Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018
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 guideline
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
28 July 2015
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Physical state/ Appearance:liquid/yellowish, clear
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: 0016044582
- Content: 91.4 /100 g (100 g/100 g minus water content)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: The stability under storage conditions over the study period was guaranteed by the sponsor.
- Solubility and stability of the test substance in the solvent/vehicle: No analysis of test substance preparation was performed because the test substance was applied minimally moistened with deionized water or PBS.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
pH value: Ca. 6 (undiluted test substance moistened with deionized water, determined in the lab prior to start of the GLP study)
Physical state / color: Liquid, high viscous to solid, waxy / colorless to light yellowish to white
Handling of the test substance: The solid test substance was grinded with pestle and mortar before application.
Storage conditions: Room temperature

In vitro test system

Test system:
human skin model
Remarks:
EpiDerm model (EPI-200)
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: MatTek, Bratislava, Slovakia; Unspecified
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm
- Tissue batch number: 25837
- Date of certificate of analysis from MatTek: 16 August 2017
- Date of initiation of testing: 15 August 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature for 25 minutes overall and 37°C for 35 minutes (Incubator)
- Temperature of post-treatment incubation: 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
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.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL MTT
- Incubation time: 3 hours
- Spectrophotometer: Sunrise Absorbance Reader
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The supplier demonstrates that each batch of the model used passes a MTT QC assay (4 hours, n=3, OD= 540-570nm).
- Reproducibility: given by historical data from Jan 2016 - Jul 2017 via given test method
- Barrier function and Quality control: The supplier demonstrates that each batch of the model used meets the defined production release criteria. MatTek determines the ET50 value following exposure to Triton X-100 (1%) for each EpiDermTM batch. The ET50 must fall within a range established based on a historical database of results.
Lower acceptance limit: ET50 = 4.0 hours
Upper acceptance limit: ET50 = 8.7 hours
- Sterility: The supplier demonstrates that each batch of the model used shows no contamination.

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- EPI-200 tissue that is killed by freezing at –20°C
- N. of replicates: 3
- Method of calculation used: In case of direct MTT reduction 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 tissues treated with the test substance (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 negative control 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: 1

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- Justification for the selection of the cut-off points if different than recommended in TG 431 and 439:

Mean tissue viability (% of negative control):
Step 1: Identification of corrosives
< 45% after 3 min exposure -> Corrosive
> 45% after 3 min exposure and < 10% after 1 h exposure -> Corrosive
45 – 55% after 3 min exposure -> Borderline (inconclusive)
> 55% after 3 min exposure and 10 – 20% after 1 h exposure -> Borderline (inconclusive)
> 55% after 3 min exposure and > 20% after 1 h exposure -> Non-corrosive

Step 2: Optional UN GHS subcategorization of corrosives identified in step 1
< 20% after 3 min exposure -> UN GHS Cat 1A
20 – 30% after 3 min exposure -> Borderline (inconclusive) for UN GHS subcategorization
> 30% after 3 min exposure -> UN GHS Cat 1B or 1C

Mean tissue viability (% of negative control):
< 45% Irritant
45 - 55% Borderline3
> 55% Non-irritant

The “borderline“ evaluation (50 ± 5%, 25 ± 5% and 15 ± 5%) was statistically determined by using historic BASF data and hence considers the variance of the test method. This evaluation is an amendment to the evaluation provided in OECD Guideline 431 and 439.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: bulk volume of ca. 25 µL ground test material (about 48 mg)

NEGATIVE CONTROL
- Amount applied: 30 µL

POSITIVE CONTROL
- Amount applied: 30 µL
- Concentration: 5% (w/v) SDS
Duration of treatment / exposure:
- under laminar flow hood at room temperature for 25 minutes overall and for 35 minutes in the incubator (37°C)
Duration of post-treatment incubation (if applicable):
Tissues were post-incubated for 24 ± 2 hours at 37°.
Number of replicates:
3

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Remarks:
Final mean viability of tissues after killed control (for direct MTT-reduction) correction as % of negative control.
Run / experiment:
Test substance treatment.
Value:
85.3
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- The test substance is not able to directly reduce MTT.

DEMONSTRATION OF TECHNICAL PROFICIENCY: The laboratory has demonstrated proficiency.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: 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 negative comtrol is >= 0.8. The mean OD570 of the negative control should not exceed 2.8.
- Acceptance criteria met for positive control: 5% SDS is used as positive control and reflects the sensitivity of the tissues used in the test conditions. A viability of <= 20% is acceptable.
- Acceptance criteria met for variability between replicate measurements: For every treatment, three tissues are treated in parallel. The inter-tissue variability is considered to be acceptable if the SD of % viability is <= 18%.
- Acceptance criteria for the killed controls (KC): The OD570 of the tissues for the KC of the negative control should be <= 0.35.
The OD570 value for direct MTT reduction of a test substance should be <= 30% of the OD570 of the negative control.

In total four test runs were performed. However the third test run is considered invalid and is
not reported.
1st test run:
The final 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 54.0%. However, due to the non-concordant replicate measurements of the test-substance treated tissues obtained (relative viability values for individual values: 76.5%, 75.9% and 9.4%), a 2nd test run was performed to clarify the result.
2nd test run:
The final mean viability of the tissues treated with the test substance was 68.0%. However, due to the non-concordant replicate measurements of the test-substance treated tissues obtained in the 2nd test run (relative viability values for single values: 77.1%, 64.3% and 40.8%), a 3rd test run was performed to clarify the result.
4th test run:
Due to technical issues at removal of the test substance during the 3rd run of the skin irritation test the test substance was applied without a metal pin in the 4th test run. A bulk volume of ca. 25 μL of the undiluted test material was formed to a flat “disc like piece” of ca. 8 mm diameter and placed atop the tissues.

The final mean viability of the tissues treated with the test substance for the 4th test run was 85.3% (relative viability values for single tissues: 96.2%, 76.7% and 83.2%). All acceptance criteria were met. Overall, the viability values of the most tissues (valid test runs) and all tissues in the 4th run are well above the cut off for skin irritation, thus it was concluded that the test substance does not indicate an irritation potential.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met