Registration Dossier

Administrative data

Description of key information

In vitro studies for skin corrosion, skin irritation and eye irritation are available.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September 2017 - March 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Test data is required for classification purposes.
Reference:
Composition 0
Qualifier:
according to
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
Version / remarks:
May 2008
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
July 2016
Deviations:
no
Qualifier:
according to
Guideline:
other: MatTek Corporation Protocol for: In Vitro Epiderm Skin Corrosion Test (EPI-200-SCT) For use with MatTek Corporation’s Reconstructed Human Epidermal Model EpiDerm; Version 07/11/2014
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
The GLP certificate is included within the attached study report
Test material information:
Composition 1
Specific details on test material used for the study:
Analytical purity – 97.60% (BaSeO4)
Physical state – Rhombohedral crystals
Colour – White
Molecular weight – 280.32 g/mol
Expiry date – 24th April 2019
Storage conditions – Room temperature
Stability under test conditions - Acceptable
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
foreskin from a single donor
Details on animal used as source of test system:
Type: Normal human epidermal keratinocytes (NHEK)

Genetic make-up: Single donor

Derived from: Neonatal-foreskin tissue (NHEK)

Alternatives: NHEK from adult breast skin

Screened for: HIV, Hepatitis-B, Hepatitis-C, mycoplasma
Justification for test system used:
EpiDerm is a proven in vitro model system for chemical, pharmaceutical and skin care product testing.
Vehicle:
physiological saline
Remarks:
phosphate buffered saline
Details on test system:
The test was carried out using the reconstituted three-dimensional human skin model EpiDermTM (MatTek). This skin model consists of normal human epidermal keratinocytes (NHEK) which have been cultured on chemically modified, collagen-coated cell culture inserts (Millicell). The EpiDermTM epidermis model exhibits in vivo-like morphological and growth characterisitcs which are uniform and highly reproducible. It consists of organised basal, spinous and granular layers and multi-layered stratum corneum analogous to patterns found in vivo.

Preparation and application of Test item:
25 mg of the test item was applied directly atop the EpiDerm tissue using an application spoon avoiding compression of the test item. To ensure good contact with the skin the test item was moistened with 25 μL of water. The test item was spread to match the size of the tissue.

Test item – 25 mg + 25 μL water
The test item was performed on a total of 4 tissues per dose group, 2 replicates for each treatment period.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ±1 oC, 5% CO2, 95% air
- Temperature of post-treatment incubation (if applicable): 37 ±1 oC, 5% CO2, 95% air
REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: approximately 20rinses with PBS (phosphate bufferred saline).
:
DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Spectrophotometer: Plate spectrophotometer 570nm no reference wavelength.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
Test item (barium selenate) – 25 mg + 25 μL water

25 mg of the test item was applied directly atop the EpiDerm tissue using an application spoon avoiding compression of the test item. To ensure good contact with the skin the test item was moistened with 25 μL of water. The test item was spread to match the size of the tissue.
Duration of treatment / exposure:
Tissue samples were transferred to 6-well plates containing 0.9 mL pre-warmed fresh assay medium, the plates were post-incubated at 37 oC, for at least 1 hour. The medium was replaced by 0.9 mL of fresh assay and the surface dyed using a sterile cotton tip. About 1 hour before the end of the first treatment period, MTT solution was prepared and pre-warmed in the incubator.
60 minute experiment: the tissues were treated with each dose group (negative control, positive control and test item) in duplicate, starting with the negative control. Start time was recorded with dosing of the first tissue. A constant time interval of e.g. 20 seconds was kept between dosing. Then the 6-well plate was incubated at 37 oC.
3 minute experiment: the tissues were treated with each dose group in duplicate, starting with the negative control. Start time was recorded with dosing of the first tissue, constant time intervals of 20 seconds were kept between dosing.
After 3 minutes of application, the first insert was removed from the 6-well plate and the tissue gently rinsed about 20 times with PBS to remove residual test item. Excess PBS was removed by gentle shaking and removing the excess with blotting paper. The insert was placed in a prepared 24-well “holding plate” containing 300 μL pre-warmed assay medium per well. All inserts were treated in the same manner. The inserts were transferred into a prepared 24-well “MTT assay plate” containing 300 μL pre-warmed MMT solution. The plate was incubated for 3 hours at 37oC
After the extraction period, the inserts were pierced to allow the extracts to run through the tissues into the corresponding wells. The inserts were discarded and extraction plates were placed on a shaker for 15 minutes.
Per tissue, 3 x 200μL aliquots of the extracts were transferred into a 96-well plate and OD (optical density) was measured at 570 nm without reference wavelength, in a plate spectrophotometer using isopropanol as a blank.
Number of replicates:
The test item was performed on a total of 4 tissues per dose group, 2 replicates for each treatment period.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Test item (barium selenate)
Vehicle controls valid:
not examined
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: No indication of corrosive effects.
Other effects / acceptance of results:
The mean relative tissue viability (% negative control) was ≥ 50% (100.7%) after 3 minutes and ≥ 15% (86.4%) after the 60 minute treatment.

