Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

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)
Version / remarks:
July 28, 2015
Deviations:
no
Qualifier:
according to
Guideline:
other: B.46: In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test; Official Journal of the European Union, No. L 193
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
The test item was homogeneous by visual inspection.
Storage conditions: Room temperature
Batch No.: VFH-2016-08

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
foreskin from multiple donors
Source strain:
not specified
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDermTM model
- Tissue batch number(s): 25863 and 25857
- Date of initiation of testing: 26 Oct 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: The tissues were kept under the laminar flow hood at room temperature for 25 minutes overall and for 35 minutes in the incubator.(37°C)
After removal of the test material, the tissues were incubated in the incubator at 37°C for 24 ± 2 hours.

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: The tissues were washed with sterile PBS to remove residual test material 1 hour after start of
application. Rinsed tissues were blotted on sterile absorbent paper and transferred into new 6-well plates,
pre-filled with 0.9 mL fresh medium. When all tissues were rinsed, the surface of each tissue was carefully dried with a sterile cotton swab.
- Observable damage in the tissue due to washing: None




FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA

Historic Range negative control (OD570)
Mean OD SD Mean + 2 SD Mean - 2 SD
2.289 0.285 2.860 1.719

Historic Range of positive control (OD570)
Mean OD SD Mean + 2 SD Mean - 2 SD
0.070 0.011 0.092 0.047

Viability (%)
Mean % SD Mean + 2 SD Mean - 2 SD
3.1 0.5 4.0 2.1


NUMBER OF REPLICATE TISSUES: Three per test group

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues and killed tissues
- Procedure used to prepare the killed tissues: freeze-kill
- N. of replicates : 3
In case of direct reduction of MTT by the test substance, the OD570 values measured in the freeze-killed control tissues (KC) will be used to correct the mean OD570 of the test-substance treated tissues (mean OD570 KC corrected). Since killed tissue might still have a residual enzyme activity that is able to produce some formazan net OD570 KC is calculated by subtracting the OD570 KC of the NC from the OD570 KC of the test substance. In case the net OD570 KC is greater than zero it is subtracted from the respective mean OD570 to result in the mean OD570 KC corrected. The mean OD570 KC corrected represents the formazan production linked to the tissue viability and therefore indicates the cytotoxic potency of the test substance.


NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 2

PREDICTION MODEL / DECISION CRITERIA
A chemical is considered as "irritant", if the mean relative tissue viability with a test material is less than or equal to 50% after a 60min exposure.

A single test composed of at least three tissue replicates should be sufficient for a test chemical when the result is unequivocal. However, in cases of borderline results, such as nonconcordant
replicate measurements and/or mean percent tissue viability equal to ±5% of the cited cut-off value, a second test should be considered, as well as a third one in case of discordant results between the first two tests.

- Observable damage in the tissue due to washing: None

MTT-Treatment:
After the postincubation period, the assay medium was replaced by 0.3 mL MTT solution and the tissues were incubated in the incubator for 3 hours.
After incubation, the tissues were washed with PBS to stop the MTT-incubation. The formazan that was metabolically produced by the tissues was extracted by incubation of the tissues in isopropanol. The optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically. Blank values were established of 4 microtiter wells filled with isopropanol for each microtiter plate.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
30 µL
Duration of treatment / exposure:
60 min

Duration of post-treatment incubation (if applicable):
24h
Number of replicates:
3

Test system

Details on study design:
Test system:
- In vitro test system on three dimensional human epidermis models. The EpiDermTM model consists of normal, human-derived epidermal keratinocytes cultured to form a multi layered, 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 test system is based on the experience that irritant chemicals produce cytotoxicity in human reconstructed epidermis after a 1-hour topical exposure and about 42 hours postincubation.

Material and technical equipment:
- EpiDerm™ 200 kit: MatTek Corp., Bratislava, Slovakia containing 24 Epi-200 tissues (reconstructed epidermis): surface 0.6 cm² cultured in Millicells® diameter 1 cm.

