(2S)-5-methoxy-N-propyl-1,2,3,4-tetrahydronaphthalen-2-aminium (2R)-(2-chlorophenyl)(hydroxy)acetate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March 27, 2013 - April 15, 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

2010

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

2003

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

(2S)-5-methoxy-N-propyl-1,2,3,4-tetrahydronaphthalen-2-aminium (2R)-(2-chlorophenyl)(hydroxy)acetate

EC Number:

814-350-4

Cas Number:

1352917-66-8

Molecular formula:

C22 H28 N Cl O4

IUPAC Name:

(2S)-5-methoxy-N-propyl-1,2,3,4-tetrahydronaphthalen-2-aminium (2R)-(2-chlorophenyl)(hydroxy)acetate

Test material form:

solid: particulate/powder

Details on test material:

- Appearance: Off-white powder with lumps
- Storage condition of test material: At room temperature in the dark

Specific details on test material used for the study:

pH: 4.4 - 4.5

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/J

Sex:

female

Details on test animals and environmental conditions:

- Source: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: Young adult animals
(main study: approx. 9 weeks old; range finding tests: approx. 10 weeks old)
- Weight at study initiation:
Main study: 19-23 g; range finding tests: 20-22 g
- Housing: Animals were group housed in labeled Makrolon cages
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS (set conditions)
- Temperature (°C): 18 – 24
- Humidity (%): 40 - 70
- Air changes (per hr): approx. 15
- Photoperiod (hrs dark / hrs light): 12/12

Variations to these conditions occurred (i.e. minimum relative humidity of 24 or 33% on a single day, respectively). Based on the laboratory’s extensive experience with variations in these parameters and absence of any clinical signs among the animals that could be associated to these variations, these were considered to have no effect on the outcome of the study.

IN-LIFE DATES: From: March 27, 2013 to April 15, 2013

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

0, 10, 25, 50%

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS
In a pre-screen test, two test substance concentrations were tested; a 25% and 50% concentration. Two young adult animals per concentration were selected. Each animal was treated with one concentration for three consecutive days. Ear thickness measurements were conducted prior to dosing on Days 1 and 3, and on Day 6. Animals were sacrificed after the final observation.

MAIN STUDY
INTERPRETATION
- Criteria used to consider a positive response: DPM values are presented for each animal and for each dose group. A Stimulation Index (SI) is calculated for each group. The SI is the ratio of the DPM/group compared to DPM/vehicle control group. If the results indicate a SI ≥ 3, the test substance may be regarded as a skin sensitizer.

ANIMAL ASSIGNMENT
Three groups of five animals were treated with one test substance concentration per group. One group of five animals was treated with vehicle.

TREATMENT PREPARATION AND ADMINISTRATION:
- Test substance preparation: The test substance formulations (w/w) were prepared within 4 hours prior to each dosing. No adjustment was made for specific gravity of the vehicle. Homogeneity was obtained to visually acceptable levels by shaking, vortexing and stirring. This resulted in a solution for the 10% concentration and in a turbid formulation for the 25% and 50% concentration.
- Rationale for vehicle: The vehicle was selected based on trial formulations performed at WIL Research.

Performed according to test guidelines:

- Days 1, 2 and 3: Induction. Topical treatment of 25 µL/ear, using a pipette or spatula (highest concentration) at approx. the same time on each day
- Day 6: Injection of 20 µCi 3H-methyl thymidine. Five hours after the injection, all animals were killed and the ear lymph nodes were 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 approximately 3 mL Phosphate buffered saline (PBS).
- Day 6: Tissue processing for radioactivity. A single cell suspension of lymph node cells was prepared in PBS by gentle separation through stainless steel gauze (diameter 125 μm). The cells were washed twice with an excess of PBS by centrifugation at 200 g for 10 minutes at 4°C. To precipitate the DNA, the cells were exposed to 5% trichloroacetic acid (TCA) and stored in the refrigerator until the next day.
- Day 7: Radioactivity measurements using a Packard scintillation counter (2800TR). Precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL of Ultima Gold cocktail (PerkinElmer Life and Analytical Sciences, Boston, US) as the scintillation fluid.

