5-methoxy-N-propyl-1,2,3,4-tetrahydronaphthalen-2-aminium chloride

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August 21, 2012 - September 3, 2012

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

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

2010

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

2009

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

(2R)-5-methoxy-N-propyl-1,2,3,4-tetrahydronaphthalen-2-amine hydrochloride

Cas Number:

93601-85-5

Molecular formula:

C14H21NO.HCl

IUPAC Name:

(2R)-5-methoxy-N-propyl-1,2,3,4-tetrahydronaphthalen-2-amine hydrochloride

Constituent 2

Reference substance name:

(2S)-5-methoxy-N-propyl-1,2,3,4-tetrahydronaphthalen-2-amine hydrochloride (1:1)

EC Number:

695-797-0

Cas Number:

93601-86-6

Molecular formula:

C14H21NO.HCl

IUPAC Name:

(2S)-5-methoxy-N-propyl-1,2,3,4-tetrahydronaphthalen-2-amine hydrochloride (1:1)

Test material form:

solid: particulate/powder

Details on test material:

- Appearance: Mauve powder
- Storage condition of test material: At room temperature in the dark

Test animals

Species:

human

Details on test animals or test system and environmental conditions:

ENVIRONMENTAL CONDITIONS
- All incubations, with the exception of the test substance incubation of 15 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 83 - 96%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.7 - 37.6°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day.
- Temporary deviations may have occurred. Based on laboratory historical data these deviations are considered not to affect the study integrity, as the OD570 of the negative control and the mean relative tissue viability of the positive control were all within the acceptability criteria of the assay.

Test system

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

TEST MATERIAL
- Amounts applied: 10.0 to 11.4 mg per skin tissue
- The test material was spread to match the size of the tissue

NEGATIVE CONTOL
- Amount applied: 25 µL Phosphate buffered saline (PBS)

POSITIVE CONTROL
- Amount applied: 25 µL
- Concentration: 5% (aq) Sodium dodecyl sulphate (SDS) in PBS

Duration of treatment / exposure:

15 minutes

Details on study design:

TEST SITE
- EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, Batch no.: 12-EKIN-031). This model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

TISSUES
- On the day of receipt the tissues were transferred to 12-well plates and preincubated with prewarmed Maintenance Medium for 24 hours at 37°C.
- Killed tissues (EPISKIN-SMTM, 0.38 cm2, Lot no.: 10-EKIN-030): Living epidermis was transferred to 12 well plates and incubated with 2 mL Milli-Q for 48 ± 1 hours. After incubation, killed epidermis was stored at ≤ -15°C. Killed tissues were thawed by placing them for 1 hour at room temperature in 12 well plates on 2 mL maintenance medium. Further use of killed tissues was similar to living tissues.

TEST FOR REDUCTION OF MTT
The test substance was checked for possible direct MTT reduction before the study was started. To assess the ability of the test substance to reduce MTT, 11.9 mg of the test substance was added to 2 mL MTT solution (0.3 mg/mL in PBS). The mixture was incubated for 3 hours at 37°C. A negative control, sterile Milli-Q water was tested concurrently.

APPLICATION/TREATMENT
- The test was performed on a total of 3 tissues per test substance together with negative and positive controls. The skin was moistened with 5 μL Milli-Q water (Millipore Corp., USA) to ensure close contact of the test substance to the tissue and the solid test substance (10.0 to 11.4 mg; with a small glass weight boat) was added into 12-well plates on top of the skin tissues. Three tissues were treated with 25 μL PBS (negative control) and 3 tissues with 25 μL 5% SDS (positive control), respectively. The positive control was re-spread after 7 minutes contact time.
- In addition three killed tissues treated with test substance and three killed non-treated tissues were used for the cytotoxicity evaluation with MTT.

REMOVAL OF TEST SUBSTANCE
- Washing: PBS
- Time after start of exposure: 15 minutes

POST INCUBATION PERIOD
- 42 hours at 37°C

CELL VIABILITY MEASUREMENT
After incubation, cell culture inserts were dried carefully to remove excess medium and were transferred into a 12-wells plate prefilled with 2 mL MTT-medium (0.3 mg/mL). The tissues were incubated for 3 h at 37°C. After incubation the tissues were placed on blotting paper to dry the tissues. Total biopsy was made by using a biopsy punch. Epidermis was separated from the collagen matrix and both parts were placed in prelabeled microtubes and extracted with 500 μL isopropanol. Tubes were stored refrigerated and protected from light for 70 hours. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.

SCORING SYSTEM:
- Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by a reduced formazan production from MTT at the end of the treatment. Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean of 3

Value:

113

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

- The test substance was checked for possible direct MTT reduction by adding the test substance to MTT medium. Because a colour change was observed it was concluded that the test substance did interact with MTT. Therefore, in addition to the normal procedure, three killed tissues treated with test substance and three killed non-treated tissues were used for the cytotoxicity evaluation with MTT. The non-specific reduction of MTT (NSMTT) by the test substance was 2.7% of the negative control tissues. The net OD of the treated killed tissues was subtracted from the ODs of the test substance treated viable tissues.

- Mean tissue viability for the test substance was > 50%, therefore the test substance is considered not to be irritant to the skin.

Applicant's summary and conclusion

Interpretation of results:

other: the substance does not need to be classified for skin irritation according to GHS and CLP

Conclusions:

An in vitro skin irritation test was conducted with the substance according to OECD 439 guideline and GLP principles. It is concluded that this test is valid and that the substance is not irritating in the in vitro skin irritation test.

Executive summary:

In an in vitro skin irritation test using a human skin model (EPISKIN Small Model), conducted according to OECD 439 and GLP principles, the influence of the substance on the viability of human skin was tested. Reliable positive and negative controls were included. The test substance was applied directly to 0.38 cm2 cultured skin (10.0 to 11.4 mg, in presence of 10 μL Milli-Q water). After 15 minutes, the substance was removed and cells were cultured for 42 hours. The viability of the cells was tested by reduction of MTT. As the substance did interact with MTT, also three killed tissues treated with test substance and three killed non-treated tissues were used for the cytotoxicity evaluation with MTT. The non-specific reduction of MTT was 2.7% of the negative control tissues. The net OD of the treated killed tissues was subtracted from the ODs of the test substance treated viable tissues. Skin irritation is expressed as the remaining cell viability after exposure to the test substance. Survival of unexposed skin was set at 100%, the positive control had a mean cell viability of 14% whereas the test substance showed cell viability of 113%. Since the mean relative tissue viability after exposure to the test substance was above 50%, it is concluded that the substance is not irritating in the in vitro skin irritation test.