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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2003-09-11 to 2003-09-29
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
other: Commission Directive 2000/32/EC, L1362000, Annex 4D (dated May 19, 2000)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Name of test material (as cited in study report): T002251
- Physical state: Solid
- Appearance: Brown powder
Specific details on test material used for the study:
Batch No.: 00410155
Aggregate State at Room Temperature: solid
Colour: brown powder
Purity: 96 %
Stability in Solvent: Not indicated by the sponsor
Storage: Room temperature
Expiration Date: 2004-05-05

Method

Target gene:
The histidine dependant strains are derived from S. typhimurium strain LT2 through a mutation in the histidine locus. Strain WP2 and its derivatives all carry the same defect in one of the genes for tryptophan biosynthesis.
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:
Phenobarbital/β-Naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
The concentration range included two logarithmic decades. The following concentrations were tested:
10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
Vehicle / solvent:
Solvent: Dimethylsulfoxide (DMSO)
Justification for choice of solvent: the solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without metabolic activation, at 10 μg/plate with TA1535 and TA100
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylene-diamine
Remarks:
without metabolic activation, at 10 μg/plate with TA 98, at 50 μg/plate with TA 1537
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without metabolic activation, at 4µL/plate with WP2 uvrA
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with metabolic activation, at 2.5 μg/plate with TA 1535, TA 1537, TA 98 and TA 100, at 10 μg/plate with WP2 uvrA
Details on test system and experimental conditions:
METHOD OF APPLICATION:
Experiment I: plate incorporation assay
Experiment II: pre-incubation assay

Experimental performance:
The following materials were mixed in a test tube and poured onto the selective agar plates:
100 μL: Test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control),
500 μL: S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation),
100 μL: Bacteria suspension (cf. test system, pre-culture of the strains),
2000 μL: Overlay agar

In the pre-incubation assay 100 μL test solution, 500 μL S9 mix / S9 mix substitution buffer and 100 μL bacterial suspension were mixed in a test tube and shaken at 37° C for 60 minutes. After pre-incubation 2.0 mL overlay agar (45° C) was added to each tube. The mixture was poured on selective agar plates.
After solidification the plates were incubated upside down for at least 48 hours at 37° C in the dark.

DURATION:
- Pre-incubation period: 60 minutes (experiment II)
- Exposure duration: After solidification the plates were incubated upside down for at least 48 hours at 37° C in the dark.
- Selection time: 48h (simultaneous with exposure)

SELECTION AGENT: tryptophan (E. coli) and histidine (S. typhimurium)

NUMBER OF REPLICATIONS: Each concentration, including controls, was tested in triplicates.

DETERMINATION OF CYTOTOXICITY
- Method: Toxicity of the test item results in 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.
Statistics:
No statistical evaluation of the data was required.

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 / choice of top concentrations:
cytotoxicity
Remarks:
see details below
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:
cytotoxicity
Remarks:
see details below
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation of the test item occurred up to the highest investigated dose.

RANGE-FINDING/SCREENING STUDIES: In the pre-experiment the concentration range of the test item was 3 – 5000 μg/plate. The pre-experiment is reported as experiment I since relevant toxic effects were only observed at high concentrations and 5000 μg/plate were chosen as maximum concentration of the
main experiments.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.
- Negative (solvent/vehicle) historical control data: In experiment I with metabolic activation, the data in the negative control of strain TA 98 and in the solvent control of strain TA 100 were slightly above the laboratory historical control range. Since this deviation is rather small, this effect is considered to be based upon biologically irrelevant fluctuations in the number of colonies.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Strong toxic effects reduced the background growth in the second experiment at 2500 and 5000 μg/plate in strain TA 98 without metabolic activation and in strain TA 100 with and without metabolic activation. Relevant toxic effects, evident as a reduction in the number of revertants below 0.5 times the corresponding control, occurred at the concentrations presented in the section "any additional information including tables".
Remarks on result:
other: all strains/cell types tested

Any other information on results incl. tables

Strong toxic effects occurred at the following concentrations (μg/plate):

Strain

Experiment 1

Experiment 2

 

Without S9 mix

With S9 mix

Without S9 mix

With S9 mix

TA1535

2500-5000

2500-5000

2500-5000

1000-5000

TA1537

2500-5000

2500-5000

2500-5000

5000

TA98

2500-5000

5000

2500-5000

/

TA100

2500-5000

2500-5000

2500-5000

2500-5000

WP2uvrA

2500-5000

1000-5000

1000-5000

1000-5000

Applicant's summary and conclusion

Conclusions:
Interpretation of results:
negative with metabolic activation
negative without metabolic activation

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.