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Diss Factsheets

Administrative data

Description of key information

An in vitro skin irritation study (OECD 439 TG ) concluded that Synthetic wollastonite should not be considered to be irritating to the skin.


An in vitro eye irritation study using RHC tissue and an in vivo eye irritation study (OECD TG 405 ) carried out on the read across substance Kieselguhr soda ash flux calcined were not considered to be irritating to the eyes. Based on read-across Synthetic wollastonite is not considered to be irritating to eyes.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 February 2021 - 20 February 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source: Imerys
- Lot/batch number of test material: 2011 20
- Purity, including information on contaminants, isomers, etc.: 100%
- Molecular weight: 116.16 g/mol
- Expiry date: 27 November 2030

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.
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Supplier: MatTek In Vitro Life Sciences Laboratories
- Model used: EPIDERM™ Reconstructed Human Epidermis
- Tissue batch number(s): 34123
- Delivery date: 17 February 2021
- Date of initiation of testing: 17 February 2021

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

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Rinsing was achieved by filling and emptying each tissue insert approximately 15 times using a constant soft stream of DPBS to gently remove any residual test item. The rinsed tissues were then submerged 3 times in 150 ml of DPBS without Ca++ and Mg++.
- Observable damage in the tissue due to washing: No
- Modifications to validated SOP: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE: yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide)
- MTT concentration: 1.0 mg/mL
- Incubation time: 1 hour
- Spectrophotometer: Labtech LT-4500 microplate reader
The upper limit of accuracy for measured absorbance of MTT Formazan at 570 nm (OD570),
filter band pass 10 nm, was determined to be at an optical density of 2.6. (Warren, N; 2017).
- Linear OD range of spectrophotometer: OD570

NUMBER OF REPLICATE TISSUES: Triplicate

PREDICTION MODEL / DECISION CRITERIA
Data Evaluation
Quantitative MTT Assessment (Percentage Tissue Viability)
For the test item the relative mean tissue viabilities obtained after the 60-Minute exposure period followed by the 42-Hour post-exposure incubation period were compared to the mean of the negative control treated tissues (n=3). The relative mean viabilities were calculated in the following way:

Relative mean viability (%) = (mean OD570 of test item/mean OD570 of negative control) x 100

Classification of irritation potential is based upon relative mean tissue viability following the
60-Minute exposure period followed by the 42-Hour post-exposure incubation period
according to the following:

Relative mean tissue viability is ≤50% = Corrosive or Irritant - H314 or H315 Category 1 or 2
Relative mean tissue viability is >50% = Non-irritant - Not classified for irritation

If the relative mean tissue viability is ≤50%, differentiation between EU CLP/UN GHS Category 1 and Category 2 will not be possible based on the results of this study in isolation.

EU CLP/UN GHS:
Hazard Category 1 - Code H314; Statement “Causes severe skin burns and eye damage”
Hazard Category 2 - Code H315; Statement “Causes Skin Irritation”

Acceptance Criteria
The results of the assay are considered acceptable if the following assay acceptance criteria are achieved:

Positive Control:
The assay establishes the acceptance criterion for an acceptable test if the relative mean tissue viability for the positive control treated tissues is ≤20% relative to the negative control treated tissues.

Negative Control:
The assay establishes the acceptance criterion for an acceptable test if the mean OD570 for the negative control treated tissues is equal or greater than 0.8 and ≤2.8.

Standard Deviation:
The assay establishes the acceptance criterion for an acceptable test if the standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues is ≤18% for each testing or control group.
Control samples:
yes, concurrent negative control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg
- Concentration (if solution): The test item was used as supplied.

NEGATIVE CONTROL: Dulbecco’s Phosphate Buffered Saline (DPBS) with Ca++ and Mg++
- Amount(s) applied (volume or weight): 30 μL
- Concentration (if solution): Used as supplied.

POSITIVE CONTROL: Sodium dodecyl sulphate
- Amount(s) applied (volume or weight): 30 μL
- Concentration (if solution): Prepared as a 5% w/v aqueous solution.

