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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:
2015-10-19 to 2015-11-02
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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2015-07-28
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
2009
Deviations:
no
Qualifier:
according to
Guideline:
other: In Vitro EpiDermTM Skin Irritation Test (EPI-200-SIT) for use with MatTek Corporation’s Reconstructed Human Epidermal Model EpiDerm (EPI-200)
Version / remarks:
Rev. 3/26/2012
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:
- Name of test material (as cited in study report): tin difluoride
- Physical state: solid, clear

Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature

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 (DPBS)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ skin model (source: MatTek Corporation, 82105 Bratislava, Slovakia)
- Tissue lot number: 23300
- Delivery date: 2015-10-27

TEMPERATURE USED FOR TEST SYSTEM
- Temperature of pre-incubation: 37 ± 1.5 °C (23.5 hours)
- 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
Tissues 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 fresh assay medium. Tissues were incubated for about 24 hours at 37 ± 1.5 °C, 5 ± 0.5 % CO2. After incubation the inserts were transferred into new plates containing fresh medium. Thereafter tissues were incubated for another approx. 18 hours at 37 ± 1.5 °C, 5 ± 0.5 % CO2. The complete incubation time was 42 hours. Following incubation, the tissue viablity was measured.

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 MTT solution was aspirated from the wells and the wells were rinsed three times with DPBS. Inserts were transferred onto new plates containing extractant solution (isopropanol) ensuring that the tissues are completely covered. The plate was sealed to inhibit the isopropanol evaporation. The formazan salt was extracted for about 70 hours without shaking in the refrigerator.
After the extraction period was completed, the inserts were pierced to allow the extract to run into the well from which the insert was taken and the inserts were 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 from the 15 minutes exposure. The optical density was determined with a spectrophotometer. Mean values were calculated from the 3 wells per tissue.
- Spectrophotometer: Versamax® Molecular Devices
- Wavelength: 570 nm
- Filter bandwidth: 1 nm

TEST FOR COLOUR INTERFERENCE
Prior to the start of the test, the test item’s colour interference potential was evaluated. About 25 mg of the test item were mixed with 300 µL of deionised water. This mixture was incubated for 60 minutes at 37 ± 1.5 °C, 5 ± 0.5 % CO2, 95 ± 5 % RH.

TEST FOR DIRECT MTT REDUCTION
For correct interpretation of results, the ability of the test item to directly reduce MTT was assessed. For this purpose, approx. 25 mg of the test item were added to 1 mL of MTT solution (resulting: 1 mg/mL). This mixture was incubated at 37 ± 1.5 °C ,5 ± 0.5 % CO2, 95 ± 5 % RH for 60 minutes.

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 after blank correction. 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(%) = (OD test item or positive control/ OD mean 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 irritauion 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 (~ 39 mg/m²) 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:
Test item: triplicates
Negative control: triplicates
Positive control: triplicates

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Remarks:
(mean)
Value:
15.6
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- OTHER EFFECTS:
- Direct-MTT reduction: optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour.
- Colour interference with MTT: the optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water did not led to a change in colour.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: after treatment with the negative control the absorbance values (2.079, 1.941, and 1.762 (mean: 1.927)) were well within the required acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 minutes treatment interval.
- Acceptance criteria met for positive control: treatment with the positive control induced a decrease in the relative absorbance as compared to the negative control to 4.8% (= ≤ 20%)..
- Acceptance criteria met for variability between replicate measurements: the relative standard deviations between the % variabilities of the test item, the positive and negative controls in the main test were below 15% (threshold of the "OECD Guideline for the Testing of Chemicals 439: In vitro Skin Irritation: Reconstructed Human Epidermis Test Method”: < 18%).
Please refer to the field "Any other information on results incl. tables" below

Any other information on results incl. tables

Table 1: Historical data

Positive Control

Negative Control [OD570]

Mean Viability

4.77%

Mean Absorption

1.77

Rel. Standard Deviation

14.9%

Rel. Standard Deviation

8.55%

Range of Viabilities

4.00% - 5.90%

Range of Absorbance

1.66 – 1.98

Mean Absorption

0.084

 

Rel. Standard Deviation

16.6%

Range of Absorbance

0.069 - 0.097

Data of 13 studies performed from July 2015 until end of October 2015

Table 2: Results after treatment with tin difluoride and the controls

Dose Group

Treatment Interval

Absorbance 570 nm
Tissue 1*

Absorbance 570 nm
Tissue 2*

Absorbance 570 nm
Tissue 3*

Mean Absorbance of 3 Tissues

Mean Rel. Absorbance

[% of Negative Control]**

Negative Control

60 min

1.932

1.954

2.059

1.982

100.0

Positive Control

60 min

0.090

0.096

0.099

0.095

4.8

Test Item

60 min

0.333

0.339

0.258

0.310

15.6

* mean of three replicate wells after blank correction

** relative absorbance per treatment group [rounded values]: (100 x (mean absorbancetestitem/positive control))/(mean absorbance negative control)

- colour interference: the colour interference pre-experiment to investigate the test item’s colour change potential in water did not led to a change in colour.

- MTT reduction: optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour.

- after treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD³0.8 and ≤ 2.8 for the 60 minutes treatment interval (range: 1.971 to 2.098) thus showing the quality of the tissues.

- treatment with the positive control induced a decrease in the relative absorbance as compared to the negative control to 4.8% (≤ 20%) thus ensuring the validity of the test system.

- the relative standard deviations between the % variabilities of the test item, the positive and negative controls in the main test were below 15% (threshold of the "OECD Guideline for the Testing of Chemicals 439:In vitroSkin Irritation: Reconstructed Human Epidermis Test Method”: < 18%), thus ensuring the validity of the study.

Applicant's summary and conclusion

Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
The test item is irritating to the skin.
According to the Regulation (EC) No 1272/2008 and subsequent regulations, the test item is classified as skin irritant (Category 2).