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EC number: - | CAS number: -
- 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
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
The test item Reaction mass of N-butylphthalimide and N-propylphthalimide and N-sec-butylphthalimide has no mutagenic activity on the applied bacterium tester strains under the test conditions used in the study OECD 471 (Ames test negative).
Reaction mass of N-butylphthalimide and N-propylphthalimide and N-sec-butylphthalimide tested up to cytotoxic concentrations, with and without mammalian metabolic activation system, induced structural chromosome aberrations and endoreduplication in Chinese Hamster lung cells.
Thus, the test item is considered clastogenic in this system (OECD 473 positive).
Based on the available in vitro data, an alkaline in vivo Comet assay (OECD 489) is proposed to assess the mutagenic properties of the test substance in vivo.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 26 April- 20 June, 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- 21st July, 1997
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- May 30, 2008
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
- Version / remarks:
- August 1998
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: ICH Guidance S2(R1): Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use, 2012
- Version / remarks:
- 2012
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- In addition to histidine and tryptophan mutation, each strain has additional mutations which enhance its sensitivity to mutagens. The uvrB (uvrA) strains are
defective in excision repair. It causes the strains to be more sensitive to the mutagenic and lethal effects of a wide variety of mutagens because they
cannot repair DNA damages. rfa mutation increases the permeability of the bacterial lipopolysaccharide wall for larger molecules. The plasmid pKM101
(TA98, TA100) carries the muc+ gene which participates in the error-prone "SOS" DNA repair pathway induced by DNA damage. This plasmid also carries an ampicillin resistance transfer factor (R-factor) which is used to identify its presence in the cell. The Escherichia coli strain used in this test (WP2uvrA) is also
defective in DNA excision repair. - Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Metabolic activation:
- with and without
- Metabolic activation system:
- post mitochondrial supernatant (S9) prepared from livers of Phenobarbital/¿-naphthoflavone-induced rats
- Test concentrations with justification for top dose:
- 3200; 1600; 500; 160; 50 and 16 µg/plate
- Vehicle / solvent:
- dimethyl sulfoxide (DMSO)
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- methylmethanesulfonate
- other: 4-Nitro-1,2-phenylenediamine, 2-aminoanthracene
- Details on test system and experimental conditions:
- The tester strains arrived to the test facility in a form of disc cultures. The origin of the following tester strains: Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coli WP2 uvrA:
Supplier: Trinova Biochem GmbH; Rathenau Str. 2; D-35394 Giessen, Germany;
Manufacturer: MOLTOX INC., P.O. BOX 1189; BOONE, NC 28607 USA.
Frozen stock cultures were prepared from the disc cultures. The identification codes and expiry dates of the actual applied stock cultures are summarized in the Table 3.
Storage of Tester Strains
The strains are stored at -80 ± 10ºC in the Laboratory of TOXI-COOP ZRT. in the form of lyophilized discs and in frozen permanent copies. Frozen permanent cultures of the tester strains are prepared from fresh, overnight cultures to which DMSO (8 % (v/v)) is added as a cryoprotective agent.
Confirmation of Phenotypes of Tester Strains
The phenotypes of the tester strains used in the bacterial reverse mutation assays with regard to membrane permeability (rfa), UV sensitivity
(uvrA and uvrB), ampicillin resistance (amp), as well as spontaneous mutation frequencies are checked regularly according to Ames.
Established procedures (Standard Operating Procedures) for the preparations of each batch of frozen stock culture and raw data and reports of phenotype confirmation are stored in the Laboratory of TOXI-COOP ZRT.
Each tester strain reverts spontaneously at a frequency that is characteristic for the strain. Spontaneous reversions of the test strains to histidine or tryptophan prototrophs are measured routinely in mutagenicity experiments and expressed as the number of spontaneous revertants per plate.
Procedure for Bacterial Cultures
The frozen bacterial cultures were thawed at room temperature and 200 µL inoculum was used to inoculate each 50 mL of Nutrient Broth No. 2 for the overnight cultures in the assay. The cultures were incubated for approximately 10-13 hours in a 37oC Benchtop Incubator Shaker.
The viability of each testing culture was determined by plating 0.1 mL of the 10-5, 10-6, 10-7 and 10-8 dilutions of cultures on nutrient agar plates. The viable cell number of the cultures was determined by manual colony counting.
The details of the used chemicals (supplier/manufacturer, batch/lot number and expiry/retest date) are summarised in Table 2. This report contains the media composition in the sections 5.4.2 - 5.4.5, 5.5.2 and 5.5.3 referring to 1000 mL.
Ready-to-use minimal glucose agar (MGA) plates were used in the study. The origin of the ready-to use MGA plates:
Supplier: VWR International;
Manufacturer: Merck Life Science GmbH, Germany.
Certificates of Analysis* were obtained from the supplier.
Typical composition (g/1000 mL) of MGA plates:
Glucose 20.0 g
Magnesium sulfate 0.2 g
Citric acid 2.0 g
di-Potassium hydrogenphosphate 10.0 g
Sodium ammonium hydrogenphosphate 3.5 g
Agar agar 13.0 g
* Batch No.: 142584; Expiry date: 22 May 2017; (used in the Informatory Toxicity Test)
143858; Expiry date: 16 August 2017; (used in the Initial and the Confirmatory Mutation Tests)
Nutrient Broth No. 2
Nutrient broth No. 2. 25.0 g
Ultrapure water ad 1000.0 mL
Sterilization for 20 minutes was performed at 121°C in an autoclave.
Nutrient Agar
Nutrient Agar 20.0 g
Ultrapure water ad 1000.0 mL
Sterilization for 20 minutes was performed at 121°C in an autoclave.
Top Agar for Salmonella typhimurium Strains
Agar solution:
Agar Bacteriological 4.0 g
NaCl 5.0 g
Ultrapure water ad 1000.0 mL
Sterilization for 20 minutes was performed at 121°C in an autoclave.
Histidine – Biotin solution (0.5 mM):
D-Biotin 122.2 mg
L-Histidine•HCl H2O 104.8 mg
Ultrapure water ad 1000.0 mL
Sterilization was performed by filtration through a 0.22 µm membrane filter.
Complete Top Agar for Salmonella typhimurium strains:
Histidine – Biotin solution (0.5 mM) 100.0 mL
Agar solution 900.0 mL
Top Agar for Escherichia coli Strain
Tryptophan solution (2 mg/mL):
L-Tryptophan 2000.0 mg
Ultrapure water ad 1000.0 mL
Sterilization was performed by filtration through a 0.22 µm membrane filter.
Complete Top Agar for Escherichia coli strain:
Nutrient Broth by (Section: 5.4.2) 50.0 mL
Tryptophan solution (2 mg/mL) 2.5 mL
Agar solution by (Section: 5.4.4) 947.5 mL
Metabolic Activation System
The test bacteria were also exposed to the test item in the presence of an appropriate metabolic activation system, which is a cofactor-supplemented post-mitochondrial fraction (S9).
Rat Liver S9 Fraction
The S9 fraction of Phenobarbital (PB) and ß-naphthoflavone (BNF)-induced rat liver was provided by Trinova Biochem GmbH (Rathenau Str. 2; D-35394 Giessen, Germany; Manufacturer: MOLTOX INC., P.O. BOX 1189; BOONE, NC 28607 USA).
The Quality Control & Production Certificate of each lot of S9 was obtained from the supplier1). The original Quality Control & Production Certificates of rat liver S9 are stored in the Laboratory of TOXI-COOP ZRT. The copies of the quality control certificates of the used S9 lots are given in Appendix VI. The following lots of the S9 were applied:
1) Lot Number: 3634; Expiry date: May 12, 2018; Protein content: 40.3 mg/mL
(used in the Informatory Toxicity and Confirmatory Mutation Test);
Lot Number: 3662; Expiry date: July 07, 2018; Protein content: 40.5 mg/mL
(used in the Informatory Toxicity Test);
Lot Number: 3712; Expiry date: November 03, 2018; Protein content: 34.3 mg/mL
(used in the Confirmatory Mutation Test);
Lot Number: 3727; Expiry date: December 01, 2018; Protein content: 33.7 mg/mL
(used in the Initial and Confirmatory Mutation Tests).
The S9 Mix (with Rat Liver S9)
Salt solution for S9 Mix Final concentration in S9 Mix
NADP Na 7.66 g 4 mM
D-glucose-6 phosphate Na 3.53 g 5 mM
MgCl2 1.90 g 8 mM
KCl 6.15 g 33 mM
Ultrapure water ad 1000 mL
Sterilized by filtration through a 0.22 µm membrane filter.
The complete S9 Mix was freshly prepared containing components as follows:
Ice cold 0.2 M sodium phosphate-buffer, pH 7.4 500 mL
Rat liver homogenate (S9) 100 mL
Salt solution for S9 Mix 400 mL
The S9 Mix was kept in an ice bath before it was added to the culture medium.
