Registration Dossier
Registration Dossier
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EC number: 300-141-0 | CAS number: 93922-04-4
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
The in vitro genetic toxicity of Barium 4-dodecylphenolate has been evaluated in a mammalian cell gene mutation assay (acc. to OECD TG 476), and in a mammalian cell cytogenicity study (acc. to OECD TG 487). These studies were performed according to the current guidelines and in accordance with GLP and are considered to be reliable without restrictions. The mutagenic potential in bacteria is addressed with existing data on the individual moieties, barium and 4-dodecylphenolate. For the moiety barium, a guideline conform bacterial reverse mutation assay was performed under GLP. For the moiety 4-dodecylphenolate, a QSAR prediction (Mutagenicity (Ames test) model - VEGA) with Phenol, dodecyl-, branched is available. Both sources are considered to be reliable without restrictions.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2019-11-14 to 2020-04-17
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)
- Version / remarks:
- Version: adopted 29 July 2016
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- Version: 2008
Remarks: "The EU method is outdated and does not reflect the latest update of the corresponding OECD method. Any new test should be performed following the updated OECD TG." Therefore, the test was performed follwing the current OECD TG and is thus in accordance to the ECHA advisory document "Three recently approved in vivo genotoxicity test guidelines" (Revised in February 2018). - Deviations:
- not specified
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- signed 2019-09-16
- Type of assay:
- in vitro mammalian cell gene mutation test using the Hprt and xprt genes
- Specific details on test material used for the study:
- STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (21 to 29°C); dry; protected from light, in the original container
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test material was suspended in n-hexane at 500 mg/mL
FORM AS APPLIED IN THE TEST (if different from that of starting material)
- Suspension in n-hexane
OTHER SPECIFICS
- measurement of pH, osmolality, and precipitate in the culture medium to which the test chemical is added:
please refer to 'any other information on results incl. tables' - Target gene:
- Hprt
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Details on mammalian cell type (if applicable):
- CELLS USED
- Type and source of cells: CHO AA8 (CHO-K1 derivative) purchased from ATCC (Batch No.: 500062)
- Suitability of cells: CHO AA8 cells are one of the recommended test systems by regulatory agencies for conducting In vitro Mammalian Gene Mutation Tests, due to their capacity to measure mutations at the X-linked Hprt locus (Thompson et al., 1980)*. These cells are chosen because of their known sensitivity toward chemical mutagens, a high cloning efficiency, a stable karyotype, and a stable spontaneous mutant frequency.
- The cultures were cleansed of pre-existing mutant cells by culturing in HAT Medium and then returned to normal growth medium.
- Cells free of mycoplasma were used for the experiment.
- Cell doubling time: 12 hours
- Modal number of chromosomes: 21
MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature: Alpha Minimal Essential Medium (MEM) without ribonucleosides containing 10% Fetal Bovine Serum (FBS) and antibiotics (1% penicillin and streptomycin) were used and the cells were incubated at 37±1°C and 5±1% CO2 in a humidified incubator.
*References
- Thompson, L.H., Fong, S., Brookman, K. (1980). Validation of conditions for efficient detection of hprt and aprt mutations in suspension-cultured Chinese hamster ovary cells. Mutat Res. 1980 Feb; 74(1):21-36. - Metabolic activation:
- with and without
- Metabolic activation system:
- Sodium phenobarbitone and β-naphthoflavone induced rat liver S9 homogenate was used as the metabolic activation system. The S9 homogenate was prepared from Wistar rats (5-6 weeks of age) induced with intraperitoneal injection of sodium phenobarbitone and β-naphthoflavone at 16 mg/mL and 20 mg/mL respectively for 3 days prior to sacrifice. The S9 homogenate was prepared and stored in the test facility at -80±10ºC until use. The batch of S9 homogenate was assessed for sterility, protein content (Modified Lowry assay, Sword and Thomson, 1980) and for its ability to metabolize the promutagens 2-aminoanthracene and benzo(a)pyrene to mutagens using Salmonella typhimurium TA100 strain.
One mL of S9 homogenate was thawed immediately before use and mixed with 9 mL of co-factor solution containing 4 mM NADP, 5 mM glucose-6-phosphate, 8 mM MgCl2 and 33 mM KCl in Phosphate Buffer Saline (PBS) of pH 7.31. The final concentration in culture was 1%. - Test concentrations with justification for top dose:
- - 0.01953125, 0.0390625, 0.078125 and 0.15625 mg/mL (based on cytotoxicity)
- Vehicle / solvent:
- - Vehicle used: n-hexane
- Justification for choice of vehicle: uniform suspension was formed in n-hexane - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- n-hexane
- True negative controls:
- yes
- Remarks:
- DMSO
- Positive controls:
- yes
- Positive control substance:
- benzo(a)pyrene
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- n-hexane
- True negative controls:
- yes
- Remarks:
- DMSO
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- Details on test system and experimental conditions:
- PRECIPITATION, OSMOLALITY AND PH TEST
Precipitation test was conducted at 0.15625, 0.3125, 0.625, 1.25, 2.5, and 5 mg/mL to determine the ability of the test item to cause precipitation in the medium through visual observation. A quantity of 100 µL of test item (15.625, 31.25, 62.5, 125, 250 and 500 mg/mL) was made up to 10 mL using culture media and incubated at 37±1ºC with 5±1% CO2 for 3 hours. After 3 hours of incubation, no change in osmolality (determined using an osmometer (Gonotec)) nor pH (determined using a pH meter (Eutech)) was observed at 0.15625, 0.3125, 0.625, 1.25, 2.5 and 5 mg/mL. No precipitation was observed at the concentrations tested at 0.15625, 0.3125, 0.625 and 1.25 mg/mL, slight precipitation was observed at 2.5 mg/mL and moderate precipitation was observed at 5 mg/mL. Thus, a test item concentration of 2.5 mg/mL was chosen as top concentration for the subsequent initial cytotoxicity test.
PREPARATION OF CULTURES
A frozen stock of cryovial was thawed immediately at 37±1°C in the water bath. The cells were transferred into a sterile flask with culture medium containing 10% FBS with antibiotics (1% penicillin and streptomycin) and incubated at 37±1°C and 5±1% CO2 for 2 to 3 days. The cell lines were trypsinized using trypsin-EDTA and the trypsinized cultures were subcultured three times (Initial cytotoxicity test) and four times (Gene mutation test) before usage in the experiment. Approximately 2×10^6 (Initial cytotoxicity test and gene mutation test) cells per culture flask were seeded using culture medium with 10% FBS with antibiotics (1% penicillin and streptomycin). Four additional flasks were seeded and kept for incubation along with flasks for treatment to determine cell count at the beginning of the treatment to determine the Adjusted Cloning Efficiency. The flasks were incubated, in a humidified incubator, at 37±1°C with 5±1% CO2 for 23 hours (Initial cytotoxicity test) and 24 hours (Gene mutation test). The cultures were cleansed of pre-existing mutant cells by culturing in HAT Medium and then returned to normal growth medium.
TEST PROCEDURE
For tests with exogenous metabolic activation (Set 1), 1 mL of S9 mix (10% v/v) was added to all the flasks. A volume of 100 µL of vehicle/different concentrations of test item was added to quadruplicate cultures to get the required test concentration per mL of the test medium and the volume of medium was made up to 10 mL. Cells were exposed to the test item for 3 hours at 37±1oC with 5±1% CO2.
