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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:
From 05 December 2018 to 22 January 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study performed according to OECD test guideline No. 429 and in compliance with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
Adopted 22 July 2010
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
Updated 06 July 2012
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Remarks:
German GLP Compliance Monitoring Programme (inspected on 13-16 July 2015 / signed on 14 September 2015)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
(±)-1-[(1RS,2SR,4SR)-BICYCLO[2.2.1]HEPT-2-YL]-5-HEXEN-2-ONE
Cas Number:
1352216-65-9
Molecular formula:
C13H20O1
IUPAC Name:
(±)-1-[(1RS,2SR,4SR)-BICYCLO[2.2.1]HEPT-2-YL]-5-HEXEN-2-ONE
Constituent 2
Chemical structure
Reference substance name:
(±)-1-[(1RS,2RS,4SR)-BICYCLO[2.2.1]HEPT-2-YL]-5-HEXEN-2-ONE
Cas Number:
1352216-63-7
Molecular formula:
C13H20O1
IUPAC Name:
(±)-1-[(1RS,2RS,4SR)-BICYCLO[2.2.1]HEPT-2-YL]-5-HEXEN-2-ONE
Test material form:
liquid
Details on test material:
Appearance: Colourless liquid
Specific details on test material used for the study:
Storage Conditions: +2 to +8 °C, light protected, under nitrogen.

In vivo test system

Test animals

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 nulliparous and non-pregnant: yes
- Age at study initiation: 10-11 weeks (Pre-test) / 9-10 weeks (Main study)
- Weight at study initiation: 17.1-21 g
- Housing: Individually housed per group, in Makrolon Type II (pre-test) / III (main study) cages, with wire mesh top furnished with granulated soft wood bedding
- Diet: ad libitum (2018C Teklad Global 18% protein rodent diet (certified), Supplier: Envigo RMS B.V., Inc).
- Water: tap water ad libitum.
- Acclimation period: at least 5 days prior to the start of dosing.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 45-65 (except for deviations: ca. 13-45% for several hours)
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From 05 December 2018 to 22 January 2019

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
100%, 30% and 10%
No. of animals per dose:
5
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: maximum attainable concentration = 100% of the undiluted test item.
- Irritation: Two mice were treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 50 and 100% once daily each on three consecutive days. Animals did not show any signs of toxicity. From day 2 to 4, animals showed an erythema of the ear skin (Score 1). On day 6, the animal treated with 50% test item concentration showed slight eschar formation. However this was considered to be unspecific since no eschar formation was observed in the animal treated with the undiluted test item, and since all other parameters (ear weights, erythema score) were well within the Guideline-recommended thresholds and did not indicate an excessive local skin irritation in this animal.
Based on this information the dose levels selected for the main test were 100%, 30% and 10% w/w in acetone/olive oil 4:1.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: individual approach, using tritiated (3H)-methyl thymidine, according to the OECD 429 test guideline.
- Additional investigation: Determination of ear thickness: The measured ear thickness of all animals treated was recorded daily from day 1 (prior to the first application of the test item) to day 6 (prior to sacrifice). The ear thickness of each ear was measured using a micrometer immediately prior to and 1-hour post application of test item on days 1 and 3 and 1-hour post application of the test item on day 2. Additional measurements were taken on days 4, 5, and 6 (day of lymph node harvest for the LLNA) to monitor for any changes in ear thickness. Group mean ear thickness changes were calculated between Days 1 to 3 and Days 1 to 6. A mean ear thickness of ≥ 25% was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitization.
- Criteria used to consider a positive response: The decision process with regard to identification of a positive response will include a SI of ≥ 3, together with consideration of dose-response, and where appropriate, statistical significance. Any test item failing to produce a SI of ≥ 3 at all test concentrations was not regarded as a skin sensitiser.

TREATMENT PREPARATION AND ADMINISTRATION:
The test item was placed into an appropriate container on a tared balance and acetone/olive oil (4+1, v/v) was added (weight per weight).
The different test item concentrations were prepared individually.
The preparations were made freshly before each dosing occasion.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
All calculations conducted on the disintegrations per minute (DPM) values were performed with a validated test script of “R”, a language and environment for statistical computing and graphics.
Within the program a statistical analysis was conducted on the DPM values and the ear thickness 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 was detected in the statistical outlier tests.

