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EC number: 935-606-2 | CAS number: 1329658-14-1
- 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:
- April 03, 2014 to April 17, 2014
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 014
- Report date:
- 2014
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
- Reference substance name:
- 5-(3-phenylprop-2-ynoyl)-1,3-dihydro-2-benzofuran-1,3-dione
- EC Number:
- 935-606-2
- Cas Number:
- 1329658-14-1
- Molecular formula:
- C17H8O4
- IUPAC Name:
- 5-(3-phenylprop-2-ynoyl)-1,3-dihydro-2-benzofuran-1,3-dione
- Test material form:
- solid: particulate/powder
- Details on test material:
- Short Name: PETA
Long Name: NNEXAMITE™ A56 (PETA)
Chemical Name: Phenylacetylene modified trimellitic anhydride
Lot Number: NEX-X61-A02
CAS Number: 1329658-14-1
Description: Yellow powder
Purity: >99 %
Molecular weight: 276.24
Manufacture date: 04 September 2013
Expiry date: 01 May 2014
Storage conditions: Room temperature (15-25 °C), protected from humidity
Safety Precautions: Routine safety precautions (gloves, goggles, face mask, lab coat) for unknown materials were applied to assure personnel health and safety
Constituent 1
- Specific details on test material used for the study:
- Not further details specified in the study report.
Method
- Target gene:
- Histidene
Species / strain
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
- Details on mammalian cell type (if applicable):
- The Salmonella typhimurium strains used in this study were mutants derived from Salmonella typhimurium LT2. The strains used in the study were obtained from Molecular Toxicology Inc., 157 Industrial Park Dr. Boone, NC 28607, U.S.A.
- Additional strain / cell type characteristics:
- not specified
- Cytokinesis block (if used):
- Not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- Unlike mammals, bacteria lack the necessary oxidative enzyme systems for metabolising foreign compounds to electrophilic metabolites capable of reacting with DNA. Sometimes these foreign compounds, when reacting with a mammalian enzyme system, yield mutagenic metabolic products. In order to test these indirectly acting mutagens, a metabolically active extract of rat liver (treated with Aroclor 1254) called S9 fraction is used. Activity of batch of S9 used in this experiment was characterized by testing a selected pre-mutagen, benzo(a)pyrene, with S. typhimurium TAI00. The S9 fraction is buffered and supplemented with the essential co-factors 13-NADP and Glucose-6-phosphate to form the "S9 mix". This mix is added to the top agar in this activated assay. The S9 fraction procured from D.R.D.O., Nagapur was used in the study.
- Test concentrations with justification for top dose:
- Cytotoxicity Test: Ten different concentrations, 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500 and 5000 μg/plate were tested for cytotoxicity.
Based on the results of the cytotoxicity test, the test concentrations of78.13, 156.25, 312.5, 625, 1250 and 2500 μg/plate of PETA both in the absence and presence (5% v/v S9 mix) of metabolic activation were selected for Trial I.
Trial II was conducted with modified concentrations i.e., 25.6, 64, 160,400, 1000 and 2500 μg/plate in the absence and presence of metabolic activation (S9 concentration was increased to 10% v/v). - Vehicle / solvent:
- Solubility and precipitation tests were performed prior to the cytotoxicity test. The test item was insoluble in sterile distilled water (Stock A), while found to be soluble in dimethyl sulfoxide (Stock B). Therefore, dimethyl sulfoxide was selected as the vehicle for treatment.
Controls
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- 2-nitrofluorene
- sodium azide
- mitomycin C
- other: 2-Aminoanthracene (2AA)
- Details on test system and experimental conditions:
- Solubility Test
Solubility and precipitation tests were performed prior to the cytotoxicity test. The test item was insoluble in sterile distilled water (Stock A), while found to be soluble in dimethyl sulfoxide (Stock B). Therefore, dimethyl sulfoxide was selected as the vehicle for treatment. A volume of 100 μL of the test item from stock B was added to 2 mL of top agar, to assess the precipitation. Slight precipitation was observed at the concentration of 5000 μg/plate. Hence, 5000 μg/plate was selected as the highest concentration to be tested for cytotoxicity, both in the absence and presence (5% v/v S9 mix) of the metabolic activation system.
Cell Viability Test
Fresh cultures for the test were prepared by inoculating frozen permanent cultures to a flask containing 10 mL of sterile nutrient broth N° 2 (Oxoid Unipath, England). The flasks were then incubated at 37.0 ± 1 °C in an orbital shaking incubator (120 rpm) for 15 h up to an early stationary or late exponential phase.
After the incubation period, the culture flasks were removed from the orbital shaking incubator. Aseptically, the cultures were diluted with oxoid nutrient broth (ONB) and the optical density was measured at 660 nm using a Spectrophotometer (Visiscan 167, manufactured by Systronics).
Oxoid nutrient broth was used as the control blank. Cell viability of the tester strains was determined prior to treatment. The optical density of the cultures was found to be in the acceptable range and so they were used for the study.
Genotype Confirmation Test
Fresh cultures for the test were prepared by inoculating frozen permanent cultures to a flask containing 10 mL of sterile nutrient broth N° 2 (Oxoid Unipath, England). The flasks were then incubated at 37.0 ± 1°C in an orbital shaking incubator for 15 h up to an early stationary or late exponential phase. The genotype of the tester strains was confirmed once in a month as per the following procedures:
Test for Histidine Requirement
A volume of 0.1 mL of culture of each strain was added to 2 mL of top agar (without histidine and biotin) maintained at 45 ± 2 °C and poured onto a Minimal Glucose Agar (MGA) plate containing an excess of biotin (0.5%). The plates were incubated at 37 ± I °C and examined after 48 h incubation.