Sodium Phosphate Buffer (0.2 M, pH 7.4)
Solution A:
Na2HPO4 x 12H2O 71.63 g
Ultrapure water ad 1000 mL
Solution B:
NaH2PO4 x H2O 27.6 g
Ultrapure water ad 1000 mL
Solution A 880 mL
Solution B 120 mL*
* The components were mixed in the above ratio; thereafter the pH was checked and corrected. The correction was performed with admixture of the solution A or B.
After the pH setting the sterilization was performed by filtration through a 0.22 µm membrane filter. - Rationale for test conditions:
- Justification of concentrations:
Choice of the concentrations was done on the basis of a Solubility Test and a concentration Range Finding Test (Informatory Toxicity Test)
Based on the results of the preliminary Concentration Range Finding Test the following concentrations of the test item were prepared and investigated in
the Initial Mutation Test: 3200; 1600; 500; 160; 50 and 16 µg/plate.
Based on the results of the Initial Mutation Test modification of the originally proposed concentrations levels, ranges was considered as necessary in the Salmonella typhimurium strains for the second main experiment, for Confirmatory Mutation Test. The following concentrations of the test item were prepared and investigated in the Confirmatory Mutation Test in Salmonella typhimurium strains: 1600, 500, 160, 50, 16 and 5 µg/plate; in E. coli WP2 uvrA: 3200, 1600, 500, 160, 50 and 16 µg/plate.
No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9 Mix) throughout the
study.
In the Initial and Confirmatory Mutation Tests, unequivocal inhibitory effect of the test item on bacterial growth was observed. The cytotoxicity was
indicated by absent or decreased revertant colony counts (most of them below the corresponding historical control data ranges) and/or affected
background lawn development: reduced or slightly reduced background lawn. In general, 500 µg/plate was considered as lowest concentration showing
cytotoxicity.
The revertant colony numbers of vehicle control (dimethyl sulfoxide (DMSO) plates with and without S9 Mix demonstrated the characteristic mean number
of spontaneous revertants that was in line with the corresponding historical control data ranges. - Evaluation criteria:
- The colony numbers on the untreated, vehicle control, positive control and the test item treated plates were determined visually by manual counting.
A test item is considered mutagenic if:
- a dose–related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.
An increase is considered biologically relevant if:
- in strain Salmonella typhimurium TA100 the number of reversions is at least twice as high as the reversion rate of the vehicle control,
- in strain Salmonella typhimurium TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times
higher than the reversion rate of the vehicle control.
According to the guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is
not regarded as necessary.
Criteria for a Negative Response:
A test item is considered non-mutagenic if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant
positive response at any of the dose groups, with or without metabolic activation. - Statistics:
- The mean values and appropriate standard deviations and mutation rates were calculated by EXCEL software.
- 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
- Untreated negative controls validity:
- valid
- 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
- Vehicle controls validity:
- valid
- Untreated negative 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
- Untreated negative 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
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- 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:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- Validity of the Performed Experiments
The tester strains used in this study demonstrated the specific phenotype characteristics , were in line with the
corresponding historical control data ranges , and showed the adequate strain culture titer.
Each batch of the S9 fraction used in this test had the appropriate biological activity and was active in the applied system
Each of the investigated reference mutagens showed the expected increase (at least a 3-fold increase) in induced revertant colonies over the mean value of the respective vehicle control in both main experimental phases and the number of revertants in most cases fell in the corresponding historical control ranges , thereby meeting the criteria for the positive control in the main experimental phases, in all tester strains .
The spontaneous revertant colony numbers of the dimethyl sulfoxide (DMSO) vehicle control plates showed characteristic mean numbers agreed with the actual historical control data ranges in all strains in both main experimental phases.
Seven concentration levels were investigated in the Informatory Toxicity Test and six in the main mutation experiments (Initial and Confirmatory Mutation
Tests)
In the performed experimental phases there were at least five analyzable concentrations and a minimum of three non-toxic and non-precipitated dose levels at each tester strain.
All criteria for the validity of the performed experiments have therefore been met.
Controls
In the performed Initial and Confirmatory Mutation Test multiple test items were tested with reference values from the common parallel controls.
In the Initial and Confirmatory Mutation Tests the revertant colony numbers of the dimethyl sulfoxide (DMSO) vehicle control plates with and without S9 Mix
were in line with the corresponding historical control data ranges.
The reference mutagen treatments (positive controls) showed the expected, biological relevant increases in induced revertant colonies in all experimental phases, in all tester strains. In the Initial Mutation Test, in the case of S. typhimurium TA100 the revertant colony numbers of
Sodium azide (SAZ) were above the corresponding historical control data range; however the higher counts were considered as acceptable without any
effect on the final conclusion of the study.
The revertant colony numbers of the untreated and ultrapure water (ASTM Type I) control plates in different experimental phases were slightly higher or
lower than the dimethyl sulfoxide (DMSO) control plates. The higher or lower revertant counts of these controls remained in the corresponding historical
control data ranges.
In summary, the actual values of untreated, vehicle and positive controls were in line with the criteria for validity of the assay.
Initial and Confirmatory Mutation Tests (Plate Incorporation and Pre-Incubation Tests)
No substantial increases were observed in revertant colony numbers of any of the five test strains following treatment with Reaction mass of
N-butylphthalimide and N-propylphthalimide and N-sec-butylphthalimide at any concentration level, either in the presence or absence of metabolic activation
(S9 Mix) in the performed experiments.
In the performed experiments, sporadically increased revertant colony numbers were observed. These increases did not show a dose-response
relationship, were of minor intensity, and all of the increases remained far below the biologically relevant thresholds for being positive. The obtained increases were therefore considered as biologically not relevant, being in the range of the biological variability of the applied test system.
The highest revertant colony number increase was observed in the Confirmatory Mutation Test (Pre-Incubation Test) in S. typhimurium TA1535 strain, at
160 µg/plate, in the presence of metabolic activation (+S9). This value however remained in the range of the corresponding vehicle historical control data
and additional concentration related increase in revertant colony counts was not noticed. The mutation rate was 1.60, which was far below the
genotoxicological threshold for being positive.
In the Initial and Confirmatory Mutation Tests, unequivocal inhibitory effect of the test item on bacterial growth was observed in all examined strains.
The cytotoxicity was indicated by absent or decreased revertant colony counts (most of them below the corresponding historical control data ranges)
and/or affected background lawn development: reduced or slightly reduced background lawn. The table contains the unequivocal cytotoxicity results, only
, where the obtained revertant colony numbers were below the vehicle (in some cases below the corresponding historical control data ranges), and/or
affected background lawn development occurred. All of the further observed lower revertant colony numbers (when compared to the revertant colony
numbers of the corresponding vehicle control) remained in the range of the biological variability of the applied test system.
In general, 500 µg/plate was considered as lowest concentration showing cytotoxicity. - Conclusions:
- The reported data of this mutagenicity assay show that under the experimental conditions applied, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
In conclusion, the test item Reaction mass of N-butylphthalimide and N-propylphthalimide and N-sec-butylphthalimide has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study. - Executive summary:
The test itemReaction mass of N-butylphthalimide and N-propylphthalimide and N-sec-butylphthalimidewas tested with regard to a potential mutagenic activity using the Bacterial Reverse Mutation Assay.
The experiments were carried out using histidine-requiring auxotroph strains ofSalmonella typhimurium(Salmonella typhimuriumTA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain ofEscherichia coli(Escherichia coliWP2uvrA) in the presence and absence of a post mitochondrial supernatant (S9) prepared from livers of Phenobarbital/b-naphthoflavone-induced rats.
The study included a Preliminary Solubility Test, a Preliminary Concentration Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Test), and a Confirmatory Mutation Test (Pre-Incubation Test).
Based on the results of the Solubility and the Concentration Range Finding Tests the test item was dissolved in dimethyl sulfoxide (DMSO). This vehicle was compatible with the survival of the bacteria and the S9 activity and appropriate historical control database is available in the testing laboratory.
Based on the results of the preliminary Concentration Range Finding Test the following concentrations of the test item were prepared and investigated in the Initial Mutation Test:3200; 1600; 500; 160; 50 and 16 µg/plate.
The selection of the concentration range was based on the recommendations in OECD 471 guideline for cytotoxic, soluble test compounds; accordingly the test item was investigated up to and including the concentration level of 3200 µg/plate.
Based on the results of the Initial Mutation Testmodification of the originally proposed concentrations levels, ranges was considered as necessary in theSalmonella typhimuriumstrains for the second main experiment, for Confirmatory Mutation Test. The following concentrations of the test item were prepared and investigated in the Confirmatory Mutation TestinSalmonella typhimuriumstrains: 1600, 500, 160, 50, 16 and 5 µg/plate; inE. coliWP2uvrA: 3200, 1600, 500, 160, 50 and 16 µg/plate.
No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9 Mix) throughout the study.
In the Initial and Confirmatory Mutation Tests, unequivocal inhibitory effect of the test item on bacterial growth was observed. The cytotoxicity was indicated by absent or decreased revertant colony counts (most of them below the corresponding historical control data ranges) and/or affected background lawn development: reduced or slightly reduced background lawn.In general, 500 µg/plate was considered as lowest concentration showing cytotoxicity.
