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EC number: 203-232-2 | CAS number: 104-74-5
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Bacterial reverse mutation test (Ames test):
Based on the results of the present study, it is concluded that N-Dodecylpyridinium Chloride [CAS No.: 104-74-5] is non-mutagenic as it does not induce (point) gene mutations by base-pair changes or frameshift in the histidine operon in any of the five tester strains of Salmonella typhimurium (TA1537, TA1535, TA98, TA100 or TA102) neither in the presence nor in the absence of metabolic activation system.
Chromosome aberration study:
Data available for structurally similar read across chemicals has been reviewed to determine the potential of the test chemical 1-Dodecylpyridinium chloride (CAS No. 104 -74 -5) to cause chromosomal aberration in mammalian cells. The studies are as mentioned below:
1. The ability of the registered substance, Tetrabutylazanium bromide (CAS No. 1643-19-2), to induce chromosome aberration in cultured Chinese Hamster Ovary (CHO) cells was tested according to OECD TG 473 both in the presence and absence of an exogenous metabolic activation system. Cofactor-supplemented S9 microsomal fraction was used as an exogenous metabolic activation system. The S9 fraction was obtained from the liver of phenobarbitone and β-naphthoflavone-induced rats. Distilled water was selected as a vehicle for the test substance. Test concentrations were selected based on the solubility, precipitation and pH checks, as well as a preliminary cytotoxicity test. In the cytotoxicity test, CHO cells were exposed to the test substance at concentrations of 0.0 (vehicle control/distilled water), 0.125, 0.25, 0.5, 1.0 and 2.0 mg/ml, both in the absence and presence of S9 metabolic activation. No cytotoxicity (defined by a Relative Increase in Cell Count [RICC] of ≤40% of the concurrent vehicle control data) was observed for the Test Item at ≤2 mg/ml, either in the presence or absence of metabolic activation. Hence, the following concentrations were employed in the chromosome aberration test: 0.0 (VC), 0.5, 1 and 2 mg/ml. Positive control substances were also included in the test, i.e., Methyl methanesulfonate (20 µg/ml, without S9) and Benzo (a) pyrene (30 µg/ml, with S9). The chromosome aberration test consisted of three phases. In Phase,s I-II, CHO cells (1 × 106 cells/flask ) were treated with test substance formulation and vehicle/ positive controls for 4 hours (short-term treatment) in the presence and absence of S9 metabolic activation which was followed by a 20 hours recovery period. In Phase III, CHO cells (1 × 106 cells/flask) were treated with the test substance and vehicle and positive controls for 24 hours (continuous treatment). The cultures were harvested 24 hours (short term treatment and continuous treatment) after the beginning of treatment. After the treatment period, CHO cells were fixed with Carnoy’s fixative (3:1 methanol: acetic acid solution) and stained with 5 % Giemsa stain. At least 300 well-spread metaphases per concentration (single culture) were analyzed using 100x magnification for the incidence of structural aberrations. Cells with structural chromosomal aberration(s) including and excluding gaps were scored. Chromatid and chromosome-type aberrations were recorded separately and classified by sub-types (breaks, exchanges). The cytotoxicity was assessed by the Relative Increase in Cell Counts (RICC) in all phases. Results: In Phase I, no significant increase in the mean percent aberrant cells at 0.5 mg/ml (the mean % aberrant cells: 0.33%, p=1.0000), 1 mg/ml (the mean % aberrant cells: 0.33%, p=1.0000), 2 mg/ml (the mean % aberrant cells: 0.67%, p=0.4992) was observed when compared to the vehicle control (the mean % aberrant cells 0.00 %). Average RICC values were 100 % (vehicle control), 84.76 % (at 0.5 mg/ml), 82.84 % (at 1 mg/ml) and 78.05 % (at 2 mg/ml). In Phase II experiment, no significant increase in mean percent aberrant cells at 0.5 mg/ml (the mean % aberrant cells: 0.33%, p=1.0000), 1 mg/ml (the mean % aberrant cells: 0.67%, p=1.0000), 2 mg/ml (the mean % aberrant cells: 0.67%, p=1.0000), was observed compared to the vehicle control (the mean % aberrant cells 0.33 %). Average RICC values were 100 % (vehicle control), 84.08 % (at 0.5 mg/ml), 81.57 % (at 1 mg/ml) and 76.74 % (at 2 mg/ml). In Phase III, the average RICC values were 100 % (vehicle control), 85.53 % (at 0.5 mg/ml), 82.13 % (at 1 mg/ml) and 76.11 % (at 2 mg/ml). No significant increase in mean percent aberrant cells at 0.5 mg/ml (the mean % aberrant cells: 0.67%, p=1.0000), 1 mg/ml (the mean % aberrant cells: 0.33%, p=1.0000), 2 mg/ml (the mean % aberrant cells: 0.67%, p=1.0000), was observed when compared to the vehicle control (the mean % aberrant cells 0.33 %). Conclusion: The registered substance, Tetrabutylazanium bromide (CAS No. 1643-19-2), did not induce chromosome aberration in cultured CHO cells up to 2 mg/ml neither in the present nor in the absence of S9 metabolic activation under the experimental conditions described.
2. In vitro mammalian chromosome aberration study was performed to determine the mutagenic nature of the test chemical Methyl nonyl ketone (CAS no. 112 -12 -9). The study was performed using CHO cells in the presence and absence of S9 metabolic activation system. The test chemical was soluble in DMSO and used at dose level of 5, 15 and 25μg/ml. Cells were exposed for a period of 4 hours. Positive control chemicals were also included in the study, Ethylmethane sulphonate and N-nitroso-dimethylamine were used as the postive controls. Cytotoxicity started at a concentration of 25μg/ml. The test substance did not induce chromosomal aberrations in CHO cells in the presence and absence of metabolic activation system.
Based on the above-summarized studies for 1-Dodecylpyridinium chloride (CAS No. 104 -74 -5) and its structurally and functionally similar read across substances, it can be concluded that the test chemical is unable to cause chromosomal aberration in mammalian cells. Comparing the above annotations with the criteria of CLP regulation, the test substance cannot be classified for genetic toxicity.
In Vitro mammalian gene mutation test:
Data available for structurally similar read across chemicals have been reviewed to determine the potential of the test chemical 1-Dodecylpyridinium chloride (CAS No. 104 -74 -5) to cause gene mutations in mammalian cells. The studies are as mentioned below:
1. In a gene toxicity test, Chinese Hamster Ovary (CHO) cells were exposed to the test chemical in the concentration of 0, 0.625, 1.25, 2.5 or 5mM with and without metabolic activation for 3 hours. The results showed that there was no evidence of cytotoxicity after treatment. In the concentrations 1.25 or 2.5mM of Tetrabutylammonium bromide concentration, the results showed evidence of gene toxicity. Therefore, it is considered that the test chemical in the concentration of 1.25 or 2.5 microM may cause genetic mutation(s) when CHO cells are exposed to the test chemical in the absence of metabolic activation.
2. In vitro mammalian cell transformation assay was performed to determine the mutagenic nature of the test chemical. The test chemical was dissolved in DMSO as the solvent and used at dose level of 0, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 20, 50, 100 or 300µg/mL. Pregnant Syrian golden hamsters were killed on days 13 and 14 of gestation for preparation of target cells and feeder-layer cells respectively. Embryos without heads and viscera were minced with scissors,a nd trypsinized with 0.25% trypsin. Inocula of 10000000 embryo cells per 75-cm’ flask were incubated at 37°C in a humidified atmosphere of 10% CO2 in air. When they became confluent primary cultures were trypsinized, dispensed in lots of 5000000 cells in glass ampules, and stored in liquid nitrogen for use as target and feeder-layer cells. The assay takes 15 days from start to finish. On Day 0, an ampule of cryopreserved primary cells prepared as feeder-layer cells was rapidly thawed and plated in a 75-cm2 flask containing 20 ml of the culture medium. The medium was changed every day. On day 3, an ampule of cryopreserved primary cells prepared as target cells was also rapidly thawed and plated in a 75-cm2 flask. On day 4, the feeder cells which were shifting from a stage of logarithmic growth to a stationary phase were irradiated with 5000 R from a linear accelerator, trypsinized, and then plated at 6 x lo4 tells/60-mm dish in 2 ml of complete medium. On day 5, the target cells which were approximately 80 -90% confluent were trypsinized, and a suspension of 500 target cells in 2 ml of complete medium was then added to each of the dishes plated the day before with irradiated feeder-layer cells. On day 6, an appropriate dose of the test chemical in a volume of 4 ml was added, giving a total volume of 8 ml of medium in each dish. Nine dishes were used for each dose level in all the experiments (in a few cases only eight or seven dishes were used for controls). On day 14, the cultures werefixed with absolute methanol for 10min and stained with Giemsa solution for 45 min or more. The stained dishes were examined with a stereoscopic dissection microscope to count normal and transformed colonies. The exposure duration was 8 days. Concurrent solvent and negative control chemicals were also included in the study. The test chemical did not induce cell transformation in the Pregnant Syrian golden hamsters embryo cell line used and hence it is not likely to classify as a gene mutant in vitro.
