3-(2-{4-[2-(4-cyanophenyl)vinyl]phenyl}vinyl)benzonitrile

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

FAT 60253/A is considered to be non-mutagenic in reverse mutation assay and non clastogenic in the chromosome aberration test.

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

No data

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)

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.

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

None

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

A key 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 ug/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 60253/A is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay. 

In another key study, FAT 60253/A, suspended in DMSO, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in two independent experiments. In each experimental group two parallel cultures were set up. Per culture 100 metaphase plates were scored for structural chromosome aberrations.

The highest applied concentration in the pre-test on toxicity should be 1250 ug/ml considering the ability to formulate a homogenous suspension of the test item in an appropriate solvent. However, due to technical reasons only 125 rng/ml were applied.Dose selection of the cytogenetic experiments was conducted considering the toxicity data and the occurrence of precipitation in accordance to OECD 473. Experiment I was conducted in two independent parts. Due to missing toxicity in experiment IA (with and without S9 mix) a higher concentration (the top concentration which could be technically applied) was tested in IB. Clear toxic effects indicated by reduced cell numbers below 50 % of control were observed in the absence of S9 after 18 and 28 hrs continuous treatment with 300 ug/ml. In addition, reduced mitotic indices were observed at interval 28 hrs after 4 hrs treatment with 400 ug/ml in the presence of S9 mix. In both independent experiments, neither a statistically significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. No increase in the frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls. Appropriate mutagens were used as positive controls. They induced statistically significant increases (p <0.05) in cells with structural chromosome aberrations. In conclusion, it can be stated that in the study described and under the experimental conditions reported, the test item did not induce structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro. Therefore, FAT 60253/A is considered to be non-clastogenic in this chromosome aberration test.

Justification for selection of genetic toxicity endpoint
GLP guideline study

Justification for classification or non-classification

FAT 60'253/A is considered to be non-mutagenic in reverse mutation assay and in chromosome aberration test.