Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames assay:

Bacterial gene mutation assay was performed to determine the mutagenic nature of the test chemical. The study was performed using Salmonella strains TA1535, TA1537, TA1538, TA98 and TA100 in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in DMSO and used at dosel levels of 0.5 to 5000µg/plate. All tests were done on duplicate plates, both with and without adding 0.5 ml S9 mix containing 0.05 ml S9 and cofactors, in the plate incorporation test and liquid preincubation test at 37°C for 20 min with shaking. The plates were counted manually after incubation in an incubator at 37°C for 48 h. When no colonies were visible in the plates after incubation with the chemicals, it was determined to be due to the toxicity of the chemicals for the test strains. Based on the observations made, the test chemical did not induce gene mutation in Salmonella strains TA1535, TA1537, TA1538, TA98 and TA100 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation. was found to be non mutagenic in the standard Ames assay . 

Chromosome aberration assay:

This study was conducted to determine the chromosomal aberration induction potential of the test chemical in human peripheral blood lymphocyte cultures. The methods followed were as per OECD guideline No. 473. The experiment was conducted using human peripheral blood lymphocytes. Blood was drawn from a healthy volunteer, by venous puncture using heparinised syringe. The experiment was performed both in the presence and in the absence of metabolic activation system after 48 h mitogenic stimulation. The test chemical was dissolved in DMSO and used at dose level of 0.00 (NC), 0.00 (VC), 0.5 (T1), 1.0 (T2) and 2.0 (T3) mg/mL in the presence and absence of S9 metabolic activation system in phase 1 and phase 2. Phase I of experiment was performed by short term treatment method both in the presence and absence of metabolic activation system(1%). Phase II of experiment was performed by short term treatment as well as long term treatment method. 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. 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 metabolic activation system. 3 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 reduction in the mitotic index in comparison with negative control. The medium of the proliferating blood culture was removed by centrifugation at 1500 rpm for 10 minutes. The cells were suspended in plain medium (medium without serum) mixed with S9 mix (Phase I - 1 % and Phase II - 2 % v/v) and in complete media mixed with phosphate buffer for the treatment in presence and in absence of metabolic activation system respectively. A volume of 7.92 mL of proliferating culture was dispensed to individual sterile culture tubes/flasks. Each tube/flask according to treatment groups was identified. Negative control tubes were treated with 80 µL of RPMI media and treatment group were treated with 80 µL of respective test item stock solution. The cultures were incubated at 37 ± 2 °C for duration (exposure period). 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 slides were dried over a slide warmer and labelled. At least two slides was made from each sample. The cells were stained with 5 % fresh Giemsa stain in phosphate buffer and mounted using DPX mountant. Evaluation of the slides was performed using microscopes with 100 x oil immersion objectives. 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.300 well spread metaphase plates per culture were scored for cytogenetic damage on coded slides. Evaluation of the slides was performed 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. 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 under the specified conditions and hence it is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation. 

Mammalian cell gene mutation assay:

Anin vitromammalian cell gene mutation study was designed and conducted to determine the genotoxicity profile of the test chemical when administered to Chinese Hamster Ovary (CHO) cells. In the genotoxicity test, the test chemical was administered to CHO cells for 3 hrs at the dose levels of 0, 0.5, 1.0, 2.5 or 5.0 mM and in the absence or presence of exogenous metabolic activation. CHO cells representing the negative controls were exposed to the vehicle. Positive controls, such asN-ethyl-N-nitrosourea (ENU) experiments without metabolic activation and 7,12-dimethylbenz(a) anthracene in experiments with metabolic activation, were also included in each test. Only the positive control ENU showed indication of gene mutations occurring while no other treatment gave rise to gene toxicity. When the mutation frequency was determined, a frequency of 3.38 x 10-4was shown after a 3 hour exposure of ENU in the absence of S9 liver microsomal fraction. Since no tested concentration of the test chemical, in the absence or presence of S9 liver microsomal fraction, resulted in colonies, it is concluded that the test chemical does not give rise to gene mutations at ≤ 5.0 mM for 3 hrs . Based on the results of the current study, it is concluded that the test chemical does not give rise to gene mutations when exposed to the test chemical at ≤ 5.0 mM for 3 hrs or more, in the presence or abscence of metabolic activation. Based on the data available, the test chemical does not exhibit gene mutaton in vitro. Hence the test chemcal is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
Data is from peer reviewed publication
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
Bacterial reverse mutation assay was performed to determine the mutagenic nature of the test chemical
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine
Species / strain / cell type:
other: TA98, TA100, TA1535, TA1537, and TA1538
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
No data
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S-9 metabolic activation system
Test concentrations with justification for top dose:
0.5 to 5000 μg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was soluble in DMSO
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
0.1 ml DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
9-aminoacridine
other:
Remarks:
All positive controls were dissolved in DMSO except for 9-aminoacridine which was dissolved in ethanol
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 20 mins
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