Based on the above-summarized studies for 1-Dodecylpyridinium chloride (CAS No. 104 -74 -5) and its structurally and functionally similar read across substances, it can be concluded that the test chemical is unable to cause gene mutation in mammalian cells. Comparing the above annotations with the criteria of CLP regulation, the test substance cannot be classified for genetic toxicity.
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 information is from experimental study report and it is as per OECD Gudeline No. 471
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- OECD 471 (Adopted 21st July 1997, Corrected: 26th June 2020)
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: N1910594
- Manufacturing data of the lot/batch: 09.04.2019
- Expiration date of the lot/batch: 07.04.2024
- Purity: 94.24%
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: The test item was stored at room temperature. (20-30 0C)
- Stability under storage conditions: The test item was stable under storage condition.
- Stability under test conditions: the test chemical was stable under test condition.
OTHER SPECIFICS
- measurement of pH, osmolality, and precipitate in the culture medium to which the test chemical is added:
- other information: - Target gene:
- Histidine Operon
- 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:
- Type and composition of metabolic activation system:
- source of S9 : A combination of phenobarbitone and β-naphthoflavone-induced rat liver microsomal enzymes (S9 homogenate) prepared in-house.
- method of preparation of S9 mix : S9 mix [Cofactors (Cofactor ingredients: D-glucose-6-phosphate, β-NADP, magnesium chloride, potassium chloride, sodium phosphate and Liver homogenate] was prepared prior to use in the study. The post-mitochondrial fraction (liver homogenate) was used at the concentration of 10 % (v/v) in the S9 mix.
- quality controls of S9: enzymatic activity - Test concentrations with justification for top dose:
- In both Trial I and Trial II, the following five concentrations of the Test Item were tested in triplicate plates along with the vehicle and positive controls.
0.00122, 0.00244, 0.00488, 0.0097656 and 0.01953125 mg/plate.
Justification: A slight reduction in the number of revertant colonies accompanied by a moderate inhibition of the background lawn growth was observed at 0.01953125 mg/plate, both in the presence (10% v/v S9 mix) and absence of metabolic activation compared to vehicle control data. No reduction in the number of revertant colonies or inhibition of the background lawn growth was observed at ≤ 0.097656 mg/plate, either in the presence (10% v/v S9 mix) or in the absence of metabolic activation when compared to the vehicle control data. Hence, considering the criteria that the highest test concentration should induce cytotoxicity determined by a decrease in the revertant colony count and/or diminution of the background lawn or precipitation formation in the final mixture under the actual test condition and evident to the unaided eye, 0.01953125 mg/plate was selected as the highest concentration of the Test Item for the main assay. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Distilled water
- Justification for choice of solvent/vehicle: The test Item was found to be soluble in distilled water (50 mg/ml). Hence, distilled water was selected as the vehicle for the study.
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- distilled water
- 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:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration: (triplicate)
- Number of independent experiments : Two
METHOD OF TREATMENT/ EXPOSURE:
- Cell density: Fresh bacterial cultures were prepared, which were in the late-log phase of growth at the time of use. The densities of the cultures were confirmed to be 1 to 2 ×109 bacteria/ml using a haemocytometer before the cultures were used in the test.
- Test substance added in medium; in agar (plate incorporation); preincubation
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition
- Evaluation criteria:
- The Test Item was deemed mutagenic 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 was observed.
A dose-dependent increase was considered biologically relevant if the threshold is was exceeded at more than one concentration.
A dose-dependent increase exceeding the threshold at only one concentration was judged as biologically relevant if reproduced in an independent experiment.
A dose-dependent increase in the number of revertant colonies below the threshold indicated a mutagenic potential if reproduced in an independent experiment. - Statistics:
- 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 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 102
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- The results of Trial I (Plate incorporation assay) and Trial II (Preincubation assay) showed no significant increases in the mean number of revertant colonies of bacterial tester strains in the presence (10 % v/v S9) or absence of the metabolic activation system. Furthermore, no trend of an increased number of revertant colonies with increased dosing of the Test Item was observed. The vehicle and the strain-specific positive control values were within the lab historical control ranges, indicating that the test conditions were appropriate, and that the metabolic activation system functioned properly.
- Remarks on result:
- other: non mutagenic
- Conclusions:
- Based on the results of the present study, it is concluded that N-Dodecylpyridinium Chloride [CAS No.: 104-74-5] is non-mutagenic as it does not induce (point) gene mutations by base-pair changes or frameshift in the histidine operon in any of the five tester strains of Salmonella typhimurium (TA1537, TA1535, TA98, TA100 or TA102) neither in the presence nor in the absence of metabolic activation system.
- Executive summary:
The mutagenicity of the test substnace, N-Dodecylpyridinium Chloride [CAS No.: 104-74-5] has been tested in Salmonella typhimurium tester strains (TA98, TA100, TA1535, TA1537, TA1538). The test was performed in two trials. Trial I was performed according to the plate incorporation method and both in the presence and absence of liver microsomal activation (S9 mix).Trial II was performed according to the preincubation method and both in the presence and absence of liver microsomal activation (S9 mix) The mammalian microsomal fraction (S9 mix) was prepared in-house from the combination of phenobarbitone and β-naphthoflavone-induced rat liver microsomal enzymes (S9 homogenate). The chemical was tested at concentrations 0.00122, 0.00244, 0.00488, 0.0097656 and 0.01953125 mg/plateboth in the presence (10 % v/v S9 mix) and absence of metabolic activation, along with the vehicle and positive controls. The maximal dose was selected based on the cytotoxicity observed in an initial dose range-finding study. The criteria used to evaluate a test were as follows: for a test chemical to be considered positive, it had to induce at least a doubling (TA98, TA100, and TA1535) and tripling (TA1537, TA1538) in the mean number of revertants per plate of at least one tester strain. This increase in the mean revertants per plate had to be accompanied by a dose-response to increasing concentrations of the test chemical. If a dose-response with a less than 3-fold increase on TA1537 was observed, the response had to be confirmed in a repeat experiment. The test substance N-Dodecylpyridinium Chloride [CAS No.: 104-74-5] failed to produce double- or triple-fold in his+revertant counts up to 0.01953125 mg/plate, either in the presence (10 % v/v S9 mix) or absence of metabolic activation when compared to the vehicle control data for both the experiment, that is trail I and trial II. The study was performed according to OECD 471 and considered to be reliable.(Klimisch 1)
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Remarks:
- Read across data
- Adequacy of study:
- weight of evidence
- Reliability:
- 1 (reliable without restriction)
- Justification for type of information:
- The study contains experimental data
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)
- Version / remarks:
- Accepted: 29th July 2016
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian chromosome aberration test
- Specific details on test material used for the study:
- Appearance :White crystalline powder
Batch Number: L217961707
Purity: 100% - Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Metabolic activation system:
- Cofactor-supplemented S9 microsomal fraction was used as an exogenous metabolic activation system. The S9 fraction was obtained from the liver of phenobarbitone and β-naphthoflavone-induced rats.
