Benzoic acid, 2,3,4,5-tetrachloro-6-cyano-, methyl ester, reaction products with 4-[2-(4-aminophenyl)diazenyl]-3-methylbenzenamine and methanol sodium salt (1:1)

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:

2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

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

Reference substance name:

(3Z)-4,5,6,7-tetrachloro-3-({3-methyl-4-[(1E)-2-(3-methylphenyl)diazen-1-yl]phenyl}imino)-2,3-dihydro-1H-isoindol-1-one; 4,5,6,7-tetrachloro-3-({3-methyl-4-[(1E)-2-{4-[(4,5,6,7-tetrachloro-1-oxo-1H-isoindol-3-yl)amino]phenyl}diazen-1-yl]phenyl}amino)-1H-isoindol-1-one; 6-acetyl-3-({4-[(1E)-2-{4-[(6-acetyl-4,5,7-trichloro-1-oxo-1H-isoindol-3-yl)amino]phenyl}diazen-1-yl]-3-methylphenyl}amino)-4,5,7-trichloro-1H-isoindol-1-one; 6-acetyl-3-({4-[(1E)-2-{4-[(7-acetyl-4,5,6-trichloro-1-oxo-1H-isoindol-3-yl)amino]phenyl}diazen-1-yl]-3-methylphenyl}amino)-4,5,7-trichloro-1H-isoindol-1-one; 7-acetyl-3-({4-[(1E)-2-{4-[(6-acetyl-4,5,7-trichloro-1-oxo-1H-isoindol-3-yl)amino]phenyl}diazen-1-yl]-3-methylphenyl}amino)-4,5,6-trichloro-1H-isoindol-1-one; 7-acetyl-3-({4-[(1E)-2-{4-[(7-acetyl-4,5,6-trichloro-1-oxo-1H-isoindol-3-yl)amino]phenyl}diazen-1-yl]-3-methylphenyl}amino)-4,5,6-trichloro-1H-isoindol-1-one; N-{4-[(1E)-2-(2-methyl-4-{[(1Z)-4,5,6,7-tetrachloro-3-oxo-2,3-dihydro-1H-isoindol-1-ylidene]amino}phenyl)diazen-1-yl]phenyl}acetamide

EC Number:

600-734-7

Cas Number:

106276-78-2

Molecular formula:

C13H14N.C9H3Cl4NO2.CH4O.Na

IUPAC Name:

(3Z)-4,5,6,7-tetrachloro-3-({3-methyl-4-[(1E)-2-(3-methylphenyl)diazen-1-yl]phenyl}imino)-2,3-dihydro-1H-isoindol-1-one; 4,5,6,7-tetrachloro-3-({3-methyl-4-[(1E)-2-{4-[(4,5,6,7-tetrachloro-1-oxo-1H-isoindol-3-yl)amino]phenyl}diazen-1-yl]phenyl}amino)-1H-isoindol-1-one; 6-acetyl-3-({4-[(1E)-2-{4-[(6-acetyl-4,5,7-trichloro-1-oxo-1H-isoindol-3-yl)amino]phenyl}diazen-1-yl]-3-methylphenyl}amino)-4,5,7-trichloro-1H-isoindol-1-one; 6-acetyl-3-({4-[(1E)-2-{4-[(7-acetyl-4,5,6-trichloro-1-oxo-1H-isoindol-3-yl)amino]phenyl}diazen-1-yl]-3-methylphenyl}amino)-4,5,7-trichloro-1H-isoindol-1-one; 7-acetyl-3-({4-[(1E)-2-{4-[(6-acetyl-4,5,7-trichloro-1-oxo-1H-isoindol-3-yl)amino]phenyl}diazen-1-yl]-3-methylphenyl}amino)-4,5,6-trichloro-1H-isoindol-1-one; 7-acetyl-3-({4-[(1E)-2-{4-[(7-acetyl-4,5,6-trichloro-1-oxo-1H-isoindol-3-yl)amino]phenyl}diazen-1-yl]-3-methylphenyl}amino)-4,5,6-trichloro-1H-isoindol-1-one; N-{4-[(1E)-2-(2-methyl-4-{[(1Z)-4,5,6,7-tetrachloro-3-oxo-2,3-dihydro-1H-isoindol-1-ylidene]amino}phenyl)diazen-1-yl]phenyl}acetamide

Test material form:

solid: nanoform

Details on test material:

- Physical state: solid/ orange
- Storage condition of test material: The test substance was stored at ambient temperature.
- BET: 57.0 m2/g
- TEM available

Method

Target gene:

his / trp

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital i.p. and β-naphthoflavone induced rat- and uninduced liver S9 fraction

Test concentrations with justification for top dose:

33 μg - 5 000 μg/plate (SPT)
33 μg - 5 000 μg/plate (Prival)

Vehicle / solvent:

Acetone

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

congo red

other: 2-aminoanthracene, benzidine

Remarks:

With hamster liver S9 mix

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

other: 4-nitro-o-phenylenediamine

Remarks:

Without S9 mix

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation
DURATION
- Preincubation period: 20 min
- Exposure duration: 37°C for 48 - 72 hours
NUMBER OF REPLICATIONS: in triplicate
NUMBER OF CELLS EVALUATED: all revertants / colonies counted
DETERMINATION OF CYTOTOXICITY
Toxicity detected by a
• decrease in the number of revertants
• clearing or diminution of the background lawn (= reduced his- or trp- background growth)
• reduction in the titer
is recorded for all test groups both with and without S9 mix in all experiments

Evaluation criteria:

The test substance was considered positive in this assay if the following criteria were met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. at least
doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and
E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA
1535 and TA 1537) of the spontaneous mutation rate in at least one tester strain either
without S9 mix or after adding a metabolizing system.
A test substance was generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the historical negative control
data range under all experimental conditions in at least two experiments carried out
independently of each other.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

A weak bacteriotoxic effect (slight decrease in the number of his+ revertants) was observed in
the standard plate test at the tester strain TA 1535 with and without S9 mix only at the
highest applied concentration.
In the prival preincubation assay bacteriotoxicity (slight decrease in the number of his+ or trp+
revertants) was occasionally observed depending on the strain and test conditions from
about 1 000 μg/plate onward.
Test substance precipitation was observed in the standard plate test from about 333 μg/plate
onward and in the prival preincubation test from about 33 μg/plate onward both with and
without S9 mix.

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions of this study, the test substance is not mutagenic in the standard plate test or in the prival preincubation test in the absence and the presence of metabolic activation.

Executive summary:

The test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay.

STRAINS: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA

DOSE RANGE: 33 μg - 5 000 μg/plate (SPT); 33 μg - 5 000 μg/plate (Prival)

TEST CONDITIONS: Standard plate test (SPT) and Prival preincubation test (Prival); both with and without metabolic activation.

SOLUBILITY: Precipitation of the test substance was found from about 33 μg/plate onward with and without S9 mix.

TOXICITY: A weak bacteriotoxic effect was occasionally observed depending on the strain and test conditions from about 1 000 μg/plate onward.

MUTAGENICITY: A biologically relevant increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the prival preincubation test either without S9 mix or after the addition of a metabolizing system.

CONCLUSION: Under the experimental conditions of this study, the test substance is not mutagenic in the standard plate test or in the prival preincubation test in the absence and the presence of metabolic activation.