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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report Date:
1998

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes
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): N,N-Di-(hydroxyethyl)-anilin
- Batch No.: 59-7325
- Purity: 97.7%
- Appearance, consistency: brown, solid
- Storage: room temperature

Method

Target gene:
- S. typhimurium: His-locus
- E. coli: Trp-locus
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Liver S9 fraction of Aroclor 1254 induced rats
Test concentrations with justification for top dose:
- 1st experiment (standard plate test, all strains): 20, 100, 500, 2500, 5000 µg/plate
- 2nd experiment (preincubation test, all stains): 4, 20, 100, 500, 1000 µg/plate
- 3rd experiment (standard plate test, TA 98 and TA 100): 20, 100, 500, 100, 2000 µg/plate
- 4th experiment (standard plate test, TA 98): 750, 1000, 1500, 2000, 2500 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
other: 2-aminoanthracene (2AA); aniline/o-toluidine; N-methyl-N'-nitro-N-nitrosoguanidine (MNNG); 4-nitro-o-phenylendiamine (NOPD)
Remarks:
With metabolic activation: 2AA (all strains); aniline/o-toluidine (tests with norharman); Without metabolic activation: MNNG (TA 100, TA1535); NOPD (TA 98); 9-aminoacridine (TA 1537); N-ethyl-N'-nitro-N-nitrosoguanidin (E.Coli)
Details on test system and experimental conditions:
-> 1st EXPERIMENT:
METHOD OF APPLICATION: in agar (plate incorporation)
DURATION: exposure duration: 48-72 hours
STRAINS TESTED: TA 1535, TA 1537, TA 98, TA 100, E.coli WP2

-> 2nd EXPERIMENT:
METHOD OF APPLICATION: preincubation
DURATION:
- Preincubation period: 20 minutes
- Exposure duration: 48-72 hours
STRAINS TESTED: TA 1535, TA 1537, TA 98, TA 100, E.coli WP2

-> 3rd EXPERIMENT:
METHOD OF APPLICATION: in agar (plate incorporation)
DURATION: exposure duration: 48-72 hours
STRAINS TESTED: TA 98, TA 100
OTHER: test with S9 mix incl. norharman

-> 3rd EXPERIMENT:
METHOD OF APPLICATION: in agar (plate incorporation)
DURATION: exposure duration: 48-72 hours
STRAINS TESTED: TA 98
OTHER: test with S9 mix incl. norharman

NUMBER OF REPLICATIONS: 3 test plates per dose or per control

DETERMINATION OF CYTOTOXICITY: Reduced his- background growth, decrease in the number of his revertants)
Evaluation criteria:
In general, a substance to be characterized as positive in the bacterial tests has to fulfil the following requirements:
- doubling of the spontaneous mutation rate (control)
- dose-response relationship
- reproducibility of the results

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:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
ADDITIONAL INFORMATION ON GENOTOXICITY
An increase in the number of his+ or trp+ revertants was not observed both in the routine standard plate test and in the preincubation test either without S9 mix or after the addition of a metabolizing system. However, after the addition of norharman there was a slight but dose-dependent increase in the number of mutant colonies from about 750 - 1500 µg/plate onward up to 2000 - 2500 µg/plate (factor 2.1 - 2.8) using the strain TA 98.

TEST-SPECIFIC CONFOUNDING FACTORS
Test substance precipitation was found from about 2500 µg/plate onward

CYTOTOXICIY
A bacteriotoxic effect (reduced his- background growth, decrease in the number of his revertants) was observed in the standard plate test depending on the strain and test conditions at doses ≥ 500 µg/plate.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results: negative