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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-01-29 to 2018-03-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2015-07-28
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2015-09-14

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
- State of aggregation: white solid
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature. Keep container tightly sealed. Protect from humidity and water

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
other: normal, human-derived epidermal keratinocytes
Cell source:
other: humans
Source strain:
not specified
Details on animal used as source of test system:
not applicable
Justification for test system used:
In an international prevalidation study performed by ECVAM, the in vitro skin irritation test using the human skin model EpiDerm™ and measurement of cell viability by dehydrogenase conversion of MTT into a blue formazan salt have turned out as a sufficiently promising predictor for skin irritancy potential.
Vehicle:
other: Dulbecco's phosphate buffered saline
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ skin model (source: MatTek Corporation, 82105 Bratislava, Slovakia)
- Tissue lot number: 25882
- Delivery date: 2018-02-21

TEMPERATURE USED FOR TEST SYSTEM
- Temperature of pre-incubation: 37 ± 1.5 °C (about 60 minues)
- Temperature used during treatment / exposure: 37 ± 1.5 °C for 35 minutes, room temperature for 25 minutes
- Temperature of post-treatment incubation: 37 ± 1.5 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
After the end of the treatment interval the inserts were rinsed with DPBS at least 15 times in order to remove any residual test material.

After the rinsing the inserts were submerged in DPBS at least three times. Afterwards the inserts were again rinsed with DPBS. The tissues were then transferred into plates with assay medium. Tissues were incubated for about 24 hours at 37 ± 1.5 °C, 5 ± 0.5 % CO2. After incubation medium will be changed (pre-warmed fresh medium). Thereafter tissues were incubated for another approximately 18 hours at 37 ± 1.5 °C, 5 ± 0.5 % CO2. The complete incubation time was 42 hours.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL (300 µL/ well)
- Incubation time with MTT: 3 hours
- Extraction of Formazan: after the incubation period, the tissues were rinsed three times with DPBS. The tissues were transferred into new plates containing extractant solution (isopropanol) in each well ensuring that the tissues were completely covered and the plate was sealed to inhibit the isopropanol evaporation. The formazan salt was extracted for about 2.5 hours while shaking at room temperature.
After the extraction period was completed, 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. The 24-well plates were placed on a shaker for 15 minutes until the solution was homogeneous in colour.
Per each tissue, 3 × 200 μL aliquots of the blue formazan solution were transferred into a 96-well flat bottom microtiter plate. The optical density was determined with a microplate reader. Mean values were calculated from the 3 wells per tissue.
- Spectrophotometer: Versamax® Molecular Devices
- Wavelength: 570 nm

TEST FOR COLOUR INTERFERENCE
Before the test started, functional check for colour interference was performed. 25 ± 2 mg of the test item were added to deionised water. The mixture was incubated in the incubator (37 ± 1.5 °C, 5 ± 0.5 % CO2) for 60 minutes. At the end of the exposure time, the mixture was shaken and the presence and intensity of the staining (if any) was evaluated.

TEST FOR DIRECT MTT REDUCTION
The test item was evaluated for its potential to interfere with MTT assay. To test if a test item directly reduces MTT, 25 ± 2 mg of the test item were added to 1 mL of the MTT-solution (1mg/mL) and was incubated in the incubator (37 ± 1.5 °C, 5 ± 0.5% CO2) for 60 minutes. Untreated MTT medium was used as control.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: tissues pass analysis for tissue viability
- Barrier function: tissues pass analysis for tissue functionality
- Morphology: presence of a functional stratum corneum, a viable basal cell layer, and intermediate spinous and granular layers
- Contamination: absence of bacteria, yeast, and other fungi (long term antibiotic, antimycotic free culture) as well as absence of HIV1- virus, Hepatitis B virus, and Hepatitis C virus
Please also refer to the field "Attached background material" below.

PREDICTION MODEL / DECISION CRITERIA
The mean optical density (OD) of the three negative control tissues was calculated. This value corresponds to 100% tissue viability in the current test. For each individual tissue treated with the test item or the positive control the individual relative tissue viability is calculated according to the following formula: relative viability(%) = (mean OD test item or positive control/ mean OD of negative control) x 100
For the test item and the positive control, the mean relative viability ± relative standard deviation of the three individual tissues was calculated and used for classification according to the following prediction model: if the mean relative tissue viability of three individual tissues is less or equal to 50% of the negative control, the test item needs to be classified and labeled for its skin irritation potential: Category 2 – irritant, H315 according to Regulation (EC) No 1272/2008.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): approx. 25 mg of the test item, wetted with vehicle

VEHICLE
- Amount(s) applied (volume or weight with unit): 25 µL DPBS

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL DPBS

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL of a 5% Sodium Lauryl Sulfate (SLS) solution
Duration of treatment / exposure:
60 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
triplicates

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Remarks:
(mean)
Value:
0.8
Vehicle controls valid:
not examined
Negative controls valid:
yes
Positive controls valid:
yes
Other effects / acceptance of results:
- OTHER EFFECTS:
- Colour interference with MTT: The test item did not reduce MTT (test for direct MTT reduction), and it did not change colour when mixed with deionised water (test for colour interference).

