Cerium gadolinium oxide

Endpoint summary

Description of key information

Skin irritation / Corrosion: relative mean tissue viability (after 15 ± 0.5 minutes) = 101% - OECD 439 – In vitro RHE test method.

Eye serious damage/irritation: 2 in vitro studies  (BCOP - OECD 437 and EpiOcular - OECD 492) led to unconclusive results. Therefore, no prediction can be made regarding the classification of the substance for the eye irritation potential.

An in vivo study will be performed when the substance tonnage reaches the next tonnage band according to Annex VIII, point 8.2, column 2: An in vivo study for eye corrosion/irritation shall be considered only if the in vitro study(ies) under point 8.2.1 in Annex VII are not applicable, or the results obtained from these study(ies) are not adequate for classification and risk assessment.

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:

From 28 APR 2021 to 10 MAY 2021

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:

26 June 2020

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

The test item was a (white) powder, which is an appropriate form to test solids in this type of test.

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from multiple donors

Source strain:

not specified

Justification for test system used:

The in vitro test system of reconstructed human epidermis closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis. This model used in this study was a three-dimensional human epidermis model, consisting of adult human-derived epidermal keratinocytes which had been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days, which resulted in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small Model (EPISKIN-SM, 0.38 cm2) - Source: SkinEthic Laboratories, Lyon, France.

- Tissue batch number: 21-EKIN-018.

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

PREPARATION OF THE TEST SYSTEM:
- On the day of receipt the tissues were transferred to 12-well plates and pre-incubated with pre-warmed Maintenance Medium for approximately 23 hours at 37°C.

ENVIRONMENTAL CONDITIONS FOR INCUBATIONS:
- All incubations, with the exception of the test item incubation of 15 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 67 - 89%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.5 - 37.2°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was 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. Based on laboratory historical data these deviations are considered not to affect the study integrity.

REMOVAL OF TEST MATERIAL AND CONTROLS
- The tissues were washed with phosphate buffered saline to remove residual test item.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL in PBS (phosphate bufferred saline).
- Incubation time: 3 hours at 37°C.
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader.

NUMBER OF REPLICATE TISSUES: 3.

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

PREDICTION MODEL / DECISION CRITERIA :
- A test item is considered irritant in the skin irritation test if the relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is < 50% of the mean viability of the negative controls.
- A test item is considered non-irritant in the in vitro skin irritation test if the relative mean tissue viability of three individual tissues alter 15 minutes of exposure to the test item and 42 hours of post incubation is > 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 applied: the skin was moistened with 5µL Milli-Q water (Millipore Corp., Bedford, Mass., USA) to ensure close contact of the test item to the tissue and the solid test item (19.1 to 37.2 mg) was added into 12-well plates on top of the skin tissues.

NEGATIVE CONTROL
- Three tissues were treated with 25 µL PBS (negative control).

POSITIVE CONTROL
- Three tissues were treated with 25 µL 5% SDS (positive control). The positive control was re-spread after 7 minutes contact time.

Duration of treatment / exposure:

The exposure period was 15 +/- 0.5 minutes.

Duration of post-treatment incubation (if applicable):

The post treatment incubation duration was 42 +/- 1 hours.

Number of replicates:

The test was performed on a total of 3 tissues per test item together with negative and positive controls.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

One experiment lasting 15 minutes +/- 0.5 minutes.

Value:

101

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

The absolute mean OD 570 of the negative control tissues was within the laboratory historical control data range.

Positive controls validity:

valid

Remarks:

The positive control had a mean cell viability of 33% after 15 ± 0.5 minutes exposure .

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- Test of color interference: the test item was checked for color interference in aqueous conditions. Addition of the test item to Milli-Q and isopropanol resulted alter subtraction of the blank in an OD of -0.0007 and 0.0053, respectively. Therefore it was concluded that the test item did not induce color interference.
- Test of MTT reduction: in addition, because no color change was observed in the presence of MTT, it was concluded that the test item did not interact with the MTT endpoint (no direct reduction of MTT).
- The standard deviation value of the percentage viability of three tissues treated identically was <14%, indicating that the test system functioned properly.

Interpretation of results:

GHS criteria not met

Conclusions:

The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues was 101%. Since the mean relative tissue viability for the test item was above 50%, the test item was considered to be non-irritant in the in vitro skin irritation test under the experimental conditions described for this study and should not be classified according to the GHS and CLP criteria.