Observations:
Mortality/Viability: Twice daily.
Body weights: On Day 1 (pre-dose) and Day 6 (prior to necropsy).
Clinical signs: Once daily on Days 1-6 (on Days 1-3 between 3 and 4 hours after dosing).
Irritation: Once daily on Days 1-6 (on Days 1-3 within 1 hour after dosing)

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:

The six-month reliability check with Alpha-hexylcinnamicaldehyde indicates that the Local Lymph Node Assay as performed at WIL Research Europe is an appropriate model for testing for contact hypersensitivity.The SI values calculated for the concentrations 5, 10 and 25% were 1.2, 1.6 and 7.7, respectively. An EC3 value of 13.4% was calculated using linear interpolation.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

2.8

Test group / Remarks:

Substance concentration 10%

Results 2

Key result

Parameter:

SI

Value:

1.9

Test group / Remarks:

Substance concentration 25%

Results 3

Key result

Parameter:

SI

Value:

1.4

Test group / Remarks:

Substance concentration 50%

Cellular proliferation data / Observations:

Mean DPM/animal values for the experimental groups treated with test substance concentrations 10, 25 and 50% were 1498, 1021 and 741 DPM respectively. The mean DPM/animal value for the vehicle control group was 539 DPM. Variability in measured DPM values was within the expected variation for this type of study.

Any other information on results incl. tables

Results Pre-screen test:

No irritation and no signs of systemic toxicity were observed in any of the animals examined. Variations in ear thickness during the observation period were less than 25% from Day 1 pre-dose values. White test substance remnants were present on the dorsal surface of the ears of both animals at 25% (Days 1 -3) and at 50% (Days 1 - 4), which did not hamper scoring of the skin reactions.

Based on these results, the highest test substance concentration selected for the main study was a 50% concentration (highest technically possible concentration for a solid).

Other results - main study:

- No irritation of the ears was observed in any of the animals examined. White test substance remnants were present on the dorsal surface of the ears of all animals at 10, 25 and 50% (Days 1, 2 and/or 3), which did not hamper scoring of the skin reactions.

- The auricular lymph nodes of three animals at 10% and three animals at 25% appeared larger in size as compared to the other animals. This increase in size did not show a clear relation to the DPM/animal values. No macroscopic abnormalities of the surrounding area were noted in any of the animals.

- 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. The body weight loss noted for some animals across the dose groups was considered not toxicologically significant since the changes were slight in nature.

Applicant's summary and conclusion

Interpretation of results:

other: the substance does not need to be classified as skin sensitiser according to GHS and CLP

Conclusions:

In an LLNA skin sensitisation study, performed according to OECD/EC test guidelines, the substance was considered not be a skin sensitiser, as the SI was shown to be < 3 when tested up to and including 50%.

Executive summary:

An LLNA skin sensitisation study was performed according to OECD 429 and according to GLP principles with the substance. Reliable positive and negative controls were included. Based on the results of a pre-screen test, three experimental groups of five female CBA/J mice were treated with test substance concentrations of 10, 25 or 50% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with vehicle alone (dimethylformamide). No irritation of the ears was observed in any of the animals examined. The auricular lymph nodes of three animals at 10% and three animals at 25% appeared larger in size as compared to the other animals. This increase in size did not show a clear relation to the DPM/animal values. No macroscopic abnormalities of the surrounding area were noted in any of the animals.

Mean DPM/animal values for the experimental groups treated with test substance concentrations 10, 25 and 50% were 1498, 1021 and 741 DPM respectively. The mean DPM/animal value for the vehicle control group was 539 DPM. The SI values calculated for the substance concentrations 10, 25 and 50% were 2.8, 1.9 and 1.4 respectively. Based on these data, the substance is considered not be a skin sensitiser, as the SI was shown to be < 3 when tested up to and including 50%. The substance does not need to be classified as skin sensitiser according to GHS and CLP.