The revertant colony numbers of vehicle control (dimethyl sulfoxide (DMSO) plates with and without S9 Mix demonstratedthe characteristic mean number of spontaneous revertantsthat was in line with the corresponding historical control data ranges.
The reference mutagen treatments (positive controls) showed the expected, biological relevant increases (more than 3-fold increase)in induced revertant coloniesand nearly all the number of revertants fell in the corresponding historical control ranges, thereby meeting the criteria for the positive controlin all experimental phases, in all tester strains.
No biologically relevant increases were observed in revertant colony numbers of any of the five test strains following treatment withReaction mass of N-butylphthalimide and N-propylphthalimide and N-sec-butylphthalimideat any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments.
The reported data of this mutagenicity assay show that under the experimental conditions applied, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
In conclusion, the test itemReaction mass of N-butylphthalimide and N-propylphthalimide and N-sec-butylphthalimidehas no mutagenic activity on the applied bacterium tester strainsunder the test conditions used in this study.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 25 August, 2017 - 15 January, 2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Version / remarks:
- 29th July, 2016
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
- Version / remarks:
- August 1998
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- other: This in vitro test is a cytogenetic test, which detects structural chromosome aberrations in somatic and/or germ cells
- Target gene:
- structural chromosome aberrations in somatic and/or germ cells
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- V79: Chinese hamster lung male
Lot. No.: 10H016
Supplier: ECACC (European Collection of Cells Cultures)
The V79 cell line is well established in toxicology studies. Stability of karyotype and morphology makes it suitable for gene toxicity assays with low background aberrations. - Cytokinesis block (if used):
- Cell cultures were treated with Colchicine (0.2 µg/mL)
- Metabolic activation:
- with and without
- Metabolic activation system:
- rodent S9 mix
- Test concentrations with justification for top dose:
- Experiment A with 3/20 h treatment/sampling time
without: 31.3, 62.5, 125 and 187.5 *g/mL test item
with S9 mix: 31.3, 62.5, 125 and 250 *g/mL test item
Experiment B with 20/20 h treatment/sampling time
without S9 mix: 15.6, 31.3, 62.5 and 125 *g/mL test item
Experiment B with 20/28 h treatment/sampling time
without S9 mix: 15.6, 31.3, 62.5 and 125 *g/mL test item
Experiment B with 3/28 h treatment/sampling time
with S9 mix: 31.3, 62.5, 125 and 250 *g/mL test item - Vehicle / solvent:
- Dimethyl sulfoxide DMSO
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- V79: Chinese hamster lung male
Lot. No.: 10H016
Supplier: ECACC (European Collection of Cells Cultures)
The V79 cell line is well established in toxicology studies. Stability of karyotype and morphology makes it suitable for gene toxicity assays with low
background aberrations. These cells were chosen because of their small number of chromosomes (diploid number, 2n=22) and because of the high
proliferation rates (doubling time 12 14 h). The V79 cell line was established after spontaneous transformation of cells isolated from the lung of a normal
Chinese hamster (male).
This cell line was purchased from ECACC (European Collection of Cells Cultures). The cell stocks were kept in liquid nitrogen. Checking for mycoplasma
infections was carried out. Trypsin-EDTA (0.25 % Trypsin, 1mM EDTA x 4 Na) solution was used for cell detachment to subculture. The laboratory
cultures were maintained in 75 cm2 plastic flasks at 37 +/- 0.5 ¿C in an incubator with a humidified atmosphere, set at 5 % CO2. The V79 cells for this
study was grown in DME (Dulbecco’s Modified Eagle’s) medium supplemented with
L-glutamine (2mM) and 1 % of Antibiotic-antimycotic solution (containing 10000 units/mL penicillin, 10 mg/mL streptomycin and 25 ¿g/mL amphoptericin-B) and heat-inactivated bovine serum (final concentration 10 %). During the 3 and 20 hours treatments with test item, negative and positive controls, the serum
content was reduced to 5%.
Mammalian Microsomal Fraction S9 Mix
An advantage of using in vitro cell cultures is the accurate control of the concentration and exposure time of cells to the test item under study. However,
due to the limited capacity of cells growing in vitro for metabolic activation of potential mutagens, an exogenous metabolic activation system is necessary.
Many substances only develop mutagenic potential when they are metabolised by the mammalian organism. Metabolic activation of substances can be
achieved by supplementing the cell cultures with liver microsome preparations (S9 mix). The protein concentrations of the S9 batch used in the experiments were 33.7 and 33.8 mg/mL.
Rat Liver S9 Fraction
The S9 fraction of phenobarbital (PB) and ß-naphthoflavone (BNF) induced rat liver was provided by Trinova Biochem GmbH (Rathenau Strasse 2, D-35394
Giessen, Germany; manufacturer: MOLTOX INC., P.O. BOX 1189, BOONE, NC 28607 USA). Certificate of Analysis was obtained from the supplier.
The Certificate of Analysis of rat liver S9 mix is stored in the laboratory.
The S9 Mix (with Rat Liver S9)
The complete S9 Mix was freshly prepared containing components with the following ratios:
S9 fraction 3 mL
HEPES* 20 mM 2 mL
KCl 330 mM 1 mL
MgCl2 50 mM 1 mL
NADP** 40 mM 1 mL
Glucose-6-phosphate 50 mM 1 mL
DME medium 1 mL
*= N-2-Hydroxyethylpiperazine-N-2-Ethane Sulphonic Acid
**= ß-Nicotinamide Adenine Dinucleotide Phosphate
Before adding to the culture medium the S9 mix was kept in an ice bath. - Rationale for test conditions:
- Acceptability of the Assay
The Chromosome Aberration Assay is considered acceptable because it meets the following criteria:
–the number of aberrations found in the negative and /or solvent controls falls within the range of historical laboratory control data, .
–concurrent positive controls induce responses that are compatible with the historical positive control data base and produce a statistically significant
increase compared with the concurrent negative control,
–cell proliferation in the solvent control is adequate,
–adequate number of cells and concentrations are analyzable,
–all requested experimental conditions were tested unless one resulted in a positive result
–the criteria for the selection of top concentration is adequate. - Evaluation criteria:
- Evaluation of Result
Treatment of results
–The percentage of cells with structural chromosome aberration(s) was evaluated.
–Different types of structural chromosome aberrations are listed, with their numbers and frequencies for experimental and control cultures.
–Gaps were recorded separately and reported, but generally not included in the total aberration frequency.
–Concurrent measures of cytotoxicity for all treated and negative control cultures in the main aberration experiment (s) were recorded.
–Individual culture data were summarised in tabular form.
–There were no equivocal results in this study.
–pH and Osmolality data were summarised in tabular form.
Interpretation of Results
Providing that all acceptability criteria are fulfilled, a test item is considered to be clearly positive because:
–one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
–the increase is dose-related when evaluated with an appropriate trend test,
–any of the results are outside the distribution of the laboratory historical negative control data.
Providing that all acceptability criteria are fulfilled, a test item is considered clearly negative if:
–none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
– there is no concentration-related increase when evaluated with an appropriate trend test,
– all results are inside the distribution of the laboratory historical negative control data - Statistics:
- For statistical analysis CHI2 test was utilized. The parameters evaluated for statistical analysis were the number of aberrations (with and without gaps) and number of cells with aberrations (with and without gaps). The number of aberrations in the treatment and positive control groups were compared to the concurrent negative control. The concurrent negative and positive controls and the treatment groups were compared to the laboratory historical controls, too.
- Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- Solubility and Dose Selection
Reaction mass of N-butylphthalimide and N-propylphthalimide and N-sec-butylphthalimide was dissolved in DMSO. A clear solution was obtained up to a
concentration of 100 mg/mL. There was no precipitation in the medium at any concentration tested. Concentration selection cytotoxicity assay was performed as part of this study to establish an appropriate concentration range for the Chromosome Aberration Assays (Experiment A and B), both in the absence
and in the presence of a metabolic activation system (rodent S9 mix). Toxicity was determined by cell counting and results noted. Based on the cell counts
Relative Increase in Cell Counts (RICC) was calculated, which is an indicator of cytotoxicity. Detailed results of the cytotoxicity assay with Reaction mass
of N-butylphthalimide and N-propylphthalimide and N-sec-butylphthalimide are presented in Table 2A - 2C. These results were used to select concentrations
of test item for the Chromosome Aberration Assays.
Based on the results of the cytotoxicity assay the following concentrations were selected for the Chromosome Aberration Assay.
All concentrations were run in duplicates (incl. negative and positive controls) and at least 300 (150-150) well-spread metaphases were assessed:
Experiment A with 3/20 h treatment/sampling time
without: 31.3, 62.5, 125 and 187.5 *g/mL test item
with S9 mix: 31.3, 62.5, 125 and 250 *g/mL test item
Experiment B with 20/20 h treatment/sampling time
without S9 mix: 15.6, 31.3, 62.5 and 125 *g/mL test item
Experiment B with 20/28 h treatment/sampling time
without S9 mix: 15.6, 31.3, 62.5 and 125 *g/mL test item
Experiment B with 3/28 h treatment/sampling time
with S9 mix: 31.3, 62.5, 125 and 250 *g/mL test item
In the performed Chromosome Aberration Assay the concentration levels were chosen mainly based on the cytotoxicity.
