Cesium tungsten oxide

Endpoint summary

Description of key information

Skin irritation/corrosion

Not irritating, OECD 439, Warren (2017)

Eye irritation/damage

Not possible to conclude, OECD 490, Henzell (2017)

Not irritating, OECD 492, Miyaura (2017)

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:

15 Nov-21 Nov 2016

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:

adopted 28 July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Commission Regulation (EC) No. 761/2009, adopted 23 July 2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom, London, England

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

The reconstructed human epidermis model showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation being used as a replacement for the Draize Skin irritation test. Test items are applied topically as the dermal route is the most likely exposure route and the results of the study are believed to be of value in predicting the likely skin irritancy potential to man.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: The EpiSkin™ model
- Tissue batch number(s): 16-EKIN-046
- Delivery date: 15 Nov 2016
- Date of initiation of testing: 15 Nov 2016

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

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: All tissues were rinsed repeatedly with DPBS with Ca++ and Mg++ for approximately 40 seconds after the exposure period.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 h
- Spectrophotometer: Anthos 2001 microplate reader
- Wavelength: 562 nm
- Filter: no reference filter was used
- Linear OD range of spectrophotometer: no data

NUMBER OF REPLICATE TISSUES: 3
CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues/killed tissues: killed tissues
- Procedure used to prepare the killed tissues: incubating in distilled water at 37 °C for 48 h
- N. of replicates: 3
- Method of calculation used: direct comparison of treated to untreated water-killed tissues

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

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be non-corrosive to skin if the viability after 15 minutes exposure period to the test substance followed by 42 hours post-exposure incubation is greater than or equal to 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 10 mg

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): DPBS, 10 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): SDS, 10 µL
- Concentration (if solution): 5% (w/v)

Duration of treatment / exposure:

15 min

Duration of post-treatment incubation (if applicable):

42 h

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

15-min exposure followed by 42-hour post-exposure incubation period

Value:

113.5

Vehicle controls validity:

not examined

Negative controls validity:

valid

Remarks:

100%

Positive controls validity:

valid

Remarks:

11.1%

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct-MTT reduction: An assessment found the test item was able to directly reduce MTT. The results of the procedure showed that no interference due to direct reduction of MTT occurred. It was therefore considered unnecessary to use the results of the water-killed tissues for quantitative correction of results or for reporting purposes.
- Colour interference with MTT: An assessment found the test item had the potential to cause colour interference with the MTT endpoint. The results of the procedure showed that no colour interference occurred. It was therefore considered unnecessary to use the results of the colour correction tissues for quantitative correction of results or for reporting purposes.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD562 for the negative control treated tissues was 0.844 and the standard deviation value of the viability was 14.2%. The negative control acceptance criteria was therefore satisfied.
- Acceptance criteria met for positive control: The relative mean tissue viability for the positive control treated tissues was 11.1% relative to the negative control treated tissues and the standard deviation value of the viability was 1.5%. The positive control acceptance criteria was therefore satisfied.
- Acceptance criteria met for variability between replicate measurements: The relative mean tissue viability for the test item treated tissues was 113.5% after a 15-minute exposure period and 42-hour post-exposure incubation period. The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 13.6%. The test item acceptance criteria was therefore satisfied.

 Mean OD562 Values and Viabilities for the Negative Control, Positive Control, and Test Items

Item	OD562 of tissues	Mean OD562 of triplicate tissues	± SD of OD562	Relative individual tissue viability (%)	Relative mean viability (%)	± SD of Relative mean viability (%)
Negative control item	0.839	0.844	0.120	99.4	100*	14.2
	0.966			114.5
	0.727			86.1
Positive control item	0.090	0.094	0.013	10.7	11.1	1.5
	0.109			12.9
	0.084			10.0
Test item	0.906	0.958	0.115	107.3	113.5	13.6
	1.090			129.1
	0.879			104.1

* = The mean viability of the negative control tissues is set at 100%

OD = Optical density

SD = Standard deviation

Interpretation of results:

other: CLP/EU GHS criteria are not met, no classification required according to Regulation (EC) No 1272/2008

Conclusions:

EU CLP: Not classified for Irritation
UN GHS: Not classified for Irritation (Category 3: cannot be determined)

Executive summary:

The skin irritation potential of the undissolved test item was assessed in accordance with OECD Guidance 439.  Triplicate tissues were exposed to the test item for ≥ 15 minutes. At the end of the exposure period the test item was rinsed and the tissues incubated for a further 42 h in the presence of maintenance solution. Each tissue was then loaded with MTT for 3 h and any resultant colour extracted. After incubation and extraction, the solutions were aliquoted for absorbance measurements. Absorbency at 562 nm for each well was measured using a spectrophotometer. The test item did not cause interference in interpretation of the MTT colourmetric assy. Mean viability of tissues exposed to the test substance after 60 minutes were > 100 %. The quality criteria required for acceptance of the results were met.  Under the conditions of this study the test substance is considered not to be irritant to the skin in accordance with EU CLP regulation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 Jan 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Version / remarks:

Adopted 26 Jul 2013

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Version / remarks:

Commission Regulation (EC) No. 440/2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom, London, England

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Local abattoir as a by-product from freshly slaughtered animals.
- Characteristics of donor animals: Adult cattle, typically 12 to 60 months old
- Storage, temperature and transport conditions of ocular tissue: Eyes were excised after slaughter and were transported on the same day (of slaughter) over ice packs in Hank's Balanced Salt Solution (HBSS) supplemented with antibiotics.
- Time interval prior to initiating testing: Corneas were prepared immediately on arrival
- indication of any existing defects or lesions in ocular tissue samples: The eyes were free of defects such as scratches, and neovascularization. Prior to testing, corneal opacity was determined by the amount of light transmitted through the cornea via a opacitometer.
- Indication of any antibiotics used: Penicillin 100 IU/mL and streptomycin 100 µg/mL in HBSS.

Vehicle:

physiological saline

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 0.75 mL
- Concentration (if solution): 20% (w/v) solution in 0.9% (w/v) sodium chloride

VEHICLE
- Amount applied: 0.75 mL
- Concentration (if solution): 0.9% sodium chloride solution
- Lot/batch no.: Aguettant Ltd. batch number 3011542
- Purity: 0.9%

Duration of treatment / exposure:

240 minutes at 32 ± 1 °C

Observation period (in vivo):

Not applicable

Duration of post- treatment incubation (in vitro):

Not applicable

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF CORNEAS: Sourced from a local abattoir as a by-product from freshly slaughtered animals.

QUALITY CHECK OF THE ISOLATED CORNEAS: The eyes were visually inspected to be free of defects such as scratches, and neovascularization. Prior to testing, corneal opacity was determined by the amount of light transmitted through the cornea via a opacitometer.

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: 0.9% sodium chloride

POSITIVE CONTROL USED: Imidazole 20% (w/v) in 0.9% (w/v) sodium chloride

APPLICATION DOSE AND EXPOSURE TIME: 0.75 mL for 240 minutes at 32 ± 1 °C

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: at least three times

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: a post-treatment opacity was measured and each cornea was visually observed prior to incubation with sodium fluorescein
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of [UV/VIS spectrophotometry / microtiter plate reader] (OD490): after 90 minutes at 32 ± 1 °C
- Others: visual observations

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: the decision criteria as indicated in the TG was used

Irritation parameter:

in vitro irritation score

Run / experiment:

mean out of all 3 eyes

Value:

14.6

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Other effects / acceptance of results:

- Acceptance criteria:
OECD 437: "A test is considered acceptable if the positive control gives an IVIS that falls within two standard deviations of the current historical mean, which is to be updated at least every three months, or each time an acceptable test is conducted in laboratories where tests are conducted infrequently (i.e., less than once a month). The negative or solvent/vehicle control responses should result in opacity and permeability values that are less than the established upper limits for background opacity and permeability values for bovine corneas treated with the respective negative or solvent/vehicle control. A single testing run composed of at least three corneas should be sufficient for a test chemical when the resulting classification is unequivocal."

- positive control criterion: Imidazole 20% (w/v) was used for positive control purposes. The test was acceptable if the positive control produced an in vitro Irritancy Score which fell within two SD of the historical mean during 2015 for this testing facility. Therefore the in vitro Irritancy score should fall within the range of 50.8 to 100.4.
- negative control criterion: Sodium chloride 0.9% (w/v) was used for negative control purposes. The test was acceptable if the negative control produced an in vitro Irritancy Score which is less than or equal to the upper limit for background opacity and permeability values during 2015 for bovine corneas treated with the respective negative control. When testing solids the negative control for opacity should be ≤ 5.4 and for permeability ≤ 0.070.

