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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:
02 September 2016 to 03 September 2016
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)
Deviations:
no
Qualifier:
according to
Guideline:
other: Method B.40bis of Commission Regulation (EC) No 440/2008, of 30 May 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Appearance/physical state: Light brown, slightly viscous, liquid
- Storage conditions: Room temperature in the dark

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
foreskin from a single donor
Source strain:
other: neonatal
Vehicle:
unchanged (no vehicle)
Details on test system:
PURPOSE OF THE TEST
- The purpose of this test is to evaluate the corrosivity potential of the test item using the EpiDerm Human Skin Model after treatment periods of 3 and 60 minutes.
- Corrosion is directly related to cytotoxicity in the EpiDerm tissue. Cytotoxicity is determined by the reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to formazan by viable cells in the test item treated tissues relative to the corresponding negative control. The results are used to make a prediction of the corrosivity potential of the test item.
- This model incorporates several features, which make it advantageous in the study of potential dermal corrosivity. The target cells are epithelial, derived from human skin, and formed into a stratified, cornified epithelium. Test items are applied to the culture surface, at the air interface, so that undiluted and/or end use dilutions can be tested directly.

PREPARATION OF NEGATIVE AND POSITIVE CONTROL ITEMS AND MTT
- The negative control item was used as supplied.
- The positive control item was used as supplied.
- A 1.0 mg/mL MTT solution was prepared from a MatTek MTT-100 kit immediately prior to use.

EPIDERM RECONSTRUCTED HUMAN EPIDERMIS MODEL KIT
- Supplier: MatTek
- Date received: 01 September 2016
- EpiDerm tissues (0.63 cm2) lot number: 23352
- Assay medium lot number: 082516ZSC
- Upon receipt of the Epiderm tissues, the sealed 24-well plate was stored in a refrigerator until use.

MTT DYE METABOLISM, CELL VIABILITY ASSAY
- The MTT assay, a colorimetric method of determining cell viability, is based on reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan dye by mitochondrial succinate dehydrogenase in viable cells.
- One limitation of the assay is possible interference of the test item with MTT. A test item may directly reduce MTT, thus mimicking dehydrogenase activity of the cellular mitochondria. This property of the test item is only a problem if at the time of the MTT test (after rinsing) there is still a sufficient amount of the test item present on or in the tissues. In this case, the true metabolic MTT reduction and the false direct MTT reduction can be differentiated and quantified.
- As specified, a test item 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 item was checked for the ability to directly reduce MTT according to the procedure below.
- Test item (50 µL) 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 item turned blue/purple relative to the control, the test item was presumed to have reduced the MTT.

ASSESSMENT OF COLOUR INTERFERENCE WITH THE MTT ENDPOINT
- A test item may interfere with the MTT endpoint if it is coloured. The MTT assay is affected only if the test item is present in the tissues when the MTT viability assay is performed.
- Test item (50 µL) 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.

PRE-INCUBATION
- The assay medium was pre-warmed before use in the main test.
- Assay medium (0.9 mL) 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.

APPLICATION OF TEST ITEM AND RINSING
- 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. Test item (50 µL) and 50 μL of 8.0 N potassium hydroxide (positive control) were also applied to the corresponding tissues in turn. Sterile water (25 μL) was added for wetting of the test item 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. Because the exposure time was so short, the tissues were dosed at regular intervals to ensure that each tissue received an equal exposure time and to allow for the time taken to rinse each tissue following exposure. Rinsing was achieved by filling and emptying each tissue under a constant soft stream of DPBS to gently remove any residual test item. 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. They 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.

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.
- Aliquots (3 x 200 µL) of the extract were transferred to the appropriate wells of a pre-labelled 96-well plate.
- Isopropanol (200 µL) alone was added to the three wells designated as blanks.
- Absorbency at 562nm (OD562) of each well was measured using the Anthos 2001 microplate reader.

