Violet sodium polysulfide aluminosilicate with a SOD-type framework structure

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:

19 July 2020 to 20 August 2020

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:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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:

Violet sodium polysulfide aluminosilicate with a SOD-type framework structure

EC Number:

701-186-2

Molecular formula:

|Na+6-x+y+z (S2•-)y (S3•-)z (S4)t|[Al6-x Si6+x O24] – SOD Where: 6 ≤ 6-x+ y+z ≤ 8 0 ≤ x ≤ 1.2 0 < y+z +t ≤ 2 t > 0 SOD = Sodalite framework structure

IUPAC Name:

Violet sodium polysulfide aluminosilicate with a SOD-type framework structure

Test material form:

solid: particulate/powder

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 cells, Batch No.5000062 procured from American Type Culture Collection (ATCC)
- Suitability of cells: The CHO AA8 cells are one of the recommended test systems by regulatory agencies for conducting in vitro mammalian gene mutation test.

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: Alpha Minimal Essential Medium (MEM) without ribonucleosides containing 10% Fetal Bovine Serum (FBS) and antibiotics (1% Penicillin and streptomycin) were used. The pH of the culture medium was 7.32 to 7.33. The media was stored at 2 to 8ºC till use thawed to room temperature before use.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: liver of male Wistar rats.
- method of preparation of S9 mix: The S9 homogenate was prepared from male wistar rats 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. 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 7.32 and 7.33.
- concentration or volume of S9 mix and S9 in the final culture medium:
1 mL, 10% (v/v) S9 mix and 0.1 mL, 1% (v/v) S9.
- quality controls of S9: Batch of S9 homogenate was assessed for sterility, protein content and for its ability to metabolize the promutagens 2-Aminoanthracene and Benzo(a)pyrene to mutagens using Salmonella typhimurium TA100 strain.

Test concentrations with justification for top dose:

0.0625, 0.125, 0.25 and 0.5 mg/mL.
The top dose was selected based on preliminary solubility and precipitation tests as well as an initial cytotoxicity test.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: based on the solubility results.
- Percentage of solvent in the final culture medium: 1% v/v.

Controls

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

benzo(a)pyrene

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: 3 (cytotoxicity and cloning efficiency in non-selective medium) and 5 (cloning efficiency of mutant colonies in selective medium)
- Number of independent experiments: 1

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): approx. 2 x 10^6 cells/culture flask (Initial cytotoxicity test and Gene mutation test).
- Test substance added in medium.

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 3 hours and 2 minutes at 37±1ºC with 5±1% CO2.
- Harvest time after the end of treatment (sampling/recovery times): 7 days

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 8 days
- Selection time (if incubation with a selective agent): 8 days.
- Method used: monolayer cultures.
- Selective agent: 10 μM of 6-Thioguanine. Flasks were incubated at 37±1°C with 5±1% CO2 for 8 days.
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: 200 x 3 per group (cytotoxicity and cloning efficiency in non-selective medium) and 4x10^5 x 5 per group (cloning efficiency of mutant colonies in selective medium). Flasks were incubated at 37±1°C with 5±1% CO2 for 8 days. Post incubation period, medium from each dish was aspirated and stained with 5% Giemsa stain, number of colonies formed were counted manually.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: relative survival (RS)
- Any supplementary information relevant to cytotoxicity: An initial cytotoxicity test was carried out at five different concentrations (0.03125, 0.0625, 0.125, 0.25 and 0.5 mg/mL of the test item). Cytotoxicity was assessed by determining the Adjusted Cloning Efficiency and Relative Survival according to formulas included in annex 2 of OECD TG 476.

Rationale for test conditions:

In preliminary solubility and precipitation tests, heavy precipitations were observed at 1 and 2 mg/mL while only slight precipitation was observed at 0.5 mg/mL which was selected as highest dose in the initial cytotoxicity test. At 0.5 mg/mL, the Relative Survival was greater than 10%. Therefore 0.5 mg/mL was selected as the highest concentration for testing in the gene mutation test.

Evaluation criteria:

A test chemical is considered to be clearly positive if, in any of the experimental conditions examined:
1) At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
2) The increase is concentration-related when evaluated with an appropriate trend test.
3) 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.

