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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:
key study
Study period:
04 July 2017 to 06 July 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
2015
Deviations:
no
Qualifier:
according to
Guideline:
other: Method B.40bis of Commission Regulation (EC) No 440/2008
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Appearance: Fine, dark grey powder

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
Recommended test system in international guidelines
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: MatTek: EpiDerm™ Reconstructed Human Epidermis Model Kit
- Tissue lot number: 25828
- Delivery date: 04 July 2017
- Date of initiation of testing: 04 July 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C
- Temperature of post-treatment incubation (if applicable): Room temperature for MTT extraction

TEST FOR DIRECT MTT REDUCTION
- A test material may interfere with the MTT endpoint if it was able to directly reduce MTT and at the same time was present on or in the tissues when the MTT viability test was performed. To identify this possible interference, the test material was checked for the ability to directly reduce MTT: 25 mg of the test material was added to 1 mL of a freshly prepared 1.0 mg/mL MTT solution. The solution was incubated in the dark at 37°C, 5% CO2 in air for 60 minutes. Untreated MTT solution was tested concurrently to act as a control. If the MTT solution containing the test material turns blue/purple relative to the control, the test material was presumed to have reduced the MTT.

ASSESSMENT OF COLOUR INTERFERENCE WITH THE MTT ENDPOINT
- 25 mg of test material was added to 300 µL of sterile water. The solution was incubated in the dark at 37°C, 5% CO2 in air for 60 minutes. A visual assessment of the colour was then made. The test material was found to produce a coloured solution which may interfere with the MTT endpoint. Therefore, colour correction tissues were incorporated into the test to correct for this possibility. These tissues were treated identically to the tissues of the main test with the exception of being placed into assay medium for three hours post-exposure instead of MTT. Two tissues were dosed with the test material and two tissues were dosed with the negative control for each exposure period.

Main test
- The assay medium was pre-warmed before use. 0.9 mL of this assay medium was pipetted into the appropriate wells of two pre-labelled 6-well plates for both the 3 minute and 60 minute exposure periods. EpiDerm™ tissues were transferred into the 6 well plates containing the assay medium. The 6 well plates containing the EpiDerm™ samples were pre-incubated (37°C, 5% CO2) for approximately 1 hour before dosing.
- Before pre-incubation was complete, a 24 well plate was prepared for use as a “holding plate” for both the 3 minute and 60 minute exposure periods. This plate was used to maintain the viability of the tissue inserts between rinsing following chemical exposure and MTT loading. Another 24 well plate was prepared for the MTT loading. 300 µL of either pre warmed assay medium (holding plate) or MTT medium (MTT loading plate) was dispensed into each well. The two plates were placed into the incubator until required.
- After pre incubation of the EpiDerm™ tissues, the medium was aspirated and replaced with 0.9 mL of fresh assay medium. The 6-well plate for the 3 minute exposure period was returned to the incubator, while the other was being dosed for the 60 minute exposure. For the 60 minute exposure period, 50 µL of sterile distilled water (negative control) was added to the first two tissues. The tissues were dosed at regular intervals to allow for the time taken to rinse each tissue following exposure and to ensure that each tissue gets an equal exposure time. 25 mg of the test material and 50 µL of 8.0 N Potassium hydroxide (positive control) were also applied to the corresponding tissues in turn. 25 µL of sterile water was added for wetting of the test material to increase tissue surface contact. The plate was returned to the incubator (37°C, 5% CO2) for the 60 minute exposure period. When dosing for the 60 minute exposure period was complete, the same procedure was repeated for the 3 minute exposure period.

REMOVAL OF TEST MATERIAL AND CONTROLS
- Rinsing was achieved by filling and emptying each tissue under a constant soft stream of DPBS to gently remove any residual test material. Excess DPBS was removed by blotting the bottom of the tissue insert with tissue paper. Each tissue was placed into the prepared holding plate until all tissues were rinsed.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- After rinsing plates were then blotted and transferred to the 24 well plate prepared for MTT loading. The plate was incubated (37 °C, 5 % CO2) for 3 hours. Once the 60 Minute exposure period was complete, the same rinsing and MTT loading procedure was repeated. After the 3 hour MTT incubation was complete, the inserts were blotted and transferred to labelled 24 well plates for MTT extraction. 2 mL of MTT extractant (isopropanol) was used to completely immerse each insert and the plate was covered with plate sealer to prevent Isopropanol evaporation. The plates stood overnight at room temperature, to allow extraction to proceed.