Results of the 3 minute experiment

Name

Negative control

Test item

Positive control

Tissue

1

2

1

2

1

2

Absolute OD570

1.356

1.471

1.479

1.554

1.616

1.639

1.484

1.533

1.544

1.473

1.586

1.558

0.220

0.236

0.231

0.235

0.231

0.235

OD570– Blank corrected*

1.312

1.427

1.435

1.510

1.572

1.595

1.440

1.489

1.500

1.429

1.542

1.514

0.176

0.192

0.187

0.191

0.187

0.191

Mean OD570of 3 aliquots (blank corrected)

1.391

1.559

1.476

1.495

0.185

0.189

SD OD570of 3 aliquots

0.068

0.046

0.038

0.058

0.025

0.024

Total mean OD570 of 2 replicate tissues (blank corrected)

1.475

1.486

0.187

Mean OD570of 2 replicate tissues

0.119

0.013

0.003

Mean relative tissue viability [%]

100.0

100.7

12.7

Coefficient of variation [%]**

8.0

0.9

1.7

 

* Corrected mean OD570of the negative control corresponds to 100% absolute tissue viability.

** Coefficient of variation (CV) (in the range 20 – 100% viability) between two tissues treated identically30%

 

Results of the 60 minute experiment

Name

Negative control

Test item

Positive control

Tissue

1

2

1

2

1

2

Absolute OD570

1.480

1.542

1.533

1.638

1.686

1.674

1.371

1.422

1.396

1.373

1.347

1.377

0.095

0.148

0.099

0.147

0.102

0.148

OD570– Blank corrected*

1.437

1.499

1.490

1.595

1.643

1.631

1.328

1.379

1.353

1.330

1.304

1.334

0.052

0.105

0.056

0.104

0.059

0.105

Mean OD570of 3 aliquots (blank corrected)

1.475

1.623

1.353

1.323

0.071

0.089

SD OD570of 3 aliquots

0.034

0.033

0.033

0.028

0.035

0.034

Total mean OD570 of 2 replicate tissues (blank corrected)

1.549

1.338

0.080

Mean OD570of 2 replicate tissues

0.105

0.022

0.013

Mean relative tissue viability [%]**

100.0

86.4

5.2

Coefficient of variation [%]***

6.8

1.6

16.4

 

* Corrected mean OD570of the negative control corresponds to 100% absolute tissue viability.

** Mean relative tissue viability of the 60 minute positive control < 15%

*** Coefficient of variation (CV) (in the range 20 – 100% viability) between two tissues treated identically30%

Interpretation of results:
other: “Non-corrosive”
Conclusions:
In this study under the given conditions the test item showed no corrosive effects. The test item is classified as “non-corrosive”.
Executive summary:

The potential for Barium Selenate to induce skin corrosion was analysed using the three-dimensional human epidermis model EpiDermTMstandard model (MatTek) comprising a reconstructed epidermis with a functional stratum corneum. The test item, Barium Selenate, was applied topically to the surface of the Epiderm tissue. Cytotoxicity was expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT after a 3 minute and 60 minute exposure period and compared to those of the concurrent negative controls.

