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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
Silicic acid (H4SiO4), zirconium (4+) salt (1:1), reaction products with sodium hydroxide did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in the tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment. In this study, acceptable responses were obtained for the negative and strain-specific positive control items indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Based on the results of this study it is concluded that Silicic acid (H4SiO4), zirconium (4+) salt (1:1), reaction products with sodium hydroxide is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-01-13 to 2016-02-04
Reliability:
1 (reliable without restriction)
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: rfa : deep rough (defective lipopolysaccharide cellcoat) gal : mutation in the galactose metabolism chl : mutation in nitrate reductase bio : defective biotin synthesis uvrB : loss of the excision repair system (deletion of the ultraviolet-repair B gene)
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Eight concentrations, 0.55, 1.7, 5.4, 17, 52, 164, 512 and 1000 µg/plate were tested in triplicate in the dose range finding test. Five concentrations, 52, 164, 512, 1000 and 2500 were used in experiment 1 and five concentrations, 200, 400, 800, 1600 and 2500 were used in experiment 2.
Vehicle / solvent:
An aqueous based vehicle could not be used as the test substance reacts with water. A solubility test was performed. In neither DMSO (10 mg/ml), ethanol (25 mg/ml), hexane (100 mg/ml), tetrahydrofuran (10 mg/ml), tween (25 mg/ml), PEG400 (25 mg/ml) nor acetone (25 mg/ml) the test item could be dissolved and sank in all vehicles quickly to the bottom of the tube. DMSO was used as vehicle in this project, since at a dose level of 10 mg/ml using a treatment with ultrasonic waves a homogeneous suspension could be obtained. At concentration of 0.52 mg/ml and higher the test item was suspended in dimethyl sulfoxide. At concentrations of 0.17 mg/ml and lower the test item was dissolved in dimethyl sulfoxide. Due to the physical properties of the test item, no workable suspension above 10 mg/ml could be achieved.
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
2-acetylaminofluorene
Remarks:
with metabolic activation
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: ICR-191 9Sigma)
Remarks:
without metabolic activation
Details on test system and experimental conditions:
Rat liver microsomal enzymes (S9 homogenate) were obtained from Trinova Biochem GmbH, Giessen, Germany and were prepared from male Sprague Dawley rats that had been injected intraperitoneally with Aroclor 1254 (500 mg/kg). Each S9 batch is characterised with the mutagens benzo-(a)-pyrene and 2-aminoanthracene, which require metabolic activation, in tester strain TA100 at concentrations of 5 µg/plate and 2.5 µg/plate, respectively.
At least five different doses (increasing with approximately half-log steps) of the test item were tested in triplicate in each strain. In the first mutation experiment, the test item was tested both in the absence and presence of 5% (v/v) S9-mix in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. In a follow-up experiment with additional parameters, the test item was tested both in the absence and presence of 10% (v/v) S9-mix in all tester strains.

The negative control (vehicle) and relevant positive controls were concurrently tested in each strain in the presence and absence of S9-mix. In the first mutation experiment an additional solvent control was tested (see deviation 1)

Top agar in top agar tubes was melted by heating to 45 ± 2°C. The following solutions were successively added to 3 ml molten top agar: 0.1 ml of a fresh bacterial culture (109 cells/ml) of one of the tester strains, 0.1 ml to 0.25 ml of a dilution of the test item in DMSO and either 0.5 ml S9-mix (in case of activation assays) or 0.5 ml 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0 °C for 48 ± 4 h. After this period revertant colonies (histidine independent (His+) for Salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli) were counted.
The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test item precipitate to interfere with automated colony counting were counted manually. Evidence of test item precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope.

Evaluation criteria:
A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
a) The vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at WIL Research Europe.
b) The selected dose range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
c) No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: only observed in strain TA98 in abscence of S9 at 2500 ug/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
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:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
All bacterial strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants in two experiments.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

Based on the results of this study it is concluded that Silicic acid (H4SiO4), zirconium (4+) salt (1:1), reaction products with sodium hydroxide is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Executive summary:

Silicic acid (H4SiO4), zirconium (4+) salt (1:1), reaction products with sodium hydroxide was tested in theSalmonella typhimuriumreverse mutation assay with four histidine-requiring strains ofSalmonella typhimurium(TA1535, TA1537, TA98 and TA100) and in theEscherichia colireverse mutation assay with a tryptophan-requiring strain ofEscherichia coli(WP2uvrA). Initially a dose range finding test was performed with the tester strains TA100 and WP2uvrA. After that the test was performed in two independent experiments in the presence and absence of S9-mix (rat liver S9-mix induced by Aroclor 1254). 

BatchCP2684of the test item was a brown powder. The test item was suspended in dimethyl sulfoxide. No workable suspension above 10 mg/ml could be obtained.

