Registration Dossier

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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 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:
key 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:
key 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:
26 March 2021 - 30 April 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 Ocular Corrosives and Severe Irritants)
Deviations:
yes
Remarks:
Please see "Principles of method if other than Guideline" for details.
Principles of method if other than guideline:
The OECD Test Guideline 437 states measurement of optical density is to be performed at 490 nm (OD490) as this is the wavelength at which peak absorbance occurs, but the 490 nm filter is unavailable for this machine. This modification to the testing is considered effective and reproducible due to the in-house observations on the corneas obtained for this study type and the significant historical control data using 492 nm rather than 490 nm. The small difference in wavelength used for measurement of the optical density is, therefore, considered to be acceptable.
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 %

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

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding):
- Preliminary purification step (if any):
- Final concentration of a dissolved solid, stock liquid or gel:
- Final preparation of a solid (e.g. stock crystals ground to fine powder using a mortar and pestle):

FORM AS APPLIED IN THE TEST (if different from that of starting material)
- Specify the relevant form characteristics if different from those in the starting material, such as state of aggregation, shape of particles or particle size distribution:
Species:
cattle
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Woolley Brothers, Sheffield
- Number of animals: See below
- Characteristics of donor animals (e.g. age, sex, weight): The BCOP test method uses isolated corneas from the eyes of freshly slaughtered cattle; cattle have a wide range of weights, depending on breed, age, and sex, so there is no recommended weight for the animal at the time of slaughter. Cattle are typically 12 to 60 months old at slaughter.
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): The eyes are excised by an abattoir employee after slaughter, and are placed in Hank's Balanced Salt Solution (HBSS) supplemented with antibiotics.
- Time interval prior to initiating testing: Refrigerated and used within 24 hours of receipt.
- Indication of any existing defects or lesions in ocular tissue samples: On receipt all eyes are carefully examined macroscopically. Any showing defects, such as opacity, scratches, pigmentation, and neovascularization are rejected.
- Indication of any antibiotics used: Penicillin at 100 IU/mL and streptomycin at 100 ug/mL
- Selection and preparation of corneas: The cornea from each selected eye was removed leaving a rim of sclera to facilitate handling. The lens and iris are them removed taking care to avoid damaging the corneal epithelium and endothelium during dissection.
- Quality check of the isolated corneas: The isolated corneas (endothelial side uppermost) were stored in HBBS until mounted in the holders. Corneas were examined to ensure there were no defects prior to mounting.
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Concentration (if solution): 20% w/v concentration in saline
VEHICLE
- Concentration (if solution): saline
- Lot/batch no. (if required):
- Purity:
Duration of treatment / exposure:
4 hours
Duration of post- treatment incubation (in vitro):
No further incubation required.
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

SOLVENT CONTROL USED (if applicable): 20 % Sodium chloride 0.9% w/v

POSITIVE CONTROL USED: 20% (w/v) imidazole

APPLICATION DOSE AND EXPOSURE TIME: 20 % concentration w/v; 4 hours

TREATMENT METHOD: open chamber

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: 3 washes

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Opacity was determined by the amount of light transmission through the cornea. Corneal opacity was measured quantitatively with the aid of an opacitometer, resulting in opacity values measured on a continuous scale. Pre-treatment opacity readings were taken at the end of the 4-hour exposure period.
- Epithelium condition: Each cornea was observed visually and pertinent observations recorded (e.g. tissue peeling, residual test item, nonuniform opacity patterns). Observations were made at the end of the 4-hour exposure period.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of [UV/VIS spectrophotometry / microtiter plate reader] (OD490): Permeability was determined by the amount of sodium fluorescein dye that penetrates all corneal cell layers (i.e. the epthelium on the outer cornea surface through the endothelium on the inner cornea surface). Following the final opacity measurement the permeability of the cornea to sodium fluorescein solution was evaluated. The medium was removed from the anterior chamber first and replaced with 1 mL of a 5 mg/mL (w/v sodium fluorescein solution. The unit was resealed and the holders incubated in a horizontal position at 32 +/- deg C for 90 +/- minutes.
Following the 90-Minute incubation period, the medium in the posterior chamber of each holder was removed ready for the optical density determination at 492 nm (OD492).
360 uL of the medium was transferred to a designated well on a 96-well plate (two wells per cornea). The first well received an undiluted media sample from each cornea tested. A further two wells were designated as blank wells and remained empty for blank subtraction purposes. The absorbance at 492 nm (OD492) of each well was measured using the LabTech LT-4500 microplate reader and LT-com analysis software. The plate reader LT-com software was set to correct the blanks.
Any OD492 value (of a control or test item media sample) that was equal to or greater than 1.3 was diluted to bring the OD492 below 1.3 using a 1 in 5 dilution factor in complete EMEM.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
The results from the two end-points, opacity and permeability, were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS) for each test item.
The IVIS was calculated using the following equation:

IVIS = mean opacity value + (15 x mean permeability OD492 value)

DECISION CRITERIA: The IVIS cut-off values for identifying test items as inducing serious eye damage and test items not requiring classification for eye irritation or serous eye damage are as follows:

IVIS UN GHS
< 3 No Category
> 3; < 55 No prediction can be made
> 55 Category 1
Irritation parameter:
in vitro irritation score
Value:
6.9
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
not determinable
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 (Warren 2010) has been performed on the analogue substance Kieselguhr soda ash flux using the SkinEthic Reconstituted Human Corneal model after a treatment period of 10 minutes.Cytotoxicity was determined by the reduction of MTT to formazan by viable cells in the test material treated tissues (quantitative measurement of tissue viability) relative to the negative control. The test material was classified according to the following criteria:i) If the percentage relative mean tissue viability ≥ 60% the test material was considered to be non-irritant (NI). ii) If the percentage relative mean tissue viability was <60% the test material was considered to be an irritant (I). The relative mean viability of the read-across substance Kieselguhr soda-ash flux calcined after a 10 minute exposure was 99.1% and it was therefore considered not to be an eye irritant.


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. As a result of this a second in vitro (OECD 492 EpiOcular Eye Irritation) study has been commissioned, the results of which should be available later this year. The expectation is that this study will confirm that Synthetic Wollastonite should not be considered to be irritating to eyes.


 


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 considerted to be irritating to eyes. No classification for eye irritation is required.