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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:
2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
Deviations:
no
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 No. 605803
- Expiration date of the lot/batch: 23 October 2016

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
- Stability under test conditions:
- Solubility and stability of the test substance in the solvent/vehicle:
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
- Preliminary purification step (if any):
- Final dilution of a dissolved solid, stock liquid or gel:
- Final preparation of a solid:

FORM AS APPLIED IN THE TEST (if different from that of starting material)

OTHER SPECIFICS:

Method

Target gene:
Histidine and tryptophan operons
Species / strain
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell lines (if applicable):
CELLS USED
- Source of cells: Trinova Biochem GmbH, Germany [Master culture from Dr. Bruce N. Ames (TA1535: 2006, TA1537: 2009, TA98: 2006, TA100: 2006; and Master culture from The National Collections of Industrial and Marine Bacteria, Aberdeen, UK (WP2uvrA: 2008)]
- Suitability of cells: Recommended test system in international guidelines (e.g. OECD, EC).
Metabolic activation:
with and without
Metabolic activation system:
Rat liver microsomal enzymes (S9 homogenate) were obtained from Trinova Biochem GmbH, Giessen, Germany and were prepared from male Sprague Dawley rats that had been injected intraperitoneally with Aroclor 1254 (500 mg/kg)
Test concentrations with justification for top dose:
Experiment 1 (direct plate assay):
Strains TA1535, TA1537 and TA98: In the absence of S9-mix: 1.7, 5.4, 17, 52, 164 and 512 μg/plate.
In the presence of S9-mix: 5.4, 17, 52, 164, 512, and 1600 μg/plate.

Experiment 2 (pre-incubation assay):
Strains TA 1535, TA1537, TA98, and TA100 in the presence and absensc ef S9-mix: 10, 25, 50, 100, 200, and 400 μg/plate.
E. coli WP2uvrA in the presence and absensc of S9-mix: 100, 200, 400, 750, and 1500 μg/plate.

Experiment 3 (pre-incubation assay):
Strains TA 1535, TA1537, TA98, and TA100 in the absence of S9-mix: 0.01, 0.1, 1, 10, and 100 μg/plate.
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Acceptable vehicle in accordance with OECD Guideline 471.
Controls
Solvent controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: 2-aminoanthracene (Sigma) and ICR-191 (Sigma)
Details on test system and conditions:
METHOD OF APPLICATION: in agar (plate incorporation) and preincubation
- Cell density at seeding (if applicable): 10^9 cells/mL (0.1 mL fresh baterial culture)

DURATION
- Preincubation period: 30 minutes
- Exposure duration:48 ± 4 hours

NUMBER OF REPLICATIONS: 3 replicates
Rationale for test conditions:
Acceptable based on protocol in OECD Guideline 471.
Evaluation criteria:
A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:

a) The vehicle control and positive control plates from each tester strain (with or without S9-mix) must
exhibit a characteristic number of revertant colonies when compared against relevant historical
control data generated at WIL Research Europe.

b) The selected dose range should include a clearly toxic concentration or should exhibit limited
solubility as demonstrated by the preliminary toxicity range-finding test or should extend to
5 mg/plate.

c) No more than 5% of the plates are lost through contamination or some other unforeseen event. If
the results are considered invalid due to contamination, the experiment will be repeated.
Statistics:
No formal hypothesis testing was done. In addition to the criteria stated below, any increase in the total number of revertants should be evaluated for its biological relevance including a comparison of the results with the historical control data range.

A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one follow up experiment.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 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 follow up experiment.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
other: TA 1535, TA1537, TA98
Remarks:
Experiment 1
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
In the direct plate test, no increase in the number of revertants was observed upon treatment under all conditions tested.
Cytotoxicity:
yes
Remarks:
A reduction of the bacterial background lawn and a biologically significant decrease in the number of revertants were observed in all tester strains.
Vehicle controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: Precipitation of the test item on the plates was observed at the start and at the end of the incubation period at the concentration of 5000 μg/plate.
Key result
Species / strain:
other: TA1535, TA1537, TA98, TA100
Remarks:
Experiment 2
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
In the pre-incubation test, no increase in the number of revertants was observed upon treatment under all conditions tested.
Cytotoxicity:
yes
Remarks:
Moderate to extreme toxicity at all tested concentrations in the absence of S9 and at test item concentrations including or above 50 μg/plate with S9. A follow-up third experiment was performed to evaluate lower concentrations in the absence of S9-mix.
Vehicle controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other:
Remarks:
Precipitation of the test item on the plates was not observed at the start or at the end of the incubation period.
Key result
Species / strain:
E. coli WP2 uvr A
Remarks:
Experiment 2
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
In the pre-incubation test, no increase in the number of revertants was observed upon treatment under all conditions tested.
Cytotoxicity:
yes
Remarks:
Moderate to extreme toxicity at all tested concentrations in the absence of S9 and at test item concentrations including or above 50 μg/plate with S9. A follow-up third experiment was performed to evaluate lower concentrations in the absence of S9-mix.
Vehicle controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other:
Remarks:
Precipitation of the test item on the plates was not observed at the start or at the end of the incubation period.
Key result
Species / strain:
other: TA1535, TA1537, TA98 and TA100
Remarks:
Experiment 3
Metabolic activation:
without
Genotoxicity:
negative
Remarks:
In the pre-incubation test, no increase in the number of revertants was observed upon treatment under all conditions tested.
Cytotoxicity:
yes
Remarks:
Moderate toxicity was observed at concentrations ≥10 μg/plate. Sufficient data was available from the lower concentrations to evaluate the mutagenicity in the absence of S9-mix.
Vehicle controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other:
Remarks:
Precipitation of MTDID 15670 on the plates was not observed at the start or at the end of the incubation period.

