Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Remarks:
No deviations ocurred that impacted study quality or result interpretation.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Batch 20160912
- Expiration date of the lot/batch: 01 August 2018
- Purity test date: 25 July 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stable until 01 August 2018
- Stability under test conditions: Stable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test article was dissolved in DMSO

Method

Target gene:
S. thyphimurius: Histidine operon
E. coli: Tryptophan operon
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9 mix
Test concentrations with justification for top dose:
1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate

The test article did not precipitate at the top dose level. The bacterial background lawn was not reduced at any of the concentrations tested.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Test article solubility and suitability for the tester strains.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
methylmethanesulfonate
other:
Details on test system and experimental conditions:
METHOD OF APPLICATION: Plate incorporation and preincubation
- Cell density at seeding (if applicable): 10^9 cells/mL

DURATION
- Preincubation period: Pre-incubation assay: 30 minutes. Direct plate: None.
- Exposure duration: 48 hours
- Expression time (cells in growth medium): 48-48.5 hours
- Selection time (if incubation with a selection agent): 48 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 48 hours

SELECTION AGENT (mutation assays): Histidine or tryptophan-minimal agar.

NUMBER OF CELLS EVALUATED: All revertant colonies were counted automatically with the Sorcerer Colony Counter.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells):

DETERMINATION OF CYTOTOXICITY
- Method: Background lawn growth.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Remarks:
Plate incorporation: The test article induced up to 15- and 12-fold dose related increases in revertant colonies compared to the vehicle control in the absence and presence of metabolic activation, respectively.
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
The test article induced 7.4-fold increases in revertant colonies in the absence of metabolic activation.
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
In the absence of metabolic activation, an increase in revertant colonies was observed in the tester strains TA1535 and TA100. In the presence of metabolic activation, an increase in revertant colonies was observed in the tester strain TA1535. All other bacterial strains showed no increase in revertant colonies in the two independent experiments.

Applicant's summary and conclusion

Conclusions:
Based on the results of the study, the test article is positive in the Ames assay.
Executive summary:

The potential for the test article to induce reverse mutations at the histidine locus in several strains of Salmonella typhimurium(S. typhimurium; TA98, TA100, TA1535, and TA1537), and at the tryptophan locus of Escherichia coli (E. coli) strain WP2uvrA in the presence or absence of an exogenous mammalian metabolic activation system (S9) was evaluated. The test was performed in two independent experiments, at first a direct plate assay was performed and secondly a pre-incubation assay. The study was conducted according to OECD 471 and was conducted in compliance with OECD GLP. The test item was dissolved in dimethyl sulfoxide. In the dose-range finding study, the test item was initially tested up to concentrations of 5000 μg/plate in the strains TA100 and WP2uvrA in the direct plate assay. MTDID 49220 did not precipitate on the plates at this dose level. In the first mutation experiment, the test item was tested up to concentrations of 5000 μg/plate in the strains TA1535, TA1537 and TA98. MTDID 49220 did not precipitate on the plates at this dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. In tester strain TA1535, the test item induced up to 15- and 12-fold dose related increases in the number of revertant colonies compared to the concurrent vehicle control in the absence and presence of S9-mix, respectively. In the second mutation experiment, the test item was tested up to concentrations of 5000 μg/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA in the pre-incubation assay. MTDID 49220 did not precipitate on the plates at this dose level. Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix. In the absence of S9-mix, the test item induced dose-related increases in the number of revertant colonies compared to the solvent control in two tester strains (TA1535 and TA100). The increases observed in the tester strains TA1535 and TA100 were above the laboratory historical control data range and were up to 7.4- and 4.4-fold the concurrent controls, respectively. No increases were observed in the presence of S9-mix. 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. In the absence of S9-mix, the test item induced dose related increases in the number of revertant colonies in the tester strains TA1535 and TA100. The increases observed in tester strain TA1535 were above the laboratory historical control data range, were up to 15-fold the concurrent vehicle control and were reproducible in both the direct plate assay and the pre-incubation assay. The increases observed in tester strain TA100 were above the laboratory historical control data range and were up to 4.4-fold the concurrent vehicle control, but were only observed in the pre-incubation assay. Based on the results of the study, the test article is positive in the Ames assay.