 

In the dose range finding test, the test item was tested up to concentrations of 1000 µg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. The test item did not precipitate on the plates at this dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

 

Based on the results of the dose range finding test, the test item was tested in the first mutation assay at a concentration range of 52 to 2500 µg/plate in the absence and presence of 5% (v/v) S9-mix in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. The test item precipitated on the plates at the top dose of 2500 μg/plate. Cytotoxicity, as evidenced by a decrease in the number of revertants, was observed in tester strain TA98 in the absence of S9-mix at the highest tested concentration.

  

In a follow-up experiment of the assay with additional parameters, the test item was tested at a concentration range of 200 to 2500 µg/plate in the absence and presence of 10% (v/v) S9-mix in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. The test item precipitated on the plates at the top dose of 2500 μg/plate. Toxicity was observed in tester strain TA98 in the absence of S9-mix at the highest tested concentration.

 

Silicic acid (H4SiO4), zirconium (4+) salt (1:1), reaction products with sodium hydroxide did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in the tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment.

 

In this study, acceptable responses were obtained for the negative and strain-specific positive control items indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

 

Based on the results of this study it is concluded that Silicic acid (H4SiO4), zirconium (4+) salt (1:1), reaction products with sodium hydroxide is not mutagenic in theSalmonella typhimuriumreverse mutation assay and in theEscherichia colireverse mutation assay.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Silicic acid (H4SiO4), zirconium (4+) salt (1:1), reaction products with sodium hydroxide did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in the tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment.

 

In this study, acceptable responses were obtained for the negative and strain-specific positive control items indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

 

Based on the results of this study it is concluded that Silicic acid (H4SiO4), zirconium (4+) salt (1:1), reaction products with sodium hydroxide is not mutagenic in theSalmonella typhimuriumreverse mutation assay and in theEscherichia colireverse mutation assay.

In addition, there are 3 supporting studies with Zirconium Acetate

Genetic toxicity in vitro:

Bacterial reverse mutation assay:

Scarcella (2013) performed an Ames test (OECD 471 and EU B.13/14) with S. typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and E. coli strain WP2 uvrA with and without metabolic activation.

Following test concentrations were applied in triplicate: 313, 625, 1250 and 2500 µg zirconium acetate/plate for experiment I; 313, 625; 1250, 2500, 5000 µg of zirconium acetate/plate and an additional dose level of 156 µg/plate with TA100 tester strain both in the absence and presence of S9 metabolism for experiment II. For experiment III:

- TA1535, TA1537, TA98 without S9 at 156, 78.1, 39.1, 19.5, 9.77, 4.88, 2.44 µg of zirconium acetate/plate
- TA100 without S9 at 78.1, 39.1, 19.5, 9.77, 4.88, 2.44, 1.22 µg of zirconium acetate/plate
- WP2 uvrA without S9 at 156, 78.1, 39.1, 19.5, 9.77, 4.88 µg of zirconium acetate/plate
- TA98 with S9 at 156, 78.1, 39.1, 19.5, 9.77, 4.88 µg of zirconium acetate/plate
- TA1535, TA1537, WP2 uvrA with S9 at 313, 156, 78.1, 39.1, 19.5, 9.77 µg of zirconium acetate/plate
- TA100 with S9 at 78.1, 39.1, 19.5, 9.77, 4.88, 2.44 µg of zirconium acetate/plate

Negative controls and positive controls were run in triplicate and were considered to be valid. Main assay: the first assay was performed using a plate-incorporation method. The second and third assays were performed using a pre-incubation method.

According to the results of the study, the test substance is not mutagenic in the Ames test with and without metabolic activation.

 

Chromosome Aberration:

Ciliutti (2013) performed an in vitro Chromosome Aberration test in Chinese hamster ovary cells (OECD 473 and EU Method B.10). Two experiments were performed using different test concentrations with and without S9 activation (3h exposure and harvested at 20h in experiment I; continuous exposure until harvest at 20h for experiment II). Both negative and positive controls were considered to be valid. On the basis of the results obtained, it was concluded that zirconium acetate did not induce structural chromosome aberrations after in vitro treatment and under the reported conditions.

In vitro mammalian cell gene mutation:

Bisini (2013) performed an in vitro mammalian gene mutation assay in L5178Y TK+/- lymphoma cells using the fluctuation method according to OECD Guideline 476 and EU Method B.17. Two experiments were performed using different concentrations with and without metabolic S9 activation (experiment I) and without metabolic S9 activation (experiment II). Plates were tested in duplicate. Plates were exposed for 3hr (experiment I) and 24 hr (experiment II). The positive and negative (vehicle) controls were considered to be valid. It was concluded that the test substance did not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation under the experimental conditions.

Genetic toxicity in vivo:

According to REACH Annex IX section 8.4, column 2, no further in vivo testing is required as no positive results were obtained in any of the three in vitro studies performed according to REACH Annexes VII and VIII section 8.4.


Justification for selection of genetic toxicity endpoint
A very recent K1 GLP study performed with the test substance

Justification for classification or non-classification

Negative results with the test substance.

The supporting studies with zirconium acetate were also negative.