3,3'-[[4-[(2,6-dichloro-4-nitrophenyl)azo]phenyl]imino]bispropiononitrile

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From April 03 to May 21, 1992

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

3,3'-[[4-[(2-chloro-4-nitrophenyl)azo]phenyl]imino]bis[propiononitrile]

EC Number:

223-765-4

EC Name:

3,3'-[[4-[(2-chloro-4-nitrophenyl)azo]phenyl]imino]bis[propiononitrile]

Cas Number:

4058-30-4

Molecular formula:

C18H15ClN6O2

IUPAC Name:

3,3'-({4-[(2-chloro-4-nitrophenyl)diazenyl]phenyl}imino)dipropanenitrile

Test material form:

solid: particulate/powder

Method

Target gene:

histidine operon

Species / strain

Species / strain / cell type:

other: TA 98, TA100, TA 1535, TA1537 and TA 1538

Details on mammalian cell type (if applicable):

Source of strains
The histidine-auxotrophic strains of Salmonella typhimurium (TA 98, TA 100, TA 1535, TA 1537 and TA 1538) were obtained from Prof. B. Ames, Berkeley, CA., U.S.A.
Preparation of the bacterial cultures
Inoculates from frozen master copies were set up monthly. They were grown in liquid NB-medium overnight and then plated on NBagar. After incubation, single colonies were taken from the plates, grown overnight in liquid NB-medium and then used for the experiment.
Control of the genotype of the strains
The characteristics of the strains were checked monthly. Histidine-auxotrophy of the strains was demonstrated by the requirement for L-histidine. The presence of the rfa character was assayed by the sensitivity for crystal-violet. The deletion of the uvrB gene was demonstrated by the sensitivity for UV-light. The
Salmonella strains containing the R-factor (TA 98 and TA 100) were additionally checked for ampicillin resistance. Furthermore, all strains were checked for their characteristic reversion properties with known mutagens (positive controls).

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Rat-liver microsomal fraction S9

Test concentrations with justification for top dose:

With and Without microsomal activation: 50, 158, 500, 1581, 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

Vehicle

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

Remarks:

Without metabolic activation

Controls 2

Untreated negative controls:

yes

Remarks:

Vehicle

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: 2-aminoanthracene

Remarks:

With metabolic activation

Details on test system and experimental conditions:

Preliminary Toxicity/Ranqe-Finding test
A toxicity test (check for reduction in the number of revertant colonies) was carried out with strain TA 100 without and with microsomal activation at six concentrations of the test substance and one negative control according to Standard Operating Procedures of Genetic Toxicology. The highest concentration
applied was 5000 µg/plate. The five lower concentrations decreased by a factor of 3. The plates were inverted and incubated for about 48 hours at 37 ± 1.5 °C in darkness. Thereafter, they were evaluated by counting the colonies and determining the background lawn. One plate per test substance concentration, as well as each negative control was used.

Mutagenicity test
The mutagenicity test was performed with strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 without and with microsomal activation according to Standard Operating Procedures of Genetic Toxicology. Each of the five concentrations of the test substance, a negative and a positive control were tested, using three plates
per test substance concentration as well as each positive and negative control with each tester strain. The highest concentration applied was 5000 µg/plate (because of weak toxicity in the range finding test) and the four lower concentrations were each decreased by a factor of V10 (3.1623). The plates were inverted and incubated for about 48 hours at 37 ± 1.5 °C in darkness. Thereafter, they were evaluated by counting the number of colonies and determining the background lawn.

Colony counting and scoring of the plates
Colonies were counted electronically with an Artek counter. The results were sent online to a computer. They were checked on a random basis by the operator.Observations indicating precipitates of the test substance in the top agar or a reduced or absent bacterial background lawn were registered additionally. Means and standard deviations for all mutagenicity assays were calculated by a previously validated computer program.

Evaluation criteria:

Assay acceptance criteria
A test is considered acceptable if the mean colony counts of the control values of all strains are within the acceptable ranges and if the results of the positive controls meet the criteria for a positive response. In either case the final decision is based on the scientific judgement of the Study Director.
Criteria for a positive response
The test substance is considered to be mutagenic in this test system if the following conditions are met:
At least a reproducible meaningful increase of the mean number of revertants per plate above that of the negative control at any concentration for one or more of the following strains:
S. typhimurium TA 98, TA 100, TA 1535, TA 1537 and TA 1538.
Generally a concentration-related effect should be demonstrable.