For tests without exogenous metabolic activation (Set 2), a volume of 100 µL of vehicle/different concentrations of test item was added to quadruplicate cultures to get the required test concentration per mL of the test medium and volume of medium was made up to 10 mL. Cells were exposed to the test item for 3 hours at 37±1°C with 5±1% CO2.
After the incubation period (Set 1 and 2), medium from each flask was aspirated and the cell monolayer was washed with DPBS. Cells were trypsinized and trypsinization was stopped by adding culture media followed by collecting the media with cells.
Quadruplicate treatments were collected in prelabelled tubes and centrifuged at 800 rpm for 10 minutes. The supernatant was discarded, and the cell pellet was retained and resuspended in culture media.
Each treatment replicate was plated in triplicate with a cell concentration of 200 cells/5 mL media in 25 cm² flasks and incubated at 37±1°C with 5±1% CO2 for 9 days.
After the incubation period, medium from each culture flask was aspirated and stained with 5% Giemsa stain. Afterwards, the number of colonies formed were counted manually.
CYTOTOXICITY TESTS
Based on the results of solubility, pH, osmolality and precipitation tests, an initial cytotoxicity test was conducted for the selection of test concentrations for the gene mutation test. Six concentrations (0.078125, 0.15625, 0.3125, 0.625, 1.25 and 2.5 mg/mL) of the test item were tested in an initial cytotoxicity test.
The treatment was carried out as described above (Test procedure). The Cytotoxicity level was determined using the following formulae:
- Cloning Efficiency (CE) = No. of colonies / No. of cell plated at low density
- Adjusted Cloning Efficiency (ACE)= CE x No. of cells at the end of treatment / No. of cell at the beginning of treatment
- Relative survival (RS) = ACE (Treated) / ACE (Vehicle control) x 100
- Cloning efficiency (CE) is the percentage of cells plated at a low density that are able to grow into a colony that can be counted.
GENE MUTATION TEST
- Treatment: The gene mutation test was carried out as described above (Test procedure). Each treatment group was maintained in quadruplicate cultures. The cells were exposed to the test item/vehicle control/reference control/positive control for 3 hours both with exogenous metabolic activation (Set 1) and without exogenous metabolic activation (Set 2) respectively in the gene mutation test at 37±1°C with 5±1% CO2. Quadruplicate treatments were pooled into a pre-labeled tube and centrifuged at 800 rpm for 10 minutes. The supernatant was discarded, and the cell pellet was retained. Each treatment replicate was plated in triplicate a with cell concentration of 200 cells/5 mL media in 25 cm² flasks and incubated at 37±1°C with 5±1% CO2 for 8 days. Cytotoxicity was estimated in parallel as described above.
- Expression: The replicate cultures were subcultured in duplicates at a density of 1×10^6 cells/culture flask. The cells were incubated at 37±1°C with 5±1% CO2, followed by sub-culturing with an interval of 2 to 3 days for the remaining 8 days of the expression period.
- Selection: After the expression period of 8 days of the mutant phenotype, each replicate treatment culture was pooled and sub-cultured in quintuplicates at a density of 4×10^5 cells per 25 cm² flask with culture media containing 10 µM of 6-Thioguanine and 200 cells/25 cm² flask in triplicates without 6-Thioguanine for the determination of the cloning efficiency. Flasks were incubated at 37±1°C with 5±1% CO2 for 11 days. After the incubation period, the medium from each dish was aspirated and stained with 5% Giemsa stain. Afterwards, the number of colonies formed was counted manually. - Evaluation criteria:
- A) A test chemical is considered to be clearly positive if, in any of the experimental conditions examined:
- At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
- The increase is concentration-related when evaluated with an appropriate trend test.
- Any of the results are outside the distribution of the historical negative/vehicle control data.
When all of these criteria are met, the test chemical is then considered able to induce gene mutations in cultured mammalian cells in this test system.
B) A test chemical is considered clearly negative if, in all experimental conditions examined:
- None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- There is no concentration-related increase when evaluated with an appropriate trend test.
- All results are inside the distribution of the historical negative/vehicle control data.
When all of these criteria are met, the test chemical is then considered unable to induce gene mutations in cultured mammalian cells in this test system. - Statistics:
- Data of mutant frequencies were analyzed for differences among vehicle control, treatment and positive control groups by performing power transformation procedure by Snee and Irr (1981)* with which, the observed mutant frequency was transformed using the formula: Y = (X + A)^B
- Y = transformed mutant frequency
- X = observed mutant frequency (=No. of mutant colonies per replicate/ACE value x 100)
- A, B = constants (viz. A = 1 and B = 0.15)]
Statistical analysis of the experimental data was carried out using SPSS Statistical package version 22.0, One-way ANOVA followed by Dunnett’s post-hoc-test and Tukey linear trend test at 95% level (p<0.05) of significance
*References:
- Snee R.D., Irr J.D. (1981). Design of a statistical method for the analysis of mutagenesis at the hypoxanthine-guanine phosphoribosyl transferase locus of cultured Chinese hamster ovary cells. Mutat Res. 1981Apr; 85(2):77-93. - Key result
- Species / strain:
- Chinese hamster Ovary (CHO)
- 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:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- SOLUBILITY, PRECIPITATION, PH AND OSMOLALITY TEST
Barium 4-dodecylphenolate formed a suspension in n-hexane at 500 mg/mL. The precipitation, pH and osmolality tests were conducted at 0.15625, 0.3125, 0.625, 1.25, 2.5 and 5 mg/mL. After 3 hours of incubation, no change in osmolality nor pH was observed at 0.15625, 0.3125, 0.625, 1.25, 2.5 and 5 mg/mL. No precipitation was observed at the concentrations tested at 0.15625, 0.3125, 0.625 and 1.25 mg/mL, slight precipitation was observed at 2.5 mg/mL and moderate precipitation was observed at 5 mg/mL.
INITIAL CYTOTOXICITY TEST
The cell monolayer was completely absent at concentrations of 0.625, 1.25 and 2.5 mg/mL of barium 4-dodecylphenolate. The RS values ranged from 0% to 31.03% in the presence of metabolic activation, and in the absence of metabolic activation, the RS values ranged from 0% to 30.00% at barium 4-dodecylphenolate concentrations of 0.078125 to 2.5 mg/mL. At a test item concentration of 0.15625 mg/mL, the RS values were 12.93 and 11.82% in presence and absence of metabolic activation, respectively. Based on the results of the initial cytotoxicity test, 0.15625 mg barium 4-dodecylphenolate/mL was selected as top concentration for the gene mutation test.
Please refer to table 1 in the field ‘Any other information on results incl. tables’.
GENE MUTATION TEST
Cultures treated for 3 hours with barium 4-dodecylphenolate showed mutant frequencies of 23.60 to 25.61 per 2×10^6 cells in the presence of metabolic activation. The concurrent vehicle control showed a mutation frequency of 24.47 per 2×10^6 cells. In the absence of metabolic activation, mutant frequencies of 24.71 to 25.61 per 2×10^6 cells were observed in cultures treated with the test item for 3 hours. Concurrently, a mutant frequency of 24.47 per 2×10^6 cells was observed in the vehicle control. There was no statistically significant increase in the mutant frequencies observed when compared with vehicle control at any of the tested concentrations. A reference control, DMSO, was also used in the experiment. The mutant frequencies observed in the reference control were comparable to the vehicle control.
