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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:
January 14-22, 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
5-Amino-1-(tetrahydro-pyran-4-yl)-1H-pyrazole-4-carbonitrile
EC Number:
821-117-0
Cas Number:
1082745-49-0
Molecular formula:
C9H12N4O
IUPAC Name:
5-Amino-1-(tetrahydro-pyran-4-yl)-1H-pyrazole-4-carbonitrile
Test material form:
solid: particulate/powder

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
with & without metabolic activation: CD 11404 BS concentrations used are 100, 300, 1000, 3000 & 5000 μg/plate

A maximum concentration of 5000 μg/plate should be investigated according to relevant
guidelines. In a non-GLP solubility pretest no precipitation was observed at the highest
concentration of 5000 μg/plate. Therefore, 5000 μg/plate was investigated as the highest
concentration.
Vehicle / solvent:
dimethylsulfoxide (DMSO)
Controls
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
mitomycin C
other: 2-Aminoanthracene
Details on test system and experimental conditions:
0.1 mL of the vehicle, test substance or positive control solution, 0.1 mL of a bacterial
shaking culture (6 h, exponential phase) and 0.5 mL phosphate buffer or S9 mix were added
to 2 mL soft agar. After vortexing, these mixtures were overlaid immediately onto minimal
medium plates in triplicate (n = 6 for the negative control).
The test was performed in the presence and absence of rat liver microsomal enzymes
(S9 mix).
No repeat experiment using preincubation was performed because the result was clearly
negative.

Revertant his+ colonies were counted using an ARTEK Counter 880 (non-computerized) after
incubation at 37°C for 2 days (TA 102: 3 days) and the values were listed manually in a word
document. For all replicate platings, the mean number of revertants per test concentration was
calculated. The condition of the background bacterial lawn (residual growth on minimum
histidine) was evaluated macroscopically for evidence of bacteriotoxicity induced by the test
substance. If extreme thinning or complete lack of the microcolony lawn compared to the
negative, vehicle control plates was observed, no revertants were counted. Evidence of test
substance precipitates in the agar overlay was recorded.

Rationale for test conditions:
The assay was considered valid since the following criteria were met:
All tester strains exhibit a characteristic number of spontaneous revertants per plate. The
addition of the metabolic activation system did not alter significantly the number of
spontaneous revertants per plate and therefore the numbers were combined and given as
ranges (see below). These ranges were taken from about 168 experiments (not filed in the raw
data) conducted in our laboratory.
TA 1535: 5 - 22
TA 1537: 3 - 29
TA 98: 16 - 68
TA 100: 57 - 197
TA 102: 252 - 531
In addition, the reference mutagens induced a distinct increase in the number of revertants,
reflecting also the activity of the metabolizing system.
Evaluation criteria:
A reproducible, concentration-dependent increase in the number of revertants of at least one
tester strain over the vehicle control value and/or outside the historical control range is
indicative of genotoxic activity.
Statistics:
No recognizable incidents that might have affected the quality or integrity of the
experimental outcome were noted during the study. The experimental findings are
summarized in Tables 8: 1 (summary) and individual values are given in Tables 8: 2 to 8: 6.

SOLUBILITY AND CYTOTOXICITY
CD 11404 BS did not precipitate and no bacteriotoxicity was observed up to the maximum
recommended concentration of 5000 μg/plate.

MUTAGENICITY
The OD600 of the individual overnight bacterial cultures varied between 2.2 and 2.8 (raw
data). These values correspond to a bacterial titer of ca. 100 millions/0.1 mL.
CD 11404 BS did not increase the number of revertant colonies in different tester strains of
S. typhimurium compared to the negative control when tested up to maximum recommended
concentration. Furthermore, metabolic activation by S9 mix did not alter the mutation
frequency of these bacterial strains.
The validity of this study is given since the vehicle control plates showed spontaneous
revertants in different tester strains of S. typhimurium at frequencies similar to those
described in the literature and within the historical control range experienced in our
laboratory.
The mutagens NaN3, 9-AA, 2-NF, MMC and 2-AA showed the expected strain specific
responses with and without metabolic activation.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
CD 11404 BS caused neither base-pair substitutions nor frameshift mutations in different
strains of S. typhimurium in the presence and absence of metabolic activation when tested up
to the maximum recommended concentration of 5000 μg/plate. Based on these results it was
concluded, that CD 11404 BS is "Ames negative".