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Genetic toxicity in vitro

Description of key information

The in vitro genetic toxicity of Zirconium zircon with encapsulated cadmium selenium sulphide has been evaluated in a bacterial reverse gene mutation assay (acc. to OECD TG 471) and in a mammalian cell gene mutation assay (acc. to OECD TG 476). These studies were performed according to the current guidelines and in compliance with GLP and were evaluated to be reliable without restrictions.

Link to relevant study records

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Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 October 2021 - 10 January 2022
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
corrected June 26, 2020
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
dated May 30, 2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
GLP certificate signed on 23 Oktober 2019
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Solubility of the test material in the solvent/vehicle: Due to the insolubility of the test material, DMSO was used to obtain a homogenous suspension.
Target gene:
hisD (TA 98), hisG (TA 100, TA 1535), hisC (TA 1537), and trpE (WP2 uvr A pKM101)
Species / strain / cell type:
S. typhimurium TA 98
Species / strain / cell type:
S. typhimurium TA 100
Species / strain / cell type:
S. typhimurium TA 1535
Species / strain / cell type:
S. typhimurium TA 1537
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9: The S9 was prepared in-house. Phenobarbital/β-naphthoflavone induced male Wistar rat liver S9 were used as the metabolic activation system.
- method of preparation of S9 mix: An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution, to result in a final concentration of approx. 10% (v/v) in the S9 mix. Cofactors were added to the S9 mix to reach the following concentrations in the S9 mix: 8 mM MgCl2, 33 mM KCl, 5 mM glucose-6-phosphate, and 4 mM NADP in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.
- concentration or volume of S9 mix and S9 in the final culture medium: 500 μL S9 mix (containing 10% (v/v) S9)
- quality controls of S9: sterility and metabolic capability
Test concentrations with justification for top dose:
- Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
- Experiment II: 33; 100; 333; 1000; 2500; and 5000 μg/plate
The top concentration was the recommended maximum test concentration for this assay.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO

- Justification for choice of solvent/vehicle:
The solvent (solvent is used as a technical term even though the formulation was a suspension) was chosen because of its solubility properties and its relative nontoxicity to the bacteria (Maron et al.; 1981)*. Importantly, treatments in this study were performed using formulations prepared as homogeneous suspensions due to the insolubility of the test item.

*References:
- Maron, D.M., J. Katzenellenbogen, and B.N. Ames (1981) Compatibility of organic solvents with the Salmonella/Microsome Test. Mutation Res. 88, 343-350.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine (4-NOPD); 2-aminoanthracene (2-AA)
Details on test system and experimental conditions:
CONCENTRATION SELECTION
In the pre-experiment the concentration range of the test item was 3 - 5000 μg/plate. The pre-experiment is reported as experiment I. Since no toxic effects were observed 5000 μg/plate were chosen as maximal concentration. The concentration range included two logarithmic decades.
The following concentrations were tested in experiment II: 33; 100; 333; 1000; 2500; and 5000 μg/plate

BACTERIAL REVERSE MUTATION ASSAY
For each strain and dose level, including the controls, three plates were used.

Experiment I (Plate Incorporation):
The following materials were mixed in a test tube and poured onto the selective agar plates: 100 μL Test suspension at each dose level (solvent or reference mutagen solution (positive control)), 500 μL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation), 100 μL bacteria suspension (cf. 3.4.3 Precultures), 2000 μL overlay agar

Experiment II (Pre-Incubation):
The following materials were mixed in a test tube and incubated at 37°C±1.5°C for 60 minutes: 100 μL Test suspension at each dose level (solvent or reference mutagen solution (positive control)), 500 μL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation), 100 μL Bacteria suspension (cf. 3.4.3 Precultures). After pre-incubation 2.0 mL overlay agar (45°C) was added to each tube.

