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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1979
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Justification for type of information:
None

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1979
Report Date:
1979

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Principles of method if other than guideline:
None
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid

Method

Target gene:
The experiments were performed to detect any properties of the test material or its metabolites to induce gene mutations in histidine-requiring strains of Salmonella typhimurium.
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 98
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 1538
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9 fraction
Test concentrations with justification for top dose:
25, 75, 225, 675 and 2025 µg/0.1 ml
Vehicle / solvent:
Dimethylsulfoxyde
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: daunorubicin-HCl for TA 98; 2-nitrofluorene for TA 1538
Remarks:
Without microsomal activation
Details on test system and experimental conditions:
Bacterial cultures were prepared from frozen stocks or from colonies on plates, and on the following days the Standard Plate Test was carried out with and without the addition of activation mixture
(rat liver microsomes and co-factors) .
The test was performed with the following concentrations of the trial substance with and without microsomal activation: 25, 75, 225, 675 and 2025 µg/0.1 ml. The substance was dissolved in DMSO. DMSO alone was used for the negative controls. Each Petri dish contained:
1) approx. 20 ml of minimum agar (Difco agar noble, Difco Laboratories, Detroit, Michigan, U.S.A., Art.No.0142-01, plus salts (Vogel-Bonner Medium E) and glucose),
2) 0.1 ml of the solution of the test substance or the vehicle and 0.1 ml of a bacterial culture (in nutrient broth: Bacto Nutrient Broth dehydrated, Difco Laboratories, Detroit, Michigan, U.S.A., Art.No.0003 0.8 % plus 0.5 % NaCl) in 2.0 ml of soft agar. The soft agar was composed of:
100 ml of 0.6 % agar solution (Difco agar noble) with 0.6 % NaCl and 10 ml of a solution of 1-histidine, 0.5 mM (Fluka, Buchs, Switzerland, Art.No.14400) and +biotin 0.5 mM (Fluka, Buchs, Switzerland,
Art .No .53320). In the experiments in which the substance was metabolically activated, 0.5 ml of an activation mixture was added also. 1 ml activation mixture contains: 0.3 ml S9 fraction of liver from rats induced with Aroclor 125 4 (Analabs, Inc., North Haven, Connecticut, U.S.A.,, No.RCS-088), 8 ymoles MgCl , 33 µmoles KCl, 5 µmoles glucose-6-phosphate, 4 µmoles NADP and 100 µmoles phosphate buffer, pH 7.4.
Positive control experiments were carried out simultaneously with the following substances: 1) for Strain TA 98: daunorubicin-HCl (DAÜNOBLASTIN , Farmitalia, Montedison Farmaceutica GmbH, Freiburg
i.Br., Germany), 5 and 10 µg/0.1 ml phosphate buffer; 2) for Strain TA 1538: 2-nitrofluorene (Fluka, Buchs, Switzerland, Art. No.73330), 5 and 10 µg/0.1 ml DMSO. The activation mixture was tested with Strain TA 15 35 and cyclophosphamide (ENDOXAN-ASTA , Asta-Werke, Bielefeld, Germany), 250 µg/0.1 ml phosphate buffer.
In the experiments with and without the addition of microsomal activation mixture three Petri dishes were prepared per strain and per group (i.e. per concentration or per control group). In the positive control experiments two Petri dishes were used per strain and per group.

The plates were incubated for about 48 hours at 37 degree C in darkness.
Evaluation criteria:
The test substance was considered to be non-mutagenic if the colony count in relation to the negative control was not doubled at any concentration .
Statistics:
When the colonies had been counted, the arithmetic mean was calculated.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
other: TA 98 and TA 1538
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
None
Remarks on result:
other: Cytotoxicity was observed at the highest tested concentration of 2000 µg/0.1 ml

Any other information on results incl. tables

In the experiments performed without microsomal activation, comparison of the number of histidine-prototrophic mutants in the controls and after treatment with FAT 31 016/C revealed no marked differences.

In the experiments with microsomal activation, on the other hand, treatment with FAT 31 016/C led to an increase in the number of back-mutant colonies of Strains TA 98 and TA 1538. This effect was observed at the concentrations of 75 µg/0.1 ml and above. At the highest concentration there was again a reduction in the number of back-mutant colonies, due to a growth-inhibiting effect of the substance on the bacteria.

Applicant's summary and conclusion

Conclusions:
FAT 31016/C was found to exert a mutagenic effect on strains TA 98 and TA 1538 in the presence of metabolic activation.
Executive summary:

FAT 31016/C was tested for mutagenic effects on histidine auxotrophic mutants of Salmonella typhimurium. The investigations were performed with the following concentrations of the trial substance with and without microsomal activation: 25, 75, 225, 675 and 2025 µg/0.1 ml. In the experiments performed without microsomal activation, comparison of the numbers of back-mutant colonies in the controls and the cultures treated with the various concentrations of FAT 31016/C revealed no marked deviations. In the experiments in which activation mixture was added to the cultures, the number of back-mutant colonies of Strains TA 98 and TA 1538 was distinctly greater after treatment with FAT 31016/C than in the controls. Hence, it can be concluded that FAT 31016/C was found to exert a mutagenic effect on strains TA 98 and TA 1538 in the presence of metabolic activation.