MTT SOLUTION
- Amount(s) applied (volume or weight): 1 mL
- Concentration (if solution): 1.0 mg/mL MTT solution.
Duration of treatment / exposure:
60 +/- 1 minutes
Duration of post-treatment incubation (if applicable):
1st period: 24 +/- 2 hours, 2nd period 18 +/- 2 hours
Number of replicates:
Triplicate
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60-Minute exposure period and 42-Hour post-exposure incubation period
Value:
100.1
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:
It was considered unnecessary to perform IL-1a analysis on the assay medium, retained from
the wells of the 24 ±2 hour post exposure incubation plate, as the results of the MTT test
were unequivocal.

Direct MTT Reduction
The MTT solution containing the test item did not turn blue or purple which indicated that the test item did not directly reduce MTT.


Assessment of Color Interference with the MTT endpoint
The solutions containing the test item were colorless. It was therefore unnecessary to run color correction tissues.


Acceptance Criteria
The relative mean tissue viability for the positive control treated tissues was 4.0% relative to the negative control treated tissues. The positive control acceptance criteria were therefore satisfied.
The mean OD570 for the negative control treated tissues was 1.813. The negative control acceptance criteria were therefore satisfied.
The standard deviation between the three tissue replicates of each treatment group did not exceed 18%. The acceptance criterion was therefore satisfied.

Interpretation of results:
GHS criteria not met
Conclusions:
In this study and under the experimental conditions reported, the test item was classified as non-irritant.
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
The study was performed between 23 March 2010 and 26 March 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 431 (In vitro skin corrosion: Human skin model test)
GLP compliance:
yes (incl. QA statement)
Species:
other: EPISKIIN™ Reconstituted Human Epidermis model
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 20 mg


VEHICLE
Test material was used as suppled
Duration of treatment / exposure:
Duplicate tissues were treated with the test material for exposure periods of 3, 60 and 240 minutes.
Irritation / corrosion parameter:
other: other: Relative mean viability %
Value:
102.8
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 3 minutes. (migrated information)
Irritation / corrosion parameter:
other: other: Relative mean viability %
Value:
111.3
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 60 minutes. (migrated information)
Irritation / corrosion parameter:
other: other: Relative tissue viability %
Value:
114.1
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 240 minutes. (migrated information)

Direct MTT reduction

The MTT solution containing the test material did not turn blue/purple. This was taken to indicate the test material did not reduce MTT

Table 1: Mean OD540 values and viabilities for the negative control, positive control material and test material

Material

Exposure period

Mean OD540of duplicate tissues

Relative mean % viability

Negative control material

240 minutes

0.142

100*

Positive control material

240 minutes

0.013

9.2

Test material

240 minutes

0.162

114.1

60 minutes

0.158

111.3

3 minutes

0.146

102.8

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

Table 2: Qualitative evaluation of tissue viability (MTT uptake visual evaluation)

Material

Exposure period

Tissue 1

Tissue 2

Negative control material

240 minutes

-

-

Positive control material

240 minutes

++

++

Test material

240 minutes

-

-

60 minutes

-

-

3 minutes

-

-

- = Blue tissue (viable)

++ = Tissue completely white (dead)

The relative mean tissue viability for the positive control treated tissues was 9.2% relative to the negative control treated tissues following the 240 -minute exposure period. The positive control acceptance criterion was therefore satisfied.

Interpretation of results:
not irritating
Conclusions:
The test material was considered to be non-corrosive to the skin
Endpoint:
skin irritation: in vivo
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Reason / purpose for cross-reference:
data waiving: supporting information
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Referenceopen allclose all

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
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)
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
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).
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
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.
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.

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.

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
Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was performed between 26 July 2010 and 05 August 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
GLP compliance:
yes (incl. QA statement)
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories U.K. Ltd., Loughborough, UK
- Age at study initiation: 12 - 20 weeks
- Weight at study initiation: 2.08 or 2.67 kg
- Housing: The animals were individually housed in suspended cages
- Diet: Teklad Global Rabbit diet supplied by Harlan Laboratories U.K. Ltd., Oxon, UK) was allowed throughout the study
- Water: Free access to mains drinking water
- Acclimation period: At least five days


ENVIRONMENTAL CONDITIONS
- Temperature: 17 to 23°C
- Humidity: 30-70 %:
- Air changes: At least fifteen changes per hour
- Photoperiod: 12 hrs dark / 12 hrs light)
Vehicle:
unchanged (no vehicle)
Controls:
other: The left eye remained untreated and was used for control purposes.
Amount / concentration applied:
A volume of 0.1 ml of the test item, which was found to weigh approximately 52 mg
Observation period (in vivo):
72 h
Number of animals or in vitro replicates:
2
Details on study design:
Scoring system: Kay and Calandra classification system.

