 3 days and then acclimated for 4 days.
- Indication of any skin lesions: no

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.5–24.4 (main study); 21.1-24.2°C (dose range finder)
- Humidity (%): 39.9–60.9 (main study); 47.5-65.0% (dose range finder)
- 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
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Dose Range Finding Study: 100, 50, 25 and 5%
Main study: 5, 2.5 and 1%
No. of animals per dose:
5
Details on study design:
PRE-SCREEN TESTS:
- Concentrations selected for the dose range finder test: 100, 50, 25, 10 and 5%
- Number of animals per dose group: 2
- Irritation: daily observations and scoring using erythema scores
- Systemic toxicity: daily observations
- Ear thickness measurements: on day 1 (pre-dose), day 3 and day 6
- Ear weight measurement: on day 6

MAIN STUDY
Based on the results of the dose range finding study, the high dose level of the main study was 5%, and two additional lower dose levels (2.5 and 1%) were selected.

TREATMENT PREPARATION AND ADMINISTRATION:
- Route: dorsum of both ear
- Method of administration: 25 μL daily for 3 consecutive days
- Negative control: vehicle
- Parameters Evaluated: clinical signs, body weight, erythema score, ear thickness, necropsy
- 5-Bromo-2-deoxyuridine (BrdU) injection: inter-peritoneally on Day 5. Approximately 24 hours later, the animals were euthanized.
- Preparation of cell suspension: for each mouse a single-cell suspension of lymph node cells (LNC) was prepared.
- Determination of cellular proliferation: BrdU was measured by ELISA using a commercial kit (Lot No.: 17267000, Roche Diagnostics GmbH., Germany). Absorbance was performed at 370 nm (Emission wavelength, em) with a reference wavelength of 492 nm (Reference wavelength, ref) was measured. The results of absorbance of each well is used to calculate the BrdU labeling index. The average BrdU labelling index of each group is then used to calculate the stimulation index (Stimulation index, SI).

ACCEPTANCE CRITERIA
- The result of the positive control should be >= 1.6.

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
Reference: Cindy A et al. Extrapolating local lymph node assay EC3 values to estimate relative sensitizing potency. Cutaneous and Ocular Toxicology, 2007, 26: 135-145.
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) and each of the test substance groups (G2–G4) or positive substance group (G5). 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) and each of the test substance groups (G2– G4) or positive substance group (G5).
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) and each of the test substance groups (G2–G4) or positive substance group (G5).
Parameter:
SI
Value:
0.98
Test group / Remarks:
5%
Parameter:
SI
Value:
0.91
Test group / Remarks:
2.5%
Parameter:
SI
Value:
0.94
Test group / Remarks:
1 %
Cellular proliferation data / Observations:
In the negative control group, the mean stimulation index was 1.00.
In the test substance groups at 1, 2.5 and 5%, the mean stimulation index was 0.94, 0.91 and 0.98, respectively. There were no significant differences when compared to the negative control group.
In the positive control group at 25%, the mean stimulation index was 3.47. There was a significant increase when compared to the negative control group (p<0.05).

PRE-SCREEN TESTS:

- Systemic toxicity: body weight was decreased by 5% at dose level of 25%

- Erythema scores: Erythema score was observed ≥3 at dose levels of 50 and 100%

MAIN STUDY:

Clinical signs:

No abnormal clinical signs or deaths in any dosing group during the observation period.

Body weights:

There were no significant differences in the mean body weights from day 1 to day 6 for the

test group animals when compared to the negative control group.

Mean Erythema Scores from day 1 to day 6:

- Negative control group: 0.0 - 0.0

- Test group 1%: 0.0 - 0.0

- Test group 2.5%: 0.0 - 0.0

- Test group 5%: 0.0 - 0.6 --> significant increases compared to negative control (p < 0.05) on days 5 - 6

- Positive control group: 0.0 - 1.8 --> significant increases compared to negative control (p < 0.01) on days 2 - 6

Group

Dose (%)

 

Erythema scores

Days after administration

1

2

3

4

5

6

G1

0

Mean

0

0

0

0

0

0

SD

0

0

0

0

0

0

N

5

5

5

5

5

5

G2

1

Mean

0

0

0

0

0

0

SD

0

0

0

0

0

0

N

5

5

5

5

5

5

G3

2.5

Mean

0

0

0

0

0

0

SD

0

0

0

0

0

0

N

5

5

5

5

5

5

G4

5

Mean

0

0

0

0.5

0.6*

0.6*

SD

0

0

0

0.5

0.4

0.4

N

5

5

5

5

5

5

G5

25

Mean

0

0.9**

1.4**

1.7**

1.8**

1.8**

SD

0

0.2

0.5

0.4

0.3

0.3

N

5

5

5

5

5

5

 

G1: Acetone: olive oil (4:1 v/v), G2-G4: test item, G5: α-hexylcinnamaldehyde

S.D.: Standard deviation
N: Number of animals


*p<0.05, Significant difference from the negative control group (G1) by Steel's t-test.

