Synthetic wollastonite

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 May 2021 to 04 June 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

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)

Deviations:

yes

Remarks:

Please see "Principles of method if other than guideline" below for details.

Principles of method if other than guideline:

In run 2 the difference in viability between the two test item treated tissues was 23.5% which was higher than the assay acceptance cut-off of 20%. This difference was considered acceptable as the overall test item group and both individual tissues are unequivocally negative when compared to the negative control.
This deviation was considered to have not affected the integrity or validity of the study.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Synthetic wollastonite

EC Number:

952-026-5

Molecular formula:

CaSiO3

IUPAC Name:

Synthetic wollastonite

Test material form:

solid: bulk

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: AGRG- 00620
- Purity, including information on contaminants, isomers, etc.: 100%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, in the dark

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- The test item was used as supplied.

- Expiry Date: 27 November 2030

Test animals / tissue source

Species:

human

Details on test animals or tissues and environmental conditions:

The EpiOcular™ EIT was validated by the European Union Reference laboratory for Alternatives to Animal Testing (EURL ECVAM) and Cosmetics Europe between 2008 and 2013. The EpiOcular™ EIT was endorsed as an in vitro test that can be used to identify those chemicals not requiring classification and labelling for eye irritation or serious eye damage in accordance with UN GHS (No Category).
The EpiOcular™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cornea.
The EpiOcular™ tissues are cultured on specially prepared cell culture inserts with a porous synthetic membrane through which nutrients can pass to the cells.

Run 1
EpiOcularTM Tissues Lot Number: 34909
Assay Medium Lot Number: 051021ISA
Run 2
EpiOcularTM Tissues Lot Number: 34912
Assay Medium Lot Number: 053121ISA

Upon receipt of the EpiOcularTM tissues, the sealed 24-well plate and the assay medium were placed into the refrigerator (2 to 10 °C) until the equilibration step.
On the day of receipt the equilibration step (15 minutes at room temperature in the 24-well shipping container) was started. An appropriate volume of EpiOcular™ Assay medium was warmed to approximately 37 °C and 1 mL of the medium aliquoted into the appropriate wells of pre-labeled 6-well plates.
Each 24-well shipping container was removed from its plastic bag under sterile conditions and its surface disinfected by wiping with ethanol soaked tissue paper. The sterile gauze was removed and each tissue inspected for air bubbles between the agarose gel and insert. Cultures with air bubbles under the insert covering greater than 50% of the insert area were not used. The tissues were carefully removed from the 24-well shipping container using sterile forceps. Any agarose adhering to the inserts was removed by gentle blotting on sterile filter paper or gauze. The insert was then transferred aseptically into the 6-well plates and pre-incubated at standard culture conditions for 1 hour in Assay Medium. After 1 hour, the Assay Medium was replaced by 1 mL of fresh Assay Medium at 37 °C and the EpiOcular™ tissues was incubated at standard culture conditions overnight (16 to 24 hours).

Test system

Controls:

yes

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 mg

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:

duplicate

Details on study design:

Application of Test Item and Rinsing
After the overnight incubation, the tissues were pre-wetted with 20 μL of Ca++ Mg++ free DPBS to mimic the wet condition of the human eye. If the Ca++ Mg++ free DPBS was not spread across the tissues, the plate was tapped to assure that the entire tissue surface was wetted. The tissues were incubated at 37 °C, 5% CO2 for 30 ±2 minutes.
50 mg of test item was applied atop duplicate cultures for an exposure period of 6 hours ±15 minutes at 37 °C, 5% CO2 followed by rinsing, a post-treatment immersion and a post-treatment incubation (described below). 50 μL of the negative and positive controls were similarly applied.
At the end of the test item exposure period, the test item was removed by extensively rinsing the tissues with Ca++ Mg++ free DPBS at room temperature. Three clean beakers (glass or plastic with minimum 150 mL capacity), containing a minimum of 100 mL each of Ca++ Mg++ free DPBS were used per test item or control with each test item or control item utilizing a different set of three beakers. The inserts containing the tissue were lifted out of the medium by grasping the upper edge of the plastic "collar" with fine forceps. The tissues were rinsed two at a time by holding replicate inserts together by their collars using forceps. The test or control items were decanted from the tissue surface onto a clean absorbent paper towel and the cultures dipped into the first beaker of DPBS, swirled in a circular motion in the liquid for approximately 2 seconds, lifted out so that the inserts were mostly filled with DPBS, and the liquid was decanted back into the container. This process was performed two additional times in the first beaker. The inserts were then rinsed in the second and third beakers of DPBS three times each in the same fashion. Finally, any remaining liquid was decanted onto the absorbent paper. Decanting was most efficiently performed by rotating the insert to approximately a 45° angle (open end down) and touching the upper lip to the absorbent paper (to break the surface tension).
After rinsing, the tissues were immediately transferred to and immersed in 5 mL of assay medium at room temperature in a pre-labeled 12-well plate for a 25 ±2 minutes immersion incubation (post‐treatment immersion) at room temperature. This incubation in assay medium was intended to remove any test item absorbed into the tissue.
At the end of the post‐treatment immersion, each insert was removed from the assay medium, the medium was decanted off the tissue, the insert was blotted on absorbent paper, and transferred to the appropriate well of the pre-labeled 6-well plate containing 1 mL of assay medium at approximately 37 °C. The tissues were incubated for a period of 18 hours ±15 minutes at 37 °C, 5% CO2 (post-treatment incubation).

Assessment of Direct Test Item Reduction of MTT
The test item was shown to directly reduce MTT in the direct MTT reduction test. There was a possibility that if the test item could not be totally rinsed off the tissues, any residual test item present on or in the tissue may directly reduce MTT and could have given rise to a false negative result. Therefore, the determination of eye irritation potential was performed in parallel on viable and non-viable, freeze-killed, tissues.
Freeze-killed tissues were prepared in-house (outside of the confines of the study) by placing untreated EpiOcularTM tissues in a freezer (-35 to -10 °C) overnight, thawing to room temperature, and then refreezing (two freeze-thaw cycles). Once killed, the tissues may be stored indefinitely in the freezer. Freeze-killed tissues were thawed for approximately 60 minutes at 37 ±2 °C, 5 ±1% CO2 in air before use.
Each MTT reducing test item was applied to two freeze-killed tissues. In addition, two freeze-killed tissues remained untreated (the untreated controls show a small amount of MTT reduction due to residual reducing enzymes within the killed tissue). The entire assay was performed on the frozen tissues in parallel to the viable tissues.
If the interference by the test item was ≤ 60% of the negative control value, the net OD of the test item treated killed control may be subtracted from the mean OD of the test item treated viable tissues to obtain the true amount of MTT reduction that reflects metabolic conversion only.
Data were corrected as follows: True viability = Viability of treated tissue – direct MTT interference from test item = OD tvt – (mean OD tkt – mean OD ukt)
Key:
OD = optical Density at 570 nm
tvt = treated viable tissues
tkt = treated killed tissues
ukt = untreated killed tissues
If the interference by the test item was greater than 60% of the negative control value the test item may be considered incompatible with this test system.
The killed tissue groups were not repeated. The values of tkt and ukt from run 1 were used for the correction of the results of both run 1 and run 2.

Assessment of Color Interference with the MTT endpoint
For non-colored test items, tests have to be performed to assess if they become colorants after contact with water or isopropanol. For this purpose 50 mg of the test item was added to 1.0 mL of water in a 6-well plate and the mixture was incubated in the dark at 37 ±1 °C in a humidified atmosphere of 5 ±1% CO2 in air for at least 1 hour. Furthermore, 50 mg of the solid test item was added to 2 mL of isopropanol, the same amount as used for MTT extraction, incubated in 6 well plates, and placed on an orbital plate shaker for 3 hours at room temperature.