Applicant's summary and conclusion

Conclusions:
Based on the results of this study it is concluded that the test article is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay with and without metabolic activation (S9-mix).
Executive summary:

The mutagenic potential of the test article was evaluated in the Bacterial Reverse Mutation Assay with S. typhimurium strains TA98, TA100, TA1535, and TA1537 and E. coli strain WP2uvrA in the presence and absence of a metabolic activation system (S9-mix rat liver induced by Phenobarbital and B-napthoflavone). The study was performed in compliance with OECD GLP (1997). The test method was based on OECD No. 471 (1997) and EC No. 440/2008 Part B (2008). The test article was dissolved in dimethyl sulfoxide. A dose range-finding test was performed with concentration up to 5000µg/plate in the absence and presence of metabolic activation in strains TA100 and WP2uvrA. Based on the results of the dose range finding test, the test article was dosed in experiment 1 (direct plate assay) at concentrations of 1.7, 5.4, 17, 52, 164, and 512µg/plate in the absence of S9 and at 5.4, 17, 52, 164, 512, and 1600µg/plate in the presence of S9 in the TA1535, TA1537 and TA98 strains. In order to obtain more information, experiment 2 (pre-incubation assay) exposed TA 1535, TA1537, TA98, and TA100 to concentrations of 10, 25, 50, 100, 200, and 400 μg/plate in the presence and absence of S9. E. coli WP2uvrA was exposed to concentrations of 100, 200, 400, 750, and 1500 μg/plate in the presence and absence of S9. A follow-up third experiment (experiment 3; pre-incubation assay) was performed to evaluate lower concentrations in the absence of S9-mix; TA1535, TA1537, TA98 and TA100 were exposed to concentrations of 0.01, 0.1, 1, 10, and 100 μg/plate in the absence of S9. Strain specific positive controls and negative (vehicle) controls were tested in parallel. All treatments were performed in triplicate. Agar plates (ø 9 cm) contained 25 ml glucose agar medium. Glucose agar medium contained per liter: 18 g purified agar in Vogel-Bonner Medium E, 20 g glucose. The agar plates for the test with the S. typhimurium strains also contained 12.5 μg/plate biotin and 15 μg/plate histidine and the agar plates for the test with the E. coli strain contained 15 μg/plate tryptophan. Top agar consisted of Milli-Q water containing 0.6% (w/v) bacteriological agar and 0.5% (w/v) sodium chloride 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 121 ± 3°C. All incubations were carried out in a controlled environment at a temperature of 37.0 ± 1.0°C (actual range 35.8 – 38.8°C). After this period, revertant colonies (His+) for S. typhimurium and (Trp+) for E. coli were counted. In experiment 1 (TA1535, TA1537 and TA98 strains via direct plate assay), precipitation of the test item on the plates was observed at the start and at the end of the incubation period at the concentration of 5000 μg/plate. A reduction of the bacterial background lawn and a biologically significant decrease in the number of revertants were observed in all tester strains. No increase in the number of revertants was observed upon treatment with and without metabolic activation. In experiment 2 (TA 1535, TA1537, TA98, and TA100 and WP2uvrA via pre-incubation assay), precipitation of the test item on the plates was not observed at the start or at the end of the incubation period. Moderate to extreme toxicity was observed at all tested concentrations in the absence of S9-mix. Moderate to extreme toxicity was also observed at test item concentrations including or above 50 μg/plate with S9-mix; experiment 3 was performed to evaluate lower concentrations in the absence of S9-mix. In the pre-incubation test, no increase in the number of revertants was observed upon treatment with and without metabolic activation. In experiment 3 (TA1535, TA1537, TA98 and TA100 without S9 via pre-incubation assay), precipitation of the test item on the plates was not observed at the start or at the end of the incubation period. Moderate toxicity was observed at concentrations ≥10 μg/plate. In the pre-incubation test, no increase in the number of revertants was observed upon treatment without metabolic activation. All criteria for a valid test were met as described in the protocol. Based on the results of this study it is concluded that the test article is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the E.coli reverse mutation assay with and without metabolic activation (S9-mix).