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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 2018-01-16 to 2018-02-20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2010
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
10-Methoxy-9,10-dihydrolysergic methylester
Cas Number:
23495-64-9
Molecular formula:
C18 H22 N2 O3
IUPAC Name:
10-Methoxy-9,10-dihydrolysergic methylester
Specific details on test material used for the study:
Batch No.: 17010ES4B5
Purity: 98.6%

In vivo test system

Test animals

Species:
mouse
Strain:
CBA:J
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Young adult animals (approximately 10 weeks old)
- Weight at study initiation:18.4 to 22.9 g
- Housing: On arrival and following assignment to the study, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages containing sterilized sawdust as bedding equipped with water bottles. Animals were separated during designated procedures/activities. Each cage was clearly labeled.
- Diet (e.g. ad libitum): Pelleted rodent diet, ad libitum throughout the study, except during designated procedures.
- Water (e.g. ad libitum): Municipal tap-water was freely available to each animal via water bottles.
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Target temperatures is 18 to 24°C, the actual is 22 to 23°C
- Humidity: Relative target humidity is 40 to 70%, the actucal is 40 to 46%
- Air changes (per hr): Ten or greater air changes per hour with 100% fresh air
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle

Study design: in vivo (LLNA)

Vehicle:
dimethyl sulphoxide
Concentration:
Pre-screen Test: 25% and 50% w/w
Main Test: 0, 5, 10 and 25 % (w/w)
No. of animals per dose:
Pre-screen Test: two animals per concentration
Main Test: five animals
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: The highest concentration was the highest concentration that could be prepared homogeneously.
- Results: At a 50% test item concentration, variation in ear thickness during the observation period were more than 25% from Day 1 pre-dose values and therefore this concentration did not meet the selection criteria.
At a 25% test item concentration, no signs of systemic toxicity were noted and no irritation was observed and therefore this concentration was selected as highest concentration for the main study.

MAIN STUDY: Three groups of five animals were treated with one test item concentration per group. One group of five animals was treated with the vehicle.
- Allocation/concetration: 0, 5, 10 and 25% w/w
- Induction - Days 1, 2 and 3: The dorsal surface of both ears was topically treated (25 µL/ear with the test item, at approximately the same time on each day. The concentrations were stirred with a magnetic stirrer immediately prior to dosing. The control animals were treated in the same way as the experimental animals, except that the vehicle was administered instead of the test item.
- Excision of the Nodes - Day 6: Each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline (PBS) containing 20 µCi of 3H-methyl thymidine. After five hours, all animals were killed by intraperitoneal injection (0.2 mL/animal) of Euthasol® 20%. The draining (auricular) lymph node of each ear was excised. The relative size of the nodes (as compared to normal) was estimated by visual examination and abnormalities of the nodes and surrounding area were recorded. The nodes were pooled for each animal in PBS.
-Tissue Processing for Radioactivity - Day 6: Following excision of the nodes, a single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel gauze (maze size: 200 μm, diameter: ± 1.5 cm). LNC were washed twice with an excess of PBS by centrifugation at 200g for 10 minutes at 4ºC. To precipitate the DNA, the LNC were exposed to 5% trichloroacetic acid (TCA) and then stored in the refrigerator until the next day.
- Radioactivity Measurements - Day 7: Precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL of Ultima Gold cocktail as the scintillation fluid. Radioactivity measurements were performed using a Packard scintillation counter (2800TR). Counting time was to a statistical precision of ± 0.2% or a maximum of 5 minutes whichever came first. The scintillation counter was programmed to automatically subtract background and convert Counts Per Minute (CPM) to Disintegrations Per Minute (DPM).

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
2.4
Test group / Remarks:
the test item concentrations 5%
Key result
Parameter:
SI
Value:
2.8
Test group / Remarks:
the test item concentrations 10 %
Key result
Parameter:
SI
Value:
7.1
Test group / Remarks:
the test item concentrations 25 %
Cellular proliferation data / Observations:
- Skin Reactions / Irritation:
No irritation was observed in any of the animals.
Brown staining of the dorsal surface of the ears by test item remnants was noted for all animals treated at 10% on Days 1, 2 and 3 and for all animals treated at 25% throughout the observation period. The staining did not hamper the scoring of the ears.
- Systemic Toxicity:
No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.
- Macroscopic Examination of the Lymph Nodes and Surrounding Area:
The majority of auricular lymph nodes were considered normal in size, except for the nodes in one animal treated at 10% and three animals treated at 25%, which were considered enlarged.
No macroscopic abnormalities of the surrounding area were noted for any of the animals.
- Radioactivity Measurements:
Mean DPM/animal values for the experimental groups treated with test item concentrations 5, 10 and 25% were 1194, 1422 and 3589 DPM, respectively. The mean DPM/animal value for the vehicle control group was 502 DPM.

Applicant's summary and conclusion

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
These results indicate that the test item could elicit a SI ≥ 3. The data showed a doseresponse and an EC3 value (the estimated test item concentration that will give a SI =3) of
10.7% was calculated. The test item would be regarded as skin sensitizer
Executive summary:

The objective of this study was to evaluate whether the test item induces skin sensitization in mice (Local Lymph Node Assay) after three epidermal exposures of the animals according to OECD 429. 

In the main study, three experimental groups of five female CBA/J mice were treated with test item concentrations of 5, 10 or 25% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with the vehicle alone (DMSO).

The majority of auricular lymph nodes were considered normal in size, except for the nodes in one animal treated at 10% and three animals treated at 25%, which were considered enlarged. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

Mean DPM/animal values for the experimental groups treated with test item concentrations 5, 10 and 25% were 1194, 1422 and 3589 DPM, respectively. The mean DPM/animal value for the vehicle control group was 502 DPM. The SI values calculated for the test item concentrations 5, 10 and 25% were 2.4, 2.8 and 7.1, respectively.

These results indicate that the test item could elicit a SI3. The data showed a dose response and an EC3 value (the estimated test item concentration that will give a SI =3) of 10.7% was calculated. The test item would be regarded as skin sensitizer