Controls:
- Negative control (NC): 30 μL PBS, sterile
- Positive control (PC): 30 µL 5% (w/v) sodium dodecyl sulfate (SDS, Sigma, Germany) in deionized water, sterile

Experimental procedure:
- Direct MTT reduction:
To assess the ability of the test material to directly reduce MTT a pretest was performed. Thereby, the test substance was added to 0.9 mL of the MTT solution. A negative control (de-ionized water) was tested concurrently. If the MTT solution colour or, in case of water-insoluble test substances the border to the water-phase, turned blue / purple, the test substance was presumed to directly reduce MTT. The direct reduction of MTT by a test substance interferes with the colour density produced by metabolic capacity of the tissue and would falsify the test results.
- Basic procedure:
Three tissues were treated with the test substance, the PC and NC, respectively.
• a volume of 30 μL of the liquid test material was applied with a pipette. A nylon mesh was placed carefully onto the tissue surface afterwards.
• 1 hour after start of application of the test material to the stratum corneum surface of the EpiDermTM tissue, residual test material was removed with sterile PBS and the surface of each tissue was dried. Subsequently, the tissues were incubated at 37°C for 24 ± 2 hours, transferred into new 6-well plates pre-filled with 0.9 mL of fresh medium and thereafter placed into the incubator for additional 18 ± 2 hours post-incubation period.
• After the postincubation period induced tissue destruction (cytotoxicity = loss of viability) was determined by measuring the metabolic activity of the tissue using a colourimetric assay. Thereby, cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity. The mitochondrial dehydrogenase reduces the yellow coloured water-soluble 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to the insoluble blue coloured formazan. After isopropanol extraction of the formazan from the tissues, the optical densitiy of the extract is determined spectrophotometrically. Optical density of the extracts of test substance treated tissues is compared to negative control values and expressed as relative tissue viability.

Acceptance criteria:
In case one of the below given acceptance criteria is not covered, repetition of the test is considered.
- Assay acceptance criterion for the negative control (NC): The absolute OD570 of the negative control tissues in the MTT-test is an indicator of tissue viability obtained in the testing laboratory after the shipping and storing procedure and under specific conditions of the assay. Tissue viability is acceptable if the mean OD570 of the NC is ≥ 1.0. The mean OD570 of the NC should not exceed 2.5.
- Assay acceptance criterion for the positive control (PC): 5% SDS is used as PC and reflects the sensitivity of the tissues used in the test conditions. A viability of ≤ 20% is acceptable.
- Assay acceptance criterion for tissue variability: For every treatment, 3 tissues are treated in parallel. The inter-tissue variability is considered to be acceptable if the SD of %-viability is ≤ 20.

Evaluation criteria:
- Irritant potential of the test materials is predicted from the mean relative tissue viabilities compared to the negative control tissues concurrently treated with sterile PBS. A chemical is considered as "irritant", if the mean relative tissue viability with a test material is less than or equal to 50%

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean of 1st run
Value:
67.1
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean of 2nd run
Value:
74.2
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
The test substance is able to reduce MTT directly. Therefore an additional MTT reduction control KC (freeze-killed control tissues) was introduced.
In a pretest it was demonstrated that the color of the test substance did not interfere with the colorimetric test.

Any other information on results incl. tables

First run (final relative mean viability of KC correction = 67.1%)
tissue 1 tissue 2 tissue 3 mean intertissue variability
negative control viable tissues OD570 1.599 1.907 1.823 1.1776
viability (% of NC) 90 107.3 10.6 100 8.9
KC tissues OD570 0.052 0.058 0.059 0.056
viability (% of NC) 2.9 3.3 3.3 3.2 0.2
test item viable tissues OD570 0.898 1.286 1.395 1.193
viability (% of NC) 50.6 72.4 78.5 67.2 14.7
KC tissues OD570 0.001 0 0.001 0.001
viability (% of NC) 0.038 0 0.066 0.034 0.03
Positive control viable tissues OD570 0.039 0.049 0.047 0.045
viability (% of NC) 2.2 2.7 2.6 2.5 0.3

