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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 December 28th, 2012 to January 29th, 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 guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
22th July, 2010
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
(3S,6E)-3,7,11-trimethyldodeca-6,10-dienal
EC Number:
810-298-1
Cas Number:
194934-66-2
Molecular formula:
C15H26O
IUPAC Name:
(3S,6E)-3,7,11-trimethyldodeca-6,10-dienal
Test material form:
liquid

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc.
- Age at study initiation: 9 weeks old.
- Weight at study initiation: 21.1 to 23.7 g
- Housing: animals were individually housed in wire mesh metal cages (W 10.0 x D 19.6 x H 13.0 cm).
- Diet: animals had free access to commercial pellet diet, CRF-1 (lot No. 120403, Oriental Yeast).
- Water: animals were allowed free access to tap water with an automatic water supply system.
- Acclimation period: one week; animals were monitored for general condition and body weight changes and acclimated to the study environment. None of the animals showed any abnormalities.

ENVIRONMENTAL CONDITIONS
- Temperature: 20 to 26 °C (actual measurement values: 22.8 to 23.2 °C).
- Humidity: 35 to 70 % RH (actual measurement values: 54.2 to 60.5 %RH)
- Air changes: 12 times or more per hour, differential air pressure: +30 Pa or more (with the door closed).
- Photoperiod: 12 hours (lights on 7 a.m., off 7 p.m.).

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Concentration:
25, 50 and 100 % test substance
No. of animals per dose:
4 animals per group
Details on study design:
RANGE FINDING TESTS
- Compound concentrations: three concentrations of 25, 50 and 100 % (100 % solution was an undiluted solution) were selected using DMF as a solvent.
- Test design: 25 µl each of the dose formulations were applied on the dorsal skin of both auricles of each animal, 2 mice for each concentration, once a day for 3 consecutive days. The general conditions including the application site condition were observed from 1 to 3 hours after the application on each day of application.
- Results: as a result, no animals showed any abnormalities. The body weights and ear thickness were measured before the initial application and on Day 6. As a result, none of the animals showed any changes deviating from the criteria of body weights. The ear thickness of the animals in 50 % group was increased more than 25 %. However, it was judged that the concentration of 100 % could be used because the ear thickness of 2 5% and 100 % groups were not increased exceeding the criteria.
- Selection for the main study: for the main study, 100 % was selected as the high concentration because it was expected not to induce any toxic general condition, 25 % or more increase of the ear thickness, dermal erythema with score of 3 or more on the auricles, or more than 5 % of body weight loss, and two lower concentrations of 50 and 25 % (three concentrations in total).

MAIN STUDY

TREATMENT PREPARATION AND ADMINISTRATION
Dose formulation
The undiluted solution of the test substance was used for 100 % dose formulation. 0.5 ml of the test substance was dissolved in the same volume of DMF to make the concentration of 50 %. 0.5 ml of this solution was diluted with the same volume of DMF to make the concentration of 25 %. 0.2 ml of the positive control substance was dissolved in 0.6 ml of DMP to make the concentration of 25 %. These formulations were prepared just before use.

ADMINISTRATION ROUTE AND METHOD
The test substance was applied to the auricles in accordance with the common administration route for LLNA.
Twenty-five microlitres each of a dose formulation were applied on the dorsal skin of auricles of each animal once a day using a MICROMAN (Model Ml100, Gilson).

ADMINISTRATION PERIOD AND SCHEDULE
The administration period was 3 consecutive days.
Day 1, 2 and 3 administration of dose solutions.
Day 4 and 5: -
Day 6: administration of 20 µCi [methyl-3H] thymidine (3HTdR); Local lymph nodes were collected and minced at 5 hours after administration of 3HTdR, and then the pooled lymph node cells (LNC) were treated with 5 % TCA overnight (approximately 18 hours).
Day 7: counting of 3HTdR incorporation into pooled LNC.

