Barium 4-dodecylphenolate

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

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

Test guideline 2

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Remarks: "The EU method is outdated and does not reflect the latest update of the corresponding OECD method.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

Test material

Test material information

Constituent 1

Reference substance name:

Barium 4-dodecylphenolate

EC Number:

300-141-0

EC Name:

Barium 4-dodecylphenolate

Cas Number:

93922-04-4

Molecular formula:

C18H30O.1/2Ba

IUPAC Name:

barium 4-dodecylphenolate

Test material form:

solid

Details on test material:

- State of aggregation: brownish, inhomogeneous solid with a chewy texture

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'

Method

Target gene:

Hprt

Species / strain

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

Controls

Controls 1

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

Controls 2

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.

Results and discussion

Test results

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.

Any other information on results incl. tables

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/ 2×10^6cells	Mutant Frequency/ 2×10^6cells
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                    

Applicant's summary and conclusion

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.