Registration Dossier
Registration Dossier
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EC number: 203-505-6 | CAS number: 107-58-4
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Bacterial reverse mutation test:
The registered substance, N-tert-butylacrylamide (CAS No. 107-58-4), was tested non-mutagenic (negative) as it does not induce (point) gene mutations by base-pair changes or frameshift in the histidine operon of Salmonella typhimurium tester strains (TA1537, TA1535, TA98, TA100 or TA102 neither in the present nor in the absence of S9 metabolic activation system. The test was performed according to OECD 471 and in compliance with OECD principles of Good Laboratory Practice.
In vitro Chromosomal Aberration test:
The registered substance, N-tert-butyl-acrylamide (CAS 107-58-5), tested non-clastogenic (negative) in human peripheral blood lymphocytes with and without the S9 metabolic activation system. The test was performed according to OECD 473 and in compliance with OECD principles of GLP.
Gene Mutation in Mammalian cells:
The registered substance, N-tert-butylacrylamide (CAS No. 107-58-4), did not induce gene mutation at the hypoxanthine-guanine phosphoribosyltransferase (Hprt) locus of CHO cells up to the highest concentration of 2 mg/ml of culture medium, either in the presence or absence of S9 metabolic activation system under the experimental conditions described. The test was performed according to OECD 476 and in compliance with the OECD principles of Good Laboratory Practice.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Justification for type of information:
- The study contains experimental data of the registered substance.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- Adopted: July 21 1997, Corrected: June 26 2020
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- Histidine
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 Homogenate
Aroclor 1254-induced rat liver microsomal enzymes (S9 homogenate) procured commercially was used for the assay. Certificate of analysis received from the supplier was included in the report. Efficiency check of S9 was performed during the main assay. The protein concentration in the S9 fraction was 34.6 mg/ml (Annexure: 1).
S9 Mix
S9 mix cofactors solution (Cofactor ingredients: D-glucose-6-phosphate, β-NADP, magnesium chloride, potassium chloride, sodium phosphate and liver homogenate] was prepared before use. The post-mitochondrial fraction (liver homogenate) was used at the concentration of 10 % (v/v) in the S9 mix. - Test concentrations with justification for top dose:
- Concentration tested were 0.3125, 0.625, 1.25, 2.5 and 5 mg/plate. These concentrations were selected based on solubility, precipitation test and preliminary cytotoxicity test.
- Vehicle / solvent:
- Dimethyl sulfoxide (DMSO) was selected as a vehicle for the test item in this study.
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- 2-nitrofluorene
- sodium azide
- benzo(a)pyrene
- mitomycin C
- Details on test system and experimental conditions:
- The mutagenic potential of N-tert-butylacrylamide [TBAA] [CAS No. 107-58-4] was tested in two independent experiments (Trial I and Trial II) and both in the presence (10% v/v cofactor supplemented post-mitochondrial S9 fraction, prepared from rat liver) and absence of metabolic activation using Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA102.
Trial I and II: 0.3125, 0.625, 1.25, 2.5 and 5 mg/plate
Trial I was performed according to the plate incorporation method, both in the presence (10% v/v S9 mix) and absence of a metabolic activation system. The Test item together with the vehicle, negative and positive controls, was tested in triplicates. The Test item doses were selected using concentration spacing factor of 2. No increase in the number of revertant colonies or inhibition in background lawn was observed up to the highest concentration of 5 mg/plate either in the presence (10% v/v S9 mix) or absence of metabolic activation when compared to the vehicle control data.
Trial II was performed to confirm the negative results observed in Trial I.
Trial II was conducted according to the preincubation method, both in the presence (10% v/v S9 mix) and absence of a metabolic activation system. The Test item together with the vehicle, negative and positive controls, was tested in all tester strains in triplicates. There was no increase in the number of revertant colonies or inhibition of the background lawn up to the highest concentration of 5 mg/plate either in the presence (10% v/v S9 mix) or absence of metabolic activation when compared to the vehicle control data.
The numbers of revertant colonies of the vehicle, negative (spontaneous revertant colonies), and positive controls (induced revertant colonies) of all the tester strains were within the range of historical data of the laboratory. The positive controls used in the study produced significant increases in the mean number of revertant colonies under both activated and non-activated conditions in all tester strains when compared to the control data confirming the validity of the mutagenicity test.
- Evaluation criteria:
- A Test item is considered as a mutagen if a biologically relevant increase in the mean number of revertants exceeding the threshold of twice (strains TA98, TA100 and TA102) or thrice (strains TA1535 and TA1537) the colony count of the corresponding solvent control is observed.
A dose-dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
A dose-dependent increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent experiment.
A dose-dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if it is reproduced in an independent experiment. - Statistics:
- The colonies were counted manually. The mean number of revertant colonies and the standard deviation within the plates for each concentration were compared to the spontaneous reversion rates of the control. Microsoft Office Excel-based calculations were used for descriptive statistical analysis.
- 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
- True negative controls validity:
- not specified
- Positive controls validity:
- not specified
- 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
- True negative controls validity:
- not specified
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 102
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not specified
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not specified
- 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
- True negative controls validity:
- not specified
- Positive controls validity:
- valid
- Remarks on result:
- other: No Mutagenic pottential
- Conclusions:
- The registered substance, N-tert-butylacrylamide (CAS No. 107-58-4) is non-mutagenic (negative) as it does not induce (point) gene mutations by base-pair changes or frameshift in the histidine operon of Salmonella typhimurium tester strains (TA1537, TA1535, TA98, TA100 or TA102 neither in the present nor in the absence of S9 metabolic activation system.
- Executive summary:
Ames test of-tert-butylacrylamide [TBAA] [CAS No.107-58-4] was conducted according to the plate incorporation and preincubation methods. This study was performed as per the OECD guideline No. 471 (Adopted: July 21 1997, Corrected: June 26 2020). Based on the solubility test, dimethyl sulfoxide (DMSO) was selected as a vehicle for the test item in the study. The mutagenic potential of N-tert-butylacrylamide [TBAA] [CAS No. 107-58-4] was tested in two independent experiments (Trial I and Trial II) and both in the presence (10% v/v cofactor supplemented post-mitochondrial S9 fraction, prepared from rat liver) and absence of metabolic activation using Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA102. The Test item was tested along with solvent-vehicle control (DMSO), negative (distilled water), and concurrent positive control substances in triplicates. A preliminary cytotoxicity assay was performed using the strains TA98 and TA100 with the following concentrations along with the vehicle, negative and positive controls both in the presence (10% v/v S9 mix) and absence of metabolic activation in triplicates: 0.0390625, 0.078125, 0.15625, 0.3125, 0.625, 1.25, 2.5 and 5 mg/plate. In the tester strains, TA98 and TA100, no reduction in the revertant count and no inhibition in background lawn were observed at any of the concentrations, either in the presence (10% v/v S9 mix) or the absence of metabolic activation, when compared to the vehicle control data. Based on the preliminary cytotoxicity test results, the main study was performed with the following concentrations: Trial I and II: 0.3125, 0.625, 1.25, 2.5, and 5 mg/plate
Trial I was performed according to the plate incorporation method, both in the presence (10% v/v S9 mix) and absence of a metabolic activation system. The Test item and the vehicle, negative and positive controls, were tested in triplicates. The Test item doses were selected using a concentration spacing factor of 2. No increase in the number of revertant colonies or inhibition in the background lawn was observed up to the highest concentration of 5mg/plate either in the presence (10% v/v S9 mix) or absence of metabolic activation when compared to the vehicle control data.
Trial II was performed to confirm the negative results observed in Trial I. Trial II was conducted according to the preincubation method, both in the presence (10% v/v S9 mix) and absence of a metabolic activation system. The Test item and the vehicle, negative and positive controls, were tested in all tester strains in triplicates. There was no increase in the number of revertant colonies or inhibition of the background lawn up to the highest concentration of 5 mg/plate either in the presence (10% v/v S9 mix) or absence of metabolic activation when compared to the vehicle control data. The numbers of revertant colonies of the vehicle, negative (spontaneous revertant colonies), and positive controls (induced revertant colonies) of all the tester strains were within the range of historical data of the laboratory. The positive controls used in the study produced significant increases in the mean number of revertant colonies under both activated and non-activated conditions in all tester strains when compared to the control data confirming the validity of the mutagenicity test.
Based on the results of this study, it is concluded that N-tert-butylacrylamide [TBAA] [CAS No. 107-58-4] is non-mutagenic as it does not induce (point) gene mutations in the histidine operon by base-pair changes or frameshifts, in the presence and the absence of metabolic activation system, in the tester strains of Salmonella typhimurium (TA1537, TA1535, TA98, TA100, and TA102).
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Justification for type of information:
- Data is from study report
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Version / remarks:
- Adopted on 29th July 2016
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian chromosome aberration test
- Target gene:
- No data
- Species / strain / cell type:
- lymphocytes: human peripheral blood lymphocytes
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: Human blood
- Suitability of cells: No data
- Cell cycle length, doubling time or proliferation index:
- Sex, age and number of blood donors if applicable:Age: 27-28 years age
- Whether whole blood or separated lymphocytes were used if applicable: Separated lymphocytes were used
- Number of passages if applicable: No data
- Methods for maintenance in cell culture if applicable: No data
- Modal number of chromosomes: No data
- Normal (negative control) cell cycle time: No data
MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Blood cultures were set up in medium containing RPMI-1640, Fetal Bovine Serum, Phytohaemagglutinin, Heparin solution, Whole Blood and Antibiotic Solution
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: No data
- Periodically checked for karyotype stability: No data
- Periodically 'cleansed' against high spontaneous background: No data - Additional strain / cell type characteristics:
- not specified
- Cytokinesis block (if used):
- No data
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 metabolic activation system
- Test concentrations with justification for top dose:
- 0.00 (NC), 0.00 (VC), 0.5 (T1), 1.0 (T2) and 2.0 (T3) mg/mL
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was soluble in DMSO - Untreated negative controls:
- yes
- Remarks:
- PBS
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): A volume of 7.92 mL of proliferating culture was dispensed to individual sterile culture tubes/flasks
DURATION
- Preincubation period: No data
- Exposure duration: Phase 1: 4 hrs (with and without metabolic activation system)
Phase 2: 4 hrs (with metabolic activation system) and 24 hrs (without metabolic activation system)
- Expression time (cells in growth medium): Phase 1: 20 hrs (with and without metabolic activation system)
Phase 2: 20 hrs (with metabolic activation system)
- Selection time (if incubation with a selection agent):No data
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hrs
SELECTION AGENT (mutation assays): No data
SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa stain in phosphate buffer
NUMBER OF REPLICATIONS: No data
METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: The cultures were incubated at 37 ± 2 °C for duration (exposure period) as mentioned. For Phase I, after incubation cells were spun down by gentle centrifugation at 1500 rpm for 10 minutes. The supernatant with the dissolved test item was discarded and the cells were re-suspended in Phosphate Buffer Saline (PBS). The washing procedure was repeated once again. After washing the cells were re-suspended in complete culture medium (RPMI-1640 with 10 % serum) and cultured at 37 ± 2 °C for 1.5 normal cell cycle lengths (22 - 25 hours). The cultures were harvested at the end of incubation of 24 hours after treatment. Before 3 hours of harvesting, 240 µL of colcemid (10 µg/mL) (final concentration: 0.3 µg/mL) was added to each of the culture tube, and kept under incubation at 37 ± 2 °C. The cultures were harvested 24 hours after beginning of treatment by centrifugation at 1500 rpm for 10 minutes. The supernatant was discarded and the cells were re-suspended in 7 mL of freshly prepared, pre-warmed (37 ± 2 °C) hypotonic solution of potassium chloride (0.075 M KCl). Then the cell suspension was allowed to stand at 37 ± 2 °C for 30 minutes in water bath. After hypotonic treatment, the culture was centrifuged and supernatant was removed. After that 5 mL of freshly prepared, chilled Carnoy’s fixative (3:1 methanol: acetic acid solution) was added and left for 5 min. The cells were collected by centrifugation and washed twice with Carnoy’s fixative. After the final centrifugation, the supernatant was removed completely, and the cell pellet resuspended in 0.5 mL of Carnoy’s fixative. The slides were prepared by dropping the cell suspension onto a clean ice-chilled microscope slide. The labelled slides were dried over a slide warmer at 50°C and labelled. At least one slide was made from each sample. The cells were stained with 5 % fresh Giemsa stain in phosphate buffer and mounted using DPX mountant.