Tool used to assess score: use of light source from a standard ophthalmoscope.
Irritation parameter:
cornea opacity score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
0
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
0
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
0
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
animal #2
Time point:
24/48/72 h
Score:
1.67
Reversibility:
fully reversible within: 48 hours
Irritation parameter:
conjunctivae score
Remarks:
redness
Basis:
animal #1
Time point:
24/48/72 h
Score:
1
Reversibility:
fully reversible within: 72 hours
Irritation parameter:
conjunctivae score
Remarks:
redness
Basis:
animal #2
Time point:
24/48/72 h
Score:
1.33
Reversibility:
fully reversible within: 72 hours
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0.33
Reversibility:
fully reversible within: 48 hours
Irritation parameter:
chemosis score
Basis:
animal #2
Time point:
24/48/72 h
Score:
1
Reversibility:
fully reversible within: 72 hours
Irritant / corrosive response data:
Individual and group mean scores for ocular irritation are given in Table 1 and Table 2.
No corneal effects were noted during the study.
Iridial inflammation was noted in one treated eye one and 24 hours after treatment.
Moderate conjunctival irritation was noted in both treated eyes one and 24 hours after treatment. Moderate conjunctival irritation was noted in one treated eye and minimal conjunctival irritation was noted in the other treated eye at the 48-Hour observation.
Both treated eyes appeared normal at the 72-Hour observation.

Table 1: Individual scores and individual total scores for ocular irritation

Rabbit number and sex

69449 Male

69482 Male

IPR = 2

IPR = 2

Time after treatment

1 h

24 h

48 h

72 h

1 h

24 h

48 h

72 h

CORNEA

E = Degree of opacity

F = Area of cornea involved

 

0

0

 

0

0

 

0

0

 

0

0

 

0

0

 

0

0

 

0

0

 

0

0

Score ( E x F) x 5

0

0

0

0

0

0

0

0

IRIS

D

 

0

 

0

 

0

 

0

 

1

 

1

 

0

 

0

Score (D x 5)

0

0

0

0

5

5

0

0

CONJUNCTIVAE

A = Redness

B = Chemosis

C = Discharge

 

2

2

2

 

2

1

1

 

1

0

0

 

0

0

0

 

2

2

2

 

2

2

2

 

2

1

1

 

0

0

0

Score (A + B + C) x 2

12

8

2

0

12

12

8

0

Total score

12

8

2

0

17

17

8

0

IPR = Initial pain reaction

Table 2: Individual total scores and group mean scores for ocular irritation

Rabbit number and sex

Individual total scores at:

1 h

24 h

48 h

72 h

69449 Male

12

8

2

0

69482 Male

17

17

8

0

Group Total

29

25

10

0

Group Mean Score

14.5

12.5

5.0

0.0

Interpretation of results:
not irritating
Conclusions:
Kieselguhr soda ash flux calcined is not irritating to eyes according to the criteria set out in CLP Regulation (EC) no. 1272/2008
Endpoint:
eye irritation: in vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
The study was performed between 26 July 2010 and 05 August 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Justification for type of information:
The use of data derived for Soda-ash flux calcined kieselghur are justified for read-across to
synthetic wollastonite. Justification for read-across is warranted given the similarities in toxicity profile and physico-chemical properties for Soda-ash flux calcined kieselghur and synthetic wollastonite.
Considering the available data:
The source substance show no concerns for the environment.
The source substance has low acute toxicity and low toxicity in repeated dose studies, is non-irritant (skin and eye), non-sensitizing, non-mutagenic to bacteria, non-cytogenic and has low toxicity for reproductive and developmental toxicity.
Please see RAAF attached in Section 13. for further details.
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
GLP compliance:
yes (incl. QA statement)
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories U.K. Ltd., Loughborough, UK
- Age at study initiation: 12 - 20 weeks
- Weight at study initiation: 2.08 or 2.67 kg
- Housing: The animals were individually housed in suspended cages
- Diet: Teklad Global Rabbit diet supplied by Harlan Laboratories U.K. Ltd., Oxon, UK) was allowed throughout the study
- Water: Free access to mains drinking water
- Acclimation period: At least five days


