Reaction mass of sodium pyrovanadate and sodium orthovanadate and diiron trioxide and sodium sulphate and sodiumironsulfide

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010-04-15 to 2010-04-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study reliable without restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: OECD 431: In vitro skin corrosion: Human Skin Model Test (Original guideline adopted 2004-04-13

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Version / remarks:

, 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2009-03-30

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of sodium pyrovanadate and sodium orthovanadate and diiron trioxide and sodium sulphate and sodiumironsulfide

EC Number:

701-306-3

IUPAC Name:

Reaction mass of sodium pyrovanadate and sodium orthovanadate and diiron trioxide and sodium sulphate and sodiumironsulfide

Test material form:

solid: particulate/powder

Details on test material:

Identity: Reaction mass of sodium pyrovanadate and sodium orthovanadate and diiron trioxide and sodium sulfate and sodiumironsulfide (GfE)
Appearance: Solid, black powder
EC/List no.: 701-306-3
Stability in Solvent: Not relvant
Storage: At room temperature, protected from moisture
Expiration Date : January 2011

Test animals

Details on test animals or test system and environmental conditions:

Not applicable - Since this is an in vitro study there is no information on test animals.

Test system

Vehicle:

water

Amount / concentration applied:

TEST MATERIAL
The test material was not crushed or ground with a mortar and a pestle since this did not improve the consistency.
- Amount(s) applied (volume or weight with unit): About 25 mg of the test material were applied to the tissues and were wetted with 25 μL deionised water. The test item was spread to uniformly cover the tissue surface.
No further infromation on the amount/concentration applied was stated.

Duration of treatment / exposure:

Exposure periods of 3 and 60 minutes

Observation period:

not applicable

Number of animals:

not applicable

Details on study design:

CELL CULTURE
EST-1000 kits (Lot No.: EST 100329-001) were purchased from CellSystems® Biotechnologievertrieb GmbH (53562 St. Katharinen; Germany). The EST-1000tissue consisted of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consisted 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 EST-1000™ tissues (surface 0.6 cm²) were cultured on specially prepared cell culture inserts (MILLICELLs®, 10 mm ∅). Generally, the tissues complied with the general and functional model conditions according to OECD 431. The supplier of the skin tissue demonstrated that the delivered batch of the tissues met defined production release criteria (Please refer to "Attached background material" for data on lot).
EST-1000 kits were shipped at 4 °C on medium-supplemented agarose gels in a 24-well plate. On day of receipt EST-1000 tissues were kept in the refrigerator at 2 - 8 °C in the refrigerator

PRE-WARMING OF EST-1000 TISSUES:
At least one hour before the treatment the EST-1000 tissues were removed from the refrigerator. Under sterile conditions using sterile forceps, the inserts were transferred into 6-well plates containing the pre-warmed assay medium. Two 24-well plates were prepared as holding plates, each well containing 300 μL assay medium per well. The holding plates were pre-warmed in an incubator (37 ± 1.5 °C, 5 ± 0.5% CO2) until use.

EXPOSURE:
The EST-1000 tissues were exposed to the test item, positive control (potassium hydroxide as a 8.0 N ready-made solution (Cat. No. 17-8, Sigma 82024 Taufkirchen, Lot No.096K6091); Volume 50 µl) and negative control (deionised water ; Volume 50 µL), respectively, in duplicates for each of the two different exposure periods: 3 minutes and 1 hour, resulting in 12 different treatments per test.
After the pre-incubation of the EST-1000 tissues (1 hour 30 minutes for the 1 hour exposure and 1 hour 10 minutes for the 3 minutes exposure) the medium in each well was replaced by 1.0 mL fresh assay medium per well. The negative control was added to the surface EST-1000 tissues in duplicates. Subsequently, the remaining tissues were exposed to the test item and the positive control in the same manner, respectively. The 6-well plates were then placed into an incubator (37 ± 1.5 °C, 5 ± 0.5% CO2).
At the end of the exposure period the tissues were removed from the 6-well plate and gently rinsed using a wash bottle containing PBS to remove any residual test material. Excess PBS was removed by gently shaking the tissue insert and blotting the lower surface with blotting paper.

MTT ASSAY:
After the exposure procedure was completed for all tissues of each time point, the cell culture inserts were transferred from the holding plates to the plates containing 300 ml MTT solution (MTT solution with a final concentration of 1 mg/ml). . After a 3 hour incubation period (37 ± 1.5 °C, 5 ± 0.5% CO2) the MTT solution was aspirated from the wells and the wells were rinsed three times with PBS. The inserts were transferred into new 24-well plates. The inserts were immersed in extractant solution by gently pipetting 2 mL of extractant solution (isopropanol) into each insert ensuring that the tissues were completely covered. The 24-well plate was sealed to minimize isopropanol evaporation. The formazan salt was extracted for 19 hours while shaking (~120 rpm) at room temperature.
After the extraction period the inserts were pierced with an injection needle to allow the extract to run into the well from which the insert was taken, and the insert was discarded. The 24-well plates were then placed on a shaker for approx. 15 minutes until the solution was homogeneous in colour.
3 × 200 μL aliquots of the blue formazan solution were transferred from each tissue into a 96-well flat bottom microtiter plate. The optical density (OD) was measured using a microplate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany, accurate and precise performance checked annually by Molecular Devices) at 570 nm (OD570) without reference filter. The mean values were calculated for each set of 3 blue formazan solution samples per tissue insert (i.e., replicate).