NUMBER OF CELLS EVALUATED: A minimum of 1000 cells were counted in different fields of slide per culture and the number of metaphases were recorded for mitotic index (MI) calculation.
NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 300 well spread metaphase plates per culture were scored for cytogenetic damage on coded slides.
CRITERIA FOR MICRONUCLEUS IDENTIFICATION: No data
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: Mitotic index
- Any supplementary information relevant to cytotoxicity: Cytotoxicity was assessed at the concentrations of 0, 0.5, 1.0 and 2.0 mg/mL of culture media.
OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
- Determination of endoreplication: Yes
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): No data
- OTHER: No data - Rationale for test conditions:
- No data
- Evaluation criteria:
- A test item can be classified as clastogenic if:
At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent vehicle control
If the increase is dose-related
Any of the results are outside the historical negative control range
A test item can be classified as non – clastogenic if:
None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control
If there is no dose-related increase
All results are within the historical negative control range
Statistical significance was confirmed by means of the non-parametric Mann Whitney Test. However, both biological and statistical significance should be considered together.
If the above mentioned criteria for the test item are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed. - Statistics:
- Statistical significance at the p < 0.05 was evaluated by means of the non-parametric Mann-Whitney test
- Species / strain:
- lymphocytes: Human perpheral blood lymphocytes
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The pH of test item in culture medium was assessed at 0 h and 4 h after incubation at 37 ± 2 °C. Significant change in pH was not observed at 0 h and 4 h when compared with negative controls.
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: There was no precipitation observed at 0.0625 mg/mL concentration
- Definition of acceptable cells for analysis: No data
- Other confounding effects: No data
RANGE-FINDING/SCREENING STUDIES: To evaluate the toxicity of the test item a cytotoxicity assay was performed both in the presence and absence of metabolic activation system. Three test concentrations (0.00025, 0.0005 and 0.001 mg/mL of culture media) based on the solubility, precipitation and pH test of the test item were tested. Cytotoxicity was determined by reduction in the mitotic index in comparison with vehicle control. The procedure for conducting cytotoxicity was the same as main experiment phase I up to the scoring of the mitotic index, except slide coding.
Before conducting the chromosomal aberration study, Methyl-2-napthyl ether (CAS no. 93-04-9) was evaluated for cytotoxicity both in the absence and presence of metabolic activation system (1%). Cytotoxicity was assessed at the concentrations of 0.016 (T1), 0.0312 (T2) and 0.0625 (T3) mg/mL at initial cytotoxicity experiment (cytotxicity experiment I). All the tested concentrations at intial cytotoxicity experiment were cytotoxic. A second cytotoxicity experiment (cytotoxicity experiment II) was conducted with 0.002 (T4), 0.004 (T5) and 0.008 (T6) mg/mL of culture media. In second cytotoxicity experiment all tested concentrations were cytotoxic.
Hence one more cytotoxicity experiment (cytotoxic experiment III) was conducted with further lower concentrations of 0.00025 (T7), 0.0005 (T8) and 0.001 (T9) mg/mL of culture media. In the absence of S9 mix, the mean mitotic index observed was 10.03 (NC), 9.95 (VC), 8.69 (T7), 6.54 (T8), 5.79 (T9) and 8.54 (PC). In the presence of S9 mix, the mean mitotic index observed was 10.05 (NC), 9.94 (VC), 8.84 (T7), 6.55 (T8), 5.74 (T9) and 8.55 (PC).
In the cytotoxicity experiment III the highest test concentration 0.001 (T9) mg/ mL of culture media show 41.8 % reduction in absence of metabolic activation and 42.18% in the presence of metabolic activation indicates slight cytotoxicity of test item. Hence 0.001 was selected as highest concentaration for main study considering the selection of test concentrations upto cytotoxicity. The mitotic index when compared to the respective vehicle control both in the presence or absence of metabolic activation.
Hence the concentrations selected for the main study are 0.00025, 0.0005 and 0.001 mg/mL. The main study was performed in two independent phases;
CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: No data
NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: No data
- Indication whether binucleate or mononucleate where appropriate: No data
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: No data
- Negative (solvent/vehicle) historical control data: Please refer table remarks section
ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: No data
- Other observations when applicable: No data - Remarks on result:
- other: No mutagenic potential
- Conclusions:
- The registered substance, N-tert-butyl-acrylamide (CAS 107-58-5), tested non-clastogenic (negative) in human peripheral blood lymphocytes with and without the S9 metabolic activation system. The test was performed according to OECD 473 and in compliance with OECD principles of GLP.
- Executive summary:
The ability of N-tert-butyl-acrylamide (CAS 107-58-5) to induce chromosomal aberration in human peripheral blood lymphocytes was tested according to OECD 473. The experiment was performed both in the presence and absence of an exogenous metabolic activation system after 48 hours of mitogenic stimulation. Cofactor-supplemented liver S9 microsomal fraction was used as a metabolic activation system. The test chemical was dissolved in dimethyl sulfoxide (DMSO). The doses for the main study were based on a preliminary cytotoxicity study. In this pre-test, the test substance was assessed at 0.0 (NC), 0.0 (VC) of 0.5, 1, and 2 mg/ml in the presence and absence of S9 metabolic activation. Cytotoxicity was determined by a reduction in the mitotic index in comparison with the vehicle control. The highest test concentration of 2 mg/ml did not induce more than 50% reduction in the mitotic index compared to the vehicle control. In the absence of S9 metabolic activation, the mean mitotic index value was 10.03 (NC), 9.93 (VC), 9.70 (at 0.5 mg/ml), 9.50 (at 1 mg/ml), 9.54 ( at 2 mg/ml) and 8.50 (PC). In the presence of S9 mix, the mean mitotic index observed was 10.10 (NC), 9.94 (VC), 9.74 (at 0.5 mg/ml), 9.60 ( at 1 mg/ml), 9.64 ( at 2 mg/ml), 8.59 (PC). Thus, in the main study, the test substance was assessed at concentrations of 0.00 (NC), 0.00 (VC), 0.5, 1.0, and 2.0 mg/ml in the presence and absence of S9 metabolic activation system in experiments of Phase I-II. Phase I was performed by short-term (4 hours) treatment method both in the presence and absence of a metabolic activation system (1%). Phase II was conducted using short-term treatment (4 hours) as well as long-term (24 hours) exposure times. Long-term treatment was performed in the absence of metabolic activation to confirm the negative results obtained in the absence of metabolic activation in Phase I. A short-term treatment method was performed with increased metabolic activation (2%) condition to confirm the negative results obtained in the presence of metabolic activation in Phase I. The cultures were harvested 24 hours after the beginning of the treatment, and they were stained with Giemsa. Three hundred well-spread metaphase plates per culture were scored for cytogenetic damage on coded slides. The slides were evaluated using microscopes with 100 x oil immersion objectives. Chromosomal and chromatid breaks, acentric fragments, deletions, exchanges, pulverization, polyploidy (including endo-reduplication), and disintegrations were recorded as structural chromosomal aberrations. Gaps were recorded as well, but they were not included in the calculation of the aberration rates. Only metaphases with 46± 2 centromere regions were included in the analysis. In the mutagenicity test, no structural abnormality of chromosomes and induction of ploidy cells were observed at any dose tested in both confirmatory trials. The percent aberrant cells were comparable in control and treated groups, both in the presence and absence of exogenous metabolic activation in Phase I and II. In Phase I experiment, the mean percent aberrant cell was 0.333 (NC), 0.333 (VC), 0.333 (at 0.5 mg/ml) 0.333 (at 1 mg/ml) 0.667 (2 mg/ml) and 0.333 (NC), 0.333 (VC), 0.333 (at 0.5 mg/ml) 0.667 (at 1 mg/ml) and 0.333 (at 2 mg/ml) and 10.333 (PC) in the absence and presence of S9 metabolic activation, respectively. In Phase II experiment, the mean percent of aberrant cells was 0.333 (NC), 0.333 (VC), 0.333 (at 0.5 mg/ml) 0.333 (at 1 mg/ml) 0.333 (2 mg/ml), 9.333 (PC) and 0.333 (NC), 0.333 (VC), 0.333 (at 0.5 mg/ml) 0.333 (at 1 mg/ml) 0.667 (2 mg/ml) and 9.667 (PC) in the absence and presence of S9 metabolic activation, respectively. Incubation with positive controls caused a significant increase in the percentage of aberrant cells in comparison to vehicle control in both phases, which confirmed the validity of the test. In the cytotoxicity experiment, the highest test concentration (2 mg/ml) showed less than 50 % reduction in the mitotic index compared with vehicle control. In conclusion, the registered substance (CAS 107-58-5) tested non-clastogenic up to 2 mg/ml both in the presence (1% and 2%) and absence of S9 metabolic activation system in cultured human peripheral blood lymphocytes.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Justification for type of information:
- The study contains experimental data of the registered substance.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)
- Version / remarks:
- Adopted July 29 2016.
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian cell gene mutation test using the Hprt and xprt genes
- Target gene:
- Hypoxanthine-guanine phosphoribosyltransferase (Hprt)
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Details on mammalian cell type (if applicable):
- CHO cells were cultured in complete RPMI-1640 medium (10 % Fetal bovine serum), 100 units Penicillin/ml, 10 µg Streptomycin/ml, and incubated at 37±2 °C, 5% CO2, in a CO2 incubator. Cells were counted, then the volume was adjusted with fresh media to obtain a cell density of 2 x 105 cells / 25 cm2 and incubated until to get 10 x 106 cells for cytotoxicity measurement and mutation frequency.
- Metabolic activation:
- with and without
- Metabolic activation system:
- Cofactor-supplemented liver S9 microsomal fraction was used. The S9 fraction was obtained from the liver of phenobarbital and beta-naphthoflavone-injected rat.
S9 Mix
Prior to treatment, a freshly prepared S9 mix was used, an appropriate quantity of S9 fraction was thawed and mixed with co-factor solution to obtain a final concentration of 1% v/v as mentioned below.
Ingredients of the S9 mix:
Glucose-6-phosphate (180 mg/ml): 1ml
NADP(25 mg/ml) : 1 ml
Potassium chloride (150 mM): 1 ml
S9 Fraction (40%): 2 ml
Final Volume: 5 ml
- Test concentrations with justification for top dose:
- Test concentrations: 0,0 (NC), 0.0 (VC), 0.25, 0.5, 1.0 and 2.0 mg/ml
Justification: Based on the solubility and precipitation test, the initial preliminary cytotoxicity testing was performed with Test Item at the concentrations of 0.125, 0.25, 0.5, 1 and 2 mg/ml in culture medium, both in the presence and absence of metabolic activation system (1 % v/v S9 mix) along with negative, vehicle controls. Cytotoxicity was determined by calculating the relative survival. At 2 mg/ml, the relative survival was 92.71% and 91.57% in the presence and absence of S9 metabolic activation - Vehicle / solvent:
- Dimethyl sulfoxide (DMSO)
- Untreated negative controls:
- yes
- Remarks:
- Distilled water
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Dimethy; sulphoxide
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- benzo(a)pyrene
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- The assay was performed employing Test Item concentrations of 2, 1, 0.5 and 0.25 mg/ml along with vehicle and positive controls using single cultures.
In the absence and presence (1% v/v) of metabolic activation, cells were treated with the Test Item, vehicle, and positive controls for 4 hours (short term treatment).
Treatment
Prior to treatment (24 hours), CHO cell cultures were prepared with a density of 10 × 106 cells /flask. On the day of treatment, media was aspirated, and a volume of 5 mL treatment media for presence (1 % v/v S9 mix), absence of metabolic activation (150 mM Potassium chloride) and 50 µL of test item/vehicle controls was dispensed in a respective pre-labelled culture flask. Flasks were incubated for a short term (4 hours) at 37±2°C, 5% CO2, in a CO2 incubator.
