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:
July 17, 2001-August 23, 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study has been performed according to OECD and GLP principles. No concentration was tested without precipitate and the independent repeat has no modification of study parameters. These deviations are considered not to influence the reliability of the study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Qualifier:
according to
Guideline:
other: ICH on Harmonisation of Technical requirements for registration of Pharmaceuticals for Human Use (1996 and 1997)
Principles of method if other than guideline:
- There was no concentration of test substance without precipitate.
- The independent repeat has no modification of study parameters.
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Physical state: dark blue solid

Method

Target gene:
Histidine gene in S. typhimurium
Tryptophan gene in E. Coli
Species / strainopen allclose all
Species / strain / cell type:
E. coli WP2 uvr A
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9
Test concentrations with justification for top dose:
Preliminary test: 6.7, 10, 33, 67, 100, 333, 667, 1000 and 3333 µg/plate
First and second test: 15, 50, 150, 400, 1250 and 3750 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: tetrahydrofuran (THF)
- Justification for choice of solvent/vehicle: based on the compatibility with the target cells and the solubility of the test article.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
tetrahydrofuran (THF)
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
pos. control for TA1537 -S9

Migrated to IUCLID6: diluted in dimethyl sulfoxide (DMSO) Conc. 75 µg/plate
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
pos. control for TA98 -S9

Migrated to IUCLID6: diluted in dimethyl sulfoxide (DMSO) Conc. 1 µg/plate
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
pos. control for TA100, TA1535 -S9

Migrated to IUCLID6: diluted in water Conc. 1 µg/plate
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene, diluted in dimethyl sulfoxide (DMSO) Conc. 1 µg/plate for all Salmonella strains and Conc. 10 µg/plate for E. Coli strain.
Remarks:
pos. control for WP2 uvrA + S9 and all Salmonella strains + S9
Positive control substance:
methylmethanesulfonate
Remarks:
pos. control for WP2 uvrA -S9

Migrated to IUCLID6: diluted in dimethyl sulfoxide (DMSO) Conc. 1000 µg/plate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 to 72 hr

NUMBER OF REPLICATIONS: all dose levels and test article, vehicle controls and positive controls were plated in triplicate.


DETERMINATION OF CYTOTOXICITY
- Method: a reduction in the background lawn, reduction in revertant colonies


OTHER:precipitation of test substance
Evaluation criteria:
The following crtiteria mus be met for the mutagenicity assay to be considered valid.
All Salmonella tester strain cultures must demonstrate the presence of the deep rough mutation (rfa) and the deletion in the uvrB gene. Cultures of tester strains TA98 and TA100 must demonstrate the presence of the pKM101 plasmid R-factor. All WP2uvrA cultures must demonstrate the deletion in the uvrA gene. All cultures must demonstrate the charcateristic mean number of spontaneous revertants in the vehicle controls as follows (inclusive): TA98, 10-50; TA100, 80-240; TA1535, 5-45; TA1537, 3-21; WP2uvrA, 10-60. To ensure that appropriate numbers of bacteria are plated, tester strain culture titers must be greater than or equal to 0.3x10^9 cells/mL. The mean of each positive control must exhibit at least a three-fold increase in the number of revertants over the mean value of the respective vehicle control. A minimum of three non-toxic dose levels are required to evaluate assay data. A dose level is considered toxic if one or both of the following criteria are met:
1. a >50% reduction in the mean number of revertants per plate as compared to the mean vehicle control value. This reduction must be accompanied by an abrupt dose-dependent drop in the revertant count.
2. a reduction in the background lawn.

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 / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: a dose level of 3750 µg/plate is the maximum dose level that can be achieved based on the solubility of the test article and the maximum plating aliquot that can be used with tetrahydrofuran.
- Precipitation: Precipitate was observed at 15 µg/plate.

RANGE-FINDING/SCREENING STUDIES:
No toxicity and mutagenicity was observed up to concentrations of 3333 µg/plate


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

Under the conditions of this study, the test substance did not cause a positive mutagenic response in either the presence or absence of Aroclor-induced rat liver S9.