There was no evidence of excessive cytotoxicity (less than 10% RS) at any of the concentrations tested both in presence and absence of metabolic activation. In the presence of metabolic activation, the RS values ranged from 14.95% to 92.52% and in the absence of metabolic activation the RS values ranged from 15.53% to 88.35% when compared to the respective vehicle control. The top concentration (0.15625 mg/mL) resulted in a RS values, which are consistent with acceptability criteria.
In the presence of metabolic activation, cultures treated with the positive control, benzo(a)pyrene, at concentrations of 3.0 and 1.5 µg/mL resulted in RS values of 75.70% and 85.05% and mutant frequencies of 258.90 and 107.41 per 2×10^6 cells, respectively. The mutant frequencies were statistically significantly increased when compared with the vehicle control. The high concentration treatment, resulted in a mutant frequency, which was within the historical positive control data range reported (please refer to 'attached background material)'.
In the absence of metabolic activation, cultures treated with the positive control, 4-nitroquinoline N-oxide, at concentrations of 1.0 and 0.5 µg/mL resulted in RS values of 69.90% and 79.61% in the absence of metabolic activation and mutant frequencies of 260.26 and 101.20 per 2×10^6 cells, respectively. The mutant frequencies obtained were statistically significantly increased when compared with the vehicle control value.
Please refer to tables 2 and 3 in the field ‘Any other information on results incl. tables’.
Based on the evaluation criteria, the test item, barium 4-dodecylphenolate is clearly negative at and up to the concentration of 0.15625 mg/mL. - Conclusions:
- Barium 4-dodecylphenolate was evaluated for the ability to induce gene mutation in CHO AA8 cells, as per the OECD guideline No. 476 adopted on 29th July 2016. Based on the results obtained, barium 4-dodecylphenolate is considered as non-mutagenic at and up to a concentration of 0.15625 mg/mL, both in the presence and absence of metabolic activation.
- Endpoint:
- in vitro cytogenicity / micronucleus study
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2019-10-05 to 2020-04-17
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
- Version / remarks:
- Version: adopted 29 July 2016
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: Commission Regulation (EC) No. 440/2008, B.49 Mutagenicity - In vitro Mammalian Cell Micronucleus Test
- Version / remarks:
- Version: 2008
Remarks: "The EU method is outdated and does not reflect the latest update of the corresponding OECD method. Any new test should be performed following the updated OECD TG." Therefore, the test was performed follwing the current OECD TG and is thus in accordance to the ECHA advisory document "Three recently approved in vivo genotoxicity test guidelines" (Revised in February 2018). - Deviations:
- not applicable
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- signed 2019-09-16
- Type of assay:
- in vitro mammalian cell micronucleus test
- Specific details on test material used for the study:
- STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
Ambient (21 to 29°C); dry; protected from light, in the original container
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
The test material was suspended in n-hexane at 500 mg/mL
FORM AS APPLIED IN THE TEST (if different from that of starting material)
- Suspension in n-hexane
OTHER SPECIFICS
- measurement of pH, osmolality, and precipitate in the culture medium to which the test chemical is added:
please refer to 'any other information on results incl. tables' - Target gene:
- not applicable
- Species / strain / cell type:
- lymphocytes: human
- Details on mammalian cell type (if applicable):
- CELLS USED
For lymphocytes:
- Sex, age and number of blood donors: Human peripheral lymphocytes from the blood of healthy, young, non-smoking male and female donors (three donors for Initial cytotoxicity studies and two donors for micronucleus test, 27 (male and female), 22 (female), and 25 (male and female)) years of age) with no known recent exposure to genotoxic chemicals or radiations were used.
- Whether whole blood or separated lymphocytes were used: whole blood
- Mitogen used for lymphocytes: phytohaemagglutinin
MEDIA USED
- Type and composition of media, CO2 concentration, temperature, if applicable: RPMI-1640 (Gibco, 2038853) with FBS (MP Biomedicals, NZ190502 and Gibco, 42A0289K), and pen-strep solution (Lonza, 18K135305) at 37±1ºC at 5±1% CO2. - Cytokinesis block (if used):
- Cytochalasin B (Sigma Aldrich, Batch No.:126094044V)
- Metabolic activation:
- with and without
- Metabolic activation system:
- Sodium phenobarbitone and β-naphthoflavone induced rat liver S9 homogenate was used as the metabolic activation system. The S9 homogenate was prepared from male Wistar rats (8 to 9 weeks of age) induced with intraperitoneal injection of sodium phenobarbitone and β-naphthoflavone at 16 mg/mL and 20 mg/mL respectively for 3 days prior to sacrifice. The S9 homogenate was prepared and stored in the test facility at -80±10ºC until use. It was assessed for sterility by streaking the supernatant fluid on Nutrient Agar plates. The plates were incubated at 37±1°C for 24 hours and the S9 homogenate was found sterile. The protein content (Modified Lowry Assay, Sword and Thomson, 1980) and its ability to metabolize the promutagens 2-aminoanthracene and benzo(a)pyrene to mutagens using Salmonella typhimurium TA100 strain were also evaluated. The metabolic activity was found to be acceptable.
One mL of S9 homogenate was thawed immediately before use and mixed with 9 mL of co-factor solution containing 4 mM NADP, 5 mM glucose-6-phosphate, 8 mM MgCl2 and 33 mM KCl in PBS of pH 7.31, 7.33 and 7.30. The final concentration in culture was 1%. - Test concentrations with justification for top dose:
- - 0.009766, 0.0195313, 0.0390625, 0.078125 and 0.15625 mg/mL (based on cytotoxicity)
- Vehicle / solvent:
- - Vehicle used: n-hexane
- Justification for choice of vehicle: uniform suspension was formed in n-hexane - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- colchicine
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- mitomycin C
- Details on test system and experimental conditions:
- CYTOTOXICITY TESTS
- Whole blood of a volume of 0.5 mL was added to each tube containing culture media of a volume of 4.5 mL supplemented with final concentration of 2% PHA-M of phytohaemagglutinin (PHA) and incubated for 44 to 48 hours at 37±1ºC at 5±1% CO2.
- After 44 to 48 hours of incubation with PHA, cells from tissue culture tube were collected along with the used medium and centrifuged at 500 g for 10 minutes, the supernatant was discarded.
- The cell pellet was resuspended with fresh media.
- The test item was prepared with n-hexane at different concentrations. Cell suspensions without any treatment were used as negative control.
- The metabolic activation and incubation details are as mentioned in the following:
• Set 1: vehicle control/untreated control/test material/positive control; with metabolic activation; 4 hours and 53 minutes (Initial cytotoxicity test) and 4 hours and 18 minutes (follow up initial cytotoxicity test);
• Set 2: vehicle control/untreated control/test material/positive control; without metabolic activation; 4 hours and 53 minutes (Initial cytotoxicity test) and 4 hours and 18 minutes (follow up initial cytotoxicity test);
• Set 3: vehicle control/untreated control/test material/positive control; without metabolic activation; 23 hours and 7 minutes (Initial cytotoxicity test and follow up initial cytotoxicity test)
- For experimental set no. 1 (+S9, with metabolic activation), the cell suspension was mixed with 50 µL of the respective concentration of the test item/50 µL vehicle control and 0.5 mL S9 mix, and subsequently, the volume was made up to 5 mL with culture media and transferred to pre-labelled tubes.
- For experimental set no. 2 (-S9; without metabolic activation), cell suspensions were mixed with 50 µL of the respective concentration of the test item or vehicle control. The volume was made up to 5 mL with culture media.