SELECTION AGENT (mutation assays): No data

SPINDLE INHIBITOR (cytogenetic assays): No data

STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: Duplicate

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: No data

NUMBER OF CELLS EVALUATED: No data

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): No data

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data
- Any supplementary information relevant to cytotoxicity: No data

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- 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:
When no colonies were visible in the plates after incubation with the chemicals, it was determined to be due to the toxicity of the chemicals for the test strains.
Statistics:
No data
Species / strain:
other: TA98, TA100, TA1535, TA1537, and TA1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
No data
Remarks on result:
other: No mutagenic potential
Conclusions:
The test chemical did not induce gene mutation in Salmonella strains TA1535, TA1537, TA1538, TA98 and TA100 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation. was found to be non mutagenic in the standard Ames assay .
Executive summary:

Bacterial gene mutation assay was performed to determine the mutagenic nature of the test chemical. The study was performed using Salmonella strains TA1535, TA1537, TA1538, TA98 and TA100 in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in DMSO and used at dosel levels of 0.5 to 5000µg/plate. All tests were done on duplicate plates, both with and without adding 0.5 ml S9 mix containing 0.05 ml S9 and cofactors, in the plate incorporation test and liquid preincubation test at 37°C for 20 min with shaking. The plates were counted manually after incubation in an incubator at 37°C for 48 h. When no colonies were visible in the plates after incubation with the chemicals, it was determined to be due to the toxicity of the chemicals for the test strains. Based on the observations made, the test chemical did not induce gene mutation in Salmonella strains TA1535, TA1537, TA1538, TA98 and TA100 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation. was found to be non mutagenic in the standard Ames assay . 

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:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Principles of method if other than guideline:
This in vitro assay was performed to assess the potential of the test chemical to induce structural chromosomal aberrations in one experiment (phase I). The induction of cytogenetic damage in human lymphocytes was assessed with and without metabolic activation. Due to the negative result in phase I, a second experiment (phase II) was performed.
GLP compliance:
not specified
Type of assay:
other: In vitro mammalian chromosome aberration assay
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:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
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
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
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

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.

Conclusions:
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 under the specified conditions and hence it is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.
Executive summary:

This study was conducted to determine the chromosomal aberration induction potential of the test chemical in human peripheral blood lymphocyte cultures. The methods followed were as per OECD guideline No. 473. The experiment was conducted using human peripheral blood lymphocytes. Blood was drawn from a healthy volunteer, by venous puncture using heparinised syringe. The experiment was performed both in the presence and in the absence of metabolic activation system after 48 h mitogenic stimulation. The test chemical was dissolved in DMSO and used at dose level of 0.00 (NC), 0.00 (VC), 0.5 (T1), 1.0 (T2) and 2.0 (T3) mg/mL in the presence and absence of S9 metabolic activation system in phase 1 and phase 2. Phase I of experiment was performed by short term treatment method both in the presence and absence of metabolic activation system(1%). Phase II of experiment was performed by short term treatment as well as long term treatment method. 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. 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 metabolic activation system. 3 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 reduction in the mitotic index in comparison with negative control. The medium of the proliferating blood culture was removed by centrifugation at 1500 rpm for 10 minutes. The cells were suspended in plain medium (medium without serum) mixed with S9 mix (Phase I - 1 % and Phase II - 2 % v/v) and in complete media mixed with phosphate buffer for the treatment in presence and in absence of metabolic activation system respectively. A volume of 7.92 mL of proliferating culture was dispensed to individual sterile culture tubes/flasks. Each tube/flask according to treatment groups was identified. Negative control tubes were treated with 80 µL of RPMI media and treatment group were treated with 80 µL of respective test item stock solution. The cultures were incubated at 37 ± 2 °C for duration (exposure period). 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 slides were dried over a slide warmer and labelled. At least two slides was made from each sample. The cells were stained with 5 % fresh Giemsa stain in phosphate buffer and mounted using DPX mountant. Evaluation of the slides was performed using microscopes with 100 x oil immersion objectives. 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.300 well spread metaphase plates per culture were scored for cytogenetic damage on coded slides. Evaluation of the slides was performed 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. 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 under the specified conditions and hence it is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation. 

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:
Data is from study report
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Principles of method if other than guideline:
The purpose of this study was to assess toxic and genotoxic effects of the test chemical on Chinese Hamster Ovary (CHO) cells by using several different in vitro-based assays, including genotoxicity tests based on the OECD Guideline No. 476 “In Vitro Mammalian Cell Gene Mutation Test”.
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
Cells deficient in hypoxanthine-guanine phosphoribosyl transferase (HPRT) due to the mutation HPRT+/- to HPRT-/- are resistant to cytotoxic effects of 6-thioguanine (TG). HPRT proficient cells are sensitive to TG (which causes inhibition of cellular metabolism and halts further cell division since HPRT enzyme activity is important for DNA synthesis), so mutant cells can proliferate in the presence of TG, while normal cells, containing hypoxanthine-guanine phosphoribosyl transferase cannot.

This in vitro test is an assay for the detection of forward gene mutations at the in hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus on the X chromosomes of hypodiploid, modal No. 20, CHO cells. Gene and chromosome mutations are considered as an initial step in the carcinogenic process.
The hypodiploid CHO cells are exposed to the test item with and without exogenous metabolic activation. Following an expression time the descendants of the treated cell population are monitored for the loss of functional HPRT enzyme.
HPRT catalyses the transformation of the purine analogues 6-thioguanine (TG) rendering them cytotoxic to normal cells. Hence, cells with mutations in the HPRT gene cannot phosphoribosylate the analogue and survive treatment with TG.

Therefore, mutated cells are able to proliferate in the presence of TG whereas the non-mutated cells die. However, the mutant phenotype requires a certain period of time before it is completely expressed. The phenotypic expression is achieved by allowing exponential growth of the cells for 7 days.
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: Ham's F-12K (Kaighn's) Medium containing 2 mM L-Glutamine supplemented with 10% Fetal Bovine Serum and 1% Penicillin-Streptomycin (10,000 U/mL).
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Not applicable
- Periodically checked for karyotype stability: Not applicable
Additional strain / cell type characteristics:
other: Hypodiploid, modal No. 20
Metabolic activation:
with and without
Metabolic activation system:
S9 liver microsomal fraction obtained from Arcolor 1254-induced male Sprague-Dawley rats
Test concentrations with justification for top dose:
0, 0.5, 1.0, 2.5 or 5.0 mM
Vehicle / solvent:
Vehicle(s)/solvent(s) used: Ethanol
Justification for choice of solvent/ vehicle: N-tert-butylacrylamide was dissolved in ethanol.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
N-ethyl-N-nitrosourea (ENU) was the positive control substance in the tests done without S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: In medium with pre-incubation

DURATION
- Pre-incubation: One week involving 3 days of incubation with Hypoxanthine-aminopterin-thymidine (HAT) in medium as a mutant cleansing stage, followed by overnight incubation with hypoxanthine-thymidine (HT) in medium prior to a 3-4 days incubation in regular cell medium. After seeding and prior to treatment, the mutant-free cells were incubated for an additional of 24 hours.
- Exposure duration: 3 hours
- Expression time: 7 days
- Selection time: 14 days
- Fixation time: 7 days (harvest of cells)

SELECTION AGENT (mutation assays): 6-thioguanine (TG)
SPINDLE INHIBITOR (cytogenetic assays): Not applicable
STAIN (for cytogenetic assays): Crystal violet

NUMBER OF REPLICATIONS: A minimum of 2 replicates per dose concentration including negative and positive control.

