(1S)-2-(benzylamino)-1-[(2R)-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 2016-01-11 to 2016-01-15

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. One deviation was recorded, that did not affect the integrity of the study : one of the positive control tissues at the 1-hour treatment of this project was lost during the procedure, therefore the positive control data from another project which was performed at same time was used. Evaluation: Since the positive control from the other project was treated identically with the same batch at the same time and the acceptance criteria of the positive control were met, this has no influence on the study integrity.

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:

yes

Remarks:

(see rationale for reliability)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Deviations:

yes

Remarks:

(see rationale for reliability)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

603-316-2

Cas Number:

129050-27-7

Molecular formula:

C18H20FNO2

IUPAC Name:

(1S)-2-(benzylamino)-1-[(2R)-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-17298957-AAA (T001592)
- 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: I15HB2929
- Expiration date of the lot/batch: 2017-08-14
- Purity: 100%
- 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: none

FORM AS APPLIED IN THE TEST: white powder

In vitro test system

Test system:

human skin model

Remarks:

human-derived epidermal keratinocytes

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: not specified

Source strain:

not specified

Details on animal used as source of test system:

- EpiDerm Skin Model (EPI-200, Lot no.:23280 kits X and J).
- 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.

Justification for test system used:

Recommended test system in international guidelines (OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

TISSUE PREPARATION
- Tissue preparation: 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).

TEMPERATURE USED FOR TEST SYSTEM
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 65 - 89 %), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.5 - 37.2°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.

TESTS FOR INTERFERENCE BY THE TEST ITEM
- 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.
- 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.
- 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.

APPLICATION/TREATMENT OF THE TEST ITEM
The skin tissues were kept in the refrigerator the day they were received. The next day, at least 1 hour before the assay was started the tissues were transferred to 6-well plates containing 0.9 ml DMEM medium per well. The level of the DMEM medium was just beneath the tissue. The plates were incubated for approximatively 1 hour at 37.0 ± 1.0ºC. The medium was replaced with fresh DMEM medium just before the test item was applied.

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 EXPOSURE
- MTT concentration: MTT concentrate (5 mg/mL) diluted (1:5) with MTT diluent (supplemented DMEM). Both supplied by MatTek Corporation.
- Incubation time: 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.
- Spectrophotometer: Magellan Tracker 7.0 (TECAN, Austria)
- Linear OD range of spectrophotometer: 570 nm

DECISION CRITERIA
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.

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: 30.90 to 40.28 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

Number of replicates:

4 tissues 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:

98

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: not corrosive

Remarks:

individual values: 97 and 100

Results 2

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean

Run / experiment:

test item after 1-hour application

Value:

78

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: not corrosive

Remarks:

individual values: 74 and 82

Other effects / acceptance of results:

The relative mean tissue viability obtained after the 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 98% (97 and 100%) and 78% (74 and 82%) respectively. 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.

NEGATIVE AND POSITIVE CONTROLS:
mean tissue viability (percentage of control):
negative control: 100% (101 and 99%) after 3-minute exposure and 100% (96 and 104%) after 1-hour exposure
positive control: 15% (12 and 18%) after 3-minute exposure and 13% (9 and 18%) after 1-hour exposure

mean optical density:
negative control: 1.609 ± 0.022 after 3-minute exposure and 1.895 ± 0.094 after 1-hour exposure
positive control: 0.242 ± 0.066 after 3-minute exposure and 0.254 ± 0.121 after 1-hour exposure

One of the positive control tissues at the 1-hour treatment of this project was lost during the procedure, therefore the positive control data from another project which was performed at same time was used. However, since the positive control from the other project was treated identically with the same batch at the same time and the acceptance criteria of the positive control were met this has no influence on the study integrity (study plan deviation).

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 maximum inter-tissue variability in viability between two tissues treated identically was less than 11% and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues was less than 6% 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 51% and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues was less than 34%, 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 is valid and the test item is not corrosive in the in vitro skin corrosion test under the experimental conditions described in the report.