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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance N,N-Di-(hydroxyethyl)-anilin was tested for its mutagenic potential based on the ability to induce back mutations in selected loci of several bacterial strains in the Ames test and in the Escherichia coli -reverse mutation assay. Test strains were TA 1535, TA 100, TA 1537, TA 98 and E.coli WP2 uvrA. The dose range was 20 µg - 5000 µg/plate (standard plate test) and 4 µg - 1000 µg/plate (preincubation test). A precipitation of the test substance was found from about 2500 µg/plate onward. A bacteriotoxic effect was observed in the standard plate test at doses >= 500 µg/plate. 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 S-9 mix or after the addition of a metabolizing system. According to the results of the present study, the test substance N,N-Di-(hydroxyethyl)-anilin is not mutagenic in the Ames test.

Link to relevant study records
Reference
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other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment
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
Target gene:
- S. typhimurium: His-locus
- E. coli: Trp-locus
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
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
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'.
Conclusions:
Interpretation of results: negative
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Justification for classification or non-classification

Based on the available studies data on genetic toxicity, the test item is no subject to classification and labelling according to Directive 67/548/EEC (DSD) and Regulation (EC) No 1272/2008 (CLP).