- Test concentrations with justification for top dose:
- Test concentrations:
0.0 (VC), 0.5, 1.0 and 2.0 mg/ml
Justification:
Test concentrations were selected based on a preliminary cytotoxicity test. In this pre-test, CHO cells were exposed to 0.0 (VC), 0.125, 0.25, 0.5, 1.0 and 2.0 mg/ml. No cytotoxicity (defined by a Relative Increase in Cell Count [RICC] of ≤40% of the concurrent vehicle control data) was observed for the Test Item at ≤2 mg/ml, either in the presence or absence of metabolic activation. RICC values were 21.43 (-S9) and 24.14 (+S9). - Vehicle / solvent:
- Distilled water
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Distilled water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- benzo(a)pyrene
- methylmethanesulfonate
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): Single
- Number of independent experiments: Phase I-III
METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 1 × 106 cells/flask
- Test substance added in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk: In medium
TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: NA
- Exposure duration/duration of treatment: Phase I-II: 4hrs, Phase IiI: 24 hrs,
- Harvest time after the end of treatment (sampling/recovery times): Recovery time for Phase I-II: 20 hrs, Harvesting time for Phase I-III: 24 hrs
FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): indicate the identity of mitotic spindle inhibitor used (e.g., colchicine), its concentration and, duration and period of cell exposure: Colchicine, final conc: 1 µg/ml, for 2 hrs
- If cytokinesis blocked method was used for micronucleus assay: indicate the identity of cytokinesis blocking substance (e.g. cytoB), its concentration, and duration and period of cell exposure: NA
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): At harvest time, CHO cells were washed with plain RPMI-1640 medium. Cells were trypsinized with Trypsin EDTA, and the cell suspensions were centrifuged. Cells were resuspended in approximately 5 ml of freshly prepared hypotonic solution (Potassium chloride 0.075 M). The cell suspensions were allowed to stand at room temperature for 10 minutes. After hypotonic treatment, the cultures were centrifuged to discard the supernatant. Carnoy’s fixative (3:1 methanol: acetic acid solution) was added to each tube and incubated at room temperature for 10 minutes. Cells were centrifuged at 1000 rpm for 10 minutes, and the cell suspension was prepared by discarding the supernatant.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): At least 300 well-spread metaphases per concentration (single culture) were analyzed using 100x magnification for the incidence of structural aberrations.
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable):NA
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): Cells with structural chromosomal aberration(s) including and excluding gaps were scored. Chromatid and chromosome-type aberrations were recorded separately and classified by sub-types (breaks, exchanges). As slide preparation procedures often result in the breakage of a proportion of metaphases with loss of chromosomes, cells which that contain the number of centromeres equal to the number 2n ± 2 were scored. In CHO cells, the number of centromeres equal to the modal number is 22 ± 2.
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition; mitotic index (MI); relative population doubling (RPD); relative increase in cell count (RICC); replication index; cytokinesis-block proliferation index; cloning efficiency; relative total growth (RTG); relative survival (RS); other: Cytotoxicity was determined by the Relative Increase in Cell Count (RICC). - Evaluation criteria:
- Acceptance of a test is based on the following criteria:
- The concurrent negative control is considered acceptable for addition to the laboratory historical negative control database.
- The positive controls induce responses that are compatible with the laboratory historical positive control database and produce a statistically significant increase compared with the concurrent negative control.
- Cell proliferation criteria in the solvent control should be fulfilled: Cytotoxicity evaluation of solvent control by Relative Increase in Cell Count both in the presence and absence of metabolic activation. Assessment of Relative Increase in Cell Count of solvent control to ensure the sufficient number of cells reached mitosis during the treatment period. Cytotoxicity evaluation of the solvent control both in the presence and absence of metabolic activation
- All three experimental conditions were tested unless one resulted in positive results.
- An adequate number of cells and concentrations are analysable. - Statistics:
- Statistical analysis was performed to assess a possible dose-dependent increase of aberrant cell frequencies using Fisher’s Exact Test (NCSS statistics software). The percentage of aberrant cells from 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:
- cytotoxicity
- Remarks:
- The cytotoxicity (%) was 78.05, 76.74 and 76.11at 2 mg/ml in the absence and presence of S9 mix (short term incubation) and without S9 mix (continuous incubation), respectively.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- Solubility, precipitation and pH checks: The test substance was found to be soluble in distilled water. No precipitation was observed at the concentration of 2 mg/ml. The pH of the test concentrations in the media was unchanged after 0 and 4 hours incubation.
Cytotoxicity: In the preliminary cytotoxicity assay, no cytotoxicity (defined by a Relative Increase in Cell Count [RICC] of ≤40% of the concurrent vehicle control data) was observed at ≤2 mg/ml, either in the presence or absence of metabolic activation. The cytotoxicity (%) was 78.05, 76.74 and 76.11at 2 mg/ml in the absence and presence of S9 mix (short term incubation) and without S9 mix (continuous incubation), respectively. - Remarks on result:
- other: non-clastogenic
- Conclusions:
- The registered substance, Tetrabutylazanium bromide (CAS No. 1643-19-2) was tested non-clastogenic (negative) up to the concentration of 2 mg/ml in cultured Chinese Hamster Ovary (CHO) cells in the presence and absence of S9 metabolic activation system. The test was performed according to OECD TG 473 and GLP.
- Executive summary:
The ability of the registered substance, Tetrabutylazanium bromide (CAS No. 1643-19-2), to induce chromosome aberration in cultured Chinese Hamster Ovary (CHO) cells was tested according to OECD TG 473 both in the presence and absence of an exogenous metabolic activation system. Cofactor-supplemented S9 microsomal fraction was used as an exogenous metabolic activation system. The S9 fraction was obtained from the liver of phenobarbitone and β-naphthoflavone-induced rats. Distilled water was selected as a vehicle for the test substance. Test concentrations were selected based on the solubility, precipitation and pH checks, as well as a preliminary cytotoxicity test. In the cytotoxicity test, CHO cells were exposed to the test substance at concentrations of 0.0 (vehicle control/distilled water), 0.125, 0.25, 0.5, 1.0 and 2.0 mg/ml, both in the absence and presence of S9 metabolic activation. No cytotoxicity (defined by a Relative Increase in Cell Count [RICC] of ≤40% of the concurrent vehicle control data) was observed for the Test Item at ≤2 mg/ml, either in the presence or absence of metabolic activation. Hence, the following concentrations were employed in the chromosome aberration test: 0.0 (VC), 0.5, 1 and 2 mg/ml. Positive control substances were also included in the test, i.e., Methyl methanesulfonate (20 µg/ml, without S9) and Benzo (a) pyrene (30 µg/ml, with S9). The chromosome aberration test consisted of three phases. In Phase,s I-II, CHO cells (1 × 106 cells/flask ) were treated with test substance formulation and vehicle/ positive controls for 4 hours (short-term treatment) in the presence and absence of S9 metabolic activation which was followed by a 20 hours recovery period. In Phase III, CHO cells (1 × 106 cells/flask) were treated with the test substance and vehicle and positive controls for 24 hours (continuous treatment). The cultures were harvested 24 hours (short term treatment and continuous treatment) after the beginning of treatment. After the treatment period, CHO cells were fixed with Carnoy’s fixative (3:1 methanol: acetic acid solution) and stained with 5 % Giemsa stain. At least 300 well-spread metaphases per concentration (single culture) were analyzed using 100x magnification for the incidence of structural aberrations. Cells with structural chromosomal aberration(s) including and excluding gaps were scored. Chromatid and chromosome-type aberrations were recorded separately and classified by sub-types (breaks, exchanges). The cytotoxicity was assessed by the Relative Increase in Cell Counts (RICC) in all phases. Results: In Phase I, no significant increase in the mean percent aberrant cells at 0.5 mg/ml (the mean % aberrant cells: 0.33%, p=1.0000), 1 mg/ml (the mean % aberrant cells: 0.33%, p=1.0000), 2 mg/ml (the mean % aberrant cells: 0.67%, p=0.4992) was observed when compared to the vehicle control (the mean % aberrant cells 0.00 %). Average RICC values were 100 % (vehicle control), 84.76 % (at 0.5 mg/ml), 82.84 % (at 1 mg/ml) and 78.05 % (at 2 mg/ml). In Phase II experiment, no significant increase in mean percent aberrant cells at 0.5 mg/ml (the mean % aberrant cells: 0.33%, p=1.0000), 1 mg/ml (the mean % aberrant cells: 0.67%, p=1.0000), 2 mg/ml (the mean % aberrant cells: 0.67%, p=1.0000), was observed compared to the vehicle control (the mean % aberrant cells 0.33 %). Average RICC values were 100 % (vehicle control), 84.08 % (at 0.5 mg/ml), 81.57 % (at 1 mg/ml) and 76.74 % (at 2 mg/ml). In Phase III, the average RICC values were 100 % (vehicle control), 85.53 % (at 0.5 mg/ml), 82.13 % (at 1 mg/ml) and 76.11 % (at 2 mg/ml). No significant increase in mean percent aberrant cells at 0.5 mg/ml (the mean % aberrant cells: 0.67%, p=1.0000), 1 mg/ml (the mean % aberrant cells: 0.33%, p=1.0000), 2 mg/ml (the mean % aberrant cells: 0.67%, p=1.0000), was observed when compared to the vehicle control (the mean % aberrant cells 0.33 %). Conclusion: The registered substance, Tetrabutylazanium bromide (CAS No. 1643-19-2), did not induce chromosome aberration in cultured CHO cells up to 2 mg/ml neither in the present nor in the absence of S9 metabolic activation under the experimental conditions described.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Remarks:
- Read across data
- Adequacy of study:
- weight of evidence
- Reliability:
- 4 (not assignable)
- Rationale for reliability incl. deficiencies:
- secondary literature
- Justification for type of information:
- Data is from secondary literature
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)
- GLP compliance:
- not specified
- Type of assay:
- in vitro mammalian chromosome aberration test
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix
- Test concentrations with justification for top dose:
- 5, 15, 25 μg/ml
- Vehicle / solvent:
- DMSO
- Untreated negative controls:
- not specified
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- N-dimethylnitrosamine
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- Not specified
- Evaluation criteria:
- Not specified
- Statistics:
- Not specified
- Key result
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at 25 μg/ml
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not specified
- True negative controls validity:
- not specified
- Positive controls validity:
- valid
- Remarks on result:
- other: Non-mutagenic
- Conclusions:
- The test substance did not induce chromosomal aberrations in CHO cells in the presence and absence of metabolic activation system.
