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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:
07-NOV-2008 to 20-NOV-2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report date:
2008

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial forward mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
2-[2-(carboxymethyl)phenoxy]-3,4-dimethylbenzoic acid
EC Number:
601-485-7
Cas Number:
117570-93-1
Molecular formula:
C17 H16 O5
IUPAC Name:
2-[2-(carboxymethyl)phenoxy]-3,4-dimethylbenzoic acid
Details on test material:
- Name of test material (as cited in study report): ASA404 C7
- Substance type: Off white powder
- Physical state: Solid
- Analytical purity: 99.9%
- Lot/batch No.: 072301
- Expiration date of the lot/batch: 18 March 2009
- Stability under test conditions: Stable
- Storage condition of test material: At room temperature in the dark

Method

Target gene:
Histidine
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535
Species / strain / cell type:
S. typhimurium TA 97
Species / strain / cell type:
S. typhimurium TA 98
Species / strain / cell type:
S. typhimurium TA 100
Species / strain / cell type:
S. typhimurium TA 102
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone
Test concentrations with justification for top dose:
Main 1
TA100: 3, 10, 33, 100, 333, 3330 and 5000 µg/plate
TA1535, TA97, TA98 and TA102: 100, 333, 3330 and 5000 µg/plate
Main 2:
All strains: 100, 333, 3330 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: dimethyl sulfoxide
- Justification for choice of solvent/vehicle: Accepted and approved by authorities and international guidelines
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cumene hydroperoxide
Remarks:
without S9

Migrated to IUCLID6: 0.1 µg/plate in DMSO for TA102
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9

Migrated to IUCLID6: 650 µg/plate in DMSO for TA100
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without S9

Migrated to IUCLID6: 5 µg/plate in saline for TA1535
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
without S9

Migrated to IUCLID6: 10 µg/plate in DMSO for TA98
Positive controls:
yes
Positive control substance:
other: ICR-191, 1 µg/plate in DMSO for TA97
Remarks:
without S9
Positive controls:
no
Positive control substance:
other: 2-aminoanthracene, TA1535, TA97, TA100, TA98
Remarks:
with S9
Positive controls:
yes
Positive control substance:
other: 1,8 dihydroxyanthraquinone , 30 µg/plate in DMSO for TA102
Remarks:
with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.

NUMBER OF CELLS EVALUATED: 10E8 bacteria per plate

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, and the reduction of the revertant colonies

OTHER EXAMINATION
- Precipitation of the test substance

Evaluation criteria:
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100, TA97 and TA102 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, and TA98 is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one independently repeated experiment.



A test substance is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100, TA97 and TA102 is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535 and, TA98 is greater than three (3) times the concurrent control.
b) In case a positive response will be repeated, the positive response should be reproducible in at least one independently repeated experiment.
Statistics:
No statistics are performed

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 97
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation on the plates was observed at the start and at the end of the incubation period at the concentration of 5000 µg/plate.


COMPARISON WITH HISTORICAL CONTROL DATA: The results of solvent and positive controls were considered valid and it can be concluded that the test conditions were adequate and that the metabolic activation system functioned properly.


Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

It is concluded that this test is valid and that ASA404 C7 is not mutagenic in the Salmonella typhimurium reverse mutation assay

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