1-Butanamine, N-butyl-, reaction products with polyethylene-polypropylene glycol ether with trimethylolpropane (3:1) acrylate

Genetic toxicity: in vitro

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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

adopted July 1997

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

adopted May 2008

Test guideline 3

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

Test material information

Constituent 1

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

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without S9

Controls 2

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.