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Toxicological information

Genetic toxicity: in vitro

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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 guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
June 2020
Deviations:
no
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

Constituent 1
Chemical structure
Reference substance name:
2-(2-(2-ethoxyethoxy)ethoxy)ethanol
EC Number:
203-978-9
EC Name:
2-(2-(2-ethoxyethoxy)ethoxy)ethanol
Cas Number:
112-50-5
Molecular formula:
C8H18O4
IUPAC Name:
2-(2-(2-ethoxyethoxy)ethoxy)ethanol
impurity 1
Chemical structure
Reference substance name:
2,2'-oxydiethanol
EC Number:
203-872-2
EC Name:
2,2'-oxydiethanol
Cas Number:
111-46-6
Molecular formula:
C4H10O3
IUPAC Name:
2,2'-oxydiethanol
Test material form:
liquid
Details on test material:
Other identified impurities, all less than 0.1% include water, triethylene glycol methyl ether and diethylene glycol ethyl ether. Balance of 0.08% not identified.
Specific details on test material used for the study:
Identity TRIETHYLENE GLYCOL MONOETHYL ETHER

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: Ineos nv, PS10193. ERBC no. 17078
- Label name: ETGEMIX
- Expiration date of the lot/batch: 27 July 2022
- Purity test date: 03 Sept 2020

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 / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not applicable
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°C then 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 resultsopen allclose all
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
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
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
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
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
- none 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.
- Mean number of revertant colonies per plate and standard deviation : provided in study report
- 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
- Negative (solvent/vehicle) historical control data: provided in study report

Applicant's summary and conclusion

Conclusions:
No mutagenic
Executive summary:

In a reliable and GLP guideline study, the substance 2 -(2 -2 -(ethoxyethoxy)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.

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