After incubation, media was aspirated, and the cells were washed with plain RPMI 1640 medium. Cell cultures were maintained to allow phenotypic expression and determining cloning efficiency.
Plating for Cloning efficiency 1
After treatment, cultures were washed with plain RPMI 1640 medium and cells were counted using a hemocytometer. The cells were diluted to attain a cell density of 330 cells/ 33 ml of cloning media, and 10 ml of cloning media containing 100 cells were dispensed in 60 mm culture plates in triplicates. Plates were incubated at 37±2°C, 5 % CO2, in a CO2 incubator for 7 days.
Plating for Expression
3x105 cells / 5 ml cells were seeded in new culture flasks from respective treated cultures and incubated at 37±2 °C, 5 % CO2 in a CO2 incubator for 7 days to allow phenotypic expression of the induced mutation. During the expression period, cell density was maintained at a density of 2x10 5cells / 5 ml culture media.
Plating for cloning efficiency (CE 2) and Mutation Frequency
Plating for Cloning efficiency 2
At the end of the expression period, cells were trypsinized and counted. Cells were diluted further to 100 cells / 10 ml of cloning media and plated in 60 mm culture plates in triplicate. The plates were incubated at 37±2 °C, 5 % CO2, in a CO2 incubator for 9 days.
Plating for Mutation Frequency
Concurrently, 2x10 5 cells / 10 ml of cloning media were seeded in the presence of 10 µg/ml of 6-thioguanine (6TG) in triplicate and incubated at 37±2 °C, 5 % CO2 in a CO2 incubator for 9 days for mutation frequency (MF).
Fixation and Staining
At the end of the incubation, media from the culture plate was aspirated, and cells in the culture plate were fixed with 2.5 % and 10 % of formaldehyde in water for 10 minutes each.
After fixation, colonies were stained with 5 % Giemsa stain for 10 minutes, followed by washing with distilled water. - Evaluation criteria:
- A test chemical is considered to be clearly positive if, in any of the experimental conditions examined:
a) at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) the increase is concentration-related when evaluated with an appropriate trend test,
c) any of the results were outside the distribution of the literature negative control data.
When all of these criteria are met, the test chemical is then considered able to induce gene mutations in cultured mammalian cells in this test system.
Providing that all acceptability criteria are fulfilled, a test chemical is considered clearly negative if, in all experimental conditions examined:
a) none of the test concentrations exhibits a significant increase compared with the concurrent negative control,
b) all results are inside the distribution of the literature negative control data.
The test chemical is then considered unable to induce gene mutations in cultured mammalian cells in this test system. - Statistics:
- Statistical analysis was performed to assess a possible dose-dependent increase of mutation frequency using Fisher's Exact Test (NCSS statistics software). The mutation frequency of the Test Item-treated group was compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05.
- Key result
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Remarks on result:
- other: Non Mutagenic
- Conclusions:
- The registered substance, N-tert-butylacrylamide (CAS No. 107-58-4) did not induce gene mutation at the hypoxanthine-guanine phosphoribosyltransferase (Hprt) locus of CHO cells up to the highest concentration of 2 mg/ml of culture medium, either in the presence or absence of S9 metabolic activation system under the experimental conditions described. The test was performed according to OECD 476 and in compliance with the OECD principles of Good Laboratory Practice.
- Executive summary:
This in vitro experiment was performed to evaluate the ability of N-tert-butylacrylamide (CAS No. 107-58-4) to cause gene mutation in the hypoxanthine-guanine phosphoribosyl transferase (Hprt) locus in cultured Chinese Hamster Ovary (CHO) cells in the presence and absence of exogenous metabolic activation system (S9) containing microsomal enzymes. The study was performed as per OECD 476 (Adopted: July 29 2016). The Test Item, N-test-butyl acrylamide (CAS No. 107-58-4), was found to be soluble in dimethyl sulfoxide. No precipitation was observed at the concentration of 2 mg/ml. Based on the solubility and precipitation test, initial preliminary cytotoxicity testing was performed with the Test Item at the concentrations of 0.125, 0.25, 0.5, 1, and 2 mg/ml in culture medium, both in the presence and absence of metabolic activation system (1 % v/v S9 mix) along with negative (distilled water), vehicle (DMSO) controls. In the absence of metabolic activation, the relative survival values were 99.34% (vehicle control), 95.65% (at 0.125 mg/ml), 95.54% (at 0.25 mg/ml), 93.81% (at 0.5 mg/ml), 93.49% (at 1 mg/ml) and 92.71% (at 2 mg/ml). In the presence of metabolic activation, the relative survival values were 96.50% (vehicle control), 99.43%, (at 0.125 mg/ml), 94.59% (at 0.25 mg/ml), 94.50% (at 0.5 mg/ml), 92.25% (at 1 mg/ml) and 91.57% (at 2 mg/ml). Thus, in the preliminary cytotoxicity assay, no cytotoxicity (<60% Relative survival [RS is cloning efficiency (CE) of cells plated immediately after treatment adjusted by any loss of cells during treatment as compared with cloning efficiency in negative controls]) was observed for the Test Item at ≥2mg/ml, either in the presence or absence of metabolic activation. Based on the preliminary cytotoxicity assay results, the gene mutation study was conducted with the Test Item concentrations of 0.25, 0.5, 1, and 2 mg/ml, both in the presence (1 % v/v S9 mix) and absence of metabolic activation system along with negative, vehicle and positive controls. In the main study, cultures were exposed to negative, vehicle, Test Item, and positive control for 4 hours (short-term exposure) in the absence and presence of metabolic activation. In the absence of metabolic activation, the relative survival values were 99.43% (vehicle control), 95.93% (at 0.25 mg/ml), 93.65% (at 0.5 mg/ml), 92.80% (at 1 mg/ml) and 91.48% (at 2 mg/ml). In the presence of metabolic activation, the relative survival values were 98.48% (vehicle control), 96.20% (at 0.25 mg/ml), 92.96% (at 0.5 mg/ml), 91.86% (at 1 mg/ml) and 91.21% (at 2 mg/ml). No significant increase in the mutation frequency (MF) either in the absence(7.85x10-6, 7.04 x10-6, 8.35 x10-6and 9.94 x10-6 at 0.25 mg/ml, 0.5 mg/ml, 1 mg/ml and 2 mg/ml, respectively) or presence of metabolic activation (7.63 x10-6, 8.02x10-6, 9.35x10-6and 9.67x10-6 at 0.25 mg/ml, 0.5 mg/ml, 1 mg/ml and 2 mg/ml, respectively) was observed when compared to vehicle control (6.61 x10-6, 6.51 x 10-6, absence and presence, respectively). There was no significant reduction in the relative survival (cytotoxicity), and no increase in the mutation frequency was observed in vehicle control (dimethyl sulfoxide) either in the presence or absence of metabolic activation. The positive controls used in the study produced statistically significant increases in mutation frequency, indicating the sensitivity of the test system to specific mutagens and confirming that the test conditions were appropriate and that the metabolic activation system functioned properly. Thus the results indicated that the Test Item, N-tert-butylacrylamide (CAS No. 107-58-4), did not induce a statistically significant or biologically relevant increase in the mutation frequency at concentrations of 0.25, 0.5, 1, and 2 mg/ml when compared to the vehicle control data neither in the present nor in the absence of S9 metabolic activation.
Based on the results of this study, it is concluded that N-tert-butylacrylamide (CAS No. 107-58-4)did not induce gene mutation in the hypoxanthine-guanine phosphoribosyl transferase (Hprt)CHO cells up to the concentration of 2 mg/ml of culture medium, either in the presence or absence of S9 metabolic activation system, under the experimental conditions described
Referenceopen allclose all
Table1 Mean Revertant Colony Count – Preliminary Cytotoxicity Assay
Test Item Concentration (mg/plate) |
TA 98 |
TA 100 |
||||||
- S9 |
+ S9 |
- S9 |
+ S9 |
|||||
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
|
NC |
20.33 |
3.21 |
22.33 |
2.89 |
103.33 |
9.50 |
103.67 |
7.02 |
VC |
20.33 |
2.52 |
21.00 |
1.73 |
100.67 |
4.73 |
102.00 |
8.00 |
T1 0.0390625 |
20.67 |
2.52 |
21.67 |
1.15 |
93.00 |
3.46 |
96.00 |
6.24 |
T2 0.078125 |
19.00 |
1.73 |
22.00 |
1.73 |
104.33 |
8.08 |
107.67 |
8.50 |
T3 0.15625 |
21.33 |
1.53 |
22.00 |
2.65 |
95.33 |
7.09 |
96.00 |
6.24 |
T4 0.3125 |
21.00 |
1.73 |
21.00 |
3.61 |
96.33 |
6.51 |
108.33 |
5.69 |
T5 0.625 |
20.33 |
2.52 |
21.67 |
2.31 |
96.00 |
6.24 |
100.33 |
8.50 |
T6 1.25 |
21.00 |
3.46 |
20.33 |
2.52 |
93.67 |
5.03 |
98.00 |
8.00 |
T7 2.5 |
19.33 |
1.15 |
21.67 |
1.15 |
109.67 |
6.66 |
91.33 |
2.52 |
T8 5.0 |
20.67 |
2.08 |
20.33 |
2.31 |
96.00 |
6.24 |
95.00 |
1.73 |
PC |
401.33 |
18.01 |
389.00 |
26.06 |
703.00 |
10.54 |
703.33 |
15.53 |
Key:NC = Negative control,VC = Vehicle control, PC = Positive Control, -S9 = Absence of metabolic activation, +S9 = Presence of metabolic activation, SD = Standard Deviation, NI = No Inhibition.
Note: Since there was no reduction in the revertant count and noinhibition of thebackground lawn observedat 5 mg/plate, Trial I (plate incorporation method) was performed with 5 mg/plate as thehighest concentration.
Positive Controls:
2-Nitrofluorene |
: |
TA98 (absence of metabolic activation) |
Sodium azide |
: |
TA100 (absence of metabolic activation) |
Benzo[a]pyrene |
: |
TA98 and TA100 (presence of metabolic activation) |
Mean Revertant Colony Count -Trial I
Plate Incorporation Method [Absence of metabolic activation (-S9)] |
|
||||||
Test Item Concentration (mg/plate) |
TA 1535 |
TA 1537 |
TA 102 |
||||
Mean |
SD |
Mean |
SD |
Mean |
SD |
||
NC |
14.00 |
1.73 |
6.67 |
1.15 |
235.67 |
7.77 |
|
VC |
12.67 |
1.53 |
7.33 |
0.58 |
238.00 |
5.00 |
|
T1(0.3125) |
11.33 |
1.53 |
5.33 |
1.15 |
228.00 |
22.61 |
|
T2(0.625) |
12.33 |
2.31 |
5.00 |
0.00 |
227.00 |
9.85 |
|
T3(1.25) |
12.33 |
3.21 |
5.00 |
1.73 |
219.33 |
3.51 |
|
T4(2.5) |
15.00 |
1.73 |
5.67 |
1.15 |
219.33 |
13.32 |
|
T5(5.0) |
13.33 |
2.52 |
5.33 |
1.15 |
215.33 |
10.21 |
|
PC |
390.00 |
18.33 |
226.67 |
9.45 |
1562.67 |
42.67 |
|
Plate Incorporation Method [Presence of metabolic activation (+S9 10 % v/v S9 Mix)] |
||||||
Test Item Concentration (mg/plate) |
TA 1535 |
TA 1537 |
TA 102 |
|||
Mean |
SD |
Mean |
SD |
Mean |
SD |
|
NC |
13.67 |
1.15 |
7.33 |
1.15 |
229.33 |
10.02 |
VC |
14.67 |
1.15 |
7.00 |
1.00 |
214.00 |
14.00 |
T1(0.3125) |
15.67 |
2.52 |
7.33 |
2.31 |
216.67 |
11.02 |
T2(0.625) |
13.67 |
0.58 |
9.33 |
1.53 |
222.33 |
17.04 |
T3(1.25) |
14.00 |
1.00 |
8.67 |
2.31 |
224.33 |
9.07 |
T4(2.5) |
13.00 |
2.00 |
6.33 |
1.53 |
225.33 |
7.77 |
T5(5.0) |
14.67 |
1.15 |
6.33 |
0.58 |
233.33 |
6.03 |
PC |
385.33 |
27.47 |
230.67 |
12.01 |
1499.67 |
20.60 |
Key:NC = Negative control, VC = Vehicle control, PC = Positive Control, -S9 = Absence of metabolic activation, +S9 = Presence of metabolic activation, SD = Standard Deviation.
