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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
No indication of genetic toxicity based on in vitro test results: bacterial reverse mutation test (Ames test)
Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20-DEC-1999 to 04-FEB-2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This GLP-compliant study was conducted in accordance with OECD guideline 471 and EU method B.13/B.14.
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Test material information:
Composition 1
Specific details on test material used for the study:
- Name of test material (as cited in study report): Perfluorosulfonyl vinyl-ether
- Impurities, purity test date: no data available
- Physical state: colourless liquid
- Lot/batch No.: 6-99
- Manufacturing date: 24 May 1999
- Expiration date of the lot/batch: May 2001
- Storage condition of test material: at room temperature
Target gene:
Histidine and tryptophan
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell lines (if applicable):
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: no data available
- Periodically checked for karyotype stability: yes; both permanent cultures and Master plates were checked to confirm the tester strain genotype.
- Periodically "cleansed" against high spontaneous background: no data available
Metabolic activation:
with and without
Metabolic activation system:
liver microsomal enzyme preparation (S9 mix)
Test concentrations with justification for top dose:
- Experiment 1 (plate incorporation - with and without metabolic activation): 5, 15, 50, 150, 500, 1500 and 5000 µg/pIate
- Experiment 2 (pre-incubation with metabolic activation; plate incorporation without metabolic activation): 50, 150, 500, 1500 and 5000 µg/plate
Vehicle:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: ethanol was the most suitable solvent for the test article.
Negative controls:
no
Solvent controls:
yes
Remarks:
100 µL/plate
True negative controls:
no
Positive controls:
yes
Remarks:
see in Table 1 below
Positive control substance:
methylmethanesulfonate
other: hydrazine sulfate, 9-aminoacridine HCl monohydrate, doxorubicine HCl, 2-aminoanthracene, 2-aminofluorene
Details on test system and conditions:
METHOD OF APPLICATION: in agar (plate incorporation) and pre-incubation
- Type and identity of media:
* Growth medium: S. typhimurium and E. coli strains were stored in growth medium supplemented with 25 µg/mL ampicillin (if ampicillin resistance bacteria) and 8% dimethyl sulfoxide, at -80°C (permanent cultures).
* Master plate: fresh bacterial cultures were subcultivated on complete medium (Master plate), and stored in a refrigerator at +4°C for up to 1 month. The liquid culture used to prepare Master plate or the first overnight culture from fresh Master plate was submitted to controls.
* Liquid growth medium: nutrient broth and NaCI dissolved in deionized water and sterilized
* Complete medium: nutrient broth, NaCI and agar dissolved in deionized water and sterilized
* Minimum medium (MM): selective medium consisting of glucose and agar dissolved in deionized water, sterilized and supplemented with a sterile solution Vogel-Bonner 50x (MgSO4.7H2O, citric acid.H2O, K2HPO4 anhydrous, NaNH4HPO4.4H2O)
* Soft agar: agar and NaCI dissolved in deionized water and sterilized
* Soft agar + His (S. typhimurium strains): soft agar supplemented with histidine and biotin
* Soft agar + Trp (E. coli strain): soft agar (at 60°C) supplemented with a filter-sterilized tryptophan solution

DURATION
- Pre-incubation period: 20 min at 37°C
- Exposure duration: 72 hours at 37°C
- Expression time (cells in growth medium): 72 hours

SELECTION AGENT: histidine and tryptophane

NUMBER OF REPLICATIONS: 3 plates per dose were prepared for each bacterial strain, both in the test without and with metabolic activation.

NUMBER OF CELLS EVALUATED: no data available

DETERMINATION OF CYTOTOXICITY
- Method: the colonies were counted using an automatic colony counter (Protos—Synoptics).

OTHER: After incubation the plates were examined for the presence of precipitation.
Evaluation criteria:
For the test to be considered valid, the following criteria must be met:
a) The S. typhimurium strains used in the test must prove to be histidine-requiring.
b) The E. coli strain used in the test must prove tryptophan-requiring.
c) The sterility check on test article and S9 must prove negative for bacterial growth.
d) The growth of all the strains must be inhibited by crystal violet; the growth of strains TA 1535 and TA 1537 must be inhibited by ampicillin, while the growth of strains TA 98 and TA 100 must not (periodic checks). The growth of all strains must be inhibited by exposure to UV rays.
e) The frequency of spontaneous reversions for each strain must fall within both the range reported in the literature and the historical range for our laboratory.
f) The activity of the microsomal preparation must be confirmed by its capability to activate the positive control (which requires a metabolic transformation in order to exert its mutagenic effect).
g) The number of reverted colonies owing to the mutagenic activity of the positive controls must be statistically greater than, and at least double the number of spontaneously reverted colonies (Student’s "t" test).

The test article was considered to have elicited a positive response when:
- the number of reverted colonies was significantly higher when compared with the number of revertants in the solvent controls (as determined by Student’s "t" test),
and
- either a dose-response could be verified, that was, a positive correlation between the number of revertants and the dose in an interval of at least 3 doses (linear regression test);
- or a statistically significant increase was recorded at one dose only, when confirmed in independent assays.
Statistics:
The mean and standard deviation were calculated for reversions read in each dosage group.
Comparison of the spontaneous reversions (in the control vehicle) with the ones in the test article plates and in the positive control plates were done by Student’s "t" test.
Significances were expressed as follows:
* p < 005
** p < 0.01
*** p < 0.001
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
Slight cytotoxicity (thinning of the background lawn) observed at 5000 µg/plate both with and without metabolic activation.
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
As expected, the reference mutagens induced a number of reverted colonies statistically greater than and at least double the mean number of spontaneous reverted colonies (Student's "t" test).

