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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type fo genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 March 2018 - 28 June 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
OECD 471

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
not specified
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
5-(Methylsulfonyl)pyridine-2-carbonitrile
EC Number:
682-451-9
Cas Number:
848141-13-9
Molecular formula:
C7 H6 N2 O2 S
IUPAC Name:
5-(Methylsulfonyl)pyridine-2-carbonitrile
Test material form:
solid
Specific details on test material used for the study:
BI 730357 Nitril was an light brown solid substance. It was received on
16 march 2018 and stored at 15-25°C, protected from light. Purity was stated as 99.2% (by
HPLC) and the expiry date was given as 31 March 2020.

Method

Target gene:
Four strains of Salmonella typhimurium bacteria (TA98, TA100, TA1535 and TA1537) and
one strain of Escherichia coli bacteria (WP2 uvrA pKM101) were used in this study. Strains
TA98, TA1535 and TA1537 were originally obtained from the UK NCTC. Strain TA100 was
derived from cultures originally obtained from Covance Laboratories Inc., USA. Strain WP2
uvrA pKM101 was obtained from MolTox Inc., USA.
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9Mix
Test concentrations with justification for top dose:
Experiment 1: concentrations of BI 730357 Nitril at 5, 16, 50, 160, 500, 1600 and 5000 μg/plate
Experiment 2 : The maximum test concentration of 5000 μg/plate was retained for all
strains. Narrowed concentration intervals were employed covering the range
80-5000 μg/plate,

Experiment 3: he maximum test concentration was reduced to 625 μg/plate based on toxicity observed in Experiment 2. Narrowed concentration
intervals were employed covering the range 300-625 μg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Controls
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
other: 2-aminoanthracene (AAN)
Details on test system and experimental conditions:
The test system was suitably labelled to clearly identify the study number, bacterial strain,
test article concentration (where appropriate), positive and vehicle controls, absence or
presence of S-9 mix.

Rationale for test conditions:
The assay was to be considered valid if the following criteria were met:
1. The vehicle control counts fell within the laboratory’s historical control ranges as defined
in Section 6.2
2. The positive control chemicals induced increases in revertant numbers of ≥2-fold (in
strains TA98, TA100 and WP2 uvrA pKM101) or ≥3-fold (in strains TA1535 and The assay was to be considered valid if the following criteria were met:
3. At least five analysable concentrations of the test article are available.
Evaluation criteria:
For valid data, the test article was considered to be mutagenic if:
1. A concentration related increase in revertant numbers was ≥2-fold (in strains TA98,
TA100 or, WP2 uvrA pKM101) or ≥3-fold (in strains TA1535 or TA1537) the concurrent
vehicle control values
2. Any observed response is reproducible.
The test article was considered positive in this assay if both of the above criteria were met.
The test article was considered negative in this assay if neither of the above criteria were met.
Results which only partially satisfied the above criteria were dealt with on a case-by-case
basis. Biological relevance was taken into account, for example consistency of response
within and between concentrations and (where applicable) between experiments.
Statistics:
Individual plate counts were recorded separately and the mean and standard deviation of the
plate counts for each treatment were determined. Control counts were compared with the
laboratory’s historical control ranges (Section 6.2 and Section 6.3). Data were considered
acceptable if the vehicle control counts fell within the calculated historical control ranges and
the positive control plate counts were comparable with the historical control ranges.
The presence or otherwise of a concentration response was checked by non-statistical
analysis, up to limiting levels (for example toxicity, precipitation or 5000 μg/plate).
However, adequate interpretation of biological relevance was of critical importance.

Results and discussion

Test resultsopen allclose all
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 specified
True negative controls validity:
other: none
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 specified
True negative controls validity:
other: none
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
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
True negative controls validity:
other: none
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 specified
True negative controls validity:
other: none
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 specified
True negative controls validity:
other: none
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
It was concluded that BI 730801 did not induce mutation in four histidine-requiring strains of
Salmonella typhimurium (TA98, TA100, TA1535 and TA1537), and one
tryptophan-requiring strain of Escherichia coli (WP2 uvrA pKM101) when tested under the
conditions of this study. These conditions included treatments at concentrations up to
5000 μg/plate (the maximum recommended concentration according to current regulatory
guidelines) in the absence and in the presence of a rat liver metabolic activation system (S-9).
Executive summary:

BI 730357 Nitril was assayed for mutation in four histidine-requiring strains (TA98, TA100,

TA1535 and TA1537) of Salmonella typhimurium, and one tryptophan-requiring strain (WP2

uvrA pKM101) of Escherichia coli, both in the absence and in the presence of metabolic

activation by phenobarbitone/β- naphthoflavone -induced rat liver post-mitochondrial fraction

(S-9), in two separate experiments using plate incorporation and pre-incubation treatment

methodology, respectively. A third experiment was performed in strains TA98, TA1535 and

TA1537 in the absence of S-9 only using pre-incubation methodology.

All BI 730357 Nitril

treatments in this study were performed using formulations prepared in

anhydrous analytical grade dimethyl sulphoxide (DMSO). The test article was completely

soluble in the aqueous assay system at all concentrations treated, in each of the experiments

performed.

Treatments of all the tester strains were performed in the absence and in the presence of S-9,

using final concentrations of BI 730357 Nitril

between 5 and 5000 μg/plate or 80 and 5000 μg/plate

in Experiment 1 and Experiment 2 treatments, respectively, plus vehicle and positive

controls. Following these treatments evidence of strain and condition-dependent toxicity was

observed starting at 1600 μg/plate or above in Experiment 1 and starting at 300 μg/plate or

above in Experiment 2.

As result of the extensive toxicity observed following Experiment 2 treatments of strains

TA98, TA1535 and TA1537 in the absence of S-9, a third experiment was performed in these

strains in the absence of S-9 at concentrations between 300 and 625 μg/plate. Following these

Experiment 3 treatments, evidence of toxicity was observed starting at 400 μg/plate or above.

The numbers of revertant colonies were all comparable with historical ranges for vehicle

controls, and were elevated by positive control treatments.

Following BI 730357 Nitril

treatments of all the test strains in the absence and presence of S-9, no

increases in revertant numbers were observed that were ≥2-fold (in strains TA98, TA100 and

WP2 uvrA pKM101) or ≥3-fold (in strains TA1535 and TA1537) the concurrent vehicle

control.

It was concluded that BI 730357 Nitril

did not induce mutation in four histidine-requiring strains of

Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and one tryptophan-requiring

strain of Escherichia coli (WP2 uvrA pKM101) when tested under the conditions of this

study using the plate incorporation and pre-incubation methodologies. These conditions

included treatments at concentrations up cytotoxic concentrations and/or to 5000 μg/plate

(the maximum recommended concentration according to current regulatory guidelines), and

testing in the absence and in the presence of a rat liver metabolic activation system (S-9).