Propylidynetrimethanol, ethoxylated, esters with acrylic acid, reaction products with diethylamine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

3 August 2012 to 12 February 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1997

Deviations:

no

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, reaction products with diethylamine

EC Number:

500-425-6

EC Name:

Propylidynetrimethanol, ethoxylated, esters with acrylic acid, reaction products with diethylamine

Cas Number:

159034-91-0

Molecular formula:

Not available for this UVCB

IUPAC Name:

Reaction product of poly(oxy-1,2-ethanediyl), .alpha.-hydro-.omega.-[(1-oxo-2-propenyl)oxy]-, ether with 2-ethyl-2-(hydroxymethyl)-1,3-propanediol (3:1) and N-ethylethanamine

Test material form:

liquid

Method

Target gene:

Mouse lymphoma L5178Y cells

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

The master stock of L5178Y tk+/- (3.7.2C) mouse lymphoma cells originated from Dr Donald Clive, Burroughs Wellcome Co. Cells supplied to Covance Laboratories Ltd. were stored as frozen stocks in liquid nitrogen. Each batch of frozen cells was purged of mutants and confirmed to be mycoplasma free. For each experiment, at least one vial was thawed rapidly, the cells diluted in RPMI 10 and incubated in a humidified atmosphere of 5±1% v/v CO2 in air. When the cells were growing well, subcultures were established in an appropriate number of flasks.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

mammalian liver post-mitochondrial fraction (S 9), prepared from male Sprague Dawley rats induced with Aroclor 1254.

Test concentrations with justification for top dose:

Positive controls:
4-nitroquinoline 1-oxide (NQO), stock solution: 0.015 and 0.020 mg/mL and final concentration: 0.15 and 0.20 µg/mL, no S-9 present.
Benzo[a]pyrene (B[a]P), stock solution: 0.200 and 0.300 mg/mL and final concentration: 2.00 and 3.00 µg/mL, S-9 present.

Range finding test: Six concentrations tested both in the presence and absence of S-9 ranging from 156.3 to 5000 µg/mL.

Main study:
Experiment 1: Twelve concentrations ranging from 0.625 to 150 µg/mL were tested in the absence of S-9 and eleven concentrations ranging from 50 to 625 µg/mL were tested in the presence of S-9.
Experiment 2: Twelve concentrations ranging from 15 to 130 µg/mL were tested in the absence of S-9 and ten concentrations were tested ranging from 50 to 450 µg/mL in the presence of S-9.

Vehicle / solvent:

DMSO diluted 100-fold in the treatment medium

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO diluted 100-fold in the treatment medium

True negative controls:

no

Positive controls:

yes

Remarks:

4-nitroquinoline-N-oxide without metabolic activation and benzo(a)pyrene with metabolic activation

Positive control substance:

4-nitroquinoline-N-oxide

benzo(a)pyrene

Details on test system and experimental conditions:

METHOD OF APPLICATION: In suspension

DURATION
- Preincubation period: NA
-Incubation period: 3 Hours
- Exposure duration: 7 Days
- Expression time (cells in growth medium): Not reported

Evaluation criteria:

For valid data, the test article was considered to induce forward mutation at the hprt locus in mouse lymphoma L5178Y cells if:
1. The mutant frequency at one or more concentrations was significantly greater than that of the negative control (p < 0.05).
2. There was a significant concentration relationship as indicated by the linear trend analysis (p < 0.05).
3. The effects described above were reproducible.

Statistics:

Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines (Robinson et al., 1990). The control log mutant frequency (LMF) was compared with the LMF from each treatment concentration and the data were checked for a linear trend in mutant frequency with test article treatment. These tests require the calculation of the heterogeneity factor to obtain a modified estimate of variance.

Results and discussion

Test results

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In Experiment 1 twelve concentrations, ranging from 0.625 to 150 µg/mL, were tested in the absence of S-9 and eleven concentrations, ranging from 50 to 625 µg/mL, were tested in the presence of S-9. No precipitate was observed in the absence and presence of S-9. Seven days after treatment, the highest three concentrations tested in the absence of S 9 (110 to 150 µg/mL) and the highest four concentrations in the presence of S-9 (450 to 625 µg/mL) were considered too toxic for selection to determine viability and 6TG resistance. In addition, the lowest three concentrations in the absence of S-9 (0.625 to 2.5 µg/mL) were not selected as there were sufficient concentrations to define the toxicity profile. All other concentrations were analysed in the absence and presence of S-9. The highest concentrations analysed were 90 µg/mL in the absence of S-9 and 400 µg/mL in the presence of S 9, which gave 17% and 13% RS, respectively.
In Experiment 2 twelve concentrations, ranging from 15 to 130 µg/mL, were tested in the absence of S-9 and ten concentrations, ranging from 50 to 450 µg/mL, were tested in the presence of S-9. Seven days after treatment, the highest three concentrations tested in the absence of S-9 (100 to 130 µg/mL) and in the presence of S-9 (410 to 450 µg/mL) were considered too toxic for selection to determine viability and 6TG resistance. All other concentrations were selected in the absence and presence of S-9. The highest concentrations selected were 95 µg/mL in the absence of S-9 and 390 µg/mL in the presence of S-9, which gave 17% and 15% RS, respectively