The test item was shown to have no corrosive effects on the Epiderm tissue. The mean relative tissue viability (expressed as % negative control) was≥ 50% after the 3 minute treatment and ≥ 15% (86.4 %) after the 60 minute treatment.

 

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September 2017 - March 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Test data is required for classification purposes.
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
July 2015
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
July 2012
Deviations:
no
Qualifier:
according to
Guideline:
other: MatTek Corporation Protocol for: In Vitro Epiderm Skin Irritation Test (EPI-200-SIT) For use with MatTek Corporation’s Reconstructed Human Epidermal Model Epiderm (EPI-200-SIT); Version 07-Nov-2014
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
The GLP certificate is included within the attached study report
Test material information:
Composition 1
Specific details on test material used for the study:
Analytical purity – 97.60% (BaSeO4)
Physical state – Rhombohedral crystals
Colour – White
Molecular weight – 280.32 g/mol
Expiry date – 24th April 2019
Storage conditions – Room temperature
Stability under test conditions - Acceptable
Test system:
human skin model
Remarks:
EpiDerm (EPI-200)
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
foreskin from a single donor
Details on animal used as source of test system:
Type: Normal human epidermal keratinocytes (NHEK)

Genetic make-up: Single donor

Derived from: Neonatal-foreskin tissue (NHEK)

Alternatives: NHEK from adult breast skin

Screened for: HIV, Hepatitis-B, Hepatitis-C, mycoplasma
Justification for test system used:
EpiDerm is a proven in vitro model system for chemical, pharmaceutical and skin care product testing.
Vehicle:
physiological saline
Remarks:
Dulbecco's phosphate buffered saline (DPBS; Gibco, cat. No. 14040-091, Lot No. 1838067)
Details on test system:
The test was carried out using the reconstituted three-dimensional human skin model EpiDermTM (MatTek). This skin model consists of normal human epidermal keratinocytes (NHEK) which have been cultured on chemically modified, collagen-coated cell culture inserts (Millicell). The EpiDermTM epidermis model exhibits in vivo-like morphological and growth characterisitcs which are uniform and highly reproducible. It consists of organised basal, spinous and granular layers and multi-layered stratum corneum analogous to patterns found in vivo.
Control samples:
yes, concurrent negative control
yes, concurrent vehicle
yes, concurrent positive control
Amount/concentration applied:
Pre-incubation, 25 μL of sterile DPBS were applied to the epidermal surface in order to improve the contact between the test item powder and the epidermis. Afterwards, 25 mg (39 mg/cm2) of the test item was applied directly on top of the EpiDerm tissue using an application spoon and avoiding compression of the test item. The test item was spread to match the size of the tissue by using a bulb-headed Pasteur pipette.
Duration of treatment / exposure:
Treated samples were incubated for 35 ±1 minutes and then placed under the sterile flow for the remaining time until 60 ±1 minutes. The tissues were then washed by filling and emptying the inserts 15 times with DPBS (Dulbecco’s phosphate buffered saline). Excess DPBS was removed using blotting paper. The inserts were then placed in new 6-well plates containing 0.9 mL pre-warmed fresh assay medium and the plates were post-incubated at 37 ±1 oC, 5% CO2, 95% air, for 24 ±2 hours. Following this incubation, the tissues were transferred to new wells containing fresh assay medium and incubated for an addition 18 ±2 hours.
Duration of post-treatment incubation (if applicable):
After post-incubation, the inserts were dried and transferred to a 24-well plate containing 300 μL pre-warmed MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) medium. The plate was incubated for 3 hours ±5 minutes at 37 ±1 oC, 5% CO2, 95% air. Following MTT incubation, the tissues were rinsed three times using DPBS and dried using blotting paper. The tissues were then transferred to 12-well plates and immersed in 2 mL isopropanol, sealed to inhibit evaporation. Extraction was carried out (protected from light) at room temperature by gentle shaking for 2 hours.
Number of replicates:
Samples were prepared in triplicate.
Irritation / corrosion parameter:
% tissue viability
Remarks:
(% tissue variability of negative control)
Run / experiment:
Test item (barium selenate)
Value:
3.4
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
The results were acceptably obtained according to the guidance.