Chromosome Aberration Assay
In Experiment A, Reaction mass of N-butylphthalimide and N-propylphthalimide and
N-sec-butylphthalimide did not induce an increase in the number of cells with aberrations without gaps at any examined concentration, neither in the
absence nor in the presence of metabolic activation. (Tables 3, 4 and 8). There were no statistically significant differences between test item treatment
and control groups and no dose-response relationship was noted.
In Experiment B, the frequency of the cells with structural chromosome aberrations showed biologically significant increase compared to concurrent
controls, up to the maximum cytotoxic concentrations without S9 mix over a prolonged treatment period of 20 hours with harvest at 20 or 28 hours
following treatment start. The results were outside the distribution of the laboratory historical negative control data.
A 3-hour treatment up to cytotoxic concentrations in the presence of S9 mix with 28-hour harvest from the beginning of treatment caused biologically
and statistically (250 µg/mL) significant increase in the number of cells showing structural chromosome aberrations. Furthermore dose-response
relationships was noted (Tables 5, 6, 7 and 9).
Tables 10-11 show the occurrence of polyploidy and endoreduplicated metaphases. No increase in the rate of polyploid was found. In the Experiment A
endoreduplication was noted at concentration of 31.3 and 250 µg/mL and in Experiment B at centrations of 15.6 and 125 µg/mL with low incidence.
Pulverization was oserved one times in Experiment A and in Experiment B, too.
pH and osmolality values of control and test item treatment solutions were measured. In Experiments A and B no significant differences between test item
treatment and control groups were observed .
In the concurrent negative control group the percentage of cells with structural aberration(s) without gap was less than 5 %, confirming the suitability of
the cell line used.
The number of aberrations found in the solvent controls was in the range of historical laboratory control data. The concurrent positive controls ethyl methanesulphonate (0.4 and 1.0 *L/mL) and Cyclophosphamide (5 *g/mL) caused the expected biologically relevant increases of cells with structural chromosome aberrations as compared to solvent controls and were compatible with the historical positive control data (Tables 3, 4, 5, 6 and 7). Thus, the study is considered valid. - Remarks on result:
- other: The test item induced structural chromosome aberrations and endoreduplication in Chinese Hamster lung cells.
- Conclusions:
- Reaction mass of N-butylphthalimide and N-propylphthalimide and N-sec-butylphthalimide tested up to cytotoxic concentrations, with and without mammalian metabolic activation system, induced structural chromosome aberrations and endoreduplication in Chinese Hamster lung cells.
Thus, the test item is considered clastogenic in this system. - Executive summary:
The test item, Reaction mass of N-butylphthalimide and N-propylphthalimide and N-sec-butylphthalimide was tested in a Chromosome Aberration Assay in V79 cells.The test item was dissolved inDMSOand the following concentrations were selected on the basis of cytotoxicity investigations made in a preliminary study (without and with metabolic activation using rodent S9 mix).In two independent experiments (both run in duplicate with concurrent negative and positive controls) at least 300 (150-150) well-spread metaphase cells were analysed at concentrations and treatment (exposure)/sampling (expression) intervals given below:
Experiment Awith 3/20 h treatment/sampling time
without: 31.3, 62.5, 125 and 187.5g/mL test item
with S9 mix: 31.3, 62.5, 125 and 250g/mL test item
Experiment Bwith 20/20 h treatment/sampling time
without S9 mix: 15.6, 31.3, 62.5 and 125g/mL test item
Experiment Bwith 20/28 h treatment/sampling time
without S9 mix: 15.6, 31.3, 62.5 and 125g/mL test item
Experiment Bwith 3/28 h treatment/sampling time
with S9 mix: 31.3, 62.5, 125 and 250g/mL test item
In the performed Chromosome Aberration Assay the concentration levels were chosen mainly based on the cytotoxicity.
Following treatment (exposure) and sampling (expression) time cells were exposed to selection agent Colchicine (0.2 µg/mL) 2.5 hours prior to harvesting. Following harvesting cells were treated with fixative for ca. 10 min. before being placed on slides and stained. Chromosome aberration frequencies were then scored for at least 300 well-spread metaphase cells.
In Experiment A, there were no biologically or statistically significant increases in the number of cells showing structural chromosome aberrations, neither in the absence nor in the presence of metabolic activation, up to the maximum cytotoxic concentrations.There were no statistical differences between treatment and concurrent solvent and historical control groups and no dose-response relationships were noted.
In Experiment B, the frequency of the cells with structural chromosome aberrations showed biologically significant increase compared to concurrent controls, up to the maximum cytotoxic concentrations without S9 mix over a prolonged treatment period of 20 hours with harvest at 20 or 28 hours following treatment start. The results were outside the distribution of the laboratory historical negative control data.
A 3-hour treatment up to cytotoxic concentrations in the presence of S9 mix with 28-hour harvest from the beginning of treatment caused biologically and statistically (250 µg/mL) significant increase in the number of cells showing structural chromosome aberrations. Furthermore dose-response relationships was noted.
There were no increases in the rate of polyploid metaphases in either experiment in the presence or absence of metabolic activation. Endoreduplicated metaphases was observed with very low incidence in Experiment A and in Experiment B.
There was no precipitation of the test item at any dose level tested. No biologically relevant changes in pH or osmolality of the test system were noted at the different dose levels tested.
The number of aberrations found in the solvent controls was in the range of historical laboratory control data. The concurrent positive controls ethyl methanesulphonate (0.4 and 1.0 L/mL) and Cyclophosphamide (5 g/mL) caused the expected biologically relevant increases of cells with structural chromosome aberrations as compared to solvent controls and were compatible with the historical positive control data. Thus, the study is considered valid.
Reaction mass of N-butylphthalimide and N-propylphthalimide and N-sec-butylphthalimidetested up to cytotoxic concentrations, with and without mammalian metabolic activation system, induced structural chromosome aberrations and endoreduplication in Chinese Hamster lung cells.
Thus, the test item is considered clastogenic in this system.
Referenceopen allclose all
Summary of the Inhibitory Tendencies in the Initial and Confirmatory Mutation Tests
Initial Mutation Test |
||||||||||
Concentrations (µg/plate) |
Salmonella typhimurium |
Escherichia coliWP2uvrA |
||||||||
TA98 |
TA100 |
TA1535 |
TA1537 |
|||||||
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
|
3200 |
B0 |
B0 |
<< B |
<< B |
B0 |
B0 |
B0 |
<< B |
<< SB |
<< SB |
1600 |
<< B |
< SB |
<< SB |
<< SB |
< SB |
< SB |
<< B |
<< B |
- |
- |
500 |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
160 |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
50 |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
16 |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
Confirmatory Mutation Test |
||||||||||
Concentrations (µg/plate) |
Salmonella typhimurium |
Escherichia coliWP2uvrA |
||||||||
TA98 |
TA100 |
TA1535 |
TA1537 |
|||||||
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
|
3200 |
|
|
|
|
|
|
|
|
< SB |
< SB |
1600 |
<< B |
<< SB |
<< SB |
<< B |
B0 |
< B |
<< B |
<< B |
< SB |
- |
500 |
<< SB |
<< |
<< SB |
<< SB |
<< SB |
SB |
B0 |
< SB |
< SB |
- |
160 |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
50 |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
16 |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
5 |
- |
- |
- |
- |
- |
- |
- |
- |
|
|
<: Revertant colony numbers significantly below the vehicle control data range
<<: Revertant colony numbers below the vehicle and historical control data ranges
B: Reduced background lawn development
SB: Slightly reduced background lawn development
B0: Reduced background lawn development and absent revertant colonies
No precipitation of the test item was observed in the Initial and Confirmatory Mutation Tests on the plates in the examined bacterial strains at any examined concentration level (±S9 Mix).