Test for Biotin Requirement
A volume of 0.1 mL of culture of each strain was added to 2 mL of top agar (without histidine and biotin) maintained at 45 ± 2 °C and poured onto an MGA plate containing an excess of histidine (0.01%). The plates were incubated at 37 ± I °C and examined after 48 h incubation.
Test for Histidine and Biotin Requirement
A volume of 0.1 mL of culture of each strain was added to 2 mL of top agar (with histidine and biotin) maintained at 45 ± 2 °C and poured onto an MGA plate without histidine and biotin. The plates were incubated at 37 ± 1 °C and examined after 48 h incubation.
Test for rfa Mutation
A volume of 0.1 mL of culture of each strain was added to 2 mL of top agar (without histidine and biotin) maintained at 45 ± 2 °C and poured onto nutrient agar plate. After the solidification of the top agar, a sterile paper disc dipped in 1 mg/mL solution of crystal violet was transferred to the plates, using sterile forceps. The plates were then incubated at 37 ± 1 °C and examined for the zone of inhibition around the impregnated paper disc after 48 h incubation.
Test for uvrB Mutation
A volume of 0.1 mL of culture of each strain was added to 2 mL of top agar (with histidine and biotin) maintained at 45 ± 2 °C and poured onto an MGA plate. After the solidification of the top agar, half of the plate was covered with sterilized aluminium foil. Plates were exposed to 15W germicidal lamp (UV radiation) at a distance of33 cm. Non R-factor strains (TA1537, TA1535) were exposed for 6 seconds and R-factor strains (TA98, TAlO0 and TA102) for 8 seconds. The plates were then incubated at 3 7 ± 1 °C and examined after 48 h incubation.
Test for R-factor Resistance (Ampicillin Resistance)
A volume of 0.1 mL of culture of each strain was added to 2 mL of top agar (without histidine and biotin) maintained at 45 ± 2 °C and poured onto an MGA plate supplemented with histidine (0.5%), biotin (0.01%) and ampicillin (24 μg/mL). The plates were incubated at 37 ± 1 °C and examined after 48 h.
Test for R-factor Resistance (Tetracycline Resistance)
A volume of 0.1 mL of culture of each strain was added to 2 mL of top agar (without histidine and biotin) maintained at 45 ± 2 °C and poured onto an MGA plate supplemented with histidine (0.5%), biotin (0.01%) and tetracycline (2 μg/mL). The plates were incubated at 37 ± 1 °C and examined after 48 h.
Cytotoxicity Test
Before commencing the mutagenicity study, PETA was tested for cytotoxicity, to Salmonella typhimurium tester strain TA 100. The experiment was conducted both in the absence and presence of metabolic activation system (5% v/v S9 mix).
A stock solution of 50000 μg/mL of PET A was prepared by dissolving 100 mg (100.04 mg i.e., 100 mg) of test item in dimethyl sulfoxide (stock A) and made up to 2 mL.
Further stock solution of concentrations, viz., 25000 (stock B), 12500 (stock C), 6250 (stock D), 3125 (stock E), 1562.5 (stock F), 781.25 (stock G), 390.63 (stock H), 195.31 (stock I) and 97.66 μg/mL (stock J) were prepared by serial dilution method. Ten different concentrations, viz., 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500 and 5000 μg/plate were tested for cytotoxicity. Volumes of 100 μL ofrelevant stock solutions A - J were used to obtain the required test concentrations for treatment both in the absence and presence (5% v/v S9 mix) of the metabolic activation system.
Tubes containing 2 mL of molten top agar with 0.5 mM histidine/biotin were maintained at 45 ± 2 °C. A volume of 500 μL of 5% v/v S9 mix was added in the presence of metabolic activation system and 500 μL of 0.2 M phosphate buffer was added in the absence of metabolic activation system. A volume of 100 μL of the relevant concentration of stock solution of the test item and dimethyl sulfoxide was used for treatment and as a negative control, respectively. Finally 100 μL of bacterial culture was added to the tubes and mixed. Cultures used were checked for cell viability prior to testing. This treatment mixture was poured on an MGA plates and allowed to solidify.
Triplicate sets were maintained for each concentration of PET A and negative control. The petriplates were incubated at 37 ± 1 °C for 48 hours and then treated plates were examined to assess the state of background bacterial growth inhibition and reduction in number of colonies.
Mutagenicity Test
The mutagenicity test was conducted as two independent experiments. In both the trials, the treatment was performed both in the absence and presence of metabolic activation system (5% v/v and I 0% v/v S9 mix in Trial I and II, respectively). The treatments were performed by plate incorporation technique as described in the cytotoxicity test. Plates were maintained in triplicates for each test concentrations of PET A, negative and positive controls.
Trial I [In the Absence and Presence of 5% v/v S9 Mix]
The tester strains were exposed to the test concentrations of78.125, 156.25, 312.5, 625, 1250 and 2500 μg/plate of PET A in the absence and presence (5% v/v S9 mix) of metabolic activation system. The first stock solution (stock A) of the test item was prepared by dissolving 250 mg (250.02 mg i.e., 250 mg)of PETA in dimethyl sulfoxide and made up to 10 mL (25000 μg/mL). For the treatment, a volume of 5 mL of stock A was added to 5 mL dimethyl sulfoxide to obtain 12500 μ/mL (stock B). Further stock solutions viz., 6250 (stock C), 3125 (stock D), 1562.5 (stock E) and 781.25 μg/mL (stock F) were prepared by serial 2 fold dilution. Volumes of 100 μL of relevant stock solutions A-F were used to obtain the required test concentrations for treatment in the absence and presence (5% v/v S9 mix) of the metabolic activation system respectively.
