Registration Dossier

Administrative data

Description of key information

- Skin corrosion: OECD 431, non-corrosive

- Skin irritation: OECD 439, irritant

-EpiOcular: at least eye-irritant

- BCOP:

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Qualifier:
according to
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
foreskin from a single donor
Justification for test system used:
Standard test system
Vehicle:
water
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Epiderm kit
- Tissue batch number(s): 25858
- Delivery date: 2017-11-14

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

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/ml
- Incubation time: 1h
- Spectrophotometer: Anthos Reader 2010 Flexi
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3

Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25-26mg

VEHICLE
- Amount(s) applied (volume or weight with unit): 25µl water

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50µl water

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50µl potassium hydroxide
- Concentration (if solution): 8M KOH
Duration of treatment / exposure:
3 minutes / 60 minutes
Number of replicates:
duplicate
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 min
Value:
ca. 84.7
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 min
Value:
ca. 45.1
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
The acceptance criteria were fulfilled, the test is considered valid.

After 3 minutes treatment with the test item, the mean value of relative tissue viability was reduced to 84.7 %. This value is above the threshold for corrosion potential (50%). After 1 hour treatment, the mean value of relative tissue viability was reduced to 45.1 %. This value, too, is above the threshold for corrosion potential (15%).

Interpretation of results:
GHS criteria not met
Conclusions:
The test item is considered non-corrosive to skin.
After 3 minutes treatment, the mean value of relative tissue viability of the test item was decreased to 84.7% This value is well above the threshold for corrosivity (50%). After 1 hour treatment the mean value of relative tissue viability of the test item was reduced to 45.1%. This value is above the threshold for cor-rosivity (15%).

The values of the negative control met the required acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for both treatment intervals, thus showing the quality of the tissues.
The positive control has met the validity criterion too, thus ensuring the validity of the test system.

For these reasons, the result of the test is considered valid.
Executive summary:

Two tissues of the human skin model EpiDermTMwere treated with the test itemAmmonium hexafluorocirconatefor 3 minutes and 1 hour, respectively. The test item was applied to each tissue and spread to match the tissue size.

Demineralised water was used as negative control, 8 M KOH was used as positive control.

After treatment, the respective substance was rinsed from the tissues. Then, cell viability of the tissues was evaluated by addition of MTT, which can be reduced to a blue formazan. Formazan production was evaluated by measuring the optical density (OD) of the resulting solution.

After treatment with the negative control, the absorbance values were within the required acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for both treatment intervals thus showing the quality of the tissues. The OD was 1.7 (3 minutes experiment) and 1.6 (1 hour experiment).

The positive control showed clear corrosive effects for both treatment intervals. The mean relative tissue viability value was reduced to 6.1 % for the 1 hour treatment.

After 3 minutes treatment with the test item, the mean value of relative tissue viability was reduced to 84.7 %. This value is above the threshold for corrosion potential (50%). After 1 hour treatment, the mean value of relative tissue viability was reduced to 45.1 %. This value, too, is above the threshold for corrosion potential (15%).

 

Therefore, the test item is considered non-corrosive to skin in the Reconstructed Human Epidermis (RHE) Test Method.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22.08.2017 - 15.09.2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
foreskin from a single donor
Justification for test system used:
Standard system for OECD 439 test.
Vehicle:
other: Dulbecco's Phosphate Buffered Saline (DPBS)
Details on test system:
SKIN DISC PREPARATION
- Procedure used: EpiDerm is a ready-to-use, highly differentiated 3D tissue model consisting of normal, human-derived epidermal keratinocytes (NHEK) cultured on specially prepared tissue culture inserts.
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C
- Temperature post-incubation period: 37°C
NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION:
PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive/irritant to skin if the tissue viability is lower or equal to 50% of the negative control.
- The test substance is considered to be non-irritant to skin if the tissue viability is higher than 50% of the negative control.
Control samples:
yes, concurrent negative control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 24.8, 25.8 and 25.3 mg