ENVIRONMENTAL CONDITIONS
- Temperature: 17 to 23°C
- Humidity: 30-70 %:
- Air changes: At least fifteen changes per hour
- Photoperiod: 12 hrs dark / 12 hrs light)
Vehicle:
unchanged (no vehicle)
Controls:
other: The left eye remained untreated and was used for control purposes.
Amount / concentration applied:
A volume of 0.1 ml of the test item, which was found to weigh approximately 52 mg
Observation period (in vivo):
72 h
Number of animals or in vitro replicates:
2
Details on study design:
Scoring system: Kay and Calandra classification system.

Tool used to assess score: use of light source from a standard ophthalmoscope.
Irritation parameter:
cornea opacity score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
0
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
0
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
0
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
animal #2
Time point:
24/48/72 h
Score:
1.67
Reversibility:
fully reversible within: 48 hours
Irritation parameter:
conjunctivae score
Remarks:
redness
Basis:
animal #1
Time point:
24/48/72 h
Score:
1
Reversibility:
fully reversible within: 72 hours
Irritation parameter:
conjunctivae score
Remarks:
redness
Basis:
animal #2
Time point:
24/48/72 h
Score:
1.33
Reversibility:
fully reversible within: 72 hours
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0.33
Reversibility:
fully reversible within: 48 hours
Irritation parameter:
chemosis score
Basis:
animal #2
Time point:
24/48/72 h
Score:
1
Reversibility:
fully reversible within: 72 hours
Irritant / corrosive response data:
Individual and group mean scores for ocular irritation are given in Table 1 and Table 2.
No corneal effects were noted during the study.
Iridial inflammation was noted in one treated eye one and 24 hours after treatment.
Moderate conjunctival irritation was noted in both treated eyes one and 24 hours after treatment. Moderate conjunctival irritation was noted in one treated eye and minimal conjunctival irritation was noted in the other treated eye at the 48-Hour observation.
Both treated eyes appeared normal at the 72-Hour observation.

Table 1: Individual scores and individual total scores for ocular irritation

Rabbit number and sex

69449 Male

69482 Male

IPR = 2

IPR = 2

Time after treatment

1 h

24 h

48 h

72 h

1 h

24 h

48 h

72 h

CORNEA

E = Degree of opacity

F = Area of cornea involved

 

0

0

 

0

0

 

0

0

 

0

0

 

0

0

 

0

0

 

0

0

 

0

0

Score ( E x F) x 5

0

0

0

0

0

0

0

0

IRIS

D

 

0

 

0

 

0

 

0

 

1

 

1

 

0

 

0

Score (D x 5)

0

0

0

0

5

5

0

0

CONJUNCTIVAE

A = Redness

B = Chemosis

C = Discharge

 

2

2

2

 

2

1

1

 

1

0

0

 

0

0

0

 

2

2

2

 

2

2

2

 

2

1

1

 

0

0

0

Score (A + B + C) x 2

12

8

2

0

12

12

8

0

Total score

12

8

2

0

17

17

8

0

IPR = Initial pain reaction

Table 2: Individual total scores and group mean scores for ocular irritation

Rabbit number and sex

Individual total scores at:

1 h

24 h

48 h

72 h

69449 Male

12

8

2

0

69482 Male

17

17

8

0

Group Total

29

25

10

0

Group Mean Score

14.5

12.5

5.0

0.0

Interpretation of results:
not irritating
Conclusions:
The analogue substance Kieselguhr soda ash flux calcined is not irritating to eyes according to the criteria set out in CLP Regulation (EC) no. 1272/2008
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
The study was performed between 26 May 2010 and 28 May 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Principles of method if other than guideline:
The purpose of this study was to determine the eye irritation potential of the test material using the SkinEthic Reconstituted Human Corneal model (HCE, SkinEthic Laboratories, Nice, France) after a treatment period of 10 minutes. The test is based on the hypothesis that irritant chemicals are able to penetrate the stratum corneum of the SkinEthic RHC model and are sufficiently cytotoxic to cause cell death in the underlying cell layers
GLP compliance:
yes (incl. QA statement)
Species:
other: Reconstituted Corneal Epithelium
Amount / concentration applied:
TEST MATERIAL
- Amount applied: Tissues were treated with 30 mg of the test material.

