Registration Dossier

Administrative data

Description of key information

Skin corrosion: not corrosive (OECD 435; GLP)

Skin irritation: irritating to skin (OECD 439; GLP). Results require classfication as skin irritating substance (CLP (Cat. 2)).

Eye irritation: not serious eye damaging (CLP (Cat 1) but the hazardous properties of the test item with regard to corneal irritation (CLP (Cat 2)) cannot be predicted (OECD 437; GLP). The eye irritation potential of zinc difluoride will be determined in a follow-up study and upon study completion, a robust study summary will be incorporated in the REACH registration dossier.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

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
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
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
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
Irritation / corrosion parameter:
% tissue viability
Remarks:
(mean)
Value:
0.8
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
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)

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)

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.
Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-12-13 to 2017-12-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 435 (In Vitro Membrane Barrier Test Method for Skin Corrosion)
Version / remarks:
2015-07-28
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. Keep tightly sealed and protected from humidity and water
Test system:
artificial membrane barrier model
Source species:
other: not specified
Cell type:
other: synthetic macromolecular bio-barrier
Cell source:
other: not specified
Source strain:
not specified
Details on animal used as source of test system:
not applicable
Justification for test system used:
Corrositex TM is a validated and accepted in vitro method to assess if a test item can produce skin corrosion and to distinguish between GHS corrosivity categories 1A, 1B, and 1C.
Vehicle:
unchanged (no vehicle)
Details on test system:
SOURCE AND COMPOSITION OF MEMBRANE BARRIER USED
- Was the Corrositex® test kit used: yes (lot no. CT120516; supplier: ROMER Labs Deutschland GmbH, 35510 Butzbach, Germany)
- Components: a synthetic macromolecular bio-barrier and a chemical detection system (CDS)
- Apparatus and preparation procedures: one day prior to testing the bio-barrier matrix was prepared. The bio-barrier powder was solved in the bio-barrier diluent and heated for 20 ± 2 minutes at 68 – 70 °C in a water bath under continuous stirring. The temperature did not exceed 70 °C. The mixture was allowed to cool in the turned-off water bath for another 10 minutes. The mixture was then filled into the membrane holders (200 μL per membrane holder). Air bubbles were avoided. The filled membrane holders were sealed with parafilm and were stored at 2 – 8 °C until further use.

WAS THE COMPATIBILITY TEST PERFORMED: yes
In order to test whether the test system is suitable for the test item, approximately 100 mg of the test item were applied into the “Qualify Test Vial”. The vial was shaken until the solution appeared homogenous, and incubated for 1 minute. Afterwards, the colour change was noted and the test proceeded.
The test item induced a change in colour in the qualify test after 1 minute incubation. Since a change in colour was visible in the “Qualify Test Vial”, the test item was considered to be suitable for the next step.

WAS THE TIMESCALE CATEGORY TEST PERFORMED: yes
In the categorisation test the observation period of the test item after application to the bio-barrier was determined:
- Category 1: observation period after application is 4 hours
- Category 2: observation period after application is 1 hour
Approximately 100 mg of the test item were applied into the “Category A Vial” as well as into the “Category B Vial”. The vials were shaken until the solution appeared homogenous. After 1 minute the colour change was monitored.
Based on the colour change obtained, a test item is assigned to a category. If an intense colour change (similar to the category 1 colour chart) is observed in “Category A Vial” or in “Category B Vial” the test item is assigned to category 1. If a less intense colour change (similar to the category 2 colour chart) is observed in “Category A Vial” or in “Category B Vial” the test item will be assigned to category 2. If no colour change is observed in either of the vials, a confirmation test is conducted. For the confirmation test two drops of the confirm reagent are added to the “Category B Vial”. The vial would be shaken for 5 seconds. The colour of the solution would match one of the colours shown in the accompanying colour chart, confirming that the test item is a category 2 substance.
The test item did not induce a change in colour neither in the Category A vial nor in the Category B vial after 1 minute incubation. A confirmation experiment was performed by adding the confirm reagent to the Category B vial. This induced a change in colour after 1 minute of incubation. Therefore, the test item was classified as category II.

TEMPERATURE USED DURING TREATMENT: room temperature

METHOD OF DETECTION
- Chemical or electrochemical detection system: chemical detection system

METHOD OF APPLICATION (CLASSIFICATION TEST):
8 vials containing the CDS were pre-warmed to room temperature (test item: 4 vials; negative control: 1 vial; positive control (sulfuric acid): 1 vial; positive control (sodium hydroxide): 1 vial; colour reference for CDS: 1 vial).
The prepared bio-barriers were placed atop the CDS vials (not longer than 2 minutes prior to application) and the test item or the respective controls were applied per bio-barrier for 1 hour, depending on the results of the categorisation test. The time interval of the possible colour change or precipitation in the CDS solution was recorded.

