Registration Dossier

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:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
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 / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535
Additional strain / cell type characteristics:
other: rfa uvrB
Species / strain / cell type:
S. typhimurium TA 1537
Additional strain / cell type characteristics:
other: rfa uvrB
Species / strain / cell type:
S. typhimurium TA 98
Additional strain / cell type characteristics:
other: rfa uvrB pKM101
Species / strain / cell type:
S. typhimurium TA 100
Additional strain / cell type characteristics:
other: rfa uvrB pKM101
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 resultsopen allclose all
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
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
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
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
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.