Registration Dossier

Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

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
Study period:
2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study in compliance with guidelines

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Wheat, ext., hydrolyzed
EC Number:
309-358-5
EC Name:
Wheat, ext., hydrolyzed
Cas Number:
100209-50-5
Molecular formula:
UVCB substance, not applicable.
IUPAC Name:
Wheat, ext., hydrolyzed
Details on test material:
Identification: Wheat Solubles, Hydrolyzed
Description Light brown-yellow powder with lumps
Batch: Cargill WSH Batch A
Purity: treated as 100% pure
Stability under storage conditions: Stable

Method

Target gene:
The objective of this study was to evaluate the test substance for its ability to induce reverse mutations in a gene of histidine-requiring Salmonella typhimurium bacterial strains resulting histidine-independent strains, and in a gene of tryptophan-requiring Escherichia coil bacterial strain resulting in a tryptophan-independent strain.
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9-mix induced by a combination of phenobarbital and B-naphthoflavone
Test concentrations with justification for top dose:
Dose range finding test
Wheat Solubles, Hydrolyzed was tested in the tester strains TA100 and WP2uvrA with concentrations of 3, 10, 33, 100, 333, 1000, 3330 and 5000 pg/plate in the absence and presence of S9-mix.

Mutation assay, Experiment 1
Based on the results of the dose range finding test, Wheat Solubles, Hydrolyzed was tested up to the dose level of 3330 pg/plate in the absence and presence of 5% (v/v) S9-mix with the Salmonella typhimurium strains, TA1535, TA1537 and TA98.

Mutation assay, Experiment 2
To obtain more information about the possible mutagenicity of Wheat Solubles, Hydrolyzed, a second mutation experiment was performed in the absence of S9-mix and in the presence of 10% (v/v) S9-mix. Based on the results of the first mutation assay, Wheat Solubles, Hydrolyzed was tested up to the dose level of 3500 pg/plate in strains TA1535, TA1537, TA98, TA100 and WP2uvrA.
Vehicle / solvent:
Dimethyl sulfoxide
Controls
Untreated negative controls:
yes
Remarks:
Dimethyl sulfoxide
Negative solvent / vehicle controls:
yes
Remarks:
same as negative control
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: sodium azide, 9-aminoacridine, 2-nitrofluorene, methylmethanesulfonate, 4-nitroquinoline N-oxide
Details on test system and experimental conditions:
Dose range finding test
Selection of an adequate range of doses was based on a dose range finding test with the strains TA100 and WP2uvrA, both with and without 5% (vlv) S9-mix. Eight concentrations, 3, 10, 33, 100, 333, 1000, 3330 and 5000 pg/plate were tested in triplicate. This dose range finding test was reported as a part of the first experiment of the mutation assay. The highest concentration of Wheat Solubles, Hydrolyzed used in the subsequent mutation assay was the level at which the test substance exhibited limited solubility.

Mutation assay
At least five different doses (increasing with approximately half-log steps) of the test substance were tested in triplicate in each strain. In the first experiment Wheat Solubles, Hydrolyzed was tested both in the absence and presence of 5% (v/v) S9-mix in tester strains TA1 535, TA1 537, and TA98. In an independent repeat of the assay with additional parameters, the test substance was tested both in the absence and presence of 10% (v/v) S9-mix in all tester strains.
The negative control (vehicle) and relevant positive controls were concurrently tested in each strain in the presence and absence of S9-mix.
Top agar in top agar tubes was melted by heating to 45°C. The following solutions were successively added to 3 ml molten top agar: 0.1 ml of a fresh bacterial culture (10 cells/mi) of one of the tester strains, 0.2 ml of a dilution of the test substance in dimethyl sulfoxide and either 0.5 ml S9-mix (in case of activation assays) or 0.5 ml 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0 °C for 48 ± 4 h. After this period revertant colonies (histidine independent (His) for Salmonella typhimurium bacteria and tryptophan independent (Trp) for Escherichia coil were counted.
Evaluation criteria:
The revertant colonies (histidine independent or tryptophan independent) were counted manually if less than 40 colonies per plate were present. If more than 40 colonies were present, these were counted automatically with a Biocount 4000 Pro-S-colony counter. Plates with abundant test article precipitate which interfered with automated colony counting were counted manually and the evidence of test substance precipitate on the plates was recorded. The condition of the bacterial background lawn was evaluated, both macroscopically and microscopically by using a dissecting microscope.
Statistics:
No formal hypothesis testing was done.

A test substance is considered negative (not mutagenic) in the test if:

a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is greater than three (3) times the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one independently repeated experiment.

