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Administrative data

Description of key information

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:
2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Envigo CRS GmbH, In den Leppsteinswiesen 19, 64380 Roßdorf, Germany
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS B.V., Inc., the Netherlands
- Females were nulliparous and non-pregnant:
- Age at study initiation: Pre-test: 9 - 10 weeks , main study: 8 - 9 weeks
- Weight at study initiation: pre-test 20g, main test 16.6 - 20.4g
- Housing: All animals belonging to the same experimental group were kept in one cage.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: al teast five days
- Indication of any skin lesions: Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 + 2°C
- Humidity (%): approx. 45-65%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): artificial light 6.00 a.m. - 6.00 p.m.
- IN-LIFE DATES: From: 20 April 2016 To: 31 May 2016 (experimental completion day)
Vehicle:
propylene glycol
Concentration:
5, 10, 25% (w/w)
No. of animals per dose:
5
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility:
A solubility experiment was performed according to the recommendations given by OECD 429. The highest test item concentration, which could be technically used, was a 25% suspension in PG. Grinding of the test item in a mortar was used to formulate the test item. At higher concentrations, an applicable formulation of the test item was not achieved, neither by the use of other vehicles nor by using additional methods to formulate the test item (e.g. vortexing, sonicating, warming to 37°C).
- Irritation: The substance was not irritating
To determine the highest non-irritant test concentration that at the same time did not induce signs of systemic toxicity, a pre-test was performed in two animals.
Two mice were treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 10 and 25% once daily each on three consecutive days. Prior to the first application of the test item and before sacrifice the body weight was determined. Clinical signs were recorded at least once daily. Eventual signs of local irritation were documented and a score was used to grade a possible erythema of the ear skin. Furthermore, prior to the first application of the test item (day 1), on day 3 and before sacrifice (day 6) the ear thickness was determined using a micrometer. Additionally, for both animals, the ears were punched after sacrifice (day 6) at the apical area using a biopsy punch (Ø 8 mm corresponding to 0.5 cm2) and were immediately pooled per animal and weighed using an analytical balance. Eventual ear irritation was considered to be excessive if an erythema of the ear skin of a score value ≥3 was observed at any observation time and/or if an increase in ear thickness of ≥25% was recorded on day 3 or day 6.
At the tested concentrations the animals did not show any signs of local skin irritation or systemic toxicity. From day 1 to day 4, redness of the ear skin could not be examined, due to the colour of the test item.
Thus, the test item in the main study was assayed at 5, 10, and 25%. The highest concentration tested was the highest level that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed in the pre-experiment.

- Systemic toxicity: not observed
- Ear thickness measurements see above
- Erythema scores: see above

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymphn node assay, incorporation of 3HTdR
- Criteria used to consider a positive response: SI >3

TREATMENT PREPARATION AND ADMINISTRATION:
The test item was placed into a mortat on a tared balance and PG was added.
The different test item concentrations were prepared individually. Homogeneity of the test item in vehicle was maintained during treatment using a magnetic stirrer.
The preparations were made freshly and used within two hours before each dosing occasion. Concentrations were in terms of material as supplied.

Each test group of mice was treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 5, 10, and 25% in PG. The application volume, 25 µL/ear/day, was spread over the entire dorsal surface (diameter ca 8 mm) of each ear once daily for three consecutive days. A further group of mice (control animals) was treated with an equivalent volume of the relevant vehicle alone (control animals).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The mean values and standard deviations were calculated in the body weight tables, for the ear weights, the lymph node weights and lymph node cell count, and for the DPM values (group mean DPM ± standard deviation).
All calculations conducted on the DPM values, the ear weights, the lymph node weights and the lymph node cell count were performed with validated program R Script.
Within the program a statistical analysis conducted on the DPM values, the ear weights, the lymph node weights and the lymph node cell count to assess whether the difference was statistically significant between the test item groups and negative control group. Statistical significance was set at the five per cent level (p < 0.05). Additionally, the Dean-Dixon-Test and Grubb’s Test were used for identification of possible outliers. No outlier value was detected.
Positive control results:
EC3 = 10.6%
Key result
Parameter:
SI
Value:
> 0.9 - < 1.4
Variability:
values of 1.1, 0.9, and 1.4
Test group / Remarks:
dose groups of 5, 10, and 25% (w/w)
Cellular proliferation data / Observations:
The animals did not show any signs of systemic toxicity during the course of the study and no cases of mortality were observed. Redness of the ear skin could partly not be examined, due to the colour of the test item. A statistically significant increase in ear weights was observed in the high dose group in comparison to the vehicle control group (p<0.05). Furthermore, for BALB/c mice, a cut-off value of 1.1 for the ear weight index was reported for a positive response regarding ear skin irritation. The index determined for the high dose group exceeded this threshold (Index of 1.29), which was also above the recommended threshold of 25% for excessive ear skin irritation as mentioned in OECD 429. This increase, however, was considered to be biologically not relevant, since the S.I. determined for this concentration was well below the threshold index of 3.

Table 1: Ear Weights after Sacrifice (main experiment)

Test item concentration Animal Ear weight Mean Ear weight (mg) SD Index
% (w/w) No. mg (Value Test Group versus
per animal Value Control)
Vehicle Control (PG) 1 23.16 23.85 1.71 1
2 26.09
3 22.8
4 25.17
5 22.02
5% 6 26.03 24.3 1.14 1.02
7 23.79
8 24.65
9 24.1
10 22.95
10% 11 22.6 23.6 0.68 0.99
12 23.22
13 24.22
14 23.83
15 24.12
25% 16 32.47 30.66 S 1.51 1.29
17 31.7
18 28.65
19 30.69
20 29.81

Sstatistically significant vs. vehicle control group (p<0.05)

Table2: lymph node weights (main experiment)

Lymph Node Weights after Sacrifice

Test item concentration
% (w/w)

Animal
No.

Lymph Node weight
mg
per animal

Mean Lymph Node weight (mg)

SD

Index
(Value Test Group versus
Value Control)

Vehicle Control (PG)

1

4.87

5.45

0.49

1.00

2

5.61

3

5.66

4

6.08

5

5.04

5%

6

8.23

5.95

1.38

1.09

7

5.55

8

4.93

9

6.19

10

4.86

10%

11

2.85

4.61

1.58

0.85

12

4.6

13

6.19

14

3.23

15

6.19

25%

16

5.66

5.76

0.28

1.06

17

5.68

18

6.25

19

5.71

20

5.52

Table 3: Lymphocyte cell count (main experiment)

Lymphocyte Cell Counts after Sacrifice

Test item concentration
% (w/w)

Animal
No.

Lymph Node Cell Count
x10E06
per animal

Mean
Lymph Node
Cell Count
x10E06
per animal

SD

Index
(Value Test Group versus
Value Control)

Vehicle Control (PG)

1

8.92

9.59

2.2

1.00

2

12.62

3

10.75

4

8.87

5

6.8

5%

6

8.57

9.75

2.08

1.02

7

11.87

8

8.05

9

12.15

10

8.1

10%

11

5.15

6.3

3.59

0.66

12

9.47

13

1

14

6.12

15

9.77

25%

16

10.17

11.81

1.95

1.23

17

9.35

18

13.45

19

12.4

20

13.67

Interpretation of results:
GHS criteria not met
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

The substance is not skin sensitizing in the LLNA (OECD 429, GLP).

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. The stimulation indeces in the LLNA (OECD 429) did not show a dose dependent increase. No EC3 would be established. As a result the substance is not considered to be classified for skin sensitization under Regulation (EC) No. 1272/2008, as amended for the seventh time in Regulation (EC) No 2015/1221 fourteenth time in Regulation (EC) No. 2020/217.