(1R)-2-(benzylamino)-1-[(2S)-6-fluoro-3,4-dihydro-2H-chromen-2-yl]ethanol

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From 2016-01-25 to 2016-01-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Well documented GLP study performed according to OECD guideline 431 and EU method B.40 BIS.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EU Method B.40 BIS (In Vitro Skin Corrosion: Human Skin Model Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

(1R)-2-(benzylamino)-1-[(2S)-6-fluoro-3,4-dihydro-2H-chromen-2-yl]ethanol

EC Number:

600-381-9

Cas Number:

1030385-16-0

Molecular formula:

C18H20FNO2

IUPAC Name:

(1R)-2-(benzylamino)-1-[(2S)-6-fluoro-3,4-dihydro-2H-chromen-2-yl]ethanol

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study reports): JNJ-17299412-AAA (T001599)
- Physical state: solid (powder)
- Appearance: white powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: I15HB2951
- Expiration date of the lot/batch: 2017-08-09 (retest date)
- Purity test date: no data
- Certificate of analysis release date: 2015-09-04

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: no data
- Solubility and stability of the test substance in the solvent/vehicle: not applicable
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not applicable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: none, the solid test item was applied neat directly on top of the skin tissue

In vitro test system

Test system:

human skin model

Remarks:

model of human-derived epidermal keratinocytes

Source species:

human

Cell type:

non-transformed keratinocytes

Source strain:

other: not applicable

Justification for test system used:

Recommended test system in international guidelines (OECD and EC)

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- EpiDerm Skin Model (EPI-200, Lot no.: 23297 kits M and N), supplied by MatTek Corporation, Ashland MA, U.S.A
- The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm tissues (surface 0.6 cm²) were cultured on polycarbonate membranes of 10 mm cell culture inserts.
- On the day of receipt the tissues were kept on agarose and stored in the refrigerator. On the next day, at least one hour before starting the assay the tissues were transferred to 6-well plates with 0.9 mL DMEM medium (Dulbecco’s Modified Eagle’s Medium) (supplemented DMEM medium, serum-free supplied by MatTek Corporation). The level of the DMEM medium was just beneath the tissue. The plates were incubated for 1 to 2.5 hours at 37.0 ± 1.0ºC. The medium was replaced with fresh DMEM medium just before the test item was applied.

ENVIRONMENTAL CONDITIONS
- All incubations, with the exception of the test item incubation of 3 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 79 - 90%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.5 - 36.7°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

REMOVAL OF TEST MATERIAL AND CONTROLS
- After the exposure period, the tissues were washed with phosphate buffered saline (Invitrogen Corporation, Breda, The Netherlands) to remove residual test item. Rinsed tissues were kept in 24 well plates on 300 μl DMEM medium until 6 tissues (= one application time) were dosed and rinsed.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT MEDIUM: MTT concentrate (5 mg/mL) diluted (1:5) with MTT diluent (supplemented DMEM). Both supplied by MatTek Corporation.
- CELL VIABILITY MEASUREMENT :
The DMEM medium was replaced by 300 μl MTT-medium and tissues were incubated for 3 hours at 37°C in air containing 5% CO2. After incubation the tissues were washed with PBS and formazan was extracted with 2 mL isopropanol (MatTek corporation) over night at room temperature. The amount of extracted formazan was determined spectrophotometrically at 570 nm in triplicate with the TECAN Infinite® M200 Pro Plate Reader.
- Cell viability was calculated for each tissue as percentage of the mean of the negative control tissues. Skin corrosion potential of the test item was classified according to remaining cell viability following exposure of the test item with either of the two exposure times.

TEST FOR INTERFERENCE OF THE TEST ITEM WITH THE MTT ENDPOINT
A test item may interfere with the MTT endpoint if it is coloured and/or it is able to directly reduce MTT. The cell viability measurement is affected only if the test item is present on the tissues when the MTT viability test is performed.

TEST FOR COLOUR INTERFERENCE BY THE TEST ITEM
The test item was checked for possible colour interference before the study was started. Some non-coloured test items may change into coloured substances in aqueous conditions and thus stain the skin tissues during the 1-hour exposure. To assess the colour interference, approximately 25 mg of the test item or 50 μl Milli-Q water as a negative control were added to 0.3 ml Milli-Q water. The mixture was incubated for approximately 1 hour at 37.0 ± 1.0°C in the dark. At the end of the exposure time the mixture was shaken and it was checked if a blue / purple colour change was observed.

