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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2012-10-23 to 2012-12-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted 1997-07-21
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
, 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2009-03-30
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
not applicable
Species / strainopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9 prepared from 8 – 12 weeks old male Wistar rats (Hsd Cpb: WU; weight approx. 220 – 320 g) induced by intraperitoneal applications of 80 mg/kg b.w. phenobarbital and by peroral administrations of β-naphthoflavone each, on three consecutive days.
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9 prepared from 8 – 12 weeks old male Wistar rats (Hsd Cpb: WU; weight approx. 220 – 320 g) induced by intraperitoneal applications of 80 mg/kg b.w. phenobarbital and by peroral administrations of β-naphthoflavone each, on three consecutive days.
Test concentrations with justification for top dose:
Pre-Experiment/Experiment I: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate (all strains; with and without metabolic activation)
Experiment Ia and II: 0.1; 0.3; 1; 3; 10; 33; 100; and 333 µg/plate (Salmonella strains; without metabolic activation)
Experiment II: 0.3; 1; 3; 10; 33; 100; 333; and 1000 µg/plate (Salmonella strains; with metabolic activation)
Experiment II: 3; 10; 33; 100; 333; 1000; 2500 and 5000 µg/plate (E. coli strain; with and without metabolic activation)
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO (first experiment:MERCK, 64293 Darmstadt/Germany; purity > 99 %; all further experiments: Acros, New Jersey, USA, purity > 99 %).
- Justification for choice of solvent/vehicle: the solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria (Maron et al., 1981)*.

*Reference:
- Maron D.M., J. Katzenellenbogen, and B.N. Ames (1981) Compatibility of organic solvents with the Salmonella/Microsome Test Mutation Res. 88, 343-350
Controlsopen allclose all
Negative controls:
yes
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Positive control without metabolic activation: dissolved in water; concentration: 10 µg/plate; strains: TA1535 and TA100
Negative controls:
yes
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylene-diamine
Remarks:
Positive control without metabolic activation: dissolved in DMSO; concentration: 10 µg/plate (strain TA98) and 50 µg/plate (strain TA1537); strains: TA98 and TA1537
Negative controls:
yes
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Positive control without metabolic activation: dissolved in deionised water; concentration: 3 µL/plate; strain: WP2 uvrA
Negative controls:
yes
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
Positive control with metabolic activation: dissolved in DMSO; concentration: 2.5 µg/plate (strains TA1535, TA1537, TA98, TA100) and 10 µg/plate (strain WP2 uvrA); strains: TA1535, TA1537, TA98, TA100 and WP2 uvrA
Details on test system and conditions:
METHOD OF APPLICATION: plate incorporation (experiment I and Ia) and preincubation (experiment II)

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
To evaluate the toxicity of the test item a pre-experiment was performed with strains TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA. Eight concentrations were tested for toxicity and mutation induction with three plates each.

DURATION
- Preincubation period: 60 minutes
- Exposure duration: at least 48 hours at 37°C

NUMBER OF REPLICATIONS: for each strain and dose level including the controls, three plates were used.

The colonies were counted using the Petri Viewer Mk2 (Perceptive Instruments Ltd, Suffolk CB9 7BN, UK) with the software program Ames Study Manager. Due to precipitation of the test item and reduced background growth the revertant colonies were partly counted manually.
Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed (Hollstein et al., 1979)*.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration (de Serres & Shelby, 1979)*.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. Whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

*References:
- de Serres F.J. and M.D. Shelby (1979) Recommendations on data production and analysis using the Salmonella/microsome mutagenicity assay. Mutation Res. 64, 159-165
- Hollstein,M., J. McCann, F.A. Angelosanto, and W.W. Nichols (1979) Short-term tests for carcinogens and mutagens. Mutation Res. 65, 133-226
Statistics:
According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: the test item precipitated in the overlay agar in the test tubes from 1000 to 5000 µg/plate. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 2500 to 5000 µg/plate. The undissolved particles had no influence on the data recording.

CYTOTOXICITY:
- plates incubated with the test item showed reduced background growth with and without metabolic activation in all experiments (please refer to the table 1 in the field "Any other information on results incl. tables" below).
- toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation (please refer to the table 2 in the field "Any other information on results incl. tables" below).

MAIN EXPERIMENTS
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.
Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.

Any other information on results incl. tables

Table 1: Plates incubated with the test item showed reduced background growth at the following concentrations (µg/plate)

Strain

Experiment I

Experiment II

 

without S9 mix

with S9 mix

without S9 mix

with S9 mix

TA 1535

33 - 5000

33 – 333 (HV1a)

333 - 5000

33 - 333

333 - 1000

TA 1537

33 - 5000

33 - 333 (HV1a)

333 - 5000

33 - 333

333 - 1000

TA 98

100 - 5000

333 - 5000

33 - 333

333 - 1000

TA 100

33 - 5000

333 - 5000

100 - 333

333 - 1000

WP2 uvrA

5000

1000 - 5000

2500 - 5000

5000

HV1a: Results for experiment Ia. Due to strong toxic effects in strains TA1535 and TA1537 in experiment I without S9 mix, this part was repeated with lower concentrations.

Table 2:

Strain

Experiment I

Experiment II

 

without S9 mix

with S9 mix

without S9 mix

with S9 mix

TA 1535

100 – 5000

100 – 333 (HV1a)

333 - 5000

33 - 333

333 - 1000

TA 1537

33 – 5000

100 – 333 (HV1a)

333 - 5000

10 - 333

333 - 1000

TA 98

100 - 5000

333 - 5000

33 - 333

333 - 1000

TA 100

100 - 5000

333 - 5000

100 - 333

333 - 1000

WP2 uvrA

2500 - 5000

1000 - 5000

2500 - 5000

2500 - 5000

HV1a: Results for experiment Ia. Due to strong toxic effects in strains TA1535 and TA1537 in experiment I without S9 mix, this part was repeated with lower concentrations.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

In conclusion, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
According to Directive 67/548 /EEC and its subsequent amendments and according to Regulation (EC) No 1272/2008 and subsequent regulations, the test substance should not be considered to have a mutagenic potential, and hence no classification or labelling is required.