The mixture was poured on minimal agar plates. After solidification the plates were incubated upside down for 72 hours at 37°C±1.5°C in the dark.
In parallel to each test a sterile control of the test item was performed and documented in the raw data. Therefore, 100μL of the stock suspension, 500 μL S9 mix / S9 mix substitution buffer were mixed with 2.0 mL overlay agar and poured on minimal agar plates.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Toxicity of the test item results in a reduction in the number of spontaneous revertants (below a factor of 0.5) or a clearing of the bacterial background lawn.

METHODS FOR MEASUREMENTS OF GENOTOXICIY
The colonies were counted using a validated computer system (Petri Viewer Sorcerer Colony Counter 3.0 (Instem, Suffolk IP33 3TA, UK) with the software program Ames Study Manager (v1.24) and Ames Archive Manager (v1.01)).
Evaluation criteria:
- A test item is considered as a mutagen if a biologically relevant increase in the number of revertants of twofold or above (strains TA 98, TA 100, and WP2 uvrA (pKM101)) or threefold or above (strains TA 1535 and TA 1537) the spontaneous mutation rate of the corresponding solvent control is observed.
- A dose dependent increase is considered biologically relevant if the threshold is reached or exceeded at more than one concentration.
- An increase of revertant colonies equal or above the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
- A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
Statistics:
Statistical analysis of the data is not mandatory
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
Experiment I: but tested up to recommended limit concentration; Experiment II: but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
Experiment I: but tested up to recommended limit concentration; Experiment II: but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
Experiment I: but tested up to recommended limit concentration; Experiment II: but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
Experiment I: but tested up to recommended limit concentration; Experiment II: but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
Experiment I: but tested up to recommended limit concentration; Experiment II: but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- The test item precipitated in the overlay agar in the test tubes from 1000 to 5000 μg/plate in both experiments. Precipitation of the test item in the overlay agar on the incubated agar plates was observed in experiment II at 5000 μg/plate. The undissolved particles had no influence on the data recording.

TOXICITY
- The plates incubated with the test item showed normal background growth up to 5000 μg/plate with and without S9 mix in all strains used.
- No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all strains with and without metabolic activation.

GENOTOXICITY RESULTS
- No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Silicic acid, zirconium salt, cadmium pigment-encapsulated at any dose level, neither in the presence nor absence of metabolic activation (S9 mix) (please refer to "Attached background material: Results"). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

ASSAY VALIDITY
- Appropriate reference mutagens were used as positive controls. They showed a distinct increase in the number of revertant colonies, which fell in the expected range (please refer to "Attached background material: Historical control data").
- Vehicle and untreated control treatments were included for all strains in both experiments. The mean number of revertant colonies fell within acceptable ranges of the historical control database.
Thus, the controls demonstrated sensitivity of the test systems and the validity of the assay.
Conclusions:
No toxicity (thinning of the background lawn or a reduction in the number of revertants) was found in both experiments. Precipitation of the test item in the overlay agar on the incubated agar plates was observed in experiment II at 5000 μg/plate. Silicic acid, zirconium salt, cadmium pigment-encapsulated (Zirconium zircon with encapsulated selenium sulphide), tested up to the recommended limit concentration and precipitating concentrations, did not induce biologically relevant increases in the number of revertant colonies. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. All validity criteria were met. The study was fully compliant with OECD 471 (2020).

Therefore, Silicic acid, zirconium salt, cadmium pigment-encapsulated (Zirconium zircon with encapsulated selenium sulphide) is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 October 2021 - 10 February 2022
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:
adopted 29 July 2016
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
dated May 30, 2008
Deviations:
no
GLP compliance:
yes
Remarks:
GLP certificate signed on 23 Oktober 2019
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Target gene:
Hprt
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: Chinese hamster lung fibroblasts (supplied by Laboratory for Mutagenicity Testing; Technical University, 64287 Darmstadt, Germany)
- Suitability of cells: The V79 cell line has been used successfully in in vitro experiments for many years. Especially the high proliferation rate and a good cloning efficiency of untreated cells both necessary for the appropriate performance of the study, recommend the use of this cell line.

