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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:
2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report date:
2000

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
3-(2-{4-[2-(4-cyanophenyl)vinyl]phenyl}vinyl)benzonitrile
EC Number:
419-060-8
EC Name:
3-(2-{4-[2-(4-cyanophenyl)vinyl]phenyl}vinyl)benzonitrile
Cas Number:
79026-02-1
Molecular formula:
Hill formula: C24 H16 N2 CAS formula: C24 H16 N2
IUPAC Name:
4-(2-{4-[2-(3-cyanophenyl)ethenyl]phenyl}ethenyl)benzonitrile
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
None

Method

Target gene:
No data
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
None
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
None
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 liver fractions of rats induced with phenobarbital/ ß-naphthoflavone.
Test concentrations with justification for top dose:
The following concentrations were tested.
33; 100; 333; 1000; 2500; and 5000 ug/plate
Vehicle / solvent:
Solvent: Dimethylformamide (DMF)
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA 1535, TA100; without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylene-diamine, 4-NOPD
Remarks:
TA 1537, TA 98; without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
WP2 uvrA; without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene, 2-AA
Remarks:
TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA; with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION:in agar (plate incorporation)

Mammalian Microsomal Fraction S9 Mix
S9 (Preparation by R C C - C C R)
The bacteria used in this assay do not possess the enzyme systems which, in mammals, are known to convert promutagens into active DNA damaging metabolites. In order to overcome this major drawback an exogenous metabolic system is added in form of mammalian microsome enzyme activation mixture.
The S9 liver microsomal fraction was obtained from the livers of 8 -12 weeks old male rats, strain Wistar Hanlbm (RCC Ltd., Biotechnology & Animal Breeding Division, CH- 4414 Füllinsdorf; weight approx. 220 - 320 g) which received daily applications of 80 mg/kg b.w. Phénobarbital i.p. dissolved in aqua deionised (Desitin; D-22335 Hamburg) and ß-Naphthoflavone orally dissolved in corn oil (Aldrich, D-89555 Steinheim) on three subsequent days. The livers were prepared 24 hours after the last treatment.
After decapitation of the anaesthetised animals the livers of the animals were removed, washed in 150 mM KCl and homogenised. The homogenate was diluted 1 +3 in KCl and centrifuged cold at 9,000 g for 10 minutes at 4° C. A stock of the supernatant containing the microsomes was frozen in ampoules and stored at -80° C. Small numbers of the ampoules are kept at - 20° C for up to one week before use. The protein content was determined using an analysis kit of Bio-Rad Laboratories, D-80939 München (Bio-Rad protein assay, Catalogue No. 5000006). The protein concentration in the S9 preparation was 37.6 mg/ml (lot no. 200400).
S9Mix
Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 co-factor solution. The amount of S9 supernatant was 15% v/v in the cultures. The concentrated co-factor solution yields the following concentrations in the S9 mix:
8 mM MgCI2
33 mM KCl
5 mM Glucose-6-phosphate
5mM NADP
in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.
During the experiment the S9 mix was stored in an ice bath. The S9 mix preparation was performed according to Ames et al.

Pre-Experiment for Toxicity
To evaluate the toxicity of the test item a prestudy was performed with strains TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA. 8 concentrations are tested for toxicity and mutation induction with 3 plates each in strains TA 98 and TA 100. Six concentrations were tested in strains TA 1535, TA 1537, and WP2 uvrA. The experimental conditions in this pre-experiment are the same as described for the experiment I below (plate incorporation test).
Toxicity of the test item results in a reduction in the number of spontaneous revenants or a clearing of the bacterial background lawn.
The pre-experiment is reported as main experiment I, if the following criteria are met:
Evaluable plates (>0 colonies) at five concentrations or more in all strains used.

Experimental Performance
For each strain and dose level including the controls, three plates were used. The following materials were mixed in a test tube and poured onto the minimal agar plates:
100 pi Test solution at each dose level, solvent (negative control) or reference mutagen
solution (positive control),
500 pi S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation),
100 pi Bacteria suspension (cf. test system, pre-culture of the strains),
2000ul Overlay agar
In the pre-incubation assay 100 pi test solution, 500 pi S9 mix / S9 mix substitution buffer and 100 pi bacterial suspension were mixed in a test tube and shaken at 37° C for 60 minutes. After pre-incubation 2.0 ml overlay agar (45e C) was added to each tube. The mixture was poured on minimal agar plates.
After solidification the plates were incubated upside down for at least 48 hours at 37° C in the dark.

Evaluation criteria:
A test item is considered positive if either a dose related increase in the number of revertants or a biologically relevant increase for at least one test concentration is induced. A test item producing neither a dose related increase in the number of revertants, nor a biologically relevant positive response at any one of the test points is considered nonmutagenic in this system.
A mutagenic response is described as follows:
A test item is considered mutagenic if in the strains TA 98, TA 100, and WP2 uvrA the number of reversions will be at least twice as high and in the strains TA 1535 and TA 1537 at least three times higher as compared to the spontaneous reversion rate .Also, a dose-dependent increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test item regardless whether the highest dose induced the above described enhancement factors or not.
Statistics:
No statistical evaluation of the data is required.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
Executive summary:

This study was performed to investigate the potential of FAT 60253/A to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. In experiment II, the colony counts of the negative and solvent controls of strain TA 1535 were far beyond our historical range in the presence and absence of metabolic activation. Therefore, the results were dismissed and an additional experiment with strain TA 1535 was performed. The results of this additional experiment are included in experiment II.


The test item was tested at the following concentrations:


33; 100; 333; 1000; 2500; and 5000 µg/plate


The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments. No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with FAT 60253/A at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, FAT 60'253/A is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.