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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 28 JUN 2002 to 23 SEP 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2002

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
impurity
Type:
impurity
Test material form:
liquid
Details on test material:
Water added up to 100 %.

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: stock cultures in the bank of "Genetic Toxicology", Aventis Pharma Germany, ProTox, prepared from the original bacterial strains
- Methods for maintenance in cell culture if applicable:
- Modal number of chromosomes: approx. 37 °C in an incubator
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: stock cultures in the bank of "Genetic Toxicology", Aventis Pharma Germany, ProTox, prepared from the original bacterial strains
- Methods for maintenance in cell culture if applicable:
- Modal number of chromosomes: approx. 37 °C in an incubator
Metabolic activation:
with and without
Metabolic activation system:
induced rat liver S9 mix (induced with Aroclor 1254)
Test concentrations with justification for top dose:
plate incorporation test: 0, 119, 381, 1190, 3810 and 11905 µg/plate; corresponding to 0, 50, 160, 500, 1600 and 5000 µg active ingredient/plate
preincubation test: 0, 38.1, 119, 381, 1190, 3810 and 11905 µg/plate; corresponding to 0, 16, 50, 160, 500, 1600 and 5000 µg active ingredient/plate
Vehicle / solvent:
Deionized water
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:
for strain TA 100 and TA 1535, without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
for strain TA1537, without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
for strain TA 98, without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
for strain WP2uvrA, without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other:
Remarks:
for strain WP2uvrA, with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION:
Experiment 1) plate incorporation assay
Experiment 2) preincubation assay

DURATION
- Preincubation period: 37 °C for 1 h
- Exposure duration: 48 h at 37 °C

NUMBER OF REPLICATIONS: 3 replicates per concentration
Rationale for test conditions:
Test conditions as described in the guideline
Evaluation criteria:
Criteria for a valid assay
The assay is considered valid if the following criteria are met:
- the solvent control data are within the laboratory's normal control range for the spontaneous mutant frequency
- the positive controls induce increases in the mutation frequency which are significant and within the laboratory's normal range

Criteria for a positive response
A test compound is classified as mutagenic if it has either of the following effects:
a) it produces at least a 2-fold increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control at complete bacterial background lawn
b) it induces a dose-related increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control in at least two to three concentrations of the test compound at complete bacterial background lawn
If the test substance does not achieve either of the above criteria, it is considered to show no evidence of mutagenic activity in this system.
Statistics:
Arithmetic means and standard deviation of the counted colonies were calculated.

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:
>= 1600 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:
>= 1600 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
>= 1600 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:
>= 1600 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: results from experiment I and II

Any other information on results incl. tables

Toxic effects, evident as incomplete or no bacterial lawn, were observed at the following concentrations of active ingredient:

 

Strain

Experiment I [μg/

plate]

 

Experiment I [μg/

plate]

 

 

Without S9 mix

With S9 mix

Without S9 mix

With S9 mix

TA 100

> 1600

> 1600

> 1600

5000

TA 1535

> 1600

5000

> 1600

5000

TA 1537

> 1600

5000

> 1600

5000

TA 98

> 1600

5000

> 1600

5000

WP2uvrA

5000

5000

5000

5000

Applicant's summary and conclusion

Conclusions:
In a guideline study according to OECD TG 471 under GLP conditions, the test item showed no mutagenic activity in a plate incorporation as well as a preincubation experiment with and without metabolic activation.
Executive summary:

In a guideline study according to OECD TG 471 under GLP conditions mutagenic activity of the test item was investigated in Salmonella typhimurium strains TA 1535, TA 1537, TA98 and TA100 as well as Escherichia coli strain WP2 uvrA with and without metabolic activation at concentrations of 0, 50, 160, 500, 1600 and 5000 μg/plate (active ingredient). The first experiment was conducted as a plate-incorporation test, the second as a preincubation test.

The test item did not reveal any mutagenic activity under the conditions tested. The appropriate reference mutagenes showed distinct positive mutagenic effects.