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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:
09 May 2017 to 08 June 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:
2016
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
Version / remarks:
2008
Deviations:
no
Qualifier:
according to
Guideline:
other: MatTek test protocol “In vitro EpiDermTM Skin Corrosion Test (EPI-200-SCT)”
Version / remarks:
2014
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: Solid, crystalline, colourless 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:
water
Remarks:
Deionised
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Epi-200 Kit
- Lot No.: 25817
- Delivery date: 06 June 2017.
- Date of initiation of testing: On day of receipt

TEST FOR DIRECT MTT REDUCTION AND COLOUR INTERFERENCE
A test material may interfere with the MTT endpoint if: a) it is coloured and/or b) able to directly reduce MTT. The MTT assay is affected only if the test material is present in the tissues when the MTT viability test is performed. Some non-coloured test materials may change into coloured test materials in wet or aqueous conditions and thus stain tissues during the 60 min exposure. Therefore, before exposure, a functional check for this possibility should be performed (step 1).
- Step 1: 25 ± 2 mg of the test material was added to 0.3 mL of deionised water (transparent glass test-tube). The mixture was incubated (37 ± 1.5°C, 5 ± 0.5% CO2) for 60 min. At the end of the exposure time, the mixture was shaken and the presence and intensity of the staining (if any) was evaluated. If the solution changed colour significantly, the test material is presumed to have the potential to stain the tissue. An additional test on viable tissues (without MTT addition) should be performed (step 2).
Since the test material did not dye water relevantly (yellow) when mixed with it, step 2 did not have to be performed.
- Step 3: All test materials (including those already evaluated in step 1 and step 2 should be further evaluated for their potential to interfere with MTT. To test if a material directly reduces MTT, 25 ± 2 mg of the test material was added to 1 mL of a MTT/DMEM solution (1 mg/mL) and was incubated (37 ± 1.5°C, 5 ± 0.5% CO2) for 60 minutes. Untreated MTT/DMEM solution (1 mg/mL) medium was used as control. If the MTT/DMEM solution (1 mg/mL) turns blue/purple, the test material reduces MTT and an additional test on freeze-killed tissues (step 4) must be performed.
Since the test material did not prove to be a MTT reducer, step 4 did not have to be performed.

PRE-WARMING OF EPIDERM TISSUES
18 to 19 hours before dosing, EpiDerm tissues were unpacked, and the inserts were transferred into 6-well plates containing the pre-warmed assay medium under sterile conditions using sterile forceps. A 24-well plate was prepared as holding plate containing 300 μL assay medium. The holding plate was pre warmed in an incubator (37 ± 1.5°C, 5 ± 0.5% CO2) until use.

TREATMENT
Duplicate EpiDerm tissues were treated with the test material, positive control or negative control for exposure times of 3 ± 0.5 minutes and 60 ± 5 minutes. After the pre-incubation of the EpiDermTM tissues was completed the DMEM-based medium in each well was replaced with 0.9 mL fresh assay medium. The 6-well plates for the 3 ± 0.5 minutes exposure periods stayed at room temperature in the sterile bench, the 6-well plates for the 60 ± 5 minutes exposure period were placed into an incubator (37 ± 1.5°C, 5 ± 0.5% CO2).

REMOVAL OF TEST MATERIAL AND CONTROLS
- At the end of the exposure period the tissues were removed from the 6-well plate and gently rinsed with DPBS to remove any residual test material (20 times). Excess DPBS was removed by gently shaking the tissue insert and blotting the lower surface with blotting paper. The tissues were placed in the prepared holding plate until all tissues were rinsed.

MTT ASSAY
Two 24-well plates were prepared prior to the end of the tissue pre-warming period. MTT solution (300 μL) was added to each well and the plates were kept in an incubator (37 ± 1.5°C, 5 ± 0.5% CO2) until required. Following rinsing, the tissues were transferred from the holding plates to the MTT-plates. After a 3 hour incubation period (37 ± 1.5°C, 5 ± 0.5% CO2) the tissues were rinsed three times with DPBS and carefully dried with blotting paper. The inserts were transferred into new 24-well plates. The tissues were each immersed in 2 mL of extractant solution (isopropanol) pipetted in each well ensuring that the tissues were completely covered. The 24-well plates were sealed to minimise isopropanol evaporation. The formazan salt was extracted for nearly 20 hours without shaking at room temperature.
After the extraction period the inserts were pierced with an injection needle to allow the extract to run into the well from which the insert was taken, and the insert was discarded. 24-well plates were then placed on a shaker for 15 minutes until the solution was homogeneous in colour. 3 × 200 μL aliquots of the blue formazan solution were transferred from each tissue into a 96-well flat bottom microtiter plate. The OD was determined in a microplate reader (Versamax®, Molecular Devices, SoftMax Pro Enterprise (version 4.7.1)) at 570 nm (OD570) without reference filter. The mean values were calculated for each set of 3 wells per tissue.