VEHICLE
Test material was used as supplied

CONTROLS:
- Amount applied: 30 µL of Solution A was applied as a negative control and 30 µL of of SDS 1.0% (w/v) as a positive control. Solution A was comprised of Na2HPO4 0.142 g/L, Glucose 1.802 g/L, HEPES 7.149 g/L, KCl 0.224 g/L and NaCl 7.597 g/L. Sodium Dodecyl Sulphate (SDS) was prepared as a 1% w/v solution in sterile distilled water.
Duration of treatment / exposure:
Cultures were exposed for 10 minutes to the test material.
Observation period (in vivo):
Skin cultures were examined after three hours.
Number of animals or in vitro replicates:
All test substances were tested in triplicate (including controls)
Irritation parameter:
mean percent tissue viability 
Remarks:
Relative mean tissue viability
Run / experiment:
10 minutes
Value:
99.1
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
The mean OD540 values and mean viabilities for each treatment group are given in Table 1.
The relative mean viability of the test material treated tissues after a 10 minute exposure was 99.1 %.
It was considered unnecessary to proceed with tissue histopathology.

The qualitative evaluation of tissue viability is presented in Table 2.
The test material and negative control material treated tissues appeared blue which was considered to be indicative of viable tissue. the positive control material treated tissues appeared blue/white which was considered to be indicative of semi viable tissue.

Table 1: Assessment of Eye Irritation Potential – Viability of RHC Tissues

Material

Mean Tissue Viability

Mean OD540

Viability (%)

Negative Control

1.036

1.006

100*

0.975

Positive Control

0.540

0.537

53.4

0.533

Test Material

0.989

0.997

99.1

1.005


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

Table 2: Qualitative Evaluation of Tissue Viability (MTT uptake visual evaluation)

Material

Score

Tissue 1

Tissue 2

Negative Control

-

-

Positive Control

+

+

Test Material

-

-

MTT Visual Scoring Scheme of SkinEthic Tissues

-     =  Blue tissue (viable)

+    =  Blue/White tissue (semi viable)

++  =  Tissue completely white (dead)

Interpretation of results:
not irritating
Conclusions:
According to the protocol followed the test material was considered to be a Non Irritant The quality criterion required for the acceptance of results in the test was satisfied
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Study period:
The study was performed between 26 May 2010 and 28 May 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Justification for type of information:
The use of data derived for Soda-ash flux calcined kieselghur are justified for read-across to
synthetic wollastonite. Justification for read-across is warranted given the similarities in toxicity profile and physico-chemical properties for Soda-ash flux calcined kieselghur and synthetic wollastonite.
Considering the available data:
The source substance show no concerns for the environment.
The source substance has low acute toxicity and low toxicity in repeated dose studies, is non-irritant (skin and eye), non-sensitizing, non-mutagenic to bacteria, non-cytogenic and has low toxicity for reproductive and developmental toxicity.
Please see RAAF attached in Section 13. for further details.
Reason / purpose for cross-reference:
data waiving: supporting information
Principles of method if other than guideline:
The purpose of this study was to determine the eye irritation potential of the test material using the SkinEthic Reconstituted Human Corneal model (HCE, SkinEthic Laboratories, Nice, France) after a treatment period of 10 minutes. The test is based on the hypothesis that irritant chemicals are able to penetrate the stratum corneum of the SkinEthic RHC model and are sufficiently cytotoxic to cause cell death in the underlying cell layers
GLP compliance:
yes (incl. QA statement)
Species:
other: Reconstituted Corneal Epithelium
Amount / concentration applied:
TEST MATERIAL
- Amount applied: Tissues were treated with 30 mg of the test material.

VEHICLE
Test material was used as supplied

CONTROLS:
- Amount applied: 30 µL of Solution A was applied as a negative control and 30 µL of of SDS 1.0% (w/v) as a positive control. Solution A was comprised of Na2HPO4 0.142 g/L, Glucose 1.802 g/L, HEPES 7.149 g/L, KCl 0.224 g/L and NaCl 7.597 g/L. Sodium Dodecyl Sulphate (SDS) was prepared as a 1% w/v solution in sterile distilled water.
Duration of treatment / exposure:
Cultures were exposed for 10 minutes to the test material.
Observation period (in vivo):
Skin cultures were examined after three hours.
Number of animals or in vitro replicates:
All test substances were tested in triplicate (including controls)
Irritation parameter:
mean percent tissue viability 
Remarks:
Relative mean tissue viability
Run / experiment:
10 minutes
Value:
99.1
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
The mean OD540 values and mean viabilities for each treatment group are given in Table 1.
The relative mean viability of the test material treated tissues after a 10 minute exposure was 99.1 %.
It was considered unnecessary to proceed with tissue histopathology.

