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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

FAT65004 is not mutagenic to histidine-auxotrophic strains of Salmonella typhimurium: TA 98, TA l00, TA 1535, TA 1537, TA 1538 and E. coli WP2 uvrA.

Link to relevant study records

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Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1980
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
None
Target gene:
histidine-auxotrophic strains of Salmonella typhimurium
Species / strain / cell type:
other: histidine- auxotrophic TA 98, TA 100, TA 1535, TA 1537 and TA 1538, strains of Salmonella typhimurium and Strain E. coli WP2 uvrA
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction of liver from rats
Test concentrations with justification for top dose:
5, 10, 50, 100, 500, 1000 and 5000 µg/0.1 ml.
Vehicle / solvent:
Dimethylsulfoxide (DMSO)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Details on test system and experimental conditions:
The tests were performed with the following concentrations of the trial substance without and with microsomal activation: 5, 10, 50, 100, 500, 1000 and 5000 µg/0.1 ml.

Preparation of test solution:
Solvent : Dimethylsulfoxide (DMSO); Merck
Amount per plate: 0.1 ml
Method for suspension of refractory substances: Ultrasonication
Time of preparation: On the day of the experiment

Media:
(1) Minimal agar Composition: Vogel-Bonner Medium E, 1,5% Agar, 2% Glucose
(2) Soft agar
Amount per plate: 2 ml
Temperature: 45°C
Composition: 0.6% agar +0.6 % NaCl in twice distilled water 400 ml + 40ml solution of histidine (0.5mM)/biotin (0.5mM) (with S. typhimurium) or 40 ml solution of tryptophan (0.5mM) (with E. coli)


Test method and conditions:
(1) Test Method: Plate Method
(2) Test conditions
Composition of the plates
Bacterial suspension: 0.1 ml/plate
Test solution : 0.1 ml/plate
Na phosphate buffered solution: -
S9 Mix where required: 0.5 ml/plate
Soft agar solution, 45°C: 2.0 ml/plate
Others Conditions:
Incubation temperature: 37°C
Incubation time: 48 - 55 hours

The tests were carried out in accordance with the method described by AMES et al
Rationale for test conditions:
None
Evaluation criteria:
The criteria of mutagenicity used in this test are a doubling of the spontaneous reversion rate and a dose-effect relationship.
Statistics:
None
Key result
Species / strain:
other: strains of Salmonella typhimurium: TA 98, TA l00, TA 1535, TA 1537, TA 1538 and E. coli WP2 uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
None

None

Conclusions:
It is concluded that FAT65004/D is not mutagenic to histidine-auxotrophic strains of Salmonella typhimurium: TA 98, TA l00, TA 1535, TA 1537, TA 1538 and E. coli WP2 uvrA
Executive summary:

FAT 65 004/D was tested for mutagenic effects on histidine-auxotrophicstrains of Salmonella typhimurium and on a tryptophan-auxotrophic strain of E. coli. The investigations were performed with the following concentrations of the trial substance with and without microsomal activation: 5, 10, 50, 100, 500, 1000 and 5000 µg/0.1 ml.

 

These tests permit the detection of point mutations in bacteria induced by chemical substances. Any mutagenic effects of the substances are demonstrable on comparison of the number of bacteriain the treated and control cultures that have undergone back-mutationto histidine- or tryptophan-prototrophism. To ensurethat mutagenic effects of metabolites of the test substanceformed in mammals would also be detected, experiments were performedin which the cultures were additionally treated with anactivation mixture (rat liver microsomes and co-factors).

In the experiment performed without and with microsomal activation,comparison of the numbers of back-mutant colonies in the controls and the cultures treated with the various concentrations of FAT 65004/D revealed no marked deviations.  

No evidence of the induction of point mutations by FAT 65004/D or by the metabolites of the substance formed as a result ofmicrosomal activation was detectable in the strains of S. typhimuriumand E. coli used in these experiments.

