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Description of key information

Skin irritation of the test item was evaluated with the EpiDerm Reconstructed Human Epidermis Model. Cell viability of the multi-layered tissue culture of highly differentiated epidermal keratinocytes topically exposed to the test substance was evaluated using the MTT assay, which measures the conversion of 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-tetrazoliumbromide (MTT) into a blue formazan salt. Undiluted test item was applied to the EpiDerm tissue for 60 minutes, alongside a negative and positive control. Following 42 hour post-exposure incubation, the mean relative absorbance value of the test item that corresponds to cell viability, did not significantly decrease (95%; threshold for irritancy: ≤ 50%), consequently the test item was not irritating to skin in vitro.

Eye irritation of the test item was evaluated in the Bovine Corneal Opacity and Permeability Test (BCOP, OECD 437), which utilises measurements of corneal opacity as an indicator of protein denaturation, swelling, vacuolation and tissue damage, and corneal fluorescein retention/leakage provides a measure of permeability in vitro. A 20% suspension of the test item was applied to bovine corneas in 750 µL of physiological saline solution (0.9% NaCl) for 4 hours, alongside positive and negative controls. The mean fluorescein retention/leakage score of the test item (mean ± standard deviation, 0.001 ± 0.014), corresponding to permeability, did not significantly vary from controls (0.020 ± 0.012, with lower and higher limits of acceptance of -0.005 and 0.045, respectively). However, the mean opacity score of 3 corneas was 10.770 ± 3.287. The calculated In Vitro Irritation Score (IVIS) of the test substance is above the cut-off value of 3 (UN GHS no category) and below the cut-off value of 55 for identifying test substances as inducing serious eye damage (UN GHS Category 1). Consequently, no direct conclusion regarding the irritant or corrosive classification for the test item can be made, and a Category 2 classification can be assumed. 

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 July 2016 - 29 July 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Elicited via a disturbance of the desquamation process and an inflammatory response (i.e. papules, vesicles, bullae and oedema), skin irritation requires penetration of the stratum corneum and elicitation of a biological response. According to Annex VII and VIII of the REACH Regulations if new test data are required, these must be derived from in vitro methods only. The EpiDerm™ human skin model (OECD 439) has been validated extensively and is an accepted in vitro test method to detect skin corrosion/irritation (Category 1 or 2) and/or the absence of effects (not classified under CLP).
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
adopted July 28, 2015;
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
Commission Regulation (EU) No. 640/2012, Method B.46: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method, adopted July 06, 2012.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No. of test material: sponsor lot# 231/08/15
- Expiration date of the lot/batch: March 2020
- Purity test date: 21 January 2016

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Composition: Bismuth oxide (Bi2O3) 83.35%; Silicon oxide (SiO) 16.63%.
- Physical characteristics: Powder
- Storage condition of test material: At ambient temperature (10 - 25°), container kept tightly closed and stored in a dry and well-ventilated place.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The optical properties of the test item were evaluated under aqueous conditions (25 mg Bismuth silicate was mixed with 300 μL deionized water and incubated at 37°C, 5% CO2 and 95% relative humidity (RH) for 60 minutes). No discolorations were noted. The test item was also evaluated for its potential to interfere with the MTT assay reagent, via reduction of 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-tetrazoliumbromide (MTT) into a blue formazan salt. Therefore, 25 mg Bismuth silicate was added to 1 mL MTT solution and incubated at 37°C, 5% CO2 and 95% relative humidity (RH) for 60 minutes. Untreated MTT solution was used as control. No change of colour was noted. The test substance was not found to interact with the MTT measurement, no additional testing was performed.