IVIS Classification
≤ 3 No Category. Not requiring classification to UN GHS or to EU CLP
> 3; ≤ 55 No prediction on eye irritation can be made.
> 55 Category 1. UN GHS or EU CLP Causes serious eye damage.
____________________________________________________________________________________________________________________________
IVIS Scores

Treatment IVIS Score
Test Item 14.6
Negative Control 1.1 The negative control gave opacity of ≤ 5.4 and permeabilty of ≤0.070, therefore acceptance criteria were satisfied.
Positive Control 93.1 The positive control IVIS was within the range of 50.8 to 100.4, therefore the acceptance criteria were satisfied.

 

Treatment	Cornea Number	Opacity				Permeability (OD)		In Vitro Irritancy Score (IVIS)
		Pre-trmt	Post-trmt	Post - Pre	Corrected		Corrected
Negative Control	1	3	3	0		0.017
	2	3	4	1		0.039
	3	3	4	1		0.024
				0.7*		0.027^@		1.1
Positive Control	4	3	81	78	77.3	1.481	1.454
	5	3	68	65	64.3	1.939	1.912
	6	2	62	60	59.3	1.876	1.849
					67.0^#		1.739^#	93.1
Test Item	7	2	13	11	10.3	0.119	0.092
	8	2	18	16	15.3	0.049	0.022
	9	2	19	17	16.3	0.025	0.000
					14.0^#		0.038^#	14.6

OD = optical density

trmt = treatment

*= mean of the post-treatment – pre-treatment values

^#= mean corrected values

^@= mean permeability

Interpretation of results:

study cannot be used for classification

Conclusions:

No prediction on eye irritation can be made

Executive summary:

The Bovine Corneal Opacity and Permeabilty (BCOP) test was conducted using undissolved test item in accordance with OECD Guideline 437. Test item was suspended in sodium chloride (20 % w/v) and 0.75 mL applied evenly to the surface of three corneas, the test time was 240 minutes . A negative and positive control group, each containing 3 corneas, were also prepared.  Measurements for corneal opacity and corneal permeability were made.  Corneal opacity and corneal permeability media solutions were analysed by a spectrophotometer at 490 nanometers (OD490). The test item did not interfere with result interpretation.   

The resulting in an In Vitro Irritation Score (IVIS) of ~14.  It was concluded that under the condition of this study the test item no prediction could be made in respect of its potential to cause eye irritation, but the test item does not cause eye damage/corrosion.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Remarks:

RhCE test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16-18 May 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:

Adopted 28 Jul 2015

Deviations:

no

GLP compliance:

yes

Species:

human

Strain:

other: Construct of normal human keratinocytes

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability
The EpiOcular™ human cell construct for eye irritation testing is a nonkeratinized epithelium prepared from normal human keratinocytes (MatTek Corporation, MA, U.S.A.). It models the corneal epithelium with progressively stratified, but not cornified cells. The ability to expose the tissue topically is essential to model the same kind of progressive injury expected in vivo. In this assay, the test item is applied to the surface of the corneal epithelial construct for a fixed period, removed, and the tissue allowed to express the resulting damage. Two construct tissues (replicates) are used for each test treatment and each control group; a 6-h exposure with an 18 hour incubation post-exposure. Relative tissue viability post-exposure is determined against the negative control-treated constructs by evaluating the reduction of MTT to a formazan product, determined spectrophotometrically (optical density). A concurrent positive control is used with each assay to determine validity of the test. Based on the "depth of injury model," the EpiOcular eye irritation test (EIT) is intended to differentiate those materials that are nonirritants (would not require a warning label in the European chemical classification systems) from those that would require labelling as either GHS eye irritant category 1 or 2.

- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live
The EpiOcular™ human cell construct for eye irritation testing (OCL-200-EIT) (Lot No. 20983) was obtained from MatTek Corporation, MA, U.S.A., and tested by MatTek for potential contaminants, sterility, barrier function and tissue viability, results for which were within acceptable ranges and inc=dicated appropriate formation of ther mucosal barrier and a viable basal cell layer..

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): 50 mg applied topically per tissue.

Duration of treatment / exposure:

6 h at 37°C in a humidified atmosphere of 5% CO2.