A test chemical is considered clearly negative if, in all experimental conditions examined:
1) None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
2) There is no concentration-related increase when evaluated with an appropriate trend test.
3) All results are inside the distribution of the historical negative/vehicle control data.
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)eB, where Y = transformed mutant frequency, X = observed mutant frequency and 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.
The statistical significances are designated by the superscripts as given below:
* Statistically significant (p<0.05) change than the vehicle control group.

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

CHO AA8 Cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: No change in pH was observed in any of the test concentrations.
- Possibility of evaporation from medium: no
- Precipitation and time of the determination: slight precipitation was observed at 0.5 mg/mL, heavy precipitation was observed at 1 and 2 mg/mL.

RANGE-FINDING/SCREENING STUDIES (if applicable):

In the initial cytotoxicity test, there was no evidence of excessive cytotoxicity (˂10% RS) up to 0.5 mg/mL in both presence of metabolic activation and absence of metabolic activation when compared to vehicle control. In the presence of metabolic activation, the RS values ranged from 50.41 % to 90.08 % and in the absence of metabolic activation, the RS values ranged from 52.54 % to 90.68 % at the concentrations of 0.03125, 0.0625, 0.125, 0.25, 0.5 mg/mL.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: mutant frequencies of 22.58 and 24.73 per 2×10^6 cells were observed in the vehicle control, with and without metabolic activation, respectively. Positive controls Benzo (a) pyrene and 4-Nitroquinoline N-oxide gave mutant frequencies of 268.13 and 264.13 per 2×10^6 cells, respectively.

Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements:
o Relative survival (RS): There was no evidence of excessive cytotoxicity (˂10% RS) at any of the concentrations both in presence and absence of metabolic activation. In the presence of metabolic activation, the RS values ranged from 50.43 to 84.35 % and in the absence of metabolic activation the RS values ranged from 51.72 to 83.62 % respectively.

- Genotoxicity results:
The test item, resulted in mutant frequencies of 22.99 to 23.60 per 2×10^6 cells in the presence of metabolic activation and mutant frequencies of 25.00 to 25.29 per 2×10^6 cells in the absence of metabolic activation.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: see Table 7
- Negative (solvent/vehicle) historical control data: see Table 7

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 (DMSO)	-	187.33±5.13	0.94	1.21	-
	Ultramarine Violet Pigment Violet 15 CI 77007	0.03125	182.67±3.06	0.91	1.09	90.08
		0.0625	183.00±3.61	0.92	1.03	85.12
		0.125	181.33±3.79	0.91	0.95	78.51
		0.25	169.33±4.73	0.85	0.79	65.29
		0.5	153.67±5.69	0.77	0.61	50.41
Set 2 -S9	Vehicle Control (DMSO)	-	185.33±5.51	0.93	1.18	-
	Ultramarine Violet Pigment Violet 15 CI 77007	0.03125	179.00±3.00	0.90	1.07	90.68
		0.0625	175.00±5.57	0.88	1.01	85.59
		0.125	171.33±3.21	0.86	0.91	77.12
		0.25	165.67±8.33	0.83	0.80	67.80
		0.5	156.00±7.94	0.78	0.62	52.54

+S9: with metabolic activation; -S9: without metabolic activation;
*Note: Cloning Efficiency = 200 cells plated for each replicate.
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.

 

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 (DMSO)	-	186.00±6.93	0.93	1.15	-
	Ultramarine Violet Pigment Violet 15 CI 77007	0.0625	174.00±4.00	0.87	0.97	84.35
		0.125	165.67±8.50	0.83	0.84	73.04
		0.25	165.67±4.93	0.83	0.74	64.35
		0.5	155.33±6.11	0.78	0.58	50.43
	Benzo(a)pyrene (Positive Control)	3 μg/mL	177.33±3.06	0.89	1.05	91.30
Set 2 -S9	Vehicle Control (DMSO)	-	187.00±3.61	0.94	1.16	-
	Ultramarine Violet Pigment Violet 15 CI 77007	0.0625	178.33±4.04	0.89	0.97	83.62
		0.125	168.00±10.58	0.84	0.88	75.86
		0.25	168.00±5.29	0.84	0.74	63.79
		0.5	155.67±4.51	0.78	0.60	51.72
	4 Nitroquinoline N-oxide (Positive Control)	1 μg/mL	182.00±9.85	0.91	1.07	92.24

