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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
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 September 1979 to 17 November 1980
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
The study predates the appropriate OECD test guideline, but was conducted in compliance with GLP. Nevertheless, the study was conducted similar to the OECD TG 482 with acceptable restrictions. The restrictions were that less cells were evaluated than demanded in the guideline.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1980
Report Date:
1980

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)
Version / remarks:
(1986)
Deviations:
yes
Remarks:
(only 30 cells evaluated)
GLP compliance:
yes
Type of assay:
other: DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
not specified

Method

Target gene:
not applicable
Species / strain
Species / strain / cell type:
hepatocytes: primary hepatocytes from one male Sprague Dawley rat (8-12 weeks of age; approximately 221 g bw)
Details on mammalian cell type (if applicable):
- Type and identity of media: Williams Medium E supplemented with 10% foetal calf serum- Properly maintained: yes- Periodically checked for Mycoplasma contamination: not applicable- Periodically checked for karyotype stability: not applicable- Periodically "cleansed" against high spontaneous background: not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
not applicable
Metabolic activation system:
primary hepatocyte cultures have sufficient metabolic capacity
Test concentrations with justification for top dose:
10 and 1 mM, 100, 10, and 1 µM, and 100, 10, and 1 nM
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: cell culture medium- Due to limited solubility of the positive control substance, cell culture medium additional supplemented with 0.1% (v/v) DMSO was used for the positive control.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
(cell culture medium with 0.1% DMSO with respect to the positive control)
True negative controls:
no
Positive controls:
yes
Remarks:
diluted in cell culture medium with 0.1% DMSO
Positive control substance:
2-acetylaminofluorene
Remarks:
10, 1, and 0.1 µM
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium containing 3H-Thymidine (10 µCi/mL) and dexamethasone (1 µM in 0.1% ethanol final concentration)DURATION- Exposure duration: 18 h- Fixation time (start of exposure up to fixation of cells): 20 hNUMBER OF REPLICATIONS: 1NUMBER OF CELLS EVALUATED: 30DETERMINATION OF CYTOTOXICITY- Method: morphological examination (detachment and/or rounded granular appearence of the cells)
Evaluation criteria:
The net number of grains in treated cells were compared to the appropriate control. Net nuclear labelling is often observed in control cells, but rarely exceeds a mean of 5 grains per nucleus. Therefore, a mean of 6 or more grains per nucleus (this may vary with experimental conditions) and statistical significance from control is required for an unequivocal positive result. In cases where nuclear labelling is very intense (approximately greater than 75 grains per nucleus) the results are reported as the best approximation of the number of grains present. In addition to statistical significance from control, a dose response relationship should exist.
Statistics:
The net number of grains in treated cells were compared to the appropriate control by analysis of variance and Dunnett's t-test, with statistical significance from control at p ≤ 0.05.

Results and discussion

Test results
Species / strain:
hepatocytes: primary hepatocytes from one male Sprague Dawley rat
Metabolic activation:
not applicable
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 1 mM and above clearly cytotoxic
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS- none stated in the study reportADDITIONAL INFORMATION ON CYTOTOXICITY:- The test item was cytotoxic to the rat hepatocyte cultures at concentrations of 10 and 1 mM as indicated by the detachment and/or rounded granular appearence of the cells after chemical treatment, however, enough cells were present after processing for UDS evaluation. The appearence of the cultures improved with decreasing concentration of the test material so that cultures treated with 0.1 mM to 1 nM approached and matched the appearence of control cultures.

Any other information on results incl. tables

Tab. 1: The effects of the test item in the Rat Hepatocyte Unscheduled DNA Synthesis (UDS) Assay

Treatment

Concentration

Net nuclear grains*

(mean±SD)

Negative control

-

-

-2±3

Test item

10 mM

1550 µg/mL

0±1 #

1 mM

155 µg/mL

1±2 #

100 µM

15.5 µg/mL

-3±4

10µM

1.55 µg/mL

-2±3

1 µM

0.155 µg/mL

-2±3

100 nM

0.0155 µg/mL

-2±2

10 nM

0.00155 µg/mL

-2±3

1 nM

0.000155 µg/mL

-3±3

Positive control

(2-AAF)**

10 µM

2.2 µg/mL

40±10***

1 µM

0.22 µg/mL

40±12***

100 nM

0.022 µg/mL

33±12***

Solvent control (with 0.1% DMSO)

-

-

-3±4

* net nuclear grains = nuclear grains-background cytoplasm grains

**2 -AAF = acetylaminofluorene

*** positive responses, 6 or more net nuclear grains and p ≤ 0.05

# treatment related cytotoxicity was observed

Applicant's summary and conclusion

Conclusions:
Interpretation of results: negative