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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:
2015-03-31 and 2015-06-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2010
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2012
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
1,4-phenylenebis(nitrilo-2,2-dimethylprop-1-yl-3-ylidene) didodecanoate
Cas Number:
1154521-93-3
Molecular formula:
C40H68N2O4
IUPAC Name:
1,4-phenylenebis(nitrilo-2,2-dimethylprop-1-yl-3-ylidene) didodecanoate
Test material form:
liquid

In vivo test system

Test animals

Species:
mouse
Strain:
CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: TOXI-COOP ZRT., Hungary
- Age at study initiation: 11 weeks
- Weight at study initiation: 16.9 -21.4 g
- Housing: Grouped caging (4 animals/cage)
- Diet: ad libitum, ssniff® SM R/M-Z+H complete diet for rats
- Water: ad libitum, tap water
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 – 70
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
25%, 10%, 5%, 2.5% (w/v)
No. of animals per dose:
4
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: The test item was miscible with Acetone: Olive oil 4:1 (v/v) mixture (AOO). Since AOO is the most preferred vehicle by the relevant guidelines no other vehicles were evaluated.
- Irritation: No signs of significant irritation (indicated by an erythema score ≥ 3 and/or an increased ear thickness of ≥ 25 % on any day of measurement) were observed in the evaluated groups (50% and 25% (w/v) test item in AOO)
- Lymph node proliferation response: No assessement of lymph node proliferation was done.
- Other: Significant systemic toxicity was observed at concentrations of 100% and 50% test item concentration

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- In the main test 24 female CBA/Ca Ola Hsd mice were allocated to 6 groups of 4 animals each:
- 4 groups received the appropriate formulation of the test substance at concentrations of 25%, 10%, 5% and 2.5%
- the negative control group received the vehicle (AOO)
- the positive control group received hexyl cinnamic aldehyde (25% (w/v) in AOO)
- Each substance was applied on the dorsal surface of each ear (25 µL) of the animals once a day for three consecutive days (days 1, 2 and 3).
- There were no treatment on days 4, 5 and 6.

- Criteria used to consider a positive response: The stimulation index of 3 or greater is an indication of a positive result.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
The positive control item (25% (w/v) in acetone olive oil 4:1) induced the appropriate stimulation over the control (stimulation index = 8.1)

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
8
Test group / Remarks:
Dose group 25 %
Key result
Parameter:
SI
Value:
3.1
Test group / Remarks:
Dose group 10 %
Key result
Parameter:
SI
Value:
2.9
Test group / Remarks:
Dose group 5 %
Key result
Parameter:
SI
Value:
1.4
Test group / Remarks:
Dose group 2.5 %
Key result
Parameter:
SI
Value:
8.1
Test group / Remarks:
Positive control group
Key result
Parameter:
SI
Value:
1
Test group / Remarks:
Vehicle control group
Cellular proliferation data / Observations:
CLINICAL OBSERVATIONS: No mortality or symptoms of systemic toxicity were observed in any treatment group. No sign of irritation (indicated by an erythema score ≥ 3) or any other local effect was observed in any treatment groups

BODY WEIGHTS: No significant, treatment related effect on the body weights was considered in any treatment group. Although ≥ 5% decrease of body weights was observed in the vehicle control group, in the 25 % and 5 % dose groups (1/4 animals in each) it was considered not relevant.

Any other information on results incl. tables

DPM in the main test

Test Group

Measured DPM/group

Group* DPM

DPN# (DPM/Node)

Vehicle control (AOO)

9786

9767.5

1220.9

Positive control (25 % HCA in AOO)

79499

79480.5

9935.1

25% test item

78576

78557.5

9819.7

10% test item

30356

30337.5

3792.2

5% test item

28735

28716.5

3589.6

2.5% test item

13456

13437.5

1679.7

HCA = α-Hexylcinnamaldehyde

AOO = Acetone: Olive oil 4:1 (v/v) mixture

 

# No. of lymph nodes per group = 8

*Group DPM = measured DPMgroup- average DPMbackground

Average DPMbackground= 18.5

No mortality was observed during the main study. No significant, treatment related effect on body weights or any other sign of systemic toxicity were observed in any treatment group. No signs of irritation or any other local effect were observed at the treatment site (ears) in any treatment group.

Applicant's summary and conclusion

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
The calculated EC3 value based on dose-response curve analysis was 7.5% (w/v) in this LLNA. Based on this value, the test item is considered to be a moderate skin sensitizer(1 ≤ EC3 < 10).
Executive summary:

The aim of this study was to determine the skin sensitization potential of the test item following dermal exposure in the Local Lymph Node Assay. Pooled treatment group approach was used in this test. Selection of test item concentrations based on the results of a formulation evaluation and also resulted from a preliminary irritation/toxicity test according to the relevant guidelines. The test item was a liquid and adequately miscible with Acetone: Olive oil 4:1 (v/v) mixture (AOO). Since AOO is the most preferred vehicle by the relevant guidelines no other vehicles were evaluated. Based on results of the preliminary irritation/toxicity test (where significant systemic toxicity was observed at concentrations of 100 % and 50 % (w/v)) the test item was examined in the main test at 25 %, 10 %, 5 % and 2.5 % (w/v) as formulations in AOO. Appropriate positive control, furthermore a negative control group dosed with the vehicle (AOO) of the test and positive control groups were employed. The positive control item (α-Hexylcinnamaldehyde [HCA]; 25 % (w/v) in AOO) induced the appropriate stimulation over the control (SI = 8.1), thus confirming the validity of the assay. No mortality was observed during the main test. No significant, treatment related effect on body weights or any other sign of systemic toxicity were observed in any treatment group. No signs of irritation or any other local effect were observed at the treatment site (ears) in any treatment group. Based on the calculated SI values significant lymphoproliferation (SI ≥ 3) was observed for the test item at concentrations of 25 % and 10 % (w/v). The corresponding stimulation index values were 8.0, 3.1, 2.9 and 1.4 at treatment concentrations of 25 %, 10 %, 5 % and 2.5 % (w/v), respectively. Statistically significant (p = 0.02) and biologically relevant dose-response relationship was observed (linear regression using the SI values). According to evaluation criteria of the relevant guidelines, the significantly increased lymphoproliferation at the maximum applicable (non-toxic, non-irritant) concentration of 25 % (w/v) and also at 10 % (w/v) concentration and the significant dose-response relationship are considered evidence that the test item is a skin sensitizer. The calculated EC3 value based on dose-response curve analysis was 7.5 % (w/v) in this LLNA. Based on this value the test item can be ranked among moderate sensitizers (1 ≤ EC3 < 10).

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