(4-cyclopropyl-6-methyl-pyrimidin-5-yl)boronic acid

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:

12 -28 Jul 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The assay design is bsed on the OECD Guideline 471

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

(4-cyclopropyl-6-methyl-pyrimidin-5-yl)boronic acid

EC Number:

826-639-2

Cas Number:

1817776-86-5

Molecular formula:

C8 H11 B N2 O2

IUPAC Name:

(4-cyclopropyl-6-methyl-pyrimidin-5-yl)boronic acid

Test material form:

solid: particulate/powder

Method

Target gene:

The tester strains used were the Salmonella typhimurium histidine auxotrophs TA98, TA100,
TA1535 and TA1537 as described by Ames et al. (1975) and Escherichia coli WP2 uvrA as
described by Green and Muriel (1976)

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:

S9 mix

Test concentrations with justification for top dose:

6.7, 10, 33, 67, 100, 333, 667, 1000, 3333, 5000 ug/ plate test substance

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene

Details on test system and experimental conditions:

Tester strains TA98 and TA1537 are reverted from histidine dependence (auxotrophy) to
histidine independence (prototrophy) by frameshift mutagens. Tester strain TA1535 is reverted
by mutagens that cause basepair substitutions. Tester strain TA100 is reverted by mutagens that
cause both frameshift and basepair substitution mutations. Specificity of the reversion
mechanism in E. coli is sensitive to basepair substitution mutations, rather than frameshift
mutations (Green and Muriel, 1976).

Each
culture was monitored spectrophotometrically for turbidity and was harvested at a percent
transmittance yielding a titer of greater than or equal to 0.3x109 cells per milliliter. The actual
titers were determined by viable count assays on nutrient agar plates.

Rationale for test conditions:

On the day of use in each assay, all tester strain cultures were checked for the appropriate genetic
markers.

Evaluation criteria:

For each replicate plating, the mean and standard deviation of the number of revertants per plate
were calculated and are reported.
For the test article to be evaluated positive, it must cause a dose-related increase in the mean
revertants per plate of at least one tester strain over a minimum of two increasing concentrations
of test article

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. 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 examined

True negative controls validity:

other:

Remarks:

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 examined

True negative controls validity:

other:

Remarks:

none

Positive controls validity:

valid

Test results 3

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:

other:

Remarks:

none

Positive controls validity:

valid

Test results 4

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:

other:

Remarks:

none

Positive controls validity:

valid

Test results 5

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 examined

True negative controls validity:

other:

Remarks:

none

Positive controls validity:

valid

Any other information on results incl. tables

All criteria for a valid study were met. IN00079056 did not cause a positive mutagenic response

with any of the tester strains in either the presence or absence of S9 activation.

Under the conditions of this study, IN00079056 was concluded to be negative in the Bacterial

Reverse Mutation Assay when tested up to maximum recommended concentrations.

Applicant's summary and conclusion

Conclusions:

In the mutagenicity assay, the dose levels tested were 33.3, 100, 333, 1000, 3333 and 5000 μg
per plate. Neither precipitate nor toxicity was observed. No positive mutagenic responses were
observed with any of the tester strains in either the presence or absence of S9 activation.
All criteria for a valid study were met. IN00079056 did not cause a positive mutagenic response
with any of the tester strains in either the presence or absence of S9 activation.
Under the conditions of this study, IN00079056 was concluded to be negative in the Bacterial
Reverse Mutation Assay when tested up to maximum recommended concentrations

Executive summary:

The test article, IN00079056, was tested in the Bacterial Reverse Mutation Assay using

Salmonella typhimurium tester strains TA98, TA100, TA1535 and TA1537 and Escherichia coli

tester strain WP2 uvrA in the presence and absence of Aroclor-induced rat liver S9. The

preliminary toxicity assay was performed to establish the dose-range for the mutagenicity assay.

The mutagenicity assay was performed to evaluate the mutagenic potential of the test article.

Both trials were performed using the plate incorporation method.

Dimethyl sulfoxide (DMSO) was the vehicle of choice based on the solubility of the test article

and compatibility with the target cells. The test article formed a clear solution in DMSO at a

concentration of approximately 100 mg/mL in the solubility test conducted at BioReliance. The

formulation prepared in the solubility test also was adjusted using a correction factor of 1.1.

In the preliminary toxicity assay, the dose levels tested were 6.67, 10.0, 33.3, 66.7, 100, 333,

667, 1000, 3333 and 5000 μg per plate. Neither precipitate nor toxicity was observed. Based

upon these results, the maximum dose tested in the mutagenicity assay was 5000 μg per plate.

In the mutagenicity assay, the dose levels tested were 33.3, 100, 333, 1000, 3333 and 5000 μg

per plate. Neither precipitate nor toxicity was observed. No positive mutagenic responses were

observed with any of the tester strains in either the presence or absence of S9 activation.

All criteria for a valid study were met. IN00079056 did not cause a positive mutagenic response

with any of the tester strains in either the presence or absence of S9 activation.

Under the conditions of this study, IN00079056 was concluded to be negative in the Bacterial

Reverse Mutation Assay when tested up to maximum recommended concentrations