Executive summary:

The objective of this study was to evaluate the test substance for its ability to induce skin irritation on a human three dimensional epidermal model (EPISKIN Small model (EPISKIN-SM)). The possible skin irritation potential of the test item was tested through topical application for 15 minutes.

The study was carried out according to the OECD guideline 439: In vitro Skin Irritation: Reconstructed Human Epidermis Test Method, (corrected 26 June 2020).

Skin tissue was moistened with 5µL of Milli-Q water and at least 10 mg of the test item was applied as such directly on top of the skin tissue for 15 ± 0.5 minutes. After a 42 ± 1 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

Skin irritation is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues was 101%. Since the mean relative tissue viability for the test item was above 50% after 15 ± 0.5 minutes treatment, the test item was considered to be non-irritant.

The positive control had a mean cell viability of 33% after 15 ± 0.5 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was < 14%, indicating that the test system functioned properly.

In conclusion, the test item was non-irritant in the in vitro skin irritation test under the experimental conditions of this study and should not be classified according to GHS and CLP criteria.

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:

weight of evidence

Study period:

FROM 01 NOV 2021 to 26 NOV 2021

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 June 18, 2019.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

The test item was a white powder, which is an appropriate form to test solids in this type of test.

Species:

human

Strain:

not specified

Details on test animals or tissues and environmental conditions:

- Justification of the test method : in the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro eye irritation tests is the EpiOcular test, which is recommended in international guidelines and scientific publications.
- Description of the cell system used: the EpiOcular tissue construct is a non-keratinized epithelium (0.6 cm2) prepared from normal human keratinocytes (MatTek).
- RhCE tissue used: OCL-200-EIT MatTek Corporation, Lot: 32878. Keratocyte strain 4F1188.
- Analysis for tissue functionality:
1)Tissue viability: MTT QC Assay, 1 hour, n=3; Result = 1.797+/- 0.121 for OD (540-570 nm) acceptance criteria of [ 1.1-3.0]
2) Barrier function: ET-50 assay, 100 µL 0.3% Triton X-100, 3 time points, n=2, MTT Assay. Result = 18.81 min for ET-50 acceptance criteria of [12.2 - 37.5 min].
3) Sterility: long term antibiotic and antimycotic free culture. Result = sterile for acceptance criteria of [no contamination].

- Environmental conditions: all incubations, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 56 - 92%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.1 - 36.9°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was 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. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

At least 50 mg (73.4 mg) of the solid test item (powder) was added into the 6-well plates on top of the tissues.

Duration of treatment / exposure:

6 hours +/- 15 minutes.

Duration of post- treatment incubation (in vitro):

After rinsing the cell culture inserts were each dried carefully and immediately transferred to and immersed in 5 mL of previously warmed Assay Medium (room temperature) in a pre-labeled 12-well plate for a 25 ± 2 minutes immersion incubation at room temperature (Post-Soak).
After the Post-Soak period cell culture inserts were each dried carefully and transferred to the 6-well plate containing 1.0 mL of warm Assay Medium and were incubated for 18 hours ± 15 minutes at 37°C

Number of animals or in vitro replicates:

2 replicates (tissues) for each of the test item, the negative control and the positive control.

Details on study design:

Details of the test procedure used:
- On the day of receipt, the tissues were equilibrated (in its 24-well shipping container) to room temperature. Subsequently, tissues were transferred to 6-well plates and incubated for 20 ± 4 hours at 37°C in 1.0 mL fresh pre-warmed Assay Medium, which was refreshed after approximately 1 hour. Assay Medium was supplied by MatTek Corporation, Ashland, USA. Before the assay was started, the entire tissues were pre-wetted with 20 μL of Ca2+Mg2+ Free D-PBS. The tissues were incubated at standard culture conditions for a minimum of 30 minutes.
- No correction was made for the purity/composition of the test item. The solid test item (73.4 mg) was applied and spread into the 6-well plates on top of the tissues, to match their size. After the exposure period with the test item (6 hours ± 15 minutes at 37.0 ± 1.0°C), the tissues were thoroughly rinsed with Ca2+Mg2+ free D-PBS (brought to room temperature) to remove residual test item. After rinsing, the cell culture inserts were each dried carefully and immediately transferred to and immersed in 5 mL of previously warmed Assay Medium (room temperature) in a pre-labeled 12-well plate for a 25 ± 2 minutes immersion incubation at room temperature (Post-Soak). After the Post-Soak period, cell culture inserts were each dried carefully and transferred to the 6-well plate containing 1.0 mL of warm Assay Medium and were incubated for 18 hours ± 15 minutes at 37°C.