OTHER SPECIFICS:
Administration of the test, negative and positive reference item: As a fine powder, 25 mg of test item was applied to the skin model with a surface area of 0.63 cm2 moistened with 25 mL of Dulbecco’s phosphate buffered saline to uniformly cover the skin surface.
Test system:
human skin model
Source species:
human
Cell type:
other: Differentiated epidermal keratinocytes
Cell source:
other: Keratinocyte strain 00267
Source strain:
other: All cells used to produce EpiDerm tissue are purchased or derived from tissue obtained from accredited institutions, from the donor or the donor's legal next of kin for use of the tissues or derivatives of the tissue for research purposes
Details on animal used as source of test system:
SOURCE ANIMAL
- Source: Human
- Tissue: normal epidermal keratinocytes
Justification for test system used:
The reconstructed human epidermis model system is suitable to test solids, liquids, semi-solids and waxes. The liquids may be aqueous or non-aqueous; solids may be soluble or insoluble in water.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ Reconstructed Human Epidermis
- Source: MatTek in vitro Life Science Laboratories (Bratislava, Slovakia)
- Tissue batch number(s): 23347
- Date of analysis of tissue functionality and quality: 27 July 2016
- Date of initiation of testing: July 2016

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

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: DPBS
- Observable damage in the tissue due to washing: None reported
- Modifications to validated SOP: None reported

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentrate: MatTek Corporation batch no# 06021MHD
- MTT diluent: MatTek Corporation batch no# 1737674
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Tecan Sunrise Magellan Version 6.4
- Wavelength: 540 nm
- Replicates: The spectrophotometer measurements were made for each of the three tissues in two replicates.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Manufacturer MTT assay, 1.833 ± 0.026 OD (acceptable range: 1.0 to 3.0)
- Barrier function: Manufacturer ET-50 assay, 4.89 hrs (acceptable range: 4.77 to 8.72 hrs)
- Contamination: Manufacturer long term antibiotic and antimycotic free culture, sterile (acceptable criteria: no contamination)

NUMBER OF REPLICATE TISSUES: 3

ASSAY ACCEPTABILITY CRITERIA
- Negative control: The absolute OD of the negative control (NC) tissues (treated with sterile PBS buffer) in the MTT test is an indicator of tissue viability obtained in the testing laboratory after shipping and storing procedures and under specific conditions of use. The assay meets the acceptance criterion if the mean OD of the NC tissues is ≥ 1.0 and ≤ 2.5.
- Positive control: A 5% SDS (in H2O) solution was used as a positive control (PC) and tested concurrently with the test chemicals. Concurrent means here that the PC has to be tested in each assay, but only one PC is required per testing day. The assay meets the acceptance criterion if the mean viability of PC tissues expressed as % of the negative control tissues is ≤ 20%.
- Standard deviation: Since in each test skin irritancy potential is predicted from the mean viability determined on 3 single tissues, the variability of tissue replicates should be acceptably low. The assay meets the acceptance criterion if the standard deviation (SD) calculated from individual % tissue viabilities of the 3 identically treated replicates is ≤ 18%.

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- Justification for the selection of the cut-off point(s): According to the EU and GHS classification (H314 or H315 / Category 1/2 or no label), an irritant is predicted if the mean relative tissue viability of three individual tissues exposed to the test substance is reduced below or equal to 50% of the mean viability of the negative controls.
mean tissue viability ≤ 50% Irritant (I), (H314 or H315 or GHS Category 1 or 29)
mean tissue viability > 50% non-irritant (NI).
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg of the test item was applied to 0.63 cm2 skin model

NEGATIVE CONTROL
- Negative control: Dulbecco’s Phosphate Buffered Saline (D-PBS)
- Source and lot/batch number: GIBCO Invitrogen GmbH (76131, Germany) batch no# 1782127
- Amount(s) applied (volume or weight): 30 µL

