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EC number: 276-940-2 | CAS number: 72846-00-5
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Genetic toxicity: in vitro
Administrative data
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2018-04-30 - 2018-05-18
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 018
- Report date:
- 2018
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
Test material
- Reference substance name:
- 1-benzyl-5-phenylbarbituric acid
- EC Number:
- 276-940-2
- EC Name:
- 1-benzyl-5-phenylbarbituric acid
- Cas Number:
- 72846-00-5
- Molecular formula:
- C17H14N2O3
- IUPAC Name:
- 1-benzyl-5-phenylpyrimidine-2,4,6(1H,3H,5H)-trione
- Test material form:
- solid: particulate/powder
Constituent 1
- Specific details on test material used for the study:
- Molecular Formula:
C17H14N2O3
Molecular Weight:
294.31
Characteristics (Physical Appearance):
White powder
CAS No.:
72846-00-5
Batch Number:
170240
Purity:
99.5%
Method
- Target gene:
- All the tester strains are defective in DNA repair capacity (uvrB), except TA102 and all tester strains have a defective lipopolysaccharide barrier on the cell wall (rfa). These two properties confer extra sensitivity to DNA damage and also increase permeability to large molecules that do not penetrate the normal cell wall.
Strains TA97a, TA98, TA100 and TA102 also contain resistance transfer factor (plasmid pKM101). This factor, which confers resistance to ampicillin, enhances the operation of an error prone repair system.
In case of TA102, it contain plasmid pAQ1, which confers resistance to tetracycline.
Strains TA1535 and TA100 are highly sensitive to base - pair substitution mutation and TA97a, TA98 are highly sensitive to frame - shift mutation.
The strain TA102 contains ochre mutation.
Species / strainopen allclose all
- Species / strain / cell type:
- S. typhimurium TA 97
- Species / strain / cell type:
- S. typhimurium TA 98
- Species / strain / cell type:
- S. typhimurium TA 100
- Species / strain / cell type:
- S. typhimurium TA 102
- Species / strain / cell type:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Test concentrations with justification for top dose:
- Maximum concentration of 5000 μg/plate, which was found non cytotoxic to TA100 in the preliminary cytotoxicity, was selected as the highest concentration for main study, with four lower concentrations viz. 1500, 500, 150 and 50 μg/plate. Triplicate plating was performed at each dose level for an adequate evaluation of the variation.
- Vehicle / solvent:
- DMSO
Controls
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- sodium azide
- methylmethanesulfonate
- other: ICR191, 2-Aminoanthracene, 2-Aminofluorene, Danthron
- Details on test system and experimental conditions:
- PREPARATION OF TEST CULTURES
Fresh cultures for mutagenicity testing were prepared by transferring 40 μl of frozen permanent of Salmonella typhimurium to sterile tubes containing 10 ml of Oxoid Nutrient Broth No 2. Inoculated cultures were incubated in orbital shaker incubator for about 16 hours at 37 ºC to achieve cell density of about 1-2×10˄9 cells / ml.
Bacterial Cell Count
The bacterial cell count was taken by hemocytometer. All the 25 small squares of RBC chamber were counted under high power (40X). Number of bacterial cells / ml of the culture was calculated by formula:
Total number of bacterial cells / ml of the culture = Sum of the cells counted in 25 small squares in the central ruled area of hemocytometer x 104 x dilution factor (10˄2).
MEDIUM
The bacterial strains were cultured in Oxoid Nutrient Broth No. 2. Minimal glucose agar contained agar, Vogel-Bonner minimal medium E and 2% glucose. The top agar contained 0.6% agar, 0.5% NaCl and 0.05 mM histidine - biotin (Maron and Ames, 1983).
METABOLIC ACTIVATION
The cofactor supplemented post-mitochondrial fraction (S9) was employed as the metabolic activation system.
Preliminary Cytotoxicity Test
Before commencing the study, the test item was assessed for cytotoxicity to the tester bacteria using tester strain TA100. Eight concentrations viz. 5000, 4000, 3000, 2000, 1000, 500, 250 and 125 μg/plate of the test item, formulated in DMSO, were tested for toxicity to bacterial cells.
