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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): UCAR™ Ester EEP (Ethyl 3-ethoxypropionate)
- Physical state: liquid
- Analytical purity: 99.9%
- Lot/batch No.: 1D03019501
- Stability under test conditions: stable
- Storage condition of test material: room temperature protected from light

Method

Target gene:
S. typhimurium TA98, TA100, TA1535, TA1537 and E. coli WP2 uvrA
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
Details on mammalian cell type (if applicable):
Not applilcable
Additional strain / cell type characteristics:
not applicable
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 and 5000 ug per plate

Confirmatory mutagenicity assay: 33, 100, 333, 1000, 3333 and 5000 ug per plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO

- Justification for choice of solvent/vehicle: compatibility with the test system and test material.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
, dimethylsulfoxide; DMSO (CAS#67-68-5)
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
other: 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 +/- 2 minutes
After an incubation period of 48 to 72 hours, plates were counted. Plates not counted immediately following the incubation period were stored at 2-8 deg C.

NUMBER OF REPLICATIONS: triplicate

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth and/or reduction of revertants per plate.

Evaluation criteria:
For the test article to be evaluated positive, it must cause a reproducible, concentration-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 TA98, TA1535, TA1537 and WP2 uvrA were judged positive if the increase in mean revertants at the peak of the response was greater than or equal to 3.0-times the mean vehicle control value. Data sets for tester strains
TA100 were judged positive if the increase in mean revertants at the peak of the response was greater than or equal to 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 was evaluated as negative, if it was neither positive nor equivocal.
Statistics:
Not applicable

Results and discussion

Test resultsopen allclose all
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
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:
Solubility Tests
Sterile water was initially selected as the solvent of choice based on the test article’s solubility in water at approximately 50 mg/mL and compatibility with the target cells. However, during dilution for use in the initial toxicity mutation assay, the test article formed oil-like droplets in sterile water at 50 mg/mL that rose to the top of the dilution. Therefore, a second solubility test was conducted, and dimethyl sulfoxide (DMSO) was selected as the solvent. The test article formed a clear solution in DMSO at approximately 500 mg/mL, the maximum concentration tested in the solubility test.

Sterility Results
No contaminant colonies were observed on the sterility plates for the vehicle control, the test article dilutions or the S9 and Sham mixes.

Initial Toxicity-Mutation Assay
In experiment B1 (initial toxicity-mutation assay), the maximum concentration tested was 5000 μg per plate, which is the maximum concentration recommended by test guidelines. This concentration was achieved using a concentration of 100 mg/mL and a plating aliquot of 50 μL. The concentrations tested were 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 μg per plate. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. A subtle change in revertant counts (1.7-fold maximum increase) was observed with TA98 in the presence of S9; however, this change did not meet the criteria required for evaluation as positive (i.e., dose-related increases with a maximum response of at least 3.0-times the mean vehicle control value). Neither precipitate nor toxicity was observed. Based on the findings of the initial toxicity-mutation assay, the maximum dose plated in the confirmatory mutagenicity assay was 5000 μg per plate.

Confirmatory Mutagenicity Assay
These data were generated in experiment B2. In experiment B2 (confirmatory mutagenicity assay), no positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. The concentrations tested were 33, 100, 333, 1000, 3333 and 5000 μg per plate. Neither precipitate nor toxicity was observed.

Dosing formulation analysis for concentration and stability of this test article was not conducted.
The study conclusion was based on the nominal dose levels as documented in the study records.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Tester Strain Titer Results

Experiment                     Tester Strain
 TA98  TA100  TA1535  TA1537  WP2 uvrA
             Titer Value (x 10E9 cells per mL)
 B1  1.3  1.7  3.7  3.1  4.2
 B2  3.6  10.7  2.6  6.9  9.7

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative , with and without metabolic activation

All criteria for a valid study were met. The results of the bacterial reverse mutation assay indicate that, under the conditions of this study, the test material, UCAR™ Ester EEP, was negative (non-mutagenic) both in the presence and absence of Aroclor-induced rat liver S9.
Executive summary:

The test article, UCAR™ Ester EEP, was tested in the bacterial reverse mutation assay using Salmonella typhimurium tester strains TA98, TA100, TA1535, TA1537 and Escherichia coli tester strain WP2 uvrA in the presence or absence of Aroclor-induced rat liver S9. The assay was performed in two phases using the preincubation method. The first phase, the initial toxicity-mutation assay, was used to establish the dose-range for the confirmatory mutagenicity assay and to provide a preliminary mutagenicity evaluation. The second phase, the confirmatory mutagenicity assay, was used to evaluate and confirm the mutagenic potential of the test article. Dimethyl sulfoxide (DMSO) was selected as the solvent. Based on the findings of the initial toxicity-mutation assay, the maximum dose plated in the confirmatory mutagenicity assay was 5000 μg per plate. In the confirmatory mutagenicity assay, no positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. The concentrations tested were 33, 100, 333, 1000, 3333 and 5000 μg per plate. Neither precipitate nor toxicity was observed.

All criteria for a valid study were met. The vehicle controls and positive controls in the initial toxicity-mutation and confirmatory mutagenicity assays were within the acceptable historical ranges and fulfilled the requirements for a valid assay. Under the conditions of this study, test article UCAR™ Ester EEP was negative (non-mutagenic) in the bacterial reverse mutation assay.