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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-02-03 to 2010-02-05
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to
Guideline:
other: "Commission Regulation (EC) No. 440/2008 B 46"
Deviations:
no
Qualifier:
according to
Guideline:
other: ECVAM international validation study on in vitro tests for acute skin irritation (Altern Lab Anim. 2007 Dec; 35 (6):559-601)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2009-03-30

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Zinc selenite
- Substance type: technical product
- Physical state: solid
- Storage condition of test material: Store product in closed containers. Keep only in the original container. Do not store together with foodstuff. Keep away from water. Metals which are attacked by an alkaline medium. Keep container tightly closed and dry in a cool, well-ventilated place.

Test animals

Details on test animals and environmental conditions:
not applicable- Since this is an in vitro study there is no information on test animals.

Test system

Vehicle:
unchanged (no vehicle)
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 15 mg of the test item were applied to each tissue (≙ 39.47 mg/cm2), spread to match the tissue size.
No further information on the amount/concentration applied was stated.
Duration of treatment / exposure:
15± 1 min
Observation period:
not applicable
Number of animals:
not applicable
Details on study design:
This in vitro study was performed to assess the irritation potential of Zinc selenite by means of the Human Skin Model Test. Three tissues of the human skin model EpiSkin™ (Lot No.: 10-EKIN-003) were treated with either the test item, the negative or the positive control.

CELL CULTURE
EpiSkin™ kits are purchased from Skinethic Laboratories (06000 Nice, France). The EpiSkin™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiSkin™ tissues (surface 0.38 cm²) are cultured on specially prepared cell culture inserts.

TREATMENT:
Negative control: Deionised water (Lot no.22.12.09) was used as the negative control. 15 µL were applied to each of triplicate tissues for 15 ± 1 minutes.
Positive Control: 5% SLS (Sodium lauryl sulphate, lot no. 1353471 51508322 (Sigma, 82024 Taufkirchen, Germany) solution in deionised water, prepared freshly prior to the performance of the experiment, was used as positive control. 15 µL were applied to each of triplicate tissues for 15 ± 1 minutes.
Test item: 15 mg of the test item were applied to each of triplicate tissues (≙ 39.47 mg/cm2).
The negative and positive control, and the test item were added into the insert atop the concerning EpiSkin™ triplicate tissues. The 12-well plates were placed into the incubator for 15 ± 1 min at 37 ± 1.5 °C, 5 ± 0.5% CO2.
After the end of the treatment interval the inserts were removed immediately from the 12-well plate. Using a wash bottle the tissues were gently rinsed with PBS to remove any residual test material. Excess PBS was removed by gently shaking the inserts and blotting the bottom with blotting paper. The inserts were placed in the plates with 2 mL maintenance medium. The tissues were incubated for 42 ± 1 hour at 37 ± 1.5 °C, 5 ± 0.5% CO2.

CELL VIABILITY TEST:
Cell viability is measured by dehydrogenase conversion of MTT [(3-4,5-dimethyl thiazole 2-yl) 2,5-diphenyl-tetrazoliumbromide], present in cell mitochondria, into a blue formazan salt that is quantitatively measured after extraction from tissues (Faller, C., Bracher, M., Dami, N., Roguet, R., (2002). Predictive ability of reconstructed human epidermis equivalents for assessment of skin irritation of cosmetics. Toxicology in vitro 16 (5), 557-552). The percent reduction of cell viability in comparison of untreated negative controls is used to predict skin irritation potential.
After the treatment procedure was completed for all tissues of each time point cell culture inserts were transferred from the holding plates to plates containing 2 mL assay medium containing 0.3 mg/mL MTT per well. . After a 3 hour incubation period (37 ± 1.5 °C, 5 ± 0.5% CO2) MTT solution was aspirated from the wells and the wells were rinsed three times with PBS. Tissue samples were cut out of the inserts with a biopsy punch and transferred into plastic vials. The tissue samples were immersed into extractant solution by gently pipetting 0.5 mL extractant solution (isopropanol) into each vial. The tissue samples were completely covered by isopropanol. The vials were sealed to inhibit isopropanol evaporation. The formazan salt was extracted for 4 hours while shaking (~120 rpm) at room temperature.
Per each tissue sample 2 x 200 µL aliquots of the formazan blue solution were transferred into a 96-well flat bottom microtiter plate. OD was read in a microplate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany) at 570 nm without reference filter. Mean values were calculated from the 2 wells per tissue sample.

EVALUATION OF RESULTS
The mean OD of the three negative control tissues was calculated. This value corresponds to 100% tissue viability in the current test. For each individual tissue treated with the test item or the positive control the individual relative tissue viability is calculated according to the following formula:
Relative viability (%) = (OD test item/OD negative control)*100.
For the test item and the positive control the mean relative viability ± standard deviation of the three individual tissues are calculated and used for classification according to the following prediction model:
For the current test, an irritation potential of a test item according to EU classification (acc. to Directive 67/548/EEC and Regulation 1272/2008/EC) is predicted if the mean relative tissue viability of three individual tissues is reduced below 50% of the negative control.

ACCEPTABILITY OF THE ASSAY:
The absolute OD 570 nm of the negative control tissues in the MTT test is an indicator of tissue viability obtained after the shipping and storing procedure and under specific conditions of the assay. Tissue viability is meeting the acceptance criterion if the mean OD of the three tissues is >= 0.8.
An assay is meeting the acceptance criterion if mean relative tissue viability of the positive control is <= 40%.

TEST FOR DIRECT MMT REDUCTION:
For correct interpretation of results it was necessary to assess the ability of the test item to directly reduce MTT. To test for this ability approximately 15 mg of the test item were added to 1 mL of MTT solution and the mixture was incubated in the dark at room temperature for 60 minutes. Untreated MTT medium was used as control. If the MTT solution colour turned blue/purple, the test item was presumed to have reduced the MTT. A colour change could not be observed.

No further information on the study design was stated.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
other:
Value:
78.6
Remarks on result:
other:
Remarks:
Basis: mean. Time point: after 15 min incubation. Reversibility: no data. Remarks: After treatment with the test item the relative absorbance values were irrelevantly decreased to 78.6%. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.
Other effects / acceptance of results:
After treatment with the test item Zinc selenite the relative absorbance values were irrelevantly decreased to 78.6%. This value is well above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.

Any other information on results incl. tables

Results after treatment with Zinc selenite

Dose group

Treatment Interval

Absorbance 570 nm
Tissue 1*

Absorbance 570 nm
Tissue 2*

Absorbance 570 nm
Tissue 3*

Mean Absorbance of 3 Tissues

Standard Deviation in %

Rel. Absorbance

[% of Negative Control]**

Negative Control

15 min

0.965

1.051

1.115

1.044

7.2

100.0

Positive Control

15 min

0.337

0.259

0.193

0.263

16.1

25.2

Zinc selenite

15 min

0.750

0.699

1.011

0.820

6.9

78.6

*       Mean of three replicate wells after blank correction
**
      relative absorbance [rounded values]: (100xabsorbance-test item)/absorbance-negative control

Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour.

Historical data:

Positive control

Negative control

Number of Studies

73

Number of Studies

73

Period

July 2007 –

March 2010

Period

July 2007 –

March 2010

Mean Viability

16.5%

Mean OD

1.081

Standard Deviation

11.0%

Standard Deviation

0.262

Range of Viabilities

3% - 36%

Range of ODs

0.7 – 1.6

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
After treatment with the test item Zinc selenite the relative absorbance values were irrelevantly decreased to 78.6%. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential. The test item should not be classified and labelled as skin irritant according to regulation (EC) No. 1272/2008 and subsequent regulations.