No cytotoxicity was observed to revertant colonies and bacterial background lawn at the concentrations from 5000 to 125 μg/plate both in the absence and presence of metabolic activation system. Hence, 5000 μg/plate was selected as the maximum test concentration for main study.
CONTROLS
Concurrent vehicle control and strain specific positive controls, both with and without metabolic activation, were included in each assay.
Vehicle Control
DMSO was employed as a vehicle. Plates containing the reaction mixture comprising of vehicle and top agar supplemented with histidine but not added with 1-Benzyl-5-Phenylbarbituric acid (BPBA) served as a control to check spontaneous revertants.
EXPERIMENTAL PROCEDURE – PRE-INCUBATION ASSAY
Maximum concentration of 5000 μg/plate, which was found non cytotoxic to TA100 in the preliminary cytotoxicity, was selected as the highest concentration for main study, with four lower concentrations viz. 1500, 500, 150 and 50 μg/plate. Triplicate plating was performed at each dose level for an adequate evaluation of the variation.
PRE-INCUBATION ASSAY WITHOUT METABOLIC ACTIVATION
Aliquots of 0.1 ml cell suspension of the tester strains, containing about 108 viable cells, were added to sets of sterile tubes. Sterile phosphate buffer (0.5 ml) and freshly prepared test item / control sample / positive control (0.1 ml) were added to the tube. All the constituents of reaction mixture were introduced into sterile test tubes on an ice bath. The tubes with reaction mixture were mixed by shaking gently and were incubated at 37 ºC for 20 minutes in orbital shaker incubator.
Finally 2 ml of top agar with L-histidine and D-biotin solution was added. The contents were mixed and poured over the surface of the petri plates containing minimal glucose VB agar and allow them to solidify.
PRE-INCUBATION ASSAY WITH METABOLIC ACTIVATION
Aliquots of 0.1 ml cell suspension of the tester strains, containing about 108 viable cells, were added to sets of sterile tubes. The S9 mixture (0.5 ml) and freshly prepared test item / control sample / positive control (0.1 ml) were added to the tube. All the constituents of reaction mixture were introduced into sterile test tubes on an ice bath. The tubes with reaction mixture were mixed by shaking gently and were incubated at 37 ºC for 20 minutes in orbital shaker incubator.
Finally 2 ml of top agar with L-histidine and D-biotin solution was added. The contents were mixed and poured over the surface of the petri plates containing minimal glucose VB agar and allow them to solidify.
INCUBATION
Contents of all the plates were allowed to solidify after which the plates were inverted and incubated at 37 ºC for about 48 hours for Experiment No. 1 and Experiment No. 2.
TEST FOR STERILITY
Sterility check was performed along with the assay for the laminar flow, vehicle, top agar, S9 mix and the highest employed dose of the test item. Nutrient agar plates were exposed in the laminar hood for 30 minutes during experimental procedure. Vehicle, S9 mix and the highest employed dose of the test item were mixed in the top agar at the same volume that was used in the assay, but in the absence of tester bacteria. These were plated on nutrient agar plates. Top agar alone was also plated on nutrient agar plates. All plating was done in triplicate. Plates were incubated for about 48 hours for Experiment No. 1 and Experiment No. 2 at 37 ºC and then assessed for presence of contamination if any, in the form of growth of colonies on the nutrient agar plates.
OBSERVATIONS
After incubation period, the plates were checked for sterility. The plates were observed for a uniform lawn of auxotrophs (his-) and the colonies for histidine revertants as the prototrophs (his+). Histidine revertant colonies per plate were counted and the mean number of colonies at each test point was calculated. - Evaluation criteria:
- Criteria for Evaluation
1. Criteria for a Positive Response
A test item is considered to be positive (mutagenic), if it induces a concentration dependent increase and / or a reproducible increase at one or more concentrations in the number of revertant colonies per plate, in at least one strain with or without metabolic activation system, which is at least 2-fold (3-fold for TA1535) of that observed in the corresponding concurrent vehicle control.
2. Criteria for a Negative Response
A test item for which the results do not meet the above criteria is considered non-mutagenic in this test. In order a substance considered to be negative if the revertant colonies cannot be greater than 2 (or 3 for strain TA 1535), or less than 0.5 in at least 5 doses for all strains tested.
