[No public or meaningful name is available]

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From January 24 to February 15, 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

2016

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

[(2-hydroxyacetyl)oxy](oxo) vanadium 2-hydroxyacetate]

IUPAC Name:

[(2-hydroxyacetyl)oxy](oxo) vanadium 2-hydroxyacetate]

Test material form:

solid

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: adult donors

Source strain:

other: 10 KERA-003 + 10-KERA-004

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKINTM(SM)
- Tissue batch number(s): Batch No.: 19-EKIN-004 in Experiment I and Batch No.: 19-EKIN-007 in Experiment II
- Expiry date: 28 January 2019 for batch used in Experiment I and 18 February 2019 fro batch used in Experiment II
- Date of initiation of testing: 24 January 2019 for Experiment I and 14 February 2019 for Experiment II

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature (24.2-24.9°C in Experiment I and 23.2-24.6°C in Experiment II)
- Temperature of post-treatment incubation (if applicable): after the incubation period, MTT solution was added to each well and the plate incubated at 37°C for 3 hours.

REMOVAL OF TEST MATERIAL AND CONTROLS
- Washing steps: after the incubation time, all test item treated tissues or also the positive control tissues were removed and rinsed thoroughly with PBS solution.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Thermo Fisher Scientific, Catalogue Number: 24072800, Serial Number: 0920-14
- Wavelength: 570 nm


NUMBER OF REPLICATE TISSUES: In Experiment I two replicates and in the Experiment II five replicates were used for test item treatment. Two negative controls and two positive controls were also run in each experiment. Furthermore, as the test item was coloured, two additional test item-treated living tissues were used for the non-specific OD evaluation in Experiment I. As the test item also had an MTT interacting potential, two additional test item-treated killed epidermis and two negative control treated killed epidermis were used in each experiment. To avoid a possible double correction for colour interference, for nonspecific colour in killed tissues, two additional disks were used in Experiment I. No colour controls were used in the Experiment II, as correction was not necessary based on the results of the first experiment.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- killed tissues
- Procedure used to prepare the killed tissues (if applicable): living epidermis units were placed in a 12 well plate with 2 mL of distilled water, then incubated at 37°C in an incubator with 5 % CO2, in a >95% humidified atmosphere for 48 hours (±1 hour). At the end of the incubation the water was discarded and the epidermis units were frozen.
- N. of replicates : 2 for test item and 2 for negative control. Furthermore, to avoid a possible double correction for colour interference, two additional control killed tissue samples were used in Experiment I.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 2 experiments to derive final prediction

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the viability after 3 minutes is less than 35 % (Sub-category 1A) or ≥ 35 % after 3 minutes and < 35 % after 60 minutes or ≥ 35 % after 60 minutes and less than 35 % after 240 minutes (Sub-categories 1B and 1C).
- The test substance is considered to be non-corrosive to skin if the viability is ≥ 35 % after 240 minutes.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 20 mg

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL of physiological saline

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL of glacial acetic acid

Duration of treatment / exposure:

4 hours

Number of replicates:

In Experiment I two replicates and in the Experiment II five replicates were used for test item treatment. Two negative controls and two positive controls were also run in each experiment. Furthermore, as the test item was coloured, two additional test item-treated living tissues were used for the non-specific OD evaluation in Experiment I. As the test item also had an MTT interacting potential, two additional test item-treated killed epidermis and two negative control treated killed epidermis were used in each experiment. To avoid a possible double correction for colour interference, for nonspecific colour in killed tissues, two additional disks were used in Experiment I. No colour controls were used in the Experiment II, as correction was not necessary based on the results of the first experiment.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

37

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Mean of two replicates

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

2

Value:

57.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Mean of two replicates

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct-MTT reduction: mean optical density (measured at 570 nm) of the two killed tissues was determined as 0.012, Non Specific Colour % (NSCkilled%) was calculated as 1.5%
- Colour interference with MTT: mean OD difference (0.277), the calculated NSMTT% is 33.8% in Experiment I and mean OD difference (0.198), the calculated NSMTT% is 29.5% in Experiment II

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The proficiency verification with 12 reference chemicals, in the in vitro skin corrosivity test, in the EPISKIN model, used in OECD No. 431 (2013) demonstrated that the laboratory is fully proficient to perform this study type. (The validity test was performed by Citoxlab Hungary Ltd., study code: 13/270-039B.).

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD value of the two negative control tissues was in the recommended range (0.820 in Experiment I and 0.668 in Experiment II).
- Acceptance criteria met for positive control: The two positive control treated tissues showed 1.2% in Experiment I and 0.8 in Experiment II viability demonstrating the proper performance of the assay.
- Acceptance criteria met for variability between replicate measurements: The difference of viability between the two test item-treated tissue samples in the MTT assay was 28.7% in Experiment I and the standard deviation of viability values of the five test item-treated tissue samples in the MTT assay was 7.4% in Experiment II.
The difference of viability between the two negative control tissue samples in the MTT assay was 4.0 % in Experiment I and 2.8% in Experiment II.
The mean OD value of the blank samples (acidified isopropanol) was 0.047 and 0.048.

Applicant's summary and conclusion

Interpretation of results:

other: Non corrosive

Remarks:

Based on the criteria set up on the OECD guideline 431

Conclusions:

Non corrosive

Executive summary:

Method

An in vitro skin corrosivity test was performed with the test item in a reconstructed human epidermis model. EPISKINTM(SM) is designed to predict and classify the corrosive potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The corrosivity of the test item was evaluated according to the OECD No. 431 guideline (2016).

In each experiment, disks of EPISKINTM(SM) (at least two units) were treated with powdered test item and incubated for 4 hours at room temperature. Exposure of test material was terminated by rinsing with Phosphate Buffered Saline solution. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

In this study two experiments were performed for the scientifically valid data. Two test item treated skin units, two NSCliving and NSCkilled control tissues and two additional controls on killed epidermis (two test item treated and two negative control treated skin units) were used in Experiment I. In Experiment II five test item treated skin units and additional controls on killed epidermis were used, but no colour control disks were used (as they were not necessary based on the results of the first experiment).

Physiological saline (0.9% (w/v) NaCl solution) and glacial acetic acid treated epidermis were used as negative and positive controls, respectively (two units / control). Two additional disks were used to provide an estimate of colour contribution (NSCliving%) from the test item. The possible MTT interaction potential of the test item was examined using two additional test item treated and two negative control treated killed epidermis units. Furthermore, to avoid a possible double correction for colour interference, a third set of controls for non-specific colour in killed tissues (NSCkilled), two additional disks were used. For each treated tissue viability was expressed as a % relative to the negative control. If the mean relative viability after 4 hours of exposure is below 35% of the negative control, the test item is considered to be corrosive to skin.

Results

In the Experiment I the individual viability values of the two replicates were 42.3% (indicating a non-corrosive result) and 31.7% (indicating a corrosive result) compared to the negative control value. Therefore, an additional experiment (Experiment II) was performed using the same experimental conditions (but using increased number of replicates) to clarify the result and to provide proper information for prediction. In the Experiment II the mean cell viability of the test item treated skin samples showed a negative result.

Following exposure with the test item, the mean cell viability was 37.0% in Experiment I and 57.9% in Experiment II compared to the negative control (after adjustment for non-specific MTT reduction). Mean value of all viability data (51.9%) was above the threshold limit of 35%, therefore the test item was considered as being non-corrosive. The experiments met the validity criteria, therefore the study was considered to be valid.

Conclusion

In conclusion, in this in vitro EPISKIN™(SM) model test with the test item, the results indicate that the test item is non-corrosive to the skin.