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Toxicological information

Genetic toxicity: in vitro

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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:
2002-11-07 to 2002-11-29
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted July 21, 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
adopted May 19, 2000
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: N-acetylmorpholine

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver (S9)
Test concentrations with justification for top dose:
Pre-incubation test (Test I) and standard plate test (Test II), both (±S9):
0 (solvent control), 20, 100, 500, 2500, 5000 µg / plate
Vehicle / solvent:
Vehicle used: water
Controls
Untreated negative controls:
yes
Remarks:
(sterility controls)
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other:
Remarks:
-S9: N-methyl-N-nitro-N-nitrosoguanidine (TA 1535; TA 100), 9-Aminoacridine (TA 1537), 4-Nitro-o-phenylendiamin (TA 98), 4-Nitroquinoline-N-oxide (WP2 uvrA); +S9: 2-Aminoanthracene (all five strains)
Details on test system and experimental conditions:
METHOD OF APPLICATION: plate incorporation test (test I) and pre-incubation test (test II)

DURATION
- Preincubation period (test II): - 20 minutes
- Expression time (cells in growth medium): 48 - 72 hours

NUMBER OF REPLICATIONS: 3 plates per concentration and strain for each individual assay (plate incorporation (test I) and pre-incubation (test II).

DETERMINATION OF CYTOTOXICITY
- other:
decrease in the number of revertants clearing or diminution of the background lawn (= reduced his or trp background growth)
reduction in the titer of viable bacteria, for all test groups both with and without S-9 mix in all experiments.
Evaluation criteria:
The test chemical is considered positive in this assay if the following criteria are met:
- a dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S-9 mix or after adding a metabolizing system.
- a test substance is generally considered non-mutagenic in this test if:
The number of revertants for all testerstrains were within the historical negative control range under all experimental conditions in two experiments carried out independently of each other.
Statistics:
Only descriptive statistics performed (arithmetic mean and standard deviation)

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 nor precipitates, but tested up to recommended limit concentrations
Remarks:
A very slight decrease in the number of revertants was occasionally observed.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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 nor precipitates, but tested up to recommended limit concentrations
Remarks:
A very slight decrease in the number of revertants was occasionally observed.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

No precipitation of the test substance was found. A very slight decrease in the number of revertants was occasionally observed. An increase in the number of his- or trp- revertants was not observed in the standard plate test or in the preincubation test either without S-9 mix or after the addition of a metabolizing system. The number of revertant colonies in the negative controls was within the normal range of the historical control data for each tester strain. The sterility controls revealed no indication of bacterial contamination. The positive control articles both with and without S-9 mix induced a significant increase in the number of revertant colonies within the range of the historical control data or above. The titer of viable bacteria was ≥10E8 / mL

Applicant's summary and conclusion