Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
From 01 JUN 2011 to 09 AUG 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: according to OECD 476 and GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay

Test material

Constituent 1
Reference substance name:
2-[(4-chloro-2-nitrophenyl)azo]-N-(2,3-dihydro-2-oxo-1H-benzimidazol-5-yl)-3-oxobutyramide
EC Number:
235-462-4
EC Name:
2-[(4-chloro-2-nitrophenyl)azo]-N-(2,3-dihydro-2-oxo-1H-benzimidazol-5-yl)-3-oxobutyramide
Cas Number:
12236-62-3
IUPAC Name:
2-[(4-chloro-2-nitrophenyl)diazenyl]-3-oxo-N-(2-oxo-2,3-dihydro-1H-benzimidazol-5-yl)butanamide

Method

Target gene:
HPRT
Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/Beta-Naphtoflavone induced Rat liver S9
Test concentrations with justification for top dose:
Experiment I:
without metabolic activation: 9.4, 18.8, 37.5, 75.0, 150, 1200 µg/mL
with metabolic activation: 9.4, 18.8, 37.5, 75.0, 150, 1200 µg/mL

Experiment II:
without metabolic activation: 6.3, 12.5, 25.0, 50.0, 100, 200, 1200 µg/mL
with metabolic activation. 3.1, 6.3, 12.5, 25.0, 50.0, 100 µg/mL
Vehicle / solvent:
DMSO
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: Experiment I: 4 hours with and without metabolic activation, Experiment II: 24 hours without metabolic activation, 4 hours with metabolic activation
- Expression time (cells in growth medium): 72 hours
- Selection time (if incubation with a selection agent): 10 days

SELECTION AGENT (mutation assays): 6-Thioguanine


NUMBER OF REPLICATIONS: 2


NUMBER OF CELLS EVALUATED: >1,5x10exp. 6


DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:
A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points.
A test item producing neither a concentration- related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-muta¬genic in this system.
A positive response is described as follows:
A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation fre¬quency at least at one of the concen¬trations in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
However, in a case by case evaluation this decision depends on the level of the correspon¬ding solvent control data. If there is by chance a low spontaneous mutation rate within the laboratory´s historical control data range, a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent con-trols within all experiments of this study was also taken into consideration.
Statistics:
A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance were considered together.

Results and discussion

Test results
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: no, but tested up to and including precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not increased
- Effects of osmolality: Not effected
- Evaporation from medium: Not examined
- Precipitation:
In the range finding pre-experiment precipitation occurred at 18.8 µg/mL and above with metabolic activation. Without metabolic activation precipitation was noted at 37.5 µg/mL and above following 4 and 24 hours treatment.
- Other confounding effects: None

RANGE-FINDING/SCREENING STUDIES:
The highest concentration used in the pre-test was 1200 µg/mL limited by the solubility of the test item in DMSO and aqueous medium. Test item concentrations between 9.4 µg/mL and 1200 µg/mL were used to evaluate toxicity in the presence (4 hours treatment) and absence (4 hours and 24 hours treatment) of metabolic activation. No relevant toxic effect occurred up to the maximum concentration with and without metabolic activation following 4 and 24 hours treatment.
The test medium was checked for precipitation at the end of each treatment period (4 or 24 hours) prior to removal to the test item. Precipitation occurred at 18.8 µg/mL and above with metabolic activation. Without metabolic activation precipitation was noted at 37.5 µg/mL and above following 4 and 24 hours treatment.
There was no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test item.
Based on the results of the pre-experiment, the individual concentrations of the main experiments were selected. A series of concentrations spaced by a factor of 2 was placed from the soluble up into the lower precipitating range. An additional large step up to the maximum possible concentration of 1200 µg/mL was added in the first experiment with and without metabolic activation and in the second experiment without metabolic activa-tion. In the second experiment with metabolic activation the maximum possible concentra-tion was not again applied in favour of more soluble concentrations in the lower range.

Doses applied in the gene mutation assay with the test substance:

Experiment I / 4 hours treatment concentration in µg/mL
without metabolic activation 9.4 18.8 37.5P 75.0 P 150.0 P 1200.0 P
with metabolic activation 9.4 18.8 P 37.5 P 75.0 P 150.0 P 1200.0 P
Experiment II / 24 hours treatment concentration in µg/mL
without metabolic activation 6.3 12.5 25.0 P 50.0 P 100.0 P 200.0 P 1200.0 P
Experiment II / 4 hours treatment
with metabolic activation 3.1 6.3 12.5 P 25.0 P 50.0 P 100.0 P

P = precipitation

In the first experiment the cultures at the lowest concentration of 9.4 µg/mL with and without metabolic activation were not continued since a minimum of only four analysable concentrations is required by the guidelines. In the second experiment the cultures at 50 and 100 µg/mL without metabolic activation and at 100 µg/mL with metabolic activation were not continued to avoid analysis of too many precipitating concentrations.