The qualitative evaluation of tissue viability is presented in Table 2.
The test material and negative control material treated tissues appeared blue which was considered to be indicative of viable tissue. the positive control material treated tissues appeared blue/white which was considered to be indicative of semi viable tissue.

Table 1: Assessment of Eye Irritation Potential – Viability of RHC Tissues

Material

Mean Tissue Viability

Mean OD540

Viability (%)

Negative Control

1.036

1.006

100*

0.975

Positive Control

0.540

0.537

53.4

0.533

Test Material

0.989

0.997

99.1

1.005


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

Table 2: Qualitative Evaluation of Tissue Viability (MTT uptake visual evaluation)

Material

Score

Tissue 1

Tissue 2

Negative Control

-

-

Positive Control

+

+

Test Material

-

-

MTT Visual Scoring Scheme of SkinEthic Tissues

-     =  Blue tissue (viable)

+    =  Blue/White tissue (semi viable)

++  =  Tissue completely white (dead)

Interpretation of results:
not irritating
Conclusions:
According to the protocol followed the analogue substance Kieselghur soda ash flux calcined was considered to be a Non Irritant The quality criterion required for the acceptance of results in the test was satisfied.
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
11 February 2021 - 11 February 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other:
Version / remarks:
Guidance Document on ‘The Collection of Tissues for Histological Evaluation and Collection of Data’. Series on Testing and Assessment, No. 160. Adopted July 6, 2018 Paris.
Deviations:
yes
Remarks:
Please see "Principles of method if other than Guideline" for details.
Qualifier:
according to guideline
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Deviations:
yes
Remarks:
Please see "Principles of method if other than Guideline" for details.
Qualifier:
according to guideline
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Version / remarks:
updated 26 June 2020
Deviations:
yes
Remarks:
Please see "Principles of method if other than Guideline" for details.
Principles of method if other than guideline:
The positive control IVIS value (83.0) was marginally below the normally accepted lower range of IVIS 83.6. However the positive control item has demonstrated the sensitivity of the assay successfully and has exhibited a Category 1 classification. The slightly low positive control IVIS value does not qualify as an outlier (≤ 75.6) and will be included in the historical control data. The result obtained is therefore considered to be valid and the integrity of the study is unaffected.
GLP compliance:
yes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier): Imerys Belgium
- Lot/batch number of test material: 2011 20
- Purity, including information on contaminants, isomers, etc.: 100%
- Expiry date: 27 November 2030

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions and during storage: Stable
- Stability in the medium: Stable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
For the purpose of this study the test item was prepared as a 20% w/v suspension in sodium chloride 0.9% w/v.
The test item was formulated within 2 hours of being applied to the test system. It is assumed that the formulation was stable for this duration.
No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.
Species:
cattle
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
Eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir as a by-product from freshly slaughtered animals. The eyes were excised by an abattoir employee after slaughter, and were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 μg/mL). They were transported to the test facility over ice packs on the same day of slaughter. The corneas were prepared immediately on arrival.

Selection and preparation of corneas:
All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.
The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders.
The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1 ºC for 60 minutes.

Quality check of the isolated corneas:
At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL/cornea
- Concentration (if solution): 20% w/v suspension in sodium chloride 0.9% w/v.

Duration of treatment / exposure:
4 hours
Duration of post- treatment incubation (in vitro):
90 minutes
Number of animals or in vitro replicates:
Triplicate
Details on study design:
NUMBER OF REPLICATES: Triplicate

NEGATIVE CONTROL USED: Sodium chloride 0.9% w/v
The negative control item, sodium chloride 0.9% w/v, was used as supplied.

POSITIVE CONTROL USED: Imidazole
The positive control item, Imidazole, was used as a 20% w/v solution in sodium chloride 0.9% w/v.

