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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:
key study
Study period:
from 2012-04-30 to 2012-08-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study according to OECD TG 471

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

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
Test concentrations with justification for top dose:
Experiment I (pre-experiment, plate incorporation test): 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate
Experiment II (pre-incubation test):
TA1535, TA98, WP2 uvra: 33, 100, 333, 1000, 2500 and 5000 µg/plate
TA1537, TA100: 1, 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria.
Controls
Untreated negative controls:
yes
Remarks:
concurrent untreated
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine, 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 60 min
- Exposure duration: 48 h

NUMBER OF CELLS EVALUATED: 10^8-10^9 cells/mL

DETERMINATION OF CYTOTOXICITY
- Method: a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.
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.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
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. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
at 5000 µ/plate (-S-9mix) and 2500-5000 µg/plate (+S-9mix)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 5000 µg/plate Experiment II
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 5000 µg/plate Experiment I
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 2500 µg/plate Experiment I and 5000 µg/plate Experiments I and II
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
at 5000 µ/plate (-S-9mix) and 2500-5000 µg/plate (+S-9mix)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 333-5000 µg/plate Experiment I
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 1000-5000 µg/plate Experiment I and 2500-5000 µg/plate Experiment II
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
at 5000 µg/plate (-S-9mix) and 2500-5000 µg/plate (+S-9mix)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid

Any other information on results incl. tables

Reduced background growth was observed in strain TA100 without S9 mix from 333 – 5000 µg/plate in experiment I. No reduction of the background growth was observed in the remaining strains and in experiment II.

Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups at the following concentrations (µg/plate):

Strain

Experiment I

Experiment II

 

without S9 mix

with S9 mix

without S9 mix

with S9 mix

TA1535

/

/

/

5000

TA1537

5000

2500, 5000

/

5000

TA98

/

/

/

/

TA100

333 – 5000

1000 – 5000

/

2500 – 5000

WP2 uvrA

/

/

/

/

/ no toxic effects observed

Applicant's summary and conclusion

Conclusions:
negative
It can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frame shifts in the genome of the strains used.
Executive summary:

This study was performed to investigate the potential of the substance to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and

TA 100, and the Escherichia coli strain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II,

TA 1535, TA 98, WP2 uvrA: 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II, TA 1537, TA 100: 1; 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Reduced background growth was observed in strain TA 100 without S9 mix from 333 - 5000 µg/plate in experiment I. No reduction of the background growth was observed in the remaining strains and in experiment II.

Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed in strains TA 1537 and TA 100 with and without S9 mix in experiment I. In experiment II, toxic effects were observed in strains TA 1535, TA 1537, and TA 100 with S9 mix.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the substance 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 and showed a distinct increase of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frame shifts in the genome of the strains used.

Therefore, the substance is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.