INTERPRETATION OF THE RESULTS:
For each vial the period until observable change in CDS solution was determined. The mean of the quadruplicate measurement was calculated. The test item was categorised according to the criteria in table 1 as presented in the field "Any other information on materials and methods incl. tables" below.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): approx. 500 mg of the neat test item

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 500 µL of citric acid (10 % (w/v) solution in deionised water)

POSITIVE CONTROL (two positive controls were used)
- Amount(s) applied (volume or weight):
1) 500 µL of sulfuric acid 95 - 97 %
2) 110 ± 15 mg of sodium hydroxide
Duration of treatment / exposure:
60 minutes
Duration of post-treatment incubation (if applicable):
not applicable
Number of replicates:
Test item: quadruplicates
Negative control: single measurement
Positive control: single measurement
Irritation / corrosion parameter:
penetration time (in minutes)
Value:
0
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Colour change was not observed up to 60 minutes (treatment period)
Other effects / acceptance of results:
QUALIFY TEST
The test item induced a change in colour in the qualify test after 1 minute incubation. Since a change in colour was visible in the “Qualify Test Vial”, the test item was considered to be suitable for the next step.

CATEGORISATION TEST:
The test item did not induce a change in colour neither in the Category A vial nor in the Category B vial after 1 minute incubation. A confirmation experiment was performed by adding the confirm reagent to the Category B vial. This induced a change in colour after 1 minute of incubation. Therefore, the test item was classified as category II.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: negative control did not induce a change in the colour of CDS reagent after 60 minutes (acceptability criteria: negative control does not induce a physical change (colour or precipitation) up to 60 minutes in the CDS solution in the classification test).

- Acceptance criteria met for positive control: the positive control sulfuric acid showed a distinct change in the colour of the CDS reagent in the time interval of 0 – 3 minutes. The positive control sodium hydroxide induced a change in the colour of the CDS reagent after 3 – 60 minutes.

Please also refer to the field "Any other information on results incl. tables" below.

CLASSIFICATION TEST:


Test Group

Time Interval of
Colour Change

UN GHS

Negative Control

Colour change was not observed up to 60 minutes

Non-corrosive

Positive Control

Sulfuric acid

1 minute 13 seconds

Corrosive

subcategory 1A

Positive Control

Sodium Hydroxide

6 minutes 30 seconds

Corrosive

subcategory 1B

Test Item

Colour change was not observed up to 60 minutes

Non-corrosive

Interpretation of results:
GHS criteria not met
Conclusions:
The test item is not corrosive to the skin.
According to the Regulation (EC) No 1272/2008 and subsequent regulations, zinc difluoride does not require classification and labelling as being corrosive to skin. However, a conclusion on "serious eye irritation" is not possible based on test result. Further testing would be necessary.
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:
2018-01-30
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 i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Version / remarks:
2017-10-09
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. Keep container tightly sealed. Protect from humidity and water
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: AB Schlachthof GmbH & Co. KG, 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 stored in HBSS containing 1% (v/v) Penicillin/Streptomycin (100 units/mL penicillin and 100 µg/mL streptomycin) in the cooled slaughter-house until transportation on the same morning to the laboratory using a Styrofoam box.
- Time interval prior to initiating testing: corneae were isolated and used on the same day after delivery of the eyes
Vehicle:
other: 0.9 % (w/v) NaCl in deionised water
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 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:
PREPARATION OF CORNEAS
- each isolated cornea was mounted according to the description given in OEDC guideline 437, i.e. in a specially designed cornea holder 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 cMEM (MEM, supplemented with sodium bicarbonate, L-glutamine, penicillin/streptomycin and 1 % fetal calf serum).
- 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 carefully examined macroscopically for defects before removing the cornea. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded.
- at the end of the equilibration period of the corneae in the holder, the basal opacity was determined (t0).
- corneas that had opacity greater than seven opacity units or equivalent for the opacitometer and cornea holders used after an initial one-hour equilibration period were discarded.

APPLICATION DOSE AND EXPOSURE TIME
- the anterior compartment received the test item suspension or negative 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 minutes).
- after exposure of the test item or control items to the corneae, they were rinsed off from the application sides with EMEM containing phenol red at least three times or more if phenol red is still discoloured (yellow or purple), or the test item is still visible.
- once the medium was free of the test item the corneas were give a final rinse with cMEM without phenol red.
- fresh cMEM was added into the anterior compartment and opacity was measured (t240).
- permeability of the corneae was determined.
- no tissue peeling, residual test chemical and non-uniform opacity patterns were observed.

