(2S-cis)-(diphenylmethyl)-N-(phenylmethyl)-1-azabicyclo[2.2.2.]octan-3-amine (1-R camphor-10-sulfonate)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

444-810-6

EC Name:

-

Molecular formula:

C27 H30 N2 * C10 H16 O4 S

IUPAC Name:

(2S-cis)-(diphenylmethyl)-N-(phenylmethyl)-1-azabicyclo[2.2.2.]octan-3-amine (1-R camphor-10-sulfonate)

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

Identity: CP-163,625-BV
Chemical name: (2-Benzhydry1-1-aza-bicyclo [2.2.2] oct-3-y1)-benzylamine
Intended use: Pharmaceutical intermediate
Appearance: White powder
Storage conditions: Room temperature in the dark
Lot No: 3 8654-1 87-3
Purity: 99%

Method

Target gene:

histidine ( S.typhimurium)
Tryptophan ( E.coli)

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

other: rfa uvrB

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1537

Additional strain / cell type characteristics:

other: rfa uvrB

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

other: rfa uvrB pKM101

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

other: rfa uvrB pKM101

Species / strain 5

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

Male SD rat liver S9 fraction, aroclor 1254 induced

Test concentrations with justification for top dose:

5, 15, 50, 150, 500, 1500, 5000 µg/plate. top dose is limit dose for assay

Vehicle / solvent:

Prior to commencing testing, the solubility of the test substance was assessed at 50 mg/nil in water in
which it was insoluble and in dimethyl sulphoxide, in which it dissolved. Therefore dimethyl sulphoxide
was used as the solvent for this study.

Controls

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-Aminoanthracene

Details on test system and experimental conditions:

First test
The test substance was added to cultures of the five tester strains at seven concentrations separated by ca
half-log10 intervals. The highest concentration of CP-163,625-BV tested was 50 mg/ml in the chosen
solvent, which provided a final concentration of 5000 pg/plate. This is the standard limit dose
recommended in the regulatory guidelines this assay follows. The negative control was the chosen solvent,
clirnethyl sulphoxide. The appropriate positive controls were also included.
An aliquot of 0.1 ml of a 10 hour bacterial culture and 0.5 ml S9 mix or 0.5 ml 0.1 M phosphate buffer
(pH 7.4) were placed in glass bottles. An aliquot of 100 p.1 of the test solution was added, followed
immediately by 2 ml of histidine/tryptophan deficient agar. The mixture was thoroughly shaken and
overlaid onto previously prepared petri dishes containing 25 ml minimal agar. Each petri dish was
individually labelled with a unique code corresponding to a sheet, identifying the dish's contents. Three
petri dishes were used for each dose level. Plates were also prepared without the addition of bacteria in
order to assess the sterility of the test substance, S9 mix and phosphate buffer. All plates were incubated at
37°C for ca 72 hours. After this period the appearance of the background bacterial lawn was examined
and revertant colonies counted using a Seescan automated colony counter.
Any toxic effects of the test substance would be detected by a substantial reduction in revertant colony
counts or by the absence of a complete background bacterial lawn. In the absence of any toxic effects the
top concentration normally used in the second test would be the same as that used in the first. If toxic
effects were observed a lower concentration may be chosen. It should be ensured that if a lower
concentration was chosen, signs of bacterial inhibition are present at the top concentration. Ideally a
minimum of three non-toxic concentrations should be obtained.

Second test
As a clear negative response was obtained in the first test, a variation to the test procedure was used for the
second. The variation used was the pre-incubation assay in which the bottles were incubated at 37°C for
30 minutes with shaking before the addition of the agar overlay. 50001Ag/plate was again chosen as the top
concentration and six dose levels were used.

Evaluation criteria:

See below

Statistics:

If no clear "positive" response can be obtained, the test data may be subjected to analysis to determine the statistical significance of any observed increases in revertant colony numbers. The statistical procedures used will be those described by Mahon et al (1989) and will usually be analysis of variance followed by Dunnett's test.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Toxicity was observed towards all the tester strains at 5000 µs/plate.

Any other information on results incl. tables

No substantial increases in revertant colony numbers of any of the tester strains were observed following treatment with CP-163,625-BV at any dose level, in the presence or absence of S9 mix, in either mutation test. The mean revertant colony counts for the solvent controls were within the historical range. The concurrent positive controls demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations by causing increases over double the concurrent solvent control.

Applicant's summary and conclusion

Conclusions:

It is concluded that, when tested in dimethyl sulphoxide, CP-163,625-BV shows no evidence of mutagenic activity in this bacterial system.

Executive summary:

In this in vitro assessment of the mutagenic potential of CP-163,625-BV, histidine dependent auxotrophic mutants of Salmonella typhimurium (strains TA 1535, TA 1537, TA98 and TA100) and a tryptophan dependent mutant of Escherichia coil . (strain CM891) were exposed to the test substance diluted in dimethyl sulphoxide, which was also used as a negative control. Two independent mutation tests were performed in the presence and absence of liver preparations from Aroclor 1254-induced rats (S9 mix). The first was a standard plate incorporation assay, the second involved a pre-incubation stage. Dose levels of up to 5000 µg/plate were tested in the mutation tests. This is the standard limit dose recommended in the regulatory guidelines this assay follows. Other dose levels used were a series of ca half-log10 dilutions of the highest concentration. Toxicity was observed towards the tester strains in the first mutation test at 5000 µg/plate and at 5000 and 1500 µg/plate in the second mutation test. No evidence of mutagenic activity was seen at any dose level of CP-163,625-BV in either mutation test. The concurrent positive controls demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations. It is concluded that, when tested in dimethyl sulphoxide, CP-163,625-BV shows no evidence of mutagenic activity in this bacterial system.