Trial II [In the Absence and Presence of 10% v/v S9 Mix]
A second trial was conducted to confirm the negative results obtained in Trial I. In Trial II, the concentration spacing was modified using a factor of 2.5 and the concentration of S9 mix was increased to 10% v/v. The tester strains were exposed to the test concentrations of 25.6, 64, 160, 400, 1000 and 2500 μg/plate in the absence and presence (I 0% v/v S9 mix) of metabolic activation. Volumes of 100 μL of relevant stock solutions were used to obtain the required test concentrations.
Plates were maintained in triplicates for each test concentration of PET A, negative and positive controls during both the trials. The number of revertant colonies was recorded after 48 h incubation period.
Treatment with 2-aminoanthracene in the absence of metabolic activation were also performed for tester strain TA 100 in both the trials to verify the efficiency of the S9 fraction used in the study. - Rationale for test conditions:
- This assay measures the ability of the test item to induce reverse mutations at specific histidine loci in the tester strains of Salmonella typhimurium i.e., TA1537, TA1535, TA98, TA100 and TA102, which are known for their reliability and reproducibility in a short term mutagenicity assay and are also recommended by the OECD and other guidelines.
- Evaluation criteria:
- Once criteria for a valid assay are met, responses observed in the assay were evaluated. The conditions necessary for determining a positive result were: there should be a dose related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing doses of the test item either in the absence or presence of the metabolic activation system.
Biological relevance of the results was considered first:
Strains TA98, TA1535, and TA1537
Data sets were judged positive, if the increase in mean revertants at the peak of the dose response was equal to or greater than 3.0-times the mean negative control value.
Strain TA100 and TA102
Data sets were judged positive, if the increase in mean revertants at the peak of the dose response was equal to or greater than 2.0-times the mean negative control value.
Statistical analysis (i.e. Simple linear regression analysis) was used as an aid in the evaluation of dose response.
A response that did not meet all three of the above criteria (magnitude, concentration responsiveness, reproducibility) was not evaluated as positive.
Negative results obtained in the first trial were confirmed by a second trial, using the same method as specified above, with an alteration in concentration spacing and metabolic activation. - Statistics:
- Simple linear regression analysis was performed for TA1537, TA1535, TA98, TAl00 and TA102, separately, to assess the dose dependent nature of any increase in revertant colonies.
Results and discussion
Test resultsopen allclose all
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- 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, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- 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, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- 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, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- Negative Control
The results of the study indicate that the values of negative control in all strains were within historical range of respective strains.
Positive Controls
2-Aminoanthracene was used as the positive control in the presence of metabolic activation for all the tester strains during both the trials. Large historical control data of this laboratory proved the efficiency and suitability of 2-aminoanthracene as a positive control in the presence of metabolic activation. The batch of S9 used in this study was characterised with benzo(a)pyrene that requires metabolic activation by microsomal enzymes by supplier. Benzo(a)pyrene exhibited clear increase in the number of revertants when compared with the concurrent negative control which demonstrated the efficiency of S9 used in this study.
Positive controls exhibited a clear increase in the number of revertants when compared with the concurrent negative controls and were within the range of historical limits. This demonstrated the efficiency of the test system and suitability of the procedures employed in the assay.
Increase in revertants were not observed in tester strain TA 100 (Trial I and II) treated with 2-aminoanthracene in the absence of metabolic activation but clear increase was observed in the presence of metabolic activation. This demonstrated the efficiency of the S9 fraction used in this assay.
Cytotoxicity Test
Cytotoxicity is characterised by inhibition of the background bacterial lawn and/or reduction in the number of revertant colonies. Complete inhibition of the background lawn with reduction in revertant colonies (83-85 % reduction) was observed at the tested concentration of 5000 μg/plate both in the absence and presence of the metabolic activation system while partial inhibition of the background lawn with reduction in revertant colonies ( 40-42 % reduction) was observed at the tested concentration of 2500 μg/plate both in the absence and presence of metabolic activation system. Normal growth was observed up to the dose level of 1250 μg/plate both in the absence and presence of the metabolic activation system (5% v/v S9 mix).
Hence, 2500 μg/plate of PET A was selected as the highest concentration to be tested in the mutagenicity test in the absence and presence of metabolic activation system, for all the tester strains.
Mutagenicity Test
Trial I [In the Absence and Presence of 5% v/v S9 mix]
Partial inhibition of the bacterial background lawn was observed at the tested concentration of 2500 μg/plate both in the absence and presence of the metabolic activation system (5% v/v S9 mix) in all the tester strains. Reduction in the number of colonies was observed at the tested concentration of 2500 μg/plate both in the absence and presence of the metabolic activation system in strains TA1535 (53.61% and 51.38%), TA98 (50.02% and 59.56%), TAlO0 (40.25% and 46.90%) and in strain TA102 (15.90% and 17.53%) while reduction in the number of revertant colonies was not observed at the tested concentration of 2500 μg/plate only in the absence and presence of metabolic action system in the strain TA1537. Normal growth was observed up to the dose of 1250 μg/plate both in the absence and presence of the metabolic activation system (5% v/v S9 mix) in the tester strains.
