[5-(methanesulfonyl)pyridin-2-yl]methanaminium chloride

Genetic toxicity: in vitro

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:

18 Nov 2016 - 21 Nov 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

OECD 471

Data source

Reference

Title:

Unnamed

Year:

2017

Report date:

2017

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

Test material information

Constituent 1

Reference substance name:

(5-methanesulfonylpyridin-2-yl)methanaminium chloride

EC Number:

946-348-5

Cas Number:

848141-14-0

Molecular formula:

C7H10N2O2S x HCl

IUPAC Name:

(5-methanesulfonylpyridin-2-yl)methanaminium chloride

Test material form:

solid

Specific details on test material used for the study:

BI 730357 Sulfon CL was a white solid. It was received on
10 July 2016 and stored at 15-25°C, protected from light. Purity was stated as 99.8% (by
HPLC).

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 Sulfon CL 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: treatments of strains TA98, TA1535 and TA1537 were performed in the
absence of S-9 using pre-incubation methodology. The maximum test concentration was reduced to 625 μg/plate based on toxicity observed in Experiment 2. Narrowed
concentrations 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
TA1537) the concurrent vehicle control confirming discrimination between different
strains, and an active S-9 preparation.
3. At least five analysable concentrations of the test article were 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 was reproducible.
The test article was considered positive in this assay if all of the above criteria were met.
The test article was considered negative in this assay if none of the above criteria were met

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 results

Test results 1

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

Test results 2

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

Test results 3

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

Test results 4

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

other: none

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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 730357 Sulfon CL
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).

Executive summary:

BI 728741 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 a phenobarbitone/b-β-naphthoflavone-induced rat liver

post-mitochondrial fraction (S-9), in two separate experiments using plate incorporation and

pre-incubation treatment methodologies.

BI 728741 was dissolved in dimethyl sulphoxide (DMSO), and all concentrations are

expressed in terms of pure free base using a correction factor of 1.2.

Experiment 1 and Experiment 2 treatments of all the tester strains were performed using final

concentrations of BI 728741 ranging from 5 to 5000 μg/plate or from 156.3-5000 μg/plate in

Experiment 1 and Experiment 2 respectively, plus vehicle and positive controls. Following

these treatments, the only evidence of possible toxicity was observed at 5000 μg/plate in

strain TA1535 in the absence of S-9 and strain TA1537 in the presence of S-9 in

Experiment 1, where a reduction in revertant numbers occurred, with no evidence of toxicity

observed in Experiment 2.

The test article was completely soluble in the aqueous assay system at all concentrations

treated, in each of the experiments performed.

The numbers of revertant colonies were all acceptable for vehicle control treatments, and

were elevated by positive control treatments in all strains in both experiments.

Following BI 728741 treatments of all the test strains in the absence and presence of S-9, no

notable and concentration-related increases in revertant numbers were observed, and none

that were ≥2-fold (in strains TA98, TA100 and WP2 uvrA pKM101) or ≥3-fold (in strains

TA1535 and TA1537) the concurrent vehicle control. This study was considered therefore to

have provided no evidence of any BI 728741 mutagenic activity in this assay system.

It was concluded that BI 728741 did not induce 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 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)

using plate incorporation and pre-incubation treatment methodologies.