Historical Control Values for Revertants/Plate (for the Period of 2008-2016)
|
Bacterial strains |
||||||
Historical control data of untreated control |
-S9 |
|
TA98 |
TA100 |
TA1535 |
TA1537 |
E. coli |
Average |
21.0 |
105.0 |
10.5 |
8.1 |
25.4 |
||
SD |
3.7 |
25.7 |
1.4 |
2.3 |
5.2 |
||
Minimum |
9 |
66 |
3 |
2 |
11 |
||
Maximum |
39 |
155 |
23 |
19 |
45 |
||
+S9 |
|
TA98 |
TA100 |
TA1535 |
TA1537 |
E. coli |
|
Average |
27.5 |
117.1 |
11.8 |
9.0 |
33.9 |
||
SD |
4.3 |
18.1 |
1.4 |
1.9 |
5.2 |
||
Minimum |
12 |
75 |
4 |
2 |
17 |
||
Maximum |
46 |
166 |
23 |
20 |
56 |
||
|
Bacterial strains |
||||||
Historical control data of DMSO control |
-S9 |
|
TA98 |
TA100 |
TA1535 |
TA1537 |
E. coli |
Average |
20.4 |
100.1 |
10.3 |
7.9 |
24.7 |
||
SD |
3.6 |
24.8 |
1.3 |
2.4 |
4.6 |
||
Minimum |
10 |
64 |
3 |
2 |
11 |
||
Maximum |
38 |
147 |
23 |
20 |
45 |
||
+S9 |
|
TA98 |
TA100 |
TA1535 |
TA1537 |
E. coli |
|
Average |
26.5 |
113.8 |
11.8 |
8.8 |
33.7 |
||
SD |
4.1 |
18.3 |
1.5 |
1.9 |
5.0 |
||
Minimum |
15 |
71 |
3 |
3 |
16 |
||
Maximum |
47 |
162 |
25 |
20 |
57 |
||
|
Bacterial strains |
||||||
Historical control data of Water control |
-S9 |
|
TA98 |
TA100 |
TA1535 |
TA1537 |
E. coli |
Average |
21.9 |
104.7 |
10.5 |
7.6 |
26.1 |
||
SD |
3.7 |
25.9 |
1.5 |
2.2 |
5.5 |
||
Minimum |
12 |
68 |
3 |
2 |
12 |
||
Maximum |
35 |
154 |
24 |
16 |
48 |
||
+S9 |
|
TA98 |
TA100 |
TA1535 |
TA1537 |
E. coli |
|
Average |
27.4 |
117.3 |
11.4 |
8.7 |
34.9 |
||
SD |
4.0 |
18.5 |
1.3 |
2.2 |
4.9 |
||
Minimum |
15 |
83 |
4 |
3 |
18 |
||
Maximum |
43 |
167 |
22 |
16 |
57 |
||
|
Bacterial strains |
||||||
Historical control data of positive controls |
-S9 |
|
TA98 |
TA100 |
TA1535 |
TA1537 |
E. coli |
Average |
260.1 |
977.2 |
847.3 |
478.6 |
724.5 |
||
SD |
31.8 |
150.6 |
126.3 |
104.5 |
65.0 |
||
Minimum |
123 |
521 |
359 |
110 |
320 |
||
Maximum |
664 |
1970 |
1855 |
1601 |
1313 |
||
+S9 |
|
TA98 |
TA100 |
TA1535 |
TA1537 |
E. coli |
|
Average |
1222.7 |
1436.4 |
164.1 |
147.0 |
257.7 |
||
SD |
274.9 |
318.3 |
33.1 |
20.1 |
72.5 |
||
Minimum |
386 |
583 |
85 |
69 |
140 |
||
Maximum |
2676 |
2988 |
498 |
399 |
477 |
Abbreviations: TA98, TA100, TA1535, TA1537: Salmonella typhimuriumTA98, TA100, TA1535,
TA1537;E. coli:Escherichia coliWP2uvrA
SD: Standard deviation; DMSO: Dimethyl sulfoxide
Summary Table of the Results of the Concentration Range Finding Test
Range Finding Test (Informatory Toxicity Test) |
|
||||||||
Concentrations (mg/plate) |
Salmonella typhimuriumtester strains |
||||||||
TA 98 |
TA 100 |
||||||||
-S9 |
+S9 |
-S9 |
+S9 |
||||||
Mean values of revertants per plate and |
Mean |
MR |
Mean |
MR |
Mean |
MR |
Mean |
MR |
|
Untreated Control |
21.7 |
0.93 |
25.7 |
1.01 |
103.7 |
1.05 |
121.0 |
1.30 |
|
DMSO Control |
23.3 |
1.00 |
25.3 |
1.00 |
99.0 |
1.00 |
93.3 |
1.00 |
|
Ultrapure Water Control |
– |
– |
– |
– |
94.0 |
1.00 |
– |
– |
|
5000 |
0.0 |
0.00 |
0.0 |
0.00 |
6.7 |
0.07 |
10.0 |
0.11 |
|
1600 |
6.0 |
0.26 |
16.7 |
0.66 |
26.7 |
0.27 |
61.0 |
0.65 |
|
500 |
21.0 |
0.90 |
24.3 |
0.96 |
81.7 |
0.82 |
105.7 |
1.13 |
|
160 |
18.3 |
0.79 |
24.7 |
0.97 |
84.3 |
0.85 |
103.0 |
1.10 |
|
50 |
18.0 |
0.77 |
26.7 |
1.05 |
87.3 |
0.88 |
93.0 |
1.00 |
|
16 |
24.3 |
1.04 |
24.0 |
0.95 |
83.7 |
0.85 |
96.7 |
1.04 |
|
5 |
24.3 |
1.04 |
21.0 |
0.83 |
90.0 |
0.91 |
109.7 |
1.18 |
|
NPD (4mg) |
218.7 |
9.37 |
– |
– |
– |
– |
– |
– |
|
SAZ (2mg) |
– |
– |
– |
– |
1944.0 |
20.68 |
– |
– |
|
2AA (2mg) |
– |
– |
1746.7 |
68.95 |
– |
– |
2360.0 |
25.29 |
|
MR:Mutation Rate
NPD:4-Nitro-1,2-phenylenediamine
SAZ:Sodium azide
2AA:2-aminoanthracene
Remarks: DMSO was applied as vehicle of the test item and the positive control substances NPD and 2AA. The ultrapure water was applied as vehicle of the positive control substance SAZ. The mutation rate of the test item, the untreated control; furthermore NPD and 2AA refers to the DMSO sample. The mutation rate of SAZ refers to ultrapure water.
Summary Table of the Results of the Initial Mutation Test
Initial Mutation Test (Plate Incorporation Test) |
||||||||||||||||||||
Concentrations (mg/plate) |
Salmonella typhimuriumtester strains |
Escherichiacoli |
||||||||||||||||||
TA 98 |
TA 100 |
TA 1535 |
TA 1537 |
WP2uvrA |
||||||||||||||||
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
|||||||||||
Mean values of revertants per plate Mutation rate (MR) |
Mean |
MR |
Mean |
MR |
Mean |
MR |
Mean |
MR |
Mean |
MR |
Mean |
MR |
Mean |
MR |
Mean |
MR |
Mean |
MR |
Mean |
MR |
Untreated Control |
25.3 |
1.36 |
36.3 |
1.43 |
97.0 |
1.27 |
101.3 |
0.96 |
10.7 |
0.91 |
13.7 |
1.00 |
7.7 |
1.10 |
11.3 |
1.17 |
27.0 |
0.95 |
37.3 |
1.13 |
DMSO Control |
18.7 |
1.00 |
25.3 |
1.00 |
76.3 |
1.00 |
105.7 |
1.00 |
11.7 |
1.00 |
13.7 |
1.00 |
7.0 |
1.00 |
9.7 |
1.00 |
28.3 |
1.00 |
33.0 |
1.00 |
Ultrapure Water Control |
– |
– |
– |
– |
82.7 |
1.00 |
– |
– |
13.0 |
1.00 |
– |
– |
– |
– |
– |
– |
33.7 |
1.00 |
– |
– |
3200 |
0.0 |
0.00 |
0.0 |
0.00 |
2.7 |
0.03 |
24.3 |
0.23 |
0.0 |
0.00 |
0.0 |
0.00 |
0.0 |
0.00 |
1.0 |
0.10 |
8.3 |
0.29 |
13.3 |
0.40 |
1600 |
4.0 |
0.21 |
14.7 |
0.58 |
37.3 |
0.49 |
53.7 |
0.51 |
6.3 |
0.54 |
6.0 |
0.44 |
0.7 |
0.10 |
1.7 |
0.17 |
24.0 |
0.85 |
35.0 |
1.06 |
500 |
17.0 |
0.91 |
19.7 |
0.78 |
82.0 |
1.07 |
100.3 |
0.95 |
10.7 |
0.91 |
7.3 |
0.54 |
5.7 |
0.81 |
10.7 |
1.10 |
26.0 |
0.92 |
37.3 |
1.13 |
160 |
18.3 |
0.98 |
27.3 |
1.08 |
79.3 |
1.04 |
93.0 |
0.88 |
12.3 |
1.06 |
11.0 |
0.80 |
6.7 |
0.95 |
11.3 |
1.17 |
22.3 |
0.79 |
32.0 |
0.97 |
50 |
18.3 |
0.98 |
19.0 |
0.75 |
86.0 |
1.13 |
97.0 |
0.92 |
11.7 |
1.00 |
14.0 |
1.02 |
6.0 |
0.86 |
8.3 |
0.86 |
31.0 |
1.09 |
31.3 |
0.95 |
16 |
19.0 |
1.02 |
25.3 |
1.00 |
88.0 |
1.15 |
97.7 |
0.92 |
12.7 |
1.09 |
11.7 |
0.85 |
5.7 |
0.81 |
10.0 |
1.03 |
27.3 |
0.96 |
38.3 |
1.16 |
NPD (4mg) |
271.3 |
14.54 |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
SAZ (2mg) |
– |
– |
– |
– |
2189.3 |
26.48 |
– |
– |
862.7 |
66.36 |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
9AA (50mg) |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
914.0 |
130.57 |
– |
– |
– |
– |
– |
– |
MMS (2mL) |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
800.0 |
23.76 |
– |
– |
2AA (2mg) |
– |
– |
2397.3 |
94.63 |
– |
– |
2205.3 |
20.87 |
– |
– |
274.7 |
20.10 |
– |
– |
152.0 |
15.72 |
– |
– |
– |
– |
2AA (50mg) |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
189.0 |
5.73 |
MR:Mutation Rate; NPD:4-Nitro-1,2-phenylenediamine;SAZ: Sodium azide;9AA:9-Aminoacridine;MMS:Methyl methanesulfonate;2AA: 2-aminoanthracene
Remarks: DMSO was applied as vehicle of the test item and positive control substances: NPD, 9AA and 2AA and the ultrapure water was applied as vehicle for the SAZ and MMS. The mutation rate of the test item and the untreated control refers to the DMSO. The mutation rate of the NPD, 9AA and 2AA refers to the DMSO and the mutation rate of the SAZ and MMS positive control refers to the ultrapure water.