Positive Controls:
2-Nitrofluorene |
: |
TA98 (absence of metabolic activation) |
Sodium azide |
: |
TA100 and TA1535 (absence of metabolic activation) |
9-Aminoacridine |
: |
TA1537 (absence of metabolic activation) |
Mitomycin-C |
: |
TA102(absence of metabolic activation) |
Benzo[a]pyrene |
: |
TA98, TA100, TA1535, TA1537 and TA102 (presence of metabolic activation) |
Table3 Mean Revertant
Colony Count -Trial II
Preincubation Method [Absence of metabolic activation] |
||||||||||
Test Item Concentration (mg/plate) |
TA 98 |
TA 100 |
TA 1535 |
TA 1537 |
TA 102 |
|||||
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
|
NC |
21.67 |
2.89 |
98.33 |
8.39 |
14.00 |
1.73 |
9.00 |
1.00 |
231.67 |
5.69 |
VC |
21.67 |
2.65 |
100.67 |
8.50 |
12.67 |
1.53 |
6.33 |
0.70 |
225.33 |
5.69 |
T1(0.3125) |
19.67 |
1.00 |
105.67 |
6.81 |
14.33 |
0.58 |
9.00 |
1.42 |
215.67 |
4.63 |
T2(0.625) |
20.67 |
3.79 |
98.00 |
4.58 |
11.67 |
1.15 |
7.67 |
1.21 |
234.33 |
5.28 |
T3(1.25) |
19.67 |
1.00 |
104.67 |
7.64 |
13.00 |
2.65 |
5.33 |
0.84 |
207.00 |
7.57 |
T4(2.5) |
22.00 |
2.00 |
102.67 |
10.02 |
12.00 |
1.73 |
6.67 |
1.05 |
217.67 |
13.01 |
T5(5.0) |
22.00 |
2.65 |
92.33 |
4.16 |
12.00 |
1.73 |
7.33 |
1.16 |
215.00 |
14.00 |
PC |
263.00 |
9.54 |
810.00 |
27.18 |
383.67 |
8.74 |
220.00 |
34.74 |
1726.00 |
6.11 |
Preincubation Method [Presence of metabolic activation (+S9 10% v/v S9 Mix)] |
||||||||||
Test Item Concentration (mg/plate) |
TA 98 |
TA 100 |
TA 1535 |
TA 1537 |
TA 102 |
|||||
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
|
NC |
22.33 |
2.89 |
102.00 |
5.57 |
15.33 |
0.58 |
7.33 |
1.00 |
216.67 |
17.62 |
VC |
21.00 |
2.65 |
103.67 |
11.37 |
13.67 |
0.58 |
6.67 |
0.91 |
224.00 |
13.00 |
T1(0.3125) |
20.00 |
1.00 |
107.33 |
9.07 |
12.67 |
0.58 |
7.67 |
1.15 |
235.33 |
6.66 |
T2(0.625) |
21.33 |
3.79 |
107.00 |
7.94 |
12.00 |
1.73 |
9.00 |
1.35 |
219.00 |
8.00 |
T3(1.25) |
20.00 |
1.00 |
98.67 |
8.02 |
12.00 |
1.73 |
7.33 |
1.10 |
237.00 |
9.54 |
T4(2.5) |
21.00 |
2.00 |
101.33 |
11.15 |
13.00 |
2.00 |
7.33 |
1.10 |
216.67 |
11.68 |
T5(5.0) |
21.00 |
2.65 |
104.33 |
7.57 |
11.67 |
1.15 |
7.00 |
1.05 |
207.00 |
4.00 |
PC |
368.00 |
9.54 |
822.33 |
31.97 |
375.00 |
11.53 |
212.67 |
31.90 |
1699.00 |
44.98 |
Key:NC = Negative control, VC = Vehicle control, PC = Positive Control, -S9 = Absence of metabolic activation, +S9 = Presence of metabolic activation, SD = Standard Deviation.
Positive Controls:
2-Nitrofluorene |
: |
TA98 (absence of metabolic activation) |
Sodium azide |
: |
TA100 and TA1535 (absence of metabolic activation) |
9-Aminoacridine |
: |
TA1537 (absence of metabolic activation) |
Mitomycin-C |
: |
TA102(absence of metabolic activation) |
Benzo[a]pyrene |
: |
TA98, TA100, TA1535, TA1537 and TA102 (presence of metabolic activation) |
Fold Increase
Trial I - Plate Incorporation Method |
||||||||||
Test Item Concentration (mg/plate) |
TA 98 |
TA 100 |
TA 1535 |
TA 1537 |
TA 102 |
|||||
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
|
NC |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
VC |
1.00 |
1.02 |
0.90 |
1.10 |
1.01 |
1.00 |
1.02 |
0.90 |
1.10 |
1.01 |
T1(0.3125) |
1.03 |
0.93 |
0.89 |
0.73 |
0.96 |
1.03 |
0.93 |
0.89 |
0.73 |
0.96 |
T2(0.625) |
1.00 |
0.93 |
0.97 |
0.68 |
0.95 |
1.00 |
0.93 |
0.97 |
0.68 |
0.95 |
T3(1.25) |
1.03 |
0.91 |
0.97 |
0.68 |
0.92 |
1.03 |
0.91 |
0.97 |
0.68 |
0.92 |
T4(2.5) |
0.95 |
1.06 |
1.18 |
0.77 |
0.92 |
0.95 |
1.06 |
1.18 |
0.77 |
0.92 |
T5(5.0) |
1.02 |
0.93 |
1.05 |
0.73 |
0.90 |
1.02 |
0.93 |
1.05 |
0.73 |
0.90 |
PC |
19.74 |
6.80 |
30.79 |
30.91 |
6.57 |
19.74 |
6.80 |
30.79 |
30.91 |
6.57 |
Trial II – Preincubation Method |
||||||||||
Test Item Concentration (mg/plate) |
TA 98 |
TA 100 |
TA 1535 |
TA 1537 |
TA 102 |
|||||
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
|
NC |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
VC |
0.94 |
0.98 |
1.07 |
0.95 |
0.93 |
0.94 |
0.98 |
1.07 |
0.95 |
0.93 |
T1(0.3125) |
1.00 |
1.06 |
1.07 |
1.05 |
1.01 |
1.00 |
1.06 |
1.07 |
1.05 |
1.01 |
T2(0.625) |
1.03 |
0.98 |
0.93 |
1.33 |
1.04 |
1.03 |
0.98 |
0.93 |
1.33 |
1.04 |
T3(1.25) |
0.97 |
0.96 |
0.95 |
1.24 |
1.05 |
0.97 |
0.96 |
0.95 |
1.24 |
1.05 |
T4(2.5) |
1.03 |
0.90 |
0.89 |
0.90 |
1.05 |
1.03 |
0.90 |
0.89 |
0.90 |
1.05 |
T5(5.0) |
0.97 |
0.93 |
1.00 |
0.90 |
1.09 |
0.97 |
0.93 |
1.00 |
0.90 |
1.09 |
PC |
18.52 |
6.90 |
26.27 |
32.95 |
7.01 |
18.52 |
6.90 |
26.27 |
32.95 |
7.01 |
Key:NC = Negative control,VC = Vehicle control, PC = Positive Control, -S9 = Absence of metabolic activation, +S9 = Presence of metabolic activation.
S9 Efficiency Check- Summary
Summary of S9 efficiency check |
||||
|
TA100 |
TA1535 |
||
Mean |
SD |
Mean |
SD |
|
VC Distilled water (-S9) |
100.67 |
4.73 |
12.67 |
1.53 |
VC Distilled water (+S9) |
102.00 |
8.00 |
14.67 |
1.15 |
PC Benzo[a]pyrene (-S9)
|
103.67 |
7.02 |
12.67 |
1.53 |
PC Benzo[a]pyrene (+S9)
|
703.33 |
15.53 |
385.33 |
27.47 |
Key:VC = Vehicle control, PC = Positive control, -S9 = Absence of metabolic activation, +S9 = Presence of metabolic activation, SD = Standard Deviation.
Individual Revertant Colony Count-Trial I
Trial I- Plate Incorporation Method- Absence of Metabolic Activation |
||||
Test ItemConcentration (mg/plate) |
R |
TA1535 |
TA1537 |
TA102 |
NC |
1 |
13 |
6 |
238 |
2 |
16 |
8 |
242 |
|
3 |
13 |
6 |
227 |
|
VC |
1 |
11 |
7 |
233 |
2 |
13 |
7 |
243 |
|
3 |
14 |
8 |
238 |
|
T1 (0.3125) |
1 |
13 |
6 |
203 |
2 |
11 |
6 |
247 |
|
3 |
10 |
4 |
234 |
|
T2 (0.625) |
1 |
11 |
5 |
219 |
2 |
11 |
5 |
238 |
|
3 |
15 |
5 |
224 |
|
T3 (1.25) |
1 |
11 |
4 |
216 |
2 |
10 |
7 |
223 |
|
3 |
16 |
4 |
219 |
|
T4 (2.5) |
1 |
14 |
5 |
208 |
2 |
14 |
5 |
216 |
|
3 |
17 |
7 |
234 |
|
T5 (5) |
1 |
11 |
6 |
211 |
2 |
13 |
4 |
208 |
|
3 |
16 |
6 |
227 |
|
PC |
1 |
410 |
216 |
1518 |
2 |
386 |
234 |
1603 |
|
3 |
374 |
230 |
1567 |
|
Key:NC = Negative control, VC = Vehicle control, PC = Positive Control, R = Replicate, TI-T5 = Test Item concentration from lower to higher concentration.
Positive Controls:
2-Nitrofluorene |
: |
TA98 (absence of metabolic activation) |
Sodium azide |
: |
TA100 and TA1535 (absence of metabolic activation) |
9-Aminoacridine |
: |
TA1537 (absence of metabolic activation) |
Mitomycin-C |
: |
TA102(absence of metabolic activation) |
Benzo[a]pyrene |
: |
TA98, TA100, TA1535, TA1537 and TA102 (presence of metabolic activation) |
Individual Revertant Colony
Count-Trial II
Trial II- Preincubation Method- Presence of Metabolic Activation |
||||||
Test Item Concentration (mg/plate) |
R |
TA98 |
TA100 |
TA1535 |
TA1537 |
TA102 |
NC |
1 |
18 |
94 |
16 |
8 |
238 |
2 |
23 |
108 |
13 |
10 |
227 |
|
3 |
24 |
93 |
13 |
9 |
230 |
|
VC |
1 |
20 |
91 |
11 |
6 |
227 |
2 |
21 |
107 |
13 |
6 |
219 |
|
3 |
24 |
104 |
14 |
7 |
230 |
|
T1 (0.3125) |
1 |
18 |
111 |
14 |
9 |
221 |
2 |
18 |
108 |
15 |
10 |
219 |
|
3 |
23 |
98 |
14 |
8 |
207 |
|
T2 (0.625) |
1 |
21 |
103 |
11 |
8 |
233 |
2 |
22 |
97 |
11 |
8 |
229 |
|
3 |
19 |
94 |
13 |
7 |
241 |
|
T3 (1.25) |
1 |
17 |
103 |
10 |
6 |
197 |
2 |
23 |
113 |
14 |
4 |
208 |
|
3 |
24 |
98 |
15 |
6 |
216 |
|
T4 (2.5) |
1 |
21 |
93 |
11 |
6 |
207 |
2 |
21 |
113 |
11 |
8 |
219 |
|
3 |
17 |
102 |
14 |
6 |
227 |
|
T5 (5.0) |
1 |
24 |
91 |
13 |
7 |
216 |
2 |
25 |
89 |
13 |
7 |
209 |
|
3 |
20 |
97 |
10 |
8 |
220 |
|
PC |
1 |
378 |
780 |
386 |
219 |
1673 |
2 |
391 |
833 |
374 |
234 |
1811 |
|
3 |
364 |
817 |
391 |
207 |
1694 |
|
Key:NC = Negative control, VC = Vehicle control, PC = Positive Control, R = Replicate, TI-T5 = Test Item concentration from lower to higher concentration.
