(1-methyl-1,2-ethanediyl)bis[oxy(methyl-2,1-ethanediyl)] diacrylate

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:

2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

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 1997

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

(1-methyl-1,2-ethanediyl)bis[oxy(methyl-2,1-ethanediyl)] diacrylate

EC Number:

256-032-2

EC Name:

(1-methyl-1,2-ethanediyl)bis[oxy(methyl-2,1-ethanediyl)] diacrylate

Cas Number:

42978-66-5

Molecular formula:

C15 H24 O6

IUPAC Name:

(1-methyl-1,2-ethanediyl)bis[oxy(methyl-2,1-ethanediyl)] diacrylate

Specific details on test material used for the study:

- Physical state: liquid
- Analytical purity: 86.3%
- Purity test date: 2014-07-30
- Lot/batch No.: 140023P040
- Expiration date of the lot/batch: 2015-07-25
- Storage condition of test material: room temperature, protected from light
- Stability under test conditions: stability was verified at room temperature in DMSO for at least 4h.

Method

Target gene:

HPRT

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 with or without (only during treatment with S9) FCS. All media were supported with 1% (v/v) penicillin/streptomycin and 1% (v/v) amphotericine
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- 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:

First experiment:
Without S9: 0.23, 0.47, 0.94, 1.88, 3.75, 7.5, 15, 30µg/mL
With S9: 1.88, 3.75, 7.5, 15, 30, 60, 120µg/mL

2nd experiment
Without S9: 0.27, 0.55, 1.09, 2.19, 4.38, 8.75, 17.5µg/mL
With S9: 2.19, 4.38, 8.75, 17.5, 35, 70, 140µg/mL

Third experiment
Without S9: 0.27, 0.55, 1.09, 2.19, 4.38, 8.75, 17.5µg/mL

In all experiments, at least four concentrations were evaluated.

Vehicle / solvent:

DMSO
Due to the insolubility of the test substance in water, dimethyl sulfoxide (DMSO) was selected as vehicle, which had been demonstrated to be suitable in the CHO/HPRT assay and for which historical control data are available.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 24h
- Exposure duration: 4h
- Expression time (cells in growth medium): 6-8 days
- Selection time (if incubation with a selection agent): 6-7 days
- Fixation time (start of exposure up to fixation or harvest of cells): 15 days

SELECTION AGENT (mutation assays): 6-thioguanine

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

Acceptance criteria
The HPRT assay is considered valid if the following criteria are met:
• The absolute cloning efficiencies of the vehicle controls should not be less than 50% (with and without S9 mix).
• The background mutant frequency in the vehicle controls should be within our historical negative control data range of 0.00 – 16.43 mutants per 106 clonable cells
• The positive controls both with and without S9 mix have to induce distinctly increased mutant frequencies (historical positive control data
• At least 4 dose levels should be tested ranging up to a toxic concentration or up to or beyond the limit of solubility under culture conditions. Freely soluble and apparently non-toxic substances are not tested at concentrations higher than 5 mg/mL or 10 mM.

Assessment criteria
A finding is assessed as positive if the following criteria are met:
• Increase in the corrected mutation frequencies (MFcorr.) both above the concurrent negative control values and our historical negative control data range
• Evidence of the reproducibility of any increase in mutant frequencies.
• A statistically significant increase in mutant frequencies and the evidence of a doseresponse relationship.
Isolated increases of mutant frequencies above our historical negative control range (i.e. 15 mutants per 106 clonable cells) or isolated statistically significant increases without a doseresponse 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 significantly increased above the concurrent negative control and is within our historical negative control data range.

Statistics:

An appropriate statistical trend test (MS EXCEL function RGP) was performed to assess a dose-related increase of mutant frequencies. The number of mutant colonies obtained for the test substance treated groups was compared with that of the respective vehicle control groups. A trend is judged as statistically significant whenever the one-sided p-value (probability value) is below 0.05 and the slope is greater than 0. However, both, biological and statistical significance will be considered together.