Summary Table of the Results of the Confirmatory Mutation Test
Confirmatory Mutation Test (Pre-Incubation Test) |
||||||||||||||||||||
Concentrations (mg/plate) |
Salmonella typhimuriumtester strains |
Escherichia coli |
||||||||||||||||||
TA 98 |
TA 100 |
TA 1535 |
TA 1537 |
WP2uvrA |
||||||||||||||||
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
|||||||||||
Mean values of revertants per plate Mutation rate (MR) |
Mean |
MR |
Mean |
MR |
Mean |
MR |
Mean |
MR |
Mean |
MR |
Mean |
MR |
Mean |
MR |
Mean |
MR |
Mean |
MR |
Mean |
MR |
Untreated Control |
15.0 |
0.94 |
22.7 |
1.19 |
113.3 |
1.11 |
113.3 |
1.00 |
13.0 |
0.87 |
14.0 |
1.20 |
8.7 |
1.37 |
8.7 |
1.08 |
30.7 |
1.05 |
34.0 |
1.10 |
DMSO Control |
16.0 |
1.00 |
19.0 |
1.00 |
102.0 |
1.00 |
113.3 |
1.00 |
15.0 |
1.00 |
11.7 |
1.00 |
6.3 |
1.00 |
8.0 |
1.00 |
29.3 |
1.00 |
31.0 |
1.00 |
Ultrapure Water Control |
– |
– |
– |
– |
109.3 |
1.00 |
– |
– |
12.3 |
1.00 |
– |
– |
– |
– |
– |
– |
33.7 |
1.00 |
– |
– |
3200 |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
13.7 |
0.47 |
18.0 |
0.58 |
1600 |
3.0 |
0.19 |
9.3 |
0.49 |
34.3 |
0.34 |
42.3 |
0.37 |
0.0 |
0.00 |
6.7 |
0.57 |
0.7 |
0.11 |
0.7 |
0.08 |
12.3 |
0.42 |
26.7 |
0.86 |
500 |
1.7 |
0.10 |
11.7 |
0.61 |
21.7 |
0.21 |
50.7 |
0.45 |
2.7 |
0.18 |
10.3 |
0.89 |
0.0 |
0.00 |
4.7 |
0.58 |
13.3 |
0.45 |
35.0 |
1.13 |
160 |
15.0 |
0.94 |
21.3 |
1.12 |
73.3 |
0.72 |
92.3 |
0.81 |
13.0 |
0.87 |
18.7 |
1.60 |
5.7 |
0.89 |
8.0 |
1.00 |
36.0 |
1.23 |
43.7 |
1.41 |
50 |
15.7 |
0.98 |
27.0 |
1.42 |
91.7 |
0.90 |
87.7 |
0.77 |
12.7 |
0.84 |
11.3 |
0.97 |
5.0 |
0.79 |
7.3 |
0.92 |
29.7 |
1.01 |
38.3 |
1.24 |
16 |
16.0 |
1.00 |
29.0 |
1.53 |
98.3 |
0.96 |
93.0 |
0.82 |
13.3 |
0.89 |
16.0 |
1.37 |
6.7 |
1.05 |
6.7 |
0.83 |
27.0 |
0.92 |
42.0 |
1.35 |
5 |
16.3 |
1.02 |
28.0 |
1.47 |
95.7 |
0.94 |
82.3 |
0.73 |
12.7 |
0.84 |
12.3 |
1.06 |
8.3 |
1.32 |
7.3 |
0.92 |
– |
– |
– |
– |
NPD (4mg) |
256.0 |
16.00 |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
SAZ (2mg) |
– |
– |
– |
– |
1584.0 |
14.49 |
– |
– |
1176.0 |
95.35 |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
9AA (50mg) |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
679.3 |
107.26 |
– |
– |
– |
– |
– |
– |
MMS (2mL) |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
1226.7 |
36.44 |
– |
– |
2AA (2mg) |
– |
– |
992.0 |
52.21 |
– |
– |
1464.0 |
12.92 |
– |
– |
211.0 |
18.09 |
– |
– |
115.0 |
14.38 |
– |
– |
– |
– |
2AA (50mg) |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
150.7 |
4.86 |
MR:Mutation Rate; NPD:4-Nitro-1,2-phenylenediamine;SAZ: Sodium azide;9AA:9-Aminoacridine;MMS:Methyl methanesulfonate;2AA: 2-aminoanthracene
Remarks: DMSO was applied as vehicle of the test item and positive control substances: NPD, 9AA and 2AA and the ultrapure water was applied as vehicle for the SAZ and MMS. The mutation rate of the test item and the untreated control refers to the DMSO. The mutation rate of the NPD, 9AA and 2AA refers to the DMSO and the mutation rate of the SAZ and MMS positive control refers to the ultrapure water.
Summarized Results of the concentration SELECTION CYTOTOXICITY ASSAY
3-hour treatment without and with S9 mix / 20-hour sampling time
Test group |
Concentration |
Parallels |
S9-mix |
Cell counts |
Mean cell counts |
Increase in cell counts |
RICC (%) |
Cytotoxicity |
|
First count |
Second count |
||||||||
Initial cell count |
- |
A |
– |
2000000 |
1850000 |
2087500 |
- |
- |
- |
- |
B |
– |
2200000 |
2100000 |
|||||
- |
C |
– |
2300000 |
2000000 |
|||||
- |
D |
– |
2100000 |
2150000 |
|||||
Solvent control (DMSO) |
- |
A |
– |
8550000 |
8750000 |
8525000 |
6437500 |
100,00 |
0,00 |
- |
B |
– |
8300000 |
8500000 |
|||||
Reaction mass of N-butylphthalimide and N-propylphthalimide and N-sec-butylphthalimide |
31.3 |
A |
– |
8300000 |
8250000 |
8275000 |
6187500 |
96,12 |
3,88 |
62.5 |
A |
– |
7050000 |
6900000 |
6975000 |
4887500 |
75,92 |
24,08 |
|
125 |
A |
– |
6750000 |
6600000 |
6675000 |
4587500 |
71,26 |
28,74 |
|
250 |
A |
– |
2600000 |
2750000 |
2675000 |
587500 |
9,13 |
90,87 |
|
500 |
A |
– |
150000 |
500000 |
325000 |
-1762500* |
-27,38** |
127,38*** |
|
1000 |
A |
– |
0 |
0 |
0 |
-2087500* |
-32,43** |
132,43*** |
|
EMS 1µL/mL |
A |
– |
5450000 |
5500000 |
5475000 |
3387500 |
52,62 |
47,38 |
|
Solvent control (DMSO) |
- |
A |
+ |
6650000 |
6500000 |
6612500 |
4525000 |
100,00 |
0,00 |
- |
B |
+ |
6600000 |
6700000 |
|||||
Reaction mass of N-butylphthalimide and N-propylphthalimide and N-sec-butylphthalimide |
31.3 |
A |
+ |
6500000 |
6500000 |
6500000 |
4412500 |
97,51 |
2,49 |
62.5 |
A |
+ |
5800000 |
6000000 |
5900000 |
3812500 |
84,25 |
15,75 |
|
125 |
A |
+ |
4800000 |
4950000 |
4875000 |
2787500 |
61,60 |
38,40 |
|
250 |
A |
+ |
4250000 |
4200000 |
4225000 |
2137500 |
47,24 |
52,76 |
|
500 |
A |
+ |
100000 |
150000 |
125000 |
-1962500* |
-43,37** |
143,37*** |
|
1000 |
A |
|
0 |
0 |
0 |
-2087500* |
-46,13** |
146,13*** |
|
Cycl. 5µg/mL |
A |
+ |
4100000 |
4250000 |
4175000 |
2087500 |
46,13 |
53,87 |
Summmarized Results of the concentration SELECTION CYTOTOXICITY ASSAY
20-hour treatment without S9 mix / 20-hour sampling time
Test group |
Concentration |
Parallels |
S9-mix |
Cell counts |
Mean cell counts |
Increase in cell counts |
RICC (%) |
Cytotoxicity |
|
First count |
Second count |
||||||||
Initial cell count |
- |
A |
– |
2000000 |
1850000 |
2087500 |
- |
- |
- |
- |
B |
– |
2200000 |
2100000 |
|||||
- |
C |
– |
2300000 |
2000000 |
|||||
- |
D |
– |
2100000 |
2150000 |
|||||
Solvent control (DMSO) |
- |
A |
– |
7600000 |
7500000 |
7587500 |
5500000 |
100,00 |
0,00 |
- |
B |
– |
7500000 |
7750000 |
|||||
Reaction mass of N-butylphthalimide and N-propylphthalimide and N-sec-butylphthalimide |
31.3 |
A |
– |
6500000 |
6500000 |
6500000 |
4412500 |
80,23 |
19,77 |
62.5 |
A |
– |
5350000 |
5450000 |
5400000 |
3312500 |
60,23 |
39,77 |
|
125 |
A |
– |
4750000 |
4750000 |
4750000 |
2662500 |
48,41 |
51,59 |
|
250 |
A |
– |
2650000 |
2800000 |
2725000 |
637500 |
11,59 |
88,41 |
|
500 |
A |
– |
0 |
0 |
0 |
-2087500* |
-37,95** |
137,95*** |
|
1000 |
A |
– |
0 |
0 |
0 |
-2087500* |
-37,95** |
137,95*** |
|
EMS 1µL/mL |
A |
– |
4600000 |
4650000 |