Positive Controls:
2-Nitrofluorene |
: |
TA98 (absence of metabolic activation) |
Sodium azide |
: |
TA100 and TA1535 (absence of metabolic activation) |
9-Aminoacridine |
: |
TA1537 (absence of metabolic activation) |
Mitomycin-C |
: |
TA102(absence of metabolic activation) |
Benzo[a]pyrene |
: |
TA98, TA100, TA1535, TA1537 and TA102 (presence of metabolic activation) |
Cytotoxicity results:
Before conducting the chromosomal aberration study, N-tert-butylacrylamide (CAS No. 107-58-4) was evaluated for cytotoxicity both in the absence and presence of metabolic activation system (1%). Cytotoxicity was assessed at the concentrations of 0.5 (T1), 1.0 (T2) and 2.0 (T3) mg/mL of culture media. Cytotoxicity was not observed in the treated concentrations both, in the absence and in the presence of metabolic activation (1%).
In the absence of S9 mix, the mean mitotic index observed was 10.03 (NC), 9.93 (VC), 9.70 (T1), 9.50 (T2), 9.54 (T3) and 8.50 (PC). In the presence of S9 mix, the mean mitotic index observed was 10.10 (NC), 9.94 (VC), 9.74 (T1), 9.60 (T2), 9.64 (T3) and 8.59 (PC).
In the cytotoxicity experiment, the highest test concentration2.0(T3)mg/ mLof culture mediadid not show more than 50% reduction the mitotic index when compared to the respective vehicle control both in the presence or absence of metabolic activation. Hence these concentrations [0.5 (T1), 1.0 (T2) and 2.0 (T3) mg/mL]were selected for the main study.
Hence, 2.0 mg/mL of culture media was selected as the highest concentration for main study both in the presence and in the absence of metabolic activation. The main study was performed in twoindependentphases;
Phase 1 results:
In the experiment, the cultures were exposed to N-tert-butylacrylamide (CAS No. 107-58-4) for a short period of time (4 h) both in the absence and in the presence of metabolic activation system (1%). The mean percentage of aberrant cells was 0.333 (NC), 0.333 (VC), 0.333 (T1), 0.333 (T2), 0.667 (T3) and 9.667 (PC) in the absence of metabolic activation and 0.333 (NC), 0.333 (VC), 0.333 (T1), 0.667 (T2), 0.333 (T3) and 10.333 (PC) in the presence of metabolic activation at the concentration of 0.00 (NC), 0.00 (VC), 0.5 (T1), 1.0 (T2) and 2.0 (T3) mg/mL and positive controls, respectively.
Treatment with Ethyl methanesulfonate at the concentration of 600 µg/mL in the absence of metabolic activation and Cyclophosphamidemonohydrate at the concentration of30 µg/mL in the presence of metabolic activation (1%) causedsignificant increase in percent aberrant cells.Even though the analysis did not reveal any statistical significance, the increase was biologically significant.
During thetreatment with test item in the absence and presence of S9 mix, there was noreduction in mitotic index observed at the tested concentrations. The observed mean mitotic indexin the absence of metabolic activation were 10.18, 9.90, 9.69, 9.50, 9.55 and 8.40 andin the presence ofmetabolic activation were 10.13, 9.95, 9.74, 9.60, 9.64 and 8.49 for NC, VC, T1, T2, T3 and PC concentrations respectively.
Phase 2 results:
The phase II experiment was performed to confirm the negative results obtained in the absence and in the presence of metabolic activation in Phase I. In the Phase II, test item concentrations used were 0.5 (T1), 1.0 (T2) and 2.0 (T3) mg/mL culture both in presence and in absence of metabolic activation (2%). The duration of exposure to the test item in presence of metabolic activation system was 4 hours and in absence of metabolic activation the duration of exposure was 24 hours. The mean percent aberrant cells were 0.333 (NC), 0.333 (VC), 0.333 (T1), 0.333 (T2), 0.333 (T3) and 9.333 (PC) in the absence of metabolic activation and 0.333 (NC), 0.333 (VC), 0.333 (T1), 0.333 (T2), 0.667 (T3) and 9.667 (PC) in the presence of metabolic activation at the concentration of 0.00 (NC), 0.00 (VC), 0.5 (T1), 1.0 (T2) and 2.0 (T3) mg/mL of culture and positive control, respectively.
Treatment with Ethyl methanesulfonate at the concentration of 600 µg/mL in the absence of metabolic activation and Cyclophosphamidemonohydrate at the concentration of30 µg/mL in the presence of metabolic activation (2%) causedsignificant increase in percent aberrant cells.Though the analysis did not reveal any statistical significance, the increase was biologically significant.
The increased frequency of aberrations observed in the concurrent positive control groups (Phase I and II) demonstrated the sensitivity of the test system, suitability of the methods and conditions employed in the experiment.
Treatment with test item in the absence and presence of S9 mix, there was noreduction in mitotic index was observed at the tested concentrations. The observed mean mitotic indexin the absence of metabolic activation were 10.07, 9.94, 9.64, 9.65, 9.60 and 8.75 andin the presence ofmetabolic activation were 10.00, 9.94, 9.63, 9.55, 9.65 and 8.64 for NC, VC, T1, T2, T3 and PC concentrations respectively.
Mitotic Index- Cytotoxic Test
Treatment |
R |
Mitotic Index (%) |
|||||||
In the Absence of Metabolic Activation (-S9) |
In the Presence of Metabolic Activation (1% S9) |
||||||||
Mitotic Index |
Mean |
SD |
Percent Reduction |
Mitotic Index |
Mean |
SD |
Percent Reduction |
||
NC |
R1 |
9.97 |
10.03 |
0.08 |
- |
10.20 |
10.10 |
0.15 |
- |
R2 |
10.09 |
9.99 |
|||||||
VC (DMSO) |
R1 |
9.89 |
9.93 |
0.06 |
- |
9.88 |
9.94 |
0.08 |
- |
R2 |
9.97 |
9.99 |
|||||||
T1 |
R1 |
9.60 |
9.70 |
0.13 |
2.37 |
9.69 |
9.74 |
0.06 |
2.01 |
R2 |
9.79 |
9.78 |
|||||||
T2 |
R1 |
9.40 |
9.50 |
0.13 |
4.38 |
9.69 |
9.60 |
0.13 |
3.42 |
R2 |
9.59 |
9.50 |
|||||||
T3 |
R1 |
9.49 |
9.54 |
0.06 |
3.97 |
9.59 |
9.64 |
0.06 |
3.02 |
R2 |
9.58 |
9.68 |
|||||||
PC |
R1 |
8.39 |
8.50 |
0.15 |
14.44 |
8.68 |
8.59 |
0.14 |
13.57 |
R2 |
8.60 |
8.49 |
|||||||
Key: R = Replicate,NC = Negative control, VC = Vehicle Control,PC = Positive control,SD = Standard Deviation,DMSO = Dimethyl Sulfoxide
Summary on mitotic index
Treatment |
Mitotic Index (%) |
|||
Phase I |
||||
In the Absence of Metabolic Activation (-S9) |
In the Presence of Metabolic Activation (1% S9) |
|||
Mean |
SD |
Mean |
SD |
|
NC |
10.18 |
0.15 |
10.13 |
0.05 |
VC (DMSO) |
9.90 |
0.14 |
9.95 |
0.08 |
T1 (0.5 mg/mL) |
9.69 |
0.15 |
9.74 |
0.08 |
T2 (1.0 mg/mL) |
9.50 |
0.15 |
9.60 |
0.15 |
T3 (2.0 mg/mL) |
9.55 |
0.08 |
9.64 |
0.06 |
PC |
8.40 |
0.14 |
8.49 |
0.26 |
Treatment |
Mitotic Index (%) |
|||
Phase II |
||||
In the Absence of Metabolic Activation (-S9) |
In the Presence of Metabolic Activation (2% S9) |
|||
Mean |
SD |
Mean |
SD |
|
NC |
10.07 |
0.13 |
10.00 |
0.15 |
VC (DMSO) |
9.94 |
0.05 |
9.94 |
0.06 |
T1 (0.5 mg/mL) |
9.64 |
0.08 |
9.63 |
0.08 |
T2 (1.0 mg/mL) |
9.65 |
0.06 |
9.55 |
0.08 |
T3 (2.0 mg/mL) |
9.60 |
0.15 |
9.65 |
0.06 |
PC |
8.75 |
0.08 |
8.64 |
0.22 |
Key:NC = Negative control,VC = Vehicle Control,PC = Positive control, MI = Mitotic Index, -S9 = In the absence of metabolic activation, +S9 = In the presence of metabolic activation
SUmmary of Percent Aberrant Cells
Treatment |
Percent Aberrant Cells |
|||
Phase I |
||||
In the Absence of Metabolic Activation (-S9) |
In the Presence of Metabolic Activation (1% S9) |
|||
Mean |
SD |
Mean |
SD |
|
NC |
0.333 |
0.471 |
0.333 |
0.471 |
VC (DMSO) |
0.333 |
0.471 |
0.333 |
0.471 |
T1 (0.5 mg/mL) |
0.333 |
0.471 |
0.333 |
0.471 |
T2 (1.0 mg/mL) |
0.333 |
0.471 |
0.667 |
0.000 |
T3 (2.0 mg/mL) |
0.667 |
0.000 |
0.333 |
0.471 |
PC |
9.667 |
0.471 |
10.333 |
0.471 |
Treatment |
Percent Aberrant Cells |
|||
Phase II |
||||
In the Absence of Metabolic Activation (-S9) |
In the Presence of Metabolic Activation (2% S9) |
|||
Mean |
SD |
Mean |
SD |
|
NC |
0.333 |
0.471 |
0.333 |
0.471 |
VC (DMSO) |
0.333 |
0.471 |
0.333 |
0.471 |
T1 (0.5 mg/mL) |
0.333 |
0.471 |
0.333 |
0.471 |
T2 (1.0 mg/mL) |
0.333 |
0.471 |
0.333 |
0.471 |
T3 (2.0 mg/mL) |
0.333 |
0.471 |
0.667 |
0.000 |
PC |
9.333 |
0.943 |
9.667 |
0.471 |
Key: NC = Negative Control,VC = Vehicle Control,SD = Standard Deviation, PC = Positive Control
Individual Observation of Slides For Mitotic ndex and Chromosome Aberrations:
Phase I [In the Absence of Metabolic Activation, (-S9)]
Treatment |
Culture No. |
Mitotic Index |
Frequencies of Aberration |
Total No. of Aberration |
Percentage of Aberrated Cells |
NC |
R1 |
10.08 |
|
0 |
0.00 |
R2 |
10.29 |
1 ctb |
1 |
0.67 |
|
VC (DMSO) |
R1 |
9.80 |
1fragment |
1 |
0.67 |
R2 |
10.00 |
- |
0 |
0.00 |
|
T1 (0. 5 mg/mL) |
R1 |
9.79 |
- |
0 |
0.00 |
R2 |
9.58 |
1 csb, 1fragment |
2 |
0.67 |
|
T2 (1.0 mg/mL) |
R1 |
9.60 |
1 ctb |
1 |
0.67 |
R2 |
9.39 |
- |
0 |
0.00 |
|
T3 (2.0 mg/mL) |
R1 |
9.60 |
1fragment |
1 |
0.67 |
R2 |
9.49 |
1 csg |
1 |
0.67 |
|
PC |
R1 |
8.49 |
6 ctb, 1 cte, 4 ctg*, 4 csb, 1 cse, 2 csg*, 1DC, 5 fragments |
24 |
9.33 |
R2 |
8.30 |
7 ctb, 2 cte, 4 ctg*, 3 csb, 2 cse, 3 csg*, 1 DC, 5 fragments |
27 |
10.00 |
Phase I [In the Presence of Metabolic Activation (1% S9)]
Treatment |
Culture No. |
Mitotic Index |
Frequencies of Aberration |
Total No. of Aberration |
Percentage of Aberrated Cells |
NC |
R1 |
10.17 |
1fragment |
1 |
0.67 |
R2 |
10.10 |
- |
0 |
0.00 |
|
VC (DMSO) |
R1 |
9.89 |
1 ctb |
1 |
0.67 |
R2 |
10.00 |
- |
0 |
0.00 |
|
T1 (0. 5 mg/mL) |
R1 |
9.68 |
1fragment |
1 |
0.67 |
R2 |
9.80 |
- |
0 |
0.00 |
|
T2 (1.0 mg/mL) |
R1 |
9.49 |
1 ctb |
1 |
0.67 |
R2 |
9.70 |
1 csb |
1 |
0.67 |
|
T3 (2.0 mg/mL) |
R1 |
9.59 |
- |
0 |
0.00 |
R2 |
9.68 |
1 ctg, 1 fragment |
2 |
0.67 |
|
PC |
R1 |
8.30 |
7 ctb, 1 cte, 3 ctg*, 3 csb, 1 cse, 3 csg*, 1 DC, 6 fragments |
25 |
10.00 |
R2 |
8.67 |
7 ctb, 1 cte, 4 ctg*, 4 csb, 1 cse, 3 csg*, 1 DC, 5 fragments |
26 |
10.67 |
Key: MI = Mitotic Index, ctg = Chromatid gap, ctb = Chromatid break, csg = Chromosomal gap, csb = Chromosomal break, DC = Dicentric, cte = Chromatid exchange, cse = Chromosome exchange, NC = Negative Control, VC = Vehicle Control, PC = Positive Control, * = Not considered in analysis of aberrant cells when the only Gap is present.
