Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 Oct 2010- 15 Nov 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
other: ICH Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals (1997)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
Name: Ethanol, 2,2'-oxybis-, reaction products with ammonia, morpholine derivs. residues
Physical state: liquid
Appearance: brown liquid
Specific details on test material used for the study:
- Name of test material (as cited in study report): Amine C8
- Purity test date: no data
- Lot/batch No.: no data
- Expiration date of the lot/batch: no data
- Stability under test conditions: no data
- Storage condition of test material: temperature: ambient; all articles will be stored in the dark

Method

Target gene:
Histidine locus, tryptophan locus and additional gene mutations
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
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9
Test concentrations with justification for top dose:
5000, 1500, 500, 150, 50, 15, 5.0 and 1.5 µg per plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene at 10 µg/plate
Remarks:
with metabolic activation, E coli strain WP2uvrA
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene at 1.0-2.0 µg/plate
Remarks:
with metabolic activation, Samonella strains
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
without metabolic activation, at 1.0 µg/plate (Salmonella strain TA98)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without metabolic activation, at 75 µg/plate (Salmonella strain TA100, TA1535)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without metabolic activation, at 1000 µg/plate (E coli WP2urvA)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: not applciable (plate incorporation method)
- Exposure duration: at the end of the working day, each inoculated flask will be placed in a shaker/incubator programmed to begin shaking at approximately 125 to 175 rpm and incubating at 37± 2°C for approximately 12 to 14 hours before the anticipated time of harvest. All cultures will be harvested by spectrophotometric monitoring of culture turbidity rather than by duration of incubation since overgrowth of cultures can cause loss of sensitivity to some mutagens. Cultures will be removed from incubation at a density of approximately 1E09 cells/mL.
- Expression time (cells in growth medium): not applicable
- Selection time (if incubation with a selection agent): 48 to 72 hours
- Fixation time (start of exposure up to fixation or harvest of cells): not applicable

SELECTION AGENT (mutation assays): not applicable
SPINDLE INHIBITOR (cytogenetic assays): not applicable
STAIN (for cytogenetic assays): not applicable

NUMBER OF REPLICATIONS:duplicate (initial toxicity - mutation assay), triplicate (confirmatory mutagenicity assay)

NUMBER OF CELLS EVALUATED:

DETERMINATION OF CYTOTOXICITY
- Method: A minimum of three non-toxic dose levels will be required to evaluate assay data. A dose level is considered toxic if it causes a > 50% reduction in the mean number of revertans per plate relative to the mean vehicle control value (this reduction must be accompanied by an abrupt dose-dependent drop in the revertant count) or a reduction in the background lawn. In the event that less than three non-toxic dose levels are achieved, the affected portion of the assay will be repeated with an appropriate change in dose levels.
Evaluation criteria:
For a 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 as specified below:
- Strains TA 1535 and TA 1537: data sets will be judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than 3.0-times the mean vehicle control value
- Strains TA98, TA100 and WP2 uvrA: data sets will be judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than 2.0-times the mean vehicle control value. An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose-responsive increase that does not achieve the respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited. A response will be evaluated as negative, if it is neither positive nor equivocal.

Results and discussion

Test resultsopen allclose all
Key result
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
Untreated negative controls validity:
not applicable
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
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
No precipitation was observed.

Applicant's summary and conclusion

Conclusions:
Under the conditions of the test,the test substance tested negative with and without metabolic activation in the bacterial reverse mutation assay. No toxicity nor precipitation was observed.