2,2'-[ethylenebis(oxyphenyl-2,1-eneazo)]bis[N-(2,3-dihydro-2-oxo-1H-benzimidazol-5-yl)-3-oxobutyramide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2 NOV 2004 to 27 DEC 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (OECD TG 476)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Dimethyl 2-[[1-[[(2,3-dihydro-2-oxo-1H-benzimidazol-5-yl)amino]carbonyl]-2-oxopropyl]azo]terephthalate

EC Number:

252-650-1

EC Name:

Dimethyl 2-[[1-[[(2,3-dihydro-2-oxo-1H-benzimidazol-5-yl)amino]carbonyl]-2-oxopropyl]azo]terephthalate

Cas Number:

35636-63-6

Molecular formula:

C21H19N5O7

IUPAC Name:

dimethyl 2-({2-oxo-1-[(2-oxo-2,3-dihydro-1H-benzimidazol-5-yl)carbamoyl]propyl}diazenyl)terephthalate

Test material form:

solid: nanoform

Method

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media:
RPMI 1640 (Hepes buffered medium) supplemented with 15% horse serum, penicillin/streptomycin (100 U/mL respectively 100 µl/mL), 220 µg/mL sodium pyruvate and 1.25 U/mL Amphotericin
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 (induced with phenobarbital/beta-naphtoflavone)

Test concentrations with justification for top dose:

Experiment I:
without S9 mix: 11.7; 23.4; 46.9; 93.8; 187.5, and 375.0 microgram/mL
with S9 mix: 23.4; 46.9; 93.8; 187.5, and 375.0 microgram/mL
Experiment II: without S9 mix: 11.7; 23.4; 46.9; 93.8; and 187.5 microgram/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility properties of the solvent and its relative non-toxicity to the cell cultures

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without metabolic activation

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in suspension
DURATION
- Exposure duration: 4 h (Experiment I: with and w/o metabolic activation) and 24 h (Experiment II w/o metabolic activation)
- Expression time (cells in growth medium): 3 days total (i.e. experiment I: 4 hours treatment period plus 68 hours expression time; experiment II: 24 hours treatment period plus 48 hours expression time)
- Selection time (if incubation with a selection agent): 10 to 15 days

SELECTION AGENT (mutation assays): trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: two independent experiments, using two parallel cultures each

NUMBER OF CELLS USED: Per culture 1 x 10EXP7 cells

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth; other: relative suspension growth

Evaluation criteria:

A test item is classified as positive if it induces either a reproducible concentration-related increase in the mutant frequency or a reproducible positive response for at least one of the test points.
A test item producing neither a reproducible concentration-related increase in the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.
A significant response is described as follows:
The test item is classified as mutagenic if it reproducibly induces a mutation frequency that is at least two times higher than the spontaneous mutation frequency (negative or solvent control) in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase in the mutation frequency. Such an evaluation may be considered independently of the enhancement factor for induced mutants.
However, in the evaluation of the test results the historical variability of the mutation rates in negative and solvent controls and the mutation rates of all negative and solvent controls of this study are taken into consideration.
Results of test groups are rejected if the relative total growth, the relative suspension growth and/or cloning efficiency 1 (survival of the cells after treatment) is less than 10 % of the solvent control or the cloning efficiency 2 (viability) after the expression period is less than 20 %.
Whenever a test item is considered mutagenic according to the above mentioned criteria, the ratio of small versus large colonies is used to differentiate point mutations from clastogenic effects. If the increase of the mutation frequency is accompanied by a reproducible and dose dependent shift in the ratio of small versus large colonies clastogenic effects are indicated.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

indicated by reduced relative cloning efficiency 1 in experiment I at 375 µg/mL in both cultures

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

experiment I: 1st culture at 375 µg/mL, 2nd culture at 187.5 µg/mL and above; experiment II: both cultures at 187.5 µg/mL and above

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:
Precipitation of the test item was observed by the unaided eye at 187.5 and 375.0 microgram/mL in all experimental parts.

COMPARISON WITH HISTORICAL CONTROL DATA:
The mutation frequency did not exceed the historical range of negative and solvent controls at any concentration with and without metabolic activation. The threshold of two times the number of mutant colonies/10EXP6 cells was reached at 46.9 microgram/mL in the second culture of experiment I (with S9 mix). Since this effect was not observed at higher concentrations or in the parallel culture, this isolated effect was judged as biologically irrelevant.
The mutation frequency of the positive control in the first culture of experiment I fell just short of the historical range of positive controls with 233 versus 292 colonies per 10EXP6 cells. Since the threshold of twice the mutation frequency of the solvent control was clearly exceeded and the mean value of both positive controls is well within the historical range, this effect was considered as irrelevant fluctuation.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In experiment I without metabolic activation relevant toxicity (relative cloning efficiency 1 and / or relative total growth of less than 50 % survival) was detected at 187.5 µg/mL and above in the second culture. In culture I a clear toxic effect occurred at the highest concentration of 375 µg/mL.
In experiment I with metabolic activation a toxic effect indicated by a reduced relative cloning efficiency 1 was observed at 375 µg/mL in both cultures.
In the second experiment (24 h treatment without metabolic activation) relevant toxic effects occurred at 187.5 µg/mL and above in both cultures.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results:
negative

The test substance did not increase the mutant frequency at the TK+/- locus in L5178Y cells and is therefore considered to be non-mutagenic under the conditions of the test.

Executive summary:

Mouse Lymphoma cells (L5178Y TK+/-) were treated with the test substance at five or six concentrations in the range of 11.7 to 375 microgram/mL. Precipitation of the test substance was detectable at dose level 187.5 and 375 microgram/mL. The solvent control gave mutant frequencies within the normal range. The positive controls gave marked increases in the mutant frequency, both in the presence or absence of metabolic activation, indicating the satisfactory performance of the test system. The test substance demonstrated no statistically significant or dose related increases in mutant frequency at any dose level, either with or without metabolic activation, thus indicating that the test item is not mutagenic under the conditions tested.