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Genetic toxicity: in vitro

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Administrative data

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:
23 November 2009 to 7 December 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test conducted in compliance with recognised guidelines and to GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Japanese Ministry of Agriculture, Forestry and Fisheries. Test Data for Registration of Agricultural Chemicals, 12 Nohsan No. 8147, Guideline 2-1-19-1, Agricultural Production Bureau, November 24, 2000.
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: ICH (1996) Guideline S2A: Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals. PAB/PCD Notification No. 444.
Deviations:
no
Principles of method if other than guideline:
other guidelines quoted in the report are:

Official notice of J MHLW, METI and ME (21 November 2003):
YAKUSHOKUHATSU No.1121002
SEIKYOKU No.2
KANPOKIHATSU No. 031121002

Official Notice of J MOL (8 February 1999)

ICH (1998) Guideline S2B: Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals. PMSB/ELD Notification No. 554.
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
2,2,3,3-tetrafluorooxetane
EC Number:
700-202-5
Cas Number:
765-63-9
Molecular formula:
C3H2F4O
IUPAC Name:
2,2,3,3-tetrafluorooxetane
Details on test material:
- Name of test material (as cited in study report): 2,2,3,3-tetrafluorooxetane (TFO)
- Physical state: Transparent liquid
- Analytical purity: 99%
- Purity test date: no data given
- Lot/batch No.: TFOS285001
- Expiration date of the lot/batch: 30 April 2010
- Stability under test conditions: no data given
- Storage condition of test material: Room temperature

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Rat liver homogenate fraction (S9 mix)
Test concentrations with justification for top dose:
5-5000 µg per plate of TFO (test 1)
5-500 µg per plate of TFO (test 2)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: TFO not miscible with water but miscible with DMSO, therefore DMSO used.
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
DMSO
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Concentration 2 µg per plate

Migrated to IUCLID6: TA100 & TA1535 without S9 mix
Positive control substance:
9-aminoacridine
Remarks:
Concentration 50 µg per plate

Migrated to IUCLID6: TA1537 without S9 mix
Positive control substance:
2-nitrofluorene
Remarks:
Concentration 2 µg per plate

Migrated to IUCLID6: TA98 without S9 mix
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
Concentration 2 µg per plate

Migrated to IUCLID6: WP2 uvrA (pKM101) without S9 mix
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene, TA100, TA1535 & WP2 uvrA (pKM101) with S9 mix
Remarks:
Concentration 5 µg per plate for TA100, TA1535, 10 µg per plate for WP2 uvrA (pKM101)
Positive control substance:
benzo(a)pyrene
Remarks:
Concentration 5 µg per plate

Migrated to IUCLID6: TA98 & TA1537 with S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 30 minutes
- Exposure duration: 72 hours

Concentrations of TFO up to 5000 µg/plate were tested initially. This is the standard limit concentration recommended in the regulatory guidelines that this assay follows. Other concentrations used were a series of ca half-log10 dilutions of the highest concentration. A lower maximum concentration was tested in some strains in the second test, in order to avoid excessive toxicity.
Evaluation criteria:
If exposure to a test substance produces a reproducible increase in revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537) the concurrent vehicle controls, with some evidence of a positive dose-response relationship, it is considered to exhibit mutagenic activity in this test system.
If exposure to a test substance does not produce a reproducible increase in revertant colony numbers, it is considered to show no evidence of mutagenic activity in this test system.
Statistics:
No statistical analysis was performed.

Results and discussion

Test resultsopen allclose all
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:

COMPARISON WITH HISTORICAL CONTROL DATA:

The mean revertant colony counts for the vehicle controls were within or close to the 99% confidence limits of the current historical control range of the laboratory. (For tabulated historical data, see attachment below).

Appropriate positive control chemicals (with S9 mix where required) induced substantial increases in revertant colony numbers with all strains in all reported tests, confirming sensitivity of the cultures and activity of the S9 mix.








Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

The absence of colonies on sterility check plates confirmed the absence of microbial contamination of the S9 mix, buffer and test substance formulation.

The viability counts confirmed that the viable cell density of the cultures of the individual organisms exceeded 109/mL in all cases, and therefore met the acceptance criteria.

For results tables, see the attachments given below.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
positive

TFO exhibited mutagenic activity in this bacterial system under the test conditions employed. Activity was enhanced following metabolic activation.

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