POSITIVE CONTROL
- Positive control: Sodium dodecyl sulphate
- Source and lot/batch number: MatTek Corporation batch no# 012616TMB
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): 5% aqueous solution
Duration of treatment / exposure:
60 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Remarks:
Results presented as decimal figure (i.e. 100% = 1)
Run / experiment:
Mean % optical density (OD540) relative to negative controls
Value:
0.952
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
other: absolute optical density (OD 540)
Run / experiment:
Tissue 1
Value:
1.742
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
other: absolute optical density (OD 540)
Run / experiment:
Tissue 2
Value:
1.719
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
other: absolute optical density (OD 540)
Run / experiment:
Tissue 3
Value:
1.34
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean optical density (OD) of 3 negative control tissues was 1.680 and was well within the acceptable range of ≥ 1.0 to ≤ 2.5.
- Acceptance criteria met for positive control: The viability of cells treated with the positive reference item, 5% SDS, was 5.5% of the negative control and fulfilled the acceptance criterion of ≤ 20%.
- Acceptance criteria met for variability between replicate measurements: The standard deviation determined for all triplicates was below the limit of acceptance of 18%.
- Range of historical values if different from the ones specified in the test guideline: Results for positive and negative control were in line with historical data. All acceptance criteria required were fulfilled.
Interpretation of results:
GHS criteria not met
Conclusions:
Under the test conditions, Bismuth silicate was non-cytotoxic to in an experiment employing an artificial three-dimensional model of human skin. The test item did not show irritant properties and should not be classified as irritant (UN GHS no category).
Executive summary:

Skin irritation of the test item was evaluated with the EpiDerm Reconstructed Human Epidermis Model. Cell viability of the multi-layered tissue culture of highly differentiated epidermal keratinocytes topically exposed to the test substance was evaluated using the MTT assay, which measures the conversion of 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-tetrazoliumbromide (MTT) into a blue formazan salt.

Undiluted test item was applied to the EpiDerm tissue for 60 minutes, alongside a negative and positive control. The mean relative absorbance value of the test item, corresponding to the cell viability did not significantly decrease (95%; threshold for irritancy: ≤ 50%), consequently the test item was not irritant to skin.

The test item passed the MTT- and the Colour Interference pre-tests. Conducted according to OECD Test Guideline 439 and GLP, the study is considered to be reliable without restriction (Klimisch 1).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 July 2016 - 27 September 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Eye irritation is primarily defined by the extent of corneal injury; substances which damage the superficial epithelium may cause slight irritation, whereas further penetration to the corneal stroma or endothelium may induce mild or severe irritation, respectively. The Bovine Corneal Opacity and Permeability Test (BCOP) utilises opacitometric and spectroscopic methods to quantitatively assess changes to bovine corneas, to determine ocular corrosivity and severe irritancy in vitro. Corneal opacity acts as an indicator of protein denaturation, swelling, vacuolation and tissue damage, whereas corneal fluorescein retention/leakage provides a measure of permeability.

The BCOP (OECD 437) has been validated extensively and is an accepted in vitro test method to detect eye corrosion/irritation (Category 1) and/or the absence of effects (not classified under CLP). Data from the BCOP can be used for Annex VII and Annex VIII requirements for serious eye damage/eye irritation.

Solid substances were correctly predicted as UN GHS Category 1 in international validation of the Bovine Corneal Opacity and Permeability Test (BCOP). Consequently, solids and partially soluble solids, such as Bismuth silicate, are not considered to be outside the applicability domain of the test method.
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Version / remarks:
Adopted July 26, 2013
Deviations:
no
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Version / remarks:
Commission Regulation (EU) No. 1152/2010 method B.47, published in the Official Journal of the European Union L324, dated December 09, 2010.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No. of test material: sponsor lot# 231/08/15
- Expiration date of the lot/batch: March 2020
- Purity test date: 21 January 2016

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Composition: Bismuth oxide (Bi2O3) 83.35%; Silicon oxide (SiO) 16.63%.
- Physical characteristics: Powder
- Storage condition of test material: At ambient temperature (10 - 25°), container kept tightly closed and stored in a dry and well-ventilated place.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Vehicle: 0.9% sodium chloride solution
- Final dilution of a dissolved solid, stock liquid or gel: 20% Bismuth silicate (w/w)

