2,2'-[[4-[(2-bromo-6-chloro-4-nitrophenyl)azo]-3-chlorophenyl]imino]bisethanol

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 September 12, 1997 to October 13, 1997

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Test Item purity < 50%

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: "Chemikaliengesetz" (Chemicals Act) of the Federal Republic of Germany, „Anhang 1" (Annexe 1) dated July 25, 1994 („BGBl. I 1994", pp. 1703)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-[N-(2-cyanoethyl)-4-[(2,6-dichloro-4-nitrophenyl)azo]anilino]ethyl acetate

EC Number:

226-070-4

EC Name:

2-[N-(2-cyanoethyl)-4-[(2,6-dichloro-4-nitrophenyl)azo]anilino]ethyl acetate

Cas Number:

5261-31-4

Molecular formula:

C19H17Cl2N5O4

IUPAC Name:

2-[(2-cyanoethyl)({4-[2-(2,6-dichloro-4-nitrophenyl)diazen-1-yl]phenyl}) amino]ethyl acetate

Test material form:

solid: particulate/powder

Method

Target gene:

histidine

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

Mammalian Microsomal Fraction S9 mix (liver)

Test concentrations with justification for top dose:

Triplicates: 0, 33, 100, 333, 1000, 2500 and 5000 ug/plate (active ingredient) (based upon the results of the pre-experiment)
(The test substance precipitated weakly at 2500 and 5000 ug/plate in the overlay agar. The undissolved particles of the test substance had no influence on the data recording.)

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

sodium azide

other: 4-nitro-o-phenylene-diamine, 2-aminoanthracene

Details on test system and experimental conditions:

Two independent Salmonella typhimurium reverse mutation assays:
Experiment I was performed as a plate incorporation assay. Since a positive result was obtained in this experiment, experiment II was performed as a plate incorporation assay as well.

Rationale for test conditions:

The Salmonella typhimurium histidine (his) reversion system measures his" -> his+ reversions. The S. typhimurium strains are constructed to differentiate between base pair (TA 1535, TA 100) and frameshift (TA 1537, TA 98) mutations.

Evaluation criteria:

The generally accepted conditions for the evaluation of the results are:
- corresponding background growth on both negative control and test plates,
- normal range of spontaneous reversion rates.

- A test substance is considered positive if either a biologically relevant and reproducible dose related increase in the number of revertants or a biologically relevant and reproducible increase for at least one test concentration is induced. A test substance producing neither a biologically relevant and reproducible dose related increase in the number of revertants nor a biologically relevant and reproducible positive response at any one of the test points is considered non-mutagenic in this system.

-A biologically relevant response is described as follows:
A test asubstance is considered mutagenic if the number of reversions is at least twice the spontaneous reversion rate in strains TA 98 and TA 100 or thrice on TA 1535 and TA 1537. 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 substance regardless whether the highest dose induced the criteria described above or not.

Statistics:

Toxicity of the test substance was evidenced by a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn. The colonies were counted. The individual and mean values of the plates for each concentration together with standard deviations and enhancement factors as compared to the spontaneous reversion rates, were measured.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

with metabolic activation

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate (a.i.)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

without metabolic activation

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate (a.i.)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

- Cytotoxicity: In experiment I, toxic effects evident as a reduction in the number of revertants occurred at the highest concentration in strain TA 1535 with S9 mix and in strain TA 1537 without S9 mix. In experiment II, toxic effects occurred at the highest concentration in strains TA 1535 and TA 1537 without S9 mix.
The plates incubated with the test substance showed normal background growth up to 5000 ug/plate with and without S9 mix in all strains used.

- Genotoxicity: In both experiments, substantial and dose dependent increases in revertant colony numbers were observed following treatment with the test substance with and without metabolic activation in strains TA 1537, TA 98 and TA 100. The number of colonies reached or exceeded the threshold of twice (strains TA 98 and TA 100) and thrice (strain TA 1537) the number of the corresponding solvent control at concentrations as low as 33 ug/plate and above. In experiment I a dose dependent increase in revertant colony numbers was observed in strain TA 1535 with and without S9 mix. In the absence of metabolic activation the threshold of thrice the number of the corresponding solvent control was not quite reached. In the presence of metabolic activation the threshold was exceeded at 2500 ug/plate. In experiment II, a dose-dependent increase was observed in strains TA 1535 and TA 1537 in the presence of metabolic activation but the threshold was not reached. In the absence of metabolic activation the threshold was exceeded at 2500 ug/plate in strain TA 1537.
- Appropriate reference mutagens were used as positive controls. They showed a distinct increase in induced revertant colonies.

Applicant's summary and conclusion

Conclusions:

Under the study conditions, the test substance was considered to be mutagenic in this Salmonella typhimurium reverse mutation assay.

Executive summary:

A study was conducted to determine the in vitro mutagenic potential of the test substance according to OECD Guideline 471, in compliance with GLP. The assay was performed in Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 in two independent experiments both with and without liver microsomal activation (S9 mix). Each concentration and the controls were tested in triplicate. The test substance was tested at the following concentrations: 33, 100, 333, 1000, 2500 and 5000 ug/plate. In Experiment I, cytotoxic effects evident as a reduction in the number of revertants occurred at the highest concentration in strain TA 1535 with S9 mix and in strain TA 1537 without S9 mix. In Experiment II, cytotoxic effects occurred at the highest concentration in strains TA 1535 and TA 1537 without S9 mix. The plates incubated with the test substance showed normal background growth up to 5000 ug/plate with and without S9 mix in all strains used. In both experiments, substantial and dose dependent increases in revertant colony numbers were observed following treatment with the test substance with and without metabolic activation in strains TA 1537, TA 98 and TA 100. Appropriate reference mutagens were used as positive control and showed a distinct increase in induced revertant colonies. Therefore, the test substance induced gene mutations by base pair changes and frameshifts in the genome of the Salmonella typhymurium strains TA98, TA100, TA1535 and TA1537. Under the study conditions, the test substance was considered to be mutagenic in this Salmonella typhimurium reverse mutation assay (Wollny, 1997).