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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro DNA damage and/or repair study
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP- and OECD testing guideline compliant study (element with metabolic activation).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1986
Report date:
1986

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)
Deviations:
no
Remarks:
optional inhibition of semi-conservative DNA replication was not included. But discrimination between cells in S-phase and repairing cells was included.
GLP compliance:
yes
Type of assay:
DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro

Test material

Constituent 1
Chemical structure
Reference substance name:
4,4'-diamino[1,1'-bianthracene]-9,9',10,10'-tetraone
EC Number:
223-754-4
EC Name:
4,4'-diamino[1,1'-bianthracene]-9,9',10,10'-tetraone
Cas Number:
4051-63-2
Molecular formula:
C28H16N2O4
IUPAC Name:
4,4'-diamino-1,1'-bianthracene]-9,9',10,10'-tetraone
Details on test material:
- Analytical purity: commercial grade
- Lot/batch No.: E.Nr.204853.59
- Stability under test conditions: ensured

Method

Species / strain
Species / strain / cell type:
hepatocytes: rat
Details on mammalian cell type (if applicable):
Primary hepatocytes are freshly isolated from a male rat (Tif.RAIf[SPF], weight: 250 g) induced with Aroclor 1254. The liver is then perfused in situ through the portal vein and carefully excised and placed into a dish containing calcium-free Hanks' solution. The cells are dispersed by gently shaking of the liver in the solution, then filtered and washed twice with calcium-free Hanks' solution. Finally, the cells are suspended in Williams' medium E and analysed for viability by trypan-blue exclusion. The viability of hepatocytes prepared in this way is generally greater than 90%. The cells are further cultivated in WILLIAMS' Medium E containing 10% foetal bovine serum and incubated in a humidified atmosphere with 5% CO2, at 37°C and treatment with test substance was performed on adhesive cells.
Metabolic activation:
not applicable
Test concentrations with justification for top dose:
- First experiment; toxicity test: 0, 1.56, 3.13, 6.25, 12.5, 25.0, 50.0 and 100.0 µg/ml
- Second experiment; DNA-Repair assay: 0.4, 2.0, 10.0 and 50.0 µg/ml (4 cultures per group)
Vehicle / solvent:
- Vehicle(s)/solvent(s) DMSO
Controls
Untreated negative controls:
yes
Remarks:
culture medium
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
other: the UDS test is regularly conducted in the testing facility
Positive controls:
yes
Positive control substance:
2-acetylaminofluorene
Remarks:
Migrated to IUCLID6: 2-AAF (200 µg/ml)
Details on test system and experimental conditions:
1) First experiment: toxicity test
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 5 hours

STAIN: after an incubation period the medium is removed and the cells are washed twice with BSS and stained with Trypan blue solution (0.2%) for five minutes

NUMBER OF CELLS EVALUATED: 100

DETERMINATION OF CYTOTOXICITY
- Method: Cell count; after washing with BSS, the cells are fixed and the percentage of unstained cells evaluated.

2) Second experiment: DNA-Repair assay
METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk

DURATION
- Exposure duration: 5 hours

STAIN (for cytogenetic assays): immediately after addition of the test substance, 6-H-thymidine is added (specific activity 21 Curies/mmol). 8 µCi in 8 µl was added to the medium. At the end of the incubation period of 5 hours the cells are washed twice with BSS and fixed with ethanol/acetic acid, 3/1, v/v. The cover-slips are mounted on microscope slides and prepared for autoradiography. The exposure time is 6 days. The autoradiographs are stained with hematoxylin-eosine.

NUMBER OF CELLS EVALUATED: from each of the treatment groups and from the positive and the negative controls 150 nuclei in altogether three slides (50 cells/slide) are scored, the number of silver grains counted, the mean values and the standard deviations calculated.

OTHER: counting of silver grains over the nuclei and cytoplasm of the hepatocytes was carried out with the aid of an electronic counter attached to a microscope at a magnification of 2000x, using an objective l00x and a projective l0x. The net values are calculated by subtracting the average grain count over the cytoplasm from the total over the nuclei. If the net value for any nucleus is less than zero (i.e. if the cytoplasmic value is greater than the nuclear value), a net value of zero is taken for the calculations. Cells which were in the DNA-synthesis phase showed more than 120 silver grains/nucleus. The percentage of such cells was about 0.2. These cells were excluded from the determination of the silver grain/nucleus count.
Evaluation criteria:
The test substance is generally considered to induce DNA damage if the mean number of silver grains per nucleus in relation to the negative controls is more than doubled at any concentration.

Results and discussion

Test results
Species / strain:
hepatocytes: rat
Metabolic activation:
not applicable
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
COMPARISON WITH HISTORICAL CONTROL DATA:
Was performed

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In a preliminary toxicity test, seven concentrations of the test substance increasing by a factor of 2 from 1.56 to 100 µg/ml were tested to determine the highest applicable concentration in the DNA-repair assay. At the three highest concentrations a clearly visible precipitation occurred. At the subsequent lower concentrations no precipitation was visible in the culture medium. From the results obtained (Table 1), the highest usable concentration was found to be 50 µg/ml.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Toxicity test

Test substance concentration (µg/ml)

Adhesion and condition of the cells (+, ±, +--, -)

Percentage of viable cells (%)

100

+--

13

50

±

76

25

±

73

12.5

±

84

6.25

±

81

3.13

+

87

1.56

±

82

DMSO (1%)

+

86

+: adequate number of adhered cells, which are in good condition; ±/+--: adequate number of adhered cells, however in bad condition; -: unadequate number or no adhered cells. If less than 25% of the cells are viable, the slide is not evaluated.

 

The DNA-repair assay was carried out with concentrations of 0.4, 2, 10 and 50 µg/ml (Table 2). At the highest tested concentration a precipitation was visible in the culture medium. Comparison of the mean number of silver grains per nucleus in the vehicle control and after treatment with test substance revealed no marked differences.

By contrast, the positive control, 2-AAF (200 µg/ml) yielded a marked increase in the mean value of silver grains per nucleus. Here, the mean value was 13.3, whereas the negative controls gave values of 0.91 and 1.32.

In addition, the net values of grains per nucleus (i.e. mean number of silver grains per nucleus minus mean number of silver grains per nucleus-equivalent area of cytoplasm) were calculated and there was no marked deviation between the treatment groups and the vehicle control.

 

Table 2: Autoradiographic DNA repair test on rat hepatocytes

Treatment groups

Concen-tration (µg/ml)

Silver grains per nucleus (±SD)

Cytoplasma silver grains per nucleus-equivalent area (±SD)

Silver grains per nucleus (net value)

Background determination outside the cells: silver grains per nucleus-equivalent area

Negative control: medium

-

0.91±0.92

0.77±0.66

0.14

0.22

Negative control: vehicle

-

1.32±1.02

0.94±0.64

0.38

0.00

Positive control: 2-AAF

200

13.3±5.68

4.09±2.80

9.23

0.28

 

Test substance

50

2.01±1.48

1.90±1.22

0.11

0.11

10

1.75±1.23

1.60±0.97

0.15

0.00

2

1.35±1.16

1.61±0.93

0.26

0.11

0.4

1.51±1.21

1.40±0.85

0.10

0.06

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative