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Toxicological information

Genetic toxicity: in vitro

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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

Constituent 1
Chemical structure
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.TreatmentConcentration (mg/mL)Average Colony Count ± SDCloning Efficiency
(CE)
Adjusted Cloning Efficiency (ACE)Relative Survival (RS) (%)
Set 1 +S9Vehicle Control
(DMSO)
-187.33±5.130.941.21-
Ultramarine Violet Pigment Violet 15 CI 770070.03125182.67±3.060.911.0990.08
0.0625183.00±3.610.921.0385.12
0.125181.33±3.790.910.9578.51
0.25169.33±4.730.850.7965.29
0.5153.67±5.690.770.6150.41
Set 2
-S9
Vehicle Control
(DMSO)
-185.33±5.510.931.18-
Ultramarine Violet Pigment Violet 15 CI 770070.03125179.00±3.000.901.0790.68
0.0625175.00±5.570.881.0185.59
0.125171.33±3.210.860.9177.12
0.25165.67±8.330.830.8067.80
0.5156.00±7.940.780.6252.54

+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.TreatmentConcentration (mg/mL)Average Colony Count ± SDCloning Efficiency
(CE)
Adjusted Cloning Efficiency (ACE)Relative Survival (RS) (%)
Set 1 +S9Vehicle Control
(DMSO)
-186.00±6.930.931.15-
Ultramarine Violet Pigment Violet 15 CI 770070.0625174.00±4.000.870.9784.35
0.125165.67±8.500.830.8473.04
0.25165.67±4.930.830.7464.35
0.5155.33±6.110.780.5850.43
Benzo(a)pyrene (Positive Control)3 μg/mL177.33±3.060.891.0591.30
Set 2
-S9
Vehicle Control
(DMSO)
-187.00±3.610.941.16-
Ultramarine Violet Pigment Violet 15 CI 770070.0625178.33±4.040.890.9783.62
0.125168.00±10.580.840.8875.86
0.25168.00±5.290.840.7463.79
0.5155.67±4.510.780.6051.72
4 Nitroquinoline N-oxide
(Positive Control)
1 μg/mL182.00±9.850.911.0792.24

+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.TreatmentConcentration (mg/mL)*Average Colony Count ± SDCloning Efficiency
in selective media
Cloning Efficiency
in non-selective media
Total number of mutant colonies / 2x106 cellsMutant Frequency/ 2x106 cells
Set 1 +S9Vehicle Control
(DMSO)
-186.67±1.150.00001050.932122.58
Ultramarine Violet Pigment Violet 15 CI 770070.0625182.00±2.000.00001050.912123.08
0.125174.67±4.160.00001000.872022.99
0.25177.67±2.520.00001050.892123.60
0.5171.00±3.610.00001000.862023.26
Benzo(a)pyrene (Positive Control)3 μg/mL182.67±7.570.00012200.91244268.13**
Set 2
-S9
Vehicle Control
(DMSO)
-186.00±4.580.00001150.932324.73
Ultramarine Violet Pigment Violet 15 CI 770070.0625181.33±4.160.00001150.912325.27
0.125175.00±5.000.00001100.882225.00
0.25175.67±5.090.00001100.882225.00
0.5174.67±7.570.00001100.872225.29
4 Nitroquinoline N-oxide
(Positive Control)
1 μg/mL183.33±5.860.00001150.92243264.13**

+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


(×106)


 
 

 


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 (×106)



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/2×106Cells



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-DMSOWith Metabolic Activation
(3 to 6 hours)
Without Metabolic Activation
(3 to 6 hours)
Mean Data of Mutant Frequency/2x106 Cells24.5125.43
Standard
Deviation
2.811.89
Margin of Error1.951.31
Upper bound26.4626.74
Lower bound22.5624.12


































Positive Control- Benzo(a)pyrene and 4- Nitroquinoline N-oxideWith 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/2x106 Cells261.94264.60
Standard
Deviation
27.2818.52
Margin of Error17.8212.10
Upper bound279.76276.70
Lower bound244.12252.50

 

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.