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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:
2019-10-22 to 2019-11-01
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021
Report date:
2019

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
30 May 2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21st July 1997
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
2-{[1,1,2-trifluoro-2-(1,1,2,2,3,3,3-heptafluoropropoxy)ethyl]sulfanyl}ethyl prop-2-enoate
EC Number:
864-951-0
Cas Number:
2170099-74-6
Molecular formula:
C10H8F10O3S
IUPAC Name:
2-{[1,1,2-trifluoro-2-(1,1,2,2,3,3,3-heptafluoropropoxy)ethyl]sulfanyl}ethyl prop-2-enoate
Test material form:
liquid

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9: rat liver homogenate from male Wistar rats, Crl: WI (HAN) (Charles River, Germany), aged 6-8 weeks that were pretreated with ß-Naphthoflavone (100 mg/kg body weight) and Phenobarbital (80 mg/kg body weight).
- method of preparation of S9 mix: livers removed and homog-enized in ice-cold 0.15 M KCl (3 mL KCl per g liver wet-weight). The homogenate was spun for 10 minutes at 9000 rpm (8784 x g, Biofuge 28 RS) and +4°C. The supernatant fluid (S9) was decanted, transferred to sterile tubes and stored in liquid nitrogen.
- concentration or volume of S9 mix and S9 in the final culture medium: 10% and 20% S9 in the S9 mix were used in the 1st and 2nd test series; 0.5 mL were used in teh final culture medium
- quality controls of S9: Each S9 batch was tested for its metabolic activity using specific substrates.
Test concentrations with justification for top dose:
5, 15.8, 50, 158, 500, 1580, 5000 µg/plate ± S9 mix
Vehicle / solvent:
- Vehicle used: DMSO

- Justification for choice of solvent/vehicle: DMSO showed best performance and was thus used for this experiment at a maximum concentration of 100 µL/plate.
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
Remarks:
2 (TA98, TA 100); 5 µg/plate (TA1535, TA 1537); 10 µg/plate (WP2 uvrA) with S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
2 µg/plate (TA 100, TA 1535) without S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
2 µg/plate (WP2 uvrA) without S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-Nitro-o-phenylenediamine
Remarks:
20 µg/plate (TA98); 60 µg/plate (TA 1537) without S9 mix
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 48 hours

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition
Evaluation criteria:
A test material was to be defined as positive or mutagenic in this assay if

- the assay is considered valid and

- a biologically relevant increase in the mean number of revertants above a threshold of 2-fold (TA 98, TA 100, WP2 uvrA) or 3-fold (TA 1535, TA 1537) as compared to the concurrent negative controls is observed

- an increase exceeding the threshold at only one concentration is considered as biologically meaningful if reproduced in a second independent experiment

- a concentration-dependent increase is considered biologically meaningful if the threshold is exceeded at more than one concentration

A test material is defined as negative or non-mutagenic in this assay if

- the assay is considered valid and
- none of the above-mentioned criteria are met
Whenever colony counts remain within the historical range of negative controls, such increases are considered as biologically not meaningful. In general, two series of experiments must be performed.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: precipitation of the test material on the agar plates occurred at a concentration of 5000 µg/plate.

STUDY RESULTS
- Concurrent vehicle negative and positive control data
The mean numbers of revertant colonies of the current negative controls were within the ränge of historical negative control values.
The strain-specific positive controls, namely sodium azide, 4-nitroquinolin-N-oxide and 4-Nitro-o-phenylenediamine in the absence of S9 mix, yielded the expected mutant frequencies that were greatly exceeding the negative controls. The genotype of the tester strains used was thus confirmed. 2-Aminoanthracene which require metabolic activation, was strongly mutagenic. This indicates that the exogenous metabolizing system used in the present investigation (S9 mix) was active.

Ames test:
- Signs of toxicity: No

HISTORICAL CONTROL DATA (see attached data)

Applicant's summary and conclusion

Conclusions:
Under the experimental conditions reported here, the test item did not induce gene mutations by base-pair or frameshift changes in the genome of the strains used. Therefore, it was concluded that with and without addition of S9 mix as the exogenous metabolizing System, the test item was not mutagenic in this Salmonella typhimurhim and Escherichia coli reverse mutation test.
Executive summary:

The test item was examined for its mutagenic activity in an in vitro bacterial reverse mutation test according to OECD TG 471 employing Salmonella typhimurium TA 98, TA 100, TA 153 5 and TA 1537, and Escherichia coli WP2 uvrA as indicator organisms.


The plate incorporation test with and without addition of liver S9 mix from Phenobarbital/ß-Naphthoflavone-pretreated rats was used. In this study, two experimental series were performed. In the experiments with S9 mix, 10% and 20% S9 in the S9 mix were used.


Treatments of all tester strains were performed using test item formulations prepared in DMSO in the absence and in the presence of S9 mix, using final concentrations between 5 and 5000 µg/plate, vehicle and positive controls.


The mean numbers of revertant colonies of the current negative controls were within the range of historical negative control values.


The strain-specific positive controls, namely sodium azide, 4-nitroquinolin-N-oxide and 4-Nitro-o-phenylenediamine in the absence of S9 mix, yielded the expected mutant frequencies that were greatly exceeding the negative controls. The genotype of the tester strains used was thus confirmed. 2-Aminoanthracene which require metabolic activation, was strongly mutagenic. This indicates that the exogenous metabolizing System used in the present investigation (S9 mix) was active. Thus, the requirements predetermined in the study plan and in section 4.6 of this report have been met in total and the study is considered valid.


Following treatment with the test item, precipitation of the test material on the agar plates occurred at a concentration of 5000 ug/plate. No toxicity to the bacteria was observed. The individual and summarized data are presented in the tables section.


Under the conditions described, there were no relevant increases in revertant numbers observed after exposure to the test item in the absence and presence of S9 mix.


According to the criteria for negative and positive results predetermined in the study plan, the test item was not mutagenic under the described experimental conditions.