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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2015-05-26 to 2015-05-29
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: EU Method B.40 BIS:"In vitro Skin Corrosion: Human Skin Model Test", 31 May 2008
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Name of test material (as stated in study reports): JNJ-4508530-AAA (T002707)
- Physical state: solid (powder)
- Appearance: white, beige powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Janssen Pharmaceutica N.V., I15BB0573
- Expiration date of the lot/batch: 8 February 2017 (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
- Solubility and stability of the test substance in the solvent/vehicle: not applicable, tested neat
- 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: the solid test item was applied directly on top of the skin tissue.

In vitro test system

Test system:
human skin model
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:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Skin Model (EPI-200) supplied by MatTek Corporation, Ashland MA, U.S.A
- Tissue batch number(s): T22251 kitAA
- 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, serumfree supplied by MatTek Corporation) per well. The level of the DMEM medium was just beneath the tissue. The plates were incubated for approximately 2 hours at 37.0 ± 1.0ºC. The medium was replaced with fresh DMEM medium just before the test item was applied.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature during 3-minute exposure and 37°C during 1-hour exposure
- Temperature of post-treatment incubation (if applicable): 37°C
- 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 66-79%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.8-36.6°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 (PBS) (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.
- 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) for 2 hours at room temperature with gentle shaking (± 200 rpm). 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 the interference 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.
FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability (OD (540-570 nm)<1.0-3.0>): 1.852 ± 0.086 (MTT QC assay, 4 hours, n=3)
- Barrier function (ET-50 <4.77-8.72 hrs>): 7.82 hours (ET-50 assay, 100 µL 1% Triton X-100, 4 time points, n=3, MTT assay)
- Sterility: sterile
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 MATERIAL
- Amount(s) applied (volume or weight with unit): 25.2 to 34.5 mg was applied directly on top of the skin tissue. The test item was spread to match the size of the tissue.
- 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.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL Milli-Q water

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL 8N KOH
Duration of treatment / exposure:
3 minutes and one 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

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3-minute application
Value:
88
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1-hour application
Value:
81
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Irritation / corrosion parameter:
other: optical density
Run / experiment:
3-minute application
Value:
1.498
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Irritation / corrosion parameter:
other: optical density
Run / experiment:
1-hour application
Value:
1.48
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Irritation / corrosion parameter:
other: coefficient of variation
Run / experiment:
3-minute application
Value:
0.7
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Irritation / corrosion parameter:
other: coefficient of variation
Run / experiment:
1-hour application
Value:
3.8
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Other effects / acceptance of results:
3-minute application:
- viability (percentage of control) (range):
negative control: 100 (103 and 97)
positive control: 14 (17 and 10)
- coefficient of variation between tissue replicates
negative control: 5.3
positive control: 39.0
- mean optical density:
negative control: 1.695
positive control: 0.229

1-hour application:
- viability (percentage of control) (range):
negative control: 100 (102 and 98)
positive control: 12 (15 and 9)
- coefficient of variation between tissue replicates
negative control: 3.1
positive control: 44.0
- mean optical density:
negative control: 1.823
positive control: 0.216

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 the 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 88% (88 and 89%) and 81% (83 and 80%) 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.
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 14% (17 and 10%) and 12% (15 and 9%) after 1 hour exposure. The maximum inter-tissue variability in viability between two tissues treated identically was less than 6% and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues was less than 3% for the negative control and test item. For the positive control, the maximum inter-tissue variability in viability between two tissues treated identically was up to 44% and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues was up to 29%, 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:
It is concluded that this test is valid and that the test item is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.