- Executive summary:
In vitro mammalian chromosome aberration study was performed to determine the mutagenic nature of the test chemical Methyl nonyl ketone (CAS no. 112 -12 -9). The study was performed using CHO cells in the presence and absence of S9 metabolic activation system. The test chemical was soluble in DMSO and used at dose level of 5, 15 and 25 μg/ml. Cells were exposed for a period of 4 hours. Positive control chemicals were also included in the study, Ethylmethane sulphonate and N-nitroso-dimethylamine were used as the postive controls. Cytotoxicity started at a concentration of 25 μg/ml. The test substance did not induce chromosomal aberrations in CHO cells in the presence and absence of metabolic activation system.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Remarks:
- Read across data
- Adequacy of study:
- weight of evidence
- Study period:
- 03-06-2014 to 12-03-2015
- 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 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Principles of method if other than guideline:
- Gene mutation toxicity study was performed to determine the mutagenic nature of the test chemica; in the absence of S9 metabolic activation system
- GLP compliance:
- no
- 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):
- - Cell line used: Chinese Hamster Ovary (CHO) cells
- 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
- Cytokinesis block (if used):
- No data
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 liver microsomal fraction obtained from Arcolor 1254-induced male Sprague-Dawley rats (Supplier: Molecular Toxicology Inc. via Trinova Biochem GmbH, Giessen, Germany)
- Test concentrations with justification for top dose:
- 0, 0.625, 1.25, 2.5 or 5 uM
- Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Phosphate-buffered saline (PBS)
Justification for choice of solvent/ vehicle:: Tetrabutylammonium bromide was easily dissolved in PBS. - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: N-ethyl-N-nitrosourea (ENU)
- 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
Cytotoxicity test
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. - Rationale for test conditions:
- No data
- Evaluation criteria:
- Chinese Hamster Ovary Cells (CHO) were observed for gene mutation caused by the test compound
- 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
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No data
- Definition of acceptable cells for analysis: No data
- Other confounding effects: No data
RANGE-FINDING/SCREENING STUDIES: Completed without S9 metabolic activation. A range of test concentrations (0, 0.01, 0.05, 0.1, 0.5, 1, 5, 10 or 20mM) was applied 24 hours after seeding to single cultures in fresh medium in 96-well plates. The cell population (control and treated cells) were assessed 24 and 48 hours after treatment using the colorimetric assay MTTand the BCA assay to assess cell viability and total protein concentration, respectively. From the basis of these results, the test concentrations of the chemical was chosen to be included in the gene toxicity test. Since cytotoxicity was evident at the tested concentration in this preliminary dose-finding test further testing concentrations were adapted to have a maximum test concentration of 5mM.
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: No data
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 test chemical in the concentration of 0, 0.625, 1.25, 2.5 or 5 microM indicated a presence of gene toxicity when CHO cells were exposed to the test chemical.
- Executive summary:
In a gene toxicity test, Chinese Hamster Ovary (CHO) cells were exposed to the test chemical in the concentration of 0, 0.625, 1.25, 2.5 or 5mM with and without metabolic activation for 3 hours. The results showed that there was no evidence of cytotoxicity after treatment. In the concentrations 1.25 or 2.5mM of Tetrabutylammonium bromide concentration, the results showed evidence of gene toxicity. Therefore, it is considered that the test chemical in the concentration of 1.25 or 2.5 microM may cause genetic mutation(s) when CHO cells are exposed to the test chemical in the absence of metabolic activation.
- Endpoint:
- in vitro transformation study in mammalian cells
- Type of information:
- experimental study
- Remarks:
- Read across data
- Adequacy of study:
- supporting 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:
- according to guideline
- Guideline:
- other: Refer below principle
- Principles of method if other than guideline:
- In vitro mammalian cell transformation 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:
- No data
- Species / strain / cell type:
- mammalian cell line, other: In vitro mammalian cell transformation assay
- Details on mammalian cell type (if applicable):
- - Type and identity of media: The standard complete culture medium used was Dulbecco’s modified Eagle medium supplemented with 2 mM+glutamine and 20% foetal bovine serum without any antibiotics. For dissociation of embryos and preparation of subcultures, 0.25% trypsin solution was used; i.e. 10 ml trypsin- EDTA solution (10 x) and 90 ml Ca2+- and Mg 2+-free solution (PBS).
- Properly maintained: No data
- 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:
- not specified
- Metabolic activation system:
- No data
- Test concentrations with justification for top dose:
- - Type and identity of media: The standard complete culture medium used was Dulbecco’s modified Eagle medium supplemented with 2 mM+glutamine and 20% foetal bovine serum without any antibiotics. For dissociation of embryos and preparation of subcultures, 0.25% trypsin solution was used; i.e. 10 ml trypsin- EDTA solution (10 x) and 90 ml Ca2+- and Mg 2+-free solution (PBS).
- Properly maintained: No data
- Periodically checked for Mycoplasma contamination: No data
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: No data - 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:
- Medium
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- other: 3-methylcholanthrene
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Preincubation period: 20 mins
- Exposure duration: 9 days
- Expression time (cells in growth medium): 9 days
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): 10 mins
SELECTION AGENT (mutation assays): No data
SPINDLE INHIBITOR (cytogenetic assays): No data
STAIN (for cytogenetic assays): Giemsa stain
NUMBER OF REPLICATIONS: Nine dishes were used for each dose level in all the experiments (in a few cases only eight or seven dishes were used for controls).
NUMBER OF CELLS EVALUATED: No data
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data
OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Other: No data
OTHER: No data - Rationale for test conditions:
- No data
- Evaluation criteria:
- Criteria for transformation: Randomly oriented three-dimensional growth with extensive crossing over of the cells at the periphery of the colony was considered to be the endpoint of morphological transformation. The centres of transformed colonies usually exhibit dense piling-up of cells. Moreover, these cells usually have an increased ratio of nucleus to cytoplasm, are more basophilic, and are variable in size.