For cell lines:
- Absence of Mycoplasma contamination: Each master cell stock is screened for mycoplasma contamination.
- Number of passages if applicable: Large stocks of the V79 cell line are stored in liquid nitrogen in the cell bank of ICCR-Roßdorf GmbH allowing the repeated use of the same cell culture batch in experiments.
- Cell doubling time: 12-16 hours in stock cultures
- Modal number of chromosomes: 22
- Periodically checked for karyotype stability: yes
- Periodically ‘cleansed’ of spontaneous mutants: yes


MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature: Thawed stock cultures were propagated at 37°C in 75 cm² plastic flasks. About 2-3x10^6 cells were seeded into each flask with 15 mL of MEM (minimal essential medium) containing Hank’s salts supplemented with 10% FBS, neomycin (5 μg/mL) and amphotericin B (1%). The cells were sub-cultured once or twice weekly. All incubations were done at 37°C with 1.5% carbon dioxide (CO2) in humidified air.
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9: The S9 was prepared in-house. Phenobarbital/β-naphthoflavone induced male Wistar rat liver S9 was used as the metabolic activation system.
- method of preparation of S9 mix: An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution, to result in a final concentration of approx. 10% (v/v) in the S9 mix. Cofactors were added to the S9 mix to reach the following concentrations in the S9 mix: 8 mM MgCl2, 33 mM KCl, 5 mM glucose-6-phosphate, and 4 mM NADP in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.
- concentration or volume of S9 mix and S9 in the final culture medium: 500 μL S9 mix (containing 10% (v/v) S9)
- quality controls of S9: sterility and metabolic capability
Test concentrations with justification for top dose:
- Pre-experiment: 7.8, 15.6, 31.3, 62.5, 125, 250, 500, and 1000 µg/mL
- Main experiment (without metabolic activation): 1.95, 3.9, 7.8, 15.6, 31.3, and 62.5 µg/mL
- Main experiment (with metabolic activation): 1.95, 3.9, 7.8, 15.6, 31.3, and 62.5 µg/mL
The maximum test item concentrations of the pre-experiment and the main experiments were chosen based on the solubility profile of the test material.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO (purity ≥ 99.9%)

- Justification for choice of solvent/vehicle: The solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures.*

- Justification for percentage of solvent in the final culture medium: The final concentration of DMSO in the culture medium was 1.0 % (v/v) based on the recommendations given in OECD 476 (2016).

*References:
- Easterbrook, J., Lu, C., Sakai, Y. and Li, A.P. (2001). Effects of organic solvents on the activities of cytochrome P450 isoforms, UDP-dependent glucuronyl transferase, and phenol sulfotransferase in human hepatocytes Drug Metabolism and Disposition, 29, 141-144
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
PRE-EXPERIMENT ON TOXICITY
A pre-test was performed in order to determine the toxicity of the test item. The osmolarity and the pH value were determined in culture medium of the solvent control and of the highest concentration.
The general culturing and experimental conditions in this pre-test were the same as described below for the mutagenicity experiment.
In this pre-test approximately 1.5 million cells were seeded in 25 cm² flasks 24 hours prior to treatment. After approximately 24 hours the test item was added and the treatment proceeds for 4 hours (with and without metabolic activation) (duplicate cultures per concentration level).
Immediately after treatment the test item was removed by rinsing with PBS. Subsequently, the cells were trypsinised and suspended in complete culture medium. After an appropriate dilution the cell density was determined with a cell counter. Toxicity of the test item is evident as a reduction of the cell density compared to a corresponding solvent control. A cell density of approximately 1.5 million cells in 25 cm² flasks is about the same as approximately 10 million cells seeded in 175 cm² bottles 24 hours prior to treatment with the main experiment.

