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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2017-03-21 to 2017-03-27
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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: wax
Details on test material:
Physical state: wax form
Appearance: clear, viscous, wax
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: I16HB3199
- Expiration date of the lot/batch: 2017-09-13 (retest date)
- Purity test date: no data

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The substance was placed in a heating chamber at 40-55°C for a period of 2 days to liquefy and aliquoted. The substance was allowed to cool down to 40°C.The liquid and homogenized test item was applied undiluted (25 μl) directly on top of the tissue. Before sampling and prior to dosing, the substance was checked on homogeneity and appearance.

Although the instructions from Janssen Pharmaceutica N.V. according to the Test Item Data Sheet were not followed appropriate (3 days at 60°C), the test substance was heated sufficiently and homogeneity was checked before applying the substance to the test system. Therefore the test results can be considered reliable.

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:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small model (EPISKIN-SMTM, 0.38 cm²) supplied by SkinEthic Laboratories, Lyon France
- Tissue batch number(s): 17-EKIN-012
- 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 a nd 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 22 hours at 37°C.
- Twenty five μl of the undiluted test item 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, ere carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 55 - 94%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.0 - 36.9°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 of 15 ± 0.5 minutes at room temperature, the tissues were washed with phosphate 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 tissues 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 69 hours. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate 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 tissues. Skin irritation potential of the test item was classified according to remaining cell viability following 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 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 direct MTT reduction and colour interference in the Skin corrosion test using EpiDerm as a skin model (project 513800). Because 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.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability (≥ 19.5%): 22.8 +/-0.4% (CV =1.8%)
- Barrier function (IC50 determination, ≥ 1.5 mg/mL): 1.9 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: 2017-03-27

NUMBER OF REPLICATE TISSUES: 3
NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 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 MATERIAL
- Amount(s) applied (volume or weight with unit): 25 μl
- Concentration (if solution): undiluted

NEGATIVE CONTROL (PBS)
- Amount(s) applied (volume or weight): 25 μL

POSITIVE CONTROL (5% SDS)
- Amount(s) applied (volume or weight): 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

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean tissue viability
Run / experiment:
1
Value:
11
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: range: 10 to 12% SD: +/- 0.6
Irritation / corrosion parameter:
other: optical density mean
Run / experiment:
1
Value:
0.075
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: range: 0.071 to 0.079 SD: +/-0.004
Other effects / acceptance of results:
mean tissue viability (percentage of control):
negative control: 100 +/- 11
positive control: 7.3 +/-1.9

mean optical density:
negative control: 0.688 +/- 0.077
positive control: 0.050 +/- 0.013

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

Results:
The test item was checked for possible direct MTT reduction and colour interference in the Skin corrosion test using EpiDerm as a skin model (project 513800). Because 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.

Initially, the absolute OD570 (optical density at 570 nm) of the negative control tissues was not within the acceptable limits of OECD 439 (lower acceptance limit ≥0.6 and upper acceptance limit ≤1.5) and the standard deviation value between tissue replicates of the negative control substance was 29% after 15 minutes exposure which is not within the acceptability criteria of the assay (≤18%). To this end, the negative control was replaced by the values of another negative control, performed at the same time using the same batch. The absolute mean OD570 of the negative control tissues was between 0.6 and 1.5.

The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues was 14% (13 to 15%). The mean cell viability following the 15 minutes exposure to the positive control was 7.3%. The standard deviation values of the percentage viability of three tissues treated identically calculated with the (second) negative control were 11%, 1.9% and 0.6% for the negative, positive control and the test item respectively, indicating that the test system functioned properly.

The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the (second) negative control tissues was 11% (10 to 12%). Since the mean relative tissue viability for the test item was below 50% after 15 ± 0.5 minutes treatment the test item is considered to be irritant

Applicant's summary and conclusion

Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
It is concluded that the test is valid and that the test item is irritant in the in vitro skin irritation test under the experimental conditions described in the report and should be classified category 2 according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations and the negative in vitro skin corrosion test (project 513800).