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

NEGATIVE CONTROL
- DPBS, 30µl

POSITIVE CONTROL
- Concentration (if solution): 5% sodium dodecyl sulphate (SDS)
Duration of treatment / exposure:
1h
Duration of post-treatment incubation (if applicable):
22 h
Number of replicates:
3 tissues were used per plate
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 min (mean)
Value:
2.5
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
All validity criteria were met.
Values for negative control and for positive control were within the range of historical data of the test facility. Therefore, the experiment is considered valid.
Conclusions:
Under the conditions of the study, the relative tissue viability was reduced to 2.5%. Therefore, the test item is considered irritant to skin.
Executive summary:

Three tissues of the human skin model EpiDermTMwere treated withAmmonium hexafluorocirconatefor 60 minutes.

The test item was applied directly to each tissue and spread to match the tissue size (0.63  cm2; as indicated by the supplier).

DPBS-buffer was used as negative control and 5% SDS solution was used as positive control.

After treatment with the negative control, the absorbance values were within the required acceptability criterion. The positive control showed clear irritating effects. The mean value of relative tissue viability was reduced to 2.5% .

The variation within the tissue replicates of negative, control, positive control and test item was acceptable.

 

After the treatment with the test item, the mean value of relative tissue viability was reduced to 2.5 %. This value is below the threshold for skin irritation potential (50%). Values below the threshold of 50% are considered irritant to skin.

 

Therefore, the test item is considered irritant to skin in the Reconstructed human Epidermis (RhE) Test Method.

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:
23.08.-25.10.2018
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:
adopted 09. Oct. 2017
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
cattle
Strain:
other: Bos primigenius Taurus (fresh bovine corneas)
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: slaughterhouse Müller Fleisch GmbH, Enzstr. 2-4, 75217 Birkenfeld, Germany
- Number of animals:
- Characteristics of donor animals (e.g. age, sex, weight): the cattle were between 12 and 60 months old.
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): the eyes were transported to the test facility in Hanks' Balanced Salt Solution with 1% Penicillin-Streptomycin solution in a suitable cooled container wihtin 1h and 30 minutes.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
The test item was tested neat. The surface of the corneas was completely and evenly covered. The amounts of the test item used where as follows:
replicate 1: 543.7 mg; replicate 2: 557.4 mg; replicate 3: 553.8 mg.
Duration of treatment / exposure:
4 hours
Duration of post- treatment incubation (in vitro):
90 minutes
Number of animals or in vitro replicates:
3 replicates
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
After having carefully cleaned and sterilised the cornea holders, they were kept in the incubation chamber at 32 ± 1 °C. On the day of the assay, the MEM without phenol red was supplemented with sodium bicarbonate, L-glutamine and 1% fetal calf serum (= complete MEM) and stored in a water bath at 32 ± 1 °C. The same was performed with the MEM with phenol red, but without addition of sodium bicarbonate. After the arrival of the corneas, they were examined and only corneas which were free
from damages were used. The corneas were excised with a scalpel and cut from the globe with a 2-3 mm ring of sclera around the outside. Each cornea was transferred to a cornea holder in which pre-warmed cMEM (32 ± 1 °C) without phenol red was filled. The holders were then incubated for 1 hour in the incubation chamber at 32 ± 1 °C.
After the initial incubation, the medium was completely changed and the baseline opacity for each cornea was recorded. None of the corneas showed tissue damage; therefore, all corneas were used. For each treatment group (negative control solution, test item and positive control), three replicates were used. After removal of the pre-incubation medium (cMEM without phenol red), 750 μL negative control solution, a defined amount of test item and 750 μL positive control solution were applied to each replicate to the epithelial side of the cornea.
After the recording of the final opacity value, the cMEM without phenol red was removed from both chambers. The posterior chamber, which interfaces with the endothelial side of the cornea was filled with fresh cMEM. Then 1 mL sodium fluorescein solution was added to the front chamber for the detection of permeability of the corneas. For solid non-surfactant test items, a sodium fluorescein solution with a concentration of 5 mg/mL was used. The chambers were then closed again and incubated for 90 minutes at 32 ± 1 °C in a horizontal position. After incubation, the content of the posterior chamber was thoroughly mixed and pipetted in a 96-well plate. Then, its optical density at 492 nm is measured with the microtiter plate photometer.