NUMBER OF CELLS EVALUATED: 5 x 10 E5 cells were plated 7 days after treatment and whatever cells left, after 14 days of incubation with the selection medium, were evaluated.

DETERMINATION OF CYTOTOXICITY
- Method: After being exposed to the test chemical for 3 hours, in the absence or presence of S9, cells were trypsinized and 0.5 x 10 E5 cells per well was seeded in duplicates from two parallel duplicate cultures into 6-well plates in fresh medium. The relative total growth and cytotoxicity was evaluated 24 and 48 hours after seeding.

OTHER EXAMINATIONS: Not applicable
- Determination of polyploidy:
- Determination of endoreplication:
- Other:

OTHER:
Rationale for test conditions:
No data
Evaluation criteria:
The cell linse was observed for gene mutation
Statistics:
No data
Species / strain:
Chinese hamster Ovary (CHO)
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:
other: Positive controls valid for study without S9 and not valid for study with S9
Additional information on results:
No data
Remarks on result:
other: No mutagenic potential
Conclusions:
The test chemical in the concentration of 0, 0.5, 1.0, 2.5 or 5.0 mM does not cause genetic mutation(s) when CHO cells are exposed to the test chemical in the presence or abscence of metabolic activation.
Executive summary:

An in vitro mammalian cell gene mutation study was designed and conducted to determine the genotoxicity profile of the test chemical when administered to Chinese Hamster Ovary (CHO) cells.

In the genotoxicity test, the test chemical was administered to CHO cells for 3 hrs at the dose levels of 0, 0.5, 1.0, 2.5 or 5.0 mM and in the absence or presence of exogenous metabolic activation. CHO cells representing the negative controls were exposed to the vehicle. Positive controls, such asN-ethyl-N-nitrosourea (ENU) experiments without metabolic activation and 7,12-dimethylbenz(a) anthracene in experiments with metabolic activation, were also included in each test.

Only the positive control ENU showed indication of gene mutations occurring while no other treatment gave rise to gene toxicity. When the mutation frequency was determined (see Table 1B), a frequency of 3.38 x 10-4was shown after a 3 hour exposure of ENU in the absence of S9 liver microsomal fraction. Since no tested concentration of N-tert-butylacrylamide, in the absence or presence of S9 liver microsomal fraction, resulted in colonies, it is concluded that N-tert-butylacrylamide does not give rise to gene mutations at ≤ 5.0 mM for 3 hrs .

Based on the results of the current study, it is concluded that the test chemical does not give rise to gene mutations when exposed to the test chemical at ≤ 5.0 mM for 3 hrs or more, in the presence or abscence of metabolic activation.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Data available for the test chemical N-tert-butylacrylamide (CAS No. 107-58-4) was reviewed to determine its mutagenic nature. The studies are as mentioned below:

Ames assay:

Bacterial gene mutation assay was performed to determine the mutagenic nature of the test chemical. The study was performed using Salmonella strains TA1535, TA1537, TA1538, TA98 and TA100 in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in DMSO and used at dosel levels of 0.5 to 5000µg/plate. All tests were done on duplicate plates, both with and without adding 0.5 ml S9 mix containing 0.05 ml S9 and cofactors, in the plate incorporation test and liquid preincubation test at 37°C for 20 min with shaking. The plates were counted manually after incubation in an incubator at 37°C for 48 h. When no colonies were visible in the plates after incubation with the chemicals, it was determined to be due to the toxicity of the chemicals for the test strains. Based on the observations made, the test chemical did not induce gene mutation in Salmonella strains TA1535, TA1537, TA1538, TA98 and TA100 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation. was found to be non mutagenic in the standard Ames assay . 

