Tetrakis(2-ethylbutyl) orthosilicate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 July 2017 - 26 Dec 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Tetrakis(2-ethylbutyl) orthosilicate

EC Number:

201-085-9

EC Name:

Tetrakis(2-ethylbutyl) orthosilicate

Cas Number:

78-13-7

Molecular formula:

C24H52O4Si

IUPAC Name:

tetrakis(2-ethylbutyl) orthosilicate

Test material form:

liquid

Details on test material:

Tetrakis(2-ethylbutyl) Orthosilicate
CAS 78-13-7
Expiration: May 2020
Clear colorless liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Gelest, Inc. OS-140 Lot# 8E-33-134
- Expiration date of the lot/batch: May 2020
- Purity test date: 07 June 2017

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: used as received

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: Stable under conditions of assay
- Solubility and stability of the test substance in the solvent/vehicle: A preliminary solubility screen was conducted for Tetrakis (2-ethylbutyl) Orthosilicate in 95% Ethanol; up to 50uL/mL Tetrakis (2-ethylbutyl) Orthosilicate produced a clear, colorless solution.

Method

Target gene:

Each S. typhimurium tester strain contains, in addition to a mutation in the histidine operon, additional mutations that enhance sensitivity to some mutagens. The rfa mutation results in a cell wall deficiency that increases the permeability of the cell to certain classes of chemicals such as those containing large ring systems that would otherwise be excluded. The deletion in the uvrB gene results in a deficient DNA excision-repair system. Tester strains TA98, TA97a and TA100 also contain the pKM101 plasmid (carrying the R-factor). It has been suggested that the plasmid increases sensitivity to mutagens by modifying an existing bacterial DNA repair polymerase complex involved with the mismatch-repair process.

TA98, and TA97a are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens. TA100 is reverted by both frameshift and base substitution mutagens and TA1535 is reverted only by mutagens that cause base substitutions.

The E. coli tester strain has an AT base pair at the critical mutation site within the trpE gene. Tester strain WP2uvrA (pKM101) has a deletion in the uvrA gene resulting in a deficient DNA excision-repair system. Tryptophan revertants can arise due to a base pair substitution at the originally mutated site or by a base pair substitution elsewhere in the chromosome causing the original mutation to be suppressed. Thus, the specificity of the reversion mechanism is sensitive to base pair substitution mutations.

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

other: his G 46

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

other: his D 3052

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

other: his G 46

Species / strain 4

Species / strain / cell type:

S. typhimurium, other: TA97a

Additional strain / cell type characteristics:

other: his D 6610

Species / strain 5

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other: T/PE

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 Supernatant (S9)

Test concentrations with justification for top dose:

A preliminary screen (5, 1, 0.5, 0.1, 0.05, 0.01, 0.005, and 0.001 uL/plate) produced no evidence of cytotoxicity in bacterial strain S. typhirmurium TA100 at any dose tested.

The following five doses were chosen for the main assay based on the recommended maximum test concentration for soluble non-cytotoxic substances:
5 µL/plate; 1 µL/plate; 0.5 µL/plate; 0.1 µL/plate; 0.05 µL/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: 95% Ethanol
- Justification for choice of solvent/vehicle: A preliminary solubility screen was conducted for Tetrakis (2-ethylbutyl) Orthosilicate in DMSO, Tissue Culture Water, and 95% Ethanol. At 50 uL/mL in tissue culture water, Tetrakis (2-ethylbutyl) Orthosilicate separated immediately into 2 clear colorless liquid phases. At 50 uL/mL in DMSO, Tetrakis (2-ethylbutyl) Orthosilicate slowly separates into 2 clear colorless liquid phases. At 50 uL/mL in 95% Ethanol, Tetrakis (2-ethylbutyl) Orthosilicate formed a clear colorless solution.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

compared to historical mutation frequency

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene (2AA), CAS # 613-13-8

Remarks:

Positive control for all bacterial strains with S9

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

compared to historical mutation frequency for E. coli WP2 uvrA

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

E. coli WP2 uvrA positive control without S9

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

compared to historical mutation frequency of TA97a

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: acridine, 6-chloro-9-(3-((2-chloroethyl)amino) propyl)amino-2-methoxy, dihydrochloride (ICR- 191), CAS # 17070-45-0

Remarks:

S. typhimurium TA97a positive control without S9

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

compared to historical mutation frequency of TA98

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Daunomycin hydrochloride, CAS #23541-50-6

Remarks:

S. typhimurium TA98 positive control without S9

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

compared to historical mutation frequency of TA1535 and TA100

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

S. typhimurium TA100 and TA1535 positive control without S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: Triplicate plating

DETERMINATION OF CYTOTOXICITY
- Method: The revertant colonies were counted manually or automatically with the Q Count Colony Counter. Evidence of test article 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. To determine the toxicity, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were evaluated.