A positive increase in the number of revertant colonies was not observed in any of the tester strains at the tested concentration of 78.13, 156.25, 312.5, 625, 1250 and 2500 μg/plate in the absence and presence (5% v/v S9 mix) of metabolic activation, when compared with the concurrent negative control.
Results revealed that there was no positive mutagenic effect in tester strains TA1537, TA1535, TA98, TA100 and TA102 at the tested concentration of78.13, 156.25, 312.5, 625, 1250 and 2500 μg/plate in the absence and presence (5% v/v S9 mix) of metabolic activation, when compared with the concurrent negative control. Statistical analysis did not reveal any significant effect.
Trial II [In the Absence and Presence of 10% v/v S9 mix]
Partial inhibition of the bacterial background lawn was observed at the tested concentration of 2500 μg/plate both in the absence and presence of the metabolic activation system (10% v/v S9 mix) in all the tester strains. Reduction in the number of colonies was observed at the tested concentration of 2500 μg/plate both in the absence and presence of the metabolic activation system in strains TA1535 (46.56% and 37.51%), TA98 (33.87% and 45.21%), TA100 (34.10% and 34.97%) and in strain TA102 (18.69% and 18.23%) while reduction in the number of revertant colonies was observed at the tested concentration of 2500 μg/plate only in the presence of the metabolic activation system in the strain TA1537 (49.95%). Normal growth was observed up to the dose of 1000 μg/plate both in the absence and presence of the metabolic activation system (10% v/v S9 mix) in the tester strains.
A positive increase in the number of revertant colonies was not observed in any of the tester strains at the tested concentration of 25.6, 64, 160, 400, 1000 and 2500 μg/plate in the absence and presence (10% v/v S9 mix) of metabolic activation, when compared with the concurrent negative control.
Results revealed that there was no positive mutagenic effect in tester strains TA1537, TA1535, TA98, TAl00 and TA102 both in the absence and presence (10% v/v S9 mix) of metabolic activation at any of the tested concentrations when compared with the concurrent negative control. Statistical analysis did not reveal any significant effect.
Any other information on results incl. tables
Mean Count of His+ Revertant Colonies in Negative Control Positive Controls and Treatment Plates in the Absence of Metabolic Activation (Trial I)
Concentration of PETA (µg/plate) |
His+ Revertant Colonies/Plate (Absence of Metabolic Activation) |
|||||||||
TA1537 |
TA1535 |
TA98 |
TA100 |
TA102 |
||||||
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
|
NC (DMSO) |
7.00 |
1.00 |
23.00 |
4.00 |
24.67 |
3.51 |
133.33 |
11.50 |
224.33 |
9.50 |
78.125 |
5.67 |
1.53 |
21.33 |
6.51 |
23.00 |
3.61 |
138.67 |
10.07 |
216.33 |
8.50 |
156.25 |
6.33 |
1.53 |
20.33 |
3.51 |
22.67 |
2.08 |
133.0 |
10.15 |
227.00 |
12.53 |
312.5 |
6.33 |
1.53 |
21.33 |
2.52 |
23.33 |
3.06 |
131.67 |
10.50 |
210.67 |
8.96 |
625 |
6.33 |
3.21 |
17.67 |
2.08 |
25.33 |
5.03 |
138.00 |
11.53 |
222.00 |
18.68 |
1250 |
6.67 |
1.53 |
20.67 |
2.08 |
27.33 |
4.04 |
137.33 |
6.03 |
217.00 |
10.54 |
2500 |
6.67 |
2.08 |
10.67 |
2.52 |
12.33 |
2.08 |
79.67 |
7.37 |
188.67 |
4.73 |
PC |
329.00 |
28.93 |
375.33 |
133.55 |
552.33 |
93.30 |
885.00 |
163.94 |
1095.33 |
174.40 |
PC-2Aa |
- |
- |
- |
- |
- |
- |
136.33 |
11.50 |
- |
- |
Key: SD = Standard deviation, NC = Negative control, DMSO = Dimethyl sulfoxide, PC = Positive control {TA1537 = 9-Aminoacridine hydrochloride hydrate (75 µg/plate), TA1535 = Sodium azide (0.5 µg/plate), TA98 = 2-Nitrofluorene (7.5 µg/plate), TA100 = Sodium azide (5 µg/plate), TA102 = Mitomycin-C (0.5 µg/plate)}, 2-Aa = 2-Aminoanthracene (5 µg/plate for TA100), - = Not applicable.