It is concluded that FAT65004/D is not mutagenic to  histidine-auxotrophic strains of Salmonella typhimurium: TA 98, TA l00, TA 1535, TA 1537, TA 1538 and E. coli WP2 uvrA

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1981
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
no strain to detect cross-linking mutagens was included
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
None
Target gene:
histidine-auxotrophic strains of Salmonella typhimurium
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction of liver from rats
Test concentrations with justification for top dose:
25, 75, 225, 675 and 2025 µg/0.1 ml.
Vehicle / solvent:
Acetone
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: (1)for Strain TA 98: daunorubicin- HCl, (2) for Strain TA l00: 4-nitroquinoline-N-oxide (3) for Strain TA 1535: N-Methyl-N'-nitro-N-nitrosoguanidine (4) for Strain TA 1537
Details on test system and experimental conditions:
The tests were performed with the following concentrations of the trial substance without and with microsomal activation: 25, 75, 225, 675 and 2025 µg/0.1 ml. The substance was dissolved in acetone. Acetone alone was used for the negative controls.
Each Petri dish contained:
l) approx. 20 ml of minimum agar (Agar, Difco Laboratories, Detroit, Michigan, U.S.A., 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, Difco Laboratories, Detroit, Michigan, u.s.A., 0.8% plus 0.5% NaCl) in 2.0 ml of soft agar. The soft agar was composed of: l00 ml of 0.6% agar solution with 0.6% NaCl and 10 ml of a solution of 1-histidine, 0.5 mM (Fluka, Buchs, Switzerland) and + biotin 0.5 mM (Fluka, Buchs, Switzerland). In the experiments in which the substance was metabolically activated, 0.5 ml of an activation mixture was added also. 1 ml activation mixture contained: 0.3 ml S9 fraction of liver from rats induced with Aroclor 1254 (Analabs., Inc., North Haven, Connecticut, U.S.A.) and 0.7 ml of a solution of co-factors.

Positive control experiments were carried out simultaneously with the following substances:
l) for Strain TA 98: daunorubicin-HCl (DAUNOBLASTIN®, Farmitalia, Montedison Farmaceutica GmbH, Freiburg i.Br., Germany), 5 and 10 µg//0.1 ml phosphate (- 5 -buffer;
2) for Strain TA l00: 4-nitroquinoline-N-oxide (Fluka, Buchs, Switzerland), 0.125 and 0.25 µg//0.1 ml phosphate buffer;
3) for Strain TA 1535: N-Methyl-N'-nitro-N-nitrosoguanidine (Serva, Heidelberg, Germany), 3 and 5 µg//0.1 ml phosphate buffer;
4) for Strain TA 1537: 9(5)aminoacridine hydrochloride monohydrate (Fluka, Buchs, Switzerland) 50 and l00 µg//0.1 ml DMSO.

The activation mixture was tested with Strain TA 1535 and cyclo.® phosphamlde (ENDOXAN-ASTA, Asta-Werke, Bielefeld, Germany), 250 µg//0.1 ml phosphate buffer. In the experiments without and with the addition of microsomal activation mixture three Petri dishes were prepared per strain and per group (i.e. per concentration or per control group).

The plates were incubated for about 48 hours at 37°C in darkness. When the colonies had been counted, the arithmetic mean was calculated. The test substance is generally considered to be non-mutagenic if the colony count in relation to the negative control is not doubled at any concentration.
Rationale for test conditions:
None
Evaluation criteria:
The criteria of mutagenicity used in this test are a doubling of the spontaneous reversion rate and a dose-effect relationship.
Statistics:
None
Key result
Species / strain:
other: strains of Salmonella typhimurium: TA 98, TA l00, TA 1535, TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
None

None

Conclusions:
It is concluded FAT65004/A is not mutagenic to histidine-auxotrophic strains of Salmonella typhimurium: TA 98, TA l00, TA 1535, TA 1537
Executive summary:

FAT 65004/A was tested for mutagenic effects on histidine-auxotrophic mutants of Salmonella typhimurium. The investigations were performed with the following concentrations of the trial substance without and with microsomal activation: 25, 75, 225, 675 and 2025 µg/0.l ml.