APPLICATION DOSE AND EXPOSURE TIME
The EMEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test item preparation or control items were applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 240 minutes.
At the end of the exposure period the test item and control items were removed from the anterior chamber and the cornea was rinsed 3 times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red. The anterior chamber was refilled with fresh complete EMEM without phenol red. A post-treatment opacity reading was taken and each cornea was visually observed.

TREATMENT METHOD:
The medium from both chambers of each holder was replaced with fresh complete EMEM.
A pre-treatment opacity reading was taken for each cornea using a calibrated opacitometer.
Three corneas were randomly allocated to the negative control. Three corneas were also allocated to the test item and three corneas to the positive control item.

POST-INCUBATION PERIOD: yes
Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (5 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 ºC for 90 minutes.

METHODS FOR MEASURED ENDPOINTS:
After incubation the medium in the posterior chamber of each holder was decanted and retained.
360 μL of media representing each cornea was dispensed into the appropriate wells of a pre-labeled 96-well plate. The optical density was measured (quantitative viability analysis) at 492 nm (without a reference filter) using the Labtech LT-4500 microplate reader.
Opacity Measurement
The change in opacity for each cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final opacity reading. These values were then corrected by subtracting the average change in opacity observed for the negative control corneas. The mean opacity value of each treatment group was then calculated by averaging the corrected opacity values of each cornea for that treatment group.
Permeability Measurement
The corrected OD492 was calculated by subtracting the mean OD492 of the negative control corneas from the OD492 value of each treated cornea. The OD492 value of each treatment group was calculated by averaging the corrected OD492 values of the treated corneas for the treatment group.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
Results from the two test method endpoints, opacity and permeability, were combined in an empirically derived formula to generate an In Vitro Irritancy Score.
The following formula was used to determine the In Vitro Irritancy Score:

In Vitro Irritancy Score = mean opacity value + (15 x mean permeability OD492 value)

Additionally, the opacity and permeability values were evaluated independently to determine whether the test item induced a response through only one of the two endpoints.

The condition of the cornea was visually assessed post treatment.

DECISION CRITERIA:
The test item was classified according to the following prediction model:
IVIS UN GHS
≤ 3 No Category
>3; ≤ 55 No stand-alone prediction can be made
> 55 Category 1
Irritation parameter:
in vitro irritation score
Value:
6.9
Negative controls validity:
valid
Remarks:
The negative control gave opacity and permeability values below the established upper limits. The negative control acceptance criteria were therefore satisfied.
Positive controls validity:
valid
Remarks:
The positive control In Vitro Irritancy Score was within an accepted range. The positive control acceptance criterion was therefore satisfied.
Remarks on result:
not determinable

Corneal Opacity and Permeability Measurement


Individual and mean corneal opacity measurements and individual and mean corneal permeability measurements are given in Table 1. below. The IVIS score obtained for Synthetic Wollastonite was predominantly due to the effects of opacity. Modest effects of test item induced permeability can be observed.


 


Table 1. Individual and Mean Corneal Opacity and Permeability Measurements
















































































































































Treatment



Cornea Number



Opacity



Permeability (OD492)



In vitro Irritancy Score



Pre-Treatment



Post-Treatment



Post-Treatment – Pre-Treatment



Corrected Value



 



Corrected Value



Negative Control



2



5



6



1



 



0.004



 



 



3



6



9



3



 



0.001



 



 



6



5



7



2



 



0.001



 



 



 



 



 



2.0*



 



0.005▪



 



2.1



Positive Control



9



6



76



70



68.0



1.510



1.505



 



11



5



64



59



57.0



0.994



0.989



 



12



6



79



73



71.0



1.048



1.043



 



 



 



 



 



65.3●



 



1.179●



83.0



Test Item



14



5



12



7



5.0



0.202



0.197



 



16



5



12



7



5.0



0.050



0.045



 



17



5



13



8



6.0



0.075



0.070



 



 



 



 



 



5.3●



 



0.104●



6.9



 


OD = Optical Density       * = Mean of the post-treatment – pre-treatment values ▪ = Mean permeability   ● = Mean corrected value


 


Corneal Epithelium Condition


The condition of each cornea is given in Table 2. below.
The corneas treated with the test item were partly cloudy post treatment. The corneas treated with the negative control item were clear post treatment. The corneas treated with the positive control item were cloudy post treatment.


