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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:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1989
Report Date:
1989

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
May 26, 1983
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535
Additional strain / cell type characteristics:
other: hisG46 base-pair substitution
Remarks:
Additional mutations: rfa, gal, chl, bio, uvrB
Species / strain / cell type:
S. typhimurium TA 1537
Additional strain / cell type characteristics:
other: hisC3076 frameshift
Remarks:
Additional mutations: rfa, gal, chl, bio, uvrB
Species / strain / cell type:
S. typhimurium TA 98
Additional strain / cell type characteristics:
other: hisD3052/R-factor frameshift
Remarks:
Additional mutations: rfa, gal, chl, bio, uvrB
Species / strain / cell type:
S. typhimurium TA 100
Additional strain / cell type characteristics:
other: hisG46/R-factor base-pair substitution
Remarks:
Additional mutations: rfa, gal, chl, bio, uvrB
Metabolic activation:
with and without
Metabolic activation system:
Rat microsomal fraction S9 mix
Test concentrations with justification for top dose:
-Experiment concentrations: 100, 333, 1000, 3330, 5000 µg/plate
Selection of an adequate range of doses was based on a preliminary toxicity test with strain TA100, both with and without S9-mix. Nine concentrations have been tested in duplicate for toxicity. The highest concentration of test substance used in the subsequent mutagenesis assay was that which gave a reduced survival on the non-selective plates. If no toxicity was observed, the highest dose level used in the mutagenesis assay was 5 mg/plate unless the test substance exhibited limited solubility or was not uniformly dispersible in the solvent of choice.
Vehicle / solvent:
Milli-Q water (Millipore Corp.,Bedford, Mass., USA)
Controlsopen allclose all
Untreated negative controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
other: daunomycine and methylmethanesulfonate
Remarks:
without metabolic activation
Untreated negative controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with metabolic activation
Details on test system and experimental conditions:
TEST SYSTEM
-Source: Dr. Bruce N. Ames, University of California at Berkeley, U.S.A. (1987)
-Storage: stock cultures of the four strains in liquid nitrogen (-196°C).
-Check: strains regularly checked for their histidine-requirement, crystal violet sensitivity, ampicillin resistance (TA98 and TA100), UV-sensitivity and the number of spontaneous revertants.

CELL CULTURE
-Preparation of bacterial cultures: samples of frozen stock cultures of bacteria transferred into enriched nutrient broth (Oxoid no. 2) and incubated in a shaking bath (37 °C, 150 spm), until the cultures reached an O.D. of 0.4 at 700 nm (109 cells/ml). Freshly grown cultures of each strain used for a test.
-Agar plates: 0 9 cm, contained 25 ml glucose agar medium. Glucose agar medium contained per liter: 18 g purified agar (Oxoid, code L28) in Vogel-Bonner Medium E (Acetylornithinase of Escherichia coli: partial purification and some properties. J. Biol. Chem. 218, 1956, 97-106), 10 g glucose, 0.5 mg biotin and 0.6 mg histidine.
-Top agar: Vogel-Bonner Medium E containing 0.6% (w/v) purified agar was heated to dissolve the agar. Samples of 3 ml top agar were transferred into 10 ml glass tubes with metal caps. Top agar tubes were autoclaved for 20 min at 120 °C. Prior to the viability determination the top agars were supplemented with 1.55 mg histidine per top agar.
-Environmental conditions: all incubations have been carried out in the dark at 37°C. The temperature was monitored during the experiment.

METABOLIC ACTIVATION SYSTEM
-Preparation of S9-homogenate: rat liver microsomal enzymes were routinely prepared from adult male Wistar or Sprague Dawley rats, which were obtained from Charles River Wiga, Sulzfeld, F.R.G. The animals were housed at RCC NOTOX in a special room under standard laboratory conditions. The rats were injected intraperitoneally with a solution (20% w/v) of Aroclor 1254 (500 mg/kg body weight) in corn oil. Five days later, they were killed by decapitation; (they were denied access to food for at least 12 hours preceding sacrifice). The livers of the rats were removed aseptically, and washed in cold (0°C) sterile 0.1 M sodium phosphate buffer (pH 7.4) containing 0.1 mM Na2-EDTA. Subsequently the livers were minced in a blender and homogenized in 3 volumes of phosphate buffer with a Potter homogenizer. The homogenate was centrifuged for 15 min at 9000 g. The supernatant (S9) was transferred into sterile ampules, which were stored in liquid nitrogen (-196 °C).
-Preparation of S9-mix: prepared immediately before use and kept on ice during the test. S9-mix contained per 10 ml: 30 mg NADP and 15.2 mg glucose-6-phosphate in 5.5 ml aqua bidest; 2 ml 0.5 M sodium phosphate buffer pH 7.4; 1 ml 0.08 M MgCl2 solution; 1 ml 0.33 M KCl solution, and 0.5 ml S9. The above solutions were mixed and filter (0.22 µm)-sterilised (apart from the S9-fraction, which was added after filter-sterilisation of the S9-mix components).
Evaluation criteria:
No formal hypothesis testing has been done.
A test substance was considered:
-negative (not mutagenic) in the Ames test if:
a) The total number of revertants in any tester strain at any concentration was not greater than two times the solvent control value, with or without metabolic activation.
b) The negative response should be reproducible in at least one independently repeated experiment.
-positive (mutagenic) in the Ames test if:
a) It induced at least a 2-fold, dose related increase in the number of revertants with respect to the number induced by the solvent control in any of the tester strains, either with or without metabolic activation. However, any mean plate count of less than 20 was considered to be not significant. If the test substance showed in the first test only a positive response at one or two concentrations, the assay was repeated with doses just below and exceeding those showing positive effects in the first test.
b) The positive response should be reproducible in at least one independently repeated experiment. The preceding criteria were not absolute and other extenuating factors might enter into the final evaluation decision.

Results and discussion

Test results
Key result
Species / strain:
S. typhimurium, other: TA1535, TA1537, TA98 and TA100 bacteria
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TOXICITY OF THE TEST SUBSTANCE
The survival of the TA100 culture is determined by comparing the number of colonies on the plates containing the test substance with those on the solvent control plate. Both in the absence and presence of S9-mix the survival of strain TA100 is not reduced up to test substance concentrations of 5000 µg/plate. Based on these data, the test substance was tested up to a concentration of 5000 µg/plate in the absence and presence of S9-mix.

THE AMES SALMONELLA/MICROSOME PLATE TEST
All bacterial strains showed negative responses over the entire dose range of the test substance, i.e. no dose-related, two-fold, increase in the number of revertants in two independently repeated experiments. The negative and strain-specific positive control values fell within laboratory background historical ranges, indicating that the test conditions were optimal and that the metabolic activation system functioned properly.

Applicant's summary and conclusion

Conclusions:
Based on the results of this study it is concluded that the test substance can be considered as not mutagenic in the Ames Salmonella/microsome assay.
Executive summary:

The test substance was tested for mutagenic effects with an in vitro bacterial reverse mutation assay, according to the OECD Guideline 471 (1983). The test was performing without and with metabolic activation in the range of concentration of 100 - 5000 μg/plate, using strains of Salmonella typhimurium (TA 98, TA 100, TA 1535 and TA 1537). Each concentration and controls were tested in triplicate. The test substance did not induce a dose-related increase in the number of revertant (His+) colonies in each of the tester strains. These results were confirmed in an independently repeated experiment. The test substance can, therefore, be considered as not mutagenic in this test system.