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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:
2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
July, 1997
Deviations:
no
Remarks:
No deviations occurred that impacted the integrity or interpretation of the study.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
C4 sulfonamido methacrylate
IUPAC Name:
C4 sulfonamido methacrylate
Details on test material:
- Name of test material (as cited in study report): 2-Propenoic acid, 2-Methyl-, 2-[Methyl[(Nonafluorobutyl)Sulfonyl]Amino]Ethyl ester, FZ-9262
- Physical state: white to grey waxy solid
- Analytical purity: 99.5%
- Storage condition of test material: at room temperature in the dark
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 3M Company, Batch 40010
- Expiration date of the lot/batch: May 2021
- Purity test date: 24 May, 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambiet temperature (15-20 C)
- Stability under test conditions: No data
- Solubility and stability of the test substance in the solvent/vehicle: No data
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No data

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test article was dissolved in DMSO
- Final preparation of a solid: The test article was dissolved in DMSO.

Method

Target gene:
S. typhimurium: Histidine operon
E. coli: Tryptophan operon
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:
S9-mix
Test concentrations with justification for top dose:
First test: 0 (vehicle control), 62, 185, 556, 1667, 5000 ug/plate
Second test: 0 (vehicle control), 125, 250, 500, 1000, 2000, 3000 ug/plate.

Top doses were chosen based on test article precipitation.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Test article solubility and test method compatibility.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
other: 2-aminoanthracene, benzo(a)pyrene, N-ethyl-N-nitrosourea
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
- Cell density at seeding (if applicable): Described as "fully grown".

DURATION
- Preincubation period: 10-16 hours
- Exposure duration: 48-72 hours.
- Expression time (cells in growth medium): 58-88 hours
- Selection time (if incubation with a selection agent): 58-88 hours.
- Fixation time (start of exposure up to fixation or harvest of cells): 58-88 hours.

SELECTION AGENT (mutation assays): Histidine or tryptophan-minimal agar.

NUMBER OF CELLS EVALUATED: All revertant colonies were counted manually and/or counted using a Sorcerer colony counter (Perceptive Instruments, UK; version 2.10)

DETERMINATION OF CYTOTOXICITY
- Method: Background lawn growth.
Rationale for test conditions:
Based on OECD Guideline 471 (1997).
Evaluation criteria:
The study was considered valid if:
- the mean colony counts of the negative control were within acceptable ranges (see Annex 5)
- if the results of the positive controls met the criteria for a positive response (see Annex 5)
- if not more than 5 % of the plates was lost through contamination or other unforeseen events
- at least three concentrations were non-toxic
Toxicity was defined as a reduction (by at least 50 %) in the number of revertant colonies and/or a clearing of the background lawn of bacterial growth as compared to the negative control and/or the occurrence of pinpoint colonies.
A test substance was considered to be positive in the bacterial gene mutation test if the mean number of revertant colonies on the test plates was increased in a concentration-related manner or if a two-fold and/or greater increase was observed compared to the negative control plates. A clear positive response would not need to be verified. Marginally or weakly positive results should be verified by additional testing.
A test substance was considered to be negative in the bacterial gene mutation test if it showed neither a dose-related increase in the mean number of revertant colonies nor a reproducible positive response at any of the concentrations tested.
Positive results from the bacterial reverse mutation test indicate that a test substance induces point mutations by base pair substitutions or frameshifts in the genome of either Salmonella typhimurium and/or Escherichia coli. Negative results indicate that, under the test conditions used, the test substance is not mutagenic in the tested strains.

Results and discussion

Test resultsopen allclose all
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
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
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
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
Positive controls validity:
valid
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
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
It is concluded that the results obtained in Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100, and in the Escherichia coli strain WP2 uvrA, in both the absence and presence of the S9-mix, indicate that the test substance is not mutagenic under the conditions used in this study.
Executive summary:

The test substance was examined for possible mutagenic activity in the bacterial reverse mutation test using the histidine-requiringSalmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and the tryptophan-requiring Escherichia coli strain WP2 uvrA, in the absence and presence of a liver fraction of Aroclor 1254-induced rats for metabolic activation (S9-mix). The study was performed according to OECD 471 (1997) and was conducted in compliance with OECD GLP. The test article was dissolved in DMSO. Two independent tests were performed. In both tests, all strains both in the absence and presence of S9-mix were tested. In the first test, five concentrations, ranging from 62 to 5000 μg/plate were tested and in the second test, six concentrations, ranging from 125 to 3000 μg/plate were tested. In the second test the maximum concentration tested was limited by the solubility of the test substance in the final treatment mix. In both tests, negative controls (solvent) and positive controls were run simultaneously with the test substance. In both tests, the mean numbers of his+ and trp+ revertant colonies of the negative controls used were within the acceptable range in all strains and the positive controls gave the expected increase in the mean numbers of revertant colonies. Therefore, both tests were considered valid. In both tests, no toxicity was observed in any strain, which was evidenced by an absence of a clearing of the background lawn of bacterial growth compared to the negative controls, absence of a decrease in the mean number of revertants was observed and absence of pinpoint colonies. In both tests, a precipitation of the test substance was observed in the final treatment mix and on the agar plates at and above 1667 μg/plate and at and above 1000 μg/plates in the first and second test, respectively. In the first test, the revertants could not be counted at a concentration of 5000 μg/plate due to the precipitation on the agar plates. In both tests, the test substance did not induce a more than 2-fold and/or dose related increase in the mean number of revertant colonies compared to the background spontaneous reversion rate observed with the negative control with strains TA 1535, TA 1537, TA 98, TA 100 and WP2 uvrA, in both the absence and presence of S9-mix. It is concluded that the results obtained in Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100, and in th eEscherichia coli strain WP2 uvrA, in both the absence and presence of the S9-mix, indicate that the test substance isnot mutagenicunder the conditions used in this study.