Results and discussion

Positive control results:
A group of five animals was treated with 50 µl (25 µl per ear) of α-Hexylcinnamaldehyde, tech., 85% as a solution in acetone/olive oil 4:1 at a concentration of 25% v/v. A further control group of five animals was treated with acetone/olive oil 4:1 alone. With a SI = 7.6, α-Hexylcinnamaldehyde, tech., 85% was considered to be a sensitiser under the conditions of the test.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
1.7
Test group / Remarks:
10%
Key result
Parameter:
SI
Value:
4.8
Test group / Remarks:
30%
Key result
Parameter:
SI
Value:
5.4
Test group / Remarks:
100%
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
0% : mean DPM / animal = 1245.3
10%: mean DPM / animal = 2100.9
30%: mean DPM / animal = 5926.3
100%: mean DPM / animal = 6742.7

CLINICAL OBSERVATIONS: No deaths occurred during the study period. No signs of systemic toxicity were observed during the study period. From days 1 to 4 and on day 6, animals showed an erythema of the ear skin (Score 1 to 2). A statistically significant increase in ear thickness was observed on day 6 in the animals treated with the high dose of the test item (see the table in section "Any other information on results incl. tables"), respectively, however this increase was biologically not relevant since it was well below the Guideline-recommended threshold of 25% increase.

BODY WEIGHTS: The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.

Any other information on results incl. tables

EC3 CALCULATION

   Test item concentration (%)  S.I.
 Test Group 2  10 (a)  1.7 (b)
 Test Group 3  30 (c)  4.8 (d)
 EC3 = (a-c) [(3 -d)/(b-d)] + c = 18.4 % (w/w)      

a,b,c,d = Co-ordinates of the two pairs of data lying immediately above and below the S.I. value of 3 on the LLNA dose response plot.

EAR THICKNESS

 Treatment Group  Concentration

 Ear Thickness Gain

Day 1 to 3

 Ear Thickness Gain

Day 1 to 6

 Test Item        10% in acetone/olive oil (4 +1, v/v)  2.0  -0.6
 30% in acetone/olive oil (4 +1, v/v)  4.9  1.8
 100%  6.7  4.8*
 Positive Control Item  25% in acetone/olive oil (4 +1, v/v)  7.4  5.5*

* Statistically significant

Applicant's summary and conclusion

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
Under these experimental conditions, the test material is classified as skin sensitiser in category 1B according to the annex I of the Regulation EC No. 1272/2008 (CLP) and to the GHS.
Executive summary:

A study was performed to assess the skin sensitisation potential of ST 03 C 18 in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear. The method was conducted according to the OECD test guideline No 429 and in compliance with GLP.

Following a preliminary screening test in which no clinical signs of toxicity were noted at the maximum attainable concentration (100%), ST 03 C 18 concentration of 100% was selected as the highest dose to be investigated in the main test.

Three groups, each of five animals, were treated with 50 μl (25 μl per ear) of the undiluted test item or the test item as a solution in acetone/olive oil 4:1 at concentrations of 30% or 10% v/v for 3 consecutive days. A further group of five animals was treated with acetone/olive oil 4:1 alone. A concurrent positive control test, using a group of five animals, was also performed with the known sensitizer, α Hexylcinnamaldehyde tech., 85%, at a concentration of 25% v/v in acetone/olive oil 4:1. The animals were allowed to rest without dosing on days 4 and 5. On day 6, the proliferation of lymphocytes in the lymph node draining the application site was measured by incorporation of 3H-Methyl Thymidine.

The irritant potential of the test item was assessed in parallel by measurement of ear thickness on days 1 to 6.

No mortality and no signs of systemic toxicity were noted in the test and controls animals during the test. From days 1 to 4 and on day 6, animals showed an erythema of the ear skin (Score 1 to 2,). A significant increase in ear thickness was observed on day 6 in the animals treated with the high dose of the test item, respectively, however this increase was well below the threshold for excessive local irritation as recommended by OECD 429.

A stimulation index of more than 3 was recorded for two concentrations of the test item (10% and 100% (v/v) in acetone/olive oil). The Stimulation Index (SI) calculated by pooled approach was respectively 1.7, 4.8 and 5.4 for the treated group at 10%, 30% and 100%, respectively. The EC3 value determined by linear interpolation of points on the dose-response curve was 18.4%.

The historical positive control, α-Hexylcinnamaldehyde, gave a SI of 7.6, when tested at 25 % v/v. The test system was therefore considered to be valid.

 

Under the test conditions, ST 03 C 18 is classified as a skin sensitiser (Category 1B) in the Local Lymph Node Assay according to the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

This study is considered as acceptable and satisfies the requirement for sensitisation endpoint.