7,8,9,10-Tetrahydro-8-(trifluoroacetyl)-6,10-methano-6H-pyrazino[2,3-h][3]benzazepine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 October - 13 November 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2,2,2-trifluoro-1-{5,8,14-triazatetracyclo[10.3.1.0²,¹¹.0⁴,⁹]hexadeca-2,4,6,8,10-pentaen-14-yl}ethan-1-one

EC Number:

810-682-9

Cas Number:

230615-70-0

Molecular formula:

C15H12F3N3O

IUPAC Name:

2,2,2-trifluoro-1-{5,8,14-triazatetracyclo[10.3.1.0²,¹¹.0⁴,⁹]hexadeca-2,4,6,8,10-pentaen-14-yl}ethan-1-one

Test material form:

solid: particulate/powder

Details on test material:

Buff coloured solid or pale brown powder depending on which batch
Storage conditions: room temperature. in the dark

Specific details on test material used for the study:

Buff coloured solid
Storage Conditions: room temperature in the dark
Batch number: 53650-10-11

Method

Target gene:

Bacteria Primary Mutation Detected
Salmonella ryphimurium TA1535 Base-pair substitution
Salmonella typhimurium TA 100 Base-pair substitution
Salmonella typhimzirium TA 1537 Frameshift
Salmonella typhimurium TA98 Frameshift
Escherichia coil WP2uvrA- Base-pair substitution
All of the above strains contain mutations in the histidine or tr-yptophan operon. thereby imposing a requirement for histidine! tryptophan in the growth medium. Three such mutations are involved in the Salmonella strains:
his G 46 in TA1535 and TA100
his C 3076 in TA1537
his D 3052 in TA98

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 mix prepared from liver of rats

Test concentrations with justification for top dose:

The highest concentration of CP-548,507 tested provided a final concentration of 5000 ug/plate.

In order to select appropriate dose levels for use in the main study a preliminary test was carried out to determine the toxicity of the test material. The concentrations tested were 0. 0.15. 0.5. 1.5. 5. 15. 50. 150. 500. 1500 and 5000 ug/plate.

Mutation Test 1: 50, 150, 500, 1500, and 5,000 ug/plate were assayed in triplicate against each tester strain, using the direct plate incorporation method.
Measured aliquots (0.1 ml) of one of the bacterial cultures were dispensed into sets of test tubes followed by 2.0 ml of molten, trace histidine or tryptophan supplemented, top agar, 0.1 ml of the test material formulation, vehicle or positive control and either 0.5 ml of S9-mix or phosphate buffer. The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated. in triplicate, for each bacterial strain and for each concentration of test material both with and without S9-mix. All of the plates were incubated at 37°C for approximately 48 hours and the frequency of revertant colonies assessed usine a Domino colony counter.

Mutation Test 2: The second experiment was performed using methodology as described for Experiment 1 using fresh bacterial cultures, test material and control solutions. The test material dose range was the same as Experiment 1 (50 to 5000 ug/plate).

Vehicle / solvent:

dimethyl sulphoxide

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

In the absence of S9 mix

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 2-Aminoanthracene

Remarks:

In the presence of S9 mix

Details on test system and experimental conditions:

The study was based on the in vitro technique described by Ames and his co-workers and Garner et al in which mutagenic activity is assessed by exposing histidine auxotrophs of Salmonella typhimurium and tryptophan auxotrophs of Escherichia coli to various concentrations of the test material. These strains have a deleted excision repair mechanism which makes them more sensitive to various mutagens and they will not grow on media which does not contain histidine. When large numbers of these organisms are exposed to a mutagen, reverse mutation to the original histidine independent form takes place. These are readily detectable due to their ability to grow on a histidine deficient medium. Using these strains of Salmonella tvphimurium revertants may be produced after exposure to a chemical mutagen, which have arisen as a result of a base-pair substitution in the genetic material (miscoding) or as a frameshift mutation in which genetic material is either added or deleted. Additionaly, a mutant strain of E coli (WP2uvrA ) which requires tryptophan and can be reverse mutated by base substitution to tryptophan independence will be used to complement the Salmonella strains. As many compounds do not exert their mutagenic effect until the have been metabolised by enzyme systems not available in the bacterial cell, the test compound and the bacteria are incubated in the presence of a liver microsomal fraction (S9) taken from an animal previously treated with a compound known to induce a high level of enzymatic activity.

Rationale for test conditions:

These test conditions are in accordance with the guidelines listed above.

Evaluation criteria:

The test material may be considered positive in this test system if the following criteria are met:
The test material should have induced a reproducible, dose-related and statistically (Dunnett's method of linear regression (5)) significant increase in the revertant count in at least one strain of bacteria.

Criteria for a Valid Assay
The following criteria will be used to determine a valid assay:
Tester Strain Integrity:
All tester strains must have demonstrated the required characteristics as determined by the strain checks performed. The tester strain cultures must exhibit a characteristic number of spontaneous revertants per plate when plated along with the vehicle under selective conditions. Although the number of spontaneous revertants can be expected to fall within the ranges, they may occasionally fall outside these. The experiment may, however, still be considered valid if the organisms respond normally to their respective positive controls.
Tester Strain Culture Density:
To demonstrate that appropriate numbers of bacterial cells have been plated, the cell count for each tester strain culture should be in the approximate range of 1 to 9.9 x 10^9 bacteria per ml.
Positive Control Values:
Acceptable positive control values demonstrate both the intrinsic sensitivity of the tester strains to mutagen exposure and the integrity of the S9-mix.
Cytotoxicity:
A minimum of four non-toxic test material dose levels will be required to evaluate assay data.
Contamination:
No evidence of excessive contamination.

Statistics:

Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS(5) can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

other: S. typhimurium TA 1535, TA1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 ug/plate. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

Applicant's summary and conclusion

Conclusions:

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies , both with and without metabolic activation. Thus. the sensitivity of the assay and the efficacy of the S9-mix were validated. The test material CP-548,507 caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was,therefore, tested up to the maximum recommended dose level of 5000 ug/plate. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. 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.

Executive summary:

CP-548,507 was considered to be non-mutagenic under the conditions of this test.