FORM AS APPLIED IN THE TEST (if different from that of starting material): Suspended fine powder

OTHER SPECIFICS: The solid test item was suspended in a 0.9% sodium chloride solution with a final concentration of 20% Bismuth silicate (w/w) as recommended in the test guideline 437 for non-surfactant solids. 0.9% NaCl solution was used as the solvent control and 20% Imidazole in 0.9% NaCl solution as the positive control item.
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
TEST MODEL
- Test model: Bovine eyes from cattle obtained from slaughterhouse at age 6-12 months
- Source: Hubert Bahlmann GmbH & Co. Versandschlachterei Speziamichfutterwerk KG 49699, Lindern, Germany
- Storage: Bovine tissues were stored in Hanks’ Balanced Salt Solution (HBSS) (Sigma Aldrich batch no# RNBD4865) containing penicillin at 100 IU/mL (GIBCO batch no# 1759447) and streptomycin at 100 µg/mL (GIBCO batch no# 1770450).
- Quality criteria: Eyes were examined for opacity, scratches and neovascularisation, only corneas from eyes free of defects were used. Prior to the assay, corneas that had opacity greater than seven opacity units or equivalent for the opacitometer and cornea holders, were also discarded.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 32 ± 1°C
Vehicle:
physiological saline
Remarks:
0.9% NaCl
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µL of the 20% test item suspension in 0.9% sodium chloride solution (w/v)
- Concentration (if solution): 20%

NEGATIVE CONTROL
- Negative control: Physiological saline solution (0.9% sodium chloride solution)
- Source and lot/batch number: B. Braun Melsungen AG batch no# 161418002
- Amount(s) applied (volume or weight): 750 µL

POSITIVE CONTROL
- Positive control: Sodium dodecyl sulphate
- Source and lot/batch number: MatTek Corporation batch no# 012616TMB
- Amount(s) applied (volume or weight): 750 µL of the 20% Imidazole suspension in 0.9% sodium chloride solution (w/v)
- Concentration (if solution): 20%
Duration of treatment / exposure:
240 minutes
Duration of post- treatment incubation (in vitro):
90 ± 5 minutes
Number of animals or in vitro replicates:
3
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
Bovine eyes from cattle in the age range of 6 to 12 months were obtained from a slaughterhouse. To minimize deterioration and bacterial contamination, on collection the eyes were completely submerged in Hanks’ Balanced Salt Solution (HBSS) containing penicillin at 100 IU/mL and streptomycin at 100 μg/mL. Upon arrival at the laboratory, the eyes were examined for defects such as but not limited to increased opacity, scratches, and neovascularisation. Only corneas from eyes free of defects were used. The quality of each cornea was also evaluated at later steps in the assay. Corneas that had opacity greater than seven opacity units or equivalent for the opacitometer and cornea holders used after an initial one hour equilibration period had to be discarded. The corneas were dissected with a 2 to 3 mm rim of sclera and mounted in corneal holders with anterior (epithelium) and posterior (endothelium) chambers. Beginning with the posterior chambers, the chambers were filled to excess with pre-warmed Eagle’s Minimum Essential Medium (EMEM), while preventing bubble formation. The corneal holder was equilibrated at 32±1°C for at least one hour.

QUALITY CHECK OF THE ISOLATED CORNEAS
After the equilibration period, fresh pre-warmed EMEM was added to both chambers and baseline opacity readings were taken for each cornea. Corneas exhibiting macroscopic tissue damage (e.g. scratches, pigmentation, neovascularisation) or an opacity >7 opacity units were discarded. The mean opacity of all equilibrated corneas was calculated by use of an opacitometer. A minimum of three corneas with opacity values close to the median value for all corneas were selected as negative control corneas. The remaining corneas were then distributed into treatment, solvent and positive control groups.