TEST FOR REDUCTION OF MTT BY THE TEST ITEM
The test item was checked for possible direct MTT reduction before the study was started. To assess the ability of the test item to reduce MTT, approximately 25 mg of the test item was added to 1 ml MTT (Sigma, Zwijndrecht, The Netherlands) solution (1 mg/ml) in phosphate buffered saline. The mixture was incubated for approximately 1 hour at 37.0 ± 1.0ºC. A negative control, sterile Milli-Q water was tested concurrently. At the end of the exposure time it was checked if a blue / purple colour change was observed.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

DECISION CRITERIA
A test item is considered corrosive in the skin corrosion test if:
a) The relative mean tissue viability obtained after the 3-minute treatment compared to the negative control tissues is decreased below 50%.
b) In addition, a test item considered non-corrosive (viability ≥ 50%) after the 3-minute treatment is considered corrosive if the relative tissue viability after 1-hour treatment with the test item is decreased below 15%.

A test item is considered non-corrosive in the in vitro skin corrosion test if:
a) The relative mean tissue viability obtained after the 3-minute treatment compared to the negative control tissues is not decreased below 50%.
b) In addition, the relative tissue viability after the 1-hour treatment is not decreased below 15%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST ITEM:
- Amount applied: 35.0 to 38.6 mg.
- The skin was moistened with 25 μl Milli-Q water (Millipore Corp., Bedford, Mass., USA) to ensure close contact of the test item to the tissue. The solid test item was added into the 6-well plates on top of the skin tissues.

NEGATIVE CONTROL:
- Amount applied: 50 μl

POSITIVE CONTROL:
- Amount applied: 50 µl

Duration of treatment / exposure:

3 minutes and 1 hour

Duration of post-treatment incubation (if applicable):

3 hours

Number of replicates:

4 replicates per test item, negative control and positive control: 2 for the 3-minute exposure and 2 for the 1-hour exposure

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Remarks:

Mean

Run / experiment:

test item after 3-minute application

Value:

85

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: individual values: 81 and 89

Results 2

Irritation / corrosion parameter:

% tissue viability

Remarks:

Mean

Run / experiment:

test item after 1-hour application

Value:

69

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: individual values: 69 and 69

Results 3

Irritation / corrosion parameter:

other: Mean absorption

Remarks:

optical density at 570 nm

Run / experiment:

test item after 3-minute application

Value:

1.842

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: individual values: 1.762 and 1.921 / SD = 0.112

Results 4

Irritation / corrosion parameter:

other: Mean absorption

Remarks:

optical density at 570 nm

Run / experiment:

test item after 1-hour application

Value:

1.378

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: individual values: 1.370 and 1.386 / SD = 0.012

Results 5

Irritation / corrosion parameter:

other: maximum inter-tissue variability in viability between two tissues treated identically

Run / experiment:

test item after 3-minute application

Value:

8.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 6

Irritation / corrosion parameter:

other: maximum inter-tissue variability in viability between two tissues treated identically

Run / experiment:

test item after 1-hour application

Value:

1.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Because the mean relative tissue viability for the test item was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment the test item is considered to be not corrosive.

OTHER EFFECTS:
The test item was checked for colour interference in aqueous conditions and possible direct MTT reduction by adding the test item to MTT medium. Because the solutions did not turn blue / purple and a blue / purple precipitate was not observed it was concluded that the test item did not interfere with the MTT endpoint.

CONTROL RESULTS
3-minute application:
- viability:
negative control: 100 (98 and 102)
positive control: 11 (9 and 12)
- mean optical density:
negative control: 2.165 +/- 0.046
positive control: 0.228 +/- 0.057
- maximum inter-tissue variability in viability:
negative control: 3.0
positive control: 30.2

1-hour application:
- viability:
negative control: 100 (98 and 102)
positive control: 9 (7 and 10)
- mean optical density:
negative control: 1.997 +/- 0.064
positive control: 0.172+/- 0.036
- maximum inter-tissue variability in viability:
negative control: 4.4
positive control: 25.6


ACCEPTANCE OF RESULTS
- The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range.
- The mean relative tissue viability following 3-minute exposure to the positive control was <30% and <15% after 1 hour exposure.
- The maximum inter-tissue variability in viability between two tissues treated identically was less than 9% and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues was less than 5% for the negative control and test item. For the positive control, the maximum inter-tissue variability in viability between two tissues treated identically was less than 31% and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues was less than 18%, however since the viabilities were below 20% the acceptability criteria were met.
- It was therefore concluded that the test system functioned properly.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test was considered to be valid and the test item was concluded to be not corrosive in the in vitro skin corrosion test under the experimental conditions described in the report.