Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
other: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From July 21 to September 27, 1993
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
read-across from supporting substance (structural analogue or surrogate)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1993
Report Date:
1993

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
, statistical analysis was not performed
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
20.5761, 61.7284 , 185.1852 ,555.5556 ,1666.6667 ,5000.0000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used:DMSO
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
mitomycin C
Remarks:
without S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
other: 2-aminoanthracene
Remarks:
with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
Inoculates from frozen master copies were set up monthly. They were grown in liquid NB-medium overnight and then plated on NB-agar. After incubation, single colonies were taken from the plates, grown overnight in liquid NB-medium and then used for the experiment.

SELECTION AGENT (mutation assays):Colonies were counted electronically with an Artek counter. The results were sent on line to a computer. They were checked on a random basis by the operator.

Control of the genotype of the strains
The characteristics of the strains were checked monthly. Histidine-auxotrophy of the strains was demonstrated by the requirement for 1-histidine. The presence of the rfa character was assayed by the sensitivity for crystal-violet. The deletion of the uvrB gene (strains TA 98, TA 100, TA 1535 and TA 1537) was demonstrated by the sensitivity for UV-light. The Salmonella strains containing the R-factor (TA 98 and TA 100) were additionally checked for ampicillin resistance. Strain TA 102 additionally was checked for tetracycline resistence (presence of multicopy plasmid pAQ1). The presence of the uvr+ gene was demonstrated by the resistence of strain TA 102 against UV-light. Furthermore, all strains were checked for their characteristic
reversion properties with known mutagens (positive controls).
Evaluation criteria:
VALIDITY CRITERIA:
A test is considered acceptable if the mean colony counts of the control values of all strains are within the acceptable ranges and if the results of the positive controls meet the criteria for a positive response. In either case the final decision is based on the scientific judgement of the Study Director.
CRITERIA FOR POSITIVE RESPONSE
The test substance is considered to be mutagenic in this test system if the following conditions are met: At least a reproducible meaningful increase of the mean number of revertants per plate above that of the negative control at any concentration for one or more of the following strains:
S. typhimurium TA 98, TA 100, TA 102, TA 1535 and TA 1537. Generally a concentration-related effect should be demonstrable.
Statistics:
In deviation to the OECD guideline a statistical analysis was not performed. At present the use of statistical methods concerning this particular test system is not generally recommended. No appropriate statistical method is available

Results and discussion

Test 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:
not determined
Vehicle controls validity:
not examined
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Non mutagenic
Executive summary:

In the original experiment carried out without and with metabolic activation, none of the tested concentrations of test item led to an increase in the incidence of histidine-prototrophic mutants by comparison with the negative control.

In the confirmatory experiment performed without and with metabolic activation, again, none of the tested concentrations of test item led to an increase in the incidence of histidine-prototrophic mutants by comparison with the negative control.

Based on the results of these experiments and on standard evaluation criteria, it is concluded that the test substance and its metabolites did not induce gene mutations in the strains of S. typhimurium used.