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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:
03-AUG-1992 to 22-OCT-1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report Date:
1992

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
other: Ames et al. (1975)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
gas under pressure: liquefied gas
Details on test material:
- Name of test material (as cited in study report): Perfluorobutadiene

Method

Target gene:
Histidine and tryptophan
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 1538
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Metabolic activation system:
S9 microsomal fraction from Aroclor 1254-treated rat liver
Test concentrations with justification for top dose:
- 1st experiment: 0.3125, 0.625, 1.25, 2.5, 5 and 7.5% (v/v)
- 2nd experiment: 0.015, 0.05, 0.15, 0.5, 1.5 and 5% (v/v)
Vehicle / solvent:
- Vehicle used: air
- Justification for choice of vehicle: the test substance is a gas
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
air
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
methylmethanesulfonate
other: 4-acetylaminofluorene (4-AAF), vinyl chloride (gaseous positive control)
Remarks:
Methylmethanesulfonate (MMS) was dissolved in sterile, ultra-pure water. 9-aminoacridine (9-AA) and 4-acetylaminofluorene (4-AAF) were dissolved in dimethylsulphoxide (DMSO).
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
For use of S. typhimurium strains, top agar was prepared by adding 5 mL of 1 mM L-histidine and 1 mM biotin solution to 100 mL of dilute agar. For use of E. coli strain, top agar was prepared by adding 1 mL of 1.35 mM L-tryptophan to 100 mL dilute agar. Each top agar was thoroughly mixed prior to use. Agar preparations were kept in a water bath at a temperature not exceeding 45°C.
In the course of testing the substance, 2 mL of soft agar was dispensed to a small, plastic sterile tube. This was followed by 0.5 mL of S9 mix or 0.05 M phosphate buffer (pH 7.4) and, finally, the bacteria (0.1 mL). Continually cooling, the tube contents were mixed and then poured in minimal medium plates prepared in-house. These plates contained 25 mL 1.5% BBL purified agar in Vogel-Bonner Medium E with 2% glucose. When the soft agar had set, the plates were inverted and metal spacers inserted under the lids. The plates were then placed in wide-necked, straight-sided flasks of known volume (6.25 L). The greased lids were replaced and the openings filled with either Quickfit Dreschel tops (T-junctions) through which the gas passed into the jars, or suba-seal caps through which the gas was injected into the jars. The latter method was used when the concentrations to be tested were very low.
The method used for dosing the toxicity test and the gaseous positive control was to take, simultaneously, hydrocarbon-free air and test gas through separate calibrated rotameters and allowing the gases to mix before they passed into the incubation jars. Approximately 25 L of gas/air mixture were allowed to flush through each incubation jar before entrance and exit taps were switched off. In all cases, before plates were removed from the jars after 48-h incubation, at least 50 L of air were passed through the system to clear the test atmosphere.
The following control groups were established, duplicate plates being poured for each mean datum point:
- Air-only controls plated in duplicate with each strain used, both in the presence and absence of S9 mix.
- Gas control: vinyl chloride tested at 30% in air with S. typhimurium TA 1535 and TA 100 in the presence and absence of S9 mix.
- With S9 mix - non-gaseous control: 4-AAF tested at 1000 µg/plate with S. typhimurium TA 1538 and TA 98.
- Without S9 mix - non-gaseous control: 9-AA tested at 80 µg/plate with S. typhimurium TA 1537 and MMS at 200 µg/plate with E. coli.

DURATION
- Exposure duration: 48 h at 37°C
- Expression time (cells in growth medium): 24 h at 37°C following the initial 48-h incubation period

NUMBER OF REPLICATES: duplicate plates were prepared for each bacterial strain and dose level in both the presence and absence of S9 mix and for each of the two independent tests.

METHOD OF EVALUATION: colonies were counted using a Biotran III automated counter at a maximum sensitivity (i.e., colonies of 0.1 mm or more in diameter).

DETERMINATION OF CYTOTOXICITY
- Method: a toxicity test using strain TA 100 only was performed in the presence and absence of S9 mix to establish suitable dose levels for the mutation tests. One plate of 10, 50 and 100% (v/v) exposure levels each was used.

