3,3'-[(4-{(E)-[2-bromo-4-nitro-6-(trifluoromethyl)phenyl]diazenyl}phenyl)imino]dipropanenitrile

Genetic toxicity: in vitro

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:

December 20, 1999 to March 28, 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: guideline test with GLP principle

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Test material form:

other: solid

Details on test material:

Name: FAT41029/A
Batch No.: ARL 123/PP 1/99 UL
Aggregate State at Room Temperature: solid
Colour: orange-red to brown
Purity: approx. 98 %
Stability in solvent: 24 hours in water, saline, polyethylene glycol, CMC, vaseline, and FCA
Storage: room temperature
Expiration Date: November 04, 2005

Method

Target gene:

The Salmonella typhimurium histidine (his) and the E. coli tryptophan (trp) reversion system measures his" — > his+ and trp" -> trp+ reversions

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

rat liver microsome

Test concentrations with justification for top dose:

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

Vehicle / solvent:

n DMF (Merck, purity >99 %).. The solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

10 µg/plate for TA 1535, TA 100

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

without metabolic activation

Positive control substance:

other: 4-nitro-o-phenylene-diamine, 4-NOPD

Remarks:

10 µg/plate in TA 98, 50 µg/plate in TA 1537

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

without metabolic activation

Positive control substance:

methylmethanesulfonate

Remarks:

5 µl/plate for WP2 uvrA

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

with metabolic activation

Positive control substance:

other: 2-aminoanthracene, 2-AA

Remarks:

2.5 µg/plate, 10 µg/plate in strain WP2 uvrA

Details on test system and experimental conditions:

The bacterial strains TA 1535, TA 1537, and TA 100 were obtained from Ames (University of California, 94720 Berkeley, U.S.A.). The bacterial strain TA 98 was obtained from E. Merck (D-64293 Darmstadt). The Escherichia coli strain WP2 uvrA was obtained from Dr. Heinz Träger, Knoll AG, D-67008 Ludwigshafen.
summarised the mutations of the TA strains and the E. coli strain
Salmonella typhimurium
Strains Genotype Type of mutations indicated
TA 1537 his C 3076; rfa-; uvrB-: frame shift mutations
TA 98 his D 3052; rfa~; uvrB-;R-factor " "
TA 1535 his G 46; rfa-; uvrB-: base-pair substitutions
TA 100 his G 46; rfa-; uvrB-;R-factor " "
Escherichia coli
WP2 uvrA trp-; uvrA-: base-pair substitutions and others

Pre-Experiment for Toxicity
To evaluate the toxicity of the test item apre-experiment was performed with strains TA 98, TA 100. Eight concentrations were tested for toxicity and mutation induction with three plates each. The experimental conditions in this pre-experiment were the same as described below for the experiment I (plate incorporation test).
Toxicity of the test item results in a reduction in the number of spontaneous revertants 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.
s9 Mix
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.
Cofactors are added to the s9 mix to reach the following concentrations in the 89 mix:
8mM MgCI2
33 mM KCI
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.(5).
Dose Selection
In the pre-experiment the concentration range of the test item was 3 - 5000 µg/plate. The pre-experiment is reported as experiment I since no relevant toxic effects were observed and 5000 µg/plate were chosen as maximal concentration.
The concentration range included two logarithmic decades. The following concentrations were tested:
33; 100; 333; 1000; 2500; and 5000 µg/plate
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 ul Test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control),
500 ul S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation),
100 ul Bacteria suspension (cf. test system, pre-culture of the strains), 2000 µl Overlay agar
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 and reproducible increase in the number of revertants or a biologically relevant and reproducible increase for at least one test concentration is induced.
A test item producing neither a reproducible and dose related increase in the number of revertants, nor a biologically relevant and reproducibly positive response at any one of the test points is considered non-mutagenic 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 (3,4).
Also, a dose-dependent and reproducible 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 results

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

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

FAT 41029/A is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

This study was performed to investigate the potential of FAT 41029/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. 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. A weak toxic effect, evident as a reduction in the number of revertants, was observed without S9 mix in strain TA 1535 at 5000 µg/plate in experiment II.

No substantial increase in revenant colony numbers of any of the five tester strains was observed following treatment with FAT 41029/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.

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.