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED
- Negative control: Physiological saline solution (0.9% sodium chloride solution)
- Source and lot/batch number: B. Braun Melsungen AG batch no# 161418002
- Amount(s) applied (volume or weight): 750 µL

POSITIVE CONTROL USED
- Positive control: Sodium dodecyl sulphate
- Source and lot/batch number: MatTek Corporation batch no# 012616TMB
- Amount(s) applied (volume or weight): 750 µL of the 20% Imidazole suspension in 0.9% sodium chloride solution (w/v)
- Concentration (if solution): 20%

APPLICATION DOSE AND EXPOSURE TIME
- Amount(s) applied (volume or weight with unit): 750 µL of the 20% test item suspension in 0.9% sodium chloride solution (w/v)
- Exposure time: 240 minutes

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After the exposure period of 240 minutes (the recommended exposure time for non-surfactant solids) the test item, the negative and positive controls, were removed from each chamber. Subsequently, the epithelium was washed with EMEM7 containing phenol red at least three times. Washing was repeated until no test item or discolouration (yellow or purple) of phenol red was visible. The corneas were rinsed a final time with EMEM only to remove any remaining phenol red from the chamber. The chamber was then filled with EMEM without phenol red.

- POST-EXPOSURE INCUBATION:
- Post-exposure preparation: To determine the corneal permeability 1 mL sodium fluorescein9 solution (5 mg/mL in 0.9% sodium chloride solution) was added to the anterior chamber (epithelial surface) while the posterior chamber (endothelial surface) was refilled with fresh EMEM.
- Incubation period: 90 ± 5 minutes
- Examination of corneas: Sodium fluorescein measurements (Tecan Sunrise Magellan Version 6.4)

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Corneal opacity was determined by the amount of light transmission through the cornea measured quantitatively with the aid of an opacitometer8 resulting in opacity values measured on a continuous scale.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of (Tecan Sunrise Magellan Version 6.4 Microtiter plate reader) (OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
After correcting the opacity and mean permeability (OD490) values for background opacity and the negative control permeability OD490 values, the mean opacity, and permeability OD490 values for each treatment group were combined in an empirically-derived formula to calculate an in vitro irritancy score (IVIS) for each treatment group as follows:
IVIS = mean opacity value + (15 x mean permeability OD490 value)
The opacity and permeability values were also evaluated independently to determine whether the test item induced corrosivity or severe irritation through only one of the two endpoints.

DECISION CRITERIA:
A test item that induces an IVIS > 55 is defined as a corrosive or severe irritant. The BCOP test method can also be used to identify chemicals that do not require classification for eye irritation or serious eye damage under the UN GHS classification system. The IVIS cut-off values for identifying test chemicals not requiring classification for irritation or serious eye damage (UN GHS No Category) is ≤3.
Irritation parameter:
cornea opacity score
Run / experiment:
mean opacity (n=3), relative to negative control
Value:
10.77
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
not determinable because of methodological limitations
Irritation parameter:
fluorescein retention score
Remarks:
(permeability)
Run / experiment:
Mean permeability score (n=3) relative to negative controls
Value:
0.001
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of irritation
Irritation parameter:
in vitro irritation score
Run / experiment:
Calculated IVIS of 3 corneas exposed to Bismuth silicate
Value:
10.79
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
not determinable because of methodological limitations
Other effects / acceptance of results:
DEMONSTRATION OF TECHNICAL PROFICIENCY:
- Range of historical values if different from the ones specified in the test guideline: The most recent control data for GLP BCOP studies at LPT (2014-2016) are as follows:

Negative control (0.9% NaCl): presented as mean ± standard deviation (limits of acceptance)
IVIS: 0.812 ± 0.762 (-0.712 - 2.336)
Opacity: 0.513 ± 0.788 (-1.064 - 2.089)
Permeability: 0.020 ± 0.012 (-0.005 - 0.045)