- For experimental set no. 3 (-S9; without metabolic activation), cell suspensions were mixed with 50 µL of the respective concentration of the test item/vehicle control and 6 µg/mL (final concentration) of cytochalasin B. The volume was made up to 5 mL with culture media.
- All test concentrations and vehicle control were maintained in duplicates.
- The vehicle control group was maintained by adding vehicle obtained based on solubility test.
- The cells were incubated both with and without S9 for 3 to 6 hours and without metabolic activation for 20 to 24 hours at 37±1ºC and 5±1% CO2 as mentioned in the table (see above).
- The treatment for set 1 and 2 tubes was terminated post 3 to 6 hours of incubation, by collecting cells from the tubes along with old media into centrifuge tubes and centrifuged at 500 g for 10 minutes.
- Culture media with 6 µg/mL (final concentration) of cytochalasin B was added to set 1 and 2 tubes, and subsequently, the content of each tube was transferred to a fresh tissue culture tube. Afterwards, the tubes were incubated for 20 to 24 hours at 37±1ºC at 5±1% CO2.
- After 20 to 24 hours of incubation with cytochalasin B, the cells were collected from each tube along with old media, transferred into pre-labelled tubes, and subsequently, centrifuged at 500 g for 10 minutes.
- The supernatant was discarded, and the cell pellet was used for slide preparation.
MICRONUCLEUS TEST
- Micronucleus test was carried out as described in the cytotoxicity tests section (see above)
- The metabolic activation and incubation details were as mentioned in the following:
• Set 1: vehicle control/untreated control/test material/positive control; with metabolic activation; 3 hours
• Set 2: vehicle control/untreated control/test material/positive control; without metabolic activation; 3 hours
• Set 3: vehicle control/untreated control/test material/positive control; without metabolic activation: 23 hours and 50 minutes
SLIDE PREPARATION
The pellet was mixed with 3 to 5 mL of freshly prepared 0.56% potassium chloride (Prepared using distilled water as solvent). Cell suspension was incubated for 10 minutes at room temperature, and subsequently, it was centrifuged at 800 g for 10 minutes. The supernatant was discarded and the cell pellet was mixed with 3 to 5 mL of freshly prepared cold acetic acid: methanol fixative (1:3). The cells were incubated for 10 minutes at room temperature, and subsequently, the suspension was centrifuged at 800 g rpm for 10 minutes. The procedure was repeated twice by adding 3 mL of cold acetic acid: methanol fixative (1:3), and afterwards, the slides were prepared. Clean slides were stored in a container with distilled water and kept in the refrigerator for 1 hour before use. The cell suspension was mixed using a pipette and few drops of the suspension was aspirated and dropped onto the chilled slide pre-labeled with study number, with (+S9) or without metabolic activation (-S9), treatment/group and slide number. The slides were air dried. The smear was stained using acridine orange stain (0.0125 mg/mL) by allowing the stain to retain for 5 minutes. Slides were evaluated for cytotoxicity by microscopical (Cx41RF, Olympus) examination at a magnification of 40x.
SLIDE EVALUATION
- Measurement of the relative frequencies of mononucleate, binucleate and multi-nucleate cells in the culture was made to determine cell proliferation and the cytostatic activity of the treatment in order to ensure that only cells that divided during or after treatment were scored.
- Slides were evaluated for the presence of micronuclei in at least 2000 binucleates per concentration (at least 1000 per culture).
- Cytotoxicity/cytostasis was quantified from the Cytokinesis-Block Proliferation Index (CBPI). CBPI was determined from 500 cells per culture. This measurement was used to estimate cytotoxicity/cytostasis by comparing values in the treated and vehicle control cultures.
SCORING CRITERIA
- Binucleate cells were analysis analysed and met the following criteria: a) The cytoplasm has remained essentially intact, and b) The daughter nuclei are of approximately equal size.
- Micronuclei were recorded and met the following criteria: a) The micronucleus had same staining characteristics and a similar morphology to the main nuclei, and b) Any micronucleus present is separate in the cytoplasm or just touching a main nucleus, and c) Micronuclei with smooth edged and smaller than approximately one third of the diameter of the main nuclei. - Rationale for test conditions:
- SOLUBILITY TEST
A solubility test was conducted to determine the maximum concentration or workable suspension solution of the test item in the vehicle compatible with the test system at 500 mg/mL. The solubility test was carried out with distilled water, DMSO, acetone, and n-hexane. The test item formed a uniform suspension in n-hexane at 500 mg/mL. Based on these results, n-hexane was used as vehicle for the study.
PRECIPITATION, OSMOLALITY AND PH TEST
The precipitation test was conducted up to a maximum concentration of 5 mg/mL to determine the ability of test item to cause precipitation in the culture medium. A quantity of 50 µL of respective test item dilutions (31.25, 62.5, 125, 250 and 500 500 mg/mL) were mixed with 5 mL with media at the concentrations of 0.3125, 0.625, 1.25, 2.5 and 5 mg/mL along with vehicle control. The contents were incubated at 37±1ºC with 5±1% CO2 for 22 hours and 41 minutes and results were recorded for change in pH (determined using a pH meter (Eutech)), osmolality (determined using an osmometer (Gonotec)) and signs of precipitation through visual observation.
INITIAL CYTOTOXICITY TEST
Based on the results of the pH, osmolality and precipitation test, an initial cytotoxicity test was conducted at barium 4-dodecylphenolate concentrations of 0.625 1.25, 2.5 and 5 mg/mL in order to select appropriate test concentrations for the micronucleus test (Main study). In the initial cytotoxicity test, 100% reduction in CBPI was observed at all test item concentrations tested both in short short-term (3 to 6 hours, with and without metabolic activation) and long long-term treatment (20 to 24 hours, without metabolic activation). Hence, a follow up initial cytotoxicity test was conducted at 0.0048, 0.0097, 0.0195, 0.0390, 0.0781, 0.15625 and 0.3125 mg/mL in order to determine appropriate concentrations for the micronucleus test. - Evaluation criteria:
- Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if:
- At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
- The increase is dose-related in at least one experimental condition when evaluated with an appropriate trend test.
- Any of the results are outside the distribution of the historical negative control data.
When all of these criteria are met, the test chemical is then considered able to induce chromosome breaks and/or gain or loss in this test system.
A test item is considered to be clearly negative if:
- None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
- There is no concentration-related increase when evaluated with an appropriate trend test.
- All results are inside the distribution of the historical negative control data.
When all of these criteria are met, the test chemical is then considered unable to induce chromosome breaks and/or gain or loss in this test system. - Statistics:
- Data was analyzed using SPSS software version 22 for differences among vehicle control, positive control and test item groups using ANOVA following Dunnett’s test at a 95% level of confidence (p≤0.05) and the statistical significance was designated by the superscripts throughout the study report as stated below.
- Key result
- Species / strain:
- lymphocytes: human
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- SOLUBILITY, PRECIPITATION, OSMOLALITY AND PH TEST
Barium 4-dodecylphenolate formed a suspension in n-hexane at 500 mg/mL. Mild and moderate precipitation was observed at 2.5 and 5 mg/mL respectively. No precipitation was observed at 0.3125, 0.625 and 1.25 mg/mL. No change in pH and osmolality was observed in any of the concentrations tested.
Please refer to table 1 in ‘any other information on results incl. tables’.
CYTOTOXICITY TESTS
- In the initial cytotoxicity test, 100% reduction in CBPI was observed at all test item concentrations tested in both short-term (3 to 6 hours, with and without metabolic activation) and long-term treatments (20 to 24 hours, without metabolic activation).
