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Administrative data

Description of key information

Skin corrosion: not corrosive (OECD 431; GLP compliant)

Skin irritation: irritating (OECD 439; GLP compliant)

Eye irritation: corrosive/severe irritant to the eyes (OECD 437, GLP compliant)

Key value for chemical safety assessment

Skin irritation / corrosion

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

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.

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).
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-10-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Version / remarks:
2013-07-26
Deviations:
no
Qualifier:
according to
Guideline:
other: Bovine Corneal Opacity and Permeability (BCOP) Assay, SOP of Microbiological Associates Ltd., UK, Procedure Details, April 1997
Version / remarks:
April 1997
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2015-09-14
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: abattoir (Schlachthof Aschaffenburg, 63739 Aschaffenburg, Germany)
- Characteristics of donor animals: at least 9 month old donor cattle
- Storage, temperature and transport conditions of ocular tissue: isolated eyes were transported to the laboratory in Hank’s Buffered Salt Solution (HBSS) supplemented with streptomycin / penicillin at ambient temperature. After removing the corneas from the eyes, they were also collected in Hank’s BSS supplemented with streptomycin / penicillin.
- Time interval prior to initiating testing: corneae were isolated and used on the same day after delivery of the eyes

Vehicle:
physiological saline
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL
- Concentration: 20% suspension (w/v) in vehicle
Duration of treatment / exposure:
240 ± 15 minutes
Observation period (in vivo):
not applicable
Duration of post- treatment incubation (in vitro):
not required
Number of animals or in vitro replicates:
Number of bovine corneae per dose:
Test item: triplicates
Negative control: triplicates
Positive control: triplicates
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
- each cornea was mounted in a specially designed cornea holder according to the description given in OEDC guideline 437, that consists of anterior and posterior compartments, which interface with the epithelial and endothelial sides of the cornea, respectively. Both compartments of the holder were filled with incubation medium.
- for equilibration, the corneae in the holder were incubated in a vertical position for about one hour at 32 ± 1 °C in a water-bath.

QUALITY CHECK OF THE ISOLATED CORNEAS
- all eyes were examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded. The isolated corneas were also examined macroscopically for defects.
- at the end of the equilibration period of the corneae in the holder, the basal opacity was determined (t0).
- each corneae with a value of the basal opacity > 7 was discarded.

APPLICATION DOSE AND EXPOSURE TIME
- the anterior compartment received the test item suspension or negative control or positive control at a volume of 0.75 mL each on the surface of the corneae.
- corneae were incubated in a horizontal position at 32 ± 1 °C in the water-bath (incubation time: 240 ± 15 minutes).
- after the incubation time, the test item or control items were rinsed off with saline
- fresh incubation medium was added into the anterior compartment and opacity was measured (t240)
- permeability of the corneae was determined.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: the opacitometer (OP_KiT opacitometer (Electro Design)) was calibrated and the opacity of each of the corneae was determined by reading each holder placed in the photoreceptor compartment for treated cornea.
Evaluation of opacity:
- the change of opacity value of each treated cornea or positive and negative control corneae is calculated by subtracting the initial basal opacity from the post treatment opacity reading (t240 – t0), for each individual cornea.
- the average change in opacity of the negative control corneae is calculated and this value is subtracted from the change in opacity of each treated cornea or positive control to obtain a corrected opacity.

- Corneal permeability: passage of sodium fluorescein dye measured with the aid of spectrophotometer (Versamax® Molecular Devices)(OD490).
- after the final opacity measurement was performed, the incubation medium was removed from the anterior compartment and replaced by 0.5% (w/v) sodium fluorescein solution in HBSS
- corneae were incubated in a horizontal position for 90 minutes in a water-bath at 32 ± 1 °C.
- incubation medium from the posterior compartment was removed, mixed and the optical density at 490 nm was determined with the spectrophotometer.
Evaluation permeability:
- the corrected OD490 value of each cornea treated with positive control and test item is calculated by subtracting the average negative control cornea value from the original permeability value for each cornea.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
The following formula was used to determine the IVIS of the negative control:
IVIS = opacity value + (15 x OD490 value)
The following formula was used to determine the IVIS of the positive control and the test item:
IVIS = (opacity value – opacity value mean negative control) + (15 x corrected OD490 value)
The mean IVIS value of each treated group was calculated from the individual IVIS values which was determined from each individual treatment and positive control cornea.
Depending on the score obtained, the test item was classified into the following category according to OECD guideline 437 (table 1 in the field "Any other information on materials and methods incl. tables" below)

DECISION CRITERIA:
The test was acceptable if
- the positive control gives an IVIS that falls within two standard deviations of the current historical mean (updated every three months), and if
- the negative control responses result in opacity and permeability values that are less than the established upper limits for background opacity and permeability values for bovine corneae treated with the respective negative control.
Irritation parameter:
in vitro irritation score
Remarks:
(mean)
Value:
440.06
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- with the negative control (saline) neither an increase of opacity nor permeability of the corneae could be observed (mean IVIS = 1.24).
- the positive control (10% (w/v) Benzalkonium chloride in saline) showed distinct opacity of the corneae (mean IVIS = 117.97) corresponding to a classification as serious eye damaging (CLP (Cat 1)).

Please refer to the field "Any other information on results incl. tables" below.
Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
Relative to the negative control, the test item caused a significant increase of the corneal opacity and permeability. In conclusion, according to the current study and under the experimental conditions reported, the substance is serious eye damaging (the calculated IVIS = 440.06). A substance with an in vitro irritation score ≥ 55 does require classification for eye irritation and serious eye damage according to OECD 437 (2013). According to the Regulation (EC) No 1272/2008 and subsequent adaptions, the substance is classified for serious eye damage (Category 1).
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin corrosion

The substance was not observed to be corrosive to the skin in a reliable in vitro skin corrosion study according to OECD 431.

Skin irritation

The substance was observed to be irritating to the skin in a reliable in vitro skin irritation study according to OECD 439.

Eye irritation:

The substance was observed to be corrosive/severe irritanting to the eyes in a reliable in vitro eye irritation study according to OECD 437.

Justification for classification or non-classification

Skin corrosion

The substance does not possess a skin corrosive potential based on an in vitro OECD 431 (2015) test and does not require classification as skin corrosive according to Regulation (EC) No 1272/2008 and its subsequent adaptations.

Skin irritation

The substance does possess an skin irritating potential based on an in vitro OECD 439 (2015) test and does require classification as skin irritating (Category 2) according to Regulation (EC) No 1272/2008 and its subsequent adaptations.

Eye irritation

The substance does possess an eye irritating potential based on an in vitro OECD 437 (2013) test and does require classification as severe eye irritant (Category 1) according to Regulation (EC) No 1272/2008 and its subsequent adaptations.

Respiratory irritation

The classification as respiratory irritant is normally covered under the endpoint specific target organ toxicity- single exposure and repeated exposure. Please refer to the endpoint summaries on acute toxicity (endpoint 7.2) for further information.