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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:
24 May 2007 to 17 August 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study followed OECD Test guideline 471 and EU Method B.13/B.14

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes (incl. certificate)
Remarks:
GLP according to German "Chemikaliengesetz" and OECD Guideline
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
his- for S. typhimurium strains
trp- for E. coli WP2 uvr A
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9 mix (phenobarbital/ß-naphthoflavone-induced rat liver S9)
Test concentrations with justification for top dose:
Experiment I (plate incorporation): 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate both with and without metabolic activation
Experiment II (pre-incubation): 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate both with and without metabolic activation
Vehicle / solvent:
DMSO, purity <99% (Merck, Darmstadt, Germany). The solvent was chosen because of its solubility properties and its relative non-toxicity to bacteria
Controls
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: see below for additional information
Details on test system and experimental conditions:
METHOD OF APPLICATION:
plate incorporation (experiment I); preincubation (experiment II). Since experiment I gave a negative result, experiment II was performed as a
preincubation assay.

DURATION
- Preincubation period: 60 minutes
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY: existence of evaluable plates (> 0 colonies) at five concentrations or more
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.
Statistics:
Not mandatory according to OECD guideline 471

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
see below for details
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: at and above 1000 µg/plate (experiment I), at and above 2500 µg/plate (experiment II), undissolved particles had no influence on data recording

RANGE-FINDING/SCREENING STUDIES:
Pre-experiment was reported as experiment I because the criterion (evaluable plates (>0 colonies) at five concentrations or more in all strains are used) was met.

COMPARISON WITH HISTORICAL CONTROL DATA:
yes

ADDITIONAL INFORMATION ON CYTOTOXICITY:
see below
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

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 plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments. Slight toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed in the absence of metabolic activation in strain TA 1535 at 5000 µg/plate and in strain TA 1537 at 2500 and 5000 µg/plate in experiment I, and in strain TA 1537 from 1000 - 5000 µg/plate in experiment II. 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 and showed a distinct increase of induced revertant cololonies.

Mean mutant number ratios treated/solvent control

Exp. I: plate incorporation method without S9 mix

Concentrations given in µg/plate

Strain -- 3 -- 10 -- 33 -- 100 -- 333 -- 1000 -- 2500 -- 5000

TA1535 -- 1.1 -- 0.9 -- 0.9 -- 1.0 -- 0.8 -- 1.0 -- 0.5 -- 0.4

TA1537 -- 1.1 -- 1.8 -- 1.3 -- 1.4 -- 1.0 -- 0.9 -- 0.4 -- 0.2

TA98 -- 1.1 -- 0.9 -- 1.0 -- 1.3 -- 1.3 -- 1.0 -- 0.8 -- 0.6

TA100 -- 0.9 -- 0.8 --0.9 -- 1.0 -- 0.9 -- 0.9 -- 0.8 -- 0.8

WP2uvrA -- 1.2 -- 1.3 -- 1.4 -- 1.2 -- 1.1 -- 0.9 -- 0.9 -- 0.7

Exp. I: plate incorporation method with rat S9 mix

Concentrations given in µg/plate

Strain -- 3 -- 10 -- 33 -- 100 -- 333 -- 1000 -- 2500 -- 5000

TA1535 -- 0.9 -- 1.1 -- 1.0 -- 0.9 -- 0.6 -- 0.7 -- 0.6 -- 0.6

TA1537 -- 0.9 -- 0.8 -- 0.9 -- 1.3 -- 1.0 -- 0.9 -- 0.8 -- 0.8

TA98 -- 1.0 -- 1.0 -- 1.0 -- 1.0 -- 0.8 -- 0.8 -- 0.8 -- 0.8

TA100 -- 1.0 -- 0.9 --1.0 -- 1.1 -- 0.9 -- 0.8 -- 0.9 -- 0.8

WP2uvrA -- 0.9 -- 1.1 -- 0.9 -- 1.0 -- 0.9 -- 0.7 -- 0.8 -- 0.9

Exp. II: pre-incubation method without S9 mix

Concentrations given in µg/plate

Strain -- 10 -- 33 -- 100 -- 333 -- 1000 -- 2500 -- 5000

TA1535 -- 1.3 -- 1.1 -- 1.6 -- 0.9 -- 1.3 -- 0.6 -- 0.5

TA1537 -- 0.3 -- 0.8 -- 1.1 -- 0.7 -- 0.4 -- 0.3 -- 03

TA98 -- 1.1 -- 1.3 -- 1.3 -- 1.2 -- 0.8 -- 0.8 -- 0.7

TA100 --0.9 -- 1.0 -- 1.0 -- 0.9 -- 0.9 -- 1.0 -- 1.1

WP2uvrA -- 1.0 -- 0.9 -- 1.1 -- 0.8 -- 0.9 -- 0.8 -- 0.8

Exp. II: pre-incubation method with rat S9 mix

Concentrations given in µg/plate

Strain -- 10 -- 33 -- 100 -- 333 -- 1000 -- 2500 -- 5000

TA1535 -- 0.9 -- 0.7 -- 0.8 -- 0.8 -- 0.9 -- 0.5 -- 0.6

TA1537 -- 1.0 -- 0.9 -- 0.7 -- 0.9 -- 0.6 -- 0.6 -- 0.6

TA98 -- 1.0 -- 1.0 -- 1.1 -- 1.0 -- 0.8 -- 0.6 -- 0.7

TA100 --0.9 -- 0.9 -- 0.9 -- 1.0 -- 0.7 -- 0.9 -- 0.8

WP2uvrA -- 1.0 -- 0.9 -- 0.9 -- 1.0 -- 0.7 -- 0.6 -- 0.6

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative both with and without metabolic activation

During the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by
frameshifts or base-pair substitutions in the genome of the strains used. Therefore, the test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.
Executive summary:
This study was performed to investigate the potential of the test item 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: 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments. Slight toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed in the absence of metabolic activation in strain TA 1535 at 5000 µg/plate and in strain TA 1537 at 2500 and 5000 µg/plate in experiment I, and in strain TA 1537 from 1000 - 5000 µg/plate in experiment II. 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 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 frameshifts in the genome of the strains used.