- Doses of control substances used:
1) negative control: 2 tissues were treated with 50 µL Milli-Q water.
2) positive control: 2 tissues were treated with 50 µL Methyl Acetate.

- Test for the Interference of the Test Item with the MTT Endpoint: the test item had been tested previously for possible direct MTT reduction and color interference in the Skin irritation test using EpiSkinTM as a skin model (see Skin Irr. in vitro KS V1 2021JACO - Study No. 20302180). The solutions were not turned blue / purple nor a blue / purple precipitate was observed in presence of MTT, therefore it was concluded that the test item did not interfere with the MTT endpoint. The Optical Density (OD) for the test item solution was ≤ 0.08, therefore it was concluded that the test item did not interact with the MTT measurement.
- Description of the method used to quantify MTT formazan: after incubation, cell culture inserts were dried carefully to remove excess medium. The cell culture inserts were transferred into a 24-wells plate prefilled with 0.3 mL MTT-medium (1.0 mg/mL). The tissues were incubated for 180 ± 10 minutes at 37°C. After incubation with MTT-medium, the tissues were placed on blotting paper to dry the tissues and then transferred to a pre-labeled 6-well plate containing 2 mL isopropanol in each well so that no isopropanol was flowing into the insert. Formazan was extracted with 2 mL isopropanol for 2 - 3 hours at room temperature with gentle shaking.
- Quantification of MTT formazan: The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.
- Evaluation criteria: cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. Eye hazard potential of the test item was classified according to remaining cell viability following exposure of the test item. The test chemical is identified as not requiring classification and labeling according to UN GHS (No Category) if the mean percent tissue viability after exposure and post-exposure incubation is > 60%. In this case no further testing in other test methods is required. The test chemical is identified as “no prediction can be made” if the mean percent tissue viability after exposure and postexposure incubation is ≤ to 60%.

- Positive and negative control means and acceptance ranges based on historical data:
Absorption OD 570:
1) negative control : Range [ 0.648 - 2.190], mean = 1.595, SD = 0.261 (n=76).
2) positive control : Range [ 0.027 - 0.720], mean = 0.372, SD = 0.172 (n=76).
Viability % :
Positive control: Range [ 1.72 - 44.55], mean = 23.55, SD = 10.40 (n=76).
- Acceptable variability between tissue replicates for negative and positive controls: the absolute mean OD570 of the two tissues of the negative control should reasonably be > 0.8 and < 2.5. and the mean relative tissue viability of the positive control should be <50% relative to the negative control.
- Acceptable variability between tissue replicates for the test chemical: the difference between the % tissue viabilities of the two identically treated replicates should be <20.

Irritation parameter:

mean percent tissue viability 

Run / experiment:

One run.

Value:

56

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range.

Positive controls validity:

valid

Remarks:

The positive control had a mean cell viability after 6 hours ± 15 minutes exposure of 20%.

Remarks on result:

not determinable

Other effects / acceptance of results:

The difference between the percentage of viability of two tissues treated identically was 18%, indicating that the test system functioned properly.

Results of the experiment are detailed below:

Table 1: Mean Absorption in the EpiOcular™ Test with the test item

 

	A (OD570)	B (OD570)	Mean +/- SD (OD570)
Negative control	1.385	1.656	1.521 +/-  0.192
Test item	0.823	0.870	0.847 +/-  0.033
Positive control	0.258	0.344	0.301 + /- 0.061

OD = optical density

SD = Standard deviation

Duplicate exposures are indicated by A and B.

In this table the values are corrected for background absorption (0.0433). Isopropanol was used to measure the background absorption.