OTHER EXAMINATIONS: the plates were examined for precipitates and, microscopically, for microcolony growth.
Rationale for test conditions:
Standard test conditions were adapted to test the gaseous substance.
Evaluation criteria:
A test was considered acceptable if for each strain:
- The bacteria demonstrated their typical responses to crystal violet, ampicillin and UV light.
- At least one of the vehicle control plates were within the following ranges: TA 1535, 4-30; TA 1537, 1-20, TA98, 10-60, TA 100, 60-200, TA 1538, 10-35 and E. coli WP2urvA (pKM101), 10-100.
- On at least one of the positive control plates there were x 2 the mean vehicle control mutant numbers per plate. If the mean colony count on the vehicle control plates was less than 10, then a value of 10 was assumed for assessment purposes. In such cases a minimum count of 20 was required before a significant mutagenic response was identified.
- No toxicity or contamination was observed in at least 4 dose levels.
- In cases where a mutagenic response was observed, that no more than one dose level was discarded before the dose which gave the highest significant mean colony number.

Where these criteria were met, a significant mutagenic response was recorded if there was:
- For S. typhimurium strains TA 1535, TA 1537, TA 1538 and TA 98 and for E. coli, at least a doubling of the mean concurrent vehicle control values at some concentration of the test substances and, for S. typhimurium strains TA 100, a 1.5-fold increase over the control value. If the mean colony count on the vehicle control plates was less than 10, than a value of 10 was assumed for assessment purposes. In such cases a minimum count of 20 was required before a significant mutagenic response was identified.
- A reproducible effect in independent tests.
Statistics:
No data available

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
At the highest dose level of 7.5% in test 1 (without S9)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
from the dose level of 1.5% in the main tests, with and without S9
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
from the dose level of 1.5% in the main tests, with and without S9
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
from the dose level of 5% in the main test, with and without S9
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
from the dose level of 1.5% in the main tests, with and without S9
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
from the dose level of 1.5% in the main tests, with and without S9
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
ADDITIONAL INFORMATION ON CYTOTOXICITY: In the preliminary toxicity assay, high toxicity to S. typhimurium TA 100 strain was observed at all 3 concentrations of test gas used (i.e., 10, 50 and 100% (v/v)). At the 10% exposure level, some remnants of the background lawn of microcolonies were apparent although no revertant colonies were present. All subsequent exposures resulted in complete killing of the bacteria.
7.5% v/v was therefore selected as top dose for the test 1 of the main assay.
5% was selected as top dose in test 2 and 3 of the main assay.

Any other information on results incl. tables

Results are presented as mean number of revertants from duplicate plates in 3 separate tests

Applicant's summary and conclusion

Conclusions:
The test substance was not mutagenic to Salmonella typhimurium or Escherichia coli when tested as a gas at exposure levels extending into the toxic range.
Executive summary:

The test substance was tested as gas for mutagenic activity in Salmonella typhimurium  and Escherichia coli according to a protocol equivalent to OECD guideline 471 (adapted for a gaseous exposure) and in compliance with good laboratory practices (GLP).

Five S. typhimurium  strains TA1535, TA1537, TA1538, TA98, TA100, and one E. coli strain WP2uvrA were used in two independent experiments performed in duplicate, using a plate incorporation method. The tests were conducted at exposure levels ranging from 0.015 to 7.5% v/v, on agar plates placed in 6.25 litres jars and exposed to the gas for 48 hours, in the presence and absence of an Aroclor 1254 induced rat liver preparation and co-factors (S9 mix) required for mixed-function oxidase activity.

A toxicity test using S. typhimurium  strain TA 100 only was performed in the presence and absence of S9 mix to establish suitable dose levels for the mutation tests. One plate of 10, 50 and 100% (v/v) exposure levels each was used. Toxicity was observed at all 3 concentrations of test gas used. At the 10% exposure level, some remnants of the background lawn of microcolonies were apparent although no revertant colonies were present. All subsequent exposures resulted in complete killing of the bacteria. 

No mutagenic activity was observed in any of the six bacterial strains used. The lowest exposure level where toxicity to the bacteria strains was recorded was 1.5%. The positive and negative controls were within respective normal ranges.

It was concluded that the test substance was not mutagenic to Salmonella typhimurium  or Escherichia coli when tested as a gas at exposure levels extending into the toxic range.