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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report date:
2002

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:
Chemical Name:3-[(2,2-Dimethyl-1 ,2-azasilolidin-1-yl)(dimethyl)silyl]-l-propanamin
Aggregate State: liquid
Colour:colourless-yellowish
Storage: refrigerator
Stability: instable in water (::=; 1.0 min.)
Safety Precautions: Routine hygienic procedures were sufficient to assure personnel health and safety

Method

Target gene:
All S. typhimurium strains contain mutations in the histidine operon, thereby imposing a require-ment for histidine in the growth medium. They contain the deep rough (rfa) mutation, which de-letes the polysaccharide side chain of the lipopolysaccharides of the bacterial cell surface. This increases cell permeability of hydrophobic substances. The other mutation is a deletion of the uvrB gene coding for the DNA excision repair system resulting in an increased sensitivity in detecting many mutagens. This deletion also includes the nitrate reductase (ch2) and biotin (bio) genes (bacteria require biotin for growth). The tester strains TA 97a, TA 98, TA 100 and TA 102 contain the R-factor plasmid, pkM101. These strains are reverted by a number of mutagens that are detected weakly or not at all with the non R-factor parent strains. pkM101 increases chemical and spontaneous mutagenesis by enhancing an error-prone DNA repair system which is normally present in these organisms.
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535
Details on mammalian cell type (if applicable):
TA 1535:
his G 46; rfa-; uvrB-: base-pair substitutions
Species / strain / cell type:
S. typhimurium TA 98
Details on mammalian cell type (if applicable):
TA 98:
his D 3052; rfa-; uvrB-; R-factor: frame shift mutations
Species / strain / cell type:
S. typhimurium TA 100
Details on mammalian cell type (if applicable):
TA 100:
his G46; rfa-; uvrB-; R-factor: base-pair substitutions
Species / strain / cell type:
S. typhimurium TA 102
Details on mammalian cell type (if applicable):
TA 102
his G 428 (PAQl); rfa-; R-factor: base-pair substitutions
Species / strain / cell type:
other: S. typhimurium TA 97a
Details on mammalian cell type (if applicable):
TA 97a:
his D661O; rfa-; uvrB-' R-factor : frame shift mutations
Metabolic activation:
with and without
Metabolic activation system:
S9 Mix
Test concentrations with justification for top dose:
Experiment I:
3.16,10.0,31.6,100,316.2,1000,2500 and 5000 µg/plate
Experiment II:
3.16,10.0,31.6,100,316.2,1000 and 2500 µg/plate,
additionally 1.0 µg/plate was evaluated for TA 102
Vehicle / solvent:
DMSO
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
For strains TA 1535, TA 100 without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylene-diamine,4-NOPD
Remarks:
For strains TA 97a, TA 98 without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
For strain TA 102 without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
For all strains with metabolic activation
Details on test system and experimental conditions:
The toxicity of the test item was determined with strains TA 98 and TA 100 in a pre-experiment. 8 concentrations were tested for toxicity and induction of mutations with 3 plates each. The ex-perimental conditions in this pre-experiment were the same as described below for the main ex-periment I (plate incorporation test). Toxicity may be detected by a reduction in the number of revertants, a clearing or diminution of the background lawn or by the degree of survival of treated cultures. The test item was tested in the pre-experiment at the following concentrations:
3.16,10.0,31.6,100,316.2,1000,2500 and 5000 µg/plate
For the pre-incubation method 100 µl of the test item solution was preincubated with the tester strains (100 µl ) and sterile buffer or the
metabolic activation system (500 µl) for 60 minutes at 37°C prior to adding the overlay agar (2000 µl ) and pouring onto the surface of a minimal agar plate. For each strain and dose level, including the controls, three plates were used.
After solidification the plates were inverted and incubated at 37 °C for atleast 48 h in the dark.
For the pre-incubation method 100 µl of the test item solution was preincubated with the tester strains (100 µl ) and sterile buffer or the metabolic activation system (500 µl) for 60 minutes at 37°C prior to adding the overlay agar (2000 µl ) and pouring onto the surface of a minimal agar plate. For each strain and dose level, including the controls, three plates were used. After solidification the plates were inverted and incubated at 37 °C for at least 48 h in the dark.
Evaluation criteria:
A test is considered acceptable if for each strain:
- the bacteria demonstrate their typical responses to crystal violet and ampicillin
- the control plates without S9 mix are within the following ranges (range of spontaneous rever-sion frequencies - historical control - data
range):
TA 1535: 5-30
TA 97a: 72-199
TA 98: 18-63
TA 100: 79-197
TA 102: 173-396

- corresponding background growth on both negative control and test plates is observed.
- the positive controls show a distinct enhancement over the control plate.

A test item is considered as mutagenic if:
- a dose-related increase in the number of revertants occurs and/or a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.

A biologically relevant increase is described as follows:
- if in strains TA 97a, TA 100 and TA 102 the number of reversions is at least twice as high
- if in strains TA 1535 and TA 98 the number of reversions is at least three times higher as com-pared to the spontaneous reversion rate

According to the OECD guidelines, the biological relevance of the results will be the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary. A test item producing neither a dose related increase in the number of revertants nor a reproduci-ble biologically relevant positive response at any of the dose groups is considered non-mutagenic in this system.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Experiment1: >=1000 µg/plate, Experiment 2: >= 31.6 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Experiment1: >=1000 µg/plate, Experiment 2: >= 31.6 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Experiment1: >=1000 µg/plate, Experiment 2: >= 31.6 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Experiment1: >=1000 µg/plate, Experiment 2: >= 31.6 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
other: S.typhimurium TA 97a
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Experiment1: >=1000 µg/plate, Experiment 2: >= 31.6 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Np precipitaion was observed.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The test matreial is not mutagenic under the conditions used.
Executive summary:

In an Ames Test according to OECD guideline 471 the test item has been tested with the Salmonella typhimurium TA 1535, TA 97a, TA 98, TA 100, TA 102 with and without metabolic activation (S9- mix) with the pre-incubation method at the following concentrations:

Experiment I: 3.16,10.0,31.6,100,316.2,1000,2500 and 5000 µg/plate

Experiment II: 3.16,10.0,31.6,100,316.2,1000 and 2500 µg/plate, additionally 1.0 µg/plate was evaluated for TA 102

The negative controls worked as expected. The positive controls showed a distinct enhancement of revertants and thus demonstrated the sensitivity of the test system and the activity of the metabolic activation system. The test item was soluble in the vehicle/test medium without precipitation. The test matreial is not mutagenic under the conditions used.