Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 February - 27 March 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well conducted and well described study in accordance with GLP and OECD Guideline 471 without any deviation.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial forward mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
impurity
Test material form:
other: liquid, may crystalize to white solid
Specific details on test material used for the study:
- Name of test material (as cited in study report): Pivacyclene
- Physical state: Clear colourless liquid
- Analytical purity: 98.4 %
- Lot/batch No.: 712204531001
- Date received: 13 February 2007
- Storage condition of test material: At room temperature in the dark

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
other: Prior to the master strains being used, characterisation checks were carried out to confirm the amino-acid requirement, presence of rfa, R factors, uvrB or uvrA mutation and the spontaneous reversion rate.
Metabolic activation:
with and without
Metabolic activation system:
10 % (v/v) S9-fraction; S9 fraction prepared from liver homogenates of male Sprague-Dawley rats induced with three consecutive daily oral doses of phenobarbitone/β-naphthoflavone (80/100 mg/kg bw/day)
Test concentrations with justification for top dose:
Preliminary Toxicity Test:
- 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate in TA100 and WP2uvrA- strains, with and without S9 mix using the direct plate incorporation method

Mutation Test- Experiment 1 (Range-finding Test):
- 50, 150, 500, 1500 and 5000 µg/plate in all tester strains, with and without S9 mix using the direct plate incorporation method

Mutation Test- Experiment 2 (Main Test):
- 50, 150, 500, 1500 and 5000 µg/plate in all tester strains, with and without S9 mix using the direct plate incorporation method
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide (DMSO)
- Justification for choice of solvent/vehicle: The test material was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in DMSO at the same concentration in solubility checks performed in-house. DMSO was therefore selected as the vehicle.
- Test substance preparation: The test material was accurately weighed and approximate half-log dilutions prepared in DMSO by mixing on a vortex mixer on the day of each experiment. Formulated concentrations were adjusted to allow for the stated water/impurity content (1.6 %) of the test material. Prior to use, the solvent was dried using molecular sieves (sodium alumino-silicate) i.e., 2 mm pellets with a nominal pore diameter of 4 x 10^-4 microns.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: N-ethyl-N'-nitro-N-nitrosoguanidine: 2 µg/plate for WP2uvrA-, 3 µg/plate for TA100 and 5 µg/plate for TA1535; 9-Aminoacridine: 80 µg/plate for TA1537; 4-Nitroquinoline-1-oxide: 0.2 µg/plate for TA98
Remarks:
without S9-mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene: 1 µg/plate for TA100, 2 µg/plate for TA1535 and TA1537, 10 µg/plate for WP2uvrA-; Benzo(a)pyrene: 5 µg/plate for TA98
Remarks:
with S9-mix
Details on test system and experimental conditions:
SOURCE OF TEST SYSTEM: Salmonella typhimurium strains were obtained from the University of California at Berkeley on culture discs on 4 August 1995 whilst Escherichia coli strain WP2uvrA- was obtained from the British Industrial Biological Research Association on 17 August 1987.

METHOD OF APPLICATION: In agar (plate incorporation)

DURATION
- Incubation period: Plates were incubated at 37 °C for approximately 48 h

NUMBER OF REPLICATIONS:
- Preliminary Toxicity Test: Single plate/dose for treatment and vehicle control
- Experiment 1 (Range-finding Test) and Experiment 2 (Main Test): 3 plates/dose for treatment, vehicle and positive controls

DETERMINATION OF CYTOTOXICITY
- Method: Toxicity was determined by growth of the bacterial background lawn.

OTHER:
- Revertant colonies were counted using a Domino colony counter.
Evaluation criteria:
- There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS (Kirkland D J (Ed) (1989)) can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response.
- A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
- Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit a definitive judgement about the test material activity. Results of this type will be reported as equivocal.
Statistics:
No data

Results and discussion

Test resultsopen allclose all
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
Remarks:
The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
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
Remarks:
The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
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
Remarks:
The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
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
Remarks:
The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
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
Remarks:
The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: A light, oily precipitate was observed at 5000 µg/plate, this did not prevent the scoring of revertant colonies.

PRELIMINARY TOXICITY TEST:
- The test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA-).

COMPARISON WITH HISTORICAL CONTROL DATA:
- Results were compared with historical data (2005-2006)

Applicant's summary and conclusion

Conclusions:
Under the test conditions, Pivacyclene is not considered as mutagenic in S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 & E. coli WP2 uvr A strains.
Executive summary:

In a reverse gene mutation assay in bacteria, performed according to the OECD Guideline 471 and in compliance with GLP, strains ofSalmonella typhimurium(TA 1535, TA 1537, TA 98 and TA 100) and Escherichia coliWP2 uvr A were exposed to Pivacyclene at the following concentrations:

Preliminary Toxicity Test:

- 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate in TA100 and WP2uvrA- strains, with and without S9 mix using the direct plate incorporation method 

Mutation Test- Experiment 1 (Range-finding Test):

- 50, 150, 500, 1500 and 5000 µg/plate in all tester strains, with and without S9 mix using the direct plate incorporation method 

Mutation Test- Experiment 2 (Main Test):

- 50, 150, 500, 1500 and 5000 µg/plate in all tester strains, with and without S9 mix using the direct plate incorporation method

 

Metabolic activation system used in this test 10 % (v/v) S9-fraction; S9 fraction prepared from liver homogenates of male Sprague-Dawley rats induced with three consecutive daily oral doses of phenobarbitone/β-naphthoflavone (80/100 mg/kg bw/day). Vehicle and positive control groups were also included in mutagenicity tests.

 

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. A light, oily precipitate was observed at 5000µg/plate, this did not prevent the scoring of revertant colonies. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.The positive and vehicle controls induced the appropriate responses in the corresponding strains indicating the validity of the study.

Under the test conditions,Pivacycleneis not considered as mutagenic in these bacterial systems.