CONCENTRATION SELECTION
Based on the results of the pre-experiment the following concentrations were applied in the main experiment: 1.95, 3.9, 7.8, 15.6, 31.3, and 62.5 µg/mL both with and without metabolic activation.

HPRT MUTAGENICITY EXPERIMENT
- Seeding: Two to four days after sub-cultivation stock cultures were trypsinised at 37°C for approximately 5 to 10 minutes. Then the enzymatic digestion was stopped by adding complete culture medium with 10% FBS and a single cell suspension was prepared. The trypsin concentration for all sub-culturing steps was 0.2% in saline. Prior to the trypsin treatment the cells were rinsed with PBS. Approximately 0.7-1.2x10^7 cells were seeded in plastic flasks. The cells were grown for 24 hours prior to treatment.
- Treatment: After 24 hours the medium was replaced with serum-free medium containing the test item, either without S9 mix or with 50 μL/mL S9 mix. Concurrent solvent and positive controls were treated in parallel. 4 hours after treatment, this medium was replaced with complete medium following two washing steps with PBS.
Immediately after the end of treatment the cells were trypsinised as described above and sub-cultivated. At least 2x10^6 cells per experimental point (concentration series plus controls) were sub-cultivated in 175 cm² flasks containing 30 mL medium.
Two additional 25 cm² flasks were seeded per experimental point with approx. 500 cells each to determine the relative survival (RS) as measure of test item induced cytotoxicity. The cultures were incubated at 37±1.5°C in a humidified atmosphere with 1.5%±0.5 CO2.
The colonies used to determine the relative survival (RS) were fixed and stained approximately 8±2 days after treatment as described below.
Three or four days after the first sub-cultivation, at least 2x10^6 cells per experimental point were again, sub-cultivated in 175 cm² flasks containing 30 mL medium.
Following the expression time of 7 days, five 75 cm² cell culture flasks were seeded with 4-5x10^5 cells each in medium containing 6-TG (11 μg/mL). Two additional 25 cm² flasks were seeded with approx. 500 cells each in non-selective medium to determine the viability. The cultures were incubated at 37°C±1.5°C in a humidified atmosphere with 1.5%±0.5 CO2.
After 8±2 days (evaluation for viability) and 9±2 days (mutation analysis) the colonies were stained with 10% methylene blue in 0.01% KOH solution.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: relative survival (RS)

METHODS FOR MEASUREMENTS OF GENOTOXICIY
Colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.

METHODS USED TO DETERMINE pH, OSMOLALITY AND PRECIPITATION
- The osmolarity and pH of the test item formulated in culture medium was determined by using an osmometer (Gonotec, Model Osmomat 030) or a pH meter (TW, Model Vario pH), respectively, in the pre-experiment without metabolic activation.
- Precipitation was evaluated at the beginning and at the end of treatment by the unaided eye and additionally with the microscope.
Evaluation criteria:
A test item is classified as clearly mutagenic if, in any of the experimental conditions examined, all of the following criteria are met:
a) at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) the increase is concentration-related when evaluated with an appropriate trend test,
c) any of the results are outside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits) (please refer to “Attached background material: Historical control data”).

A test item is classified as clearly non-mutagenic if, in all experimental conditions examined, all of the following criteria are met:
a) none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) there is no concentration-related increase when evaluated with an appropriate trend test,
c) all results are inside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits) (please refer to “Attached background material: Historical control data”).

There is no requirement for verification of a clearly positive or negative response. In case the response is neither clearly negative nor clearly positive as described above or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgement and/or further investigations.
In rare cases, even after further investigations, the data set will preclude making a conclusion of positive or negative results, and therefore the test chemical response will be concluded to be equivocal.
Statistics:
The statistical analysis was performed on the mean values of culture I and II for the main experiment, additionally the linear regression was determined for the individual culture I and II for the experimental part without S9 mix.

A linear regression (least squares, calculated using a validated excel spreadsheet) was performed to assess a possible concentration dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05.

