Registration Dossier

Diss Factsheets

Administrative data

Type of information:
experimental study
Adequacy of study:
key study
Study period:
From June 11 to July 01, 2003
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
other: ISO 10993 (Biological Evaluation of Medical Devices" Part 5: Tests for cytotoxicity: in vitro methods"
GLP compliance:
yes (incl. QA statement)
Type of method:
in vitro

Test material

Constituent 1
Reference substance name:
Fluorescent Brightener 367
Fluorescent Brightener 367

Test animals

Details on test animals or test system and environmental conditions:
Reasons for the Choice of the Gell Line L929
The ATCC, CCL 1 NCTC clone 929 (clone of strain L, mouse connective tissue) cell line has been used for many years in in vitro experiments with success. Especially the high and established proliferation time rate (doubling time: 16 h) and a good viability of untreated cells (as a rule more than 70 %), both necessary for the appropriate performance of the study, recommend the use of this cell line.

Cell Cultures
Large stocks of the L929 cell line (supplied by LMP, Technical University Darmstadt, D-64287 Darmstadt) were stored in liquid nitrogen in the cell bank of testing laboratory allowing the repeated use of the same cell culture batch in experiments.
Therefore, the parameters of the experiments remain similar, because of the reproducible characteristics of the cells.
Thawed stock cultures were propagated at 37 °C in plastic flasks (GREINER, D-72632Frickenhausen). Seeding was done with about 2 x 10^5 cells per flask in 6 ml of RPMI 1640 supplemented with 10 % fetal calf serum (FKS; PAA Laboratories GmbH, D-35091 Cölbe). The cells were subcultured twice weekly. The cell cultures were incubated at 37 °C and 4.5 % carbon dioxide atmosphere.

Administration / exposure

Details on exposure:
On the day of the experiment, the test item was suspended with DMSO. The final concentration of DMSO did not exceed 1.0 % (v/v) in the culture medium (RPMI-medium with 10 % FCS).
Duration of treatment / exposure:
8 hours of incubation period, followed by a second inbubation period of 16 hours.
Doses / concentrations
39.1, 78.1, 156.3, 312.5, 625, 1250, 2500 and 5000 µg/ml
Control animals:
yes, concurrent no treatment
yes, concurrent vehicle
Details on study design:
TEST MEDUM: RPMI-medium with 10 % FCS

Due to an experimental error during preparation of the samples for scintillation measurements, the experiment had to be performed twice. Only the results of the second experiment are reported.

Seeding of the Cultures
Exponentially growing stock cultures more than 50 % confluent were rinsed with Ca-Mg-free salt solution and treated with Trypsin at 37 °C for 5 minutes (Gibco BRL Trypsin/EDTA Solution 10x Kat.Nr. 35400-019). Then the enzymatic digestion was stopped by adding complete culture medium and a single cell suspension was prepared.
The Ca-Mg-free salt solution was composed as follows (per litre): NaCl 8000 mg, KCI 400 mg, Glucose 1000 mg, NaHCO3 350 mg.
lndividual wells of a 96-well tissue-culture microtitre plate (Greiner) were inoculated with 0.1 ml medium containing 1000 cells. The medium was RPMI 1640 + 10 % FCS (complete medium). The plates were incubated for 24 hours to enable cellular attachment.

Thereafter, the medium was removed and the cells were re-fed with 0.1 ml treatment medium containing different concentrations of the test item, negative, solvent, and positive control, respectively. All incubations were done at 37 °C in a humidified atmosphere with 4.5 % CO2.


After an incubation period of 8 h, 3HTdR (5 µCi/ml culture medium, specific activity 20 Ciimmol; New England Nuclear, D-63033 Dreieich) was added to each well. The cells were incubated forfurther 16 h. Subsequently, the cells were harvested using a 12- well cell harvester (Skatron Instruments, USA) and one filter per culture (Filter MAT, Skatron lnstruments, USA). The filters were washed with deionised water. The filters were dried (at approx. 100 °C) overnight. Each filter was placed in a vial containing 3 ml rotiszint eco plus solution (Roth, D-76185 Karlsruhe). The amount of 3HTdR on the filter was measured using a LKB Wallac 1219 Rackbeta Liquid Scintillation Counter.
Positive control:
- Positive control: sodium dodecyl sulfate
- Concentrations: 2.5, 5, 10, 15, 25, 50, 100 and 200 µg/ml

Results and discussion

Details on results:
EC50-value for the test item could not be determined as viability was not reduced.

EC50-value: 70.5 µg/ml

Any other information on results incl. tables

Test Group Concentration in µg/ml Scintillation counts* Standard Deviation Chem. Blanks Scintillation counts in % of solvent control**
Negative control - 7584 2596.8 103.3 106.2
Solvent control - 7136.5 1809.3 94.2 100
Test item 39.1 5875.3 1693.2 130.5 81.6
Test item 78.1 5248.1 871.4 104.5 73
Test item 156.3 6054.5 1854.3 86.6 84.7
Test item 312.5 6590.6 1371.6 67.1 92.6
Test item 625 6180.1 1885.1 61.1 86.9
Test item 1250 6396.3 1079.9 69.1 89.8
Test item 2500 6998.2 1308.3 37.3 98.8
Test item 5000 5580.2 2115.8 48.3 78.6

* mean scintillation counts (absolute) of at least 6 wells

** relative scintillation: (100x(counts specimers-counts chem.blanks)) / (counts solv.control - counts chem.blank)

Applicant's summary and conclusion

No EC50-value could be determined as viability was not reduced.
Executive summary:

The in vitro study was performed to assess the cytotoxicity potential of the test item by means of the cytotoxicity in vitro test for soluble test items. This test was carried out with the mouse cell line L929. The study was conducted in accordance with the ISO 10993 (Biological Evaluation of Medical Devices" Part 5: Tests for cytotoxicity: in vitro methods".

The following concentrations of the test item were tested: 39.1, 78.1, 156.3, 312.5,625, 1250,2500, and 5000 µg/ml. Culture medium (RPMI containing 10 % (v/v) FCS) was used as negative control; the solvent control was also RPMI medium containing 10 % (vlv) FCS and 1.0 % DMSO. Sodium dodecyl sulfate was used as positive control.

The incubation time was 24 hours at 37 °C.

The negative control and the solvent control showed no reduction in cell viability and cell proliferation. The positive control (SDS) induced a reduction in cell viability and cell proliferation. The calculated EC50 value was 70.5 µg/ml.

No relevant cytotoxic effects were observed following incubation with test item up to the highest tested concentration (5000 µg/ml). Due to the lack of cytotoxicity, no EC50 value could be calculated.


No EC50-value could be determined as viability was not reduced.