Results of the test item Barium Selenate

Name

Negative control

Positive control

Test item

Tissue

1

2

3

1

2

3

1

2

3

Absolute OD570

1.956

1.969

1.874

1.899

1.856

1.889

0.096

0.097

0.102

0.103

0.099

0.102

0.106

0.105

0.105

0.107

0.105

0.106

OD570(Blank corrected)

1.913

1.926

1.832

1.856

1.814

1.846

0.053

0.055

0.059

0.061

0.057

0.059

0.063

0.062

0.062

0.064

0.062

0.063

Mean OD570of the blank duplicates (Blank Corrected)

1.920

1.844

1.830

0.054

0.060

0.058

0.063

0.063

0.063

Total Mean OD570of 3 Replicate Tissues (Blank Corrected)

1.865*

0.057

0.063

SD OD570

0.048

0.003

0.000

Relative Tissue Viability [%]

103.0

98.9

98.1

2.9

3.2

3.1

3.4

3.4

3.4

Mean Relative Tissue Viability [%]

100.0

3.1**

3.4

SD Tissue Viability [%]***

2.6

0.2

0.0

CV [% Viabilities]

2.6

5.4

0.5

 

* Blank-corrected mean OD570nm of the negative control corresponds to 100% absolute tissues viability.

** Mean relative tissue viability of the three positive control tissues is20%

*** Standard deviation (SD) obtained from the three concurrently tested tissues is18%

Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
In this study under the given conditions the test item showed irritant effects. The relative mean tissue viability after 60 minutes of exposure and 42 hours post-incubation was ≤ 50%. The test item is therefore classified in accordance with UN GHS “Category 2”.
Executive summary:

The potential for Barium Selenate to induce skin irritation was analysed using the three-dimensional human epidermis model EpiDermTMstandard model (MatTek) comprising a reconstructed epidermis with a functional stratum corneum. This model was used as areplacement for the Draize Skin Irritation test (OECD TG 404), to distinguish between UN GHS “Category 2” skin irritating test substances and not categorised test substances, which may be considered as non-irritants. 

In the present study Barium Selenate was applied topically to the EpiDermTMtissue for 60 minutes followed by a 42 hour post-incubation period and immediate determination of cytotoxic effects via MTT reduction assay.

A mixture comprising 25 mg test item per 1 mL MTT medium showed no reduction of MTT compared to the solvent. The mixture did not turn blue/purple. Therefore, NSMTT equalled 0%.

The mixture of 25 mg of test item per 300μL aqua dest. and/or isopropanol showed no colouring detected by unaided eye-assessment. Therefore NSC equalled 0%.

The test item showed irritant effects. The mean relative tissue variability (% negative control) was50% (3.4 %) after 60 minutes and 42 hour post-incubation.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (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:
September 2017 - March 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Test data is required for classification purposes.
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:
October 2017
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
The GLP certificate is included within the attached study report
Test material information:
Composition 1
Specific details on test material used for the study:
Analytical purity – 97.60% (BaSeO4)
Physical state – Rhombohedral crystals
Colour – White
Molecular weight – 280.32 g/mol
Expiry date – 24th April 2019
Storage conditions – Room temperature
Stability under test conditions - Acceptable
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
The assay uses isolated corneas obtained as a by-product from animals freshly slaughtered at the abattoir A. Moksel AG, Buhloe, Germany.
Vehicle:
other: RPMI 1640 without phenol red
Remarks:
(RPMI = Roswell Park Memorial Institute)
Amount / concentration applied:
750 μL of the test item (barium selenate) or control substance (positive control – 20% imidazole in physiological saline 0.9%; negative control – physiological saline 0.9% NaCl)
Duration of treatment / exposure:
Samples were mounted in the chambers of the corneal holder and filled with RPMI (without phenol red) containing 1% FBS and 2 mM L-glutamine (complete RPMI). The posterior chamber was always filled first. The corneas were incubated for 1 hour at 32 ±1 oC.
Duration of post- treatment incubation (in vitro):
Not applicable.
Number of animals or in vitro replicates:
3 for each group (test item, negative control and positive control)
Irritation parameter:
in vitro irritation score
Run / experiment:
Test item (barium selenate)
Value:
24.13
Vehicle controls valid:
not examined
Negative controls valid:
other: The negative control responses should result in opacity and permeability values that are less than the established upper limits for the background bovine corneas treated with the respective negative control.
Positive controls valid:
yes
Other effects / acceptance of results:
The eye irritancy potential of Barium Selenate was investigated in the bovine opacity and permeability assay.
The test item was suspended with physiological saline 0.9% NaCl (see 10.3) to give a 20% concentration.
All 3 corneas treated with Barium Selenate showed opacity of the tissue
The following mean in vitro irritation score was calculated: 24.13
No prediction can be made regarding the classification of the test substance Barium Selenate according to the evaluation criteria.
The in vitro irritation score obtained with the positive control fell within two standard deviations of the current historical mean and therefore this assay is considered to be valid.
The negative control responses should result in opacity and permeability values that are less than the established upper limits for the background bovine corneas treated with the respective negative control.

