CTO

Endpoint summary

Description of key information

Cesium Tungsten Oxide is considered non-irritant to skin in the Reconstructed human Epidermis (RhE) Test Method.

Cesium Tungsten Oxide is considered non-eye irritant in the EpiOcular(TM) Eye Irritation Test.

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:

01 to 15 December 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No. of test material: B0345
- Expiration date of the lot/batch: 08. Feb. 2022
- Purity: >99%
- Appearance: dark blue powder

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Details on animal used as source of test system:

The EpiDerm tissue consists of human-derived epidermal keratinocytes which have been cultured to form a multi-layered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers representing main lipid classes analogous to those found in vivo. The EpiDerm tissues are cultured on specially prepared cell culture inserts.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: commercially available EpiDerm™ -Kit, procured by MatTek. Designation of kit: EPI-200-SIT
- Tissue batch number(s): 25864
- Delivery date: 12. December 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1°C
- Temperature of post-treatment incubation: 37 ± 1°C

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: Tissue 1: 24.7 mg; Tissue 2: 25.1 mg; Tissue 3: 25.6 mg
- test item was applied directly to each tissue and spread to match the tissue size (0.63 cm²; as indicated by the supplier).

NEGATIVE CONTROL
- Amount(s) applied: 30 µL per tissue

POSITIVE CONTROL
- Amount(s) applied: 30 µL per tissue
- Concentration: solution in demineralised water containing 5% Sodium dodecyl sulphate (SDS)

Duration of treatment / exposure:

60 minutes

Duration of post-treatment incubation (if applicable):

19 hours and 5 minutes

Number of replicates:

One plate (3 tissues) each for the test item, for negative control, and for positive control

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

One valid experiment was performed

Value:

113.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The following validity criteria and results are provided:

Criterion: OD of negative control
Demanded: ≥ 0.8 and ≤ 2.8
Result: 1.5

Criterion: % tissue viability of positive control SDS
Demanded: ≤ 20% of negative control
Result: 2.2%

Criterion: SD of mean viability of the tissue replicates (%)
Demanded: ≤ 18%
Results: 1.6% (negative control), 0.2% (positive control), 2.3% (test item)

All validity criteria were met. Values for negative control and for positive control were within the range of historical data of the test facility.

Interpretation of results:

GHS criteria not met

Conclusions:

The test item, Cesium Tungsten Oxide, is considered as non-irritant to skin. After the treatment, the mean value of relative tissue viability was increased to 113.2%. This value is above the threshold for skin irritation (50%). All validity criteria were met.

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:

December 01 to 14, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

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:

2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No. of test material: B0345
- Expiration date of the lot/batch: 08. Feb. 2022
- Purity: >99%
- Appearance: dark blue powder

Species:

human

Details on test animals or tissues and environmental conditions:

The EpiOcularTM tissue consists of normal, human-derived keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cornea. It consists of highly organized basal cells. These cells are not transformed or transfected with genes to induce an extended life span. The EpiOcularTM tissues are cultured in specially prepared cell culture inserts with a porous membrane through which nutrients can pass to the cells. The tissue surface is 0.6 cm2.

Source: EpiOcularTM tissues were procured from MatTek In Vitro Life Science Laboratories, Mylnské Nivy 73, 82105 Bratislava, Slovakia.
Designation of the kit: OCL-200-EIT
Day of delivery: 12. December 2017
Batch no.: 27017

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: Tissue 1: 53.0 mg; Tissue 2: 52.5 mg

Duration of treatment / exposure:

6 hrs

Duration of post- treatment incubation (in vitro):

18 hrs

Number of animals or in vitro replicates:

2 replicates (Tissue 1 and Tissue 2)

Details on study design:

- Details of the test procedure used: EpiOcular ™ Eye Irritation Test (EIT) which predicts the acute ocular irritation potential of chemicals by measurement of its irreversible tissue damage caused by cytotoxic effects in the human cornea model.

- RhCE tissue construct used, including batch number: normal, human-derived keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cornea. It consists of highly organized basal cells. Batch No.: 27017

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods: The assay medium was warmed in the water bath to 37 ± 1°C. Viable tissues were transferred in the prepared 6-well-plate and incubated at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity for 1 hour. After the pre-incubation, the medium was replaced and the wells were filled with 1 mL fresh assay medium. All 6-well-plates were incubated at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity for 16 hours. After overnight incubation, the tissues were pre-wetted with 20 μL DPBS buffer and the tissues were incubated at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity for 30 min. After dosing the last tissue, all plates were transferred into the incubator for 6 hours at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity. At the end of exposure time, the tissues were immediately transferred into 5 mL of assay medium in pre-labelled 12-well plate for 25 minutes post soak at room temperature. After that, each insert was blotted on absorbent material and transferred into a pre-labelled 6-well plate, containing 1 mL assay medium. For post-treatment incubation, the tissues were incubated for 18 hours at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity. After the post-treatment incubation, the MTT Assay was performed.