Second run (final relative mean viability of KC correction = 74.2%)
tissue 1 tissue 2 tissue 3 mean intertissue variability
negative control viable tissues OD570 1.887 1.829 1.82 1.845
viability (% of NC) 102.3 99.1 98.6 100 2
KC tissues OD570 0.047 0.049 0.045 0.047
viability (% of NC) 2.5 2.7 2.4 2.5 0.1
test item viable tissues OD570 0.968 1.343 1.8 1.37
viability (% of NC) 52.5 72.8 97.6 74.3 22.6
KC tissues OD570 0.001 0 0.002 0.001
viability (% of NC) 0.036 0 0.09 0.042 0
Positive control viable tissues OD570 0.052 0.045 0.041 0.046
viability (% of NC) 2.8 2.4 2.2 2.5 0.3

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In-vitro testing showed that the substance is not irritating to skin (OECD 439, GLP).
Executive summary:

The objective was to assess the skin corrosion and irritation potential of the test substance. Using the methods currently available, a single in vitro assay is not sufficient to cover the full range of skin irritating/corrosion potential including transport classification. Therefore, three in vitro assays were proposed for this in vitro skin irritation and corrosion test strategy including transport classification: The Skin Corrosion Test (SCT), Skin Irritation Test (SIT) and In vitro Membrane Barrier Test (Corrositex®). However, in the current case, the results derived with SIT and SCT were sufficient for a final assessment. Therefore, further testing in Corrositex® was waived.

A GLP conform guideline study was conducted.The potential of the test substance to cause dermal corrosion/irritation was assessed by a single topical application of 50μL (corrosion test) or 30μL (irritation test) undiluted test substance to a reconstructed three-dimensional, human epidermis model (EpiDerm™).

For the corrosion test, two EpiDerm™ tissues were incubated with the test substance for 3 minutes and 1 hour each. The irritation test was performed with three EpiDerm™ tissues for each test run, which were incubated with the test substance for 1 hour followed by a 42-hour post-incubation period. Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation by using a colorimetric test. The reduction of mitochondrial dehydrogenase activity measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the epidermal tissues treated with the test substance is compared to that of negative control tissues. The quotient of the values indicates the relative tissue viability.

The following results were obtained in the EpiDerm™ skin corrosion/irritation test:

The test substance is able to reduce MTT directly. Therefore, an additional MTT reduction control KC (freeze-killed control tissues) was introduced.

Corrosion test: The final relative mean viability of the tissues treated with the test substance determined after an exposure period of 3 minutes was 99.0%, and it was 91.4% after an exposure period of 1 hour.

Irritation test: Two test runs of the EpiDermTM skin irritation test were performed. 1st test run: The final relative mean viability of the tissues treated with the test substance determined after an exposure period of 1 hour with an about 42-hour post-incubation was 67.1% (values for individual tissues (without KC correction): 50.6%, 72.4% and 78.5%). Because high inter-tissue variability of the test-substance treated tissues was obtained, a second experiment was performed to verify the result. 2nd test run: The final relative mean viability of the tissues treated with the test substance for the 2nd test run was 74.2% (values for individual tissues (without KC correction): 52.5%, 72.8% and 97.6%). Again, high inter-tissue variability of the test-substance treated tissues was obtained. In addition, the SD of % viability of the test-substance treated tissues was out of the acceptance range.However, since all other quality criteria of the test were met and the viability values of four out of six tissues are well above the cutoff for skin irritation and the viability values of two tissue lie within the borderline range, this deviation is not considered to affect the evaluation of this study adversely.Overall, the obtained results of both test runs did not indicate an irritation potential of the test substance.

Based on the results observed and by applying the evaluation criteria it was concluded that the test substance does not show a skin irritation potential in the EpiDerm™ in vitro skin irritation and corrosion test strategy including transport classification under the test conditions chosen.