A t-test test was performed to evaluate a significant increase of the mutation frequency at test points exceeding the 95% confidence interval. A t-test is judged as significant if the p-value is below 0.05. Here, a t-test was performed only for the positive controls since all mean mutant frequencies of the groups treated with the test item were well within the 95% confidence interval of our laboratory’s historical negative control data.

However, both, biological and statistical significance will be considered together.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
PRE-EXPERIMENT ON TOXICITY
- Test item concentrations between 7.8 µg/mL and 1000 µg/mL were used in the pre-experiment with and without metabolic activation following 4 hours treatment. The maximum concentration in the pre-experiment was chosen by the solubility properties of the test item.
- The test medium was checked for phase separation and precipitation at the end of the treatment period (4 hours) before the test item was removed. Precipitation occurred at 31.3 µg/mL and above with and without metabolic activation observed with the unaided eye and at 62.5 µg/mL and above with and without metabolic activation observed with the unaided eye and by microscopical analysis.
- No relevant cytotoxic effect, indicated by a relative cloning efficiency of 50% or below was observed
- There was no relevant shift of the pH of the medium even at the maximum concentration of the test item measured in the pre-experiment. A shift of the osmolarity of > 50 mOsm to the solvent control was observed, therefore the osmolarity was determined in the main experiment for the highest applied concentration, also.

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH and osmolality: There was no relevant shift of osmolarity and pH of the medium even at the maximum concentration of the test item measured in the main experiment.
- Precipitation and time of the determination: Precipitation of the test item was observed by the unaided eye at 62.5 µg/mL after the 4-hour exposure period.

HPRT MUTAGENICITY ASSAY
Main Experiment (without metabolic activation):
- Based on the results of the pre-experiment the following concentrations were applied in the main experiment: 1.95, 3.9, 7.8, 15.6, 31.3, and 62.5 µg/mL
- No relevant cytotoxic effects indicated by a relative adjusted cloning efficiency I (survival rate) below 50% (mean value of both parallel cultures) were noted up to the maximum concentration.
- Precipitation of the test item was observed at 62.5 µg/mL. Consequently, the concentrations of 3.9 to 62.5 µg/mL were evaluated for mutagenicity in the absence of metabolic activation.
- The mutation frequency (MF) of the solvent control was 6.4 mutants per 10^6 cells and the MF range of the treated groups was 6.0 - 10.0 mutants per 10^6 cells (please refer to “Attached background material: Results”). The observed mean mutant frequency (MF) of the solvent control and all evaluated concentrations was within the 95% control limits of the solvent historical control data (2.9 - 22.4 mutants per 106 cells; please refer to “Attached background material: Historical control data”).
- Linear regression analysis showed a statistically significant trend (p ≤ 0.001) for the mean value of both cultures but not for the single cultures runs (please refer to “Attached background material: Results”). Separate analysis of culture I and II treatment revealed p-values of 0.345 and 0.078, respectively. Since all values are well within the 95% control limit and the significant trend is based on a rather low MF of the solvent control, this statistically significant trend for the mean value is judged as incidental finding, and thus, biologically irrelevant.

Main Experiment (with metabolic activation):
- Based on the results of the pre-experiment the following concentrations were applied in the main experiment: 1.95, 3.9, 7.8, 15.6, 31.3, and 62.5 µg/mL
- No relevant cytotoxic effects indicated by a relative adjusted cloning efficiency I (survival rate) below 50% (mean value of both parallel cultures) were noted up to the maximum concentration.
- Precipitation of the test item was observed at 62.5 µg/mL. Consequently, the concentrations of 3.9 to 62.5 µg/mL were evaluated for mutagenicity in the absence of metabolic activation.
- The mutation frequency (MF) of the solvent control was 6.5 mutants per 10^6 cells and the mutation MF range of the treated groups was 8.1 - 10.7 mutants per 10^6 cells (cf. Table 3). The observed mean MF of the solvent control and all evaluated test item concentrations was within the 95% control limits of the solvent historical control data (2.9-23.7 mutants per 10^6 cells).
- Linear regression analysis showed no statistically significant trend.