+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 / 2x10^6 cells	Mutant Frequency/ 2x10^6 cells
Set 1 +S9	Vehicle Control (DMSO)	-	186.67±1.15	0.0000105	0.93	21	22.58
	Ultramarine Violet Pigment Violet 15 CI 77007	0.0625	182.00±2.00	0.0000105	0.91	21	23.08
		0.125	174.67±4.16	0.0000100	0.87	20	22.99
		0.25	177.67±2.52	0.0000105	0.89	21	23.60
		0.5	171.00±3.61	0.0000100	0.86	20	23.26
	Benzo(a)pyrene (Positive Control)	3 μg/mL	182.67±7.57	0.0001220	0.91	244	268.13**
Set 2 -S9	Vehicle Control (DMSO)	-	186.00±4.58	0.0000115	0.93	23	24.73
	Ultramarine Violet Pigment Violet 15 CI 77007	0.0625	181.33±4.16	0.0000115	0.91	23	25.27
		0.125	175.00±5.00	0.0000110	0.88	22	25.00
		0.25	175.67±5.09	0.0000110	0.88	22	25.00
		0.5	174.67±7.57	0.0000110	0.87	22	25.29
	4 Nitroquinoline N-oxide (Positive Control)	1 μg/mL	183.33±5.86	0.0000115	0.92	243	264.13**

+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 4. Individual data of initial cytotoxicity test.

Set No.	Treatment	Concentration (mg/mL)	Replicate	No. of Colonies/200 Cells
				R1	R2	R3
Set 1 +S9	Vehicle Control (DMSO)	-	3	193	186	183
	Ultramarine Violet Pigment Violet 15 CI 77007	0.03125	3	180	186	182
		0.0625	3	184	186	179
		0.125	3	177	184	183
		0.25	3	173	164	171
		0.5	3	149	152	160
Set 2 -S9	Vehicle Control (DMSO)	-	3	191	180	185
	Ultramarine Violet Pigment Violet 15 CI 77007	0.03125	3	182	179	176
		0.0625	3	176	180	169
		0.125	3	175	169	170
		0.25	3	159	163	175
		0.5	3	159	162	147

 

Set No.	Treatment	Concentration (mg/mL)		Cell count (×10^6)
Set 1 +S9	End of Treatment	Vehicle Control  (DMSO)		27.60
		Ultramarine Violet Pigment Violet 15 CI 77007	0.03125	25.80
			0.0625	24.00
			0.125	22.50
			0.25	20.10
			0.5	16.95
Set 2    -S9	End of Treatment	Vehicle Control  (DMSO)		27.30
		Ultramarine Violet Pigment Violet 15 CI 77007	0.03125	25.50
			0.0625	24.60
			0.125	22.65
			0.25	20.70
			0.5	17.10
Beginning of the Treatment				21.50

 

Table 5. Individual data of parallel cytotoxicity test.

Set No.	Treatment	Concentration (mg/mL)	Replicate	No. of Colonies/200 Cells
				R1	R2	R3
Set 1 +S9	Vehicle Control  (DMSO)	-	3	190	190	178
	Ultramarine Violet Pigment Violet 15 CI 77007	0.0625	3	170	174	178
		0.125	3	169	172	156
		0.25	3	160	168	169
		0.5	3	154	150	162
	Benzo(a)pyrene                     (Positive Control)	3 µg/mL	3	180	178	174
Set 2 -S9	Vehicle Control  (DMSO)	-	3	190	188	183
	Ultramarine Violet Pigment Violet 15 CI 77007	0.0625	3	179	182	174
		0.125	3	176	172	156
		0.25	3	170	162	172
		0.5	3	151	156	160
	4 Nitroquinoline N-oxide             (Positive Control)	1 µg/mL	3	190	185	171

 

Set No.	Treatment	Concentration (mg/mL)		Cell Count (×10^6)
Set 1 +S9	End of Treatment	Vehicle Control  (DMSO)		27.30
		Ultramarine Violet Pigment Violet 15 CI 77007	0.0625	24.45
			0.125	22.35
			0.25	19.65
			0.5	16.35
		Positive Control Benzo(a)pyrene (3 µg/mL)		25.95
Set 2    -S9	End of Treatment	Vehicle Control  (DMSO)		27.15
		Ultramarine Violet Pigment Violet 15 CI 77007	0.0625	24.00
			0.125	22.95
			0.25	19.50
			0.5	16.80
		Positive Control 4 Nitroquinoline N-oxide (1 µg/mL)		25.80
Beginning of the Treatment				22.00