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals:
Assessment of Direct Reduction of MTT by the Test Item:
The test item was tested for the ability of direct MTT reduction. To test for this ability, 53.1 mg of the solid test item were added to 1 mL of MTT solution in a 6-well plate and the mixture was incubated in the dark at 37 ± 1 °C, 5.0 ± 1 % CO2 and 80 – 100 % relative humidity for 3 hours. 1 mL of MTT solution plus 50 μL of H2O demin. was used as negative control. The MTT solution did not change its colour; therefore, direct MTT reduction had not taken place, and no data correction was necessary.

Assessment of Coloured or Staining Test Items:
53.0 mg of the test item were added to 2 mL isopropanol, incubated in 6-well plates on an orbital shaker for 3 hours at room temperature. Then, two 200 μL aliquots of the resulting solution and two 200 μL aliquots of neat isopropanol were transferred into a 96-well plate and measured with a plate reader at 570 nm. After subtraction of mean OD for isopropanol, the mean OD of the test item solution was 0.0025 (≤ 0.08). Therefore, the main test was performed without colourant controls.

- Number of tissue replicates used per test chemical and controls: 2 tissue replicates used for each (test item, positive control, negative control)

- Description of the method used to quantify MTT formazan:
A 24-well-plate was prepared with 300 μL freshly prepared MTT solution in each well. The tissue inserts were blotted on absorbent material and then transferred into the MTT solution. The plate was incubated for 190 minutes at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity. At last, each insert was thoroughly dried and set into a pre-labelled 6-well-plate, containing 2 mL isopropanol, taking care that no isopropanol is flowing into the tissue insert. The plate was firmly sealed to avoid evaporation of the solvent and then shaken for 2 hours at room temperature, protected from light. The inserts were removed from the 6-well plate and discarded. The content of each well was thoroughly mixed in order to achieve homogenisation. From each well, two replicates with 200 μL solution (each) were pipetted into a 96-wellplate. Eight wells with 200 μL isopropanol were pipetted also. The plate was read in a plate spectrophotometer at 570 nm.

Irritation parameter:

other: Eye irritation potential

Remarks:

Relative tissue viability

Run / experiment:

EpiOcular(TM) Eye Irritation Test

Value:

102.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Remarks:

Results expressed as %

Other effects / acceptance of results:

The following validity criteria and results are provided:

Criterion: OD of negative control
Demanded: >0.8 and <2.5
Result: 1.7

Criterion: % mean relative viability of positive control
Demanded: <50% of negative control
Result: 38.8%

Criterion: Variation within replicates
Demanded: <20%
Results: 3.9% (negative control), 2.0% (positive control), 3.1% (test item)

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.

Interpretation of results:

GHS criteria not met

Conclusions:

Cesium Tungsten Oxide is considered non-eye irritant in the EpiOcular(TM) Eye Irritation Test. After treatment with the test item, the mean value of relative tissue viability was increased to 102.3%. This value is above the threshold for eye irritation potential (≤ 60%). All validity criteria were met.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Additional information

Three tissues of the human skin model EpiDerm(TM) were treated with Cesium Tungsten Oxide for 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). After the treatment with the test item, the mean value of relative tissue viability was increased to 113.2%. This value is above the threshold for skin irritation potential (50%). Test items that induce values above the threshold of 50% are considered non-irritant to skin. Therefore, Cesium Tungsten Oxide is considered non-irritant to skin in the Reconstructed human Epidermis (RhE) Test Method. All validity criteria were met.

Cesium Tungsten Oxide is considered non-eye irritant in the EpiOcular(TM) Eye Irritation Test. After treatment with the test item, the mean value of relative tissue viability was increased to 102.3%. This value is above the threshold for eye irritation potential (≤ 60%). All validity criteria were met.

Justification for classification or non-classification

Cesium Tungsten Oxide is considered non-irritant to skin in the Reconstructed human Epidermis (RhE) Test Method. Therefore, cesium tungsten oxide is not classified for skin corrosion/irritation according to the criteria of the CLP Regulation (EC) No 1278/2008 as amended.

Cesium Tungsten Oxide is considered non-eye irritant in the EpiOcular(TM) Eye Irritation Test. Therefore, cesium tungsten oxide is not classified for serious eye damage/eye irritation according to the criteria of the CLP Regulation (EC) No 1278/2008 as amended.