- In the follow up initial cytotoxicity test, the average cytotoxicity observed was 1.69, 23.73, 25.42, 30.51, 33.90, 37.29, 54.25 and 79.66% at 0, 0.004883, 0.009766, 0.0195313, 0.0390625, 0.078125, 0.15625 and 0.3125 mg/mL, respectively, in presence of metabolic activation (short-term treatment). In the absence of a metabolic activations system (short-term treatment), the average cytotoxicity observed was 1.61, 25.81, 29.03, 27.42, 33.87, 37.10, 51.61 and 82.26% at 0, 0.004883, 0.009766, 0.0195313, 0.0390625, 0.078125, 0.15625 and 0.3125 mg/mL, respectively.
The long-term treatment (without metabolic activation) resulted in an average cytotoxicity of 0.00, 21.31, 19.67, 26.23, 24.59, 36.07, 50.82 and 83.61% at barium 4-dodecylphenolate concentrations of 0, 0.004883, 0.009766, 0.0195313, 0.0390625, 0.078125, 0.15625 and 0.3125 mg/mL, respectively.
Please refer to tables 2a-c in ‘any other information on results incl. tables’.
MICRONUCLEUS TEST
- In the micronucleus test, barium 4-dodecylphenolate concentrations of 0.009766, 0.0195313, 0.0390625, 0.078125 and 0.15625 mg/mL were tested for the presence of micronuclei. The test item induced, in absence of a metabolic activation system, 50.0% cytotoxicity both in the short-term and long-term treatment. Test item treatment in combination with metabolic activation resulted in 48.28% cytotoxicity. It has to be mentioned that this value is below the acceptability range of 55±5%. However, the preceding cytotoxicity tests revealed the test item showed steep concentration response curves which complicates exact titration of the cytotoxicity. Notably, the response was only marginally below the acceptability range and the concentration exhibited an acceptable cytotoxicity level (54.24%) in the follow up cytotoxicity test. Based on these findings, the cytotoxicity value observed in the main experiment is considered to be acceptable. In general, the average percentage of cytotoxicity among the different doses tested were within the range of 17.19 to 50.00% when compared to vehicle control.
- In the short-term treatment, test item concentrations of 0.009766, 0.0195313, 0.0390625, 0.078125 and 0.15625 mg/mL resulted in average micronucleus frequencies of 0.15, 0.19, 0.15, 0.15 and 0.20% (in presence of metabolic activation), respectively, and 0.14, 0.15, 0.19, 0.24 and 0.25% (in the absence of metabolic activation), respectively.
- The long-term treatment (in the absence of metabolic activation) with barium 4-dodecylphenolate resulted in micronucleus frequencies of 0.15, 0.20, 0.15, 0.20 and 0.20% at concentrations of 0.009766, 0.0195313, 0.0390625, 0.078125 and 0.15625 mg/mL, respectively. There was no statistically significant difference in the micronucleus frequency (%) in any of the treated groups, when compared to the vehicle control. There was no concentration-related increase when evaluated with an appropriate trend test. Moreover, the responses in micronucleus frequency observed fell within acceptable ranges with regard to the historical control data (please refer to attached background material - historical control data).
- In the short-term treatment (in presence of metabolic activation), the positive control, cyclophosphamide monohydrate, resulted in average micronucleus frequencies of 1.07 and 1.64%, when tested at concentrations of 10 and 15 µg/mL, respectively. Colchicine at 0.03 and 0.012 µg/mL resulted in average micronucleus frequencies of 1.08 and 0.70%, respectively, in the short-term treatment without metabolic activation. Long-term treatment (in absence of metabolic activation) with mitomycin C resulted in 1.04 and 1.43% micronuclei at 0.075 and 0.15 µg/mL, respectively.
- The positive controls resulted in a statistically significant increase in the micronuclei frequency with statistical significance at 95% level of confidence (p<0.05) under identical conditions, when compared with the vehicle control. Moreover, the response observed were well within historical control data ranges reported (please refer to attached background material - historical control data). This demonstrated the sensitivity of the test system and confirmed that the test conditions were adequate.
Please refer to tables 3a-c in ‘any other information on results incl. tables’. - Conclusions:
- In an in vitro mammalian cells micronucleus test, barium 4-dodecylphenolate was tested at concentrations of 0.009766, 0.0195313, 0.0390625, 0.078125 and 0.15625 mg/mL for short-term (6 hours) in presence and absence of metabolic activation and for long-term (20 to 24 hours) in the absence of a metabolic activation system. The test item induced cytotoxicity at and up to a concentration of 0.15625 mg/mL when compared to vehicle control. No statistically significant change in the percentage of micronuclei in binucleated cells were observed in any of the concentrations tested when compared to the vehicle control, and there was no concentration-related increase when evaluated with an appropriate trend test. The positive controls resulted in increase in the micronuclei frequency with statistical significance at the 95% level of confidence (p<0.05) under identical conditions, when compared with the vehicle control. This demonstrated the sensitivity of the test system and confirmed that the test conditions were adequate.
Based on the results obtained, it is concluded that the test item, barium 4-dodecylphenolate, is non-clastogenic and non-aneugenic in cultured human lymphocytes at and up to concentrations of 0.15625 mg/mL in short- and long-term treatments, both in presence and absence of metabolic activation, as it showed no evidence of increase in the induction of micronuclei under the test conditions. - Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Guideline conform study with GLP.
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Species / strain / cell type:
- S. typhimurium TA 97
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix
- Test concentrations with justification for top dose:
- 100, 333, 1000, 3333, 10000 µg/plate, duplicate cultures
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: no data - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-Aminoanthracene
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 4-nitro-o-phenylenediamine
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: pre-incubation; barium chloride dihydrate was incubated with the Salmonella typhimurium tester strains
either in buffer or S9 mix for 20 minutes at 37°C. Top agar supplemented with I-histidine and d-biotin was added, and the contents of the tubes were mixed and poured onto the surfaces of minimal glucose agar plates.
DURATION
- Exposure duration: 20 minutes
- Expression time (cells in growth medium): 2 days at 37°C
NUMBER OF REPLICATIONS: Each trial consisted of triplicate plates of concurrent positive and negative controls, and of at least five doses of barium chloride dihydrate.
No further details are given. - Evaluation criteria:
- In this test, a positive response was defined as a reproducible, dose-related increase in histidin-independent (revertant) colonies in any one strain/activation combination. An equivocal response was defined as an increase in revertants that was not dose related, not reproducible, nor was of sufficient magnitude to support a determination of mutagenicity. A negative response was obtained when no increase in revertant colonies was observed following chemical treatment. There was no minimum percentage or fold increase required for a chemical to be judged positive or weakly positive.
- Statistics:
- Statistical analysis is not mandatory for the bacterial reverse mutation assay.
- Species / strain:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- other: slight toxicity at concentration from 333 to 10000 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 97
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- other: slight toxicity at the highest tested concentration
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- no data
- Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results (migrated information):
negative
No increased induction of revertant colonies at all concentrations in all strains with and without metabolic activation. - Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- (Q)SAR
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification
- Justification for type of information:
- This endpoint study record refers to a QSAR prediction. The prediction of the mutagenic potential of phenol, dodecyl-, branched is sufficient to fulfil the information requirements as further explained in the provided endpoint summary. The reliable documentation of the applied method (QMRF) and the prediction (QPRF) is attached under “Attached justification” in this endpoint summary.
