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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:
25 - 31 Jan 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
22 July 2010
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
30 May 2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
OGYÉI, Országos Gyógyszerészeti és Élelmezés-egészségügyi Intézet, Budapest, Hungary
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
Ethyl 9-oxo-9H-thioxanthene-2-carboxylate
EC Number:
280-960-7
EC Name:
Ethyl 9-oxo-9H-thioxanthene-2-carboxylate
Cas Number:
83817-60-1
Molecular formula:
C16H12O3S
IUPAC Name:
ethyl 9-oxo-9H-thioxanthene-2-carboxylate
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature, dry conditions, protected from heat and direct sunlight

FORM AS APPLIED IN THE TEST (if different from that of starting material)
liquid

In vivo test system

Test animals

Species:
mouse
Strain:
CBA/Ca
Remarks:
Ola Hsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Toxi-Coop ZRT., Budapest, Hungary
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 12 weeks
- Weight at study initiation: 18.5 - 21.7 g
- Housing: 4 animals per cage in Type II. Polypropylene / polycarbonate cages with deep wood sawdust bedding
- Diet: ssniff® Rat/Souris-Elevage E complete diet for rats and mice (ssniff Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 21 d
- Indication of any skin lesions: no

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 – 70
- Photoperiod (hrs dark / hrs light): 12 / 12
- IN-LIFE DATES: From: 25 To: 31 Jan 2016

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Concentration:
0.25, 0.5, 1 and 2.5 %
No. of animals per dose:
4 (controls), 4 (test groups)
Details on study design:
PRE-SCREEN TESTS:
In the pre-screen test, three concentrations (0.5, 1 and 2.5% solution in DMF) were selected, and 25 μl each dose formulation were applied on the ears of each animal, 2 mice for each concentration, once a day for 3 consecutive days. The general conditions of the animal including an observation of the application site were performed during the preliminary test. As a result, no animals showed any abnormalities.
The body weights and ear thickness were measured before the initial application and on Day 6, the ear thickness additionally on Day 3. None of the animals showed any signs of significant irritation (indicated by an erythema score ≥ 3) or any changes deviating from the criteria of ear thickness (less than 25% increase in ear thickness). 1 out of 2 animals of the 1% treatment group showed a body weight decrease of 6% (more than the criteria of body weights (less than 5%)), but the effect was considered not treatment related (the only significant body weight changes observed in all dose groups).
From the results mentioned above and due to solubility reasons, 2.5% (w/v) was selected as the maximum concentration for the main study. Three lower concentrations of 0.25, 0.5 and 1% were tested additionally.

- Compound solubility: 2.5%
- Irritation: no
- Systemic toxicity: no
- Ear thickness measurements:yes, less than 25% increase in ear thickness
- Erythema scores: 0 (each test group, each time point)

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: 3H-methyl-thymidine incorporation, determined by β-scintillation
- Criteria used to consider a positive response: A substance is regarded as a sensitizer in the LLNA, if the Stimulation Index (SI) is ≥ 3.

TREATMENT PREPARATION AND ADMINISTRATION:
25 μl of each dose formulation were applied to the dorsal skin of each ear of each animal once a day for 3 days. On day 6, 20 μCi 3H-methyl-thymidine, contained in 250 μL of 1 x PBS, was administered intravenously to each mouse via the tail vein with a 1 mL sterile syringe. 5 h (± 30 minutes) after administration, local lymph nodes of each group were pooled and collected separately. Then a single cell suspension of lymph node cells was prepared by gentle mechanical disaggregation of the lymph nodes through a cell strainer using the plunger of a disposable syringe. After pelletation and two washing steps with PBS, the macromolecules were precipitated with 3 mL of 5% trichloroacetic acid at 2 - 8 °C for approximatively 18 hours. The amount of 3HTdR incorporation was measured by a liquid scintillation analyzer (Tri-Carb 3100TR).

ASSAY ACCEPTANCE CRITERIA
- Lymph nodes from all 4 animals in each dose groups were pooled for a valid experiment.
- All 4, but at least 3, test item concentrations are available.
- Skin sensitizing effect was observed at the applied concentration of the positive control (SI ≥ 3)

EVALUATION OF THE RESULTS
DPM (disintegration per minute) was measured for each treatment group. The measured DPM values were corrected with the background DPM value (named as Group DPM): the average of the two measured DPM values of 5 % (w/v) TCA solution was used as the background DPM value. The results were expressed as DPM/mouse (Group DPM values devided by the number of animals (actually 4) per groups). The stimulation index (SI = the DPM/mouse of a treated (positive control or test item) group divided by the DPM/mouse of the respective negative control group) for each treatment group was also calculated. A stimulation index of 3 or greater is an indication of a positive result. Dose-response relationship was evaluated by linear regression using SI values. All calculations were made by Microsoft Excel Software.
Based on the results an EC3 value (dose calculated to induce a stimulation index of 3) of the test item was not calculated.

INTERPRETATION OF RESULTS
The test item is considered as a skin sensitizer, if:
Exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR to at least 3-fold or greater than recorded in control mice (indicated by SI ≥ 3). However, the strength of the dose-response, the statistical significance and the consistency of the solvent/vehicle and positive control responses may also be used when determining whether a borderline result is declared positive.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Mean values and standard deviations were calculated.