 

Table 2: Mean Tissue Viability in the EpiOcular™ Test with the test item

 

	Mean tissue viability (percentage of control)	Difference between two tissues (percentage)
Negative control	100	18
Test item	56	3.1
Positive control	20	5.7

 

Interpretation of results:

study cannot be used for classification

Remarks:

Eye hazard potential is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 6 hours ± 15 minutes treatment with the test item compared to the negative control tissues was 56%. Since the mean relative tissue viability for the test item was below or equal to 60% after 6 hours ± 15 minutes treatment, the test item is considered to be potentially irritant or corrosive to the eye.

Conclusions:

In conclusion, the test item was identified as no prediction can be made regarding the classification in the EpiOcular™ test under the experimental conditions described for this study.

Executive summary:

The objective of this study was to evaluate the eye hazard potential of Cerium gadolinium oxide. For this purpose Cerium gadolinium oxide was topically applied on the Reconstructed Human EpiOcular™ Model. The study procedures were based on the OECD test guideline n° 492 and the study was performed according to GLP.

The test item (73.4 mg) was applied as such directly on top of 2 EpiOcular tissues for 6 hours ± 15 minutes. Positive (Methyl Acetate) and negative (Sterile Milli-Q water) controls were added in this study (2 tissues each) using the same experimental conditions than the test item.

After exposure the cornea epithelial construct was thoroughly rinsed to remove the test substances and transferred to fresh medium for an immersion incubation. Afterwards, the tissues were transferred to fresh medium and incubated for 18 hours at standard culture conditions, prior to determination of the cytotoxic (irritancy) effect using a MTT assay.

The positive control  (Methyl Acetate) had a mean cell viability of 20% after 6 hours ± 15 minutes exposure. The absolute mean OD₅₇₀ (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The difference between the percentage of viability of two tissues treated identically was 18%, indicating that the test system functioned properly.

Eye hazard potential is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 6 hours ± 15 minutes treatment with the test item compared to the negative control tissues was 56%. Since the mean relative tissue viability for the test item was below or equal to 60% after 6 hours ± 15 minutes treatment, the test item was considered to be potentially irritant or corrosive to the eye.

In conclusion, the test item was identified as no prediction can be made regarding the classification in the EpiOcular™ test under the experimental conditions described for this study.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From 29 APRIL 2021 to 01 JUNE 2021.

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 i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

26 June 2020.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

The test item was a white powder.

Species:

cattle

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: bovine eyes from young cattle were obtained from the slaughterhouse (Vitelco, -'s Hertogenbosch, The Netherlands), where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter.
- Characteristics of donor animals : the animals were ± 6 -12 months old.
- Storage, temperature and transport conditions of ocular tissue : eyes were collected and transported in physiological saline in a suitable container under cooled conditions.
- Time interval before treatment: the eyes were tested the day of arrival in the laboratory.
- Indication of any existing defects or lesions in ocular tissue samples: the eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded.
- Preparation of corneas: the isolated corneas were stored in a petri dish with cMEM (Eagle’s Minimum Essential Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-glutamine (Life Technologies) and 1% (v/v) Fetal Bovine Serum (Life Technologies)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of Duratec Analysentechnik GmbH (Hockenheim, Germany) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 ± 1°C. The corneas were incubated for a minimum of 1 hour at 32 ± 1°C.
- Selection of corneas: after the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer (BASF-OP3.0, Duratec GmbH). The opacity of each cornea was read against a cMEM filled chamber and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used. Three corneas were selected at random for each treatment group.

Vehicle:

unchanged (no vehicle)

Remarks:

Since no workable suspension of the test item in physiological saline could be obtained, the test item was used as delivered (powder) and added as such on top of the corneas.

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

The test item was weighed in a bottle and applied directly on the corneas in such a way that the cornea was completely covered (Experiment 1: 605.7, 334.4, 379.1 mg and Experiment 2: 343.4, 357.5, 320.0).

Duration of treatment / exposure:

Corneas were incubated in a horizontal position for 240 ± 10 minutes.

Duration of post- treatment incubation (in vitro):

A 90 ± 5 minutes post-treatement incubation was carried out after the opacity measurement, when the Na-fluorescein had been applied and before determining the permeability.

Number of animals or in vitro replicates:

3 replicates (corneas) for the test item and for each of the controls.

Details on study design:

TREATMENT METHOD: no correction was made for the purity/composition of the test item. The medium from the anterior compartment was removed and 750 µL of the negative control and 20% (w/v) of the Imidazole solution (positive control) were introduced onto the epithelium of the cornea. The test item was weighed in a bottle and applied directly on the corneas in such a way that the cornea was completely covered. The holder was slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the solutions or test item over the entire cornea. Corneas were incubated in a horizontal position for 240 ± 10 minutes at 32 ± 1°C.

