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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:
July 20, 2001 till August 17, 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study according OECD Guideline 471 (Bacterial Reverse Mutation Assay) under GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report date:
2001

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Identity: ZP-TIX 1014
Batch: NW-01-028
Purity > 99%,
Appearance: Solid
Expiration date: 30.12.2001
Stability in Solvent: not indicated by the sponsor
Storage: At room temperature
Color: white

Method

Target gene:
uvrB
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 1537
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 98
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 100
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 102
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 liver microsomal activation (Wistar rat , Phenobarbital/ß-Naphthoflavone)
Test concentrations with justification for top dose:
The test item was tested at the following concentrations:
33; 100; 333; 1000; 2500; 5000 µg/plate
Vehicle / solvent:
The test item was dissolved in DMSO (purity > 99%, Merck, D-64293 Darmstadt).
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
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Migrated to IUCLID6: 2-aminoanthracene, 4-nitro-o-phenylene diamine, methyl methane sulfonate
Details on test system and experimental conditions:
METHOD OF APPLICATION:
Testsolution, S9 mix or S9 mix substitution buffer, bacteria suspension were mixed. After pre incubation overlay agar was added to each tube. The mixture was poured on minimal agar plates.

DURATION
Preculture period: 4h incubation in water bath (37°C)
Preincubation period: 60 min (37°C)
Exposure duration: 48h (in the dark, 37°C) plus 1h pre incubation

SELECTION AGENT (mutation assays): Histidine

NUMBER OF REPLICATIONS: 3
NUMBER OF EXPERIMENTS: 2

OTHER EXAMINATIONS:
regualar background growth in negative and solvent control
spontaneous reversion rates in the negative and solvent control


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 TA98, TA100 and TA 102) or thrice (strains TA 1535 and TA 1537) the colony 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:
A statistical analysis of the data is not required.

Results and discussion

Test results
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation. The plates incubated with the test item showd normal backround growth up to 5000 µg/plate with and without S9 mix in all strains used.
No substantial increase in the revertant colony numbers of any of the five tester strains was observed following treatment with ZP-TIX 1014 at any concentration 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 or biological relevance.
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.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
No toxic effects, no substantial increase in revertant colony numbers of any of the five strains was observed following treatment with ZP-TIX 1014 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.
Executive summary:

This study was performed to investigate the potential of ZP-TIX 1014 to induce gene mutations according to the plate incorporation test and the pre-incubation test using Salmonella typhimurium strains TA1535, TA 1537, TA98, TA100, TA102.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concnetration, including the controls, was tested in triplicate. The test item was tested at the following concentrations: 33;100;333;1000;2500 and 5000 µg/plate. No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation.

The plates incubated with the test item showd normal background growth up to 5000 µg/plate with and without S9 mix in all strains used.

No substantial increase in revertant colony numbers of any of the five strains was observed following treatment with ZP-TIX 1014 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 showd a distinct increase of induced revertant colonies.