QUANTITATIVE MTT ASSESSMENT (PERCENTAGE TISSUE VIABILITY)
- The corrosivity potential of the test item 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.
- The relative mean viabilities were calculated using the equation relative mean viability (%) = (mean OD562 of test item / mean OD562 of negative control) x 100.

QUALITY CRITERIA
- The results of the assay are considered acceptable if the following assay acceptance criteria are achieved:
(i) Negative control: The absolute OD562 of the negative control treated 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. The mean OD562 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.
(ii) Positive control: Potassium hydroxide 8.0 N solution is used as a positive control. An assay meets the acceptance criterion if mean relative tissue viability of the 60 minute positive control is < 15%.
(iii) Coefficient of variation: In the range 20 and 100% viability, the Coefficient of Variation between tissue replicates should be ≤ 30%.

MAJOR COMPUTERISED SYSTEMS
- Delta Building Monitoring System
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
50 µL
Duration of treatment / exposure:
3 minutes and 60 minutes
Duration of post-treatment incubation (if applicable):
3 hours
Number of replicates:
Two

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3-minute exposure
Value:
97.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: mean relative viability (% of negative control)
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60-minute exposure
Value:
95.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: mean relative viability (% of negative control)
Other effects / acceptance of results:
DIRECT MTT REDUCTION
- The MTT solution containing the test item did not turn blue/purple. This was taken to indicate the test item did not reduce MTT.

ASSESSMENT OF COLOUR INTERFERENCE WITH THE MTT ENDPOINT
- The solution containing the test item did not become coloured.
- This was taken to indicate the test item did not have the potential to cause colour interference.

TEST ITEM, POSITIVE CONTROL ITEM AND NEGATIVE CONTROL ITEM
- Mean OD562 values and viabilities for the negative control, positive control and test item are given in Appendix 1 (attached).
- The relative mean viabilities for each treatment group are shown in the table below.

QUALITY CRITERIA
- The mean OD562 for the negative control treated tissues was 1.557 for the 3-Minute exposure period and 1.503 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 4.1 % 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

RELATIVE MEAN VIABILITIES FOR EACH TREATMENT GROUP

Exposure period

Percentage viability negative control*

Percentage viability positive control

Percentage viability test item

3 minute

100

4.1

97.7

60 minutes

100

4.1

95.2

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

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The relative mean viability of the test item treated tissues was 97.7 % after 3 minutes exposure and 95.2 % after 60 minutes exposure.
Executive summary:

GUIDELINE

The study was performed in compliance with the OECD Guideline for the Testing of Chemicals No 431 In Vitro Skin Corrosion: Reconstructed Human EpiDermis (RHE) Test Method (28 July 2015) and Method B.40bis of Commission Regulation (EC) No 440/2008 of 30 May 2008, laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH). The purpose of the test was to evaluate the corrosivity potential of the test item using the EpiDerm Human Skin Model after treatment periods of 3 and 60 minutes. Corrosion is directly related to cytotoxicity in the EpiDerm tissue and cytotoxicity is determined by reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) to formazan by viable cells in the test item-treated tissues relative to the corresponding negative control. The results are used to make a prediction of the corrosivity potential of the test item.

 

METHODS

Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes. Negative and positive control groups were treated for each exposure period. At the end of the exposure period the test item 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 mL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density (OD) was measured at 562 nm (OD562). Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

 

RESULTS

Mean viability of the negative control tissues was set at 100 % and quality criteria for acceptance of results were satisfied. Relative mean viabilities after 3 minutes exposure were determined to be 100 % (negative control), 4.1 % (positive control) and 97.7 % (test item). Relative mean viabilities after 60 minutes exposure were determined to be 100 % (negative control), 4.1 % (positive control) and 95.2 % (test item).

 

CONCLUSION

The relative mean viability of the test item treated tissues was 97.7 % after 3 minutes exposure and 95.2 % after 60 minutes exposure.