Chromosome aberration assay:

This study was conducted to determine the chromosomal aberration induction potential of the test chemical in human peripheral blood lymphocyte cultures. The methods followed were as per OECD guideline No. 473. The experiment was conducted using human peripheral blood lymphocytes. Blood was drawn from a healthy volunteer, by venous puncture using heparinised syringe. The experiment was performed both in the presence and in the absence of metabolic activation system after 48 h mitogenic stimulation. The test chemical was dissolved in DMSO and used at dose level of 0.00 (NC), 0.00 (VC), 0.5 (T1), 1.0 (T2) and 2.0 (T3) mg/mL in the presence and absence of S9 metabolic activation system in phase 1 and phase 2. Phase I of experiment was performed by short term treatment method both in the presence and absence of metabolic activation system(1%). Phase II of experiment was performed by short term treatment as well as long term treatment method. 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. 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 metabolic activation system. 3 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 reduction in the mitotic index in comparison with negative control. The medium of the proliferating blood culture was removed by centrifugation at 1500 rpm for 10 minutes. The cells were suspended in plain medium (medium without serum) mixed with S9 mix (Phase I - 1 % and Phase II - 2 % v/v) and in complete media mixed with phosphate buffer for the treatment in presence and in absence of metabolic activation system respectively. A volume of 7.92 mL of proliferating culture was dispensed to individual sterile culture tubes/flasks. Each tube/flask according to treatment groups was identified. Negative control tubes were treated with 80 µL of RPMI media and treatment group were treated with 80 µL of respective test item stock solution. The cultures were incubated at 37 ± 2 °C for duration (exposure period). 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 slides were dried over a slide warmer and labelled. At least two slides was made from each sample. The cells were stained with 5 % fresh Giemsa stain in phosphate buffer and mounted using DPX mountant. Evaluation of the slides was performed using microscopes with 100 x oil immersion objectives. 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.300 well spread metaphase plates per culture were scored for cytogenetic damage on coded slides. Evaluation of the slides was performed 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. 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 under the specified conditions and hence it is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation. 

Mammalian cell gene mutation assay:

An in vitro mammalian cell gene mutation study was designed and conducted to determine the genotoxicity profile of the test chemical when administered to Chinese Hamster Ovary (CHO) cells. In the genotoxicity test, the test chemical was administered to CHO cells for 3 hrs at the dose levels of 0, 0.5, 1.0, 2.5 or 5.0 mM and in the absence or presence of exogenous metabolic activation. CHO cells representing the negative controls were exposed to the vehicle. Positive controls, such asN-ethyl-N-nitrosourea (ENU) experiments without metabolic activation and 7,12-dimethylbenz(a) anthracene in experiments with metabolic activation, were also included in each test. Only the positive control ENU showed indication of gene mutations occurring while no other treatment gave rise to gene toxicity. When the mutation frequency was determined, a frequency of 3.38 x 10-4was shown after a 3 hour exposure of ENU in the absence of S9 liver microsomal fraction. Since no tested concentration of the test chemical, in the absence or presence of S9 liver microsomal fraction, resulted in colonies, it is concluded that the test chemical does not give rise to gene mutations at ≤ 5.0 mM for 3 hrs . Based on the results of the current study, it is concluded that the test chemical does not give rise to gene mutations when exposed to the test chemical at ≤ 5.0 mM for 3 hrs or more, in the presence or abscence of metabolic activation. Based on the data available, the test chemical does not exhibit gene mutaton in vitro. Hence the test chemcal is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.

Based on the data available, N-tert-butylacrylamide (CAS No. 107-58-4) does not exhibit gene mutaton in vitro. Hence the test chemcal is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.

Justification for classification or non-classification

Based on the data available, N-tert-butylacrylamide (CAS No. 107-58-4) does not exhibit gene mutaton in vitro. Hence the test chemcal is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.