Duration of post- treatment incubation (in vitro):

18 h

Number of animals or in vitro replicates:

Two tissue replicates were exposed per treatment with the test material, positive control and negative control.

Details on study design:

- Details of the test procedure used: EpiOcular™ was delivered and each cultured tissue was removed, inspected and transferred to plates containing assay medium and incubated for 60 minutes, followed by transferring out the assay medium, adding fresh assay medium, and pre-incubating overnight at 37°C, in a humidified atmosphere of 5% CO2. At day 2, the tissues were pre-treated by wetting with PBS and incubated at standard culture conditions for 30 minutes. The negative and positive controls were tested by applying 50 µL topically on the tissues. The test material was tested by applying topically onto the tissue surface at 50 mg per tissue; two tissues (replicates) were used per treatment with test material, negative and positive controls. The cultures were returned to the incubator for 6 hours at 37°C, in a humidified atmosphere of 5% CO2. After the 6-hour exposure time, tissues were rinsed with PBS (three times) to remove any residual test material. After rinsing, the tissues were immediately transferred to and immersed in assay medium for a 25-minute immersion incubation (post-soak). The tissues were then returned to assay medium, and post-incubated for an additional 18 hours at 37°C, in a humidified atmosphere of 5% CO2. Then the cultures were transferred to 0.3 mL of MTT reagent (1 mg/mL) and incubated for 3 hours. After incubation, the cultures were transferred to new tissue well-plates, and extracted in 2 mL of 2-propanol for 2 hours, with plate shaking. After 2 hours, the extracts were mixed to obtain homogeneous solutions, and duplicate volumes of 200 μL of each extraction solution were transferred to a 96-well plate and their absorbances (ODs) were recorded, while 200 μL of 2-propanol was used as the blank.
- RhCE tissue construct used, including batch number: The EpiOcular™ human cell construct for eye irritation testing (OCL-200-EIT) (Lot No. 20983), MatTek Corporation, MA, U.S.A.
- Doses of test chemical and control substances used: test material: 50 mg of AF-378; negative control: 50 µL of distilled water (water for injection, Lot K6G73, Otsuka Pharmaceutical Factory); positive control: 50 µL of Methyl acetate (Lot 050117ALA, MatTek Corporation)
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable): Exposure: 6 hours at 37°C in a humidified atmosphere of 5% CO2; Post-exposure immersion: 25 min not further specified; Post-exposure incubation: 18 hours at 37°C in a humidified atmosphere of 5% CO2.
- Justification for the use of a different negative control than ultrapure H2O (if applicable): Water for injection was used for the negative control article
- Justification for the use of a different positive control than neat methyl acetate (if applicable): Methyl acetate was used
- Description of any modifications to the test procedure: None
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable): The test material was tested for tissue-binding in assay medium without MTT, with resultant staining being below 60%. Therefore Mean cell viability of the test substance group was reduced by the Mean staining ratio. The test material was tested for direct MTT-reduction and results were negative.
- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable): Two replicates per treatment: test material, positive and negative controls
- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer): Wavelength of 570 nm, measured on a spectrophotometer
- Description of the method used to quantify MTT formazan: Incubation in 0.3 mL of MTT reagent (1 mg/mL) at 37°C, in a humidified atmosphere of 5% CO2.
- Acceptable variability between tissue replicates for positive controls: The difference in viability in the positive control group must be <50%
- Acceptable variability between tissue replicates for negative controls: Mean OD in the negative control group was >0.8 and <2.5.
- Acceptable variability between tissue replicates for the test material: The difference in viabilities in each treatment group (test material, positive control, negative control) must be <20%

Irritation parameter:

other: percent viability

Run / experiment:

two tissue replicates per treatment

Value:

ca. 63.3

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Negative control OD >0.8 and <2.5
- Acceptance criteria met for positive control: Mean relative viability of the Positive control at 6 hours exposure is <60% of the Negative control viability
- Acceptance criteria for all materials tested: The difference of viability between the two replicate tissues of the test material, positive control, and negative control is <20%

Eye irritation potential of YM-01A after 6-hours exposure in the human eye irritation test model: EpiOcular^TM

Treatment group	Tissue Mean OD	Cell Viability (%)	Corr. value^a	Difference^b	Category
Negative control (distilled water)	1.594	100	--	2.8
Positive control (Methyl acetate)	0.475	29.8	--	0.8
Test substance (YM-01A)	1.019	63.9	63.3	12.2	Non-irritant

^acell viability corrected for tissue staining

^bdifference of cell viability between the two tissues

Interpretation of results:

other: CLP/EU GHS criteria are not met, no classification required according to Regulation (EC) No 1272/2008

Conclusions:

The mean relative absorption value of the tissues corresponding to the cornea viability was ≥ 60 % compared with the value of the negative control (threshold for irritancy: ≤ 60%). In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item did not possess an eye irritating potential.  Therefore does not need to be classified as an irritant under the CLP regulation (Regulation (EC) 1272/2008).