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- other: REACH guidance on QSARs R.6, May 2008
- Principles of method if other than guideline:
- VEGA for estimating the in vitro gene mutation in bacteria (Ames Test). The model extends the following models:
Mutagenicity (Ames test) model (CAESAR) – v. 2.1.13
Mutagenicity (Ames test) model (SarPy/IRFMN) – v. 1.0.7
Mutagenicity (Ames test) model (ISS) – v. 1.0.2
Mutagenicity (Ames test) model (KNN/Read-Across) – v. 1.0.0 - GLP compliance:
- no
- Specific details on test material used for the study:
- OC1=CC=C(CCC(C)C(C)C(C)CC)C=C1
- Key result
- Remarks on result:
- no mutagenic potential (based on QSAR/QSPR prediction)
- Conclusions:
- Based on the outcome of the VEGA models for estimating the in vitro gene mutation in bacteria (Ames Test)., phenol, dodecyl-, branched is not considered to cause gene mutation. The substance under concern is within the applicability domain of all independent prediction models within VEGA. The prediction is reliable to replace an Ames test to fulfill requirements for Regulation (EC) No 1907/2006 (REACH) Annex VII section 8.4.1.
Referenceopen allclose all
Table 1. Summary of initial cytotoxicity test
Set No. |
Treatment |
Concentration (mg/mL) |
Average Colony Count± SD |
Cloning Efficiency (CE) |
Adjusted Cloning Efficiency (ACE) |
Relative Survival (RS) (%) |
Set 1 +S9 |
Vehicle Control (n-Hexane) |
- |
183.67±9.61 |
0.92 |
1.16 |
- |
Barium 4-dodecylphenolate |
0.078125 |
91.00±9.00 |
0.46 |
0.36 |
31.03 |
|
0.15625 |
46.67±9.29 |
0.23 |
0.15 |
12.93 |
||
0.3125 |
22.33±6.81 |
0.11 |
0.02 |
1.72 |
||
0.625 |
- |
- |
- |
- |
||
1.25 |
- |
- |
- |
- |
||
2.5 |
- |
- |
- |
- |
||
Set 2 -S9 |
Vehicle Control (n-Hexane) |
- |
180.33±10.50 |
0.90 |
1.10 |
- |
Barium 4-dodecylphenolate |
0.078125 |
90.67±13.20 |
0.45 |
0.33 |
30.00 |
|
0.15625 |
61.00±8.19 |
0.31 |
0.13 |
11.82 |
||
0.3125 |
19.67±6.03 |
0.10 |
0.03 |
2.73 |
||
0.625 |
- |
- |
- |
- |
||
1.25 |
- |
- |
- |
- |
||
2.5 |
- |
- |
- |
- |
+S9: with metabolic activation; -S9: without metabolic activation;
Adjusted CE = CE × Number of cells at the end of treatment/number of cells at the beginning of treatment.
RS = Adjusted CE in treated culture/Adjusted CE in the vehicle control × 100.
CE = Number of colonies/Number of cells plated.
-: cell monolayer completely washed out
Table 2. Summary of parallel cytotoxicity test – Gene mutation test
Set No. |
Treatment |
Concentration (mg/mL) |
Average Colony Count ± SD |
Cloning Efficiency (CE) |
Adjusted Cloning Efficiency (ACE) |
Relative Survival (RS) (%) |
Set 1 +S9 |
Vehicle Control (n-Hexane) |
- |
181.00 ± 2.65 |
0.91 |
1.07 |
- |
Reference Control (DMSO) |
- |
188.00 ± 5.57 |
0.94 |
1.15 |
107.48 |
|
Barium 4-dodecylphenolate |
0.01953125 |
175.00 ± 9.64 |
0.88 |
0.99 |
92.52 |
|
0.0390625 |
147.00 ± 20.66 |
0.74 |
0.73 |
68.22 |
||
0.078125 |
95.00 ± 9.64 |
0.48 |
0.39 |
36.45 |
||
0.15625 |
55.00 ± 6.00 |
0.28 |
0.16 |
14.95 |
||
Benzo(a)pyrene (Positive Control) |
3 µg/mL |
145.00 ± 19.00 |
0.73 |
0.81 |
75.70 |
|
1.5 µg/mL |
158.67 ± 15.50 |
0.79 |
0.91 |
85.05 |
||
Set 2 -S9 |
Vehicle Control (n-Hexane) |
- |
178.33 ± 17.10 |
0.89 |
1.03 |
- |
Reference Control (DMSO) |
- |
184.67 ± 7.02 |
0.92 |
1.11 |
107.77 |
|
Barium 4-dodecylphenolate |
0.01953125 |
169.67 ± 10.02 |
0.85 |
0.91 |
88.35 |
|
0.0390625 |
152.33 ± 7.77 |
0.76 |
0.67 |
65.05 |
||
0.078125 |
127.67 ± 6.66 |
0.64 |
0.42 |
40.78 |
||
0.15625 |
65.67 ± 7.09 |
0.33 |
0.16 |
15.53 |
||
4 Nitroquinoline N-oxide (Positive Control) |
1 µg/mL |
134.33 ± 4.51 |
0.67 |
0.72 |
69.90 |
|
0.5 µg/mL |
148.33 ± 4.04 |
0.74 |
0.82 |
79.61 |
+S9: with metabolic activation; -S9: without metabolic activation;
*Note: Cloning Efficiency = 200 cells plated for each replicate.
RS = Adjusted CE in treated culture/Adjusted CE in the vehicle control × 100.
CE = Number of colonies/Number of cells plated.
Adjusted CE = CE × Number of cells at the end of treatment/number of cells at the beginning of treatment.
Table 3. Summary of gene mutation test
Set No. |
Treatment |
Concentration (mg/mL) |
*Average Colony Count ± SD |
Cloning Efficiency in selective media |
Cloning Efficiency in non-selective media |
Total number of Mutant Colonies/ 2×106cells |
Mutant Frequency/ 2×106cells |
Set 1 +S9 |
Vehicle Control (n-Hexane) |
- |
188.67 ± 1.53 |
0.0000115 |
0.94 |
23 |
24.47 |
Reference Control (DMSO) |
- |
182.33±12.01 |
0.0000110 |
0.91 |
22 |
24.18 |
|
Barium 4-dodecylphenolate |
0.01953125 |
178.67 ± 5.03 |
0.0000105 |
0.89 |
21 |
23.60 |
|
0.0390625 |
177.33 ± 4.93 |
0.0000110 |
0.89 |
22 |
24.72 |
||
0.078125 |
163.67 ± 4.51 |
0.0000105 |
0.82 |
21 |
25.61 |
||
0.15625 |
152.00 ± 13.45 |
0.0000095 |
0.76 |
19 |
25.00 |
||
Benzo(a)pyrene (Positive Control) |
3 µg/mL |
146.67 ± 10.26 |
0.0000945 |
0.73 |
189 |
258.90** |
|
1.5 µg/mL |
161.00±6.56 |
0.0000435 |
0.81 |
87 |
107.41** |
||
Set 2 -S9 |
Vehicle Control (n-Hexane) |
- |
187.33 ± 7.02 |
0.0000115 |
0.94 |
23 |
24.47 |
Reference Control (DMSO) |
- |
183.00±5.57 |
0.0000115 |
0.92 |
23 |
25.00 |
|
Barium 4-dodecylphenolate |
0.01953125 |
174.33 ± 13.43 |
0.0000110 |
0.87 |
22 |
25.29 |
|
0.0390625 |
169.67 ± 6.03 |
0.0000105 |
0.85 |
21 |
24.71 |
||
0.078125 |
164.33 ± 3.06 |
0.0000105 |
0.82 |
21 |
25.61 |
||
0.15625 |
163.67 ± 5.69 |
0.0000105 |
0.82 |
21 |
25.61 |
||
4 Nitroquinoline N-oxide (Positive Control) |
1 µg/mL |
155.33 ± 11.50 |
0.0001015 |
0.78 |
203 |
260.26** |
|
0.5 µg/mL |
166.67±4.16 |
0.0000420 |
0.83 |
84 |
101.20** |
+S9: with metabolic activation; -S9: without metabolic activation
*Note: Cloning efficiency = 200 cells plated for each replicate.
