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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:
2006-05-29 to 2006-06-09
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Only 3 strains are tested, however an expert statement is added in 'Overall Remarks' to justify the fact that no further testing is necessary.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
ninth addendum
Deviations:
yes
Remarks:
only three S. typhimurium (TA98, TA100, and TA 102) strains were used.
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
yes
Remarks:
only three S. typhimurium (TA98, TA100, and TA 102) strains were used
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
White to light yellow powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 00405251 ZT000841PUA421
- Expiration date of the lot/batch: 2007-06-30 (expiry date)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: Not indicated
- Solubility and stability of the test substance in the solvent/vehicle: Not indicated

Method

Target gene:
histidine locus
Species / strain
Species / strain:
other: TA 98, TA 100, and TA 102
Details on mammalian cell lines (if applicable):
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/B-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Pre-Experiment / Experiment I (with and without metabolic activation for all tester strains): 0, 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate;
Experiment II (with and without metabolic activation for all tester strains): 0, 16, 50, 166, 500, 1660 and 5000 µg/plate;

Since the test item was soluble in DMSO up to the standard limit concentration recommended in the regulatory guidelines that this assay followed (5000 µg/plate), the highest tested concentration in the Pre-experiment/Experiment I was 5000 µg/plate.
The highest tested concentration in the mutation experiment 2 was selected based on the toxicity of the test item.
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.
Controlsopen allclose all
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Without S9-mix; at 10 µg/plate (TA100)
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylene-diamine
Remarks:
Without S9-mix; at 10 µg/plate (TA98)
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9-mix; at 4.0 µL/plate (TA102)
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracine
Remarks:
With S9-mix; at 2.5 µg./plate (TA98 and TA100); at 10 µg/plate (TA102)
Details on test system and conditions:
METHOD OF APPLICATION: plate incorporation (Experiment I) ; preincubation (Experiment II)
- Plate incorporation (Experiment I):
In the plate incorporation assay, 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. After solidification the plates will be incubated upside down for at least 48 hours at 37° C in the dark.
- Pre-incubation (Experiment II):
In the pre-incubation method, 100 µL test solution, 500 µL S9 mix / S9 mix substitution buffer and 100 µL bacteria suspension were mixed in a test tube and incubated at 37°C for 60 minutes. After pre-incubation 2.0 mL overlay agar (45°) was added to each tube. The mixture was poured on selective agar plates. After solidification the plates will be incubated upside down for at least 48 hours at 37° C in the dark.

DURATION
- Preincubation period: 60 minutes (Experiment II)
- Exposure duration: at least 48 hours (Experiments I and II)
- Selection time (if incubation with a selection agent): at least 48 hours (Experiments I and II) simultaneous with exposure duration
- Fixation time (start of exposure up to fixation or harvest of cells): at least 48 hours

SELECTION AGENT (mutation assays): histidine (S. typhimurium strains)

NUMBER OF REPLICATIONS: triplicate

DETERMINATION OF CYTOTOXICITY
- Method: Toxic effects of the test substance were detected by a substantial reduction in mean revertant colony counts or by the sparse or absent background bacterial lawn.
Rationale for test conditions:
Solubility limitations: Since the test item was fully soluble in DMSO, the highest tested concentration for the Mutation assay was 5000 μg/plate.
Evaluation criteria:
A test substance was considered a mutagen if a biologically relevant increases in the number of revertants exceeding the threshold of twice the colony count of the corresponding solvent control was observed.
A dose dependent increase was considered biologically relevant if the threshold was exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration was judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold was 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 was not considered biologically relevant.
Statistics:
A statistical analysis of the data is not required.

Results and discussion

Test resultsopen allclose all
Species / strain:
other: TA 98, TA 100, and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
in all strains: at 333 µg/plate and above (without S9-mix), and at 1000 µg/plate and above (with S9-mix)
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: Experiment I
Species / strain:
other: TA 98, TA 100, and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
in all strains: at 1660 µg/plate and above with and without S9-mix
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: Experiment II
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: no data
- Precipitation:
Pre-Experiment/Experiment I: at 2500 µg/plate and above with S9-mix in all strains;
Experiment II: no precipitation observed up to the highest tested concentration (5000 µg/plate)
- Other confounding effects: no data

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 the criteria mentioned above were met and 5000 µg/plate were chosen as maximum concentration. Based on the results of experiment I, 5000 μg/plate was selected as the highest test item concentration for experiment II.

COMPARISON WITH HISTORICAL CONTROL DATA:
- Vehicle control: The vehicle control values were within the laboratory's historical control range.
- Positive control: Appropriate reference mutagens were used as positive controls. They showed a distinct increase in induced revertant colonies.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Toxic effects, evident as a reduction in the number of revertants, occurred with all strains with and without metabolic activation at the following concentrations: Experiment I - without S9 mix: 333-5000 µg/plate and with S9 mix: 1000-5000 µg/plate; Experiment II - with and without S9 mix: 1660 and 5000 µg/plate.

Applicant's summary and conclusion

Conclusions:
Interpretation of results: negative with and without metabolic activation
It was concluded that during the described mutagenicity test and under the experimental conditions reported, the test substance did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.