Registration Dossier

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Aluminum Ammonium Superphosphate (Batch No. 38771-55-3), a white powder stored at room temp in a dark, dry, closed container.

Method

Target gene:
Histidine synthesising
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:
S9-Mix
Test concentrations with justification for top dose:
Preliminary test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Mutation test 1&2: 50, 150, 500, 1500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test material formed the best doseable suspension in DMSO, therefore, this solvent was selected as the vehicle
Controls
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: N-ethyl-N-nitro-N-nitrosoguanidine, 9-aminoacridine, 4-nitroquinoline-N-oxide, 2-Aminoanthracene, benzo(a)pyrene
Details on test system and experimental conditions:
Experiment 1:
Measured aliquots (0.1 ml) of one of the bacterial cultures were dispensed into sets of test tubes followed by 2.0 ml of molten, trace histidine or tryptophan supplemented, top agar, 0.1 ml of the test material formulation, vehicle or positive control and either 0.5 ml of S9-mix or phosphate buffer. The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test material both with and without S9-mix
All of the plates were incubated at 37°C for approximately 48 hours and the frequency of revertant colonies assessed using a Domino colony counter.
Experiment 2:
The second experiment was performed using fresh bacterial cultures, test material and control solutions. The test material dose range was the same as Experiment 1 (50 to 5000 µg/plate).
The test material formulations and vehicle control were dosed using the pre-incubation method as follows:
Measured aliquots (0.1 ml) of one of the bacterial cultures were dispensed into sets of test tubes followed by 0.5 ml of S9-mix or phosphate buffer and 0.1 ml of the vehicle or test material formulation and incubated for 20 minutes at 37°C prior to the addition of 2.0 ml of molten, trace histidine or tryptophan supplemented, top agar. The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test material both with and without S9-mix
The positive and untreated controls were dosed using the standard plate incorporation method described in Experiment 1.
Evaluation criteria:
There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response.
A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

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:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
During the preliminary toxicity test the test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA-). The test material formulation and S9-mix used in this experiment were both shown to be sterile.
No toxicological significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation or exposure method. Small, statistically significant increases in TA100 revertant colony frequency were observed (presence of S9-mix only) at 50 and 500 µg/plate, in the range-finding and main tests, respectively. These increases were considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant counts at each statistically significant dose level were within the in-house historical untreated/vehicle control range for the tester strain and the fold increases were less than two times the concurrent vehicle controls.
All the positive control chemicals used in the test induced marked increase in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The test material was considered to be non-mutagenic under the conditions of this test.