4625000 |
2537500 |
46,14 |
53,86 |
Table 2/C
Summarized Results of the concentration SELECTION CYTOTOXICITY ASSAY
20-hour treatment without S9 mix and 3-hour treatment with S9 mix / 28-hour sampling time
Test group |
Concentration |
Parallels |
S9-mix |
Cell counts |
Mean cell counts |
Increase in cell counts |
RICC (%) |
Cytotoxicity |
|
First count |
Second count |
||||||||
Initial cell count |
- |
A |
– |
2000000 |
1850000 |
2087500 |
- |
- |
- |
- |
B |
– |
2200000 |
2100000 |
|||||
- |
C |
– |
2300000 |
2000000 |
|||||
- |
D |
– |
2100000 |
2150000 |
|||||
Solvent control (DMSO) |
- |
A |
– |
9600000 |
9600000 |
9600000 |
7512500 |
100,00 |
0,00 |
- |
B |
– |
9700000 |
9500000 |
|||||
Reaction mass of N-butylphthalimide and N-propylphthalimide and N-sec-butylphthalimide |
31.3 |
A |
– |
8150000 |
8150000 |
8150000 |
6062500 |
80,70 |
19,30 |
62.5 |
A |
– |
6200000 |
6450000 |
6325000 |
4237500 |
56,41 |
43,59 |
|
125 |
A |
– |
5800000 |
5850000 |
5825000 |
3737500 |
49,75 |
50,25 |
|
250 |
A |
– |
3000000 |
3350000 |
3175000 |
1087500 |
14,48 |
85,52 |
|
500 |
A |
– |
0 |
0 |
0 |
-2087500* |
-27,79** |
127,79*** |
|
1000 |
A |
– |
0 |
0 |
0 |
-2087500* |
-27,79** |
127,79*** |
|
EMS 1µL/mL |
A |
– |
5350000 |
5650000 |
5500000 |
3412500 |
45,42 |
54,58 |
|
Solvent control (DMSO) |
- |
A |
+ |
8700000 |
8450000 |
8562500 |
6475000 |
100,00 |
0,00 |
- |
B |
+ |
8600000 |
8500000 |
|||||
Reaction mass of N-butylphthalimide and N-propylphthalimide and N-sec-butylphthalimide |
31.3 |
A |
+ |
8250000 |
8250000 |
8250000 |
6162500 |
95,17 |
4,83 |
62.5 |
A |
+ |
7350000 |
7200000 |
7275000 |
5187500 |
80,12 |
19,88 |
|
125 |
A |
+ |
6250000 |
6300000 |
6275000 |
4187500 |
64,67 |
35,33 |
|
250 |
A |
+ |
5250000 |
5250000 |
5250000 |
3162500 |
48,84 |
51,16 |
|
500 |
A |
+ |
100000 |
250000 |
175000 |
-1912500* |
-29,54** |
129,54*** |
|
1000 |
A |
+ |
0 |
0 |
0 |
-2087500* |
-32,24** |
132,24***b |
|
Cycl. 5µg/mL |
A |
+ |
5200000 |
5400000 |
5300000 |
3212500 |
49,61 |
50,39 |
Results of
|
MEAN NUMBER OF CELLS WITH STRUCTURAL
CHROMOSOME ABERRATION(S)
MEAN NUMBER OF CELLS WITH STRUCTURAL
CHROMOSOME ABERRATION(s) EXPERIMENT A
Concentration |
S9 mix |
Treatment |
Harvesting time |
Mean aberrant cells/150cells |
|
incl. gaps |
excl. gaps |
||||
Negative (Solvent) control |
- |
3 h |
20 h |
7 |
3 |
Reaction mass of N-butylphthalimide and N-propylphthalimide and N-sec-butylphthalimide |
|||||
31.3 µg/mL |
- |
3 h |
20 h |
10 |
4 |
62.5 µg/mL |
- |
3 h |
20 h |
14 |
5 |
125 µg/mL |
- |
3 h |
20 h |
11 |
5 |
187.5 µg/mL |
- |
3 h |
20 h |
15 |
4 |
Pos. Control |
- |
3 h |
20 h |
42** |
31** |
Negative (Solvent) control |
+ |
3 h |
20 h |
8 |
3 |
Reaction mass of N-butylphthalimide and N-propylphthalimide and N-sec-butylphthalimide |
|||||
31.3 µg/mL |
+ |
3 h |
20 h |
15 |
5 |
62.5 µg/mL |
+ |
3 h |
20 h |
14 |
4 |
125 µg/mL |
+ |
3 h |
20 h |
18* |
3 |
250 µg/mL |
+ |
3 h |
20 h |
15 |
4 |
Pos. Control (Cyclophosphamide) |
+ |
3 h |
20 h |
49** |
41** |
Positive control (-S9): Ethyl methanesulphonate (1.0L/mL)
Positive control (+S9): Cyclophosphamide (5.0g/mL)
** = p < 0.01 to the concurrent negative control and to the historical control
* = p < 0.05 to the concurrent negative control
NUMBER OF CELLS WITH STRUCTURAL
CHROMOSOME ABERRATION(s) EXPERIMENT B
Concentration |
S9 mix |
Treatment |
Harvesting time |
Mean aberrant cells/150cells |
|||
incl. gaps |
excl. gaps |
||||||
|
Negative (Solvent) control |
- |
20 h |
20 h |
8 |
3 |
|
|
Reaction mass of N-butylphthalimide and N-propylphthalimide and N-sec-butylphthalimide |
|
|||||
|
15.6 µg/mL |
- |
20 h |
20 h |
14 |
5 |
|
|
31.3 µg/mL |
- |
20 h |
20 h |
13 |
7 |
|
|
62.5 µg/mL |
- |
20 h |
20 h |
16 |
7 |
|
|
125 µg/mL |
- |
20 h |
20 h |
18* |
7 |
|
|
Pos. Control |
- |
20 h |
20 h |
48** |
43** |
|
|
Negative (Solvent) control |
- |
20 h |
28 h |
8 |
3 |
|
|
Reaction mass of N-butylphthalimide and N-propylphthalimide and N-sec-butylphthalimide |
|
|||||
|
15.6 µg/mL |
- |
20 h |
28 h |
15 |
7 |
|
|
31.3 µg/mL |
- |
20 h |
28 h |
15 |
6 |
|
|
62.5 µg/mL |
- |
20 h |
20 h |
16 |
6 |
|
|
125 µg/mL |
- |
20 h |
28 h |
17 |
8 |
|
|
Pos. Control |
- |
20 h |
28 h |
44** |
36** |
|
Positive control (-S9): Ethyl methanesulphonate (0.4L/mL)
** = p < 0.01 to the concurrent negative control and to the historical control
* = p < 0.05 to the concurrent negative control
MEAN NUMBER OF CELLS WITH STRUCTURAL
CHROMOSOME ABERRATION(s) EXPERIMENT B
Concentration |
S9 mix |
Treatment |
Harvesting time |
Mean aberrant cells/150cells |
|
|
incl. gaps |
excl. gaps |
|
||||
Negative (Solvent) control |
+ |
3 h |
28 h |
8 |
3 |
|
Reaction mass of N-butylphthalimide and N-propylphthalimide and N-sec-butylphthalimide |
||||||
31.3 µg/mL |
+ |
3 h |
28 h |
20* |
8 |
|
62.5 µg/mL |
+ |
3 h |
28 h |
19* |
6 |
|
125 µg/mL |
+ |
3 h |
28 h |
18* |
8 |
|
250 µg/mL |
+ |
3 h |
28 h |
31** |
13* |
|
Pos. Control (Cyclophosphamide) |
+ |
3 h |
28 h |
48** |
40** |
|
Cyclophosphamide: 5.0g/mL
** = p < 0.01 to the concurrent negative control and to the historical control
* = p < 0.05 to the concurrent negative control
NUMBER OF POLYPLOID AND ENDOREDUPLICATED CELLS
NUMBER OF POLYPLOID CELLS AND ENDOREDUPLICATED CELLS
EXPERIMENT A
Concentration |
S9 mix |
Treatment/Harvesting |
Polyploid Cells (mean) |
Endoredup-lication (mean) |
Negative (Solvent) control |
- |
3/20 h |
0.0 |
0.0 |
Reaction mass of N-butylphthalimide and N-propylphthalimide and N-sec-butylphthalimide |
||||
31.3 µg/mL |
- |
3/20 h |
0.0 |
0.5 |
62.5 µg/mL |
- |
3/20 h |
0.0 |
0.0 |
125 µg/mL |
- |
3/20 h |
0.0 |
0.0 |
187.5 µg/mL |
- |
3/20 h |
0.0 |
0.0 |
Pos. Control |
- |
3/20 h |
0.0 |
0.0 |
Negative (Solvent) control |
+ |
3/20 h |
0.0 |
0.0 |
Reaction mass of N-butylphthalimide and N-propylphthalimide and N-sec-butylphthalimide |
||||
31.3 µg/mL |
+ |
3/20 h |
0.0 |
0.0 |
62.5 µg/mL |
+ |
3/20 h |
0.0 |
0.0 |
125 µg/mL |
+ |
3/20 h |
0.0 |
0.0 |
250 µg/mL |
+ |
3/20 h |
0.0 |
0.5 |
Pos. Control (Cyclophosphamide) |
+ |
3/20 h |
0.0 |
0.0 |
Ethyl methanesulphonate: 1.0mL/mL
Cyclophosphamide: 5.0g/mL
The number of polyploid and endoreduplicated cells was determined
in
300 cells of each test group.