Executive summary:

This in vitro study was performed to assess the eye irritation potential of the test item. Tissues of the human cornea model were treated with the test item, the positive and the negative control for 6 hours for each in duplicate. The test item was applied topically. The test was considered valid. The mean relative absorption value of the tissues corresponding to the cornea viability was ≥ 60 % compared with the value of the negative control (threshold for irritancy: ≤ 60%). In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item did not possess an eye irritating potential.  Therefore does not need to be classified as an irritant under the CLP regulation (Regulation (EC) 1272/2008).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin Irritation / Corrosion

OECD 439 (2017) - The skin irritation potential of the undissolved test item was assessed in accordance with OECD Guidance 439.  Triplicate tissues were exposed to the test item for ≥ 15 minutes. At the end of the exposure period the test item was rinsed and the tissues incubated for a further 42 h in the presence of maintenance solution. Each tissue was then loaded with MTT for 3 h and any resultant colour extracted. After incubation and extraction, the solutions were aliquoted for absorbance measurements. Absorbency at 562 nm for each well was measured using a spectrophotometer. The test item did not cause interference in interpretation of the MTT colourmetric assy. Mean viability of tissues exposed to the test substance after 60 minutes were > 100 %. The quality criteria required for acceptance of the results were met.  Under the conditions of this study the test substance is considered not to be irritant to the skin in accordance with EU CLP regulation.

 

Eye Irritation

OECD 437 (2017) - The Bovine Corneal Opacity and Permeabilty (BCOP) test was conducted using undissolved test item in accordance with OECD Guideline 437. Test item was suspended in sodium chloride (20 % w/v) and 0.75 mL applied evenly to the surface of three corneas, the test time was 240 minutes . A negative and positive control group, each containing 3 corneas, were also prepared.  Measurements for corneal opacity and corneal permeability were made.  Corneal opacity and corneal permeability media solutions were analysed by a spectrophotometer at 490 nanometers (OD490). The test item did not interfere with result interpretation.   

The resulting in an In Vitro Irritation Score (IVIS) of ~14.  It was concluded that under the condition of this study the test item no prediction could be made in respect of its potential to cause eye irritation, but the test item does not cause eye damage/corrosion.

 

OECD 492 (2017) - This in vitro study was performed to assess the eye irritation potential of the test item. Tissues of the human cornea model were treated with the test item, the positive and the negative control for 6 hours for each in duplicate. The test item was applied topically. The test was considered valid. The mean relative absorption value of the tissues corresponding to the cornea viability was ≥ 60 % compared with the value of the negative control (threshold for irritancy: ≤ 60%). In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item did not possess an eye irritating potential.  Therefore does not need to be classified as an irritant under the CLP regulation (Regulation (EC) 1272/2008).

 

The studies were conducted on the particulate nanoform (i.e. not the dissolved form). Based on dissolution study results (Rosenfeldt, 2021, Section 4) this aligns with the draft ECHA guidance (Appendix R7-1 for nanoforms applicable to Chapter R7a and R7c Endpoint specific guidance, draft v3.0, 2021), whereby testing on the nanoform should be conducted if the nanoform is not highly soluble in water (>33,3 g/L) and/or does not have a half-life of water dissolution ≤ 10 min (Appendix R7-1 for nanoforms applicable to Chapter R7a and R7c Endpoint specific guidance, draft v3.0, 2021; Figure 1).  This is further supported by the lack of dissolution seen in any vehicle. 

Justification for classification or non-classification

CLP (Regulation (EC) No 1272/2008) criteria are not met, no classification required for skin or eye irritation. 

Data used: 

OECD 439 Test item results: cell viability = > 100 %, Warren, 2017.

OECD 437 IVIS score: 14.6, Henzell, 2017.

OECD 492 cell viability: 63.3 %, Miyuara, 2017.