These tests permit the detection of point mutations in bacteria induced by chemical substances. Any mutagenic effects of the substances are demonstrable on comparison of the numbers of bacteriain the treated and control cultures that have undergone back-mutation to histidine-prototrophism. To ensure that mutagenic effectsof metabolites of the test substances formed in mammals would also be detected, experiments were performed in which the cultures were additionally treated with an activation mixture (rat liver microsomes andco-factors).

In the experiments performed without and with microsomal activation on Strain TA 1537, comparison of the number of back-mutant colonies in the controls and the cultures treated with the various concentrations of FAT 65004/A revealed a reduction in the colony count due to a growth-inhibiting effect of the compound at the concentrations of 225 µg/0.1 ml and above.

No evidence of the induction of point mutations by FAT 61004/A or by the metabolites of the substance formed as a result of microsomal activation was detectable in the strains of S. typhimurium used in these experiments.

It is concluded that FAT65004/A is not mutagenic to  histidine-auxotrophic strains of Salmonella typhimurium: TA 98, TA l00, TA 1535, TA 1537.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Data are available for two Ames test performed to investigate the potential of FAT65004 to induce gene mutations according to the plate incorporation test using the Salmonella typhimurium strains.

In a key studyFAT 65004/A was tested for mutagenic effects on histidine-auxotrophic mutants of Salmonella typhimurium. The investigations were performed with the following concentrations of the trial substance without and with microsomal activation: 25, 75, 225, 675 and2025 µg/0.l ml.

These tests permit the detection of point mutations in bacteria induced by chemical substances. Any mutagenic effects of the substances are demonstrable on comparison of the numbers of bacteriain the treated and control cultures that have undergone back-mutation to histidine-prototrophism. To ensure that mutagenic effectsof metabolites of the test substances formed in mammals would also be detected, experiments were performed in which the cultures were additionally treated with an activation mixture (rat liver microsomes andco-factors).

In the experiments performed without and with microsomal activation on Strain TA 1537, comparison of the number of back-mutant colonies in the controls and the cultures treated with the various concentrations of FAT 61004/A revealed a reduction in the colonycount due to a growth-inhibiting effect of the compound at the concentrations of 225 µg/0.1 ml and above.

No evidence of the induction of point mutations by FAT 65004/A or by the metabolites of the substance formed as a result ofmicrosomal activation was detectable in the strains of S. typhimuriumused in these experiments.

 

In another key study FAT 65004/D was tested for mutagenic effects on histidine-auxotrophicstrains of Salmonella typhimurium and on a tryptophan-auxotrophic strain of E. coli. The investigations were performed with the following concentrations of the trial substancewith and without microsomal activation: 5, 10, 50, 100. 500, 1000 and 5000 µg/0.1 ml.

These tests permit the detection of point mutations in bacteria induced by chemical substances. Any mutagenic effects of the substancesare demonstrable on comparison of the number of bacteriain the treated and control cultures that have undergone back-mutationto histidine- or tryptophan-prototrophism. To ensurethat mutagenic effects of metabolites of the test substanceformed in mammals would also be detected, experiments were performedin which the cultures were additionally treated with anactivation mixture (rat liver microsomes and co-factors).

In the experiment performed without and with microsomal activation,comparison of the numbers of back-mutant colonies in the controls and the cultures treated with the various concentrationsof FAT 65004/D revealed no marked deviations.

No evidence of the induction of point mutations by FAT 65004/D or by the metabolites of the substance formed as a result of microsomal activation was detectable in the strains of S. typhimuriumand E. coli used in these experiments.

 

 

Based on the data from the two key studies it can be concluded that FAT65004 is not mutagenic to histidine-auxotrophic strains of Salmonella typhimurium: TA 98, TA l00, TA 1535, TA 1537, TA 1538 and E. coli WP2 uvrA.

Justification for classification or non-classification

FAT65004 is not mutagenic to histidine-auxotrophic strains of Salmonella typhimurium: TA 98, TA l00, TA 1535, TA 1537, TA 1538 and E. coli WP2 uvrA.

Hence, the substanceis not classified.