Registration Dossier

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:
14.01.2008 to 03.03.2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Study report

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

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
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
The characteristics of the different Salmonella typhimurium strains were as follows:
Strain Histidine mutation Mutation type
TA 1537 hisC3076 Frameshift
TA 98 hisD3052/R-factor* Frameshift
TA 1535 hisG46 Base-pair substitutions
TA 100 hisG46/R-factor* Base-pair substitutions
*: R-factor = plasmid pKM101 (increases error-prone DNA repair)
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
In the dose range finding test, DCM was tested up to concentrations of 5000 IJg/plate in the absence and presence of 5% (v/v) 89-mix
in the strains TA100 and WP2uvrA. DCM did not precipitate on the plates at this dose level. The bacterial background lawn was not reduced at
any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. Results of this dose range finding
test were reported as part of the first experiment of the mutation assay.

Based on the results of the dose range finding test, DCM was tested in the first mutation assay at a concentration range of 1 00 to 5000 µg/plate
in the absence and presence of 5% (v/v) S9-mix in tester strains TA1535, TA1537 and TA98. The bacterial background lawn was not
reduced at any of the concentrations tested and no biologically relevant decrease in the number
of revertants was observed.

In an independent repeat of the assay with additional parameters, DCM was tested at the same concentration range as the first assay
in the absence and presence of 10% (v/v) S9-mix in tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. Toxicity was only
observed in tester strains TA1537 and TA100 in the presence of S9-mix.

Since more severe toxicity was observed in tester strain TA1537 and TA100 in the presence of 10% (v/v) S9-mix in comparison with the
other strains, a third mutation experiment was performed with these strains in the presence of 1 0% (v/v) S9-mix. DCM was tested at a
concentration range of 100 to 5000 µg/plate. Toxicity was again observed in both tester strains.

DCM did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains
(TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence
of S9-metabolic activation. These results were confirmed in independently repeated experiments.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Saline
True negative controls:
no
Positive controls:
yes
Remarks:
5 µg/plate
Positive control substance:
sodium azide
Remarks:
TA 1535 without metabolic activation (-S9 mix)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Milli-Q water
True negative controls:
no
Positive controls:
yes
Remarks:
60 µg/plate
Positive control substance:
9-aminoacridine
Remarks:
TA 1537 without metabolic activation (-S9 mix)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
10 µg/plate
Positive control substance:
2-nitrofluorene
Remarks:
TA 98 without metabolic activation (-S9 mix)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
650 µg/plate
Positive control substance:
methylmethanesulfonate
Remarks:
TA 100 without metabolic activation (-S9 mix)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
10 µg/plate
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
WP2uvrA without metabolic activation (-S9 mix)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
1 µg/plate
Positive control substance:
other: 2-aminoanthracene
Remarks:
TA 1535 with metabolic activation (+S9 mix)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
2.5 µg/plate and 5 µg/plate
Positive control substance:
other: 2-aminoanthracene
Remarks:
TA 1537 with metabolic activation (+S9 mix)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
1 µg/plate
Positive control substance:
other: 2-aminoanthracene
Remarks:
TA 98 with metabolic activation (+S9 mix)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
1 µg/plate and 2.5 µg/plate
Positive control substance:
other: 2-aminoanthracene
Remarks:
TA 100 with metabolic activation (+S9 mix)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
10 µg/plate
Positive control substance:
other: 2-aminoanthracene
Remarks:
WP2uvrA with metabolic activation (+S9 mix)
Details on test system and experimental conditions:
Test system: Salmonella typhimurium bacteria and Escherichia coli bacteria
Rationale: Recommended test system in international guidelines (e.g. OECD, EEC).
Source:
Salmonella typhimurium strains:
Trinova Biochem GmbH, Germany (Master culture from Dr. Bruce N. Ames):
TA100 received on 14-06-2006, used batch: TA100.210807
TA98 received on 14-06-2006, used batch: TA98.210807
TA1537 received on 14-06-2006, used batch: TA1537.210807
TA1535 received on 14-06-2006, used batch: TA1535.210807
Escherichia coli strain:
Trinova Biochem GmbH, Germany (Master culture from The National
Collections of Industrial and Marine Bacteria, Aberdeen, UK)
WP2uvrA received on 14-06-2006, used batch: EC.0'70307

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
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
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 with metabolic activation
negative without metabolic activation

All bacterial strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of
revertants in two independently repeated experiments.
The negative and strain-specific positive control values were within our laboratory historical control data ranges indicating that
the test conditions were adequate and that the metabolic activation system functioned properly.
Based on the results of this study it is concluded that DCM is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the
Escherichia coli reverse mutation assay.