2-(2-(2-butoxyethoxy)ethoxy)ethanol

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:

September 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

June 2020

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-(2-(2-butoxyethoxy)ethoxy)ethanol

EC Number:

205-592-6

EC Name:

2-(2-(2-butoxyethoxy)ethoxy)ethanol

Cas Number:

143-22-6

Molecular formula:

C10H22O4

IUPAC Name:

2-[2-(2-butoxyethoxy)ethoxy]ethanol

impurity 1

Reference substance name:

2-(2-butoxyethoxy)ethanol

EC Number:

203-961-6

EC Name:

2-(2-butoxyethoxy)ethanol

Cas Number:

112-34-5

Molecular formula:

C8H18O3

IUPAC Name:

2-(2-butoxyethoxy)ethanol

impurity 2

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

water

impurity 3

Reference substance name:

2,2'-(ethylenedioxy)diethanol

EC Number:

203-953-2

EC Name:

2,2'-(ethylenedioxy)diethanol

Cas Number:

112-27-6

Molecular formula:

C6H14O4

IUPAC Name:

2,2'-[ethane-1,2-diylbis(oxy)]diethanol

Test material form:

liquid

Details on test material:

Other identified impurities, all less than 0.1% include n-butanol, monoethylene glycol, diethylene glycol and tetraethylene glycol butyl ether. Balance of 0.59% not identified.

Specific details on test material used for the study:

Identity TRIETHYLENE GLYCOL MONOBUTYL ETHER

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: Ineos nv, PS10202. ERBC no. 17077
- Label name: BTGEMIX
- Expiration date of the lot/batch: 27 July 2022
- Purity test date:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, protected from light and under nitrogen
- Stability under storage conditions: stable
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium: fully misciible in water
- Reactivity of the test substance with the solvent/vehicle /test medium (if applicable): not reactive

Method

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : SD Rat liver, Phenobarbital and 5,6-Benzoflavone induced. Supplier MOLTOX,Molecular Toxicology, Inc. (Batch Number 4209)
- method of preparation of S9 mix : S9 tissue fraction 1.0mL, NADP (100 mM) 0.4mL, G-6-P (100 mM) 0.5mL, KCl (330 mM) 1.0mL, MgCl2 (100 mM) 0.8mL,
Phosphate buffer (pH 7.4, 200 mM) 5.0mL, Distilled Water 1.3mL. S9 tissue fraction 10% of S9 mix.
- concentration or volume of S9 mix and S9 in the final culture medium. Plate incorporation method: 0.5ml S9 mix in 2.7ml. Pre-incubation method: 0.5mL in 2.75mL.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability). Yes, details provide in production and quality control certificate from supplier. Testeed for the presence of adventitious agents (contaminating microorganisms) and efficacy via the ability to activate ethidium bromide and cyclophosphamide to give positive results with TA98 and TA1535 respectively.

Test concentrations with justification for top dose:

5.00, 2.50, 1.25, 0.625, 0.313 μL/plate with all tester strains. The preliminary toxicity test showed no toxicity up to 5μL/plate, which is the maximum recommended concentration to use with this assay.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Sterile water for substance. DMSO for the positive controls that are not water soluble.
- Justification for choice of solvent/vehicle: recommended for assay.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments : two, plate incorporation and pre-incubation assays

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium; in agar (plate incorporation); preincubation.
- Plates incubated for approximately 72 hours at 37°Cthen immediately scored by counting the number of revertant colonies on each plate.

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 30 mins

Evaluation criteria:

Positive result must show both:
- at least two-fold increase in mean revertant numbers at two consecutive dose levels or at highest dose only
- evidence of a dose-response relationship (increasing numbers of mutant colonies with increasing dose levels.)

Statistics:

Mean and standard error reported

Results and discussion

Test results

Test results 1

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

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

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

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

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

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

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

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

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

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- non identified

RANGE-FINDING/SCREENING STUDIES (if applicable): no toxicity up to maximum dose recommended for assay

STUDY RESULTS
- Signs of toxicity : no
- Individual plate counts : provided in study report. See attachment to this record.
- Mean number of revertant colonies per plate and standard deviation : provided in study report. See attachment to this record.
- Overall: No strain, with our without metabolic activation, showed any evidence of a dose response relationship and no individual result at any concentration came close to exceeding the criteria of a 2 fold increase in revertant colonies that would trigger a possible concern.

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: provided in study report. See attachment to this record.
- Negative (solvent/vehicle) historical control data: provided in study report. See attachment to this record.

Applicant's summary and conclusion

Conclusions:

No mutagenic

Executive summary:

In a reliable and GLP guideline study, the substance 2 -(2 -2 -(butoxyethoxy)ethoxy)ethanol showed no evidence of mutagenicity in a bacterial reverse mutation (Ames) assay in Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and Escherichia coli strain WP2uvra, with or without metabolic activation.