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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:
June 2016
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
Test material form:
liquid
Details on test material:
- Appearance: colourless liquid
- Storage conditions: at room temperature

Method

Target gene:
- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by Aroclor 1254
Test concentrations with justification for top dose:
Experiment 1:
Preliminary test (without and with 5% (v/v) S9-mix) (TA100 and WP2uvrA): 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate
Main study (TA1535, TA1537 and TA98):
Without S9-mix: 5.4, 17, 52, 164, 512 and 1600 µg/plate
With 5% (v/v) S9-mix: 52, 164, 512, 1600 and 5000 µg/plate

Experiment 2:
TA1535, TA1537 and TA100:
Without S9-mix: 87, 154, 275, 492, 878 and 1568 μg/plate
With 10% (v/v) S9-mix: 492, 878, 1568, 2800 and 5000 μg/plate
TA98 and WP2uvrA:
Without S9-mix: 275, 492, 878, 1568, 2800 and 5000 μg/plate
With 10% (v/v) S9-mix: 492, 878, 1568, 2800 and 5000 μg/plate

Doses were selected based on the dose range finding test (observed precipitation and toxicity).
Vehicle / solvent:
- Vehicle used: DMSO
- Justification for choice of vehicle: A homogeneous suspension could be obtained in DMSO and DMSO is accepted and approved by authorities and international guidelines as solvent.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without S9; 5 µg/plate in saline for TA1535
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: ICR-191
Remarks:
without S9; 2.5 µg/plate in DMSO for TA1537
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-nitrofluorene
Remarks:
without S9; 10 µg/plate in DMSO for TA98
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9; 650 µg/plate in DMSO for TA100
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without S9; 10 µg/plate in DMSO for WP2uvrA
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
in DMSO; with 5% S9-mix, 2.5 µg/plate for TA1535, TA1537, 1 µg/plate for TA98, TA100, 15 µg/plate for WP2 uvrA; with 10% S9-mix, 2.5 µg/plate for TA1535, 5 µg/plate for TA1537, 1 µg/plate for TA98, 2 µg/plate for TA100, 15 µg/plate for WP2 uvrA
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hour

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.

NUMBER OF CELLS EVALUATED: 10E8 per plate

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies was determined.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.
Rationale for test conditions:
Recommended test conditions in international guidelines (e.g. OECD, EC).
The first mutation experiment was performed with 5% (v/v) S9-mix. To obtain more information about the possible mutagenicity of Dibutyl itaconate, the second mutation experiment was performed in the absence and presence of 10% (v/v) S9-mix.
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 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 the 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, TA98 is greater than three (3) times the concurrent vehicle control.
b) 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.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
Precipitation observed at highest concentration tested (5000 μg/plate) in experiment 2
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at highest tested concentration (5000 μg/plate) in experiment 1; precipitation was observed at 2800 μg/plate and above in experiment 2
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at the highest tested concentration tested (1600 μg/plate) in experiment 1; precipitation was observed at highest concentration tested (1568 μg/plate) in experiment 2
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
Precipitation observed at highest concentration tested (1568 μg/plate) in experiment 2
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at highest concentration tested (1568 μg/plate); preciptation was observed at highest tested concentration (1568 μg/plate) in experiment 2
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
Precipitation observed at highest tested concentration (5000 μg/plate) in experiment 2
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
Precipitation observed at 2800 μg/plate and above
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 878 μg/plate and above; precipitation was observed at highest concentration tested (1568 μg/plate) in experiment 2
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
Precipitation observed at highest concentration tested (5000 μg/plate) in experiment 2
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Slight precipitation was observed at the highest concentration tested except in tester strain TA98 in the presence of S9-mix, where no precipitation was observed. In addition, in tester strains TA98 and WP2uvrA (absence of S9-mix), precipitation was also observed at 2800 μg/plate and above.

RANGE-FINDING/SCREENING STUDIES:
- In tester strain TA100, toxicity was observed at dose levels of 512 μg/plate and above in the absence of 5% (v/v) S9-mix and at 1600 and 5000 μg/plate in the presence of 5% (v/v) S9-mix, respectively. In tester strain WP2uvrA, toxicity was only observed at the highest concentration (5000 μg/plate) in the absence of S9-mix.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
TA1535: without S9: 1600 µg/plate, with S9: no toxicity
TA1537: without S9: 1568 µg/plate and above, with S9: no toxicity
TA98: without S9: no toxicity , with S9: no toxicity
TA100: without S9: 878 µg/plate and above, with S9: no toxicity
WP2uvrA: without S9: no toxicity , with S9: no toxicity

ACCEPTANCE OF RESULTS:
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 Charles River Den Bosch.
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.

- Acceptance criteria met for vehicle control: Yes
- Acceptance criteria met for positive control: Yes

Any other information on results incl. tables

In strains TA1537 (absence and presence of S9-mix, experiment 1+2) and TA98 (absence of S9-mix, experiment 1), fluctuations in the number of revertant colonies below the laboratory historical control data range were observed. However, since no dose-relationship was observed, these reduction are not considered to be caused by toxicity of the test item. It is more likely these reductions are caused by an incidental fluctuation in the number of revertant colonies.

Applicant's summary and conclusion

Conclusions:
In an AMES test, performed according to OECD/EC guidelines and GLP principles, Dibutyl itaconate was found not to be mutagenic with or without metabolic activation.
Executive summary:

An AMES test was performed with Dibutyl itaconate according to OECD/EC guidelines and GLP principles. All bacterial strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants with or without metabolic activation in two experiments was seen. In the second experiment the test item precipitated on the plates in the absence and presence of S9-mix, except in tester strain TA98 in the presence of S9-mix, where no precipitation was observed. Cytotoxicity, as evidenced by a decrease in the number of revertants and/or bacterial background lawn, was observed in tester strains TA1535, TA1537 and TA100 in the absence of S9-mix. The negative and 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.

Based on the results of this study it is concluded that Dibutyl itaconate is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay with or without metabolic activation.