NUMBER OF POLYPLOID CELLS AND ENDOREDUPLICATED CELLS
EXPERIMENT B
Concentration |
S9 mix |
Treatment/Harvesting |
Polyploid Cells (mean) |
Endoredup-lication (mean) |
Negative (Solvent) control |
- |
20/20 h |
0.0 |
0.0 |
Reaction mass of N-butylphthalimide and N-propylphthalimide and N-sec-butylphthalimide |
||||
15.6 µg/mL |
- |
20/20 h |
0.0 |
0.0 |
31.3 µg/mL |
- |
20/20 h |
0.0 |
0.0 |
62.5 µg/mL |
- |
20/20 h |
0.0 |
0.0 |
125 µg/mL |
- |
20/20 h |
0.0 |
0.5 |
Pos. Control |
- |
20/20 h |
0.0 |
0.0 |
Negative (Solvent) control |
- |
20/28 h |
0.0 |
0.0 |
Reaction mass of N-butylphthalimide and N-propylphthalimide and N-sec-butylphthalimide |
||||
15.6 µg/mL |
- |
20/28 h |
0.0 |
0.5 |
31.3 µg/mL |
- |
20/28 h |
0.0 |
0.0 |
62.5 µg/mL |
- |
20/28 h |
0.0 |
0.0 |
125 µg/mL |
- |
20/28 h |
0.0 |
0.0 |
Pos. Control |
- |
20/28 h |
0.0 |
0.0 |
Positive control (-S9):Ethyl methanesulphonate(0.4L/mL)
The number of polyploid and endoreduplicated cells was determined
in
300 cells of each test group.
NUMBER OF POLYPLOID CELLS AND ENDOREDUPLICATED CELLS
EXPERIMENT B
Concentration |
S9 mix |
Treatment/Harvesting |
Polyploid Cells (mean) |
Endoredup-lication (mean) |
Negative (Solvent) control |
+ |
3/28 h |
0.0 |
0.0 |
Reaction mass of N-butylphthalimide and N-propylphthalimide and N-sec-butylphthalimide |
||||
31.3 µg/mL |
+ |
3/28 h |
0.0 |
0.0 |
62.5 µg/mL |
+ |
3/28 h |
0.0 |
0.0 |
125 µg/mL |
+ |
3/28 h |
0.0 |
0.0 |
250 µg/mL |
+ |
3/28 h |
0.0 |
0.0 |
Pos. Control |
+ |
3/28 h |
0.0 |
0.0 |
Cyclophosphamide: 5.0g/mL
The number of polyploid and endoreduplicated cells was determined
in
300 cells of each test group.
X V
pH AND OSMOLALITY DATA
pH AND OSMOLALITY DATA
Pre-test on Toxicity (Concentration selection)
Concentration |
pH |
Osmolality (mmol/kg) |
|
3-hour treatment /without S9 Mix/20-hour sampling times |
|||
Negative (solvent) Control |
8.08 |
479 |
|
Reaction mass of N-butylphthalimide and N-propylphthalimide and N-sec-butylphthalimide
|
31.3 |
8.05 |
483 |
62.5 |
8.05 |
478 |
|
125 |
8.06 |
480 |
|
250 |
8.05 |
475 |
|
500 |
8.02 |
471 |
|
1000 |
7.99 |
466 |
|
EMS |
7.93 |
341 |
|
20-hour treatment /without S9 Mix/20 and 28-hour sampling times |
|||
Negative (solvent) Control |
8.08 |
479 |
|
Reaction mass of N-butylphthalimide and N-propylphthalimide and N-sec-butylphthalimide |
31.3 |
8.05 |
483 |
62.5 |
8.05 |
478 |
|
125 |
8.06 |
480 |
|
250 |
8.05 |
475 |
|
500 |
8.02 |
471 |
|
1000 |
7.99 |
466 |
|
EMS |
7.98 |
340 |
|
3-hour treatment with S9 Mix / 20 and 28-hour sampling time |
|||
Negative (solvent) Control |
7.95 |
469 |
|
Reaction mass of N-butylphthalimide and N-propylphthalimide and N-sec-butylphthalimide |
31.3 |
7.96 |
476 |
62.5 |
7.88 |
474 |
|
125 |
7.89 |
477 |
|
250 |
7.93 |
475 |
|
500 |
7.93 |
470 |
|
1000 |
7.88 |
465 |
|
Cycl. |
7.89 |
327 |
EMS:Ethyl methanesulphonate(1L/mL, 0.4L/mL )
Cycl.:Cyclophosphamide (5.0 g/mL)
Experiment A
Concentration |
pH |
Osmolality (mmol/kg) |
|
3-hour treatment without S9 Mix / 20-hour sampling time |
|||
Negative (solvent) Control |
7.91 |
367 |
|
Reaction mass of N-butylphthalimide and N-propylphthalimide and N-sec-butylphthalimide |
31.3 |
7.94 |
366 |
62.5 |
7.86 |
365 |
|
125 |
7.80 |
368 |
|
187.5 |
7.78 |
365 |
|
EMS |
7.88 |
354 |
|
3-hour treatment with S9 Mix / 20-hour sampling time |
|||
Negative (solvent) Control |
7.90 |
370 |
|
Reaction mass of N-butylphthalimide and N-propylphthalimide and N-sec-butylphthalimide |
31.3 |
7.82 |
374 |
62.5 |
7.79 |
378 |
|
125 |
7.82 |
376 |
|
250 |
7.81 |
376 |
|
Cycl. |
7.85 |
340 |
EMS:Ethyl methanesulphonate(1L/mL)
Cycl.:Cyclophosphamide (5.0 g/mL)
TABLE 14
pH AND OSMOLALITY DATA
Experiment BC
ncentration |
pH |
Osmolality (mmol/kg) |
|
20-hour treatment without S9 Mix / 20-hour and 28-hor sampling times |
|||
Negative (solvent) Control |
7.89 |
367 |
|
Reaction mass of N-butylphthalimide and N-propylphthalimide and N-sec-butylphthalimide |
15.6 |
7.95 |
368 |
31.3 |
7.85 |
366 |
|
62.5 |
7.78 |
367 |
|
125 |
7.80 |
363 |
|
EMS |
7.87 |
353 |
|
3-hour treatment with S9 Mix / 28-hour sampling time |
|||
Negative (solvent) Control |
7.91 |
371 |
|
Reaction mass of N-butylphthalimide and N-propylphthalimide and N-sec-butylphthalimide |
31.3 |
7.81 |
375 |
62.5 |
7.79 |
378 |
|
125 |
7.80 |
375 |
|
250 |
7.81 |
376 |
|
Cycl. |
7.88 |
343 |
EMS:Ethyl methanesulphonate(0.4L/mL )
Cycl.:Cyclophosphamide (5.0 g/mL)
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed (positive)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available (further information necessary)
Additional information
Justification for classification or non-classification
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