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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From June 13th to July 07th, 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted on 21st July 1997
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Qualifier:
according to
Guideline:
other: Notification No. 700 of the Environmental Agency, No. 1039 ofthe Ministry of Health and Welfare and No. 1014 of Ministry of International Trade and Industry, 9th December 1986.
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 / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell type (if applicable):
- Supplier: B.N. Ames Laboratory (University of Califomia, Berkeley, USA).
- Storage: in a cryoprotective medium (1 ml nutrient broth and 0.09 ml dimethylsulfoxide) in liquid nitrogen.
- Inoculumn: the day before treatment, cultures were inoculated from frozen permanents; a scrape was taken under sterile conditions and put into approximately 6 ml of nutrient broth. The nutrient broth was then placed under agitation in an incubator at 37 °C for about 14 hours, to produce bacterial suspensions.
- Histidine check: each strain derived from Salmonella typhimurium LT 2 contains one mutation in the histidine operon, resulting in a requirement for histidine.
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
- Supplier: S. Venitt's Laboratory (ICR, Sutton, England).
- Storage: stored in a cryoprotective medium (1 ml nutrient broth and 0.09 ml dimethylsulfoxide) in liquid nitrogen.
- Inoculumn: the day before treatment, cultures were inoculated from frozen permanents: a scrape was taken under sterile conditions and put into approximately 6 ml of nutrient broth. The nutrient broth was then placed under agitation in an incubator at 37 °C for about 14 hours, to produce bacterial suspensions.
- Tryptophan check: the strain of Escherichia coli contains one mutation in the tryptophan operon, resulting in a requirement for tryptophan.
Metabolic activation:
with and without
Metabolic activation system:
liver microsomal fraction (S9 fraction)
Test concentrations with justification for top dose:
PRELIMINARY TOXICITY TEST: 10, 100, 500, 1000,2500 and 5000 μg/plate.
MUTAGENICITY EXPERIMENTS: 312.5, 625, 1250, 2500 and 5000 µg/plate.
Vehicle / solvent:
- Vehicle: water for injections
- Formulation: the test item was dissolved in the vehicle at a concentration of 50 mg/ml for the preliminary toxicity test and for both mutagenicity experiments.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
water for injections
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
mitomycin C
other:
Details on test system and experimental conditions:
TREATMENT
The test item was tested in a preliminary test and two mutagenicity experiments. The preliminary test, both experiments without S9 mix and the first experiment with S9 mix were performed according to the direct plate incorporation method. The second experiment with S9 mix was performed according to the preincubation method. The direct plate incorporation method was performed as follows:
test item solution (0.1 ml), S9 mix when required or phosphate buffer pH 7.4 (0.5 ml) and bacterial suspension (0.1 ml) were mixed with 2 ml of overlay agar (containing traces of the relevant aminoacid and biotin and maintained at 45 °C). After rapid homogenization, the mixture was overlaid onto a Petri plate containing minimum medium.
The preincubation method was performed as follows: test item solution (0.1 ml), S9 mix (0.5 ml) and the bacterial suspension (0.1 ml) were incubated for 60 minutes at 37 °C under shaking before adding the overlay agar and pouring onto the surface of a minimum agar plate. After 48 to 72 hours of
incubation at 37 °C, revertants were scored with an automatic counter (Cardinal counter. Perceptive Instruments, Suffolk CB9 7 BN, UK).

PRELIMINARY TOXICITY TEST
To assess the toxicity of the test item to the bacteria, six dose-levels (one plate/dose-level) were tested in the TA 98, TA 100, TA 102 and WP2 uvrA strains, with and witiiout S9 mix. The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn. Since the test item was freely soluble and non-toxic in the preliminary test, the highest dose-level was 5000 µg/plate, according to the criteria specified in the intemational guidelines.

MUTAGENICITY EXPERIMENTS
In two independent experiments, using three plates/dose-level, each strain was tested, with and without S9 mix, with:
- at least five dose-levels of the test item,
- the vehicle control,
- the appropriate positive control.
The sterility of the S9 mix was checked before the beginning and at the end of each experiment and was found to be satisfactory.

METABOLIC ACTIVATION SYSTEM
The S9 mix consists of induced enzymatic systems contained in rat liver post-mitochondrial firaction (S9 fraction) and the cofactors necessary for their function. S9 firaction was purchased from Moltox (Molecular Toxicology, INC, USA) and obtained from the liver of rats treated with Aroclor 1254 (500 mg/kg) by the intraperitoneal route. The S9 fraction was preserved in sterile tubes at -80 °C, until use.
The S9 mix was prepared at 4 °C immediately before use and maintained at this temperature until added to the overlay agar.
The composition of S9 mix was as follows: glucose-6-phosphate 5 mM, NADP 4 mM, KCl 33 mM, MgCl2 8mM, sodium phosphate buffer pH 7.4 100 mM, S9 fraction (protein concentrations: 43 and 38.8 mg/ml, respectively) 10 % (v/v) and water to volume.

ACCEPTANCE CRITERIA
This study is considered valid if the following criteria are fully met:
- the number of revertants in the vehicle controls is consistent with the historical data of the testing facility,
- the number of revertants in the positive
Evaluation criteria:
A reproducible 2-fold increase (for the TA 98, TA 100, TA 102 and WP2 uvrA strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive result. Reference to historical data, or other considerations of biological relevance may also be taken into account in the evaluation of the data obtained.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
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
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
MAIN TEST
No toxicity was noted towards all the strains used, both with and without S9 mix.
The test item did not induce any noteworthy increase in the number of revertants, both with and without S9 mix, in any of the six strains.

CONTROLS
The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid.

PRELIMINARY TOXICITY TEST
The test item was freely soluble in the vehicle (water for injections) at 50 mg/ml.
Consequently, with a treatment volume of 100 µl/plate, the dose-levels were 10, 100, 500, 1000, 2500 and 5000 µg/plate. No precipitate was observed in the Petri plates when scoring the revertants at all dose-levels. A yellow coloration was noted in the plates when scoring the revertants at dose-levels > 500 µg/plate. No noteworthy toxicity was noted towards the four strains used, with and without S9 mix.

Applicant's summary and conclusion

Conclusions:
Under the experimental conditions, the test item did not show mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium and Escherichia coli.
Executive summary:

Method

The substance was tested for mutagenic effects in vitro in Salmonella typhimurium and Escherichia coli strains. Five strains of bacteria Salmonella typhimurium: TA 1535, TA 1537, TA98, TA100 and TA 102 and one strain of Escherichia coli WP2 uvrA were used. Each strain was exposed to five dose-levels of the test item (three plates/dose-level). After 48 to 72 hours of incubation at 37 °C, the revertant colonies were scored. The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

Results

The number of revertants for the vehicle and positive controls was in the acceptance criteria. The study was therefore considered valid. Since the test item was freely soluble and non-toxic in the preliminary test, the highest dose-level was 5000 μg/plate, according to the criteria specified in the intemational guidelines. The selected treatment-levels were: 312.5, 625, 1250, 2500 and 5000 μg/plate, for both mutagenicity experiments with and without S9 mix. No toxicity was noted towards all the strains used, both with and without S9 mix. The test item did not induce any noteworthy increase in the number of revertants, both with and without S9 mix, in any of the six strains.

Under the experimental conditions, the test item did not show mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium and Escherichia coli.