Mean Count of His+ Revertant Colonies in Negative Control Positive Controls and Treatment Plates in the Presence of Metabolic Activation (Trial I)
Concentration of PETA (µg/plate) |
His+ Revertant Colonies/Plate (Presence of Metabolic Activation [5% v/v S9 mix]) |
|||||||||
TA1537 |
TA1535 |
TA98 |
TA100 |
TA102 |
||||||
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
|
NC (DMSO) |
9.00 |
1.00 |
24.00 |
4.00 |
31.33 |
2.52 |
134.33 |
8.50 |
230.00 |
16.64 |
78.125 |
7.00 |
1.00 |
23.33 |
3.21 |
26.33 |
3.21 |
134.00 |
6.56 |
221.00 |
8.54 |
156.25 |
8.33 |
1.53 |
20.67 |
3.79 |
27.67 |
4.51 |
131.00 |
6.00 |
233.00 |
14.11 |
312.5 |
7.33 |
1.53 |
22.67 |
5.69 |
30.00 |
3.61 |
130.67 |
10.79 |
226.00 |
13.75 |
625 |
8.00 |
1.00 |
24.67 |
3.06 |
26.00 |
3.61 |
136.00 |
6.56 |
222.67 |
18.15 |
1250 |
7.67 |
1.53 |
22.00 |
4.00 |
26.67 |
3.21 |
129.00 |
9.85 |
230.33 |
19.76 |
2500 |
8.00 |
2.65 |
11.67 |
3.79 |
12.67 |
2.08 |
71.33 |
7.51 |
189.67 |
6.43 |
PC-2Aa |
401.67 |
90.67 |
428.67 |
83.29 |
806.67 |
145.95 |
1291.00 |
346.22 |
1031.33 |
100.85 |
Key: SD = Standard deviation, NC = Negative control, DMSO = Dimethyl sulfoxide, PC = Positive control, 2-Aa = 2-Aminoanthracene (10 µg/plate for TA1537, TA1535, TA102 and 5 µg/plate for TA98 and TA100).
Mean Count of His+ Revertant Colonies in Negative Control Positive Controls and Treatment Plates in the Absence of Metabolic Activation (Trial II)
Concentration of PETA (µg/plate) |
His+ Revertant Colonies/Plate (Absence of Metabolic Activation) |
|||||||||
TA1537 |
TA1535 |
TA98 |
TA100 |
TA102 |
||||||
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
|
NC (DMSO) |
6.33 |
1.15 |
19.33 |
2.08 |
20.67 |
1.53 |
131.00 |
7.81 |
224.67 |
12.50 |
25.6 |
6.33 |
3.06 |
20.67 |
3.06 |
20.00 |
2.00 |
135.00 |
7.55 |
227.33 |
7.23 |
64 |
5.67 |
0.58 |
18.00 |
1.00 |
21.67 |
2.52 |
136.00 |
4.00 |
224.33 |
13.87 |
160 |
6.00 |
3.46 |
17.33 |
0.58 |
23.00 |
2.65 |
135.33 |
2.89 |
223.00 |
6.00 |
400 |
3.33 |
0.58 |
20.00 |
1.00 |
22.67 |
2.52 |
131.67 |
10.60 |
225.33 |
6.51 |
1000 |
4.67 |
0.58 |
18.00 |
1.00 |
20.33 |
1.15 |
127.33 |
3.06 |
222.33 |
14.01 |
2500 |
5.33 |
2.08 |
10.33 |
2.52 |
13.67 |
1.53 |
86.33 |
8.74 |
182.67 |
9.29 |
PC |
247.67 |
18.23 |
234.00 |
15.10 |
335.00 |
38.30 |
758.67 |
58.50 |
1204.67 |
131.50 |
PC-2Aa |
- |
- |
- |
- |
- |
- |
134.00 |
6.56 |
- |
- |
Key: SD = Standard deviation, NC = Negative control, DMSO = Dimethyl sulfoxide, PC = Positive control {TA1537 = 9-Aminoacridine hydrochloride hydrate (75 µg/plate), TA1535 = Sodium azide (0.5 µg/plate), TA98 = 2-Nitrofluorene (7.5 µg/plate), TA100 = Sodium azide (5 µg/plate), TA102 = Mitomycin-C (0.5 µg/plate)}, 2-Aa = 2-Aminoanthracene (5 µg/plate for TA100), - = Not applicable.
Mean Count of His+ Revertant Colonies in Negative Control Positive Controls and Treatment Plates in the Presence of Metabolic Activation (Trial II)
Concentration of PETA (µg/plate) |
His+ Revertant Colonies/Plate (Presence of Metabolic Activation [10% v/v S9 mix]) |
|||||||||
TA1537 |
TA1535 |
TA98 |
TA100 |
TA102 |
||||||
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
|
NC (DMSO) |
9.33 |
2.08 |
21.33 |
1.53 |
24.33 |
2.08 |
135.33 |
10.21 |
230.33 |
14.64 |
25.6 |
6.67 |
2.08 |
21.67 |
4.04 |
23.00 |
4.58 |
134.00 |
10.15 |
230.33 |
10.07 |
64 |
7.00 |
2.00 |
21.33 |
1.53 |
24.67 |
1.53 |
133.00 |
9.00 |
222.33 |
13.05 |
160 |
7.33 |
0.58 |
20.33 |
2.08 |
24.33 |
3.06 |
137.00 |
7.55 |
233.33 |
13.05 |
400 |
7.33 |
2.31 |
18.00 |
1.00 |
22.00 |
1.73 |
134.33 |
6.66 |
233.67 |
8.50 |
1000 |
5.67 |
0.58 |
19.33 |
1.53 |
22.00 |
2.65 |
134.00 |
6.56 |
228.67 |
12.50 |
2500 |
4.67 |
2.08 |
13.33 |
4.16 |
13.33 |
1.53 |
88.00 |
10.54 |
188.33 |
11.02 |
PC-2Aa |
271.33 |
25.79 |
290.00 |
23.00 |
435.00 |
48.28 |
954.33 |
97.29 |
1456.33 |
107.75 |
Key: SD = Standard deviation, NC = Negative control, DMSO = Dimethyl sulfoxide, PC = Positive control, 2-Aa = 2-Aminoanthracene (10 µg/plate for TA1537, TA1535, TA102 and 5 µg/plate for TA98 and TA100).