- Statistics:
- No data
- Species / strain:
- mammalian cell line, other: Pregnant Syrian golden hamsters embryo cell line
- Metabolic activation:
- not specified
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- No data
- Remarks on result:
- other: No mutagenic potential
- Conclusions:
- The test chemical did not induce cell transformation in the Pregnant Syrian golden hamsters embryo cell line used and hence it is not likely to classify as a gene mutant in vitro.
- Executive summary:
In vitro mammalian cell transformation assay was performed to determine the mutagenic nature of the test chemical. The test chemical was dissolved in DMSO as the solvent and used at dose level of 0, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 20, 50, 100 or 300µg/mL. Pregnant Syrian golden hamsters were killed on days 13 and 14 of gestation for preparation of target cells and feeder-layer cells respectively. Embryos without heads and viscera were minced with scissors,a nd trypsinized with 0.25% trypsin. Inocula of 10000000 embryo cells per 75-cm’ flask were incubated at 37°C in a humidified atmosphere of 10% CO2 in air. When they became confluent primary cultures were trypsinized, dispensed in lots of 5000000 cells in glass ampules, and stored in liquid nitrogen for use as target and feeder-layer cells. The assay takes 15 days from start to finish. On Day 0, an ampule of cryopreserved primary cells prepared as feeder-layer cells was rapidly thawed and plated in a 75-cm2 flask containing 20 ml of the culture medium. The medium was changed every day. On day 3, an ampule of cryopreserved primary cells prepared as target cells was also rapidly thawed and plated in a 75-cm2 flask. On day 4, the feeder cells which were shifting from a stage of logarithmic growth to a stationary phase were irradiated with 5000 R from a linear accelerator, trypsinized, and then plated at 6 x lo4 tells/60-mm dish in 2 ml of complete medium. On day 5, the target cells which were approximately 80 -90% confluent were trypsinized, and a suspension of 500 target cells in 2 ml of complete medium was then added to each of the dishes plated the day before with irradiated feeder-layer cells. On day 6, an appropriate dose of the test chemical in a volume of 4 ml was added, giving a total volume of 8 ml of medium in each dish. Nine dishes were used for each dose level in all the experiments (in a few cases only eight or seven dishes were used for controls). On day 14, the cultures werefixed with absolute methanol for 10min and stained with Giemsa solution for 45 min or more. The stained dishes were examined with a stereoscopic dissection microscope to count normal and transformed colonies. The exposure duration was 8 days. Concurrent solvent and negative control chemicals were also included in the study. The test chemical did not induce cell transformation in the Pregnant Syrian golden hamsters embryo cell line used and hence it is not likely to classify as a gene mutant in vitro.
Referenceopen allclose all
Table1 Mean Revertant Colony Count – Preliminary Cytotoxicity Assay-I
Test Item Concentration (mg/plate) |
TA 98 |
TA 100 |
||||||
- S9 |
+ S9 |
- S9 |
+ S9 |
|||||
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
|
VC (Distilled water) |
19 (NI) |
0.58 |
21 (NI) |
3.00 |
97 (NI) |
3.06 |
100 (NI) |
3.06 |
T1 (0.0390625) |
5 (CI) |
1.53 |
4 (CI) |
0.58 |
19 (CI) |
2.65 |
17 (CI) |
1.15 |
T2 (0.078125) |
0 (CI) |
0.00 |
0 (CI) |
0.00 |
0 (CI) |
0.00 |
0 (CI) |
0.00 |
T3 (0.15625) |
0 (CI) |
0.00 |
0 (CI) |
0.00 |
0 (CI) |
0.00 |
0 (CI) |
0.00 |
T4 (0.3125) |
0 (CI) |
0.00 |
0 (CI) |
0.00 |
0 (CI) |
0.00 |
0 (CI) |
0.00 |
T5 (0.625) |
0 (CI) |
0.00 |
0 (CI) |
0.00 |
0 (CI) |
0.00 |
0 (CI) |
0.00 |
T6 (1.25) |
0 (CI) |
0.00 |
0 (CI) |
0.00 |
0 (CI) |
0.00 |
0 (CI) |
0.00 |
T7 (2.5) |
0 (CI) |
0.00 |
0 (CI) |
0.00 |
0 (CI) |
0.00 |
0 (CI) |
0.00 |
T8 (5.0) |
0 (CI) |
0.00 |
0(CI) |
0.00 |
0 (CI) |
0.00 |
0 (CI) |
0.00 |
PC |
330 (NI) |
10.60 |
339 (NI) |
7.55 |
703 (NI) |
12.86 |
732 (NI) |
8.50 |
Key: VC = Vehicle control, PC = Positive Control, -S9 = Absence of metabolic activation, +S9 = Presence of metabolic activation, SD = Standard Deviation, T1-T8 = Test Item concentration from lower to higher, NI = No Inhibition, CI = Complete inhibition, MI = Moderate inhibition.
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) |
Table2 Mean Revertant Colony Count – Preliminary Cytotoxicity Assay-II
Test Item Concentration (mg/plate) |
TA 98 |
TA 100 |
||||||
- S9 |
+ S9 |
- S9 |
+ S9 |
|||||
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
|
VC (Distilled water) |
20 (NI) |
2.89 |
19 (NI) |
1.15 |
93 (NI) |
3.21 |
93 (NI) |
3.21 |
T1 (0.000) |
21 (NI) |
2.00 |
21 (NI) |
2.52 |
92 (NI) |
3.61 |
93 (NI) |
5.13 |
T2 (0.00030) |
19 (NI) |
1.15 |
18 (NI) |
0.58 |
93 (NI) |
3.79 |
95 (NI) |
3.51 |
T3 (0.00061) |
19 (NI) |
2.31 |
19 (NI) |
1.15 |
94 (NI) |
4.51 |
96 (NI) |
3.06 |
T4 (0.00122) |
21 (NI) |
2.52 |
20 (NI) |
2.00 |
94 (NI) |
1.00 |
92 (NI) |
5.57 |
T5 (0.00244) |
17 (NI) |
1.53 |
21 (NI) |
2.00 |
93 (NI) |
4.16 |
90 (NI) |
3.06 |
T6 (0.00488) |
19 (NI) |
1.15 |
18 (NI) |
0.58 |
92 (NI) |
2.65 |
87 (NI) |
7.55 |
T7 (0.0097656) |
19 (NI) |
2.31 |
19 (NI) |
1.73 |
84 (NI) |
2.52 |
85 (NI) |
3.51 |
T8 (0.01953125) |
12 (MI) |
2.08 |
12 (MI) |
0.58 |
62 (MI) |
3.51 |
55 (MI) |
4.00 |
PC |
319 (NI) |
11.15 |
315 (NI) |
7.00 |
673 (NI) |
22.85 |
665 (NI) |
8.33 |
Key: VC = Vehicle control, PC = Positive Control, -S9 = Absence of metabolic activation, +S9 = Presence of metabolic activation, SD = Standard Deviation, T1-T8 = Test Item concentration from lower to higher, NI = No Inhibition, MI = Moderate inhibition.
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) |
Table3 Mean Revertant Colony Count in Trial I(Plate Incorporation Method)
Absence of metabolic activation (-S9) |
Presence of metabolic activation (+S9 10 % v/v S9 Mix) |
|||||||||||
Test Item Concentration (mg/plate) |
TA 1535 |
TA 1537 |
TA 102 |
TA 1535 |
TA 1537 |
TA 102 |
||||||
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
|
VC (Distilled water) |
13 |
1.73 |
6 |
0.00 |
234 |
4.73 |
13 |
2.00 |
6 |
1.15 |
229 |
4.93 |
T1 (0.00122) |
13 |
2.00 |
6 |
1.15 |
223 |
4.73 |
12 |
0.58 |
7 |
1.15 |
228 |
9.54 |
T2 (0.00244) |
13 |
0.58 |
7 |
1.15 |
227 |
9.07 |
14 |
1.53 |
6 |
0.58 |
233 |
6.43 |
T3 (0.00488) |
12 |
1.53 |
7 |
1.73 |
227 |
9.07 |
13 |
0.58 |
6 |
1.73 |
228 |
8.19 |
T4 (0.0097656) |
12 |
1.73 |
5 |
1.15 |
227 |
8.54 |
12 |
1.73 |
5 |
0.58 |
219 |
6.51 |
T5 (0.01953125) |
10 |
1.15 |
2 |
1.15 |
207 |
9.54 |
9 |
1.15 |
3 |
1.15 |
189 |
5.13 |
PC |
326 |
9.07 |
191 |
5.13 |
1668 |
21.93 |
316 |
4.16 |
205 |
7.37 |
1690 |
16.80 |
Key: VC = Vehicle control, PC = Positive Control, -S9 = Absence of metabolic activation, +S9 = Presence of metabolic activation, SD = Standard Deviation, T1-T5 = Test Item concentration from lower to higher.