MTT Assay
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 placed into the 24-well plate containing 0.3 mL of 1.0 mg/mL MTT solution. Once all the tissues were placed into the 24-well plate, the plate was incubated at 37 °C, 5% CO2 in air for 3 hours.
A procedure was used which only extracted from beneath the tissue, since residual test item may remain on the tissue and could contaminate the isopropanol. Inserts were removed from the 24‐well plate after approximately 3 hours. The bottom of the insert was blotted on absorbent paper and then transferred to a pre‐labeled 6-well plate containing 2 mL isopropanol in each well so that no isopropanol flowed into the insert. The plates were sealed with a film sealer (between the plate cover and upper edge of the wells) or a standard plate sealer and stored overnight at 2 to 10 °C in the dark.

Absorbance/Optical Density Measurements
At the end of the extraction period, using a pipette fitted with a 1000 μL tip, the extraction solution was forced vigorously up and down to thoroughly mix. The tissues and empty inserts were discarded.
For each tissue, duplicate 200 μL samples were transferred to the appropriate wells of a pre-labeled 96 well plate. 200 μL of isopropanol alone was added to eight wells designated as ‘blanks’. All wells were examined and any air bubbles were removed. The absorbance at 570 nm (OD570) of each well was measured using the LabTech LT-4500 microplate reader and LT-com analysis software.
The plate reader LT-com analysis software was set to correct for blanks and calculate the mean OD570 values of the duplicate wells representing each tissue. The mean OD570 values of the duplicate tissues were manually calculated.

Acceptability Criteria
The results of the assay are considered acceptable if the following assay acceptance criteria are achieved:
1) The negative control OD is > 0.8 and < 2.8.
2) The mean relative viability of the positive control is below 50% of the negative control viability for either the 30 minutes or 6 hours exposures.
3) The difference of viability between the two relating tissues of a single group of duplicate tissues is < 20% in the same run (for positive and negative control tissues and tissues of test items). This applies also to the killed controls (test items and negative killed control) and the colorant controls which are calculated as percent values related to the viability of the relating negative control.
Major Computerized Systems
The following computerized systems were used in the study:
Labtech LT-4500 microplate reader and LT-com analysis software (Version 7.0)
Veeva QMS Electronic communication system

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

mean percent tissue viability 

Run / experiment:

1

Value:

60.9

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Remarks:

The relative mean viability of the test item treated tissues was 60.9% after a 6-Hour exposure period and 18-Hour post-exposure incubation period. Due to a borderline result the test was repeated with both runs being reported here.

Results 2

Irritation parameter:

mean percent tissue viability 

Run / experiment:

1

Value:

86.6

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Remarks:

The relative mean viability of the test item treated tissues was 86.6% after a 6-Hour exposure period and 18-Hour post-exposure incubation period.

Any other information on results incl. tables

Direct MTT Reduction
An assessment found the test item was able to directly reduce MTT. Therefore, an additional procedure using, non-viable, freeze-killed tissues was performed. The results of the freeze-killed tissues were subtracted from the OD of the test item treated viable tissues to obtain the true amount of MTT reduction that reflects metabolic conversion only.

Assessment of Color Interference with the MTT endpoint
The test item did not become colored in either the water or isopropanol solutions. It was therefore unnecessary to run color correction tissues.

Acceptance Criteria
Run 1
The relative mean tissue viability for the positive control treated tissues was 31.3% relative to the negative control treated tissues. The positive control acceptance criterion was therefore satisfied.
The mean OD570 for the negative control treated tissues was 1.869. The negative control acceptance criterion was therefore satisfied.
The difference in viability between the two relating tissues in each treatment group was <20%. This acceptance criterion was therefore satisfied.

Run 2
The relative mean tissue viability for the positive control treated tissues was 40.8% relative to the negative control treated tissues. The positive control acceptance criterion was therefore satisfied.
The mean OD570 for the negative control treated tissues was1.554. The negative control acceptance criterion was therefore satisfied.
The difference in viability between the two relating tissues in each treatment group was <20% for the negative and positive control groups only. This acceptance criterion was therefore not fully satisfied, this is reported as a deviation.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was classified as non-irritant to the eye. The following classifications apply:
Both EU CLP and UN GHS No Category