**p<0.01, Significant difference from the negative control group (G1) by Steel's t-test.

Mean ear thickness from day 1 to day 6:

- Negative control group: 0.18 - 0.20 mm

- Test group 1%: 0.19 - 0.20 mm

- Test group 2.5%: 0.19 - 0.21 mm --> significant increase compared to negative control (p < 0.01) on day 6

- Test group 5%: 0.19 - 0.21 mm --> significant increase compared to negative control (p < 0.01) on day 6

- Positive control group: 0.19 - 0.22 mm --> significant increases compared to negative control (p < 0.05) on days 3 and (p < 0.01) on day 6

Mean ear weights:

- Negative control group: 13.0 mg

- Test group 1%: 13.2 mg

- Test group 2.5%: 13.2 mg

- Test group 5%: 13.1 mg

- Positive control group: 13.8 mg

No significant differences observed when compared to the negative control group.

Mean Stimulation Index:

- Negative control group: 1.00

- Test group 1%: 0.94

- Test group 2.5%: 0.91

- Test group 5%: 0.98

- Positive control group: 3.47

There were no significant differences when the test groups were compared with the negative control.

The positive control group showed a significant increase (p < 0.05) when compared with the negative control.

Group

Dose (%)

 

BrdU labelling index

 

Stimulation index

G1

0

Mean

0.11

1.00

SD

0.01

0.12

N

5

5

G2

1

Mean

0.11

0.94

SD

0.02

0.13

N

5

5

G3

2.5

Mean

0.10

0.91

SD

0.01

0.08

N

5

5

G4

5

Mean

0.11

0.98

SD

0.02

0.20

N

5

5

G5

25

Mean

0.40

3.47*

SD

0.04

0.38

N

5

5

 

G1: Acetone: olive oil (4:1 v/v), G2-G4: test item, G5: α-hexylcinnamaldehyde

S.D.: Standard deviation
N: Number of animals


*p<0.05, Significant difference from the negative control group (G1) by Steel's t-test.

 

Conclusions:
The test item does not have to be classified as skin 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 study was according to OECD 442B and GLP.

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 erythema score was 3 at dose levels of 50 and 100%, and the body weight was decreased by 5% at dose level of 25%. Based on the results of the dose range finding study, the high dose level of the main study was selected to be 5%, and two additional lower dose levels (2.5 and 1%) were selected.

In the main study positive and negative control groups were included. No animals died and no abnormal clinical signs were observed in the main study. In the positive control group, the body weights and ear weights were not significantly different compared to the negative control group and the erythema score, ear thickness and stimulation index were significantly different compared to the negative control group. In the test substance groups, the body weights, ear weights and stimulation index were not significantly different compared to the negative control group. However, the erythema score and ear thickness were significantly different compared to the negative control group.

In the negative control group, the mean stimulation index was 1.00. In the test substance groups at 1, 2.5 and 5%, the mean stimulation index was 0.94, 0.91 and 0.98, respectively. There were no significant differences when compared to the negative control group. In the positive control group at 25%, the mean stimulation index was 3.47. There was a significant increase when compared to the negative control group (p<0.05).

The SI values were measured and below 1.6 at all concentrations, thus EC1.6 was not calculated. EC1.6 values are normally used for classification, however, in the current test this was not applicable and based on the result of this study the test substance did not show evidence of skin sensitization under the conditions of this study.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

For this endpoint there is one study available assessing the skin sensitization potential of the test substance in a LLNA study according to OECD 442B and GLP.

The dose range finding study with dose levels of 5, 10, 25, 50 and 100% revealed that doses 50 and 100% induced an erythema score 3 and at 25% the body weight decreased with 5%. Based on this information the dose levels for the main study were selected to be 1, 2.5 and 5%.

In the main study positive and negative control groups were included. No animals died and no abnormal clinical signs were observed in the main study.

In the positive control group, the erythema score, ear thickness and stimulation index (3.47) were significantly different compared to the negative control group (1.00).

In the test substance groups, the body weights, ear weights and stimulation index (at 1, 2.5 and 5% 0.94, 0.91 and 0.98 respectively) were not significantly different compared to the negative control group. However, the erythema score and ear thickness were significantly different compared to the negative control group, which indicates the susbtance has skin irritant effect. The SI values were measured and below 1.6 at all concentrations, thus EC1.6 was not calculated. EC1.6 values are normally used for classification, however, in the current test this was not applicable and based on the result of this study the test substance did not show evidence of skin sensitization under the conditions of this study.

Justification for classification or non-classification

The test item should not be classified for skin sensitisation. In an LLNA test the stimulation index was at 1, 2.5 and 5% 0.94, 0.91 and 0.98 respectively and thus not significantly different compared to the negative control group. As the SI values were below 1.6 the EC1.6 was not calculated. Nevertheless, in the current test the test substance did not show evidence of skin sensitization.