POST-INCUBATION PERIOD: no post incubation period before measuring the opacity but a 90 ± 5 minutes incubation period after application of the Na-fluorescein and before determining the permeability.

REMOVAL OF TEST SUBSTANCE : After the incubation the solutions and the test item were removed and the epithelium was washed at least three times with MEM (Eagle’s Minimum Essential Medium Life Technologies) containing phenol red to evaluate and then, record possible pH effects of the test item on the corneas. Each cornea was inspected visually for dissimilar opacity patterns. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM and the opacity determinations were performed.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: the opacity of a cornea was measured by the diminution of light passing through the cornea. The light was measured as illuminance (I = luminous flux per area, unit: lux) by a light meter. The opacity value was measured with the device OP-KIT.
- Corneal permeability: following the final opacity measurement, permeability of the cornea to Na-fluorescein (Sigma-Aldrich, Germany) was evaluated. The medium of both compartments (anterior compartment first) was removed. The posterior compartment was refilled with fresh cMEM. The anterior compartment was filled with 1 mL of 5 mg Na-fluorescein/mL cMEM solution (Sigma-Aldrich Chemie GmbH, Germany). The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the sodium-fluorescein solution over the entire cornea. Corneas were incubated in a horizontal position for 90 ± 5 minutes at 32 ± 1°C. After the incubation period, the medium in the posterior compartment of each holder was removed and placed into a sampling tube labelled according to holder number. 360 µL of the medium from each sampling tube was transferred to a 96-well plate. The optical density at 490 nm (OD490) of each sampling tube was measured in triplicate using a microplate reader (TECAN Infinite® M200 Pro Plate Reader).

SCORING SYSTEM: In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value).

DECISION CRITERIA: as indicated in OECD 437, i.e.:
In vitro irritancy score range / UN GSH
≤ 3 => No category
> 3 => No prediction can be made
> 55 => Category 1

Irritation parameter:

in vitro irritation score

Run / experiment:

2 runs.

Value:

> 3 - < 55

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Other effects / acceptance of results:

- In the first experiment, the individual IVIS for the negative controls ranged from -0.6 to 1.4. The corneas treated with the negative control item were clear after the 240 minutes of treatment. The individual positive control IVIS ranged from 139 to 280. The corneas treated with the positive control were turbid after the 240 minutes of treatment. No pH effects of the negative and positive control were observed on the rinsing medium.
- In the second experiment, the IVIS scores for the negative controls ranged from 0.2 to 1.3. The corneas treated with the negative control item were clear after the 240 minutes of treatment. The individual positive control IVIS ranged from 125 to 180. The corneas treated with the positive control were turbid after the 240 minutes of treatment. No pH effects of the negative and positive control were observed on the rinsing medium.

The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean IVIS of the positive control (20% (w/v) Imidazole) was 188 and 154 in the first and second experiment, respectively, and within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

In the first experiment, the corneas treated with the test item showed opacity values ranging from 0.4 to 5.2 and permeability values ranging from 0.027 to 0.101. Two of the three corneas were translucent after the 240 minutes of treatment with the test item. No pH effect of the test item was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from 0.8 to 6.8 after 240 minutes of treatment with the test item (see Table 1 below for details on the IVIS).

In the second experiment, the corneas treated with the test item showed opacity values ranging from 0.6 to 5.1 and permeability values ranging from 0.012 to 0.089. The 3 corneas were translucent after the 240 minutes of treatment with the test item. No pH effect of the test item was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from 2.0 to 5.7 after 240 minutes of treatment with the test item (see Table 1 below for details on the IVIS).

The mean in vitro irritancy score was of the test item was 3.2 and 3.4 after 240 minutes of treatment with the test item in the first and second experiment respectively. therefore, no conclusion can be drawn for the eye irritation or serious eye damage of the test  item i the experimental condition of this study.