 

Table 6. Individual data of mutant phenotype

Set No.	Treatment	Concentration (mg/mL)	No. of Colonies/200 Cells			No. of Mutant Colonies/2×10^6Cells
			Replicate 1	Replicate 2	Replicate 3	Replicate 1	Replicate 2	Replicate 3	Replicate 4	Replicate 5
Set 1 +S9	Vehicle Control   (DMSO)	-	186	188	186	5	4	4	6	2
	Ultramarine  Violet Pigment Violet 15 CI 77007	0.0625	180	184	182	4	4	2	8	3
		0.125	176	170	178	0	6	5	4	5
		0.25	180	178	175	3	8	2	6	2
		0.5	175	170	168	2	2	10	1	5
	Benzo(a) pyrene                (Positive Control)	3 µg/mL	188	186	174	43	48	52	55	46
Set 2  -S9	Vehicle Control (DMSO)	-	185	191	182	8	3	5	5	2
	Ultramarine   Violet Pigment Violet 15 CI 77007	0.0625	178	186	180	5	4	3	7	4
		0.125	180	170	175	2	1	7	9	3
		0.25	176	179	175	4	6	5	2	5
		0.5	180	178	166	9	1	7	0	5
	4 Nitroquinoline N-oxide (Positive Control)	1 µg/mL	190	179	181	52	47	43	49	52

Note: For cloning efficiency 200 cells were plated for each replicate and the No. of Mutant Colonies mentioned in the table is obtained from 2 x106 cells (that is from 5 replicates).

 

Table 7. Historical data.

Vehicle-DMSO	With Metabolic Activation (3 to 6 hours)	Without Metabolic Activation (3 to 6 hours)
Mean Data of Mutant Frequency/2x10^6 Cells	24.51	25.43
Standard Deviation	2.81	1.89
Margin of Error	1.95	1.31
Upper bound	26.46	26.74
Lower bound	22.56	24.12

Positive Control- Benzo(a)pyrene and 4- Nitroquinoline N-oxide	With Metabolic Activation (3 to 6 hours) [Benzo(a)pyrene]	Without Metabolic Activation (3 to 6 hours)[4 Nitroquinoline N-oxide]
Mean Data of Mutant Frequency/2x10^6 Cells	261.94	264.60
Standard Deviation	27.28	18.52
Margin of Error	17.82	12.10
Upper bound	279.76	276.70
Lower bound	244.12	252.50

 

Applicant's summary and conclusion

Conclusions:

In an in vitro HPRT gene mutation test using CHO AA8 cells, the test item is considered as non-mutagenic up to the concentration of 0.5 mg/mL, both in presence and absence of metabolic activation.

Executive summary:

A gene mutation study at HPRT gene using CHO AA8 cells was performed for the test item, with and without metabolic activation (±S9), according to OECD 476 Guideline (GLP study). Based on the results of the solubility, pH and precipitation tests, the test item was formulated in DMSO as vehicle and 0.5 mg/mL was selected as the highest concentration in an initial cytotoxicity test. This test was conducted at concentrations of 0.03125, 0.0625, 0.125, 0.25 and 0.5 mg/mL. There was no evidence of excessive cytotoxicity (˂10% RS) at and up to 0.5 mg/mL in both presence and absence of metabolic activation when compared to vehicle control. Therefore, 0.5 mg/mL was selected as the highest concentration in the gene mutation test. The main test was conducted at the concentrations of 0.0625, 0.125, 0.25 and 0.5 mg/mL with an exposure time of 3 h and 2 min both in the presence and absence of metabolic activation and 6-Thioguanine as the selective agent. DMSO alone was tested as solvent control and Benzo(a) pyrene and 4 Nitroquinoline N-oxide were used as positive controls. Parallel cytotoxicity was assessed by determining the Adjusted Cloning Efficiency and Relative Survival. There was no statistically significant increase in number of mutant colonies at any of the concentrations tested when compared with the vehicle control. All results were inside the distribution of the historical vehicle control data. Positive controls resulted in mutant frequencies, which were statistically significant when compared with the vehicle control and within the historical positive control data. Based on these results, the test item is considered as non-mutagenic up to the concentration of 0.5 mg/mL, both in the presence and absence of metabolic activation under the tested conditions.