2,2'-[[5-acetamido-4-[(2-bromo-4,6-dinitrophenyl)azo]-2-methoxyphenyl]imino]diethyl diacetate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1980

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Not conducted under GLP, no information on test substance purity

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1980

Report date:

1980

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2,2'-[[5-acetamide-4-[(2-bromo-4,6-dinitrophenyl)azo]-2-ethoxyphenyl]imino]diethyl diacetate

EC Number:

235-475-5

EC Name:

2,2'-[[5-acetamide-4-[(2-bromo-4,6-dinitrophenyl)azo]-2-ethoxyphenyl]imino]diethyl diacetate

Cas Number:

12239-34-8

Molecular formula:

C24H27BrN6O10

IUPAC Name:

2,2'-[[5-acetamide-4-[(2-bromo-4,6-dinitrophenyl)azo]-2-ethoxyphenyl]imino]diethyl diacetate

Test material form:

solid

Method

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

0, 10, 50, 100, 500, 1000 and 5000 µg/plate

Vehicle / solvent:

The substance was dissolved 50 mg/ml in dimethylsulfoxide and further diluited in the same solvent according the need.

Controls

Negative solvent / vehicle controls:

yes

Details on test system and experimental conditions:

Media: Vogel-Bonner E agar medium (VB) has the following compositlon:

MgSO4 x 7H20 0.2 g/l
citric acid x H2O 2 g/l
K2HPO4 10 g/l
NaNH4HPO4 x 4 H2O 3.5 g/l
glucose 2 g/l
Agar 15 g/l

It was prepared by mixing 750 ml of 2% molten autoclave sterillzed water agar wlth 250 ml of.4 times concentrated salt solution and 10 ml of 20 % glucose solution autoclaved separately.
For the antibacterlal test, histidine was also added to give a final concentratlon of 10 µg/ml.
10 cm plastic petri dishes were prepared containing 20 ml each of VB agar and used within 3 days from preparation.
Top agar was prepared by mlxing 100 ml of autoclave sterilised 0.6 % agar and 0.5 % NaCl in water with 10 ml of a millipore filter sterillzed 0.5 mM histidine, 0.5 mM blotln solutlon. Top agar was dlspensed in 2 ml amounts into 16x100 mm glass tubes kept at 42 °C.

TA1535, TA1537, TA1538 Ames strains were kept at 4 °C in deep soft nutrient agar, strains TA100 and TA98 were kept at -80 °C in nutrient broth plus ampicillin (20 µg/ml) and dimethysulfoxide (80 µg/ml).
The day before the test, fresh cultures were prepared by inoculating 10 ml of nurtrient broth with each strain, which were incubated overnight at 37 °C. Concurrently with each series of test, cultures were checked for relevant characters, namely:
-histidine requirement
-biotin requirement
- uvrB character
-rfa charcter
- presence or absence of the pKM101 prototrophy
-induced reversion rate by known mutagens

For testing the antibacterial activity :
0.1 ml of solvent containing the required amount of substance + 0.1 ml of a 10^-5 dilution of an overnight culture of TA1535 or TA98 containing between 100 and 500 colony forming units (CFU) + if requested S9 mix
were added in sequence to each top agar tube. Controls received 0.1 ml of the solvent mixture.
The contents of each tube were quickly mixed and poured on the surface of a histidine containing VB plate.
Colonies were counted after 24 hours of incubation at 37 °C. Tests were carried put inm triplicate.

For testing mutagenicity:
0.1 ml of solvent containing the required amounts of substance or 0.1 ml of sovlent alone + 0.1 ml of overnight culture of each strain containing 10^7 bacteria and if reuired 0.5 ml of S9 mix were added in sequence to each top agar tube. The contents of each tube were quickly mixed and pured on the surface of a VB plate.
Revertant colonies were counted after 2 days of incubation at 37 °C. Tests were carried out in triplicate.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

up to 5000 ug/plate

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Test results 3

Key result

Species / strain:

S. typhimurium, other: TA1535, TA1537

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Test results 4

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Applicant's summary and conclusion

Conclusions:

Under the study conditions, the substance was mutagenic in strains TA98 and TA100 and ambiguous in strain TA1538 with metabolic activation.

Executive summary:

A study was conducted to determine the mutagenic potential of the test substance (purity not given), according to OECD Guideline 471. Five strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537 and TA 1538) were exposed to the test substance, dissolved in dimethylsulfoxide, at concentrations of 0, 10, 50, 100, 500, 1000 and 5000 µg/plate with or without metabolic activation (S-9 mix) for 48 h. Negative and positive control experiments were valid. Under the experimental conditions, the substance was mutagenic in strains TA98 and TA100, and ambiguous in TA1538 with metabolic activation. The mutagenicity is of frame shift type (Archroma, 1980).