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EC number: 826-639-2 | CAS number: 1817776-86-5
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:
- 2 016
- 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
- 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
Constituent 1
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 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 examined
- True negative controls validity:
- other:
- Remarks:
- 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 examined
- True negative controls validity:
- other:
- Remarks:
- 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 examined
- True negative controls validity:
- other:
- Remarks:
- 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 examined
- True negative controls validity:
- other:
- Remarks:
- 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 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
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