COMPARISON WITH HISTORICAL CONTROL DATA: Complies


ADDITIONAL INFORMATION ON CYTOTOXICITY: None
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Summary Table
  relative relative relative mutant   relative relative relative mutant  
conc. P S9 cloning cell cloning colonies/ induction cloning cell cloning colonies/ induction
µg/mL mix efficiency I density efficiency II 106cells factor efficiency I density efficiency II 106cells factor
        % % %     % % %    
Column 1 2 3 4 5 6 7 8 9 10 11 12 13
Experiment I / 4 h treatment     culture I          culture II
Solvent control with DMSO - 100.0 100.0 100.0 13.3 1.0 100.0 100.0 100.0 18.9 1.0
Positive control (EMS) 150.0 - 108.7 116.7 77.5 116.8 8.8 91.8 80.0 82.7 101.2 5.4
Test item 9.4 - 102.9 culture was not continued# 90.9 culture was not continued#
Test item 18.8 - 103.0 78.0 91.3 14.2 1.1 96.4 99.2 73.8 19.1 1.0
Test item 37.5 P - 90.6 106.2 92.0 18.5 1.4 88.7 112.8 112.8 10.1 0.5
Test item 75.0 P - 99.4 85.7 83.7 12.3 0.9 93.5 103.3 78.6 40.2 2.1
Test item 150.0 P - 91.1 112.1 76.2 22.0 1.7 75.9 87.6 73.7 20.6 1.1
Test item 1200.0 P - 97.1 82.2 90.5 20.0 1.5 88.6 112.8 70.2 25.8 1.4
Solvent control with DMSO + 100.0 100.0 100.0 14.4 1.0 100.0 100.0 100.0 18.8 1.0
Positive control (DMBA) 1.1 + 102.4 92.8 58.0 1320.1 91.7 105.9 91.9 78.4 919.6 49.0
Test item 9.4 + 99.9 culture was not continued# 95.8 culture was not continued#
Test item 18.8 P + 98.5 78.0 83.2 16.4 1.1 105.3 92.9 92.1 11.7 0.6
Test item 37.5 P + 105.6 91.1 106.4 6.9 0.5 100.4 91.8 111.5 12.9 0.7
Test item 75.0 P + 99.4 101.3 75.7 15.6 1.1 93.1 104.6 88.6 22.4 1.2
Test item 150.0 P + 97.9 80.7 112.0 10.3 0.7 94.0 98.8 80.7 11.9 0.6
Test item 1200.0 P + 99.4 87.8 79.2 23.5 1.6 93.8 85.3 80.5 21.2 1.1
Experiment II / 24 h treatment     culture I          culture II
Solvent control with DMSO   - 100.0 100.0 100.0 18.2 1.0 100.0 100.0 100.0 17.5 1.0
Positive control (EMS) 150.0 - 92.1 90.3 71.5 244.9 13.5 66.2 87.1 89.8 334.0 19.0
Test item 6.3 - 98.3 86.9 100.6 6.1 0.3 83.3 106.8 110.1 11.5 0.7
Test item 12.5 - 99.9 98.8 108.0 10.3 0.6 88.0 111.0 112.7 13.2 0.8
Test item 25.0 P - 99.4 81.7 99.1 14.4 0.8 83.4 99.6 92.3 17.0 1.0
Test item 50.0 P - 100.6 culture was not continued## 92.5 culture was not continued##
Test item 100.0 P - 99.6 culture was not continued## 71.1 culture was not continued##
Test item 200.0 P - 100.1 76.4 99.5 9.8 0.5 72.1 99.1 100.7 16.3 0.9
Test item 1200.0 P - 95.8 81.3 95.5 23.3 1.3 66.3 93.8 91.9 15.2 0.9
Experiment II / 4 h treatment        
Solvent control with DMSO   + 100.0 100.0 100.0 18.1 1.0 100.0 100.0 100.0 16.2 1.0
Positive control (DMBA) 1.1 + 42.2 55.7 104.1 815.4 45.0 51.5 62.7 77.9 1004.5 61.8
Test item 3.1 + 94.4 82.4 124.1 15.8 0.9 102.4 84.7 90.3 11.0 0.7
Test item 6.3 + 95.1 74.8 130.1 14.4 0.8 101.3 107.0 95.1 14.7 0.9
Test item 12.5 P + 96.0 78.8 128.6 15.0 0.8 103.2 81.8 96.4 15.0 0.9
Test item 25.0 P + 93.2 94.1 138.4 13.5 0.7 102.1 81.3 94.4 13.6 0.8
Test item 50.0 P + 95.6 84.8 131.2 26.4 1.5 102.1 94.8 95.0 23.3 1.4
Test item 100.0 P + 97.3 culture was not continued## 102.3 culture was not continued##

#    culture was not continued since a minimum of only four analysable concentrations is required
##   culture was not continued to avoid analysis of too many precipitating concentrations

P = precipitation

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test substance is considered to be non-mutagenic in this HPRT assay.

Executive summary:

The test item was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster.

The study was performed in two independent experiments, using identical experimental procedures. In the first experiment the treatment period was 4 hours with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.

The highest applied concentration of 1200 µg/mL was limited by the solubility properties of the test item in DMSO and aqueous medium.

No relevant cytotoxic effects defined as a reduction of the relative cloning efficiency I and/or relative cell density to values below 50% in both parallel cultures were noted up to the maximum concentration with and without metabolic activation.

No relevant and reproducible increase in mutant colony numbers/106cells was observed in the main experiments up to the maximum concentration. The mutation frequency remained within the historical range of solvent controls.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. No significant dose dependent trend of the mutation frequency indicated by a probabilityvalue of <0.05 was determined in any of the experimental groups.

In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 13.3 up to 18.9 mutants per 106cells; the range of the groups treated with the test item was from 6.1 up to 40.2 mutant colonies per 106cells.

EMS (150 µg/mL) and DMBA (1.1 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.