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:
19 May - 2 June 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD and EC guidelines and according to GLP principles.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

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
Details on test material:
- Name of test material (as cited in study report): Aquapel® 203
- Substance type: yellow fatty liquid
- Physical state: liquid
- Analytical purity: 88.7%
- Lot/batch No.: G11AN059
- Expiration date of the lot/batch: 21 January 2010
- Stability under storage conditions: stable
- Storage condition of test material: at room temperature in the dark

Method

Target gene:
histidine in Salmonella
tryptophan in Escherichia Coli
Species / strainopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone
Species / strain:
E. coli WP2 uvr A
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:
Dose range finding: 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate (+/- S9)
Mutation assay: 33, 100, 333, 1000, 3330 µg/plate (+/- S9)
Vehicle:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: not indicated
Controlsopen allclose all
Solvent controls:
yes
Remarks:
ethanol
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
pos. control for TA1535 (-S9)

Migrated to IUCLID6: diluted in saline 5 µg/plate
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
pos. control for TA1537 (-S9)

Migrated to IUCLID6: diluted in water conc. 60 µg/plate
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
pos. control for TA98 (-S9)

Migrated to IUCLID6: diluted in DMSO conc. 10 µg/plate
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
pos. control for TA100 (-S9)

Migrated to IUCLID6: diluted in DMSO conc. 650 g/plate
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
pos. control for WP2uvrA

Migrated to IUCLID6: diluted in DMSO conc. 10 µg/plate
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene diluted in DMSO
Remarks:
Conc. 1 µg/plate for strains TA98 (5 & 10% S9) and TA100 (5% S9); Conc. 2.5 µ/plate for strains TA1535 (5 & 10% S9), TA1537 (5% S9) and TA100 (10% S9); Conc. 5 µg/plate for strain TA1537 (10% S9); Conc. 10 µg/plate for strain WP2uvrA (5 & 10% S9),
Details on test system and conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: At least five different doses (increasing with approximately half-log steps) of the test substance were tested in triplicate in each strain.

DETERMINATION OF CYTOTOXICITY
- Method: the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined.

OTHER EXAMINATIONS:
- Other: precipitation
Evaluation criteria:
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA 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 is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is greater than three (3) times the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one independently repeated experiment.
The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to precipitating concentrations
Vehicle controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to precipitating concentrations
Vehicle controls valid:
yes
Positive controls valid:
yes
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Aquapel® 203 precipitated on the plates at the top dose of 3330 μg/plate.


RANGE-FINDING/SCREENING STUDIES:
Aquapel® 203 was tested in the tester strains TA100 and WP2uvrA with concentrations of 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate in the absence and presence of S9-mix.
Precipitation of Aquapel® 203 on the plates was observed at the start and at the end of the incubation period at concentrations of 3330 and 5000 µg/plate.
No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed.
In the dose range finding test, no increase in the number of revertants was observed upon treatment with Aquapel® 203 under all conditions tested.


COMPARISON WITH HISTORICAL CONTROL DATA:
The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating 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

Based on the results of this study it is concluded that Aquapel® 203 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.