D-Glucitol, 1,5-anhydro-1-C-[3-[[5-(4-fluorophenyl)-2-thienyl]methyl]-4-methylphenyl]-, 2,3,4,6-tetrakis(2,2-dimethylpropanoate), (1S)-

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 2016-03-07 to 2016-03-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Well documented GLP study performed according to OECD guideline 439 and EU method B.46. There were no deviations from the study plan.

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

[(2R,3R,4R,5S,6S)-3,4,5-tris[(2,2-dimethylpropanoyl)oxy]-6-(3-{[5-(4-fluorophenyl)thiophen-2-yl]methyl}-4-methylphenyl)oxan-2-yl]methyl 2,2-dimethylpropanoate

EC Number:

695-878-0

Cas Number:

1283129-18-9

Molecular formula:

C44H57FO9S

IUPAC Name:

[(2R,3R,4R,5S,6S)-3,4,5-tris[(2,2-dimethylpropanoyl)oxy]-6-(3-{[5-(4-fluorophenyl)thiophen-2-yl]methyl}-4-methylphenyl)oxan-2-yl]methyl 2,2-dimethylpropanoate

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study reports): JNJ-42808415-AAA (T003422)
- 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: I15AB0305
- Expiration date of the lot/batch: 2017-01-23 (retest date)
- Purity test date: no data

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: no data

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

In vitro test system

Test system:

human skin model

Remarks:

of human-derived epidermal keratinocytes

Source species:

human

Cell type:

non-transformed keratinocytes

Source strain:

other: not applicable

Justification for test system used:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC)

Vehicle:

unchanged (no vehicle)

Details on test system:

TEST SYSTEM
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2) supplied by SkinEthic Laboratories, Lyon, France.
- Tissue batch number(s): 16-EKIN-010
- 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 prewa rmed Maintenance Medium for 24 hours at 37°C.
- The skin was moistened with 5 µl Milli-Q water (Millipore Corp., Bedford, Mass., USA) to ensure close contact of the test item to the tissue and the solid test item (18.2 to 31.5 mg) was added into 12-well plates on top of the skin tissues.

TEMPERATURE USED FOR TEST SYSTEM - Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): 37°C
- All incubations, with the exception of the test item 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 71 - 92%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.4 - 37.2C).
- 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 of 15 ± 0.5 minutes at room temperature, the tissues were washed with ph osphate buffered saline (PBS) to remove residual test item. After rinsing the cell culture inserts were each dried carefully and moved to a new well on 2 mL pre-warmed maintenance medium until all t is sues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37°C.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- After incubation, cell culture inserts were dried carefully to remove excess medium and were transfer red into a 12-wells plate prefilled with 2 mL MTT-medium (0.3 mg/mL). The tissues were in cubated 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 (Merck, Darmstadt, Germany). Tubes were stored refrigerated and protected from light for approximately 69 hours. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplic ate with the TECAN Infinite® M200 Pro Plate Reader.
- Cell viability was calculated for each tissue as a percentage of the mean of the negative control t issues. Skin irritation potential of the test item was classified according to remaining cell viability foll owing exposure of the test item.
- Test for reduction of MTT by test item: A test item may interfere with the MTT endpoint if it is coloure d 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 direct MTT colour interference before the study was started. Because solutions did not turn blue / purple and a blue / purple pre cipitate was not observed it was concluded that the test item did not interfere with the MTT endpoint.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability (≥ 19.5%): 22.3 +/-0.7% (CV =3.1%)
- Barrier function (IC50 determination, ≥ 1.5 mg/mL): 2.3 mg/mL
- Morphology: well-differentiated epidermis consisting of a basal layer, several spinous and granular layers and a thick stratum corneum.
- Contamination:
- on blood of the same donors, the following was verified: the absence of HIV1 and 2 antibodies, the absence of hepatitis C antibodies, the absence of hepatitis B antigen HBs;
- on epidermal cells of the same donors, the following was verified: the absence of bacteria, fungus and mycoplasma.
- Expiration date: 2016-03-16

NUMBER OF REPLICATE TISSUES: 3

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

INTERPRETATION
- A test item is considered to be irritant in the skin irritation test (category 2) if: the relative mean tissue viability of three individual tussues after 15 minutes of exposure to the test item and 42 hours of post incubation is ≤ 50% of the mean viability of the negative controls.
- A test item is considered to be non-irritant in the in vitro skin irritation test (no category) if the relative mean tissue viability after 15 minutes of exposure to the test item 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 ITEM
- Amount applied: 18.2 to 31.5 mg

NEGATIVE CONTROL (PBS)
- Amount applied: 25 μl

POSITIVE CONTROL (5% SDS)
- Amount applied: 25 μl

Duration of treatment / exposure:

15 +/- 0.5 minutes (the positive control was re-spread after 7 minutes contact time)

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3 replicates per test item together with negative and positive controls

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean tissue viability

Run / experiment:

1

Value:

94

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: range: 85 to 106% SD: +/-10.9

Results 2

Irritation / corrosion parameter:

other: optical density

Remarks:

mean

Run / experiment:

1

Value:

1.11

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: range 0.997 to 1.248 SD: +/-0.128

Other effects / acceptance of results:

mean tissue viability (percentage of control):
negative control: 100 +/- 8.1
positive control: 19 +/- 6.9

mean optical density:
negative control: 1.176 +/- 0.096
positive control: 0.227 +/- 0.081

Interpretation: The viabilities of all replicates were within one category.

Results:
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.

The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compa red to the negative control tissues was 94% (85 to 106%). Since the mean relative tissue viability for the test item was above 50% the test item is considered to be non-irritant.

The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 19% (15 to 27%). The absolute mean OD570 of the negative control tissues was within 0.6 and 1.5. The standard deviation values of the percentage viability of three tissues treated identically were 8%, 7% and 11% for the negative, positive control and the test item respectively, indicating that the test system functioned properly.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

It is concluded that this test is valid and that the test item is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report.