6.4 INDIVIDUAL OBSERVATION OF SLIDES FOR MITOTIC INDEX AND CHROMOSOME ABERRATIONS(Contd.)
Phase II [In the Absence of Metabolic Activation (-S9)]
Treatment |
Culture No. |
Mitotic Index |
Frequencies of Aberration |
Total No. of Aberration |
Percentage of Aberrated Cells |
||||||
NC |
R1 |
9.98 |
1 fragment |
1 |
0.67 |
||||||
R2 |
10.17 |
- |
0 |
0.00 |
|||||||
VC (DMSO) |
R1 |
9.90 |
1 ctb |
1 |
0.67 |
||||||
R2 |
9.97 |
- |
0 |
0.00 |
|||||||
T1 (0. 5 mg/mL) |
R1 |
9.58 |
1 ctg, 1 fragment |
2 |
0.67 |
||||||
R2 |
9.69 |
- |
0 |
0.00 |
|||||||
T2 (1.0 mg/mL) |
R1 |
9.69 |
- |
0 |
0.00 |
||||||
R2 |
9.60 |
1 ctb |
1 |
0.67 |
|||||||
T3 (2.0 mg/mL) |
R1 |
9.49 |
1 fragment |
1 |
0.67 |
||||||
R2 |
9.70 |
- |
0 |
0.00 |
|||||||
PC |
R1 |
8.69 |
7 ctb, 1 cte, 3 ctg, 2 csb, 1 cse, 2 csg, 1 Dc, 7 fragments |
24 |
10.00 |
||||||
R2 |
8.80 |
5 ctb, 2 cte, 4 ctg, 2 csb, 1 cse, 2 csg, 1 Dc, 5 fragments |
22 |
8.67 |
Phase II [In the Presence of Metabolic Activation (2% S9)]
Treatment |
Culture No. |
Mitotic Index |
Frequencies of Aberration |
Total No. of Aberration |
Percentage of Aberrated Cells |
||||||
NC |
R1 |
10.10 |
- |
0 |
0.00 |
||||||
R2 |
9.89 |
1 fragment |
1 |
0.67 |
|||||||
VC (DMSO) |
R1 |
9.98 |
1 ctb |
1 |
0.67 |
||||||
R2 |
9.89 |
- |
0 |
0.00 |
|||||||
T1 (0. 5 mg/mL) |
R1 |
9.69 |
- |
0 |
0.00 |
||||||
R2 |
9.57 |
1 ctg, 1 fragment |
2 |
0.67 |
|||||||
T2 (1.0 mg/mL) |
R1 |
9.60 |
- |
0 |
0.00 |
||||||
R2 |
9.49 |
1 ctb |
1 |
0.67 |
|||||||
T3 (2.0 mg/mL) |
R1 |
9.60 |
1 fragment |
1 |
0.67 |
||||||
R2 |
9.69 |
1 cte |
1 |
0.67 |
|||||||
PC |
R1 |
8.80 |
6 ctb, 1 cte, 4 ctg, 3 csb, 1 cse, 3 csg, 1 Dc, 5 fragments |
24 |
10.00 |
||||||
R2 |
8.48 |
5 ctb, 2 cte, 5 ctg, 3 csb, 1 cse, 3 csg, 1 Dc, 6 fragments |
26 |
9.33 |
Key: MI = Mitotic Index, ctg = Chromatid gap, ctb = Chromatid break, csg = Chromosomal gap, csb = Chromosomal break, DC = Dicentric, cte = Chromatid exchange, cse = Chromosome exchange, NC = Negative Control, VC = Vehicle Control, PC = Positive Control, * = Not considered in analysis of aberrant cells when the only Gap is present.\
Historical data
HISTORICAL DATA FOR IN VITRO MAMMALIAN CHROMOSOMAL ABERRATION TEST (CA-H) |
|||||||||||||||||||||
S.No. |
Study No. |
Vehicle |
Phase I |
Phase II |
|||||||||||||||||
Absence of S9 |
Presence of S9 |
Absence of S9 |
Presence of S9 |
||||||||||||||||||
Vehicle Control |
Positive Control |
Vehicle Control |
Positive Control |
Vehicle Control |
Positive Control |
Vehicle Control |
Positive Control |
||||||||||||||
1 |
1151 |
DMSO |
0.000 |
9.000 |
0.000 |
10.5 |
0.000 |
9.000 |
0.000 |
8.000 |
|||||||||||
2 |
1333 |
DMSO |
0.000 |
8.000 |
0.000 |
7.500 |
0.5 |
8.500 |
0.5 |
9.000 |
|||||||||||
3 |
2060 |
DMSO |
0.5 |
8.000 |
0.000 |
7.000 |
1.500 |
6.500 |
0.000 |
9.000 |
|||||||||||
4 |
2450 |
DMSO |
0.000 |
10.000 |
0.000 |
10.5 |
0.000 |
11.500 |
0.000 |
12.000 |
|||||||||||
5 |
2452 |
DMSO |
0.000 |
10.000 |
0.000 |
8.500 |
0.000 |
9.500 |
0.000 |
8.500 |
|||||||||||
6 |
3000 |
PBS |
0.000 |
7.500 |
0.000 |
8.500 |
0.000 |
11.000 |
0.000 |
10.000 |
|||||||||||
7 |
3313 |
DMSO |
0.000 |
8.000 |
0.000 |
10.5 |
0.5 |
9.500 |
0.000 |
9.500 |
|||||||||||
8 |
3422 |
DMSO |
0.000 |
9.000 |
0.5 |
10.000 |
1.000 |
9.500 |
1.000 |
8.500 |
|||||||||||
9 |
3665 |
RPMI |
0.5 |
8.500 |
0.000 |
7.500 |
0.000 |
8.500 |
0.5 |
8.000 |
|||||||||||
10 |
3801 |
Sodium Phosphate Buffer |
1.500 |
9.500 |
1.000 |
9.000 |
1.000 |
9.500 |
0.5 |
9.500 |
|||||||||||
11 |
3862 |
DMSO |
1.500 |
9.500 |
1.000 |
9.000 |
1.000 |
9.500 |
0.5 |
9.500 |
|||||||||||
12 |
4792 |
PBS |
0.5 |
7.500 |
0.5 |
8.500 |
0.5 |
8.500 |
0.000 |
8.000 |
|||||||||||
13 |
4938 |
DMSO |
0.5 |
8.500 |
1.000 |
8.500 |
0.5 |
8.000 |
1.000 |
8.000 |
|||||||||||
14 |
5123 |
DMSO |
0.333 |
9.000 |
0.667 |
8.667 |
0.333 |
9.667 |
0.333 |
9.000 |
|||||||||||
15 |
5739 |
Ethyl alcohol |
0.333 |
10.333 |
0.333 |
10.000 |
0.333 |
9.667 |
0.333 |
10.667 |
|||||||||||
16 |
5824 |
PBS |
0.333 |
10.000 |
0.333 |
11.000 |
0.333 |
9.333 |
0.333 |
10.000 |
|||||||||||
17 |
6461 |
PBS |
0.333 |
10.000 |
0.333 |
9.667 |
0.333 |
9.000 |
0.333 |
10.000 |
|||||||||||
18 |
6196 |
RPMI |
0.333 |
11.000 |
0.333 |
10.000 |
0.333 |
10.667 |
0.333 |
10.000 |
|||||||||||
19 |
6121 |
DMSO |
0.667 |
8.667 |
0.667 |
9.667 |
0.667 |
9.667 |
0.667 |
9.333 |
|||||||||||
20 |
6678 |
DMSO |
0.333 |
10.333 |
0.333 |
10.000 |
0.333 |
9.667 |
0.333 |
10.667 |
|||||||||||
21 |
6687 |
DMSO |
0.333 |
11.333 |
0.333 |
11.333 |
0.333 |
12.333 |
0.333 |
12.000 |
|||||||||||
22 |
6221 |
DMSO |
0.333 |
9.667 |
0.333 |
10.667 |
0.333 |
9.667 |
0.333 |
10.333 |
|||||||||||
23 |
6834 |
DMSO |
0.333 |
10.333 |
0.333 |
11.333 |
0.333 |
11.333 |
0.333 |
10.667 |
|||||||||||
24 |
6759 |
PBS |
0.667 |
10.667 |
0.000 |
10.000 |
0.333 |
12.000 |
0.333 |
11.333 |
|||||||||||
25 |
6430 |
DMSO |
0.333 |
9.000 |
0.333 |
10.000 |
0.667 |
9.667 |
0.667 |
9.667 |
|||||||||||
26 |
7703 |
DMSO |
0.333 |
10.000 |
0.333 |
10 |
0.333 |
10.333 |
0.333 |
10.333 |
|||||||||||
27 |
7576 |
RPMI |
0.333 |
10.000 |
0.333 |
10.667 |
0.333 |
10.333 |
0.333 |
10.333 |
|||||||||||
28 |
7572 |
DMSO |
0.667 |
10.333 |
0.667 |
10 |
0.667 |
9.667 |
0.333 |
10 |
|||||||||||
29 |
7574 |
Ethyl alcohol |
0.333 |
10.333 |
0.333 |
10.333 |
0.333 |
10.000 |
0.333 |
10.333 |
|||||||||||
Mean |
0.391 |
9.448 |
0.345 |
9.615 |
0.442 |
9.724 |
0.345 |
9.730 |
|||||||||||||
SD |
0.371 |
1.041 |
0.312 |
1.142 |
0.346 |
1.201 |
0.267 |
1.106 |
|||||||||||||
Mean + 2SD |
1.132 |
11.530 |
0.968 |
11.899 |
1.134 |
12.127 |
0.879 |
11.942 |
|||||||||||||
Mean - 2SD |
-0.351 |
7.367 |
-0.279 |
7.331 |
-0.249 |
7.322 |
-0.189 |
7.518 |
|||||||||||||
SD - Standard Deviation
Note: Thepresent study no. 7708 was 32ndstudy. Number of CA-H studies conducted before finalizing present study was 31 (2 study under finalization). Out of 29 studies, 18 studies using DMSO, 5 studies with PBS, 3 studies using RPMI, 2 studies using ethyl alcohol and 1 study using Sodium Phosphate Buffer were conducted. However, positive control data upto S. No. 13 studies were available for 200 metaphases but from S. No. 14 performed for 300 metaphases for aberrant cells as per the revised OECD guideline 473 adopted on 29thJuly 2016.
