[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:

13 July 2016 to 15 July 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EU Method B40 bis (In Vitro Skin Corrosion Human Skin Model Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Test material form:

liquid

Details on test material:

- Appearance/physical state: Colourless to yellow 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.
- MTT solution (1.0 mg/mL) was prepared from a MatTek MTT-100 kit immediately prior to use.

EPIDERM RECONSTRUCTED HUMAN EPIDERMIS MODEL KIT
- Supplier: MatTek
- Date received: 12 July 2016
- EpiDerm Tissues (0.63 cm2) lot number: 23345
- Assay medium lot number: 080716ZSB
- Upon receipt of the EpiDerm tissues, the sealed 24-well plate was stored in a refrigerator until use.

PRE-TEST PROCEDURE - MTT DYE METABOLISM CELL VIABILITY ASSAY
- The MTT assay is a colorimetric method of determining cell viability and is based on reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) to a blue formazan dye by mitrochondrial 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.

PRE-TEST PROCEDURE - TEST FOR DIRECT MTT REDUCTION
- To identify possible interference, the test item was checked for ability to directly reduce MTT.
- Test material (50 µL) was added to 1 mL of a freshly prepared 1.0 mg/L MTT solution.
- The solution was incubated in the dark at 37 °C for 60 minutes in an atmosphere of 5 % CO2 in air.
- Untreated MTT solution was tested concurrently 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.

PRE-TEST PROCEDURE - 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 for 60 minutes in an atmosphere of 5 % CO2 in air. A visual inspection of the colour was then made.

MAIN TEST – PRE-INCUBATION
- The assay medium was pre-warmed before use.
- Assay medium (0.9 mL) was pipetted into the appropriate wells of two pre-labelled 6-well plates for 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.

MAIN TEST – 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 after chemical exposure and MTT loading.
- Another 24-well plate was prepared for the MTT loading.
- Pre-warmed assay medium (300 µL) was dispensed into each well of the holding plate and the plate was placed into the incubator until required.
- MTT medium (300 µL) was dispensed into each well of the MTT loading plate and the plate was 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 whilst 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. Tissues were dosed at regular intervals to allow for the time taken to rinse each tissue following exposure and to ensure that each tissue was subjected to 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. 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 period was so short the tissues were dosed at regular intervals to ensure that each tissue received equal exposure 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. The tissues were then blotted and transferred to the 24-well plate prepared for MTT loading. The plate was incubated (37 °C, 5 % CO2) for three hours.
- The same rinsing and MTT loading procedure was repeated after the 60-minute exposure period was complete.
- After the 3-hour MTT incubation was complete, the inserts were blotted and transferred to labelled 24-well plates for MTT extraction. The MTT extractant (isopropanol, 2 mL) was used to completely immerse each insert and the plate was covered with plate sealer to prevent isopropanol evaporation. The plates were allowed to stand overnight at room temperature to allow evaporation 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 of the extract (3 x 200 µL) were transferred to the appropriate wells of a pre-labelled 96-well plate.
- Isopropanol alone (200 µL) was added to the three wells designated as blanks.
- Absorbance at 562 nm (OD562) was measured for each well using an Anthos 2001 microplate reader.

QUANTITATIVE MTT ASSESSMENT (PERCENTAGE TISSUE VIABILITY)
- Corrosivity potential of the test item was predicted from the relative mean tissue viabilities obtained after the 3-minute and 60-minute exposure periods, compared to the mean of the negative control tissues (n=2) treated with sterile distilled water.
- 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
- Results of the assay were considered acceptable if the assay acceptance criteria for negative control, positive control and coefficient of variation were achieved.
- 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 storage 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.
- Positive control: Potassium hydroxide 8.0 N solution is used as a positive control. An assay meets the acceptance criteria if mean relative tissue viability of the 60-minute positive control is < 15 %.
- Coefficient of variation: In the range 20 to 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 of test item

Duration of treatment / exposure:

3 minutes and 60 minutes

Duration of post-treatment incubation (if applicable):

3 hours

Number of replicates:

Duplicate tissues

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3-minute exposure

Value:

98.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: mean relative viability (% of negative control)

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60-minute exposure

Value:

103.2

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 result was taken to indicate that 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 result 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).
- Relative mean viabilities for each treatment group are shown in the table below.

QUALITY CRITERIA
- Mean OD562 for the negative control treated tissues was 1.773 for the 3-minute exposure period and mean OD562 for the negative control treated tissues was 1.727 for the 60-minute exposure period. The negative control acceptance criteria were therefore satisfied.
- Relative mean tissue viability for the positive control treated tissues was 4.8 % relative to the negative control following the 60-minute exposure period. The positive control acceptance criteria 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 minutes	100*	5.2	98.7
60 minutes	100*	4.8	103.2

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

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

see classification criteria given in Appendix 2 (attached)

Conclusions:

Relative mean viability of cells after exposure to test item was 98.7 % at 3 minutes and 103.2 % at 60 minutes.

Executive summary:

GUIDELINE

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 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. 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 B40bis 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).

 

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 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 562 nm. Data were presented in the form of percentage viability (MTT reduction in the test item-treated tissues relative to negative control tissues).

 

RESULTS

For the 3-minute exposure period, relative mean cell viabilities were 100 % (negative control), 5.2 % (positive control) and 98.7 % (test item). For the 60-minute exposure period, relative mean viabilities were 100 % (negative control), 4.8 % (positive control) and 103.2 % (test item). The quality criteria for acceptance of results in the test were satisfied.

 

CONCLUSION

Relative mean viability of cells after exposure to test item was 98.7 % at 3 minutes and 103.2 % at 60 minutes.