TREATMENT METHOD: open chamber
In order to apply the test item, the window-locking ring and glass window of the anterior chamber was unscrewed to remove the glass disc. The test item was given on the epithelium that the cornea was evenly covered with test
item. Exposure time of the controls and test item on the corneas was 4 hours at 32 ± 1 °C. After thorough rinsing the anterior chamber with cMEM with phenol red and final rinsing with cMEM without phenol red, the anterior chamber was filled with cMEM without phenol red and the final opacity value of each cornea was recorded.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The change of opacity value of each treated cornea with test item, positive control and negative control was calculated by subtracting the initial basal opacity from the post treatment opacity reading for each cornea. The average change in opacity of the negative control cornea was calculated and this value was subtracted from the change in opacity of each treated cornea with test item and positive control to obtain a corrected opacity.
- Corneal permeability: The corrected OD492 value of each cornea treated with test item and positive control was calculated by subtracting the mean negative control cornea value from the original permeability value for each cornea. The mean OD492 value for each treatment group (test item, positive control and negative control) was determined by averaging the final corrected OD492 values of the treated corneas for one treatment group.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
Irritation parameter:
in vitro irritation score
Run / experiment:
mean of three replicates
Value:
70.7
Vehicle controls validity:
not applicable
Remarks:
Test item was tested neat.
Negative controls validity:
valid
Positive controls validity:
valid
Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
The test item was tested neat. Under the conditions of this test, the test item Ammonium hexafluorozirconate induced serious eye damage on the cornea of the bovine eye. The calculated mean IVIS was 70.70.
According to OECD Guideline no. 437 (Oct. 2017), a substance with an IVIS > 55 induces serious eye damage, that should be classified as UN GHS Category I.
Executive summary:

This in vitro study was performed to assess corneal damage potential of Ammonium hexafluorozirconate by quantitative measurements of changes in opacity and permeability in a bovine cornea.

Six experiments were performed, but two of them were invalid, because one validity criterion of the negative control was not met. These results are not reported but kept with the raw data in the GLP-archive of the test facility. Further three experiments were performed, which led to equivocal results. This is why a sixth experiment was performed with the undiluted test item, because it was assumed that the suspension was not homogeneous. Only this valid experiment is described in detail.

 

The test item Ammonium hexafluorozirconate was brought onto the cornea of a bovine eye which previously had been incubated with cMEM without phenol red at 32 ± 1 °C for 1 hour and whose opacity had been determined. The test item was incubated on the cornea for 4 hours at 32 ± 1 °C. After removal of the test item, opacity and permeability values were measured.

 

The test item was tested neat. Under the conditions of this test, the test item Ammonium hexafluorozirconate induced serious eye damage on the cornea of the bovine eye. The calculated mean IVIS was 70.70.

According to OECD Guideline no. 437 (Oct. 2017), a substance with an IVIS > 55 induces serious eye damage, that should be classified as UN GHS Category I.

 

The negative control (HBSS) and the positive control (20% imidazole solution) have met the validity criteria.

 

No observations were made which might cause doubts concerning the validity of the study outcome. The test is considered valid.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation/corrosion:

The test substance was tested for its skin irritation potential in an in-vitro test system according to OECD 439 (Epiderm). Three tissues of the human skin model EpiDermTMwere treated with the test item for 60 minutes. The test item was applied directly to each tissue. DPBS-buffer was used as negative control and 5% SDS solution was used as positive control.

After treatment with the negative control, the absorbance values were within the required acceptability criterion. The positive control showed clear irritating effects. The mean value of relative tissue viability was reduced to 2.5% .

After the treatment with the test item, the mean value of relative tissue viability was reduced to 2.5 %. This value is below the threshold for skin irritation potential (50%). The test item was considered a skin irritant under the conditions of the study.