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) |
Table 4 Mean Revertant Colony Count in Trial II (Preincubation Method)
Absence of metabolic activation |
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 |
TA 98 |
TA 100 |
TA 1535 |
TA 1537 |
TA 102 |
||||||||||
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
|
VC (Distilled water) |
20 |
2.89 |
93 |
3.21 |
13 |
1.73 |
6 |
0.00 |
234 |
4.73 |
19 |
1.15 |
93 |
3.21 |
13 |
2.00 |
6 |
1.15 |
229 |
4.93 |
T1 (0.00122) |
21 |
2.52 |
94 |
1.00 |
13 |
2.00 |
6 |
1.15 |
223 |
4.73 |
20 |
2.00 |
92 |
5.57 |
12 |
0.58 |
7 |
1.15 |
228 |
9.54 |
T2 (0.00244) |
17 |
1.53 |
93 |
4.16 |
13 |
0.58 |
7 |
1.15 |
227 |
9.07 |
21 |
2.00 |
90 |
3.06 |
14 |
1.53 |
6 |
0.58 |
233 |
6.43 |
T3 (0.00488) |
19 |
1.15 |
92 |
2.65 |
12 |
1.53 |
7 |
1.73 |
227 |
9.07 |
18 |
0.58 |
87 |
7.55 |
13 |
0.58 |
6 |
1.73 |
228 |
8.19 |
T4 (0.0097656) |
19 |
2.31 |
84 |
2.52 |
12 |
1.73 |
5 |
1.15 |
227 |
8.54 |
19 |
1.73 |
85 |
3.51 |
12 |
1.73 |
5 |
0.58 |
219 |
6.51 |
T5 (0.01953125) |
12 |
2.08 |
62 |
3.51 |
10 |
1.15 |
2 |
1.15 |
207 |
9.54 |
12 |
0.58 |
55 |
4.00 |
9 |
1.15 |
3 |
1.15 |
189 |
5.13 |
PC |
319 |
11.15 |
673 |
22.85 |
326 |
9.07 |
191 |
5.13 |
1668 |
21.93 |
315 |
7.00 |
665 |
8.33 |
316 |
4.16 |
205 |
7.37 |
1690 |
16.80 |
Key: VC = Vehicle control, PC = Positive Control, -S9 = Absence of metabolic activation, +S9 = Presence of metabolic activation, SD = Standard Deviation,T1-T5 = Test Item concentration from lower to higher.
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) |
Table 5 Fold Increase
Trial I - Plate Incorporation Method |
Trial II – Preincubation Method |
|||||||||||||||||||
Test Item Concentration (mg/plate) |
TA 98 |
TA 100 |
TA 1535 |
TA 1537 |
TA 102 |
TA 98 |
TA 100 |
TA 1535 |
TA 1537 |
TA 102 |
||||||||||
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
|
VC (Distilled water) |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
T1 (0.00122) |
1.05 |
1.07 |
1.01 |
0.99 |
1.00 |
0.95 |
0.94 |
1.18 |
0.95 |
0.99 |
1.05 |
1.07 |
1.01 |
0.99 |
1.00 |
0.95 |
0.94 |
1.18 |
0.95 |
0.99 |
T2 (0.00244) |
0.88 |
1.13 |
1.00 |
0.97 |
0.97 |
1.05 |
1.11 |
1.12 |
0.97 |
1.02 |
0.88 |
1.13 |
1.00 |
0.97 |
0.97 |
1.05 |
1.11 |
1.12 |
0.97 |
1.02 |
T3 (0.00488) |
0.95 |
0.95 |
0.99 |
0.94 |
0.90 |
0.97 |
1.17 |
1.06 |
0.97 |
0.99 |
0.95 |
0.95 |
0.99 |
0.94 |
0.90 |
0.97 |
1.17 |
1.06 |
0.97 |
0.99 |
T4 (0.0097656) |
0.98 |
1.02 |
0.90 |
0.91 |
0.92 |
0.92 |
0.89 |
0.94 |
0.97 |
0.96 |
0.98 |
1.02 |
0.90 |
0.91 |
0.92 |
0.92 |
0.89 |
0.94 |
0.97 |
0.96 |
T5 (0.01953125) |
0.59 |
0.63 |
0.67 |
0.59 |
0.74 |
0.67 |
0.39 |
0.59 |
0.88 |
0.82 |
0.59 |
0.63 |
0.67 |
0.59 |
0.74 |
0.67 |
0.39 |
0.59 |
0.88 |
0.82 |
PC |
16.24 |
16.88 |
7.27 |
7.17 |
25.05 |
24.33 |
31.89 |
36.24 |
7.12 |
7.37 |
16.24 |
16.88 |
7.27 |
7.17 |
25.05 |
24.33 |
31.89 |
36.24 |
7.12 |
7.37 |
Key: VC = Vehicle control, PC = Positive Control, -S9 = Absence of metabolic activation, +S9 = Presence of metabolic activation, T1-T5 = Test Item concentration from lower to higher.
Table 6 S9 Efficacy Check- Summary
Summary of S9 efficacy check |
||||
|
TA100 |
TA1535 |
||
Mean |
SD |
Mean |
SD |
|
VC Distilled water (-S9) |
97 |
3.06 |
13 |
1.73 |
VC Distilled water (+S9) |
100 |
3.06 |
13 |
2.00 |
PC Benzo[a]pyrene (-S9) |
94 |
2.52 |
15 |
1.15 |
PC Benzo[a]pyrene (+S9) |
732 |
8.50 |
316 |
4.16 |
Key: VC = Vehicle control, PC = Positive control, -S9 = Absence of metabolic activation, +S9 = Presence of metabolic activation, SD = Standard Deviation.
Appendix 1: Relative Increase in Cell Counts – Preliminary Cytotoxicity Assay
Dose Level |
Conc. (mg/ml) |
Absence of Metabolic activation |
Presence of Metabolic activation |
||||||
Cell count |
RICC |
% Cytotoxicity |
Cell count |
RICC |
% Cytotoxicity |
||||
Starting |
Final |
Starting |
Final |
||||||
VC |
- |
1000000 |
3240000 |
100.00 |
0.00 |
1000000 |
3220000 |
100.00 |
0.00 |
T1 |
0.125 |
1000000 |
3160000 |
96.43 |
3.57 |
1000000 |
3084000 |
93.87 |
6.13 |
T2 |
0.25 |
1000000 |
2980000 |
88.39 |
11.61 |
1000000 |
2986000 |
89.46 |
10.54 |
T3 |
0.5 |
1000000 |
2968000 |
87.86 |
12.14 |
1000000 |
2846000 |
83.15 |
16.85 |
T4 |
1 |
1000000 |
2846000 |
82.41 |
17.59 |
1000000 |
2824000 |
82.16 |
17.84 |
T5 |
2 |
1000000 |
2760000 |
78.57 |
21.43 |
1000000 |
2684000 |
75.86 |
24.14 |
Appendix 2: Relative Increase in Cell Counts- Main Study
Dose Level |
Conc.
