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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
adopted July 1997
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
adopted May 2008
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Version / remarks:
adopted August 1998
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
propylidynetrimethanol, ethoxylated, esters with acrylic acid and its reactions products with dibutylamine
EC Number:
606-330-7
Molecular formula:
Not specified UVCB - Reaction product of ethoxylated (0-7 EO) propylidenetrimethanol with acrylic acid and dibutylamine (propylidenetrimethanol with acrylic acid : dibutylamine = 5:1)
IUPAC Name:
propylidynetrimethanol, ethoxylated, esters with acrylic acid and its reactions products with dibutylamine
Details on test material:
- Name of test material (as cited in study report): Laromer LR 8869
- Physical state: clear, colorless liquid
- Analytical purity: The test substance is a complex mixture of isomers and homologous components (see analytical report, study code 11L00263).
- Purity test date: 2012-01-11
- Lot/batch No.: 110009P040
- Expiration date of the lot/batch: 11/2012
- Stability under test conditions: stable in DMSO for 4h (verified analytically)
- Storage condition of test material: room temperature, below 25°C, protected from light

Method

Target gene:
hypoxanthine-guanine phosphoribosyl transferase (HGPRT)
Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media:
Ham's F12 medium, containing 1% (v/v) penicillin/streptomycin, 1% (v/v) amphotericine B; 10% (v/v) FCS was added except during the 4 hour exposure period
Treatment and culture medium:
+ stable glutamine and hypoxanthine
Pretreatment medium:
+ hypoxanthine (13.6µg/ml), aminopterin (0.18µg/ml), thymidine (3.88µg/ml)
Selection medium:
+ 6-thioguanine (10µg/ml), 1%(v/v) stable glutamine

- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital and β-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
1st experiment
without S9, 4h exposure: 0.16, 0.31, 0.63, 1.25, 2.5, 5, 10µg/ml
with S9, 4h exposure: 3.13, 6.25, 12.5, 25, 50, 100, 150µg/ml

2nd experiment
without S9, 24h exposure: 0.16, 0.31, 0.63, 1.25, 2.5, 5, 10µg/ml
with S9, 4h exposure: 4.38, 8.75, 17.5, 35, 70, 140µg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: good solubility of the test substance and available historical control data
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
Remarks:
with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4h or 24h
- Expression time (cells in growth medium): 7-9 days
- Selection time (if incubation with a selection agent): 6-7 days
- Fixation time (start of exposure up to fixation or harvest of cells): 13-16 days

SELECTION AGENT (mutation assays): 6-thioguanine

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

OTHER:
check for changes in pH, osmolarity, precipitation of test substance, cell morphology
Evaluation criteria:
The HPRT assay is considered valid if the following criteria are met:
• The absolute cloning efficiencies of the negative/vehicle controls should not be less than 50% (with and without S9 mix).
• The background mutant frequency in the negative/vehicle controls should fall within our historical negative control data range of 0 – 16.43 mutants per 106 clonable cells.
• The positive controls both with and without S9 mix must induce distinctly increased mutant frequencies.
• At least 4 dose levels ranging up to a toxic concentration or up to or beyond the limit of solubility under culture conditions should be tested. Freely soluble and apparently non-toxic substances are not tested at concentrations higher than 5 mg/mL or 10 mM.

A finding is assessed as positive if the following criteria are met:
• Increase of the corrected mutation frequencies (MFcorr.) both above the concurrent negative control values and our historical negative control data range.
• Evidence of reproducibility of any increase in mutant frequencies.
• A statistically significant increase in mutant frequencies and the evidence of a dose response relationship. Isolated increases of mutant frequencies above our historical negative control range (i.e. 15 mutants per 10^6 clonable cells) or isolated statistically significant increases without a dose-response relationship may indicate a biological effect but are not regarded as sufficient evidence of mutagenicity.

The test substance is considered non-mutagenic according to the following criteria:
• The corrected mutation frequency (MFcorr.) in the dose groups is not statistically significant increased above the concurrent negative control and is within our historical negative control data range.

Results and discussion

Test results
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
without S9: at and above 5µg/ml; with S9: at and above 70µg/ml
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no effect at the concentrations tested
- Effects of osmolality: no effect at the concentrations tested
- Precipitation: no precipitation at the concentrations tested

COMPARISON WITH HISTORICAL CONTROL DATA: results for all groups were within the respective (negative or positive) historical control data.

Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Under the experimental conditions of this study, the test substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.