However, reproducibility of negative results was confirmed by repeat experimentation.
3. Criteria for an Equivocal Response
Occasionally, a test item cannot be judged to be positive or negative [e.g., concentration dependent increases that fail to reach 2-fold (3-fold for TA1535) control values, or 2-fold (3-fold for TA1535) increases that do not appear to be concentration dependent]. In these rare instances, the results may be classified as equivocal. Equivocal or weak positive results may indicate the need to repeat the test, possibly with a modified study plan such as appropriate spacing of dose levels.
Results and discussion
Test resultsopen allclose all
- Key result
- Species / strain:
- S. typhimurium TA 97
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 102
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
Applicant's summary and conclusion
- Conclusions:
- Salmonella typhimurium, Reverse Mutation Assay of 1-Benzyl-5-Phenylbarbituric acid (BPBA) was carried out in compliance with the Organization for Economic co-operation and Development (OECD) Guidelines for Testing of Chemicals (Guideline No. 471, Section 4: Health Effects) on conduct of "Bacterial Reverse Mutation Test", adopted by the council on 21 July 1997.
Under the conditions described for this study, it is concluded that, 1-Benzyl-5-Phenylbarbituric acid (BPBA) is non-mutagenic in Salmonella typhimurium strains TA97a, TA98, TA100, TA102 and TA1535. - Executive summary:
‘Salmonella typhimurium, Reverse Mutation Assay of 1-Benzyl-5-Phenylbarbituric acid (BPBA) was carried out in compliance with the OECD Guidelines for Testing of Chemicals (Guideline No. 471, Section 4: Health Effects) on conduct of "Bacterial Reverse Mutation Test", adopted 21 July 1997 and as per mutually agreed study plan.
1-Benzyl-5-Phenylbarbituric acid (BPBA) was evaluated in Ames test to determine its ability to induce reverse mutation at selected histidine loci in five tester strains of Salmonella typhimurium viz. TA1535, TA97a, TA98, TA100 and TA102 in the presence and absence of metabolic activation system (S9). Based upon the preliminary tests conducted to assess the solubility / precipitation and cytotoxicity of 1-Benzyl-5-Phenylbarbituric acid (BPBA), the tester strains were exposed to the test item in triplicate cultures at the concentrations of 5000, 1500, 500, 150 and 50 μg/plate, both in the presence and absence of metabolic activation system (S9). Liver S9, induced in Wistar rats with sodium phenobarbitone and - naphthoflavone, was used for this purpose.
DMSO was used as a vehicle. The exposed bacteria were plated onto minimal glucose agar medium supplemented with L-histidine and D-Biotin solution. The plates were incubated at 37 ºC for about 48 hours for Experiment No. 1 and experiment No. 2 after which the histidine revertant colonies were counted and their frequency was compared with that in the vehicle control group. Concurrent positive control groups were also included in the experiment, as specified by the test guideline.
Results of this test indicated that the frequencies of histidine revertant colonies at all concentrations of 1-Benzyl-5-Phenylbarbituric acid (BPBA) in strains TA1535, TA97a, TA98, TA100 and TA102, in the presence and absence of a metabolic activation system, were comparable to those observed in the vehicle control group, as per the criteria employed for evaluation of mutagenic potential, and this observation was confirmed by repetition of the experiments.
Plate counts for the spontaneous histidine revertant colonies in the vehicle control groups were found to be within the frequency ranges expected from the laboratory historical control data . They also compared well with the range reported in the literature. Concurrent positive controls demonstrated sensitivity of the assay with and without metabolic activation
Analysis of the samples formulated for experiment No. 1 revealed that the average measured concentrations of 1-Benzyl-5-Phenylbarbituric acid (BPBA) were 52.99 and 0.49 mg/ml against the nominal concentrations of 50 and 0.5 mg/ml respectively. The small observed difference of 5.98% and -2.00% respectively for above mentioned doses confirmed that the test system received an adequate dose of the Test Item.
It is concluded that, under the conditions of this study, 1-Benzyl-5-Phenylbarbituric acid (BPBA) is non-mutagenic in Salmonella typhimurium strains TA1535, TA97a, TA98, TA100 and TA102.
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