Appendix 1: Relative Survival – Preliminary Cytotoxicity Assay:Absence of metabolic activation
Dose level |
Concentration |
No. of Cells |
No. of colonies |
Mean Colony count |
No. of cells seeded |
CE |
Adjusted CE |
RS |
|||
Before |
After |
R1 |
R2 |
R3 |
|||||||
NC |
Distilled water |
20000000 |
23240000 |
238 |
230 |
224 |
230.67 |
100 |
2.307 |
2.680 |
100.00 |
VC |
Dimethyl sulfoxide |
20000000 |
23120000 |
224 |
235 |
232 |
230.33 |
100 |
2.303 |
2.663 |
99.34 |
T1 |
0.125 mg/ml |
20000000 |
22980000 |
224 |
220 |
221 |
221.67 |
100 |
2.217 |
2.547 |
95.65 |
T2 |
0.25 mg/ml |
20000000 |
22780000 |
214 |
224 |
232 |
223.33 |
100 |
2.233 |
2.544 |
95.54 |
T3 |
0.5 mg/ml |
20000000 |
22640000 |
220 |
221 |
221 |
220.67 |
100 |
2.207 |
2.498 |
93.81 |
T4 |
1 mg/ml |
20000000 |
22260000 |
233 |
220 |
218 |
223.67 |
100 |
2.237 |
2.489 |
93.49 |
T5 |
2 mg/ml |
20000000 |
22140000 |
214 |
225 |
230 |
223.00 |
100 |
2.230 |
2.469 |
92.71 |
Key: NC = Negative Control, VC = Vehicle Control, T5- T1= Test Item concentration from higher to lower, R = Replicate, CE = Cloning Efficiency, RS = Relative Survival, mg = milligram, ml = milliliter.
Appendix2: Relative Survival – Preliminary Cytotoxicity Assay:Presenceof metabolic activation
Dose level |
Concentration |
No. of Cells |
No. of colonies |
Mean Colony count |
No. of cells seeded |
CE |
Adjusted CE |
RS |
|||
Before |
After |
R1 |
R2 |
R3 |
|||||||
NC |
Distilled water |
20000000 |
23540000 |
241 |
238 |
232 |
237.00 |
100 |
2.370 |
2.789 |
100.00 |
VC |
Dimethyl sulfoxide |
20000000 |
23240000 |
234 |
233 |
228 |
231.67 |
100 |
2.317 |
2.692 |
96.50 |
T1 |
0.125 mg/ml |
20000000 |
23140000 |
230 |
228 |
236 |
231.33 |
100 |
2.313 |
2.677 |
99.43 |
T2 |
0.25 mg/ml |
20000000 |
22940000 |
231 |
214 |
221 |
222.00 |
100 |
2.220 |
2.546 |
94.59 |
T3 |
0.5 mg/ml |
20000000 |
22820000 |
219 |
227 |
225 |
223.67 |
100 |
2.237 |
2.552 |
94.80 |
T4 |
1 mg/ml |
20000000 |
22680000 |
220 |
216 |
221 |
219.00 |
100 |
2.190 |
2.483 |
92.25 |
T5 |
2 mg/ml |
20000000 |
22580000 |
220 |
214 |
221 |
218.33 |
100 |
2.183 |
2.465 |
91.57 |
Key: NC = Negative Control, VC = Vehicle Control, T5- T1= Test Item concentration from higher to lower, R = Replicate, CE = Cloning Efficiency, RS = Relative Survival, mg = milligram, ml = milliliter.
Appendix3: Relative Survival –Main Study: Absence of metabolic activation
Dose level |
Concentration |
No. of Cells |
No. of colonies |
Mean Colony count |
No. of cells seeded |
CE |
Adjusted CE |
RS |
|||
Before |
After |
R1 |
R2 |
R3 |
|||||||
NC |
Distilled water |
20000000 |
23520000 |
234 |
230 |
238 |
234.00 |
100 |
2.340 |
2.752 |
100.00 |
VC |
Dimethyl sulfoxide |
20000000 |
23420000 |
233 |
232 |
236 |
233.67 |
100 |
2.337 |
2.736 |
99.43 |
T1 |
0.25 mg/ml |
20000000 |
23160000 |
227 |
229 |
224 |
226.67 |
100 |
2.267 |
2.625 |
95.93 |
T2 |
0.5 mg/ml |
20000000 |
23120000 |
221 |
224 |
220 |
221.67 |
100 |
2.217 |
2.562 |
93.65 |
T3 |
1 mg/ml |
20000000 |
22980000 |
220 |
224 |
219 |
221.00 |
100 |
2.210 |
2.539 |
92.80 |
T4 |
2 mg/ml |
20000000 |
22860000 |
219 |
218 |
220 |
219.00 |
100 |
2.190 |
2.503 |
91.48 |
PC |
400 µg/ml |
20000000 |
22680000 |
231 |
224 |
226 |
227.00 |
100 |
2.270 |
2.574 |
94.08 |
Key: NC = Negative Control, VC = Vehicle Control, PC = Positive Control (Ethylmethanesulfonate), T4-T1= Test Item concentration from higher to lower, R = Replicate, CE = Cloning Efficiency, RS = Relative Survival, mg = milligram, µg = microgram, ml = milliliter.
Relative Survival –Main Study:Presenceof metabolic activation
Dose level |
Concentration |
No. of Cells |
No. of colonies |
Mean Colony count |
No. of cells seeded |
CE |
Adjusted CE |
RS |
|||
Before |
After |
R1 |
R2 |
R3 |
|||||||
NC |
Distilled water |
20000000 |
23640000 |
238 |
234 |
237 |
236.33 |
100 |
2.363 |
2.793 |
100.00 |
VC |
Dimethyl sulfoxide |
20000000 |
23480000 |
234 |
236 |
233 |
234.33 |
100 |
2.343 |
2.751 |
98.48 |
T1 |
0.25 mg/ml |
20000000 |
23250000 |
229 |
230 |
224 |
227.67 |
100 |
2.277 |
2.647 |
96.20 |
T2 |
0.5 mg/ml |
20000000 |
23180000 |
218 |
224 |
220 |
220.67 |
100 |
2.207 |
2.558 |
92.96 |
T3 |
1 mg/ml |
20000000 |
22940000 |
219 |
223 |
219 |
220.33 |
100 |
2.203 |
2.527 |
91.86 |
T4 |
2 mg/ml |
20000000 |
22880000 |
217 |
219 |
222 |
219.33 |
100 |
2.193 |
2.509 |
91.21 |
PC |
30 µg/ml |
20000000 |
22560000 |
230 |
225 |
232 |
229.00 |
100 |
2.290 |
2.583 |
93.89 |
Key: NC = Negative Control, VC = Vehicle Control, PC = Positive Control (Benzo[a]pyrene), T4-T1= Test Item concentration from higher to lower, R = Replicate, CE = Cloning Efficiency, RS = Relative Survival, mg = milligram, µg = microgram, ml = milliliter.
Cloning Efficiency(Non-selective medium)Main Study: Absence of metabolic activation
Dose level |
Non Selective medium |
||||||
Concentration |
No. of cells seeded |
No. of colonies |
Mean No. of colonies |
CE |
|||
R1 |
R2 |
R3 |
|||||
NC |
Distilled water |
100 |
206 |
206 |
199 |
204 |
2.04 |
VC |
Dimethyl sulfoxide |
100 |
205 |
199 |
201 |
202 |
2.02 |
T1 |
0.25 mg/ml |
100 |
198 |
189 |
186 |
191 |
1.91 |
T2 |
0.5 mg/ml |
100 |
188 |
190 |
190 |
189 |
1.89 |
T3 |
1 mg/ml |
100 |
178 |
182 |
179 |
180 |
1.80 |
T4 |
2 mg/ml |
100 |
170 |
164 |
169 |
168 |
1.68 |
PC |
400 µg/ml |
100 |
170 |
167 |
169 |
169 |
1.69 |
Key: NC = Negative Control, VC = Vehicle Control, PC = Positive Control (Ethylmethanesulfonate), T4-T1= Test Item concentration from higher to lower, R = Replicate, CE = Cloning Efficiency, RS = Relative Survival, mg = milligram, µg = microgram, ml = milliliter.
Cloning Efficiency(Non-selective medium)Main Study: Presenceof metabolic activation
Dose level |
Non Selective medium |
||||||
Concentration |
No. of cells seeded |
No. of colonies |
Mean No. of colonies |
CE |
|||
R1 |
R2 |
R3 |
|||||
NC |
Distilled water |
100 |
208 |
208 |
206 |
207 |
2.07 |
VC |
Dimethyl sulfoxide |
100 |
210 |
202 |
202 |
205 |
2.05 |
T1 |
0.25 mg/ml |
100 |
199 |
194 |
197 |
197 |
1.97 |
T2 |
0.5 mg/ml |
100 |
190 |
183 |
188 |
187 |
1.87 |
T3 |
1 mg/ml |
100 |
180 |
176 |
179 |
178 |
1.78 |
T4 |
2 mg/ml |
100 |
168 |
175 |
174 |
172 |
1.72 |
PC |
30 µg/ml |
100 |
177 |
172 |
175 |
175 |
1.75 |
Key: NC = Negative Control, VC = Vehicle Control, PC = Positive Control (Benzo[a]pyrene), T4-T1= Test Item concentration from higher to lower, R = Replicate, CE = Cloning Efficiency, RS = Relative Survival, mg = milligram, µg = microgram, ml = milliliter.
Cloning Efficiency(Selective medium): Absenceof metabolic activation
Dose level |
Selective medium |
||||||
Concentration |
No. of cells seeded |
No. of colonies |
Mean No. of colonies |
CE |
|||
R1 |
R2 |
R3 |
|||||
NC |
Distilled water |
200000 |
3 |
2 |
3 |
2.67 |
0.00001333 |
VC |
Dimethyl sulfoxide |
200000 |
3 |
2 |
3 |
2.67 |
0.00001333 |
T1 |
0.25 mg/ml |
200000 |
3 |
4 |
2 |
3.00 |
0.00001500 |
T2 |
0.5 mg/ml |
200000 |
2 |
4 |
2 |
2.67 |
0.00001333 |
T3 |
1 mg/ml |
200000 |
3 |
2 |
4 |
3.00 |
0.00001500 |
T4 |
2 mg/ml |
200000 |
3 |
3 |
4 |
3.33 |
0.00001667 |
PC |
400 µg/ml |
200000 |
80 |
77 |
79 |
78.67 |
0.00039333 |
Key: NC = Negative Control, VC = Vehicle Control, PC = Positive Control (Ethylmethanesulfonate), T4-T1= Test Item concentration from higher to lower, R = Replicate, CE = Cloning Efficiency, RS = Relative Survival, mg = milligram, µg = microgram, ml = milliliter.
Cloning Efficiency(Selective medium)Phase I: Presence of metabolic activation
Dose level |
Selective medium |
|
|||||
Concentration |
No. of cells seeded |
No. of colonies |
Mean No. of colonies |
CE |
|||
R1 |
R2 |
R3 |
|||||
NC |
Distilled water |
200000 |
3 |
3 |
2 |
2.67 |
0.00001333 |
VC |
Dimethyl sulfoxide |
200000 |
4 |
2 |
2 |
2.67 |
0.00001333 |
T1 |
0.25 mg/ml |
200000 |
3 |
3 |
3 |
3.00 |
0.00001500 |
T2 |
0.5 mg/ml |
200000 |
2 |
3 |
4 |
3.00 |
0.00001500 |
T3 |
1 mg/ml |
200000 |
3 |
4 |
3 |
3.33 |
0.00001667 |
T4 |
2 mg/ml |
200000 |
4 |
2 |
4 |
3.33 |
0.00001667 |
PC |
30 µg/ml |
200000 |
83 |
85 |
84 |
84.00 |
0.00042000 |
Key: NC = Negative Control, VC = Vehicle Control, PC = Positive Control (Benzo[a]pyrene), T4-T1= Test Item concentration from higher to lower, R = Replicate, CE = Cloning Efficiency, RS = Relative Survival, mg = milligram, µg = microgram, ml = milliliter.