Opacity of treated bovine corneas

Cornea No.

Test item

Initial opacity

Final opacity

Change of opacity value

Corrected opacity value

1

2

3

Mean value (MV)

Negative control

0.73

0.39

0.52

0.55

1.36

0.49

1.19

1.01

0.63

0.10

0.66

0.46

 

4

5

6

MV

Positive control

1.15

1.19

1.26

1.20

98.20

84.99

100.26

94.49

97.05

83.81

99.01

93.29

96.59

83.34

98.54

92.82

7

8

9

MV

Barium selenate test item

1.04

-0.02

-0.38

0.22

27.13

33.82

13.97

24.63

26.09

33.84

13.97

24.63

25.63

33.37

13.51

24.17

 

Permeability

Cornea No.

Test item

OD490

Corrected OD490 value

1

2

3

Mean value (MV)

Negative control

0.012

0.010

0.006

0.009

 

4

5

6

MV

Positive control

2.110

1.745

1.443

1.766

2.101

1.736

1.434

1.757

7

8

9

MV

Barium selenate test item

0.010

0.005

0.005

0.007

0.001

-0.004

-0.004

-0.003

 

In vitro irritation score

Cornea No.

Test item

OD490

Corrected OD490 value

IVIS

1

2

3

Mean value (MV)

Negative control

0.63

0.10

0.66

0.46

0.,012

0.010

0.006

0.009

 

 

 

0.060

4

5

6

MV

Positive control

96.59

83.34

98.54

92.82

2.101

1.736

1.434

1.757

 

 

 

119.17

7

8

9

MV

Barium selenate test item

25.63

33.37

13.51

24.17

0.001

-0.004

-0.004

-0.003

 

 

 

24.13

 

Historical mean in in vitro irritation score of the positive control

 

IVIS positive control – imidazole 20%

Mean value (MV)

Standard deviation (SD)

MV-2xSD

MV+2xSD

123.84

17.61

88.61

159.07

Number of replicates providing historical mean 30

 

 

Interpretation of results:
study cannot be used for classification
Conclusions:
Based on the results of this test, no prediction can be made regarding the classification of the test substance Barium Selenate, according to the evaluation criteria.
Executive summary:

The eye irritancy potential of Barium Selenate was investigated in the bovine corneal opacity and permeability assay. The test item (barium selenate) was suspended with physiological saline 0.9% NaCl, to give a 20% concentration.

The three corneas treated with the test item showed opacity of the tissue – the calculated mean in vitro irritation score is 24.13. No prediction could be made regarding the classification of the test substance, according to the evaluation criteria.

 

The in vitro irritation score obtained with the positive control fell within the two standard deviations of the current historical mean and therefore this assay is considered to be valid.

The negative control responses resulted in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control.

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

A positive result for the in vitro skin irritation study (Gehrke, Study 176660) was observed therefore classification as skin irritant Cat 2 applies.

The result for the in vitro eye irritation study (Bachmann, Study 176661) demonstrated that the test substance was not corrosive to eyes but could not conclude on the eye irritancy potential of the substance. On the basis that the substance tested positive for skin irritancy, the classification for eye irritant Cat 2 has been applied.