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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:
02-Dec-15 to 07-Mar-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study has been performed according to OECD and/or EC guidelines and according to GLP principles.

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
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
Adopted July 21, 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes
Remarks:
Except for the quality environment in which the characterisation of the test item was performed was not known.
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
605-263-0
EC Number:
605-263-0
Cas Number:
161611-74-1
Molecular formula:
C4F6O3
IUPAC Name:
605-263-0
Test material form:
liquid
Details on test material:
- Physical state: Colourless liquid; odorless
- Storage condition of test material: At room temperature

Method

Target gene:
- S. typhimurium: Histidine gene
Species / strain
Species / strain / cell type:
other: S. Typhimurium (TA98, TA100, TA102, TA1535 and TA1537)
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not applicable
Cytokinesis block (if used):
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by Aroclor 1254
Test concentrations with justification for top dose:
Preliminary test (without and with S9) TA100: 200, 500, 1000, 2000 and 5000 µg/plate

Main study:
- Experiment 1 (pre incubation): TA1535, TA1537, TA102 and TA98: Without and with S9-mix: 200, 500, 1000, 2000 and 5000 µg/plate
- Experiment 2 (pre incubation): TA1535, TA1537, TA98, TA100 and TA102: Without and with S9-mix: 200, 500, 1000, 2000 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: tetrahydrofuran
- Justification for choice of solvent/vehicle:
Test compound was soluble in tetrahydrofuran and tetrahydrofuran has been accepted and approved by authorities and international guidelines.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
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
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
without S9: 10 µg/plate in DMSO for TA98 and 15 µg/plate for TA1537
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Tert-butyl hydroperoxide (TBH)
Remarks:
without S9: 250 µg/plate in DMSO for TA102
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
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
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene in DMSO for all tester strains
Remarks:
with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: pre incubation

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.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.


ACCEPTABILITY OF THE ASSAY:
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 WIL Research Europe.
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.
Evaluation criteria:
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or TA102 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 control.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive if:
a) The total number of revertants in the tester strains TA100 and TA102 is greater than two (2) times the concurrent control, or the total number of revertants in the tester strains TA1535, TA1537 or TA98 is greater than three (3) times the concurrent 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.

Results and discussion

Test results
Key result
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed up to and including the top dose of 5000 µg/plate

RANGE-FINDING/SCREENING STUDIES:
- No toxicity or mutagenicity was observed up to and including the top dose of 5000 µg/plate

COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative and strain-specific positive control values were within our laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- No toxicity or mutagenicity was observed up to and including the top dose of 5000 µg/plate
Remarks on result:
other: not mutagenic

Applicant's summary and conclusion

Conclusions:
Under the test conditions, test substance is not considered as mutagenic with and without metabolic activation in S. typhimurium strains TA1535, TA1537, TA98, TA100 and TA102.
Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471, EU Method B.13/14 and in compliance with GLP, strains of Salmonella typhimurium (TA 1535, TA 1537, TA 98 TA100 and TA 102) were exposed to the test substance diluted in tetrahydrofuran at the following concentrations both in the presence and absence of metabolic activation system (S9 -mix).

Preliminary test (without and with S9) TA100: 200, 500, 1000, 2000 and 5000 µg/plate

Main study:

- Experiment 1 (pre incubation): TA1535, TA1537, TA102 and TA98: Without and with S9-mix: 200, 500, 1000, 2000 and 5000 µg/plate

- Experiment 2 (pre incubation): TA1535, TA1537, TA98, TA100 and TA102: Without and with S9-mix: 200, 500, 1000, 2000 and 5000 µg/plate

Negative and positive control groups were also included in mutagenicity tests.

The negative control values were within the laboratory historical control data ranges, except the responses for TA102 (absence of S9-mix, first experiment) and TA1535 (absence of S9-mix, second experiment). However since the mean number of revertant colonies showed a characteristic number of revertant colonies (237 and 3 revertant colonies, respectively) when compared against relevant historical control data (248 and 5 relevant colonies, respectively), the validity of the test was considered to be not affected.

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, except the response for TA1535, TA1537 and TA98 in the second experiment. The purpose of the positive control is as a reference for the test system, where a positive response is required to check if the test system functions correctly. Since the value was more than 3 times greater than the concurrent solvent control values, this deviation in the mean plate count of the positive control had no effect on the results of the study.

No cytotoxic effect was observed. All bacterial strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants in two independently repeated experiments.

Under the test conditions, test substance is not considered as mutagenic with and without metabolic activation in S. typhimurium strains TA1535, TA1537, TA98, TA100 and TA102.

This study is considered as acceptable and satisfies the requirement for reverse gene mutation endpoint.