**: Statistically significant (p˂0.05).
Mutant Frequency = Cloning efficiency of mutant colonies in selective medium/Cloning efficiency in non-selective medium
Table 1. Summary of pH, precipitation, and osmolality test
Treatment |
Concentration (mg/mL) |
Initial Observation |
Results (Post incubation) |
||||
pH |
Precipitation |
Osmolality (mmol/kg) |
pH |
Precipitation |
Osmolality (mmol/kg) |
||
Vehicle Control |
50 µL of n-hexane |
7.34 |
No precipitation |
302 |
7.33 |
No precipitation |
303 |
Barium 4-dodecylphenolate |
0.3125 |
7.33 |
No precipitation |
299 |
7.35 |
No precipitation |
301 |
0.625 |
7.33 |
No precipitation |
301 |
7.31 |
No precipitation |
302 |
|
1.25 |
7.31 |
Mild precipitation |
301 |
7.30 |
No precipitation |
303 |
|
2.5 |
7.32 |
Moderate precipitation |
298 |
7.33 |
Mild precipitation |
297 |
|
5 |
7.32 |
Heavy precipitation |
299 |
7.30 |
Moderate precipitation |
300 |
|
Table 2a. Summary - Follow up initial cytotoxicity test
Treatment |
Metabolic Activation |
Concentration (mg/mL) |
Duration (hours) |
Replicates No. |
Total Number of Cells |
CBPI |
Mean CBPI |
% of Cytotoxicity |
Vehicle Control |
With S9 |
0 |
3 to 6 |
1 |
505 |
1.60 |
1.59 |
0.00 |
2 |
514 |
1.58 |
||||||
Negative control |
0 |
1 |
532 |
1.57 |
1.58 |
1.69 |
||
2 |
514 |
1.59 |
||||||
Barium 4-dodecylphenolate |
0.004883 |
1 |
500 |
1.45 |
1.45 |
23.73 |
||
2 |
504 |
1.45 |
||||||
0.009766 |
1 |
509 |
1.42 |
1.44 |
25.42 |
|||
2 |
502 |
1.45 |
||||||
0.0195313 |
1 |
502 |
1.40 |
1.41 |
30.51 |
|||
2 |
501 |
1.42 |
||||||
0.0390625 |
1 |
503 |
1.39 |
1.39 |
33.90 |
|||
2 |
514 |
1.39 |
||||||
0.078125 |
1 |
516 |
1.37 |
1.37 |
37.29 |
|||
2 |
505 |
1.37 |
||||||
0.15625 |
1 |
505 |
1.25 |
1.27 |
54.24 |
|||
2 |
513 |
1.29 |
||||||
0.3125 |
1 |
502 |
1.12 |
1.12 |
79.66 |
|||
2 |
508 |
1.12 |
Table 2b. Summary - Follow up initial cytotoxicity test
Treatment |
Metabolic Activation |
Concentration (mg/mL) |
Duration (hours) |
Replicates No. |
Total Number of Cells |
CBPI |
Mean CBPI |
% of Cytotoxicity |
Vehicle Control |
Without S9 |
0 |
3 to 6 |
1 |
510 |
1.64 |
1.62 |
0.00 |
2 |
514 |
1.61 |
||||||
Negative control |
0 |
1 |
519 |
1.63 |
1.61 |
1.61 |
||
2 |
520 |
1.59 |
||||||
Barium 4-dodecylphenolate |
0.004883 |
1 |
505 |
1.47 |
1.46 |
25.81 |
||
2 |
511 |
1.46 |
||||||
0.009766 |
1 |
520 |
1.43 |
1.44 |
29.03 |
|||
2 |
513 |
1.45 |
||||||
0.0195313 |
1 |
504 |
1.45 |
1.45 |
27.42 |
|||
2 |
522 |
1.44 |
||||||
0.0390625 |
1 |
510 |
1.42 |
1.41 |
33.87 |
|||
2 |
517 |
1.41 |
||||||
0.078125 |
1 |
537 |
1.39 |
1.39 |
37.10 |
|||
2 |
546 |
1.39 |
||||||
0.15625 |
1 |
505 |
1.31 |
1.30 |
51.61 |
|||
2 |
504 |
1.29 |
||||||
0.3125 |
1 |
505 |
1.10 |
1.11 |
82.26 |
|||
2 |
512 |
1.11 |
Table 2c. Summary - Follow up initial cytotoxicity test
Treatment |
Metabolic Activation |
Concentration (mg/mL) |
Duration (hours) |
Replicates No. |
Total Number of Cells |
CBPI |
Mean CBPI |
% of Cytotoxicity |
Vehicle Control |
Without S9 |
0 |
20 to 24 |
1 |
531 |
1.61 |
1.61 |
0.00 |
2 |
545 |
1.61 |
||||||
Negative control |
0 |
1 |
523 |
1.61 |
1.61 |
0.00 |
||
2 |
525 |
1.61 |
||||||
Barium 4-dodecylphenolate |
0.004883 |
1 |
519 |
1.49 |
1.48 |
21.31 |
||
2 |
506 |
1.48 |
||||||
0.009766 |
1 |
532 |
1.49 |
1.49 |
19.67 |
|||
2 |
517 |
1.49 |
||||||
0.0195313 |
1 |
510 |
1.44 |
1.45 |
26.23 |
|||
2 |
506 |
1.46 |
||||||
0.0390625 |
1 |
519 |
1.45 |
1.46 |
24.59 |
|||
2 |
533 |
1.47 |
||||||
0.078125 |
1 |
520 |
1.40 |
1.39 |
36.07 |
|||
2 |
545 |
1.38 |
||||||
0.15625 |
1 |
511 |
1.29 |
1.30 |
50.82 |
|||
2 |
518 |
1.32 |
||||||
0.3125 |
1 |
502 |
1.09 |
1.10 |
83.61 |
|||
2 |
517 |
1.10 |
Table 3a. Summary of micronuclei incidence – Micronucleus test
Treatment |
Concentration (mg/mL) |
Average CBPI |
Average % of Cytotoxicity |
Total No. of Binucleates |
Total No. of Micronucleus |
Average Percentage of Micronucleus |
With Metabolic Activation - 3 to 6 Hours |
||||||
NegativeControl |
0 |
1.59 |
0.00 |
2072 |
4 |
0.19 |
VehicleControl |
0 |
1.58 |
1.89 |
2106 |
3 |
0.14 |
Barium 4-dodecylphenolate |
0.009766 |
1.47 |
18.97 |
2029 |
3 |
0.15 |
0.0195313 |
1.46 |
20.69 |
2066 |
4 |
0.19 |
|
0.0390625 |
1.42 |
27.59 |
2046 |
3 |
0.15 |
|
0.078125 |
1.39 |
32.76 |
2026 |
3 |
0.15 |
|
0.15625 |
1.30 |
48.28 |
2026 |
4 |
0.20 |
|
Cyclophosphamide Monohydrate |
10 µg/mL |
1.52 |
10.34 |
2055 |
22 |
1.07* |
15 µg/mL |
1.49 |
15.52 |
2014 |
33 |
1.64* |
|
Note: For cytotoxicity and percentage of micronucleus calculation, vehicle control compared with negative control and test material values compared with vehicle control.
