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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:
22 November to 24 November 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: Human Skin Model Test)
Version / remarks:
2016
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
-White powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: YBF1002/16
- Expiration date of the lot/batch: 17 October 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature over silica gel, in the dark

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: EpiDerm, MatTek
- Tissue lot number: 23380
- Delivery date: 22 November 2016, upon receipt of the EpidermTM tissues, the sealed 24-well plate was stored in a refrigerator until use.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C
- Temperature of post-treatment incubation: 37 °C

NUMBER OF REPLICATE TISSUES: 2

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 according to the procedure below:
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:
A test material may interfere with the MTT endpoint if it is coloured. The MTT assay is affected only if the test material is present in the tissues when the MTT viability assay is performed. 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.

MAIN TEST
PRE-INCUBATION:
The assay medium was pre-warmed before use, 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 MATERIAL 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. 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. 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 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. 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. 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 562nm (OD562) of each well was measured using the Anthos 2001 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 OD562 of test material / 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:
-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.
-Potassium Hydroxide 8.0N 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 %.
-In the range 20 and 100 % viability, the Coefficient of Variation between tissue replicates should be ≤ 30 %.
Control samples:
yes, concurrent vehicle
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 25 mg wetted with 25 µL of sterile water

VEHICLE CONTROL
- Amount applied: 50 µL

POSITIVE CONTROL
- Amount applied: 50 µL
- Concentration (if solution): 8.0 N
Duration of treatment / exposure:
3 minutes and 60 minutes
Duration of post-treatment incubation (if applicable):
3 hours with MTT
Number of replicates:
The test was performed in duplicate.

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 minutes exposure
Value:
95.7
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 minutes exposure
Value:
95.8
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
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 did not become coloured. This was taken to indicate the test material did not have the potential to cause colour interference.

RELATIVE MEAN VIABILITIES - TEST MATERIAL
The relative mean viabilities for each treatment group are shown in Table 2.

QUALITY CRITERIA
-The mean OD562 for the negative control treated tissues was 1.709 for the 3 minute exposure period and 1.692 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 3.1% 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

Table 2: relative mean viabilities for each treatment group

Exposure Period

Percentage Viability

Negative Control

Positive Control

Test Material

3 minute

100*

3.1

95.7

60 minute

100*

2.5

95.8

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

Table 3: Mean OD562 Values and Viabilities for the Negative Control, Positive Control and Test Material

Tissue

Exposure Period

Mean OD562 of individual tissues

Mean OD562 of duplicate tissues

Standard Deviation

Coefficient of Variation

(%)

Relative Mean Viability (%)

Negative Control

3 Minutes

1.720

1.709

0.016

1.0

100*

1.697

60 Minutes

1.649

1.692

0.060

3.6

1.734

Positive Control

3 Minutes

0.058

0.053

0.007

N/A

3.1

0.048

60 Minutes

0.055

0.043

0.018

N/A

2.5

0.030

Test Material

3 Minutes

1.658

1.635

0.033

2.0

95.7

1.611

60 Minutes

1.621

1.620

0.001

0.1

95.8

1.619

 

Relative mean % tissue viability = (mean OD562 of test material / mean OD562 of negative control) x 100

Coefficient of variation = (standard deviation / mean OD562 of duplicate tissues) x 100


Applicant's summary and conclusion

Interpretation of results:
other: Not corrosive in accordance with EU criteria
Conclusions:
Under the conditions of the study the test material was considered to be non-corrosive to the skin.
Executive summary:

The potential of the test material to cause skin corrosion was determined in accordance with the standardised guidelines OECD 431 and EU method B40.bis, under GLP conditions using the EpiDerm Human Skin Model.

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 material treated tissues relative to the corresponding negative control. The results are used to make a prediction of the corrosivity potential of the test material.

Before the main test was performed the test material was tested for 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. The test material was also assessed for its potential to cause colour interference with the MTT endpoint. The solution containing the test material did not become coloured. This was taken to indicate the test material did not have the potential to cause colour interference.

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. 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 562 nm (OD562).

The negative control, positive control and coefficient of variation were all within the acceptability criteria and so the study was valid.

Under the conditions of the study the test material was considered to be non-corrosive to the skin.