ADDITIONAL INFORMATION ON CYTOTOXICITY: at 5000 µg/plate the test substance proved to be slightly cytotoxic on the test system, causing thinning of the background lawn, both with and without metabolic activation. At the other concentrations tested with metabolic activation no significant cytotoxic effects were detected.
Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

The test article did not induce any significant increase in the number of mutant clones in Salmonella typhimurium TA 1535, TA 1537, TA 98 and TA 100 or Escherichia coli WP2 uvrA strains up to the concentration of 5000 µg/plate, in the absence and in the presence of metabolic activation, in two independent experiments.
Under the experimental conditions applied, the test article is not mutagenic in the Ames test.
Executive summary:

The mutagenic potential of the test substance was investigated using Salmonella typhirnurium TA 1535, TA 1537, TA 98 and TA 100 and Escherichia coIi WP2 uvrA as tester strains. The test method was in accordance with EU method B.13/B.14 and OECD guideline 471 and in compliance with good laboratory practices (GLP).

 

The study was performed using the plate incorporation assay with and without liver homogenate (S9 Mix). S9 fraction (9000 g supernatant) was prepared from adult male Sprague Dawley rats pre-treated by intraperitoneal route at 80 mg/kg (5 mL/kg) of a mixture of Phenobarbital Na and 3-Naphthoflavone, S9 Mix consisted of S9 plus cofactors.

 

Two independent experiments were performed, setting up triplicate plates for each experimental point. Hydrazine sulfate, 9-Aminoacridine HCI monohydrate, Doxorubicine HCI, Methylmethane sulfonate, 2-Aminofluorene and 2-Aminoanthracene served as positive controls to test the mutagenicity of the S. typhimurium and E. coli bacterial strains as well as the activity of the metabolizing system. The negative control was the test article solvent, i.e. ethanol

 

The preliminary toxicity test was performed as part of the first study, using the plate incorporation assay. In this trial seven test article concentrations, spaced approximately at half-log intervals, ranging from 5 to 5000 µg/plate, were tested, both with and without metabolic activation.

 

At 5000 µg/plate the test substance proved to be slightly cytotoxic on the test system, causing thinning of the background lawn, both with and without metabolic activation. At the other concentrations tested with metabolic activation no significant cytotoxic effects were detected.

 

A second trial was carried out following the pre-incubation method with metabolic activation and the plate method without metabolic activation.

 

In the concentration range investigated, the test substance did not show any mutagenic activity with or without the addition of S9 liver homogenate fractions. The known reversion properties were determined for the tester strains with the control substances; the positive responses confirmed the good metabolic activity of the liver homogenate fractions.

 

In conclusion, the test article did not induce any significant increase in the number of mutant clones in Salmonella typhimurium TA 1535, TA 1537, TA 98 and TA 100 or Escherichia coli WP2 uvrA strains up to the concentration of 5000 µg/plate, in the absence and in the presence of metabolic activation, in two independent experiments.

Under the experimental conditions applied, the test article is not mutagenic in the Ames test.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

The mutagenic potential of the test substance was investigated using Salmonella typhirnurium TA 1535, TA 1537, TA 98 and TA 100 and Escherichia coIi WP2 uvrA as tester strains. The test method was in accordance with EU method B.13/B.14 and OECD guideline 471 and in compliance with good laboratory practices (GLP).

 

Two independent experiments were performed: the first experiment was carried out according to the plate incorporation assay, with and without metabolic activation (S9 mix); the second trial was carried out following the pre-incubation method with metabolic activation and the plate method without metabolic activation.

 

At the highest dose (5000 µg/plate) the test substance proved to be slightly cytotoxic on the test system, causing thinning of the background lawn, both with and without metabolic activation. At the other concentrations tested with metabolic activation no significant cytotoxic effects were detected.

 

In the concentration range investigated, the test substance did not show any mutagenic activity with or without the addition of S9 liver homogenate fractions. The known reversion properties were determined for the tester strains with the control substances; the positive responses confirmed the good metabolic activity of the liver homogenate fractions.

 

In conclusion, the test article did not induce any significant increase in the number of mutant clones in Salmonella typhimurium TA 1535, TA 1537, TA 98 and TA 100 or Escherichia coli WP2 uvrA strains up to the concentration of 5000 µg/plate, in the absence and in the presence of metabolic activation, in two independent experiments.

According to REACh annex VII requirement, since no positive results were obtained under in vitro gene mutation study in bacteria, no further mutagenicity studies need be considered for the tonnage band of registration.

Justification for selection of genetic toxicity endpoint
The key study was conducted on the test item in compliance with GLP and according to OECD guideline 471 and EU method B.13/B.14. Well described and meeting the generally acceptable scientific principles, this study was awarded a reliability score of 1 in accordance with the Klimisch cotation criteria (1997).

Justification for classification or non-classification

No alerts for the presence of mutagenic properties have been identified for the substance under in vitro gene mutation study in bacteria. According to REACh annex VII requirements, no further mutagenicity studies need be considered for the tonnage band of registration.

No classification for germ cell mutagenicity according to the criteria of Regulation (EC) No. 1272/2008 is required.