Mutation Frequency: Absence of metabolic activation
Dose level |
Absence of metabolic activation |
||
Concentration |
Mutation Frequency |
MF x 10-6 |
|
NC |
Distilled water |
0.00000655 |
6.55 |
VC |
Dimethyl sulfoxide |
0.00000661 |
6.61 |
T1 |
0.25 mg/ml |
0.00000785 |
7.85 |
T2 |
0.5 mg/ml |
0.00000704 |
7.04 |
T3 |
1 mg/ml |
0.00000835 |
8.35 |
T4 |
2 mg/ml |
0.00000994 |
9.94 |
PC |
400 µg/ml |
0.00023320 |
233.20 |
Dose level |
Presence of metabolic activation |
||
Concentration |
Mutation Frequency |
MF x 10-6 |
|
NC |
Distilled water |
0.00000643 |
6.43 |
VC |
Dimethyl sulfoxide |
0.00000651 |
6.51 |
T1 |
0.25 mg/ml |
0.00000763 |
7.63 |
T2 |
0.5 mg/ml |
0.00000802 |
8.02 |
T3 |
1 mg/ml |
0.00000935 |
9.35 |
T4 |
2 mg/ml |
0.00000967 |
9.67 |
PC |
30 µg/ml |
0.00024046 |
240.46 |
Key: NC = Negative Control, VC = Vehicle Control, PC = Positive Control (Absence of metabolic activation- Ethylmethanesulfonate, Presence of metabolic activation- Benzo[a]pyrene), T4-T1= Test item concentration from higher to lower, MF = Mutation Frequency, mg = milligram, µg = microgram, ml = milliliter.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Bacterial Gene Mutation test:
Ames test of-tert-butylacrylamide [TBAA] [CAS No.107-58-4] was conducted according to the plate incorporation and preincubation methods. This study was performed as per the OECD guideline No. 471 (Adopted: July 21 1997, Corrected: June 26 2020). Based on the solubility test, dimethyl sulfoxide (DMSO) was selected as a vehicle for the test item in the study. The mutagenic potential of N-tert-butylacrylamide [TBAA] [CAS No. 107-58-4] was tested in two independent experiments (Trial I and Trial II) and both in the presence (10% v/v cofactor supplemented post-mitochondrial S9 fraction, prepared from rat liver) and absence of metabolic activation using Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA102. The Test item was tested along with solvent-vehicle control (DMSO), negative (distilled water), and concurrent positive control substances in triplicates. A preliminary cytotoxicity assay was performed using the strains TA98 and TA100 with the following concentrations along with the vehicle, negative and positive controls both in the presence (10% v/v S9 mix) and absence of metabolic activation in triplicates: 0.0390625, 0.078125, 0.15625, 0.3125, 0.625, 1.25, 2.5 and 5 mg/plate. In the tester strains, TA98 and TA100, no reduction in the revertant count and no inhibition in background lawn were observed at any of the concentrations, either in the presence (10% v/v S9 mix) or the absence of metabolic activation, when compared to the vehicle control data. Based on the preliminary cytotoxicity test results, the main study was performed with the following concentrations: Trial I and II: 0.3125, 0.625, 1.25, 2.5, and 5 mg/plate
Trial I was performed according to the plate incorporation method, both in the presence (10% v/v S9 mix) and absence of a metabolic activation system. The Test item and the vehicle, negative and positive controls, were tested in triplicates. The Test item doses were selected using a concentration spacing factor of 2. No increase in the number of revertant colonies or inhibition in the background lawn was observed up to the highest concentration of 5mg/plate either in the presence (10% v/v S9 mix) or absence of metabolic activation when compared to the vehicle control data.
Trial II was performed to confirm the negative results observed in Trial I. Trial II was conducted according to the preincubation method, both in the presence (10% v/v S9 mix) and absence of a metabolic activation system. The Test item and the vehicle, negative and positive controls, were tested in all tester strains in triplicates. There was no increase in the number of revertant colonies or inhibition of the background lawn up to the highest concentration of 5 mg/plate either in the presence (10% v/v S9 mix) or absence of metabolic activation when compared to the vehicle control data. The numbers of revertant colonies of the vehicle, negative (spontaneous revertant colonies), and positive controls (induced revertant colonies) of all the tester strains were within the range of historical data of the laboratory. The positive controls used in the study produced significant increases in the mean number of revertant colonies under both activated and non-activated conditions in all tester strains when compared to the control data confirming the validity of the mutagenicity test.
Based on the results of this study, it is concluded that N-tert-butylacrylamide [TBAA] [CAS No. 107-58-4] is non-mutagenic as it does not induce (point) gene mutations in the histidine operon by base-pair changes or frameshifts, in the presence and the absence of metabolic activation system, in the tester strains of Salmonella typhimurium (TA1537, TA1535, TA98, TA100, and TA102).
In vitro Chromosomal Aberration Study:
An in vitro mammalian chromosome aberration test (OECD 473) was conducted to chromosomal aberration induction potential of the test chemical in human peripheral blood lymphocyte cultures. The experiment was performed both in the presence and in the absence of a metabolic activation system after 48 hours of mitogenic stimulation. The test chemical was dissolved in DMSO and used at a dose level of 0.00 (NC), 0.00 (VC), 0.5, 1.0, and 2.0 mg/ml in the presence and absence of S9 metabolic activation system in experiments of Phase I-II. The experiment of Phase I was performed by short-term (4 hours) treatment method both in the presence and absence of a metabolic activation system (1%)The experiment of Phase II was conducted using short-term treatment (4 hours) as well as long-term (24 hours) exposure times. Long-term treatment was performed in absence of metabolic activation to confirm the negative results obtained in the absence of metabolic activation in Phase I. A short-term treatment method was performed with increased metabolic activation (2%) condition to confirm the negative results obtained in the presence of metabolic activation in Phase I. The doses for the main study were based on the cytotoxicity study conducted both in the presence and absence of a metabolic activation system. The test concentrations (0.5, 1, and 2mg/mL of culture media) based on the solubility, precipitation, and pH test of the test item were tested. Cytotoxicity was determined by a reduction in the mitotic index in comparison with the negative control. The cultures were harvested 24 hours after the beginning of the treatment, and they were stained with Giemsa.300 well-spread metaphase plates per culture were scored for cytogenetic damage on coded slides. The slides were evaluated using microscopes with 100 x oil immersion objectives. Chromosomal and chromatid breaks, acentric fragments, deletions, exchanges, pulverization, polyploidy (including endo-reduplication), and disintegrations were recorded as structural chromosomal aberrations. Gaps were recorded as well, but they were not included in the calculation of the aberration rates. Only metaphases with 46± 2 centromere regions were included in the analysis. In the mutagenicity test, no structural abnormality of chromosomes and induction of ploidy cells were observed at any dose tested in both confirmatory trials. The percent aberrant cells were comparable in control and treated groups, both in the presence and in the absence of exogenous metabolic activation. Incubation with positive controls caused a significant increase in the percentage of aberrant cells in comparison to vehicle control in both phases, which confirmed the validity of the test. In the cytotoxicity experiment, the highest test concentration (2 mg/ml) showed less then 50 % reduction in the mitotic index when compared with vehicle control. In conclusion, the test chemical is non-clastogenic at the highest tested concentration of 2 mg/ml both in the presence (1% and 2%) and in the absence of metabolic activation in vitro mammalian chromosome aberration test and under the described experimental conditions.
Gene Mutation in Mammalian cells:
This in vitro experiment was performed to evaluate the ability of N-tert-butylacrylamide (CAS No. 107-58-4) to cause gene mutation in the hypoxanthine-guanine phosphoribosyl transferase (Hprt) locus in cultured Chinese Hamster Ovary (CHO) cells in the presence and absence of exogenous metabolic activation system (S9) containing microsomal enzymes. The study was performed as per OECD 476 (Adopted: July 29 2016). The Test Item, N-test-butyl acrylamide (CAS No. 107-58-4), was found to be soluble in dimethyl sulfoxide. No precipitation was observed at the concentration of 2 mg/ml. Based on the solubility and precipitation test, initial preliminary cytotoxicity testing was performed with the Test Item at the concentrations of 0.125, 0.25, 0.5, 1, and 2 mg/ml in culture medium, both in the presence and absence of metabolic activation system (1 % v/v S9 mix) along with negative (distilled water), vehicle (DMSO) controls. In the absence of metabolic activation, the relative survival values were 99.34% (vehicle control), 95.65% (at 0.125 mg/ml), 95.54% (at 0.25 mg/ml), 93.81% (at 0.5 mg/ml), 93.49% (at 1 mg/ml) and 92.71% (at 2 mg/ml). In the presence of metabolic activation, the relative survival values were 96.50% (vehicle control), 99.43%, (at 0.125 mg/ml), 94.59% (at 0.25 mg/ml), 94.50% (at 0.5 mg/ml), 92.25% (at 1 mg/ml) and 91.57% (at 2 mg/ml). Thus, in the preliminary cytotoxicity assay, no cytotoxicity (<60% Relative survival [RS is cloning efficiency (CE) of cells plated immediately after treatment adjusted by any loss of cells during treatment as compared with cloning efficiency in negative controls]) was observed for the Test Item at ≥2mg/ml, either in the presence or absence of metabolic activation. Based on the preliminary cytotoxicity assay results, the gene mutation study was conducted with the Test Item concentrations of 0.25, 0.5, 1, and 2 mg/ml, both in the presence (1 % v/v S9 mix) and absence of metabolic activation system along with negative, vehicle and positive controls. In the main study, cultures were exposed to negative, vehicle, Test Item, and positive control for 4 hours (short-term exposure) in the absence and presence of metabolic activation. In the absence of metabolic activation, the relative survival values were 99.43% (vehicle control), 95.93% (at 0.25 mg/ml), 93.65% (at 0.5 mg/ml), 92.80% (at 1 mg/ml) and 91.48% (at 2 mg/ml). In the presence of metabolic activation, the relative survival values were 98.48% (vehicle control), 96.20% (at 0.25 mg/ml), 92.96% (at 0.5 mg/ml), 91.86% (at 1 mg/ml) and 91.21% (at 2 mg/ml). No significant increase in the mutation frequency (MF) either in the absence(7.85x10-6, 7.04 x10-6, 8.35 x10-6and 9.94 x10-6 at 0.25 mg/ml, 0.5 mg/ml, 1 mg/ml and 2 mg/ml, respectively) or presence of metabolic activation (7.63 x10-6, 8.02x10-6, 9.35x10-6and 9.67x10-6 at 0.25 mg/ml, 0.5 mg/ml, 1 mg/ml and 2 mg/ml, respectively) was observed when compared to vehicle control (6.61 x10-6, 6.51 x 10-6, absence and presence, respectively). There was no significant reduction in the relative survival (cytotoxicity), and no increase in the mutation frequency was observed in vehicle control (dimethyl sulfoxide) either in the presence or absence of metabolic activation. The positive controls used in the study produced statistically significant increases in mutation frequency, indicating the sensitivity of the test system to specific mutagens and confirming that the test conditions were appropriate and that the metabolic activation system functioned properly. Thus the results indicated that the Test Item, N-tert-butylacrylamide (CAS No. 107-58-4), did not induce a statistically significant or biologically relevant increase in the mutation frequency at concentrations of 0.25, 0.5, 1, and 2 mg/ml when compared to the vehicle control data neither in the present nor in the absence of S9 metabolic activation.
Based on the results of this study, it is concluded that N-tert-butylacrylamide (CAS No. 107-58-4)did not induce gene mutation in the hypoxanthine-guanine phosphoribosyl transferase (Hprt)CHO cells up to the concentration of 2 mg/ml of culture medium, either in the presence or absence of S9 metabolic activation system, under the experimental conditions described
Justification for classification or non-classification
The mutagenic nature of the test chemical was thoughtfully investigated by a combination of in vitro genotoxicity tests: bacterial reverse mutation test (OECD 471), in vitro mammalian cell chromosome aberration test (OECD 473) and in vitro gene mutation tests in mammalian cells (OECD 476). Conclusive evidence from these in vitro genotoxicity studies summarized above indicated that N-tert-butylacrylamide (CAS No. 107-58-4) did not induce alterations in genetic material such as gene and chromosome mutations, structural chromosome aberrations in vitro in somatic cells and, consequently the test chemical is Not classified for Germ cell mutagenicity according to the criteria of Regulation (EC) No 1272/2008 on the classification, labelling and packaging of substances and mixtures (CLP Regulation).
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