CBPI:Cytokinesis Block Proliferation Index, *: Statistically Significant.
Table 3b. Summary of micronuclei incidence – Micronucleus test
Treatment |
Concentration (mg/mL) |
Average CBPI |
Average % of Cytotoxicity |
Total No. of Binucleates |
Total No. of Micronucleus |
Average Percentage of Micronucleus |
Without Metabolic Activation - 3 to 6 Hours |
||||||
NegativeControl |
0 |
1.63 |
0.00 |
2221 |
4 |
0.18 |
VehicleControl |
0 |
1.64 |
0.00 |
2139 |
3 |
0.14 |
Barium 4-dodecylphenolate |
0.009766 |
1.53 |
17.19 |
2138 |
3 |
0.14 |
0.0195313 |
1.50 |
21.88 |
2019 |
3 |
0.15 |
|
0.0390625 |
1.48 |
25.00 |
2080 |
4 |
0.19 |
|
0.078125 |
1.42 |
34.38 |
2092 |
5 |
0.24 |
|
0.15625 |
1.32 |
50.00 |
2032 |
5 |
0.25 |
|
Colchicine |
0.03 µg/mL |
1.53 |
17.19 |
2029 |
22 |
1.08* |
0.012 µg/mL |
1.56 |
12.5 |
2131 |
15 |
0.70* |
|
Note: For cytotoxicity and percentage of micronucleus calculation, vehicle control compared with negative control and test material values compared with vehicle control.
CBPI:Cytokinesis Block Proliferation Index, *: Statistically Significant.
Table 3c. Summary of micronuclei incidence – Micronucleus test
Treatment |
Concentration (mg/mL) |
Average CBPI |
Average % of Cytotoxicity |
Total No. of Binucleates |
Total No. of Micronucleus |
Average Percentage of Micronucleus |
Without Metabolic Activation - 20 to 24 Hours |
||||||
NegativeControl |
0 |
1.60 |
0.00 |
2063 |
3 |
0.15 |
VehicleControl |
0 |
1.64 |
0.00 |
2105 |
3 |
0.14 |
Barium 4-dodecylphenolate |
0.009766 |
1.53 |
17.19 |
2051 |
3 |
0.15 |
0.0195313 |
1.50 |
21.88 |
2028 |
4 |
0.20 |
|
0.0390625 |
1.44 |
31.25 |
2002 |
3 |
0.15 |
|
0.078125 |
1.42 |
34.38 |
2043 |
4 |
0.20 |
|
0.15625 |
1.32 |
50.00 |
2021 |
4 |
0.20 |
|
Mitomycin C |
0.075 µg/mL |
1.53 |
15.87 |
2025 |
21 |
1.04* |
0.15 µg/mL |
1.49 |
22.22 |
2025 |
29 |
1.43* |
|
Note: For cytotoxicity and percentage of micronucleus calculation, vehicle control compared with negative control and test material values compared with vehicle control.
CBPI:Cytokinesis Block Proliferation Index, *: Statistically Significant.
VEGA - v. 1.1.4 predicted that phenol, dodecyl-, branched does not cause gene mutation.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
In vitro gene mutation in bacteria:
In order to evaluate the potential of the substance Barium 4 -dodecylphenolate to induce gene mutation in bacteria, information on the moieties barium and 4 -dodecylphenolate were considered. For a documentation and justification of that approach, please refer to the separate document attached to section 13, namely Read Across Assessment Report for Barium 4 -dodecylphenolate.
Barium:
In a NTP study (1994), Barium dichloride dihydrate was evaluated for its ability to induce reverse mutations in histidine-requiring tester strains of Salmonella typhimurium. Tester strains TA 97, TA 98, TA 100, TA 1535, and TA 1537 were exposed to Barium dichloride dihydrate at dose levels up to 10000 µg per plate both in absence and presence of a S9 metabolic activation system. Following Barium dichloride dihydrate treatments of all the tester strains in the absence and presence of S9, no increases in revertant colony numbers were observed.
4 -Dodecylphenolate:
Based on a QSAR prediction (Mutagenicity (Ames test) model - VEGA), phenol, dodecyl-, branched is not considered to cause gene mutation in bacteria. Phenol, dodecyl-, branched is within the applicability domain of all independent prediction models within VEGA. The prediction is reliable and adequate to replace an Ames test to fulfil requirements for Regulation (EC) No 1907/2006 (REACH) Annex VII section 8.4.1.
In vitro micronucleus in mammalian cells:
Barium 4 -dodecylphenolate:
Rashmi (2020) investigated the potential clastogenic and aneugenic activity of Barium 4-dodecylphenolate in an in vitro mammalian cell micronucleus test according to OECD TG 487 (2016) and under GLP. Human peripheral blood lymphocytes were exposed to Barium 4 -dodecylphenolate up to a cytotoxic concentration of 0.15625 mg/mL both in absence (for 3 or ca. 24 hours) and presence (for 3 hours) of metabolic activation system (S9 fraction from phenobarbitone/β-naphthoflavone induced rat livers). None of the treatments with Barium 4 -dodecylphenolate resulted in a biologically relevant increase in the micronucleus frequency. Appropriate positive controls demonstrated the sensitivity of the test system.
In vitro gene mutation in mammalian cells:
Barium 4 -dodecylphenolate:
Jagadeesh (2020) examined the gene mutation potential of Barium 4 -dodecylphenolate in an in vitro mammalian cell gene mutation test at the Hprt locus according to OECD TG 476 (2016) and under GLP. Chinese hamster ovarian AA8 cells were exposed for 3 hours to Barium 4 -dodecylphenolate up to a cytotoxic concentration of 0.15625 mg/mL with and without metabolic activation (S9 fraction from phenobarbitone/β-naphthoflavone induced rat livers). No positive mutagenic responses were observed at any concentration in either the absence or presence of S9 metabolic activation. Appropriate positive controls demonstrated the sensitivity of the test system.
Overall conclusion:
Barium 4 -dodecylphenolate is not expected to be genotoxic, since Barium 4 -dodecylphenolate has neither shown any gene mutation potential nor clastogenic or aneugenic activity in mammalian cells. A mutagenic potential in bacteria is not anticipated, since the two moieties, barium and 4 -dodecylphenolate have not shown any evidence for mutagenic activity in a bacterial reverse mutation assay or in the VEGA mutagenicity (Ames test) prediction model, respectively. Further testing is not required. Thus, Barium 4-dodecylphenolate is not to be classified according to regulation (EC) 1272/2008 and its subsequent amendments as genetic toxicant. For further information on the toxicity of the individual constituents, please refer to the relevant sections in the IUCLID and CSR.
Justification for classification or non-classification
Barium 4 -dodecylphenolate showed no mutagenicity in a mammalian cell gene mutation test (Hprt assay) or in an in vitro micronucleus test. Moreover, data from both moieties, barium and 4-dodecylphenolate, indicated no gene mutation potential in bacteria.
The classification criteria according to regulation (EC) 1272/2008 and its subsequent amendments as germ cell mutagen are not met, thus no classification is required.
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