|
Phase I -Absence of Metabolic activation |
|||
Cell count |
RICC |
% Cytotoxicity |
|||
Starting |
Final |
||||
VC |
- |
1000000 |
3342000 |
100.00 |
0.00 |
T1 |
0.5 mg/ml |
1000000 |
2985000 |
84.76 |
15.24 |
T2 |
1 mg/ml |
1000000 |
2940000 |
82.84 |
17.16 |
T3 |
2 mg/ml |
1000000 |
2828000 |
78.05 |
21.95 |
PC |
20 µg/ml |
1000000 |
2568000 |
66.95 |
33.05 |
Dose Level |
Conc. |
Phase II -Presence of Metabolic activation |
|||
Cell count |
RICC |
% Cytotoxicity |
|||
Starting |
Final |
||||
VC |
- |
1000000 |
3236000 |
100.00 |
0.00 |
T1 |
0.5 mg/ml |
1000000 |
2880000 |
84.08 |
15.92 |
T2 |
1 mg/ml |
1000000 |
2824000 |
81.57 |
18.43 |
T3 |
2 mg/ml |
1000000 |
2716000 |
76.74 |
23.26 |
PC |
30 µg/ml |
1000000 |
2545000 |
69.10 |
30.90 |
Dose Level |
Conc. |
Phase III -Absence of Metabolic activation |
|||
Cell count |
RICC |
% Cytotoxicity |
|||
Starting |
Final |
||||
VC |
- |
1000000 |
3294000 |
100.00 |
0.00 |
T1 |
0.5 mg/ml |
1000000 |
2962000 |
85.53 |
14.47 |
T2 |
1 mg/ml |
1000000 |
2884000 |
82.13 |
17.87 |
T3 |
2 mg/ml |
1000000 |
2746000 |
76.11 |
23.89 |
PC |
20 µg/ml |
1000000 |
2532000 |
66.78 |
33.22 |
Appendix 3: Individual Data on Chromosome Aberrations- Phase I: Absence of metabolic activation (short term)
Dose level & Concentration |
No. of Metaphases |
Frequencies of Aberration |
Total No of Aberrant cells |
|
with gap |
without gap |
|||
VC 0 mg/ml |
300 |
1 Ctg |
1 |
0 |
T1 0.5 mg/ml |
300 |
1 Ctb, 2 Ctg |
3 |
1 |
T2 1 mg/ml |
300 |
1 dic |
1 |
1 |
T3 2 mg/ml |
300 |
2 dic |
2 |
2 |
PC 20 µg/ml |
300 |
1 Ctg, 11 Ctb, 2 fragments, 1 ring, 6 dic, 2 minute |
21 |
20 |
Appendix 4: Individual Data on Chromosome Aberrations- Phase II:Presence of metabolic activation (short term)
Dose level & Concentration |
No. of Metaphases |
Frequencies of Aberration |
Total No of Aberrant cells |
|
with gap |
without gap |
|||
VC 0 mg/ml |
300 |
1 dic |
1 |
1 |
T1 0.5 mg/ml |
300 |
1 Ctg, 1 dic |
2 |
1 |
T2 1 mg/ml |
300 |
1 Ctb, 1Csb, 1 ring |
2 |
2 |
T3 2 mg/ml |
300 |
1 Csg, 1Ctb, 1fragment |
2 |
2 |
PC 30 µg/ml |
300 |
1 Ctg,3 Csg, 5 Ctb,3 Csb, 3 fragment, 1 ring, 1 exchange, 10 dic |
21 |
20 |
Appendix 5: Individual Data on Chromosome Aberrations- Phase III: Absence of metabolic activation (Continuous)
Dose level & Concentration |
No. of Metaphases |
Frequencies of Aberration |
Total No of Aberrant cells |
|
with gap |
without gap |
|||
VC 0 mg/ml |
300 |
1 Ctb |
1 |
1 |
T1 0.5 mg/ml |
300 |
1 fragment, 1 Csb |
2 |
2 |
T2 1 mg/ml |
300 |
1 dic |
1 |
1 |
T3 2 mg/ml |
300 |
1 Csg, 1 exchange |
2 |
2 |
PC 20 µg/ml |
300 |
11 Ctb, 3 fragements, 2 rings, 9 dic, 3 minute |
25 |
25 |
Key: VC = Vehicle Control (distilled water), PC = Positive Control (methyl methanesulfonate), mg = milligram, µg = microgram, ml = milliliter, T3-T1 = Test Item concentration from higher to lower, Ctg = Chromatid gap, Csg = Chromosome gap, Ctb = Chromatid break, Csb = Chromosome break, dic = dicentric.
Appendix 6: Summary Dataon Chromosome Aberrations - Phase I
Dose Level |
Concentration
|
Absence of metabolic activation |
||
Total No. of Aberrant cells without gap |
Percent aberrant cells |
|
||
VC |
- |
0 |
0.00 |
|
T1 |
0.5 mg/ml |
1 |
0.33 |
|
T2 |
1 mg/ml |
1 |
0.33 |
|
T3 |
2 mg/ml |
2 |
0.67 |
|
PC |
20 µg/ml* |
20 |
6.67 |
|
Appendix 7: Summary Data on Chromosome Aberrations - Phase II
Dose Level |
Concentration
|
Presence of metabolic activation |
||
Total No. of Aberrant cells without gap |
Percent aberrant cells |
|
||
VC |
- |
1 |
0.33 |
|
T1 |
0.5 mg/ml |
1 |
0.33 |
|
T2 |
1 mg/ml |
2 |
0.67 |
|
T3 |
2 mg/ml |
2 |
0.67 |
|
PC |
30 µg/ml* |
20 |
6.67 |
|
Appendix 8: SummaryData on Chromosome Aberrations - Phase III
Dose Level |
Concentration |
Absence of metabolic activation |
||
Total No. of Aberrant cells without gap |
Percent aberrant cells |
|
||
VC |
- |
1 |
0.33 |
|
T1 |
0.5 mg/ml |
2 |
0.67 |
|
T2 |
1 mg/ml |
1 |
0.33 |
|
T3 |
2 mg/ml |
2 |
0.67 |
|
PC |
20 µg/ml* |
25 |
8.33 |
|
Table 1. In vitro cell transformation of the test compound and the respective control chemicals
Compound |
No. of transformed colonies/No. of surviving colonies |
|||||||||||||||
0.2% DMSO |
3MC (µg/mL) |
Dose of the test chemical (µg/mL) |
||||||||||||||
0.1 |
0.5 |
1.0 |
0 |
0.01 |
0.05 |
0.1. |
0.5 |
1 |
5 |
10 |
20 |
50 |
100 |
300 |
||
Test chemical |
0/603 |
0/538 |
1/527 |
0/500 |
0/620 |
0/583 |
0/606 |
0/576 |
0/496 |
- |
- |
- |
- |
- |
- |
- |
DMSO = Dimethylsulphoxide
MC = 3-Methylcholanthrene
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Bacterial reverse mutation test (Ames test):
Based on the results of the present study, it is concluded that N-Dodecylpyridinium Chloride [CAS No.: 104-74-5] is non-mutagenic as it does not induce (point) gene mutations by base-pair changes or frameshift in the histidine operon in any of the five tester strains of Salmonella typhimurium (TA1537, TA1535, TA98, TA100 or TA102) neither in the presence nor in the absence of metabolic activation system.