3HTdR SOLUTION
- Preparation: the [methyl-3H] thymidine (3HTdR; specific radioactivity: 2.0 Ci/mmol, concentration: 1.0 mCi/ml) was diluted with phosphate buffered saline (PBSn pH 7.2) to a concentration of 80 µCi/ml just before use.
- Confirmation of concentration of 3HTdR solution: eighty microlitres of 3HTdR solution were diluted to 200 ml with water for injection, and 1 ml of this solution was mixed with 10 ml of liquid scintillator. Amounts of radioactivity were counted with the liquid scintillation counter. Because the actual amount of radioactivity of the 3HTdR solution was within 120 % of the calculated value, it was confirmed that the 3HTdR solution was appropriately prepared (actual measurement value: 1048 Bq, calculated value: 1184 Bq).
- Incorporation of 3HTdR: on Day 6, all animals in the same dosage group were transferred to polycarbonate cages and transported to the RI laboratory. Twenty µCi of 3HTdR (250 µl of 80 µCi/ml 3HTdR solution) were administered intravenously to each mouse via the tail vein with disposable syringes and 27G needles.

PREPARATION OF SINGLE CELL SUSPENSION
At approximately 5 hours alter 3HTdR injection, all mice were sacrificed by dislocation of cervical vertebra under isoflurane anesthetizing. The auricular lymph nodes were immediately removed, and the lymph nodes for each animal were weighed with an electronic balance, then these tissues were collected into 1.0 ml of a phosphate buffered saline (PBS) solution for each experimental group (4 animals/group). A single cell suspension (SCS) of pooled lymph node cells (LNC) was prepared and collected into the base of a culture dish by gentle mechanical disaggregating of pooled lymph nodes through a nylon mesh using the plunger of a 2.5 ml disposable syringe. The cell strainer was washed with 4-5 ml of PBS and the rinse was added into me base of the culture dish and the SCS was transferred into a 10 ml graduated round-bottomed centrifuge tube. A final volume of 10 ml of SCS was made with rinsing the culture dish with PBS. This procedure was repeated. Pooled LNC was pelleted by centrifugation at 1500 rpm (approx. 438 x g, RCF) for 10 min. After centrifugation, each supernatant was removed by aspiration, leaving 1-2 ml of supernatant above each pellet.
Each pellet was gently agitated and mixed with PBS to make a 10-ml LNC suspension. The washing procedure was repeated twice.

OBSERVATIONS, MEASUREMENTS AND EXAMINATIONS
- Determination of incorporated 3HTdR: after the final wash, each supernatant was removed leaving just a small volume (< 0.5 ml) of supernatant above each pellet. Each pellet was gently agitated before re-suspending the LNC in 3 ml of 5 % trichloroacetic acid (TCA). After incubation with 5 % TCA at 4 °C for about 18 hours, each precipitate was recovered by centrifugation at 1500 rpm for 10 minutes, removing each supernatant and re-suspending in l ml of 5 % TCA. Each precipitate was transferred to a 20 ml-scintillation vial with 10 ml of liquid scintillator and thoroughly mixed. The vials were loaded into a liquid scintillation counter and after approximately 30 minutes, 3HTdR incorporation was measured. The 3HTdR incorporation was expressed as the number of radioactive disintegrations per minute (DPM). Similarly, background 3HTdR level was also measured with 1 ml of 5 % TCA.
- Observation of general condition: general conditions of mice were observed once a day from the first administration to the anatomy. All signs were recorded.
- Body weight: the mice were weighed with an electronic balance before the first administration and before transportation to the RI facility.
- Observation of auricles: both auricles of each mouse were observed for erythema and scored on every application day and before transportation to the RI facility.
Erythema Scores: no erythema 0; very slight erythema (barely perceptible) 1; well-defined erythema 2; moderate to severe erythema 3; severe. erythema (beet redness) to eschar formation preventing grading of erythema 4.
- Ear thickness measurement: thickness of the right ear of each animal was measured three times continuously with a thickness gauge (Mitutoyo) before the first administration and before transportation to the RI facility.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
The SI of the positive control group was 3.1.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
2.5
Test group / Remarks:
25% test item
Key result
Parameter:
SI
Value:
6.3
Test group / Remarks:
50% test item
Key result
Parameter:
SI
Value:
6.9
Test group / Remarks:
100% test item
Cellular proliferation data / Observations:
The DPM/animal of the vehicle control group and 3 test substance groups (25, 50 and 100 %) were 733.0, 1831.5, 4648.8 and 5023.0, respectively. DPM/animal of the positive control group was 2300.1

Any other information on results incl. tables

No animals showed abnormal clinical signs due to test substance administration during the observation period.

