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Toxicological information

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:
20 - 28 Mar 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
adopted in 2010
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
adopted in 2008
GLP compliance:
yes (incl. certificate)
Remarks:
GYEMSZI National Institute for Quality- and Organizational Development in Healthcare and Medicines, Budapest, Hungary
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/J Rj
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Elevage Janvier, Genest-St-Isle, France
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 10 weeks (main study), 11 weeks (preliminary experiment)
- Weight at study initiation: 20.3 - 21.7 g (main study), 20.5 - 22.8 g (preliminary experiment)
- Housing: groups in type II polypropylene/ polycarbonate cages, Lignocel® 3/4-FASERN Hygienic Animal Bedding (J. Rettenmaier & Söhne GmbH & Co.KG, Rosenberg, Germany), glass tunnel-tubes
- Diet: ssniff® SM Rat/Mouse (ssniff Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: tap water from the municipal supply, ad libitum
- Acclimation period: 20 days
- Indication of any skin lesions: no

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.5 - 24.8
- Humidity (%): 30 - 73
- Air changes (per hr): 15 - 20
- Photoperiod (hrs dark / hrs light):12/12

Study design: in vivo (LLNA)

Vehicle:
dimethyl sulphoxide
Concentration:
25 and 50% (w/v) (preliminary irritation/toxicity test)
10, 25 and 50% (w/v) (main test)
No. of animals per dose:
2 (preliminary irritation/toxicity test)
4 (main test)
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: The solubility of the test item was examined in a short preliminary compatibility test. Due to the physical characteristics of the test item, 100% concentration was not achievable. No proper formulation was achieved at 50% (w/v) concentration using acetone: olive oil 4:1 (v/v) mixture, N,N-dimethylformamide, methyl ethyl ketone, propylene glycol or 1% aqueous pluronic PE9200 as vehicle. However, the formulation at 50% (w/v) using dimethyl sulfoxide as vehicle was suitable for the test.
- Irritation: A preliminary irritation/toxicity test was conducted in 2 animals per dose at concentrations of 25 and 50% (w/v) in DMSO. The preliminary experiment was conducted in a similar experimental manner to the main study, but it was terminated on Day 6 and no radioactive proliferation assay was performed. All mice were observed daily for local irritation at the application site. Alopecia was observed in the 50% (w/v) dose group on Day 6. Test item precipitate was observed on the ears for the both animals in the 50% (w/v) group on Days 1-6 and in the 25% (w/v) dose group on Days 1-4. Rigid ears was observed in the 50% (w/v) dose group on Days 2-5, and in the 25% (w/v) dose group on Days 2-3 which may have been caused by test item remaining on the ears. No irritation was observed.
- Systemic toxicity: All mice were observed daily for any clinical signs of systemic toxicity. No mortality or clinical signs of systemic toxicity were observed.
- Ear thickness measurements: Ear thickness was measured using a thickness gauge on Day 1 (pre-dose), Day 3 (before treatment, approximately 48 h after the first dose) and Day 6. Additional quantification of the ear thickness was performed by ear punch weight determination after the euthanasia of the experimental animals. The revealing ear punch weights were over the historical control range which may have been caused by test item remaining on the ears.
- Erythema scores: No erythema was observed.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: tritiated thymidine (³HTdR) incorporation determined by beta-scintillation
- Criteria used to consider a positive response: The test item is regarded as a sensitizer if both of the following criteria are fulfilled: Exposure to at least one concentration of the test item resulted in an incorporation of ³HTdR at least 3-fold or greater than recorded in control mice, as indicated by the stimulation index and the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

TREATMENT PREPARATION AND ADMINISTRATION: On Day 1, 25 µL of the test substance freshly diluted with DMSO was topically applied to the dorsal surface of each ear of each mouse. The application was repeated on Days 2 and 3. There was no treatment on Days 4, 5 and 6. On day 6 an intravenous injection of 250 µL phosphate buffered saline (PBS) containing approximately 20 µCi of ³HTdR was made into the tail vein of each experimental mouse. 5 h (± 30 min) later, the draining auricular lymph node of each ear was excised into PBS. A single cell suspension of pooled lymph node cells was prepared from each mouse and collected by gentle mechanical disaggregating of the lymph nodes through a cell strainer using the plunger of a disposable syringe. The cell strainer was washed with PBS. Pooled LNCs were washed twice via pelleting using centrifugation and resuspension in PBS. Pellets were resuspended in 5% trichloroacetic acid and precipitation was conducted overnight at 2 - 8°C. Following centrifugation pellets were resuspended in 5% trichloroacetic acid and dispersed by using an ultrasonic bath.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
The positive control (25% (w/v) alpha-hexylcinnamaldehyde in DMSO) induced a significant lymphoproliferative response with a stimulation index value of 13.2. No mortality, cutaneous reactions or signs of toxicity were observed in the positive control group. Thus, the results of the positive control group demonstrated the appropriate performance of the assay.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
1.6
Test group / Remarks:
10% (w/v)
Key result
Parameter:
SI
Value:
3.4
Test group / Remarks:
25% (w/v)
Key result
Parameter:
SI
Value:
5.6
Test group / Remarks:
50% (w/v)
Key result
Parameter:
EC3
Value:
21.7
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA: Appearance of the lymph nodes was normal in the 25 and 10% (w/v) dose groups and in the negative control group. Larger than normal lymph nodes were observed in the 50% (w/v) dose group and in the positive control group. The number of radioactive disintegrations per minute (DPM) were measured and corrected with the background DPM value consisting of 5% (w/v) TCA solution measurements. The results were expressed as DPN (DPM divided by the number of lymph nodes) and are shown in table 1.

DETAILS ON STIMULATION INDEX CALCULATION: The stimulation index was calculated by division of the DPN value of a treated group with the DPN value of the negative control group. The SI values for the respective treatment groups are shown in table 1.

EC3 CALCULATION: The calculation of the EC3 value was conducted by linear interpolation according to the equation: EC3 = c + [(3-d)/(b-d)] x (a-c), where the data points lying immediately above and below the SI value of 3 on the LLNA dose-response plot have the co-ordinates (a,b) and (c,d) respectively. The calculated EC3 value for the test substance is 21.7% (w/v).

CLINICAL OBSERVATIONS: No mortality or signs of systemic toxicity were observed during the study. There were no indications of any irritancy at the site of application. Alopecia was observed in the 50% (w/v) dose group on Days 2 - 6 and in the 25% (w/v) dose group on Days 3 - 6. Test item precipitate was observed on the ears for the animals in all treated group on Days 1 - 6. Rigid ears were observed in the 50% (w/v) dose group on Days 2 - 6.

BODY WEIGHTS: No treatment-related effects were observed on animal body weights.

Any other information on results incl. tables

Table 1: Results of the LLNA

Test group

Measured DPM / group

DPM

Number of lymph nodes

DPN

Stimulation Index

Background (5% (w/v) TCA)

34

-

-

-

-

Vehicle control (DMSO)

1610

1576.0

8

197.0

1.0

Test substance 50% (w/v)

8925

8891.0

8

111.4

5.6

Test substance 25% (w/v)

5427

5393.0

8

674.1

3.4

Test substance 10% (w/v)

2522

2488.0

8

311.0

1.6

Positive control (25% HCA in DMSO)

20873

20839.0

8

2604.9

13.2

TCA = trichloroacetic acid, HCA = alpha-Hexylcinnamaldehyde 

Applicant's summary and conclusion

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
CLP: Skin sens 1B, H317