NUMBER OF REPLICATE TISSUES: 2
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied: 25 ± 2 mg (39.7 mg/cm²) of test material, applied with 25 μL of deionised water

NEGATIVE CONTROL
- Amount(s) applied: 50 µL

POSITIVE CONTROL
- Amount(s) applied: 50 µL
Duration of treatment / exposure:
3 ± 0.5 minutes and 60 ± 5 minutes
Duration of post-treatment incubation (if applicable):
3 hours with MTT
Number of replicates:
Duplicate plates were treated for the test material, positive control and negative control.

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 minutes exposure
Value:
97
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 minutes exposure
Value:
104.6
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Other effects / acceptance of results:
MTT INTERFERENCE POTENTIAL
- The optical pre-experiment (colour interference pre-experiment) to investigate the test material’s colour change potential in water did not lead to a relevant change in colour.
- Optical evaluation of the MTT-reducing capacity of the test material after 1 hour incubation with MTT-reagent did not show blue colour.

MAIN TEST
The test material is considered to be non-corrosive to skin since the viability after 3 minutes exposure is greater than 50% and the viability after 1 hour exposure is greater than 15%. Results can be seen in Table 1.

ACCEPTABILITY OF THE TEST
The acceptance criteria are met:
- the mean OD of the tissue replicates treated with the negative control is ≥ 0.8 and ≤ 2.8 for every exposure time (range: 1.455 to 1.575)
- the mean viability of the tissue replicates treated with the positive control for 1 hour, is <15% compared to the negative control (3.2%)
- the Coefficient of Variation (CV) in the range 20 to 100% viability between tissue replicates is ≤ 30% (range: 0.2 to 3.5%)

Any other information on results incl. tables

Table 1: Results of main study after treatment with test material and positive control

Treatment

3 minute relative absorbance

(% of negative control)

60 minute relative absorbance

(% of negative control)

Test material

97.0

104.6

Positive control

22.6

3.2

Negative control

100.0

100.0

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:

The corrosivity potential of the test material to skin was investigated in accordance with the standardised guidelines OECD 431 and EU Method B40 under GLP conditions, by means of the Human Skin Model Test with an EpiDerm™ tissue model.

The test material did not reduce MTT (pre-test for direct MTT reduction), and it did not dye deionised water significantly (yellow), when mixed it (pre-test for colour interference). Therefore, additional tests with freeze-killed or viable tissues (without MTT addition) to determine correction factors for calculating the true viability in the main experiment were not necessary.

Independent duplicate tissues of EpiDerm™ were exposed to the test material, the negative control (deionised water) or the positive control (8.0 N KOH) for 3 minutes and 1 hour, respectively. Afterwards, the test and the control materials were rinsed off the tissues, and a 3 hour incubation period (37 ± 1°C, 5 ± 0.5% CO2) with MTT solution followed. MTT solution was then aspirated from the wells and the wells were rinsed with DPBS. Inserts were transferred into new 24 well plates. The formazan salt was extracted for nearly 20 hours at room temperature.

After exposure to the negative control the absorbance values met the required acceptability criterion of a mean OD570 ≥ 0.8 and ≤ 2.8 for both treatment intervals thereby confirming the acceptable quality of the tissues.Exposure to the positive control induced a decrease in the relative absorbance as compared to the negative control, both for the 3 minutes exposure period (22.6%) and for the 1 hour exposure period (3.2%) thus confirming the validity of the test system and the specific batch of tissue models.

After exposure to the test material the relative absorbance value decreased slightly to 97.0% after 3 minutes exposure. After 1 hour exposure the relative absorbance value was not reduced (104.6%). Both values did not fall below the threshold for corrosivity which is defined to be 50% after the 3 minutes exposure and 15% after the 1 hour exposure. Therefore, under the conditions of this study, the test material is not corrosive in the in vitro skin corrosion test.