Applicant's summary and conclusion

Conclusions:

It is concluded that Diethylamine modified ethoxylated trimethylolpropane triacrylate (CAS Number 159034-91-0) induced mutation at the hprt locus of L5178Y mouse lymphoma cells when tested up to toxic concentrations in the absence of a rat liver metabolic activation system (S-9), but did not induce increases in mutant frequency in the same test system when tested up to toxic concentrations in the presence of S-9.

Executive summary:

Diethylamine modified ethoxylated trimethylolpropane triacrylate (CAS Number 159034-91-0) was assayed for the ability to induce mutation at the hypoxanthine guanine phosphoribosyl transferase (hprt) locus (6 thioguanine [6TG] resistance) in mouse lymphoma cells using a fluctuation protocol. The study consisted of a cytotoxicity Range-Finder Experiment followed by two independent experiments, each conducted in the absence and presence of metabolic activation by an Aroclor 1254 induced rat liver post mitochondrial fraction (S-9). The test article was formulated in anhydrous analytical grade dimethyl sulphoxide DMSO.

A 3 hour treatment incubation period was used for all experiments.

In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9, ranging from 156.3 to 5000 µg/mL (an acceptable maximum concentration for in vitro genetic toxicology studies according to current regulatory guidelines). The highest concentration to provide >10% relative survival (RS) in the presence of S-9 was 312.5 µg/mL, which gave 40% RS. In the absence of S-9 all concentrations analysed gave < 10% RS, with the lowest concentration tested (156.3 µg/mL) giving 1% RS.

In Experiment 1 twelve concentrations, ranging from 0.625 to 150 µg/mL, were tested in the absence of S-9 and eleven concentrations ranging from 50 to 625 µg/mL, in the presence of S-9. Seven days after treatment, the highest concentrations analysed to determine viability and 6TG resistance were 90 µg/mL in the absence of S-9 and 400 µg/mL in the presence of S-9, which gave 17% and 13% RS, respectively.

In Experiment 2 twelve concentrations, ranging from 15 to 130 µg/mL, were tested in the absence of S 9 and ten concentrations, ranging from 50 to 450 µg/mL were tested in the presence of S-9. Seven days after treatment, the highest concentrations analysed to determine viability and 6TG resistance were 95 µg/mL in the absence of S-9 and 390 µg/mL in the presence of S-9, which gave 17% and 15% RS, respectively.

Negative (vehicle) and positive control treatments were included in each Mutation Experiment in the absence and presence of S-9. Mutant frequencies in negative control cultures fell within acceptable ranges and clear increases in mutation were induced by the positive control chemicals 4 nitroquinoline 1-oxide (without S-9) and benzo(a)pyrene (with S-9). Therefore the study was accepted as valid.

In the absence of S-9, significant increases in mutant frequency over the concurrent control were observed following treatment with Diethylamine modified ethoxylated trimethylolpropane triacrylate (CAS Number 159034-91-0) at the highest concentrations analysed in Experiments 1 and 2 (90 and 95 µg/mL, respectively) and statistically significant linear trends were observed in both experiments. Although the increases were observed towards the upper limit of cytotoxicity (10-20% RS) and there was some variation between experiments in the magnitude of the induced mutagenic response, the criteria for a positive result were fulfilled in both experiments.

In the presence of S-9, no significant increases in mutant frequency were observed in Experiments 1 and 2. A statistically significant linear trend was observed in Experiment 1 but as there were no significant increases in mutant frequency at any concentration analysed in this experiment, the observation was considered not biologically relevant.

It is concluded that Diethylamine modified ethoxylated trimethylolpropane triacrylate (CAS Number 159034-91-0) induced mutation at the hprt locus of L5178Y mouse lymphoma cells when tested up to toxic concentrations in the absence of a rat liver metabolic activation system (S-9), but did not induce increases in mutant frequency in the same test system when tested up to toxic concentrations in the presence of S-9.