Statistics:

In deviation to the OECD guideline a statistical analysis was not performed. At present the use of statistical methods concerning this particular test system is not generally recommended.
No appropriate statistical method is available.

Results and discussion

Test results

Test results 1

Species / strain:

other: TA 98, TA 100, TA 1535, TA 1537, and TA 1538

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

other: TA 98, TA 100, TA 1537, and TA 1538

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Toxicity test/Range finding test

Six concentrations of the test substance ranging from 20.6 - 5000 µg/plate were tested with strain S. typhimurium TA 100 to determine the highest concentration to be used in the mutagenicity assay. The experiments were performed with and without microsomal activation. Normal background growth was observed. The numbers of revertant colonies were not reduced. From the results obtained, the highest concentration suitable for the mutagenicity test was selected to be 5000 µg/plate without and with activation.

Mutagenicity test, original experiment

In the experiment performed without microsomal activation, treatment with the test substance lead to a concentration dependend increase in the number of revertant colonies with strains TA 98, TA 100, TA 1537, and TA 1538. A slight increase was registered with strain TA 1537 at the concentrations of 1581.1 to 5000 µg/plate, a distinct increase with strain TA 100 at the concentrations of 500 to 5000 µg/plate and a strong increase with strains TA 98 and TA 1538 at the concentrations of 158.1 and 5000 µg/plate. In the experiments with activation a similar effect occurred on the same strains. A slight increase was seen with strain TA 1537 at the concentration of 5000 µg/plate, a distinct increase with strain TA 1538 at the concentration of 5000 µg/plate, a strong increase with strain TA 98 at the concentrations of 500 to 5000 µg/plate and with strain TA 100 at the concentrations of 1581.1 to 5000 µg/plate.

Mutagenicity test, confirmatory experiment

In the experiments performed without microsomal activation, treatment with the test substance lead to increased back-mutant counts with strains TA 98, TA 100 and TA 1538. A distinct increase was observed with strain TA 100 at the concentrations of 500 to 5000 µg/plate, and a strong increase with strain TA 98 at the concentrations of 500 to 5000 µg/plate and with strain TA 1538 at the concentrations of 158.1 and 5000 µg/plate. In the experiments with activation increased numbers of revertants occurred on strains TA 98, TA 100 TA 1535 and TA 1538. A slight increase was seen with strain TA 1535 at the concentration of 5000 µg/plate, a distinct increase with strain TA 100 at the concentrations of 1581.1 to 5000 µg/plate, with strain TA 1538 at the concentration of 5000 µg/plate and strong increase with strain TA 98 at the concentrations of 50 to 5000 µg/plate. In the mutagenicity tests normal background growth was observed with all strains at all concentrations. The numbers of revertant colonies were not reduced. The test substance exerted no toxic effect on the growth of the bacteria. The various mutagens, promutagens, sterility checks, sensitivity and resistance tests, etc., employed to ensure the test system was acceptable, all produced results within our established limits. There were no known circumstances or occurrences in this study that were considered to have affected the quality or integrity of the data.

Applicant's summary and conclusion

Conclusions:

Mutagenic

Executive summary:

Method

The substance was tested for mutagenic effects in vitro in histidine-requiring strains of Salmonella typhimunum, according to the OECD Guideline 471. The following strains of Salmonella typhimunum were used: TA 98, TA 100, TA 1535, TA 1537 and TA 1538.

The concentration range of the test substance to be tested in the mutagenicity test was determined in a preliminary toxicity test . Thus, the test substance was tested for mutagenic effects without and with metabolic activation at five concentrations in the range of 50 to 5000 µg/plate. In order to confirm the results, the experiments were repeated in an independent experiment with the same concentrations.

 

Results

Toxicity test/Range finding test

In this test no signs of toxicity of the test substance on the bacteria were observed up to the concentration of 5000 µg/plate.

Mutagenicity test, original experiment

In the original experiment performed without and with metabolic activation, treatment with the test substance led to an increase of revertant growth with strains TA 98, TA 100, TA 1537 and TA 1538 at the upper concentrations.

Mutagenicity test, confirmatory experiment

In the confirmatory experiment performed without metabolic activation, an increase of revertant growth was observed with strains TA 98, TA 100, and TA 1538 at the upper concentrations. In the experiment carried out with activation this effect was seen with strains TA 98, TA 100, TA 1535, and TA 1538.

 

Conclusion

Based on the results of these experiments and on standard evaluation criteria, it is concluded that the test substance and its metabolites exerted a strong mutagenic effect in this test system.