No animals showed erythema on the auricles during the observation period. There were no body weight increases or decreases due to test substance or positive control substance administration during the sensitization period. The weights of lymph nodes in all test substance groups and the positive control group were higher man that in the vehicle control group.

The ear thickness of the animals in 50 % test substance, 100 % test substance and positive control group were increased during the sensitization period.

Stimulation index of each compound group

Group Compound N. animals DPM/animal Stimulation Index Response GHS classification
1 DMF 4 733.0 - - -
2 25 % test item 4 1831.5 2.5 Negative Sub-category 1B
(EC3: 27.5 %)
3 50 % test item 4 4648.8 6.3 Positive
4 100 % test item 4 5023.0 6.9 Positive
5 25 % HCA 4 2300.1 3.1 Positive -

Stimulation Index: DPM/animal of group 2 - 5 / DPM/animal of group 1

Response: the cases showing three or greater SI values were defined as positive

DMF: N,N-dimethylformamide

HCA: α-Hexylcinnamaldehyde (positive control)

The classification of skin sensitization was conformed to the "Globally Harmonized System of Classification and Labelling of Chemicals (GHS)" (4th revised edition, United Nations, 2011).

Mean body weights and lymph node weights

Group Compound N. animals Body weight (g) Lymph node weight
(mg)
Day 1 Day 6
1 DMF 4 22.4±0.9 23.8 ± 1.1 4.4 ± 0.3
2 25 % test item 4 22.4 ± 0.7 23.2 ± 0.9 7.2 ± 0.5
3 50 % test item 4 22.3 ± 0.9 22.5 ± 0.5 10.2 ± 0.9
4 100 % test item 4 22.4 ± 1.1 22.0 ± 0.7 11.3 ± 0.7
5 25 % HCA 4 22.4 ± 1.0 23.3 ± 1.3 8.8 ± 1.7

Mean ear thickness values

Group Compound N. animals mm
Day 1 Day 6
1 DMF 4 0.13±0.01 0.14 ± 0.01
2 25 % test item 4 0.13 ± 0.01 0.15 ± 0.01
3 50 % test item 4 0.13 ± 0.01 0.18 ± 0.02
4 100 % test item 4 0.13 ± 0.01 0.17 ± 0.02
5 25 % HCA 4 0.13 ± 0.01 0.19 ± 0.03

Applicant's summary and conclusion

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
The test substance showed SI higher than 3 at the concentrations of 50 % or over; therefore, it was judged to be a sensitizer under the conditions of this study, and was categorized in sub-category 1B of GHS classification for skin sensitization.
Executive summary:

A mouse local lymph node assay (LLNA) was conducted to investigate the potential of (3S,6E)-3,7,11-Trimethyl-6,10-dodecadienal to induce allergic contact dermatitis.

The DPM/animal of the vehicle control group and 3 test substance groups (25, 50 and 100 %) were 733.0, 1831.5, 4648.8 and 5023.0, respectively. The SI for the 25, 50 and 100 % test item groups were calculated to be 2.5, 6.3 and 6.9, respectively, therefore, it was judged to be a sensitizer under the conditions of this study, and was categorized in sub-category 1B of GHS classification for skin sensitization.

No animals showed abnormal clinical signs due to test substance administration during the observation period.

No animals showed erythema on the auricles during the observation period. There were no body weight increases or decreases due to test substance or positive control substance administration during the sensitization period. The weights of lymph nodes in all test substance groups and the positive control group were higher man that in the vehicle control group.

The ear thickness of the animals in 50 % test substance, 100 % test substance and positive control group were increased during the sensitization period.

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