Results and discussion

Test resultsopen allclose all
Species / strain:
other: TA100 and WP2uvrA
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
other: TA1535, TA1537 and TA98
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
other: TA1535, TA1537, TA98, TA100 and WP2uvrA
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
DOSE RANGE FINDING TEST

Precipitate
Precipitation of Wheat Solubles, Hydrolyzed on the plates was observed at the start and at the end of the incubation period at concentrations of 3330 and 5000 pg/plate.

Toxicity
No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed.

Mutaqenicity
In tester strain WP2uvrA, Wheat Solubles, Hydrolyzed induced up to 2.3- and 2.6-fold dose related increases in the number of revertant colonies compared to the solvent control in the absence and presence of S9-mix, respectively. In tester strain TA100, no increase in the number of revertants was observed upon treatment with Wheat Solubles, Hydrolyzed.


MUTATION ASSAY, EXPERIMENT 1

Precipitate
Precipitation of Wheat Solubles, Hydrolyzed on the plates was observed at the start and at the end of the incubation period at a concentration of 3330 pg/plate.

Toxicity
No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all tester strains in the absence and presence of S9-mix was observed.

Mutagenicity
In tester strain TAI 535, Wheat Solubles, Hydrolyzed induced up to 3.8- and 3.6-fold dose related, increases in the number of revertant colonies compared to the solvent control in the absence and presence of S9-mix, respectively. In tester strain TA1537, Wheat Solubles, Hydrolyzed induced up to 8.3- and 5.5-fold dose related, increases in the number of revertant colonies compared to the solvent control in the absence and presence of S9-mix, respectively. In tester strain TA98 up to 1.5- and 2.2-fold increases in the number of revertants was observed upon treatment with Wheat Solubles, Hydrolyzed.


MUTATION ASSAY, EXPERIMENT 2

Precipitate
Precipitation of Wheat Solubles, Hydrolyzed on the plates was observed at the start and at the end of the incubation period at concentrations of 2000 and 3330 pg/plate.

Toxicity
No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all tester strains in the absence and presence of S9-mix was observed.

Mutaqenicity
In tester strain TA1 535, Wheat Solubles, Hydrolyzed induced up to 4.1- and 4.6-fold dose related, increases in the number of revertant colonies compared to the solvent control in the absence and presence of S9-mix, respectively. In tester strain TA1537, Wheat Solubles, Hydrolyzed induced up to 8.8- and 7.0-fold dose related, increases in the number of revertant colonies in the absence and presence of S9-mix, respectively. In tester strain TA98 in the absence and presence of S9-mix, up to 2.1- and 3.3-fold increases in the number of revertants was observed upon treatment with Wheat Solubles, Hydrolyzed. In addition an up to 1.5-fold increase in the number of revertants was observed in the absence of S9-mix in tester strain TA100. In tester strain WP2uvrA, Wheat Solubles, Hydrolyzed induced up to 3.1- and 2.3-fold dose related, increases in the number of revertant colonies compared to the solvent control in the absence and presence of S9-mix, respectively.
Remarks on result:
other: other: Dose range finding test
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

In the absence of S9-mix, Wheat Solubles, Hydrolyzed induced dose related increases in all tester strains (TA1 535, TA1 535, TA98, TA1 00 and WP2uvrA). The increases observed in tester strains TA1535 and TA1537 were above the laboratory historical control data range, in two independently repeated experiments, dose dependent and up to 4.1 and 8.8-fold the concurrent controls, respectively.

 

The increases observed in tester strain WP2uvrA were dose dependent, up to 3.1-fold and above the historical control data range in the second mutation experiment only. The increases observed in tester strains TA98 and TA100 were dose dependent, however not above the laboratory historical control data range.

 

In the presence of S9-mix, Wheat Solubles, Hydrolyzed induced dose related increases in four tester strains (TA1535, TA1535, TA98 and WP2uvrA). The increases observed in tester strains TA1535 and TA1537 were above the laboratory historical control data range, in two independently repeated experiments, dose dependent and up to 4.6 and 7.0-fold the concurrent controls, respectively. The increases observed in tester strain WP2uvrA were dose dependent, in two independently repeated experiments, above the historical control data range, but were not greater than three times the concurrent vehicle control (up to 2.6-fold). The increases observed in tester strain TA98 were dose dependent and up to 3.3-fold the concurrent controls, however not above the historical control data range in both independently repeated experiments.

 

Since the mutagenic effect was observed in two independent repeat experiments, in several tester strains, the observed increases were outside or just below the historical control data range and were dose-related, these increases are considered to be biologically relevant and Wheat Solubles, Hydrolyzed is considered to be mutagenic.

 

The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

 

Based on the results of this study it is concluded that Wheat Solubles, Hydrolyzed is mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coil reverse mutation assay mainly at precipitating dose levels.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
positive