In summary, the outcome of the main experiment was clearly negative in the presence and absence of metabolic activation.

ASSAY VALIDITY
- The observed mean mutant frequencies of the solvent controls were within the 95% control limits of the solvent historical control data.
- EMS and DMBA were used as positive controls and induced a distinct increase in the mutant frequency, which fell well within the historical control data range. The concurrent solvent control cultures showed mutant frequency, which were well within the acceptable ranges of the historical control data base.
Thus, the controls demonstrated sensitivity of the test systems and the validity of the assay.
Conclusions:
No relevant toxicity (10 to 20% relative survival) was found in the experiments. Precipitation of the test item was observed at ≥62.5 μg/mL both in absence and presence of metabolic activation. Silicic acid, zirconium salt, cadmium pigment-encapsulated (Zirconium zircon with encapsulated selenium sulphide), tested up to precipitating concentrations, did not induce biologically relevant increases in the mutant frequency. In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the Hprt locus in V79 cells. All validity criteria were met. The study was fully compliant with OECD 476 (2016).

Therefore, Silicic acid, zirconium salt, cadmium pigment-encapsulated (Zirconium zircon with encapsulated selenium sulphide) is considered to be non-mutagenic in this HPRT assay.
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:

Chang (2022) examined the mutagenic potential of Zirconium zircon with encapsulated selenium sulphide in a bacterial reverse mutation assay according to OECD TG 471 (2020) under GLP. The test was performed in S. typhimurium TA 98, TA 100, TA 1535, and TA 1537 as well as E. coli WP2 uvrA (pKM101) in two independent experiments using triplicate cultures. The cell cultures were exposed to the test material, both in absence and presence of metabolic activation, up to the recommended maximum concentration of 5 mg/plate. The two independent experiments were performed according to the plate incorporation (experiment I) and pre-incubation (experiment II) procedure. In experiment II, precipitation of the test material was observed at the top concentration. Toxic effects were not evident under any condition tested. Treatment with Zirconium zircon with encapsulated selenium sulphide did not result in a mutagenic response at all concentrations tested both in absence and presence of S9 metabolic activation. Appropriate positive controls demonstrated the activity of the metabolic activation system and the sensitivity of the test system.

 

In vitro gene mutation in mammalian cells:

Sokolowski (2022) investigated on the gene mutation potential Zirconium zircon with encapsulated selenium sulphide in an in vitro mammalian cell gene mutation test at the Hprt locus according to OECD TG 476 (2016) and under GLP. Chinese hamster lung fibroblasts (V79) were exposed for 4 hours to Zirconium zircon with encapsulated selenium sulphide up to a precipitating concentration of 62.5 µg/mL both with and without metabolic activation (S9 fraction from phenobarbital/β-naphthoflavone induced rat livers). The outcome of this study was judged as clearly negative both in the absence and presence of S9 metabolic activation. Appropriate positive controls demonstrated the activity of the metabolic activation system and the sensitivity of the test system.

 

 

Overall conclusion:

Zirconium zircon with encapsulated cadmium selenium sulphide is not considered to be genotoxic, since Zirconium zircon with encapsulated cadmium selenium sulphide has neither shown any gene mutation potential in bacteria or mammalian cells. Thus, Zirconium zircon with encapsulated cadmium selenium sulphide is not to be classified according to regulation (EC) 1272/2008 as genetic toxicant.

Justification for classification or non-classification

Genotoxicity studies with Zirconium zircon with encapsulated cadmium selenium sulphide did not show any effects in a bacterial reverse mutation test or in a mammalian cell gene mutation test (HPRT assay).

 

The classification criteria acc. to regulation (EC) 1272/2008 as germ cell mutagen are not met, thus no classification is required.