Results and discussion

Positive control results:
The positive control substance (25% α-Hexylcinnamaldehyde in AOO) induced a positive reaction, determined by a DPM/animal of 17605.9 compared to 1287.9 DPM/animal in the vehicle control group of the positive control, leading to a SI of 13.7. No abnormal clinical signs, erythema on the ears or body weight changes were observed. The lymph nodes were however larger than those in the respective vehicle control group.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
1
Test group / Remarks:
vehicle control group for test item: DMF
Key result
Parameter:
SI
Value:
0.5
Test group / Remarks:
0.25% test group
Key result
Parameter:
SI
Value:
1
Test group / Remarks:
0.5% test group
Key result
Parameter:
SI
Value:
0.8
Test group / Remarks:
1 % test group
Key result
Parameter:
SI
Value:
0.5
Test group / Remarks:
2.5 % test group
Parameter:
SI
Value:
1
Test group / Remarks:
vehicle control group for positive control: AOO
Parameter:
SI
Value:
13.7
Test group / Remarks:
positive control group
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA: Visually larger lymph nodes compared to the respective vehicle controls (AOO or DMF) were observed in the positive control group only. Appearance of the lymph nodes was normal in both vehicle control groups and in the test groups.
No significant lymphoproliferation (i.e. no SI ≥ 3) was observed for the test item at treatment concentrations of 0.25, 0.5, 1 and 2.5% (w/v). No dose-related response was observed.

DETAILS ON STIMULATION INDEX CALCULATION: SI = DPM/mouse of a treated group divided by the DPM/mouse of the respective negative control group (DPM/Mouse = Group DPM/4; Group DPM = measured DPMgroup - average DPMbackground; Average DPMbackground = 23.5)

EC3 CALCULATION: Based on the results of the test item (SI = 0.5 - 1) no EC3 value (dose calculated to induce a stimulation index of 3) of the test item was calculated.

CLINICAL OBSERVATIONS:
No mortality or symptoms of systemic toxicity were observed in any treatment group. No signs of irritation (indicated by an erythema score ≥ 3) or any other local effect were observed in any treatment group. Lymph node appearance was not altered.

BODY WEIGHTS:
Body weights, decreased by ≥ 5 %, were observed only in 2/4 animals of the vehicle control group for the positive control substance (AOO: -5 and -6%).

Any other information on results incl. tables

Table 1: Stimulation index in mice after application of the vehicles (AOO and DMF), test substance (0.25, 0.5, 1.0, 2.5% in DMF) or positive control substance (25% HCA in AOO)

Compound

Concentration [%]

DPM/ animal

Stimulation index

Judgement

DMF

100

1143.1

1.0

-

Test substance

0.25

582.6

0.5

Negative

0.5

1120.4

1.0

Negative

1.0

930.6

0.8

Negative

2.5

612.1

0.5

Negative

AOO

100

1287.9

1.0

-

HCA

25

17605.9

13.7

Positive

AOO = Acetone: Olive oil 4:1 (v/v) mixture (vehicle used for the positive control substance HCA)

DMF = N,N -Dimethylformamide (vehicle used for the test substance)

HCA = α-Hexylcinnamaldehyde

- = Not applicable

Table 2: Body weight after application of the vehicle (AOO/DMF), test substance (0.25, 0.5, 1.0, 2.5% in DMF) or positive control substance (25% HCA in AOO)

Compound

Concentration [%]

Animal ID No.

Body weight

Day 1 [g]

Day 6 [g]

Change [%]

DMF

100

1

20.4

21.6

6

2

19.4

19.8

2

3

20.6

19.9

-3

4

18.5

17.7

-4

Mean ± SD

19.7 ± 1.0

19.8 ± 1.6

0

Test substance

0.25

5

21.4

20.6

-4

6

19.3

18.9

-2

7

18.6

18.7

1

8

19.8

20.1

2

Mean ± SD

19.8 ± 1.2

19.6 ± 0.9

-1

0.5

9

21.7

21.8

0

10

19.7

20.1

2

11

18.9

18.5

-2

12

19.4

19.2

-1

Mean ± SD

19.9 ± 1.2

19.9 ± 1.4

0

1

13

21.3

21.7

2

14

19.0

19.5

3

15

19.8

20.0

1

16

19.1

19.1

0

Mean ± SD

19.8 ± 1.1

20.1 ± 1.1

1

2.5

17

21.7

22.4

3

18

18.9

18.7

-1

19

19.8

19.1

-4

20

19.3

18.8

-3

Mean ± SD

19.9 ± 1.2

19.8 ± 1.8

-1

AOO

100

21

20.5

19.9

-3

22

18.7

17.6

-6

23

20.8

19.8

-5

24

19.4

20.1

4

Mean ± SD

19.9 ± 1.0

19.4 ± 1.2

-3

HCA

25

25

19.1

19.1

0

26

18.5

18.6

1

27

20.9

21.3

2

28

20.0

19.5

-3

Mean ± SD

19.6 ± 1.1

19.6 ± 1.2

0

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

DMF = N,N -Dimethylformamide

HCA = α-Hexylcinnamaldehyde

SD = Standard deviation

Applicant's summary and conclusion

Interpretation of results:
other: CLP/ EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008
Conclusions:
CLP not classified