Results and discussion

Test results

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

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: with S9 mix, the pH was lowered at concentrations at and above 3012.5µg/mL
- Precipitation: precipitation in the culture medium (but not in the vehicle) occured at concentrations of 1506.3µg/mL and above (with or without S9 mix)
These concentrations are well above those used in the study due to high cytotoxicity. No influence on the result is expected.

RANGE-FINDING/SCREENING STUDIES: The doses were selected to reduce cloning efficiency to about 20% or less.

COMPARISON WITH HISTORICAL CONTROL DATA:
The positive control (EMS) without S9 mix did not produce the expected increase in mutant colonies. That's why this experiment was repeated - designated experiment 3. All other positive and negative control values were within the historical range. Also, mutant frequencies of the treatment groups did not exceed the historical negative control data.

Any other information on results incl. tables

Experimenta result without S9
Experiment	Test group	Mutant frequency [per 10^6cells]	Cytotoxicity CE1 [%]	Cytotoxicity CE2 [%]
1	vehicle control	0.61	100.0	100.0
	0.23	n.c.	97.7	n.c.
	0.47	n.c.	102.4	n.c.
	0.94	0.32	96.9	95.6
	1.88	0.63	96.8	95.9
	3.75	5.20	90.7	91.1
	7.50	1.73	49.9	65.5
	15.00	n.c.	0.1	n.c.
	30.00	n.c.	0.0	n.c.
	positive control	111.10	96.8	96.0
2	vehicle control	5.72	100.0	100.0
	0.27	n.c.	95.3	n.c.
	0.55	6.06	96.9	116.5
	1.09	1.55	86.5	105.1
	2.19	1.99	84.1	109.1
	4.38	0.35	87.9	96.2
	8.75	n.c.	1.1	n.c.
	17.50	n.c.	0.3	n.c.
	positive control	5.18	88.5	119.8
	Positive control did not fulfill acceptance criteria
3	vehicle control	6.41	100.0	100.0
	0.27	n.c.	86.5	n.c.
	0.55	n.c.	97.2	n.c.
	1.09	3.93	86.2	85.8
	2.19	5.29	79.9	83.2
	4.38	1.78	77.5	86.6
	8.75	0.00	35.8	74.2
	17.50	n.c.	0.0	n.c.
	positive control	111.19	83.9	73.2

Experimenta result with S9
Experiment	Test group	Mutant frequency [per 10^6cells]	Cytotoxicity CE1 [%]	Cytotoxicity CE2 [%]
1	vehicle control	1.71	100.0	100.0
	1.88	n.c.	102.5	n.c.
	3.75	n.c.	99.6	n.c.
	7.50	4.00	98.2	93.1
	15.00	2.65	94.4	102.4
	30.00	10.43	66.7	95.1
	60.00	2.07	30.6	102.7
	120.00	n.c.	0.0	n.c.
	positive control	120.67	101.6	102.0
2	vehicle control	1.73	100.0	100.0
	2.19	n.c.	94.0	n.c.
	4.38	1.35	83.6	102.4
	8.75	1.00	50.8	100.5
	17.50	3.55	38.7	100.2
	35.00	7.01	24.1	93.3
	70.00	n.c.	0.2	n.c.
	140.00	n.c.	0.2	n.c.
	positive control	89.81	107.1	93.0

Vehicle controls (culture medium, DMSO, acetone, ethanol, tetrahydrofurane)

	Without S9		With S9
Mutant frequency	uncorrected	corrected*	uncorrected	corrected*
Mean	2,51	3,09	2,29	2,84
Min	0,00	0,00	0,00	0,00
Max	13,33	16,43	14,44	16,12
SD	2,57	3,18	2,35	2,94
Number of experiments	131		239

* = mutant frequency (per 1 million cells) corrected with the cloning efficiency at the end of the expression period

Applicant's summary and conclusion

Conclusions:

Thus, 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.