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Administrative data

Description of key information

Skin irritation

In an in vitro skin irritation study (EpiSkin, according to OECD 439, Klimisch 1, Orovecz, 2017), tungsten zirconium (hydroxide) oxide was considered not to be irritating to skin.

Eye irritation

In an in vitro eye irritation study (EpiOcular, according to OECD 492, Klimisch 1, Grignard-Racinet, 2018), tungsten zirconium (hydroxide) oxide was considered not to be irritating to eyes.

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:
2017-08-23 to 2017-08-25
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:
28 July 2015
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
6 July 2012
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled room temperature (15-25°C, below 70 RH%)

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: None; the test item was applied as supplied, no formulation was required.
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: not specified (adult)
Source strain:
other: not applicable
Justification for test system used:
The EPISKIN (SM) model has been validated for irritation testing in an international validation study and its use is recommended by the relevant OECD guideline for irritation testing (OECD No. 439). Therefore, it was considered to be suitable for this study.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKINTM (SM) (0.38 cm²), SkinEthic, France
- Tissue batch number(s): 17-EKIN-034
- Expiry date: 28 August 2017
- Date of initiation of testing: August 23, 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 24.8-27.6°C
- Temperature of post-treatment incubation (if applicable): 37.0 ± 1.0°C
- All incubations were carried out in a humid atmosphere (> 95 RH%) containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C. Temperature and humidity were continuously monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Any variation to these conditions were evaluated and maintained in the raw data.

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: After 15 min incubation time, the EPISKIN (SM) units were removed and rinsed thoroughly with phosphate buffered saline (PBS) to remove any remaining material from the epidermal surface as much as possible. The rest of the PBS was removed from the epidermal surface with a pipette (without touching the epidermis).
- Observable damage in the tissue due to washing: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: yes, plate reader, not further specified
- Wavelength: 570 nm

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues: As the test item appeared to be an MTT-interacting substance, in addition to the normal procedure, two test item treated killed epidermis and two negative control treated killed epidermis were used in the study (untreated killed tissues may exhibit little residual NADH and dehydrogenase associated activity). The batch of killed tissues was different than the batch of the living tissues. The same treatment steps were followed in case of the killed tissues as in case of the living tissues.
As the test item had an intrinsic colour, further evaluation to detect colouring potential was necessary. Non Specific Colour % (NSCliving %) was determined in order to evaluate the ability of the test item to stain the epidermis by using additional control tissues. Therefore, in addition to the normal procedure, two additional test item treated living tissues were used for the non-specific OD evaluation. This tissue followed the same test item application and all steps as for the other tissues, except for the MTT step: MTT incubation was replaced by incubation with fresh Assay Medium to mimic the amount of colour from the test item that may be present in the test disks.
To avoid a possible double correction for colour interference, a third set of control for non-specific colour in killed tissues (NSCkilled) was performed.
- Procedure used to prepare the killed tissues (if applicable): For killed epidermis, living epidermis units (Manufacturer: SkinEthic, France, Batch No.:17-EKIN-024, Expiry Date: 19 June 2017) were placed in a 12 well plate with 2 mL of distilled water, then incubated at 37°C in an incubator with 5% CO2, in a > 95% humidified atmosphere for 48 hours (± 1 hour). At the end of the incubation the water was discarded and the dead epidermis units were frozen on 16 June 2017 (the frozen units can be used up to 6 months). Before use, the killed tissues were thawed at room temperature (at least 30 minutes in 2 mL of Maintenance Medium). Further use of killed tissues was similar to living tissues.
- Number of replicates: 2 for the test item and 2 for the negative control
- Method of calculation used: For test substances detected as able to stain the tissues and interfere with MTT a third set of controls is required before calculation of the “true” viability %.

Non Specific Colour % with killed tissues (NSCkilled%):
NSCkilled % = (mean ODCTK / mean ODNC)×100
ODCTK: test substance treated killed tissues (not incubated with MTT)
ODNC: negative control OD (incubated with MTT)

In that case the true MTT metabolic conversion (TODTT) will be undertaken as follows:
TODTT = [ODTT – (ODKT – ODKNC) – mean ODCTV + mean ODCTK]
ODTT: test substance treated viable tissues (incubated with MTT)
ODKT: test substance treated killed tissues OD
ODKNC: negative control killed tissues OD
ODCTV: test substance treated viable tissues (not incubated with MTT)
ODCTK: test substance treated killed tissues (not incubated with MTT)

The % relative viability (% RV) for each test substance replicate is calculated relative to the mean negative control:
% RV 1 = [TODTT1 / mean ODNC] × 100
% RV 2 = [TODTT2 / mean ODNC] × 100
% RV 3 = [TODTT3 / mean ODNC] × 100

The mean value of the 3 individual relative viability % for the test substance is calculated as follows:
Mean Relative Viability % = (% RV 1 + % RV 2 + % RV 3) / 3

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

DECISION CRITERIA
- The test substance is considered to be non-irritant to skin if the relative mean viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours post incubation is more than 50% of the mean viability of the negative controls.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 mg. As the test item was solid, first an appropriate amount (10 µL) of distilled water was applied to the epidermal surface in order to improve further contact between test item and epidermis.

