N,N'-(2-chloro-1,4-phenylene)bis(3-oxobutaneamide)

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:

1999-12-06 to 2000-01-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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)

Version / remarks:

adopted: July 21st, 1997

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

443-010-4

EC Name:

-

Cas Number:

53641-10-4

Molecular formula:

C14H15ClN2O4

IUPAC Name:

N-[3-chloro-4-(3-oxobutanamido)phenyl]-3-oxobutanamide

Test material form:

solid

Method

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:

S9-mix from rat liver

Test concentrations with justification for top dose:

plate incorporation test:
with and without metabolic activation:
50, 160, 500, 1600 and 5000 µg/plate

preincubation test:
with and without metabolic activation:
16, 50,160,500, 1600 and 5000 µg/plate

The test compound did not precipitate on the plates up to the highest investigated dose
of 5000 µg/plate.
In the plate incorporation test toxicity was observed with and without metabolic
activation with the strain TA 100 at a concentration of 5000 µg/plate.
In the preincubation test toxicity was not observed with and without metabolic
activation.

Vehicle / solvent:

Solvent: DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-aminoanthracene

Details on test system and experimental conditions:

Two independent mutation tests were performed unless clearly positive or dose-related
activity was observed in the first test. Where results were negative or equivocal, a
second test was conducted. This included a pre-incubation step if the first test was
clearly negative. Pre-incubation involved incubating the test substance, S9-mix and
bacteria for a short period before pouring this mixture onto plates of minimal agar.
Each test was performed in both the presence and absence of S9-mix using all
bacterial tester strains and a range of concentrations of the test substance. Positive
and negative controls as well as solvent controls were included in each test. Triplicate
plates were used.
The highest concentration in the first mutation experiment was usually 50 mg/ml of the
test substance in the chosen solvent, which provided a final concentration of
5000 µg/plate. Further dilutions of 1600, 500, 160 and 50 µg/plate were used. Suitable
dose levels used in the second experiment may be different depending on any toxicity
seen in the first experiment. A reduction in the number of spontaneously occurring
colonies and visible thinning of the bacterial lawn were used as toxicity indicators.
Thinning of the bacterial lawn was evaluated microscopically.
In both tests top agar was prepared which, for the Salmonella strains, contained 100 ml
agar (0.6 % (w/v) agar, 0.5 % (w/v) NaCl) with 10 ml of a 0.5 mM histidine-biotin
solution. For E. coli histidine was replaced by tryptophan (2.5 ml, 0.5 mM). The
following ingredients were added (in the following order) to 2 ml of molten top agar at
approx. 48 °C:
0.5 ml S9-mix (if required) or buffer
0.1 ml of an overnight nutrient broth culture of the bacterial tester strain
0.1 ml test compound solution (dissolved in DMSO)
In the second mutagenicity test if appropriate these top-agar ingredients were
preincubated by shaking for approximately 20 minutes at approx. 30°C.
After mixing, and preincubation if appropriate, the liquid was poured into a petri dish
containing a 25 ml layer of minimal agar (1.5 % (w/v) agar, Vogel-Bonner E medium
with 2 % (w/v)glucose). After incubation for approximately 48 hours at approx. 37 °C in
the dark, colonies (his+ and trp+ revertants) were counted by hand or by a suitable
automatic colony counter.
The counter was calibrated for each test by reading a test pattern plate to verify the
manufacturer's requirements for the counter's sensitiveness.

Evaluation criteria:

Criteria for a valid assay
The assay is considered valid if the following criteria are met:
- the solvent control data are within the laboratory's normal control range for the
spontaneous mutant frequency
- the positive controls induce increases in the mutation frequency which are
significant and within the laboratory's normal range

Criteria for a positive response
A test compound is classified as mutagenic if it has either of the following effects:
a) it produces at least a 2-fold increase in the mean number of revertants per plate of
at least one of the tester strains over the mean number of revertants per plate of
the appropriate vehicle control at complete bacterial background lawn
b) it induces a dose-related increase in the mean number of revertants per plate of at
least one of the tester strains over the mean number of revertants per plate of the
appropriate vehicle control in at least two to three concentrations of the test
compound at complete bacterial background lawn.

If the test substance does not achieve either of the above criteria, it is considered to
show no evidence of mutagenic activity in this system.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(> 5000 µg/plate)

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

Remarks:

(>5000 µg/plate)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(> 5000 µg/plate)

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

Remarks:

( 5000 µg/plate)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(>5000 µg/plate)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

The results lead to the conclusion that the test item is not mutagenic in these bacterial
test systems either in the absence or in the presence of an exogenous metabolizing
system.

Executive summary:

The test item was tested for mutagenicity with the strains TA 100,
TA 1535, TA 1537 and TA 98 of Salmonella typhimurium and with Escherichia coli
WP2uvrA.
Two independent mutagenicity studies were conducted (one plate incorporation test
and one preincubation test), each in the absence and in the presence of a metabolizing
system derived from a rat liver homogenate.
For both studies, the compound was dissolved in DMSO, and each bacterial strain was
exposed to 5 dose levels, in the preincubation test to 6 dose levels.
The test compound did not precipitate on the plates up to the highest investigated dose
of 5000 μg/plate.
The concentrations for the plate incorporation test were 50, 160, 500, 1600 and
5000 μg/plate.
Because of toxicity in the plate incorporation test dose levels from 16 to 5000 μg/plate
were chosen for the pre incubation test.
Control plates without mutagen showed that the number of spontaneous revertant
colonies was within the laboratory's historical control range. All the positive control
compounds showed the expected increase in the number of revertant colonies.
Toxicity: In the plate incorporation test toxicity was observed with and without metabolic
activation with the strain TA 100 at a concentration of 5000 μg/plate.
In the preincubation test toxicity was not observed with and without metabolic
activation.
Mutagenicity: In the absence and in the presence of the metabolic activation system
the test item did not result in relevant increases in the number of revertants in any of the
bacterial strains.
Summarizing, it can be stated that the test item was not mutagenic in this bacterial
mutation test at any dose level either in the absence or presence of an exogenous
metabolic activation.