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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:
22 March, 2010 to 14 May, 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted according to OECD Guideline 471, EC 440/2008 B.13/14 and EPA OPPTS 870.5100 and with GLP compliance

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
not specified
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
Salmonella typhimurium
Species / strainopen allclose all
Species / strain / cell type:
other: TA 100, TA 1535
Details on mammalian cell type (if applicable):
Not applicable
Species / strain / cell type:
other: TA 98, TA 1537
Details on mammalian cell type (if applicable):
Not applicable
Species / strain / cell type:
S. typhimurium TA 102
Details on mammalian cell type (if applicable):
Not applicable
Species / strain / cell type:
other: TA 98, TA 100, TA 102, TA 1535, TA 1537
Details on mammalian cell type (if applicable):
Not applicable
Metabolic activation:
without
Metabolic activation system:
Not applicable
Test concentrations with justification for top dose:
Experiment 1:
- 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 2500 μg/plate
TA 98, TA 100, TA 1535, TA 102 with and without metabolic activation; TA 1537 with metabolic activation

- 0.3.6, 1.0, 3.16, 10.0, 31.6, 100, 316, 1000 and 2500 μg/plate
TA 1537 without metabolic activation.

Experiment 2:
- 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 2500 μg/plate
TA 98, TA 100, TA 102 with and without metabolic activation; TA 1535 and TA 1537 with metabolic activation.

- 0.1, 0.316, 1.0, 3.16, 10.0, 31.6, 100, 316 and 1000 μg/plate
TA 1535 without metabolic activation

- 0.316, 1.0, 3.16, 10.0, 31.6, 100, 316 and 1000 μg/plate
TA 1537 without metabolic activation
Vehicle / solvent:
TA 100, TA 1535: distilled water
TA 98, TA 1537: DMSO
TA 102: distilled water
TA 98, TA 100, TA 102, TA 1535, TA 1537: DMSO

- Justification for choice of solvent/vehicle: The solvent was compatible with the survival of the bacteria and the S9 activity
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA 100 and TA 1535
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylene-diamine (4-NOPD)
Remarks:
TA 98 and TA1537
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
TA 102
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (2-AA)
Remarks:
TA98, TA 100, TA 1535, TA 1537 and TA 102 (with metabolic activation)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: 1 h (experiment 2)
- Exposure duration: 48 h
- Expression time (cells in growth medium): 48 h
- Selection time (if incubation with a selection agent): Not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): Not applicable

SELECTION AGENT (mutation assays): Not applicable
SPINDLE INHIBITOR (cytogenetic assays): Not applicable
STAIN (for cytogenetic assays): Not applicable

NUMBER OF REPLICATIONS: Triplicate

NUMBER OF CELLS EVALUATED: Not applicable

DETERMINATION OF CYTOTOXICITY
- Method: By detecting clear or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control.

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

A biologically relevant increase is described as follows:
- if a tester strains TA 100 and TA 102 the number of reversions is at least twice as high
- if in tester strains TA 98, TA 1535 and TA 1537 the number of reversions is at least three times higher
as compared to the reversion rate of the solvent control.
Statistics:
According to the OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results in not regarded as neccesary.

A test substance producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.

Results and discussion

Test results
Species / strain:
other: TA 98, TA 100, TA 102, TA 1535, TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Precipitation of the test item was observed in all tester strains used in experiment 1 and 2 with and without metabolic activation.

Toxic effects of the test item was observed in all tester strains used in experiment 1 and 2 (with and without metabolic activation).

The reduction of in the number of revertants down to a mutation factor of 0.5 found in experiment 2 in tester strain TA 1535 at a dose of 0.1 μg/plate (without metabolic activation) was regarded as not biologically relevant due to lack of a dose-response relationship.

No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with the test substance at any concentration level, neither in the presence nor absence of metabolic activation in experiment 1 and 2.

The reference mutagens induced a distinct increase of revertant colonies indicating the validity of the experiments.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Under the experimental conditions, it can be stated that the substance does not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used. The test substance is therefore not considered as mutagenic in this bacterial mutation assay.
Executive summary:

An Ames test was conducted with the following tester strains: Salmonella typhimurium: TA 98, TA 100, TA 102, TA 1535 and TA 1537. In two independent experiments, several concentrations of NPEO were used. Each assay was conducted with and without metabolic activation. The concentrations, including the controls (negative and/or solvent as well as positive control), were tested in triplicateup to 2,500 µg/plate. Precipitation of the test substance was observed in all tester strains used in experiments 1 and 2 (with and without metabolic activation). Toxic effects were noted in all tester strains used in the experiments. No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with the test substance at any concentration level, neither in the presence nor absence of metabolic activation in experiments 1 and 2. The reference mutagens induced a distinct increase of revertant colonies indicating the validity of the experiments. Under the experimental conditions, NPEO did not cause gene mutations by base pair changes or frame shifts in the genome of the tester strains used. NPEO was considered not mutagenic in this bacterial mutation assay (Wallner B, 2010).