Positive control (Imidazol 20%): presented as mean ± standard deviation (limits of acceptance)
IVIS: 86.039 ± 10.607 (64.826 - 107.253)
Opacity: 63.051 ± 10.709 (41.634 - 84.468)
Permeability: 1.533 ± 0.463 (0.607 - 2.458)

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The corneas treated with the negative control item 0.9% sodium chloride solution revealed a mean opacity value of -0.385 ± 0.852 and a mean permeability value of 0.030 ± 0.006. The calculated IVIS value of 0.070 ± 0.899 was well below the cut-off value of 3 (UN GHS no category).
- Acceptance criteria met for positive control: The corneas treated with the positive control item 20% Imidazole in 0.9% NaCl solution revealed a mean opacity value of 64.395 ± 6.850 and a mean permeability value of 1.483 ± 0.223 compared to the solvent control. The calculated IVIS value of 86.645 ± 8.731 was within two standard deviations of the current historical mean and well above the cut-off value of 55.
- The acceptance criteria for the test were fulfilled.
Interpretation of results:
Category 2 (irritating to eyes) based on GHS criteria
Remarks:
Indirect classification based upon an IVIS that was above the cut-off value of 3 (UN GHS no category) and below the cut-off value of 55 for identifying test substances as inducing serious eye damage (UN GHS Category 1).
Conclusions:
The BCOP (OECD 437) has been validated extensively and is an accepted in vitro test method to detect eye corrosion/irritation (Category 1) and/or the absence of effects (not classified under CLP). Solids and partially soluble solids, such as Bismuth silicate, are not considered to be outside the applicability domain of the test method. Consequently, data from the BCOP can be used to fulfil Annex VII and Annex VIII requirements for serious eye damage/eye irritation potential.

Under the test conditions, the calculated in vitro irritation score (IVIS) for Bismuth silicate of 10.790 ± 3.078 was above the cut-off value for no category, but below the cut-off value of 55 for test substances classified as inducing serious eye damage (Category 1). The BCOP test is not recommended for distinction between irritancy (Category 2 or 2A) and mild irritancy (Category 2B). Conducted according to the aforementioned guidelines and GLP, the BCOP passed all validity criteria and was considered to be reliable without restriction (Klimisch 1). In the absence of further in vitro and/or in vivo testing, the BCOP result suggests classification as Category 2.
Executive summary:

Eye irritation is primarily defined by the extent of corneal injury; substances which damage the superficial epithelium may cause slight irritation, whereas further penetration to the corneal stroma or endothelium may induce mild or severe irritation, respectively. Eye irritation of the test item was evaluated in the Bovine Corneal Opacity and Permeability Test (BCOP, OECD 437), which utilises measurements of corneal opacity as an indicator of protein denaturation, swelling, vacuolation and tissue damage, and corneal fluorescein retention/leakage provides a measure of permeability in vitro.

A 20% suspension of the test item was applied to bovine corneas in 750 µL of physiological saline solution (0.9% NaCl) for 4 hours, alongside positive and negative controls. The mean fluorescein retention/leakage score of the test item (mean ± standard deviation, 0.001 ± 0.014), corresponding to permeability, did not significantly vary from controls (0.020 ± 0.012, with lower and higher limits of acceptance of -0.005 and 0.045, respectively). However, the mean opacity score of 3 corneas was 10.770 ± 3.287. The calculated In Vitro Irritation Score (IVIS) of the test substance is above the cut-off value of 3 (UN GHS no category) and below the cut-off value of 55 for identifying test substances as inducing serious eye damage (UN GHS Category 1). Consequently, no direct conclusion regarding the irritant or corrosive classification for the test item can be made, and a Category 2 classification can be assumed.

