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

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 21 August, 2012 to 27 August, 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:

(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

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

other: epidermal keratinocytes

Cell source:

other: SkinEthic Laboratories, Lyon, France

Source strain:

other: Not applicable

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small ModelTM (Skinethic Laboratories, Lyon, France)
- Tissue batch number: 12-EKIN-30
- 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.
- 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.Maintenance medium and Assay medium were supplied by Skinethic Laboratories, Lyon, France.

PRE-TEST PROCEDURE
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, 10.7 mg of the test substance was added to 2 mL MTT solution (0.3 mg/mL in Phosphate buffered saline (PBS)). The mixture was incubated for 3 hours at 37°C. A negative control, sterile Milli-Q water was tested concurrently.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): 36.7 - 37.8°C

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

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 approx. 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.

PREDICTION MODEL / DECISION CRITERIA
- A test substance is considered irritant in the in vitro skin irritation test if:
The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test substance and 42 hours of post incubation is ≤ 50% of the mean viability of the negative controls.
- A test substance is considered non-irritant in the in vitro skin irritation test if:
The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test substance and 42 hours of post incubation is > 50% of the mean viability of the negative controls.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amounts applied: 10.4 to 12.1 mg per skin tissue
- The skin was moistened with 5 µL Milli-Q water (Millipore Corp., USA) to ensure close contact of the test substance to 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

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean of 3

Value:

112

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Negative control = 100%; Positive control = 6%

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct-MTT reduction: Because no colour change was observed it was concluded that the test substance did not interact with MTT.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The absolute mean OD570(optical density at 570 nm) of the three tissues of the negative control (0.727) was within the laboratory historical control data range (0.597 – 1.207).
- Acceptance criteria met for positive control: The mean relative tissue viability of the positive control was ≤ 40% (6%) relative to the negative control and the Standard Deviation value (SD) of the % viability was ≤18 (2).
- Acceptance criteria met for variability between replicate measurements:
The SD calculated from individual % tissue viabilities of the three identically treated replicates of the negative control (PBS) was 19%. As this was only slightly above 18 and the other acceptance criterium for the negative control was met (see above), it was concluded that this did not affect the study integrity.
The SD calculated from individual % tissue viabilities of three identically treated replicates was ≤18 for the positive control (2) and for the test substance (18).

Any other information on results incl. tables

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 (10.4 to 12.1 mg, in presence of 5 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. 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 6% whereas the test substance showed cell viability of 112%. 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. Based on the results, the substance does not need to be classified for skin irritation according to Regulation (EC) No. 1272/2008 (CLP Regulation).