Reaction mass of disodium metasilicate and sodium hydroxide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Based on the results of this study it is concluded that Reaction mass of disodium metasilicate and 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:

2016-01-25 to 2016-02-15

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, 1.7, 5.4, 17, 52, 164, 512 and 1600 and 5000 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, 492, 878, 1568, 2800 and 5000 were used in experiment 2.

Vehicle / solvent:

Sterile water.

Negative solvent / vehicle controls:

yes

Remarks:

water

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

Remarks:

with metabolic activation

Negative solvent / vehicle controls:

yes

Remarks:

water

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: Slight to extreme toxicity was observed at the test item concentration of 5000 µg/plate in the absence and presence of S9-mix in all tester strains, with the exception of tester strain TA1535 and TA98 in the presence of S9-mix where no toxicity was seen

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 nor precipitates, but tested up to recommended limit 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 the reaction mass of disodium metasilicate and sodium hydroxide is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

The reaction mass of disodium metasilicate and 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).

The test was performed in two independent experiments in the presence and absence of S9-mix (rat liver S9-mix induced by Aroclor 1254).

In the dose range finding test, the test item was tested up to concentrations of 5000 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. Toxicity was observed in both tester strains. Results of this dose range finding test were reported as part of the first mutation assay.

 

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 5000 µg/plate in the absence and presence of 5% (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. Toxicity was observed in all tester strains, except in the tester strains TA1535 and TA98 in the presence of S9-mix.

 

In a follow-up experiment of the assay with additional parameters, the test item was tested at a concentration range of 492 to 5000 µg/plate in the absence and presence of 10% (v/v) S9-mix in the tester strains TA1535, TA1537, TA98, TA100 and WP₂uvrA. 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.

 

Reaction mass of disodium metasilicate and 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.

Based on the results of this study it is concluded that Reaction mass of disodium metasilicate and 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:

Reaction mass of disodium metasilicate and 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). The test was performed in two independent experiments in the presence and absence of S9-mix (rat liver S9-mix induced by Aroclor 1254).

 

The study procedures described in this report were based on the most recent OECD and EC guidelines.

 

BatchSO103567of Reaction mass of disodium metasilicate and sodium hydroxide was a colourless liquid. A correction factor of 6.66 was used for the purity of 15% actual solid contents. The vehicle of the test item was Milli-Q water.

 

In the dose range finding test, the test item was tested up to concentrations of 5000 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. Toxicity was observed in both tester strains. Results of this dose range finding test were reported as part of the first mutation assay.

 

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 5000 µg/plate in the absence and presence of 5% (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. Toxicity was observed in all tester strains, except in the tester strains TA1535 and TA98 in the presence of S9-mix.

 

In a follow-up experiment of the assay with additional parameters, the test item was tested at a concentration range of 492 to 5000 µg/plate in the absence and presence of 10% (v/v) S9-mix in the tester strains TA1535, TA1537, TA98, TA100 and WP₂uvrA. 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.

 

Reaction mass of disodium metasilicate and 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.

 

The negative and strain-specific positive control values were within the laboratory historical control data ranges 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 Reaction mass of disodium metasilicate and sodium hydroxide is not mutagenic in theSalmonella typhimuriumreverse mutation assay and in theEscherichia colireverse mutation assay. Supporting information:

Sodium Hydroxide: Source - the sodium hydroxide summary risk assessment report JRC EC 2008

Both the in vitro and the in vivo genotoxicity studies indicated no evidence for a mutagenic activity. Furthermore NaOH is not expected to be systemically available in the body under normal handling and use conditions and for this reason additional testing is considered unnecessary

Sodium Silicate:

The in-vitro genetic toxicity of disodium metasilicate nonahydrate was investigated in the Ames test (BASF SE, 2012).The standard plate test and the preincubation test were conducted each with S. typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvrA in the presence and absence of a metabolic activation system. Concentrations up to 5000 µg/plate were used for the standard plate test and concentrations up to 2500 µg/plate for the preincubation test. The test substance did not induce reversions in any of the S. typhimurium strains or in E. coli WP2 uvrA with or without metabolic activation.All other in vitro mutagenicity tests with bacteria were negative. Sodium silicate (MR = 3.3) also did not induce chromosomal aberrations and HPRT mutations in V79 mammalian cells in vitro, both in the presence and the absence of metabolic activation. In vivo, sodium metasilicate did not induce chromosome aberrations in the bone marrow of mice. From the available results it can be concluded that there is no evidence of a genotoxic potential for sodium silicate

Justification for selection of genetic toxicity endpoint
A K1 GLP study run on the test substance in 2016

Justification for classification or non-classification

Based on the results of this study it is concluded that Reaction mass of disodium metasilicate and sodium hydroxide is not mutagenic in theSalmonella typhimuriumreverse mutation assay and in theEscherichia colireverse mutation assay.