METABOLIC ACTIVATION SYSTEM
Rat liver microsomal enzymes (S9 homogenate) were obtained from Molecular Toxicology Inc., Boone, NC. and were prepared from male Sprague Dawley rats that had been pretreated with Aroclor 1254. The S9 used in this study was tested by the manufacturer for its ability to activate an S9 dependent mutagen. The S9 was found to be acceptable for use in mutation tests.  

PREPARATION OF S9
The S9 was stored at <-80oC until just prior to use. NADPH REGENSYS B (used with S9) is stored at approximately -20ºC. NADPH REGENSYS A (also used with S9) and bacteria discs are stored at approximately 4ºC. The rat S9 mix was prepared the day of dosing and was stored on ice until used. Lyophilized REGENESYS B is reconstituted in REGENESYS A buffer and Aroclor 1254 Supernatant (S9) is added to make a 10% S9 for use in the assay. The aliquots are refrigerated until use.
 
TEST SYSTEM
Test System: Salmonella typhimurium and Escherichia coli bacteria
Rationale: Recommended test system in international guidelines (e.g. OECD, EC).
Source: Molecular Toxicology Inc. (Moltox), Boone, NC.

Bacterial strains were as follows:
Strain - Histidine mutation - Mutation type
TA98 -his D3052/uvrB/rfa/R-factor* - Frameshift mutations
TA1535 -his G46/uvrB/rfa - Base-pair substitutions
TA100 -his G46/uvrB/rfa/R-factor* - Base-pair substitutions and
Frameshift mutations
TA97a -his D 6610/uvrB/rfa/R-factor* - Frameshift mutations
WP2uvrA - trpE/uvrA/R-factor* - Base pair substitutions
*: R-factor = plasmid pKM101 (increases error-prone DNA repair)
 
PREPARATION OF BACTERIAL CULTURES
Frozen tester strains were thawed and inoculated into a nutrient broth culture one day prior to dosing and incubated at 37±2°C until an appropriate density is reached.

Rationale for test conditions:

The tester strains were exposed to the test article via the plate incorporation methodology originally described by Ames et al (1975) and Maron and Ames (1983). This methodology has been shown to detect a wide range of classes of chemical mutagens.

Evaluation criteria:

TESTER STRAIN INTEGRITY: All Salmonella typhimurium tester strains must exhibit sensitivity to crystal violet and to ultraviolet light to demonstrate the presence ofrfa mutation and uvrB mutation, respectively. The Escherichia coli tester strain must exhibit sensitivity to ultraviolet light to demonstrate the presence of uvrA mutation. Salmonella typhimurium strains TA98 and TA100 and Escherichia coli strain WP2uvrA (pKM101) must exhibit resistance to ampicillin to demonstrate the presence of the plasmid R-factor.The spontaneous reversion rates in the solvent/vehicle control must be in the range of in-house historical data.

TEST SUBSTANCE: For all tester strains, an individual dose was considered positive if the mean revertant colony count on the test plates was equal to or greaterthan two times the mean number of spontaneous revertants on the vehicle control plates. A positive result for the assay was defined as a dose-related increase in the mean number of revertant colonies over at least three concentrations of test substance, including at least one positive dose.

POSITIVE CONTROL: Positive control treatment for each tester strain of bacteria must result in at least a 2-fold increase of revertants over the mean negative control value (vehicle). The effectiveness of the metabolic activation mix will be demonstrated by the positive response of the control.

VEHICLE CONTROL: The spontaneous reversion rate for each strain of bacteria will be calculated and compared to in-house historical ranges. Any deviations from those ranges will be taken into account when evaluating the data.

Statistics:

The mean revertant colony count and standard deviation were determined for each dose point.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

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:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 98

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:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 100

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:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium, other: TA97a

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:

not examined

Positive controls validity:

valid

Test results 5

Key result

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:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Evaporation from medium: No evaporation from medium was noted
- Water solubility: Test substance was soluble up to highest dose tested
- Precipitation: No precipitation was observed

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 independently repeated experiments. Based on the results of this study it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay.

Remarks on result:

other: Main Assay

Applicant's summary and conclusion

Conclusions:

Tetrakis (2-ethylbutyl) Orthosilicate is not mutagenic in Salmonella typhimurium or Escherichia coli bacteria. This finding does not warrant classification of Tetrakis (2-ethylbutyl) Orthosilicate as a mutagen under the Regulation (EC) 1272/2008 on classification, labeling and packaging of substances and mixtures (CLP).

Executive summary:

Tetrakis(2-ethylbutyl) Orthosilicate was tested for its mutagenic potential based on the ability to induce mutations in selected loci in Salmonella typhimurium strains TA 1535, TA 100, TA 98, TA 97a and E. coli WP2uvrA- at concentrations ranging from 0.05 to 5 µL/plate in the Ames test according to OECD TG 471. The assay was performed using the Standard plate test with and without metabolic activation (Arochlor 1254 induced rat liver microsomes), respectively. The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5 µL/plate. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation. These findings do not warrant classification of Tetrakis (2-ethylbutyl) Orthosilicate as a mutagen under the Regulation (EC) 1272/2008 on classification, labeling and packaging of substances and mixtures (CLP).