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

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL
- Concentration (if solution): 5% (w/v)
Duration of treatment / exposure:
15 minutes (± 0.5 min)
Duration of post-treatment incubation (if applicable):
42 hours (± 1 h)
Number of replicates:
3 per test group (test item, negative control and poitive control)
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1 / mean of 3 replicates
Value:
86.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Remarks:
range of values: 82.6 - 91.2
Irritation / corrosion parameter:
other: optical density (TODTT)
Remarks:
corrected for non-specific MTT reduction (by subtracting the NSMTT value of 0.014)
Run / experiment:
1 / mean of 3 replicates
Value:
0.657
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: range of values: 0.624 - 0.690
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: no
- Colour interference with MTT: Yes, as the test item was coloured, two additional test item treated living tissues were used for the non-specific OD evaluation. The mean optical density (measured at 570 nm) of tissues was 0.007, Non Specific Colour % was calculated as 0.9%. This value was below 5%, therefore additional data calculation was not necessary.
- Direct-MTT reduction: Yes, as colour change (purple precipitate) was observed after three hours of incubation of the test item in MTT working solution, the test material might interact with MTT. Therefore, additional controls and data calculations were necessary to exclude the false estimation of viability. Based on these observed mean OD (0.014), the calculated NSMTT% was 1.8%. This was considered to be significant, thus correction with NSMTT was made.
- As the test item was showed being an MTT-interacting substance and the test item had an intrinsic colour, two additional test item treated killed tissues were used for the non-specific OD evaluation. The mean optical density (measured at 570 nm) of these tissues was determined as 0.015, Non Specific Colour % (NSCkilled%) was calculated as 1.9%. Because the NSCliving% was not used, correction with NSCkilled% was not necessary.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes. The mean OD value of the three negative control tissues was in the recommended range (0.756). Standard deviation of the viability results for negative control samples was 6.4%.
- Acceptance criteria met for positive control: Yes. The positive control treated tissues showed 6.1% viability demonstrating the proper performance of the assay. The standard deviation of the viability results for positive control samples was 1.4%.
- Acceptance criteria met for variability between replicate measurements: Yes, the standard deviation of viability values of the three test item-treated tissue samples in the MTT assay was 4.3%.
- The mean OD value of the blank samples (acidified isopropanol) was 0.047.
All these parameters met the acceptability criteria, therefore the study was considered to be valid.

Optical Density (OD) and the calculated Non Specific Colour % (NSCliving%) of the Additional Control Tissues

 Additional control    OD Measured  OD Blank corrected  NSC% (living)
 Treated with test item  1  0.053  0.006  
   2  0.055  0.008  0.9
   mean  ---  0.007  

Notes:

1.  Mean blank value was 0.047.

2. Optical density means the mean value of the duplicate wells for each sample (rounded to three decimal places).

Optical Density (OD) and the calculated relative viability % of the samples

 Substance     OD Measured  OD Blank corrected  TODTT  Viability (% RV)
 Negative Control:  1  0.850  0.803  ---  106.2
Phosphate buffered saline  2  0.753  0.706  ---  93.4
   3  0.806  0.759  ---  100.4
   mean  ---  0.756  ---  100.0
 Positive Control:  1  0.103  0.056  ---  7.4
 5% (w/v) SDS solution  2  0.095  0.048  ---  6.4
   3  0.082  0.055  ---  4.7
   mean  ---  0.046  ---  6.1
   1  0.717  0.670  0.657  86.9
 Test Item  2  0.685  0.638  0.624  82.6
   3  0.750  0.703  0.690  91.2
   mean  ---  0.670  0.657  86.9

Notes:

1. Mean blank value was 0.047.

2. Optical density means the mean value of the duplicate wells for each sample (rounded to three decimal places).

3. TODTT: the measured values were corrected for non-specific MTT reduction (by subtracting the NSMTT value of 0.014)

Interpretation of results:
GHS criteria not met
Conclusions:
Following exposure to the test item, the mean cell viability was 86.9% compared to the negative control (after adjustment for non-specific MTT reduction). This is above the threshold of 50%, therefore the test item was considered as being non-irritant to skin. The experiment met the validity criteria.
In conclusion, in the in vitro EPISKINTM (SM) model test, the results indicate that the test item is non-irritant to skin.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not 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:
2017-11-16 to 2017-12-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
Version / remarks:
28 July 2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability under test conditions: no data

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: None, the test item was used in its original form.
Species:
human
Strain:
other: Reconstructed human Cornea-like Epithelium
Details on test animals or tissues and environmental conditions:
- Source: MatTek, Bratislava, Slovak Republic.
- Selection: At receipt, the tissues were inspected for obvious defects prior to use.
- Storage conditions: At receipt, the tissues were equilibrated (in the 24-well shipping container) to room temperature for at least 15 minutes. Each tissue was inspected as specified in an internal procedure (a check of the batch number and of any information relating to the characterisation and integrity of the test system was performed and recorded in CiToxLAB France files). They were then removed from the 24-well shipping containers and placed in a 6-well plate containing 1 mL of pre-warmed assay medium. Tissues were then incubated for 1 hour at 37°C (± 2°C), 5% CO2 in a humidified incubator. After the pre-incubation period, the assay medium was removed and replaced by 1 mL of fresh assay medium before incubation overnight (16-24h) at 37°C (± 2°C), 5% CO2 in a humidified incubator.
- Description: The EpiOcularTM model consists of an airlifted, living, multilayered ocular tissue construction (surface 0.60 cm²), reconstructed from normal (non-transformed) human-derived keratinocytes. This is a non-keratinised epithelium which models the cornea epithelium with progressively stratified, but not cornified cells. The cells are cultured in proprietary serum-free culture media, which induces corneal differentiation and the formation of the organotypic 3D cornea-like model. The 3D tissue consists of highly organised cell layers similar to that found in the cornea. The model features a normal ultra-structure and is functionally equivalent to human in vivo tissue.
- Expiry date: The EpiOcular tissues were used within 72 hours of their production.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 51 mg (± 1 mg)

NEGATIVE CONTROL:
- Amount(s) applied (volume or weight with unit): 50 μL

POSITIVE CONTROL:
- Amount(s) applied (volume or weight with unit): 50 μL
Duration of treatment / exposure:
6 hours (± 15 minutes)
Duration of post- treatment incubation (in vitro):
18 hours (± 15 minutes)
Number of animals or in vitro replicates:
2 per test group (test item, negative control and positive control)
Details on study design:
Details of the test procedure used:
- RhCE tissue construct used, including batch number: EpiOcular tissue, MatTek, Bratislava, Slovak Republic. Batch number documented in a certificate of analysis archived in the study files.
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods: Exposure for 6 hours (± 15 minutes) at 37°C (± 2°C). The rinsed tissues were transferred to new wells of a pre-labeled 12-well plate containing 5 mL of assay medium pre-warmed at room temperature and then incubated for 25 minutes (± 2 minutes) at room temperature. This incubation in assay medium was intended to remove any test article from the tissue. However, residual amounts of test item were still noted on both test item treated tissues. At the end of the post-soak immersion, each insert of test item, negative and positive controls was blotted on absorbent material and transferred to an appropriate well of the pre-labeled 6-well plate containing 1 mL of assay medium. The tissues were then incubated for 18 hours (± 15 minutes) at 37°C (± 2°C), 5% CO2 in a humidified incubator.
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals: As the test item was found in the preliminary test not to have any colouring potential or any direct MTT-reducing properties, no additional controls were run during the main test.
- Number of tissue replicates used per test chemical and controls (positive control, negative control): 2
- Wavelength used for quantifying MTT formazan: 570 nm
- Description of the method used to quantify MTT formazan: Following the post-treatment incubation, a volume of 0.3 mL of a freshly prepared MTT solution (1.0 mg/mL) was added into new wells of pre-labeled 24-well plates. At the end of the post-treatment incubation, each insert was removed from the 6-well plate and gently blotted on absorbent paper. The tissues were then transferred to the MTT pre-filled wells and incubated for 3 hours (± 10 minutes) at 37°C (± 2°C), 5% CO2 in a humidified incubator. At the end of the 3-hour incubation period, the underside of each tissue was blotted on absorbent paper to dry. Each tissue was examined with the naked eye and the degree of MTT staining was evaluated. Again, at this step, residual amounts of test item were observed on both test item-treated tissues.
As the test item was a solid, the inserts of each test item, negative control and positive control treated tissues were transferred to a 6-well plate containing 2 mL of isopropanol in each well so that no isopropanol was flowing into the insert. This avoided any potential contamination of the isopropanol solution with any test item that may have remained on the tissue. Plates were surrounded with parafilm to prevent evaporation. Formazan extraction was performed overnight at +2-8°C and protected from light. At the end of the formazan extraction period, the plates were placed under orbital shaking at room temperature for 11 minutes. Since the test item was a solid, the tissues (test item, negative and positive control treated tissues) were not pierced. The extract solution was mixed using a pipette and two 200 µL aliquots were transferred to the appropriate wells of a pre-labeled 96-well plate. One 96-well plate was used for the negative and positive controls (placed at the opposite end of the plate), and a separate 96-well plate was used for test item treated tissues. For each 96-well plate, the average Optical Density value (OD) of 4 wells containing 200 µL of isopropanol only was used as the blank. The OD was measured at 570 nm using a plate reader.
- Acceptable variability between tissue replicates for positive and negative controls: Negative control acceptance criteria: mean cOD between 0.8 and 2.5. Positive control acceptance criteria: relative mean viability of the positive control is < 50% of the relative mean viability of the negative control.
- Acceptable variability between tissue replicates for the test chemical: Acceptable if the difference of viability between the two tissue replicates is < 20%.
- Data interpretation: A test substance is predicted as ocular irritant, if the mean relative tissue viability (%) of two tissues exposed to the test item is ≤ 60%.
Irritation parameter:
other: relative viability in %
Run / experiment:
1 / mean of 2
Value:
99
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Remarks:
individual values of % viability: 99-100
Irritation parameter:
other: optical density
Run / experiment:
1 / mean of 2
Value:
1.64
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: individual values: 1.628 - 1.652
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: no