NUMBER OF REPLICATE TISSUES: 2

ABSORBANCE/ OPTICAL DENSITY MEASUREMENTS
- After extraction, each tissue was pierced with a pipette fitted with a 1000 µL tip and the extraction solution was forced vigorously up and down to form a homogenous solution. 3 x 200 µL aliquots of the extract were transferred to the appropriate wells of a pre labelled 96 well plate. 200 µL of isopropanol alone was added to the three wells designated as blanks. Absorbency at 570 nm (OD570) of each well was measured using the Labtech LT 4500 microplate reader.

QUANTITATIVE MTT ASSESSMENT (% TISSUE VIABILITY)
- The corrosivity potential of the test material was predicted from the relative mean tissue viabilities obtained after the 3 and 60 minute exposure periods, compared to the mean of the negative control tissues (n=2) treated with sterile distilled water. Relative mean viability (%) = (mean OD570 of the test material/ mean OD570 of negative control) x 100
- Classification of corrosivity potential is based on relative % viabilities for both exposure times according to the following relative mean tissue viability (% of negative control):
3 min: < 50%: H314 Category 1A
3 min: ≥ 50%, 1 hour: < 15%: H314 Category 1B or 1C
3 min: ≥ 50%, 1 hour: ≥ 15: Not classified for corrosivity.

QUALITY CRITERIA
- Negative Control: The mean OD570 of the two negative control tissues should be ≥ 0.8 and ≤ 2.8 for each exposure time, which ensures that the tissue viability meets the acceptance criteria.
- Positive Control: An assay meets the acceptance criterion if mean relative tissue viability of the 60 minute positive control is < 15%.
- Coefficient of Variation: In the range 20 and 100 % viability, the Coefficient of Variation between tissue replicates should be ≤ 30%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied: 25 mg

NEGATIVE CONTROL
- Amount(s) applied: 50 µL

POSITIVE CONTROL
- Amount(s) applied: 50 µL
- Concentration: 8.0 N
Duration of treatment / exposure:
3 minutes of exposure and 1 hour of exposure
Duration of post-treatment incubation (if applicable):
Incubated for 3 hours with MTT
Number of replicates:
2 per exposure time

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 minute exposure
Value:
111.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 minute exposure
Value:
96.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- Direct MTT Reduction: The MTT solution containing the test material did not turn blue/purple. This was taken to indicate the test material did not reduce MTT.
- Assessment of Colour Interference with the MTT endpoint: The solution containing the test material was a black to dark grey colour, therefore additional colour correction tissues were incorporated into the testing procedure. However, the results obtained showed that no colour interference occurred. It was therefore considered unnecessary to use the results of the colour correction tissues for quantitative correction of results or for reporting purposes.
- Test Material, Positive Control Material and Negative Control Material: Results are summarised in Table 1.

- Quality Criteria:
The mean OD570 for the negative control treated tissues was 1.596 for the 3 minute exposure period and 1.812 for the 60 minute exposure period. The negative control acceptance criteria were therefore satisfied.
The relative mean tissue viability for the positive control treated tissues was 3.3% relative to the negative control following the 60 minute exposure period. The positive control acceptance criterion was therefore satisfied.
In the range 20 to 100% viability the Coefficient of Variation between the two tissue replicates of each treatment group did not exceed 30%. The acceptance criterion was therefore satisfied.

Any other information on results incl. tables

Table 1: Summary of results

Exposure period

Percentage viability

Negative control

Positive control

Test material

3 minutes

100*

4.7

111.7

60 minutes

100*

3.3

96.2

*The mean viability of the negative control tissues is set at 100%

Applicant's summary and conclusion

Interpretation of results:
other: Not corrosive in accordance with EU criteria
Conclusions:
Under the conditions of this study, the test material is not corrosive in the in vitro skin corrosion test.
Executive summary:

An in vitro skin corrosion test was carried out with the test material using a human skin model in accordance with the standardised guidelines OECD 431 and EU Method B.40 BIS under GLP conditions.

During the study, duplicate tissues were treated with the test material for exposure periods of 3 and 60 minutes. Negative and positive control groups were treated for each exposure period. The test material was found to have the potential to cause colour interference with the MTT end-point therefore additional tissues were incorporated into the testing to correct for this. At the end of the exposure period the test material was rinsed from each tissue before each tissue was taken for MTT loading. After MTT loading each tissue was placed in 2 mL isopropanol for MTT extraction.

At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 µL samples were transferred to the appropriate wells of a pre-labelled 96 well plate. The optical density (OD) was measured at 570 nm (OD570).

Under the conditions of the study the percentatge viability of the tissues was 111.7 at 3 minutes and 96.2 after 60 minutes. The quality criteria required for acceptance of results in the test were satisfied.

The test material was therefore not considered to be corrosive to the skin, in vitro.