TEST FOR DIRECT MTT REDUCTION BY TEST ITEM:
To check the MTT reducing capability of the test item two times a solution of MTT in DMEM (1.0 mg/mL) was prepared and one time each about 25 mg and one time each about 50 mg of the test item were added to 1 mL MTT medium. If the mixture turned blue/purple after about 1 hour at room temperature, the test material would have been presumably to have reduced the MTT. No colour change was observed when 25 mg or 50 mg of the test item were applied in the present study.

INTERPRETATION OF RESULTS:
The mean OD values obtained for the duplicate tissues per test item were used to calculate the percent viability relative to the negative control, which was arbitrarily set at 100%.
The test item is considered to be corrosive to skin:
(1) if the viability after 3 minutes exposure is less than 50%, or
(2) if the viability after 3 minutes exposure is greater or equal than 50% and the viability after 1 hour exposure is less than 15%.
The test item is considered to be non-corrosive to skin:
(1) if the viability after 3 minutes exposure is greater or equal than 50% and the viability after 1 hour exposure is greater than or equal to 15%.

ACCEPTABILITY OF THE ASSAY:
The absolute OD570 of the negative control tissues in the MTT-test is an indicator of tissue viability obtained in the testing laboratory after the shipping and storing procedure and under specific conditions of the assay. The tissue viability would meet the acceptance criterion if the mean OD570 of the two negative control tissue replicates would be ≥ 0.8.
The assay would meet the internal acceptance criterion if mean relative tissue viability of the positive control would be ≤ 30%.
No further information on the study design was stated.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

other: other: relative viability (%)

Value:

23.9

Remarks on result:

other:

Remarks:

Basis: mean. Time point: after 3 minute treatment. Reversibility: no data. (migrated information)

Results 2

Irritation / corrosion parameter:

other: other: relative viability (%)

Value:

2

Remarks on result:

other:

Remarks:

Basis: mean. Time point: after 1 hour treatment. Reversibility: no data. (migrated information)

Other effects / acceptance of results:

After exposure to the test item (GfE), the relative absorbance decreased to 23.9% after 3 minutes. After the 1 hour exposure, relative absorbances were reduced to 2.0%. The decrease in relative absorbances exceeded the threshold for corrosivity of 50% for the 3-minutes exposure and of 15% for the 1-hour exposure. Therefore, the test item was considered to be corrosive.

Any other information on results incl. tables

Results

Table 1: Results after treatment with Reaction mass of sodium pyrovanadate and sodium orthovanadate and diiron trioxide and sodium sulfate and sodiumironsulfide (GfE)

Dose group	Exposure interval	Absorbance 570 nm tissue 1	Absorbance 570 nm tissue 2	Mean absorbance of 2 tissues	Relative absorbance (% of negative control) per tissue	Rel. absorbance (% of negative control)
Negative control	3 min	1.288	1.217	1.253	102.8 97.2	100.0
Positive control	3 min	0.096	0.089	0.093	7.7 7.1	7.4
Slags, steelmaking, vanadium (GfE)	3 min	0.221	0.379	0.300	17.6 30.3	23.9
Negative control	1 hour	1.268	1.053	1.160	109.3 90.7	100.0
Positive control	1 hour	0.020	0.006	0.013	1.7 0.5	1.1
Slags, steelmaking, vanadium (GfE)	1 hour	0.028	0.017	0.023	2.4 1.5	2.0

No other effects are observed.

The optical evaluation of the MTT-reducing capacity of the test item after the 1-hour incubation with MTT-reagent did not show evidence of a blue colour. Thus, the test item was not considered to be an MTT reducer.

Historical data:

3 minutes treatment:

Positive control		Negative control
Number of studies	59	Number of studies	59
Period	March 2009 – May 2010	Period	March 2009 – May 2010
Mean viability	12.0 %	Mean OD	1.278
Standard deviation	7.06 %	Standard deviation	0.241
Range of viabilities	33 % - 5 %	Range of ODs	0.9 – 2.0

60 minutes treatment:

Positive control		Negative control
Number of studies	59	Number of studies	59
Period	March 2009 – May 2010	Period	March 2009 – May 2010
Mean viability	2.19 %	Mean OD	1.235
Standard deviation	3.43 %	Standard deviation	0.233
Range of viabilities	16% - 0.5 %	Range of ODs	0.8 – 1.8

Applicant's summary and conclusion

Interpretation of results:

corrosive

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test item Reaction mass of sodium pyrovanadate and sodium orthovanadate and diiron trioxide and sodium sulfate and sodiumironsulfide (GfE) was corrosive to skin.