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Toxicological information

Genetic toxicity: in vitro

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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:
18 September 2014 to 29 September 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This USFDA and OECD GLP compliant study was performed according to OECD 471 (1997).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report date:
2014

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
MTDID 13999
IUPAC Name:
MTDID 13999
Test material form:
other: Liquid
Details on test material:
- Name of test material (as cited in study report): MTDID 13999
- Substance type: Mono-constituent
- Physical state: Liquid
- Analytical purity: 99.0%
- Purity test date: 09 February 2009
- Lot/batch No.: Lot: 529783

Method

Target gene:
Histidine 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:
Aroclor induced rat liver S9
Test concentrations with justification for top dose:
Initial toxicity-mutation assay: 1.5, 5.0, 15, 50, 150, 500, 1500, and 5000 ug/plate.
Confirmatory mutagenicity assay: 0.15, 0.5, 1.5, 5.0, 15, 50, and 150 ug/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: Based on solubility information from the sponsor and compatability with target cells.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
other:
Remarks:
All Strains with S9 Mix: 2-aminoanthracene. Without S9 Mix: TA98: 2-nitrofluorene, TA100 and TA1535: sodium azide, TA1537: 9-aminoacridine, WP2 uvrA: methyl methanesulfonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorportation)

DURATION
- Preincubation period: Overnight
- Exposure duration: Initial assay: 56.92 hours, Confirmatory assay: 61.03 hours
- Expression time (cells in growth medium): Approximately 69-73 hours
- Selection time (if incubation with a selection agent): Initial assay: 56.92 hours, Confirmatory assay: 61.03 hours
- Fixation time (start of exposure up to fixation or harvest of cells):Initial assay: 56.92 hours, Confirmatory assay: 61.03 hours

SELECTION AGENT (mutation assays): Histidine minimal augar

NUMBER OF CELLS EVALUATED: All colonies counted by a Sorcerer Colony Counter utilizing Ames Study Manager software.

DETERMINATION OF CYTOTOXICITY
- Method:Bacterial background lawn condition.
Evaluation criteria:
For the test article to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test article. Data sets for tester strains TA1535 and TA1537 were judged positive if the increase in mean revertants at the peak of the dose response was greater than or equal to 3.0-times the mean vehicle control value. Data sets for tester strains TA98, TA100, and WP2 uvrA were judged positive if the increase in mean revertants at the peak of the dose response was greater than or equal to 2.0-times the mean vehicle control value.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Cytotoxicity was observed at test article doses greater than 15 ug per plate.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation was observed at 1500 ug/plate and higher.

RANGE-FINDING/SCREENING STUDIES: An initial toxicity-mutation assay was conducted up to 5000 ug/plate to determine the appropriate doses for the confirmatory mutagenicity assay.

COMPARISON WITH HISTORICAL CONTROL DATA: Controls were within historical ranges.

ADDITIONAL INFORMATION ON CYTOTOXICITY: Cytotoxicity was observed at test article doses greater than 15 ug per plate.
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

Under the conditions of this study, the test material was not mutagenic in the Bacterial Reverse Mutation Assay.
Executive summary:

The mutagenic potential of the test article (Clear colorless liquid, Lot 529783, 99% CASRN 4303-67-7) was evaluated in the Bacterial Reverse Mutation Assay with S. typhimurium strains TA98, TA100, TA1535, and TA1537 and E. coli strain WP2 uvrA in the presence and absence of a metabolic activation system (Aroclor induced S9-mix). The study was performed in compliance with FDA GLP 21 CFR Part 58 and OECD GLP regulations. The test method was based on OECD 471 (1997). Ethanol was used as the vehicle at all test article doses. A toxicity-mutation assay was conducted with the test article up to 5000 ug/plate with all tester strains in the presence and absence of metabolic activation to establish the dose-range for the confirmatory mutagenicity assay. In the confirmatory mutagenicity assay, the test article was tested up to 150 ug/plate in the absence of S9 and up to 500 ug/plate in the presence of S9 with all tester strains. Strain specific positive controls and vehicle controls were tested in parallel.  No increase in revertant colonies was observed in either the presence or absence of metabolic activation in any strain tested. All criteria for a valid test were met as described in the protocol.  Under the conditions of this study, the test material was not mutagenic in the Bacterial Reverse Mutation Assay.