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: After treatment with the negative control the absorbance values were well within the required range of the acceptability criterion of mean OD  0.8 and ≤ 2.8 for the 60 minutes treatment interval, thus assuring the quality of the tissues
- Acceptance criteria met for positive control: Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control for the 60 minutes treatment interval, and thus assuring the validity of the test system.
- Acceptance criteria met for variability between replicate measurements: Criterion 1 (negative control): The absolute OD 570 nm of the negative control tissues in the MTT test is an indicator of tissue viability obtained after the shipping and storing procedure and under specific conditions of the assay. Tissue viability is meeting the acceptance criterion if the mean OD570 of the negative control tissues is  0.8 and ≤ 2.8 in accordance with OECD TG 439.
Criterion 2 (positive control): An assay is meeting the acceptance criterion if mean relative tissue viability of the positive control is  20%.
Criterion 3: Standard deviation: The SD of 3 concurrently tested tissue replicates should be < 18.
Criterion 4: OD values should not be below historically established boundaries.
Concurrent negative controls (NC) and positive controls (PC) will be used in each run to demonstrate that viability (with the NC), barrier function and resulting tissue sensitivity (with the PC) of the tissues are within a defined historical acceptance range.
Historical data (see annex 1) and the quality certificate of the supplier of the test kit (see annex 3) demonstrating its robustness of the test system, including quality control data (determined by MatTek Corporation, 82105 Bratislava, Slovakia) of the respective EpiDermTM lot. According to the OECD TG 439, the acceptance limit of the ET50 should be between 4.8 hours and 8.7 hours after treatment with 1% Triton X-100 (QC batch release criteria)

Any other information on results incl. tables

Results after treatment with Zinc fluoride and the controls:

Treatment Group

Tissue No.

OD 570 nm
Well 1

OD 570 nm
Well 2

OD 570 nm
Well 3

Mean OD of 3 Wells

Mean OD

of 3 Wells blank

corrected

Mean

OD

of 3 tissues

blank corrected

Rel. Viability [%] Tissue
1, 2 + 3*

Standard Deviation

Mean Rel. Viability

[%]**

Blank

 

0.038

0.038

0.037

0.037

 

 

 

 

 

Negative Control

1

1.707

1.603

1.613

1.641

1.603

1.706

94.0

5.4

100.0

2

1.796

1.776

1.746

1.773

1.735

101.7

3

1.862

1.809

1.782

1.817

1.780

104.3

Positive Control

1

0.080

0.079

0.078

0.079

0.042

0.047

2.5

0.4

2.8

2

0.093

0.093

0.094

0.093

0.056

3.3

3

0.082

0.082

0.079

0.081

0.043

2.5

Test Item

1

0.055

0.057

0.056

0.056

0.019

0.014

1.1

0.2

0.8

2

0.047

0.048

0.048

0.048

0.010

0.6

3

0.050

0.051

0.052

0.051

0.014

0.8

*                      Relative viability [rounded values]:

 **                    Mean relative viability [rounded values]:

Historical data:

Positive Control; OD at 570 nm after
exposition to 5 % SDS solution in deionised
water (MatTek)

Negative Control OD at 570 nm
DPBS (MatTek)

MeanViability

4.28%

Mean Absorption

1.66

Standard Deviation

 1.00 p.p.

Standard Deviation

0.20

Rel. Standard Deviation

23.44%

Rel. Standard Deviation

11.96%

Range of Viabilities

2.24%—6.19%

Range of Absorbance*

1.28—2.00

Mean Absorption

0.07

* should be 0.8—2.8 (OECD 439)
or 1.0—2.5 (MatTek)

Standard Deviation

0.02

Rel. Standard Deviation

25.96%

Range of Absorbance

0.03—0.11

Data of 36 sets of controls shared between 147 studies performed from January 2017 until January 2018. (p.p.—percentage points)

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
A study on skin irritation in accordance with OECD 429 was performed with zinc difluoride resulting in C&L as skin irritant Cat. 2 according to Regulation (EC) No 1272/2008 and its subsequent adaptations.