 


Table 2. Corneal Epithelium Condition Post Treatment


















































Treatment



Cornea Number



Observation Post Treatment



Negative Control



2



Clear



3



Clear



6



Clear



Positive Control



9



Cloudy



11



Cloudy



12



Cloudy



Teat Item



14



Partly Cloudy



16



Partly Cloudy



17



Partly Cloudy


Interpretation of results:
study cannot be used for classification
Conclusions:
An in vitro eye irritation study (OECD TG 437) has been performed on the substance Synthetic wollastonite using the Bovine Corneal Opacity Permeability (BCOP) Assay for identifying Ocular corrosives and severe irritants.This produced an in vitro irritation score of 6.9. It was not possible to determine a classification from this result and there was a suggestion that the test item adhered to the cornea and could not be rinsed away completely, causing a small change in opacity. No effect on permeability was observed. 
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation:


An in vitro skin irritation study using using the EpiskinTM reconstituted human epidermis model has been performed (Frosdick 2021). In the skin irritation study triplicate tissues were treated with the test material for an exposure period of 60 minutes. The relative mean viability of the test material treated tissues was 100.1% after a 60 minute exposure and was therefore not considered to be a skin irritant.


Read-Across Justification for eye irritation


Synthetic wollastonite is a UVCB substance, the main constituents of which are amorphous in nature. It also contains crystalline silica. The percentage of crystalline silica may range up to 1.5% (<=0.21% respirable). The eye irritation information has been read-across from the analogue substance Kieselguhr, soda ash flux-calcined. The analogue has been chosen for its similarity in structure and properties to Synthetic wollastonite. The main difference in structure between Synthetic wollastonite and Kieselguhr, soda ash flux-calcined is the presence of a calcium ion in Synthetic wollastonite. It is clear from a number of studies carried out on amorphous and crystalline silica and the analogue substance Silicic acid, calcium salt that results were consistent between the two substances and that there was no effect from the presence of the calcium ion. Moreover, it is well documented that these substances have a low potential for hazard to health and the environment. 


The toxicological properties of both forms are well described and may be used to predict the eye irritation effects of exposure to Synthetic wollastonite, and to support the available data for this substance.


Eye irritation (in vitro):


An in vitro eye irritation study (OECD 492) has been performed on the substance Synthetic wollastonite using the Reconstructed human Cornea-like Epithelium (RhCE) test method. The relative mean viability of the test item treated tissues was 60.9% in run 1 and 86.6% in run 2.The test item was classified as non-irritant to the eye.


An in vitro eye irritation study (OECD TG 437) has been performed on the substance Synthetic wollastonite using the Bovine Corneal Opacity Permeability (BCOP) Assay for identifying Ocular corrosives and severe irritants.This produced an in vitro irritation score of 6.9. It was not possible to determine a classification from this result and there was a suggestion that the test item adhered to the cornea and could not be rinsed away completely, causing a small change in opacity. No effect on permeability was observed.


 


Eye irritation (in vivo):


An in vivo eye irritation study was performed on the analogue substance Kieselguhr soda ash flux, according to OECD Guideline 205. A single application of the test item to the non-irrigated eye of two rabbits produced iridial inflammation and moderate conjunctival irritation. Both treated eyes appeared normal after the 72 h observation. The test material was not considered to be irritating to eyes in accordance with CLP criteria.

Justification for classification or non-classification

Skin irritation/corrosion:


An in vitro skin corrosion and irritation study have been performed which confirm that the substance is not corrosive or irritating to skin. The in vitro method using the EpiSkinTM test method is considered for the classification of substances for skin irritancy according to CLP criteria (ECVAM/ESAC, 2009). No classification for skin irritancy is required.


(An in vitro skin corrosion (OECD TG 431) and in vitro skin irritation study B46 have been performed on an analogue substance. Kieselguhr soda ash flux calcined is not irritating to skin.)


Eye irritation:


An in vitro eye irritation study using RHC tissue and an in vivo eye irritation study (OECD TG 405 ) carried out on the read across substance Kieselguhr soda ash flux calcined were not considered to be irritating to the eyes.


Based on read-across Synthetic wollastonite the test substance is not considered to be irritating to eyes. No classification for eye irritation is required.