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 microplate reader (Versamax® Molecular Devices)(OD490).
- after the final opacity measurement was performed, the incubation medium was removed from both chambers. The posterior chamber was filled with fresh cMEM first. Then the anterior compartment was filled with 0.5% (w/v) sodium fluorescein solution in HBSS.
- corneae were incubated in a horizontal position for 90 ± 10 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 a microplate reader.
Evaluation permeability:
- the corrected OD490 value of each cornea treated with positive control or 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 is used to determine the IVIS of the negative control:
IVIS = opacity value + (15 x OD490 value)
The following formula is used to determine the IVIS of the positive control and the test item:
IVIS = (opacity value – corrected opacity value mean negative control) + (15 x corrected OD490 value)
The mean IVIS value of each treated group is calculated from the IVIS values of each individual treatment and positive control cornea.
Depending on the IVIS score obtained, the test item is classified into the following category according to OECD guideline 437 (please refer to table 1 in the field "Any other information on material and methods incl. tables" below).

DECISION CRITERIA:
The test will be 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.
- a single testing run composed of at least three corneas should be sufficient for a test chemical when the resulting classification is unequivocal. However, in cases of borderline results in the first testing run as described in the OECD guideline 437, a second testing run should be considered as well as a third one in case of discordant mean IVIS results between the first two testing runs.
Irritation parameter:
in vitro irritation score
Value:
10.21
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- after exposure to the negative control (physiological saline) an increase of opacity or permeability of the corneae was not observed (mean IVIS = 1.52).
- exposure to the positive control (10% (w/v) benzalkonium chloride in saline) resulted in clear opacity and distinctive permeability of the corneae (mean IVIS =121.96) corresponding to the classification serious eye damaging (EU CLP/ UN GHS (Category 1)).
- the test is valid since the IVIS of the positive control falls within two standard deviations of the current historical mean. Further, opacity and permeability of the negative control are less than the respective established upper limits for background opacity and permeability.

Please refer to the field "Any other information on results incl. tables" below.

Table 1: Results after 240 Minutes Treatment Time


Test Group

Opacity value = Difference (t240-t0) of Opacity

Permeability at 490 nm (OD490)

IVIS

Mean IVIS

Proposed in vitro Irritancy Score

 

 

Mean

 

Mean

 

 

 

Negative Control

0

0.33

0.053

0.079

0.80

1.52

No Category

-1

0.084

0.26

2

0.101

3.52

Positive Control

98.67*

0.101**

100.18

121.96

Category 1

104.67*

0.092**

106.04

158.67*

0.066**

159.65

Zinc fluoride

6.67*

-0.030**

6.21

10.21

No prediction can be made

9.67*

-0.029**

9.23

15.67*

-0.032**

15.18

* final corrected opacity ((t240 - t0) - average-corrected negative control opacity value)

** final corrected OD490 (OD490 - average-corrected negative control OD490 value)

Table 2: Historical Data

 

Positive Control

Negative Control

Mean IVIS (MV)

115.47

1.31

Standard Deviation of IVIS (SD)

8.96

0.21

Range of IVIS

98.30—130.61

0.86—1.64

95 % Control limits of IVISpos

(MV ± 2xSD)

97.55—133.39

 

Mean Opacity t240min

114.26

0.22

Standard Deviation of
Opacity t240min

12.06

0.21

Range of Opacity t240min

82.00—135.00

0.00—0.67

Mean Permeability

0.08

0.07

Standard Deviation of Permeability

0.11

0.01

Range of Permeability

-0.01—0.52

0.06—0.09

Values of 45 studies with solid test items sharing 24 sets of controls, performed between January 2016 and October 2017.

Interpretation of results:
other: test item is not serious eye damaging (CLP (Cat 1) but the hazardous properties of the test item with regard to corneal irritation cannot be predicted.
Conclusions:
The test substance is not serious eye damaging according to the Regulation (EC) No 1272/2008 and subsequent adaptions (Category 1), but no further prediction regarding the eye irritation potential can be made according to the evaluation criteria.

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 irritation study according to OECD 435. However, a study on skin irritation in accordance with OECD 429 was performed resulting in C&L as skin irritant Cat. 2.

Eye irritation:

According to a OECD 437 guideline study (GLP), zinc difluoride is not serious eye damaging (CLP (Cat 1) but the hazardous properties of the test item with regard to corneal irritation (CLP (Cat 2)) cannot be predicted. The eye irritation potential of zinc difluoride will be determined in a follow-up study and upon study completion, a robust study summary will be incorporated in the REACH registration dossier.

Justification for classification or non-classification

Skin corrosion:

The substance does not possess a skin corrosive potential based on an in vitro OECD 435 (2015) test and does not require classification as skin corrosive according to Regulation (EC) No 1272/2008 and its subsequent adaptations.

However, a study on skin irritation in accordance with OECD 429 was performed resulting in C&L as skin irritant Cat. 2 according to Regulation (EC) No 1272/2008 and its subsequent adaptations.

Eye irritation:

According to a OECD 437 guideline study (GLP), zinc difluoride is not serious eye damaging (CLP (Cat 1) but the hazardous properties of the test item with regard to corneal irritation (CLP (Cat 2)) cannot be predicted. The eye irritation potential of zinc difluoride will be determined in a follow-up study and upon study completion, a robust study summary will be incorporated in the REACH registration dossier.