Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March - April 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Method

Target gene:
Histidine
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell type (if applicable):
N/A
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
10% S9: S9-mix from the livers of male Sprague Dawley rats
Test concentrations with justification for top dose:
In order to choose the range of concentraion to be tested, the bacteriostatic activity of the test substance is evaluated.
Preliminary cytotoxicity testing study: 0; 50;150; 500; 1500; 5000 μg/plate, with and without S9-mix in
TA 100 strain
Main study - Direct plate incorporation method: 0; 50;150; 500; 1500; 5000 μg/plate, with and without
S9-mix in TA 1535, TA 1537, TA 98, TA 100, TA 102
Confirmatory assay - pre-incubation method: 0; 50;150; 500; 1500; 5000 μg/plate, with and without
S9-mix in TA 1535, TA 1537, TA 98, TA 100, TA 102
Vehicle / solvent:
Apyrogen physiological serum (NaCl 0.9% (w/v)
Controls
Untreated negative controls:
yes
Remarks:
absolute negative control: spontaneous reversion rate
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
mitomycin C
other: 2-Anthramine (without S9)
Details on test system and experimental conditions:
SOURCE OF TEST SYSTEM
- Bacteria used in the test was obtained from Unité de Programmation moléculaire et toxicologie génétiuqe CNRS UA 144 (Institut Pasteur)
METHOD OF APPLICATION: in agar (plate incorporation); preincubation
DURATION
- Preincubation period: 37 °C for 20 minutes
- Exposure duration: Plates were incubated at 37 ± 1 °C for 48-72 h
NUMBER OF REPLICATIONS: Triplicate plates per dose level
Evaluation criteria:
The result of the test is considered as negative if the revertant number is below three fold the number of spontaneous reversions for TA 1535 and TA 1537 strains, and below two fold the number of spontaneous reversions fo TA 98, TA 100 and TA 102 strains with and/or without metabolic activation.
The validity criteria are:
- bacteriostatic activity of the highest concentration shall be equal to or less than 75%
- the spontaneous reversion rate of the absolute negative control shall comply with the laboratory's historical control data.
- the mean number of revertant colonies obtained for each strain and the corresponding positive control, with and/or without metabolic activation shall comply with laboratory's historical control data
The result of the test is considered positive if a concentration- related increase is obtained in one, or several of the 5 strains, with and/or without metabolic activation; a mutagenic effect is taken into acount for a given concentration of the test substance if the number of revertant colonies is at least two fold that of spontaneous revertant colonies number for TA 98, TA 100 and TA102 and 3 fold for TA1535 and TA 1537.
All results must be confirmed in an independent experiment

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
The test substance does not induce any mutagenic change in Salmonella typhimurium TA 1535, TA1537, TA 98, TA 100 and TA 102, without or with metabolic activation according to the OECD Guidelines n" 471and to the method B13/B14 of the directive 2000/32/EC.
Executive summary:

There is no significant difference between the number of spontaneous reversions, the number of reversions obtained in the positive controls (without and with metabolic activation), and the mean of corresponding experimental historic values obtained in the laboratory.

There is no evidence of any increase in the number of revertant colonies in the presence of the test substance (5 000, 1 500, 500, 150 and 50 pg/plate) without and with metabolic activation f'or bacterial strains in salmonella typhimurium TA 1535, TA1537, TA 98, TA 100 and TA 102.

Results were confirmed in a second independent experiment.

The test substance does not induce any mutagenic change in Salmonella typhimurium TA 1535,TA1537, TA 98, TA 100 and TA 102, without or with metabolic activation according to the OECD Guidelines n" 471and to the method B13/B14 of the directive 2000/32/EC.