Individual Plate Count (Trial I)
Absence of Metabolic Activation
Concentration of PETA (µg/plate) |
Number of Revertant Colonies |
||||||||||||||
TA1537 |
TA1535 |
TA98 |
TA100 |
TA102 |
|||||||||||
R1 |
R2 |
R3 |
R1 |
R2 |
R3 |
R1 |
R2 |
R3 |
R1 |
R2 |
R3 |
R1 |
R2 |
R3 |
|
NC (DMSO) |
8 |
6 |
7 |
23 |
27 |
19 |
21 |
28 |
25 |
145 |
122 |
133 |
234 |
215 |
224 |
78.125 |
4 |
6 |
7 |
28 |
21 |
15 |
26 |
19 |
24 |
148 |
128 |
140 |
208 |
225 |
216 |
156.25 |
5 |
8 |
6 |
20 |
17 |
24 |
22 |
25 |
21 |
122 |
142 |
135 |
239 |
228 |
214 |
312.5 |
8 |
5 |
6 |
19 |
21 |
24 |
20 |
24 |
26 |
121 |
142 |
132 |
206 |
221 |
205 |
625 |
10 |
4 |
5 |
16 |
20 |
17 |
20 |
30 |
26 |
139 |
149 |
126 |
225 |
239 |
202 |
1250 |
8 |
7 |
5 |
23 |
19 |
20 |
28 |
31 |
23 |
138 |
131 |
143 |
228 |
207 |
216 |
2500 |
6 |
9 |
5 |
13 |
8 |
11 |
13 |
14 |
10 |
74 |
77 |
88 |
194 |
187 |
185 |
PC |
341 |
296 |
350 |
340 |
523 |
263 |
445 |
614 |
598 |
735 |
860 |
1060 |
946 |
1053 |
1287 |
PC-2Aa |
- |
- |
- |
- |
- |
- |
- |
- |
- |
136 |
125 |
148 |
- |
- |
- |
Presence of Metabolic Activation (5% v/v S9 mix)
Concentration of PETA (µg/plate) |
Number of Revertant Colonies |
||||||||||||||
TA1537 |
TA1535 |
TA98 |
TA100 |
TA102 |
|||||||||||
R1 |
R2 |
R3 |
R1 |
R2 |
R3 |
R1 |
R2 |
R3 |
R1 |
R2 |
R3 |
R1 |
R2 |
R3 |
|
NC (DMSO) |
10 |
8 |
9 |
28 |
24 |
20 |
29 |
31 |
34 |
126 |
143 |
134 |
223 |
249 |
218 |
78.125 |
7 |
8 |
6 |
22 |
27 |
21 |
24 |
30 |
25 |
133 |
128 |
141 |
222 |
212 |
229 |
156.25 |
7 |
10 |
8 |
18 |
25 |
19 |
23 |
32 |
28 |
137 |
131 |
125 |
235 |
218 |
246 |
312.5 |
9 |
7 |
6 |
29 |
18 |
21 |
27 |
34 |
29 |
143 |
123 |
126 |
229 |
238 |
211 |
625 |
8 |
9 |
7 |
24 |
22 |
28 |
23 |
25 |
30 |
135 |
143 |
130 |
242 |
206 |
220 |
1250 |
6 |
9 |
8 |
18 |
22 |
26 |
28 |
29 |
23 |
121 |
126 |
140 |
248 |
209 |
234 |
2500 |
7 |
11 |
6 |
9 |
16 |
10 |
12 |
15 |
11 |
64 |
71 |
79 |
197 |
185 |
187 |
PC-2Aa |
318 |
389 |
498 |
370 |
392 |
524 |
783 |
963 |
674 |
1684 |
1031 |
1158 |
926 |
1041 |
1127 |
Key: R = Replicate, NC = Negative control, DMSO = Dimethyl sulfoxide, PC = Positive control {TA1537 = 9-Aminoacridine hydrochloride hydrate (75 µg/plate), TA1535 = Sodium azide (0.5 µg/plate), TA98 = 2-Nitrofluorene (7.5 µg/plate), TA100 = Sodium azide (5 µg/plate), TA102 = Mitomycin-C (0.5 µg/plate)}, 2-Aa = 2-Aminoanthracene (10 µg/plate for TA1537, TA1535, TA102 and 5 µg/plate for TA98 and TA100), - = Not applicable.