Table 1: In vitro irritancy scrore – First experiment

Treatment	Final Opacity²	Final OD490²	In vitro Irritancy Score^1
Negative control	-0.7	0.006	-0.6
	1.3	0.012	1.4
	-0.6	0.002	-0.6
Positive control	258	1.468	280
	111	1.848	139
	124	1.303	144
Test item	5.2	0.101	6.8
	0.4	0.027	0.8
	1.5	0.039	2.1

1 In vitro irritancy score (IVIS) = opacity value + (15 x OD490 value).

2 Positive control and test item are corrected for the negative control.

 

In vitro irritancy scrore – Second experiment

Treatment	Final Opacity²	Final OD490²	In vitro Irritancy Score^1
Negative control	1.1	0.014	1.3
	1.0	0.018	1.3
	0.0	0.009	0.2
Positive control	143	2.500	180
	133	1.550	157
	105	1.317	125
Test item	2.4	0.012	2.6
	5.1	0.043	5.7
	0.6	0.089	2.0

1 In vitro irritancy score (IVIS) = opacity value + (15 x OD490 value).

2 Positive control and test item are corrected for the negative control.

Interpretation of results:

study cannot be used for classification

Remarks:

In the first experiment, the mean IVIS was 3.2 after 4 hours of treatment with the test item. Since the results were spread over 2 categories (IVIS of 6.8, 0.8 and 2.1, respectively), the test was inconclusive and a repeat experiment was performed. In the second experiment, the mean IVIS was 3.4 after 4 hours of treatment with the test item. Since the results were spread over 2 categories (IVIS of 2.6, 5.7 and 2.0, respectively) the test was inconclusive again.

Conclusions:

In conclusion, since the test item showed IVIS results that were spread over 2 categories in both experiments, no conclusion can be drawn for the eye irritation or serious eye damage potential hazard of this substance.

Executive summary:

The objective of this study was to evaluate the eye hazard potential of the test item as measured by its ability to induce opacity and increase permeability in an isolated bovine cornea using the Bovine Corneal Opacity and Permeability test (BCOP test).

The eye damage potential of the test item was tested through topical application on isolated bovine corneas for approximately 240 minutes. The study procedures were based on the OECD test guideline 437. Two independant experiments were performed.

The test item was a white powder. Since no workable suspension in physiological saline could be obtained, the test item was used as delivered and added as such on top of the corneas.

In both experiments, the negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy scores of the positive control (20% (w/v) Imidazole) were 188  and 154, in the Experiments 1 and 2, respectively, and within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

In the First experiment, the mean in vitro irritancy score was 3.2 after 4 hours of treatment with the test item. Since the results were spread over 2 categories (IVIS of 6.8, 0.8 and 2.1, respectively), the test was inconclusive and a repeat experiment was performed.

In the 2d experiment, the mean in vitro irritancy score was 3.4 after 4 hours of treatment with the test item. Since the results were spread over 2 categories (IVIS of 2.6, 5.7 and 2.0, respectively) the test was inconclusive again.

In conclusion, since the test substance showed IVIS results that were spread over 2 categories in the both experiments, no conclusion can be drawn for the eye irritation or serious eye damage potential hazard of this substance.

Justification for classification or non-classification

Skin irritation/corrosion : The potential of the test item to induce skin irritation was tested on a human three dimensional epidermal model (EPISKIN Small model (EPISKIN-SM)) according to OECD guideline 439 and in compliance to GLP.  The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues was 101%. Since the mean relative tissue viability for the test item was above 50% after 15 ± 0.5 minutes treatment, the test item was considered to be non-irritant. Therefore, the test subtance does not need to be classified according to GHS and CLP criteria.

Eye serious damage/irritation: The potential of the test item to induce skin irritation was tested in two in vitro assaies performed according to GLP. Firts, an in vitro Bovine Corneal Opacity and Permeability test (BCOP) was initally performed according to OECD guideline 437 with the test item but the results were inconclusive (IVIS scores of 3.2 and 3.4 after 4 hours of treatment with the test item in 2 independant experiments and thus, spread over 2 categories). Subsequently an in vitro EpiOcular cornea test was performed acording to OECD guideline 492, but the results led to the conclusion that no prediction could be made regarding the classification of the test substance for potential Eye Serious damage/irritation properties since the mean relative tissue viability for the test item was 56 % after 6 hours of treatment. Consequently an in vivo Eye serious damage/eye irritation study will be perfomed when the substance tonnage reaches the next tonnage band according to Annex VIII, point 8.2, column 2.