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Toxicological information

Skin irritation / corrosion

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Administrative data

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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
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

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
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

In vitro test system

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.

Results and discussion

In vitro

Results
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.

Any other information on results incl. tables

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%

Applicant's summary and conclusion

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.