According to Regulation (EC) No 1272/2008 (CLP Regulation or CLP) all available information relevant for the evaluation of the specific hazard should be considered in a weight of evidence (WoE) assessment, when the criteria cannot be applied directly (Article 9 (3), CLP). The BCOP (EU B.47; OECD 437) is an intentionally accepted in vitro method to detect serious eye damage (Category 1 under CLP) and/or absence of effects requiring classification for serious eye damage/eye irritation (i.e. not classified under CLP), as described in the Annex to the EU Test Methods (TM) Regulation (Council Regulation (EC) No 440/2008). Conducted according to the aforementioned guidelines and GLP, the BCOP passed all validity criteria and was considered to be reliable without restriction (Klimisch 1).

As specified in Section 8.2 of Column 2 of Annex VIII to the REACH Regulation, for substances manufactured or imported in quantities of ≥10 tpa in vivo testing must only be considered only if the in vitro study under Section 8.2.1 in Annex VII is not applicable for the substance, or the results of the study are not adequate for classification and risk assessment. There are currently no validated in vitro eye irritation test methods available that could be used for direct identification of Eye irritants Category 2 under CLP, thus, in order to distinguish between irritancy (UN GHS Category 2 or 2A) and mild irritancy (UN GHS Category 2B), in vivo testing would be required (OECD 405). In order to avoid in vivo testing and in line with the principles of the Replacement, Refinement and Reduction (3Rs) of the use of animals for scientific purposes, no further testing has been initiated and a Category 2 classification has been assumed. 

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Elicited via a disturbance of the desquamation process and an inflammatory response (i.e. papules, vesicles, bullae and oedema), skin irritation requires penetration of the stratum corneum and elicitation of a biological response. According to Annex VII and VIII of the REACH Regulations if new test data are required, these must be derived from in vitro methods only. The EpiDerm™ human skin model (OECD 439) has been validated extensively and is an accepted in vitro test method to detect skin corrosion/irritation (Category 1 or 2) and/or the absence of effects (not classified under CLP). Bismuth silicate was non-cytotoxic in an in vitro experiment employing the EpiDerm™ artificial three-dimensional model of human skin. The test item did not show irritant properties and should not be classified as an irritant (UN GHS no category). Conducted according to OECD Test Guideline 439 and GLP, the study was considered to be reliable without restriction (Klimisch 1) and sufficient for classification.

According to Regulation (EC) No 1272/2008 (CLP Regulation or CLP) all available information relevant for the evaluation of the specific hazard should be considered in a weight of evidence (WoE) assessment, when the criteria cannot be applied directly (Article 9 (3), CLP). The BCOP (EU B.47; OECD 437) is an internationally accepted in vitro test method to detect serious eye damage (Category 1 under CLP) and/or absence of effects requiring classification for serious eye damage/eye irritation (i.e. not classified under CLP), as described in the Annex to the EU Test Methods (TM) Regulation (Council Regulation (EC) No 440/2008). Conducted according to the aforementioned guidelines and GLP, the BCOP passed all validity criteria and was considered to be reliable without restriction (Klimisch 1). However, the calculated In Vitro Irritation Score (IVIS) of the test substance is above the cut-off value of 3 (UN GHS no category) and below the cut-off value of 55 for identifying test substances as inducing serious eye damage (UN GHS Category 1). Consequently, no direct conclusion regarding the irritant or corrosive classification for the test item can be made, and a Category 2 classification can be assumed. There are currently no validated in vitro eye irritation test methods available that could be used for direct identification of Eye irritants Category 2 under CLP, thus, in order to distinguish between irritancy (UN GHS Category 2 or 2A) and mild irritancy (UN GHS Category 2B), in vivo testing would be required (OECD 405). In order to avoid in vivo testing and in line with the principles of the Replacement, Refinement and Reduction (3Rs) of the use of animals for scientific purposes, no further testing has been initiated and a Category 2 classification has been assumed. 

Bismuth silicate is a low solubility inorganic powder. In the absence of functional groups associated with chemical irritancy, skin irritancy and corneal permeability, the potential eye irritation is believed to be as a result of the physical properties of the test substance (i.e. dustiness).