Individual Plate Count (Trial II)
Absence of Metabolic Activation
Concentration of PETA (µg/plate) |
Number of Revertant Colonies |
||||||||||||||
TA1537 |
TA1535 |
TA98 |
TA100 |
TA102 |
|||||||||||
R1 |
R2 |
R3 |
R1 |
R2 |
R3 |
R1 |
R2 |
R3 |
R1 |
R2 |
R3 |
R1 |
R2 |
R3 |
|
NC (DMSO) |
7 |
5 |
7 |
20 |
21 |
17 |
22 |
19 |
21 |
135 |
136 |
122 |
237 |
225 |
212 |
25.6 |
9 |
7 |
3 |
24 |
20 |
18 |
22 |
18 |
20 |
143 |
134 |
128 |
219 |
232 |
231 |
64 |
6 |
5 |
6 |
19 |
18 |
17 |
22 |
19 |
24 |
132 |
136 |
140 |
209 |
228 |
236 |
160 |
4 |
4 |
10 |
17 |
18 |
17 |
20 |
25 |
24 |
137 |
132 |
137 |
229 |
217 |
223 |
400 |
3 |
4 |
3 |
19 |
20 |
21 |
23 |
20 |
25 |
130 |
122 |
143 |
225 |
219 |
232 |
1000 |
4 |
5 |
5 |
18 |
19 |
17 |
19 |
21 |
21 |
128 |
124 |
130 |
211 |
238 |
218 |
2500 |
7 |
3 |
6 |
10 |
8 |
13 |
14 |
12 |
15 |
79 |
84 |
96 |
180 |
175 |
193 |
PC |
251 |
264 |
228 |
218 |
236 |
248 |
302 |
326 |
377 |
700 |
759 |
817 |
1205 |
1073 |
1336 |
PC-2Aa |
- |
- |
- |
- |
- |
- |
- |
- |
- |
133 |
141 |
128 |
- |
- |
- |
Presence of Metabolic Activation (10% v/v S9 mix)
Concentration of PETA (µg/plate) |
Number of Revertant Colonies |
||||||||||||||
TA1537 |
TA1535 |
TA98 |
TA100 |
TA102 |
|||||||||||
R1 |
R2 |
R3 |
R1 |
R2 |
R3 |
R1 |
R2 |
R3 |
R1 |
R2 |
R3 |
R1 |
R2 |
R3 |
|
NC (DMSO) |
10 |
11 |
7 |
21 |
23 |
20 |
25 |
22 |
26 |
131 |
128 |
147 |
246 |
217 |
228 |
25.6 |
5 |
9 |
6 |
26 |
21 |
18 |
28 |
22 |
19 |
125 |
145 |
132 |
221 |
241 |
229 |
64 |
5 |
7 |
9 |
23 |
21 |
20 |
23 |
25 |
26 |
124 |
133 |
142 |
222 |
211 |
234 |
160 |
7 |
8 |
7 |
18 |
21 |
22 |
25 |
21 |
27 |
144 |
138 |
129 |
221 |
232 |
247 |
400 |
6 |
6 |
10 |
19 |
18 |
17 |
23 |
20 |
23 |
136 |
127 |
140 |
242 |
234 |
225 |
1000 |
5 |
6 |
6 |
18 |
19 |
21 |
23 |
24 |
19 |
133 |
128 |
141 |
229 |
216 |
241 |
2500 |
7 |
4 |
3 |
18 |
10 |
12 |
13 |
12 |
15 |
98 |
89 |
77 |
189 |
177 |
199 |
PC-2Aa |
250 |
264 |
300 |
264 |
313 |
290 |
486 |
390 |
429 |
963 |
853 |
1047 |
1471 |
1342 |
1556 |
Key: R = Replicate, NC = Negative control, DMSO = Dimethyl sulfoxide, PC = Positive control {TA1537 = 9-Aminoacridine hydrochloride hydrate (75 µg/plate), TA1535 = Sodium azide (0.5 µg/plate), TA98 = 2-Nitrofluorene (7.5 µg/plate), TA100 = Sodium azide (5 µg/plate), TA102 = Mitomycin-C (0.5 µg/plate)}, 2-Aa = 2-Aminoanthracene (10 µg/plate for TA1537, TA1535, TA102 and 5 µg/plate for TA98 and TA100), - = Not applicable.
Results of Cytotoxicity Test (TA100) in Absence and presence of Metabolic Activation
Concentration of PETA (µg/plate) |
In the Absence of Metabolic Activation |
In the Presence (5% v/v S9 mix) if Metabolic Activation |
||||
R1 |
R2 |
R3 |
R1 |
R2 |
R3 |
|
NC (DMSO) |
NI (137) |
NI (122) |
NI (140) |
NI (130) |
NI (123) |
NI (148) |
9.77 |
NI (127) |
NI (136) |
NI (139) |
NI (127) |
NI (143) |
NI (133) |
19.53 |
NI (142) |
NI (135) |
NI (123) |
NI (141) |
NI (125) |
NI (136) |
39.06 |
NI (133) |
NI (123) |
NI (141) |
NI (139) |
NI (129) |
NI (144) |
78.13 |
NI (129) |
NI (138) |
NI (137) |
NI (143) |
NI (134) |
NI (123) |
156.25 |
NI (144) |
NI (133) |
NI (128) |
NI (140) |
NI (127) |
NI (130) |
312.5 |
NI (139) |
NI (143) |
NI 9125) |
NI (137) |
NI (128) |
NI (144) |
625 |
NI (137) |
NI (125) |
NI (141) |
NI (133) |
NI (141) |
NI (127) |
1250 |
NI (129) |
NI (139) |
NI (137) |
NI (129) |
NI (134) |
NI (141) |
2500 |
PT (71) |
PI (79) |
PI (83) |
PI (74) |
PI (85) |
PI (80) |
5000 |
I (17) |
I (21) |
I (23) |
I (19) |
I (27) |
I (22) |
Key: DMSO = Dimethyl sulfoxide, I = Inhibition, NC = Negative control, NI = No inhibition, PI = Partial inhibition, R = Replicate
Note: Number of revertant colonies observed/plate are provided in parentheses
AMES TEST – HISTORICAL CONTROL DATA (November 2012 to February 2014)
Negative Control (Distilled Water) without S9 Mix |
|||||
Strain |
TA1537 |
TA1535 |
TA98 |
TA100 |
TA102 |
Mean Revertants per Plate |
7.62 |
18.07 |
24.57 |
143.71 |
236.28 |
Standard Deviation |
2.34 |
3.37 |
4.22 |
10.98 |
15.72 |
Maximum |
14 |
30 |
34 |
175 |
298 |
Minimum |
2 |
9 |
14 |
107 |
201 |
Negative Control (Distilled Water) with S9 Mix |
|||||
Mean Revertants per Plate |
8.35 |
20.22 |
26.14 |
147.00 |
241.53 |
Standard Deviation |
2.49 |
3.70 |
4.57 |
11.72 |
13.95 |
Maximum |
15 |
31 |
36 |
207 |
297 |
Minimum |
3 |
9 |
13 |
104 |
202 |
Negative Control (Dimethyl Sulfoxide) without S9 Mix |
|||||
Mean Revertants per Plate |
