Reaction mass of Cobaltate(2-), [2-[[[4-hydroxy-3-[[2-oxo-1-[(phenylamino)carbonyl]propyl]azo]phenyl]sulfonyl]amino]benzoato(3-)][2-[[2-hydroxy-5-[(phenylamino)sulfonyl]phenyl]azo]-3-oxo-N-phenylbutanamidato(2-)]-, disodium and Cobaltate(3-), bis[2-[[[4-hydroxy-3-[[2-oxo-1-[(phenylamino)carbonyl]propyl]azo]phenyl]sulfonyl]amino]benzoato(3-)]-, trisodium and sodium bis[2-[[2-hydroxy-5-[(phenylamino)sulphonyl]phenyl]azo]-3-oxo-N-phenylbutyramidato(2-)]cobaltate(1-)

Specific investigations: other studies

Administrative data

Endpoint:

cytotoxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From March the 14th to the 15th, 2007

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: Guideline ISO 10993 "Biological Evaluation of Medical Device" Part 5: "Tests for cytotoxicityz In vitro methods“

Version / remarks:

1992

GLP compliance:

yes (incl. QA statement)

Type of method:

in vitro

Test material

Test material information

Results and discussion

Details on results:

Toxic effects were observed following incubation with the test item from 312.5 µg/ml up to the highest tested concentration (5000 µg/ml). The calculated XTT50 value is 289.5 µg/ml. Even after stringent washing not all of the test item could be removed from the cells in the higher concentrations leading to high chemical blank values and therewith freak high viability values.

CONTROLS
The negative control and the solvent control showed no reduction in cell viability. The positive control (SDS) induced a distinct dose-related reduction in cell viability.

Any other information on results incl. tables

Results with test item

Test group	Absorbance*	SD	Chem. Blanks	Absorbance of solvent control (%)**
Negative control	0.730	0.024	0.113	106.4
Solvent control	0.690	0.018	0.109	100.0
Test item 39.1 µg/ml	0.693	0.021	0.115	99.5
Test item 78.1 µg/ml	0.675	0.017	0.115	96.4
Test item 156.3 µg/ml	0.620	0.017	0.122	85.8
Test item 312.5 µg/ml	0.391	0.012	0.137	43.7
Test item 625 µg/ml	0.262	0.007	0.148	19.7
Test item 1250 µg/ml	0.340	0.016	0.180	27.6
Test item 2500 µg/ml	0.393	0.021	0.235	27.4
Test item 5000 µg/ml	0.615	0.022	0.331	49.1

*Mean absorbance (absolute) of 7 wells

**Relative absorbance (rounded values)

Positive control

Test group	Absorbance*	SD	Chem. Blanks	Absorbance of solvent control (%)**
Negative control	1.085	0.132	0.116	112.4
Solvent control	0.974	0.059	0.112	100.0
SDS 3.125 µg/ml	1.062	0.075	0.109	110.6
SDS 6.25 µg/ml	1.012	0.092	0.110	104.7
SDS 12.5 µg/ml	0.973	0.070	0.110	100.2
SDS 25 µg/ml	0.940	0.066	0.106	96.7
SDS 50 µg/ml	0.722	0.014	0.104	71.8
SDS 100 µg/ml	0.117	0.003	0.103	1.7
SDS 125 µg/ml	0.118	0.003	0.106	1.5
SDS 250 µg/ml	0.126	0.003	0.102	2.8

*Mean absorbance (absolute) of 7 wells

**Relative absorbance (rounded values)

Applicant's summary and conclusion

Conclusions:

The test item possesses a cytotoxic potential.

Executive summary:

The in vitro study was performed to assess the cytotoxic potential of test item by means of the XTT test using the mouse cell line L929.

The following concentrations of the test item were tested: 39.1, 78.1, 156.3, 312.5, 625, 1250, 2500, 5000 µg/ml.

Complete medium (RPMI containing 10 % (v/v) FCS) was used as negative control. The solvent control for the positive control was also RPMI medium containing 10 % (v/v) FCS and 10.0 % (v/v) deion. water.

SDS was used as positive control. The following concentrations were applied: 3.125, 6.25, 12.5, 25, 50, 100, 125, 250 µg/ml.

The incubation time was 24 hours at 37 ± 1.5 °C.

The negative control and the solvent control showed no reduction in cell viability. The positive control (SDS) induced a distinct dose~re|ated reduction in cell viability.

Toxic effects were observed following incubation with the test item from 312.5 µg/ml up to the highest tested concentration (5000 µg/ml). The calculated XTT50 value is 289.5 µg/ml. Even after stringent washing not all of the test item could be removed from the cells in the higher concentrations leading to high chemical blank values and therewith freak high viability values.

Conclusion

It can be stated that under the experimental conditions reported, the test item possesses a cytotoxic potential.