- Test for direct MTT reduction with the test item:
The MTT solution containing the test item did not turn blue/purple when compared with the negative control. The test item was therefore considered not to have direct MTT reducing properties. As a result, no additional controls were performed on freeze-dead tissues in parallel to the main test.

- Test for the detection of the colouring potential of the test item:
During this test, as both water and isopropanol solutions containing the test item did not change colour, the test item was found not to have a colouring potential. As a result, no additional controls were used in parallel to the main test.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes. Mean cOD for the two replicate tissues was 1.636 and 1.662.
- Acceptance criteria met for positive control: Yes. 27 and 19% viability was observed for both tissue replicates in the positive control, whereas 99 and 100% viability was observed for the two tissue replicates in the negative control. The relative mean viability in the positive control is hence < 50% of that in the negative control.
- Acceptable variability between replicate tissues treated with test item: Yes. A difference of only 2%, 8% and 1% was obtained between the replicate tissues of the negative control, positive control and test item, respectively.
Interpretation of results:
GHS criteria not met
Conclusions:
Under the experimental conditions of the study, the test item is considered to be non-irritant to Reconstructed human Cornea-like Epithelium.
The substance does not need to be classified according to the CLP Regulation.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation/corrosion

One reliable (Klimisch 1) in vitro study is available (Orovecz, 2017). This study was performed according to OECD guideline 439 (Reconstructed Human Epidermis Test method, EPISKIN) and conform GLP requirements. The mean % tissue viability in the three tissue replicates exposed to the test item was 86.9%. A test item is considered to be non-irritant to skin if the mean relative viability of three individual tissues after 15 min exposure to the test item and 42 hours post-incubation is more than 50% of the mean viability of the negative controls. Since the mean viability in the negative controls was 100%, the test item was concluded to be non-irritant under the conditions of the study. Based on these results, the test item is considered not classified according to the CLP Regulation. The study of Orovecz (2017) is considered as the key study for endpoint coverage.

Eye irritation

One reliable (Klimisch 1) in vitro study is available (Grignard-Racinet, 2018). In this study, the test substance was tested for its eye irritation potential in a Reconstructed Human Cornea-like Epithelium assay performed according to OECD guideline 492 and conform GLP requirements. Since mean viability in the treated tissues after MTT reduction was 99%, which is higher than the cut-off level of 60%, the test results met the criteria for a non-irritant response. Therefore, the test substance could be concluded not to be classified for eye irritation under the CLP Regulation. This study is considered the key study for endpoint coverage.

Justification for classification or non-classification

Skin irritation/corrosion

The test item was demonstrated not to be irritant to skin in an in vitro study performed according to OECD guideline 439 and is therefore not to be classified according to the CLP regulation.

Eye irritation

The test item was demonstrated not to be irritant to eyes in an in vitro study performed according to OECD guideline 492 and is therefore not to be classified according to the CLP Regulation.