7.39 |
18.44 |
24.65 |
142.26 |
233.99 |
Standard Deviation |
2.30 |
3.43 |
4.10 |
10.15 |
13.35 |
Maximum |
13 |
37 |
36 |
168 |
268 |
Minimum |
3 |
9 |
12 |
115 |
184 |
Negative Control (Dimethyl Sulfoxide) with S9 Mix |
|||||
Mean Revertants per Plate |
8.34 |
20.33 |
26.21 |
146.25 |
241.84 |
Standard Deviation |
2.44 |
3.41 |
4.12 |
12.11 |
14.57 |
Maximum |
14 |
35 |
35 |
178 |
296 |
Minimum |
2 |
8 |
16 |
109 |
201 |
Positive Control without S9 Mix |
|||||
Mean Revertants per Plate |
324.78 |
426.99 |
567.00 |
939.15 |
1139.80 |
Standard Deviation |
172.50 |
160.32 |
179.90 |
164.06 |
193.54 |
Maximum |
1587 |
1151 |
1487 |
1782 |
2116 |
Minimum |
86 |
185 |
188 |
491 |
677 |
Positive Control with S9 Mix |
|||||
Mean Revertants per Plate |
342.77 |
469.93 |
684.25 |
1035.23 |
1253.88 |
Standard Deviation |
153.84 |
163.98 |
283.92 |
182.42 |
250.30 |
Maximum |
1355 |
1181 |
1999 |
1893 |
2520 |
Minimum |
138 |
161 |
224 |
420 |
670 |
Negative control
Dimethyl sulfoxide
Positive controls in the absence of metabolic activation
TA1537 = 9-Aminoacridine hydrochloride hydrate (75 µg/plate), TA1535 = Sodium azide (0.5 µg/plate), TA98 = 2-nitrofluorene (7.5 µg/plate), TA100 = Sodium azide (5 µg/plate), TA102 = Mitomycin-C (0.5 µg/plate)
Positive control in the presence of metabolic activation
2Aa = 2-Aminoanthracene (10 µg/plate for TA1537, TA1535, TA102 and 5 µg/plate for TA98 and TA100)
Applicant's summary and conclusion
- Conclusions:
- From the results of the study, it is concluded that PETA is non-mutagenic to any of the five strains of Salmonella typhimurium viz., TA1537, TA1535, TA98, TA100 and TA102 when tested under the specified conditions.
- Executive summary:
This study was performed to evaluate the mutagenic activity of PETA (Supplied by Nexam Chemical AB, Sweden) by the bacterial reverse mutation test, using five histidine deficient (his-) mutant tester strains of Salmonella typhimurium, TA1537, TA1535, TA98, TA100 and TA102. The method followed was as per the guidelines of the OECD No. 471 (July 1997).
The treatments were performed by the plate incorporation technique both in the absence and presence of metabolic activation (S9 mix). The S9 mix of 5% and 10% v/v consisted of an S9 fraction (Aroclor 1254 induced rat liver homogenate) supplemented with cofactors.
Before conducting the mutagenicity test, PETA was evaluated for cytotoxicity in tester strain TA100, both in the absence and presence of S9 mix (5% v/v S9 mix). Complete inhibition of the background lawn with reduction in revertant colonies (83-85% reduction) was observed at the tested concentration of 5000 μg/plate both in the absence and presence of the metabolic activation system while partial inhibition of the background lawn with reduction in revertant colonies (40-42% reduction) was observed at the tested concentration of 2500 μg/plate both in the absence and presence of metabolic activation system. Normal growth was observed up to the dose level of 1250 μg/plate both in the absence and presence of the metabolic activation system (5% v/v S9 mix).
Hence, 2500 μg/plate of PETA was selected as the highest concentration to be tested in the mutagenicity test both in the absence and presence of a metabolic activation system, for all the tester strains.
Based on the results of the cytotoxicity test, the test concentrations of 78.13, 156.25, 312.5, 625, 1250 and 2500 μg/plate of PETA both in the absence and presence (5% v/v S9 mix) of metabolic activation were selected for Trial I. Trial I did not show any positive mutagenic response when compared to the negative control at any of the tested concentrations. Trial II was conducted to confirm the negative results of Trial I with modified concentrations i.e., 25.6, 64, 160,400, 1000 and 2500 μg/plate in the absence and presence of metabolic activation (S9 concentration was increased to 10% v/v). A positive mutagenic response was not observed in Trial II confirming the results of Trial I. The efficiency of the test system was demonstrated by a clear increase in revertant colonies observed with the positive controls both in the absence and presence of metabolic activation.
From the results of this study, under the specified experimental conditions, PETA is concluded to be non-mutagenic in the bacterial reverse mutation assay using Salmonella typhimurium.
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