Registration Dossier

Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

in vitro gene mutation study in mammalian cells
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and OECD guideline compliant study with well characterized test material

Data source

Reference Type:
study report

Materials and methods

Test guideline
according to guideline
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Tetramethyl 2,2'-[1,4-phenylenebis[imino(1-acetyl-2-oxoethane-1,2-diyl)azo]]bisterephthalate
EC Number:
EC Name:
Tetramethyl 2,2'-[1,4-phenylenebis[imino(1-acetyl-2-oxoethane-1,2-diyl)azo]]bisterephthalate
Cas Number:
Molecular formula:
tetramethyl 2,2'-{1,4-phenylenebis[imino(1,3-dioxobutane-2,1-diyl)diazene-2,1-diyl]}diterephthalate
Test material form:
solid: nanoform, no surface treatment


Target gene:
TK locus
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 medium including stable glutamine supplemented with
1% (v/v) penicillin/streptomycin (10 000 IU / 10 000 μg/mL)
1% (v/v) sodium pyruvate (10 mM) (= RPMI-0)
For 4-hour exposure interval:
RPMI-0 supplemented with 5% (v/v) fetal calf serum (FCS) (= RPMI-5)
For 24-hour exposure interval and subculturing cells:
RPMI-0 is supplemented with 10% (v/v) fetal calf serum (= RPMI-10)
For cloning efficiency and selection medium:
RPMI-0 supplemented with 20% (v/v) fetal calf serum (= RPMI-20)
Metabolic activation:
with and without
Metabolic activation system:
liver S9 fraction from rats pretreated with phenobarbital i.p. and β-naphthoflavone orally
Test concentrations with justification for top dose:
1st Experiment
without S9 mix (4-hour exposure period)
0; 0.31; 0.63; 1.25; 2.5; 5.0; 10.0; 20.0 μg/mL

with S9 mix (4-hour exposure period)
0; 0.31; 0.63; 1.25; 2.5; 5.0; 10.0; 20.0 μg/mL

2nd Experiment
without S9 mix (24-hour exposure period)
0; 0.31; 0.63; 1.25; 2.5; 5.0; 10.0 μg/mL

with S9 mix (4-hour exposure period)
0; 0.25; 0.5; 1.0; 2.0; 4.0; 8.0 μg/mL
Vehicle / solvent:
Controlsopen allclose all
Untreated negative controls:
Negative solvent / vehicle controls:
True negative controls:
Positive controls:
Positive control substance:
without metabolic activation
Untreated negative controls:
Negative solvent / vehicle controls:
True negative controls:
Positive controls:
Positive control substance:
with metabolic activation
Details on test system and experimental conditions:

- Preincubation period: 4 - 5 days
- Exposure duration: 4h or 24h
- Expression time (cells in growth medium): 2 days

SELECTION AGENT (mutation assays): trifluorthymidine


- Method: cloning efficiency, relative growth

OTHER: During the week prior to treatment, spontaneous TK deficient mutants (TK-/-) were eliminated from the stock cultures by incubating 1.5 x 10E6 cells per 75 cm² flask (5 x 104 cells/mL) for 1 day in “THMG" medium (pretreatment medium A), and for the following 3 days in “THG" medium (pretreatment medium B).

Evaluation criteria:
The MLTK assay is considered valid if the following criteria are met considering the international guidelines and the current recommendations of the IWGT:
- The absolute cloning efficiency obtained at the time of mutant selection (CE2) of the negative/vehicle controls should fall in the range of 65 - 120%.
- The suspension growth (SG) of the negative/vehicle controls referring to the expression period following treatment should fall in the range of 8 - 32 for 4-hour exposure and 32 - 180 for 24-hour exposure.
- The mutant frequency of the negative/vehicle controls should fall within the range of 50 - 170 x 10E-6 colonies.
- The positive controls should yield an absolute increase in total MF that is an increase above the spontaneous background MF (an induced MF [IMF]) of at least 300 x 10E-6 colonies. The small colony MF should account for at least 40% of that IMF, means a small colony IMF of at least 120 x 10E-6 colonies. Alternatively, the positive controls should induce at least 150 small colonies. The upper limit of cytotoxicity observed in the positive controls should have a relative total growth (RTG) that is greater than 10%.
- The highest applied concentration of the test substance should be 5 mg/mL, 5 μL/mL or 10 mM, unless limited by cytotoxicity or solubility of the test substance. If toxicity occurs, the highest concentration should lower the cloning efficiency 1 (CE1) or the relative total growth (RTG) to 10 to 20% of survival. If precipitation occurs, the highest evaluated concentration should be the lowest concentration where precipitation is observed by the unaided eye.

Mutagenicity criteria:
- MF exceeds a threshold of 126 mutant colonies per 10exp6 cells above the concurrent negative/vehicle control value.
- Evidence of reproducibility of any increase in mutant frequencies
- A statistically significant dose-related increase in mutant frequencies
An appropriate statistical trend test (SAS procedure PROC REG; 9) was performed to assess a possible dose-related increase of mutant frequencies. The number of mutant colonies obtained for the test substance treated groups was compared with that of the respective negative/vehicle control groups. A trend was judged as statistically significant whenever the p-value (probability value) was below 0.10 and the slope was greater than 0.
However, both, biological and statistical significance has been considered together.

Results and discussion

Test results
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
Untreated negative controls validity:
not examined
Positive controls validity:
Additional information on results:
- Effects of pH: pH value was not influenced by test substance treatment.
- Effects of osmolality: Osmolarity was not influenced by test substance treatment.
- Precipitation: was found from 4µg/plate onwards

ADDITIONAL INFORMATION ON CYTOTOXICITY: In both experiments, no cytotoxic effects indicated by reduced cloning efficiencies or reduced relative total growth of below 20% of control were observed neither in the presence nor in the absence of S9 mix.
Remarks on result:
other: all strains/cell types tested
Migrated from field 'Test system'.

Any other information on results incl. tables

According to the results of the present in vitro study, the test substance did not lead to a relevant increase in the number of mutant colonies either without S9 mix or after the addition of a metabolizing system in two experiments performed independently of each other. The mutant frequencies at any concentration were close to the range of the concurrent negative control values and within the range of our recent negative control data set.

Unfortunately, not all acceptance criteria mentioned were fully fulfilled. In the 1st Experiment in the absence of S9 mix the absolute cloning efficiency of the vehicle control determined at the time of mutant selection (CE2) was slightly higher than the proposed upper border of 120% as recommended by IWGT. However, due to missing cytotoxicity and the fact that dose selection was based on the solubility properties of the test substance this finding has no impact on the outcome of this experiment. In addition, either in the 1st Experiment or in the 2nd Experiment in the absence of metabolic activation the corrected mutation frequencies of the vehicle controls (35.7 x 10E-6 colonies or 46.6 x 10E-6 colonies, respectively) were slightly lower than proposed by IWGT (50 – 170 x 10E-6 colonies). However, the mutation frequencies of the vehicle control groups were clearly within our laboratory’s historical negative control data range and, thus, these observations have to be regarded as irrelevant.

Finally, it has to be considered that the minor deviations from the criteria of acceptance described above have no detrimental impact on the validity of this study. The increase in the frequencies of mutant colonies induced by the positive control substances MMS and CPP clearly demonstrated the sensitivity of the test method and of the metabolic activity of the S9 mix employed. The values were within or above the range of the historical positive control data and, thus, fulfilled the acceptance criteria of this study.

Applicant's summary and conclusion

Interpretation of results (migrated information):