Chromosome aberration study:
Data available for structurally similar read across chemicals has been reviewed to determine the potential of the test chemical 1-Dodecylpyridinium chloride (CAS No. 104 -74 -5) to cause chromosomal aberration in mammalian cells. The studies are as mentioned below:
1. The ability of the registered substance, Tetrabutylazanium bromide (CAS No. 1643-19-2), to induce chromosome aberration in cultured Chinese Hamster Ovary (CHO) cells was tested according to OECD TG 473 both in the presence and absence of an exogenous metabolic activation system. Cofactor-supplemented S9 microsomal fraction was used as an exogenous metabolic activation system. The S9 fraction was obtained from the liver of phenobarbitone and β-naphthoflavone-induced rats. Distilled water was selected as a vehicle for the test substance. Test concentrations were selected based on the solubility, precipitation and pH checks, as well as a preliminary cytotoxicity test. In the cytotoxicity test, CHO cells were exposed to the test substance at concentrations of 0.0 (vehicle control/distilled water), 0.125, 0.25, 0.5, 1.0 and 2.0 mg/ml, both in the absence and presence of S9 metabolic activation. No cytotoxicity (defined by a Relative Increase in Cell Count [RICC] of ≤40% of the concurrent vehicle control data) was observed for the Test Item at ≤2 mg/ml, either in the presence or absence of metabolic activation. Hence, the following concentrations were employed in the chromosome aberration test: 0.0 (VC), 0.5, 1 and 2 mg/ml. Positive control substances were also included in the test, i.e., Methyl methanesulfonate (20 µg/ml, without S9) and Benzo (a) pyrene (30 µg/ml, with S9). The chromosome aberration test consisted of three phases. In Phase,s I-II, CHO cells (1 × 106 cells/flask ) were treated with test substance formulation and vehicle/ positive controls for 4 hours (short-term treatment) in the presence and absence of S9 metabolic activation which was followed by a 20 hours recovery period. In Phase III, CHO cells (1 × 106 cells/flask) were treated with the test substance and vehicle and positive controls for 24 hours (continuous treatment). The cultures were harvested 24 hours (short term treatment and continuous treatment) after the beginning of treatment. After the treatment period, CHO cells were fixed with Carnoy’s fixative (3:1 methanol: acetic acid solution) and stained with 5 % Giemsa stain. At least 300 well-spread metaphases per concentration (single culture) were analyzed using 100x magnification for the incidence of structural aberrations. Cells with structural chromosomal aberration(s) including and excluding gaps were scored. Chromatid and chromosome-type aberrations were recorded separately and classified by sub-types (breaks, exchanges). The cytotoxicity was assessed by the Relative Increase in Cell Counts (RICC) in all phases. Results: In Phase I, no significant increase in the mean percent aberrant cells at 0.5 mg/ml (the mean % aberrant cells: 0.33%, p=1.0000), 1 mg/ml (the mean % aberrant cells: 0.33%, p=1.0000), 2 mg/ml (the mean % aberrant cells: 0.67%, p=0.4992) was observed when compared to the vehicle control (the mean % aberrant cells 0.00 %). Average RICC values were 100 % (vehicle control), 84.76 % (at 0.5 mg/ml), 82.84 % (at 1 mg/ml) and 78.05 % (at 2 mg/ml). In Phase II experiment, no significant increase in mean percent aberrant cells at 0.5 mg/ml (the mean % aberrant cells: 0.33%, p=1.0000), 1 mg/ml (the mean % aberrant cells: 0.67%, p=1.0000), 2 mg/ml (the mean % aberrant cells: 0.67%, p=1.0000), was observed compared to the vehicle control (the mean % aberrant cells 0.33 %). Average RICC values were 100 % (vehicle control), 84.08 % (at 0.5 mg/ml), 81.57 % (at 1 mg/ml) and 76.74 % (at 2 mg/ml). In Phase III, the average RICC values were 100 % (vehicle control), 85.53 % (at 0.5 mg/ml), 82.13 % (at 1 mg/ml) and 76.11 % (at 2 mg/ml). No significant increase in mean percent aberrant cells at 0.5 mg/ml (the mean % aberrant cells: 0.67%, p=1.0000), 1 mg/ml (the mean % aberrant cells: 0.33%, p=1.0000), 2 mg/ml (the mean % aberrant cells: 0.67%, p=1.0000), was observed when compared to the vehicle control (the mean % aberrant cells 0.33 %). Conclusion: The registered substance, Tetrabutylazanium bromide (CAS No. 1643-19-2), did not induce chromosome aberration in cultured CHO cells up to 2 mg/ml neither in the present nor in the absence of S9 metabolic activation under the experimental conditions described.
2. In vitro mammalian chromosome aberration study was performed to determine the mutagenic nature of the test chemical Methyl nonyl ketone (CAS no. 112 -12 -9). The study was performed using CHO cells in the presence and absence of S9 metabolic activation system. The test chemical was soluble in DMSO and used at dose level of 5, 15 and 25μg/ml. Cells were exposed for a period of 4 hours. Positive control chemicals were also included in the study, Ethylmethane sulphonate and N-nitroso-dimethylamine were used as the postive controls. Cytotoxicity started at a concentration of 25μg/ml. The test substance did not induce chromosomal aberrations in CHO cells in the presence and absence of metabolic activation system.
Based on the above-summarized studies for 1-Dodecylpyridinium chloride (CAS No. 104 -74 -5) and its structurally and functionally similar read across substances, it can be concluded that the test chemical is unable to cause chromosomal aberration in mammalian cells. Comparing the above annotations with the criteria of CLP regulation, the test substance cannot be classified for genetic toxicity.
In Vitro mammalian gene mutation test:
Data available for structurally similar read across chemicals have been reviewed to determine the potential of the test chemical 1-Dodecylpyridinium chloride (CAS No. 104 -74 -5) to cause gene mutations in mammalian cells. The studies are as mentioned below:
1.In a gene toxicity test, Chinese Hamster Ovary (CHO) cells were exposed to the test chemical in the concentration of 0, 0.625, 1.25, 2.5 or 5mM with and without metabolic activation for 3 hours. The results showed that there was no evidence of cytotoxicity after treatment. In the concentrations 1.25 or 2.5mM of Tetrabutylammonium bromide concentration, the results showed evidence of gene toxicity. Therefore, it is considered that the test chemical in the concentration of 1.25 or 2.5 microM may cause genetic mutation(s) when CHO cells are exposed to the test chemical in the absence of metabolic activation.
2. In vitro mammalian cell transformation assay was performed to determine the mutagenic nature of the test chemical. The test chemical was dissolved in DMSO as the solvent and used at dose level of 0, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 20, 50, 100 or 300µg/mL. Pregnant Syrian golden hamsters were killed on days 13 and 14 of gestation for preparation of target cells and feeder-layer cells respectively. Embryos without heads and viscera were minced with scissors,a nd trypsinized with 0.25% trypsin. Inocula of 10000000 embryo cells per 75-cm’ flask were incubated at 37°C in a humidified atmosphere of 10% CO2 in air. When they became confluent primary cultures were trypsinized, dispensed in lots of 5000000 cells in glass ampules, and stored in liquid nitrogen for use as target and feeder-layer cells. The assay takes 15 days from start to finish. On Day 0, an ampule of cryopreserved primary cells prepared as feeder-layer cells was rapidly thawed and plated in a 75-cm2 flask containing 20 ml of the culture medium. The medium was changed every day. On day 3, an ampule of cryopreserved primary cells prepared as target cells was also rapidly thawed and plated in a 75-cm2 flask. On day 4, the feeder cells which were shifting from a stage of logarithmic growth to a stationary phase were irradiated with 5000 R from a linear accelerator, trypsinized, and then plated at 6 x lo4 tells/60-mm dish in 2 ml of complete medium. On day 5, the target cells which were approximately 80 -90% confluent were trypsinized, and a suspension of 500 target cells in 2 ml of complete medium was then added to each of the dishes plated the day before with irradiated feeder-layer cells. On day 6, an appropriate dose of the test chemical in a volume of 4 ml was added, giving a total volume of 8 ml of medium in each dish. Nine dishes were used for each dose level in all the experiments (in a few cases only eight or seven dishes were used for controls). On day 14, the cultures werefixed with absolute methanol for 10min and stained with Giemsa solution for 45 min or more. The stained dishes were examined with a stereoscopic dissection microscope to count normal and transformed colonies. The exposure duration was 8 days. Concurrent solvent and negative control chemicals were also included in the study. The test chemical did not induce cell transformation in the Pregnant Syrian golden hamsters embryo cell line used and hence it is not likely to classify as a gene mutant in vitro.
Based on the above-summarized studies for 1-Dodecylpyridinium chloride (CAS No. 104 -74 -5) and its structurally and functionally similar read across substances, it can be concluded that the test chemical is unable to cause gene mutation in mammalian cells. Comparing the above annotations with the criteria of CLP regulation, the test substance cannot be classified for genetic toxicity.
Justification for classification or non-classification
Based on the data available for the test chemical, and its structurally similiar read across chemicals, it does not exhibit gene mutation in In-Vitro Bacterial reverse mutation test (Ames test), In-Vitro chromosomal assay and In-Vitro mammalian cell gene mutation assay. Hence the test chemical is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.
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