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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:
2017-08-21 to 2017-09-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

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

Test material

Constituent 1
Chemical structure
Reference substance name:
Diethyl ethoxymethylenemalonate
EC Number:
201-725-7
EC Name:
Diethyl ethoxymethylenemalonate
Cas Number:
87-13-8
Molecular formula:
C10H16O5
IUPAC Name:
diethyl ethoxymethylenemalonate
Test material form:
liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: JC170405
- Expiration date of the lot/batch: 2018-04-12 (retest date)
- Purity test date: 2017-04-13

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: not indicated
- Solubility and stability of the test substance in the solvent/vehicle: not indicated

OTHER SPECIFICS: correction factor is 1.00

Method

Target gene:
Histidine locus (histidine-dependent S. typhimurium strains); Tryptophan locus (tryptophan-dependent E. coli strains)
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver metabolic activation system
Test concentrations with justification for top dose:
Dose-range finding test: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate in TA100 and WP2uvrA with and without 5%(v/v) S9-mix.
Mutation experiment I: 17, 52, 164, 512, 1600 and 5000 μg/plate in TA1535, TA1537 and TA98 with and without 5% (v/v) S9-mix (top dose selected based on the dose range finding test results).
Mutation experiment II: 275, 492, 878, 1568, 2800 and 5000 µg/plate with and without 10%(v/v) S9-mix (based on the results of the first mutation experiment).

Based on the findings of the solubility test, DMSO was selected as vehicle and 5000 μg/plate was selected as the maximum final concentration for the dose range finding test.
Based on the results of the dose range finding test, 5000 µg/plate was selected as the top dose level for the first mutation experiment.
Based on the results of the dose range finding test and the first mutation experiment, 5000 µg/plate was selected as the top dose level for the second mutation experiment.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test item was observed to be insoluble in water at 50 mg/ml. In DMSO, the test item formed a clear solution at 50 mg/ml (5000 μg/plate). DMSO was selected as vehicle and 5000 μg/plate was selected as the maximum final concentration for the dose range finding test.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Without metabolic activation, 5 μg/plate (TA1535)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: ICR-191
Remarks:
Without metabolic activation, 2.5 μg/plate (TA1537)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
Without metabolic activation, 10 μg/plate (TA98)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Without metabolic activation, 650 μg/plate (TA100)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
Without metabolic activation, 10 μg/plate (WP2uvrA)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
With S9-mix; 2.5μg/plate (TA1535 with 5 and 10% S9-mix, TA1537 with 5% S9-mix), 5μg/plate ( TA1537 with 10% S9-mix), 1μg/plate(TA98 with 5 and 10% S9-mix, TA100 with 5% S9-mix), 2μg/ plate(TA100 with 10% S9-mix), 15μg/plate(WP2uvrA with 5 and 10% S9-mix)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation)
Top agar in top agar tubes was melted by heating to 45 ± 2°C. The following solutions were successively added to 3 ml molten top agar:
- 0.1 ml of a fresh bacterial culture (10^9 cells/ml) of one of the tester strains
- 0.1 ml of a dilution of the test item in DMSO and
- either 0.5 ml S9-mix (in case of activation assays) or 0.5 ml 0.1 M phosphate buffer (in case of nonactivation assays).
The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0 °C for 48 ± 4 h. After this period revertant colonies were counted.

DURATION
- Exposure duration: 48 ± 4 h
- Selection time (if incubation with a selection agent): 48h (simultaneous with exposure)

SELECTION AGENT (mutation assays): Histidine (S. Typhimurium histidine-dependent strains); Tryptophan (E. coli tryptophan-dependent strains)

NUMBER OF REPLICATIONS: all concentrations for all experiments were tested in triplicate

DETERMINATION OF CYTOTOXICITY
- Method: Reduction in bacterial background lawn; increase in size of the microcolonies; reduction of revertant colonies
Rationale for test conditions:
Recommended test system in international guidelines (e.g. OECD and EC).
Solubility limitations: Since the test item was fully soluble in DMSO, the highest tested concentration for the Mutation assay was 5000 μg/plate.
Evaluation criteria:
A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent vehicle control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent vehicle 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 vehicle control, or the total number of revertants in tester strain TA1535, TA1537 or TA98 is greater than three (3) times the concurrent vehicle control.
b) A concentration related effect is observed.
c) In case a follow up 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.
Statistics:
No formal hypothesis testing was done.
In addition to the criteria stated above, any increase in the total number of revertants was evaluated for its biological relevance including a comparison of the results with the historical control data range.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium, other: TA98, TA100, TA1535, TA1537 bacteria
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Exp.1: in TA98 with S9-mix and in TA1537, TA100 with and without S9-mix; Exp. 2: in TA1537 with S9-mix, TA98 without S9-mix and TA100 with and without S9-mix at 5000 μg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Exp. I: with and without S9-mix at 5000 μg/plate; Exp. II: without S9-mix at 5000 μg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test item was observed to be insoluble in water at 50 mg/ml.
- Precipitation: Precipitation of the test item on the plates was not observed at the start or at the end of the incubation period in any tester strain in any of the experiments.

RANGE-FINDING/SCREENING STUDIES:
In the dose-range finding test, the test item was tested at a concentration range of 1.7 to 5000 μg/plate in the absence and presence of 5% (v/v) S9-mix in the tester strains TA100 and WP2uvrA. The test item did not precipitate on the plates up to the concentration of 5000 μg/plate. Cytotoxicity, as evidenced by a decrease in the number of revertants and/or reduction of the bacterial background lawn, was observed in both tester strains in the absence and presence of S9-mix at 1600 μg/plate and upwards. Results of the dose range finding test were reported as part of the first mutation experiment.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: The 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.
- Negative (solvent/vehicle) historical control data: The vehicle control values were within the laboratory historical control data ranges


ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed

Applicant's summary and conclusion

Conclusions:
Interpretation of the results: negative with and without metabolic activation
All bacterial strains showed negative responses over the entire dose range, i.e. no biologically relevant and/or significant dose-related increase in the number of revertants in any of the experiments.
Based on the results of this study it is concluded that the test item is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

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