The test item was tested for skin corrosion potential in an in-vitro test according to OECD 431. Two tissues of the human skin model EpiDermTMwere treated with the test item for 3 minutes and 1 hour, respectively. The test item was applied to each tissue and spread to match the tissue size. Demineralised water was used as negative control, 8 M KOH was used as positive control.

After treatment, the respective substance was rinsed from the tissues. Then, cell viability of the tissues was evaluated by addition of MTT, which can be reduced to a blue formazan. Formazan production was evaluated by measuring the optical density (OD) of the resulting solution. After treatment with the negative control, the absorbance values were within the required acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for both treatment intervals thus showing the quality of the tissues. The OD was 1.7 (3 minutes experiment) and 1.6 (1 hour experiment).

The positive control showed clear corrosive effects for both treatment intervals. The mean relative tissue viability value was reduced to 6.1 % for the 1 hour treatment.

After 3 minutes treatment with the test item, the mean value of relative tissue viability was reduced to 84.7 %. This value is above the threshold for corrosion potential (50%). After 1 hour treatment, the mean value of relative tissue viability was reduced to 45.1 %. This value, too, is above the threshold for corrosion potential (15%). Therefore, the test item is considered non-corrosive to skin in the Reconstructed Human Epidermis (RHE) Test Method.

Eye irritation:

EpiOcular:

The test itemwas applied to a three-dimensional human cornea tissue model in duplicate for an exposure time of 6 hours. After treatment, the respective substance was rinsed from the tissue; then, cell viability of the tissues was evaluated by addition of MTT, which can be reduced to formazan. The formazan production was evaluated by measuring the optical density (OD) of the resulting solution. Demineralised water was used as negative control and methyl acetate was used as positive control. The controls showed the following results: After treatment with the negative control, the absorbance values were within the required acceptability criterion of mean OD>0.8 and < 2.5, OD was 1.7. The positive control showed clear eye irritating effects, the mean value of the relative tissue viability was 40.0% (< 50%). Variation within tissue replicates was acceptable (< 20%).

After treatment with the test item, the mean value of relative tissue viability was 1.9%.

This value is well below the threshold for eye irritation potential (≤ 60%). Test items that induce values below the threshold are considered either eye irritant or inducing serious eye damage.

 According to the OECD Guideline 492, the EpiOcularTMEye Irritation Test does not allow discrimination between eye irritation/reversible effects on the eye (Category 2) and serious eye damage/irreversible effects on the eye (Category 1). For these purposes, further testing with other suitable test methods is required. Under the conditions of the test,Ammonium hexafluorozirconateis considered either eye irritant or inducing serious eye damage in the EpiOcularTMEye Irritation Test.

BCOP:

This in vitro study was performed to assess corneal damage potential of Ammonium hexafluorozirconate by quantitative measurements of changes in opacity and permeability in a bovine cornea.

Six experiments were performed, but two of them were invalid, because one validity criterion of the negative control was not met. These results are not reported but kept with the raw data in the GLP-archive of the test facility. Further three experiments were performed, which led to equivocal results. This is why a sixth experiment was performed with the undiluted test item, because it was assumed that the suspension was not homogeneous. Only this valid experiment is described in detail.

 The test item Ammonium hexafluorozirconatewas brought onto the cornea of a bovine eye which previously had been incubated with cMEM without phenol red at 32 ± 1 °C for 1 hour and whose opacity had been determined. The test item was incubated on the cornea for 4 hoursat32 ± 1 °C. After removal of the test item, opacity and permeability values were measured.

The test item was tested neat. Under the conditions of this test, the test itemAmmonium hexafluorozirconateinduced serious eye damage on the cornea of the bovine eye. The calculated mean IVIS was 70.70.

According to OECD Guideline no. 437 (Oct. 2017), a substance with an IVIS > 55 induces serious eye damage, that should be classified as UN GHS Category I.

 

Justification for classification or non-classification

According to Regulation (EU) No 1272/2008 (CLP) ammonium hexafluorozirconate is classified in Category 2 regarding skin irritation and Category 1 for eye irritation/damage.