1-methyltrimethylene dimethacrylate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1979-11-26 to 1980-03-24

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Remarks:

Relevant methodological deficiencies: the independent repeats were not consistent; results confunded by precipitation

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1985

Report date:

1985

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

1-methyltrimethylene dimethacrylate

EC Number:

214-711-0

EC Name:

1-methyltrimethylene dimethacrylate

Cas Number:

1189-08-8

Molecular formula:

C12H18O4

IUPAC Name:

4-[(2-methylprop-2-enoyl)oxy]butan-2-yl 2-methylprop-2-enoate

Test material form:

other: liquid

Details on test material:

- chemical name: 1,3-Butanediol dimethacrylate

Method

Target gene:

thymidine kinase (TK)

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media:
growth medium: Fisher's mouse leukemia medium, supplemented with L-glutamine, sodium pyruvate and 10% horse serum;
cloning medium: growth medium + 0.35% agar
selection medium: cloning medium + 100 µg/mL BrdU or 3 µg/mL TFT
- 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:

S9 mix

Test concentrations with justification for top dose:

1.95 nL/mL to 1.0 µL/mL in twofold dilution steps

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: the test substance was immiscible with water at 100 µL/mL, but soluble in DMSO

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Controls 2

Positive controls:

yes

Positive control substance:

other: dimethylnitrosamine

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 h
- Expression time (cells in growth medium): 2-3 d
- Selection time (if incubation with a selection agent): 10 d
- Fixation time (start of exposure up to fixation or harvest of cells): 12-13 d

SELECTION AGENT (mutation assays): 3 µg/mL trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: 1; seeded in triplicates for selection

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency / relative growth

Evaluation criteria:

A test substance is considered to be mutagenic if:
- for any treatment the mutant frequency exceeds 150% of the concurrent background control by at least 1E-06 (= minimum criterion)
- a concentration-related or toxicity-related increase in mutant frequency is observed
- an increase of 2 times the minimum criterion is observed in a single concentration near the highest testable concentration

Results and discussion

Test results

Test results 1

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
The test substance separated from the culture medium to form suspended globules at concentrations of 250 nL/mL and higher; in the presence of S9 mix increased cloudiness was observed at 62.5 nL/mL and higher

RANGE-FINDING/SCREENING STUDIES:
in a preliminary test for cytotoxicity treatment with 125 nL/mL reduced the cell growth after 24 h, 250 nL/mL were almost completely lethal

Any other information on results incl. tables

		Rel. Suspension growth [%]	Rel. cloning efficiency[%]	percent rel. Growth[%]	mutant frequency x10E-06	evaluation
Experiment 1, without metabolic activation
solvent control		100.0	100.0	100.0	33.1	-
solvent control		100.0	100.0	100.0	24.2	-
untreated control		60.9	84.1	51.2	50.0	-
Positive control (EMS)		42.6	36.3	15.5	768.5	+
test item [nL/mL]	15.6	31.5	91.0	28.7	23.8	-
	31.3	36.4	124.5	45.3	22.6	-
	62.5	63.3	72.2	45.7	44.1	-
	125	57.5	61.2	35.2	46.0	-
	250	15.5	98.0	15.2	25.4	-
Experiment 1, with metabolic activation
solvent control		100.0	100.0	100.0	26.9	-
solvent control		100.0	100.0	100.0	34.3	-
untreated control		139.9	76.0	106.3	35.4	-
Positive control (DMN)		60.9	41.0	24.9	248.6	+
test item [nL/mL]	3.9	63.6	72.2	46.2	40.1	-
	31.3	145.7	83.0	121.0	41.3	-
	62.5	112.7	99.3	111.6	35.7	-
	125	58.7	97.4	57.2	47.0	-
	250	68.4	89.7	43.4	47.7	-
Experiment 2, with metabolic activation
solvent control		100.0	100.0	100.0	24.2	-
solvent control		100.0	100.0	100.0	21.2	-
untreated control		102.9	82.0	84.4	28.3	-
Positive control (DMN)		35.2	20.6	7.3	194.5	+
test item [nL/mL]	250	52.4	78.7	41.2	31.0	-
	300	44.0	89.9	39.5	32.9	-
	400	42.6	89.9	38.3	36.7	-
	400	48.1	96.3	46.3	40.9	-
	500	30.3	77.6	23.5	48.8	+
	500	39.8	67.4	26.8	51.7	+
	600	18.9	86.9	16.5	59.5	+
Experiment 3, with metabolic activation
solvent control		100.0	100.0	100.0	14.6	-
solvent control		100.0	100.0	100.0	20.1	-
untreated control		127.0	86.7	112.7	16.8	-
Positive control (DMN)		45.8	10.1	4.6	317.2	+
test item [nL/mL]	400	24.8	67.6	16.8	45.6	+
	400	43.2	86.7	37.5	32.0	-
	500	50.8	88.4	44.2	25.5	-
	500	38.0	54.4	20.7	49.7	+
	600	26.0	49.6	12.9	100.0	+
Experiment 4, with metabolic activation
solvent control		100.0	100.0	100.0	18.9	-
solvent control		100.0	100.0	100.0	15.4	-
untreated control		89.0	84.6	75.3	27.2	-
Positive control (DMN)		42.0	16.4	6.9	175.0	+
test item [nL/mL]	400	24.9	46.4	11.6	62.8	+
	400	18.4	46.2	8.3	71.8	+
	500	17.0	27.5	4.7	94.0	+
	600	6.2	16.8	1.0	120.0	+
Experiment 5, with metabolic activation
solvent control		100.0	100.0	100.0	34.0	-
solvent control		100.0	100.0	100.0	17.8	-
untreated control		119.2	38.1	105.0	19.2	-
Positive control (DMN)		27.5	35.0	9.6	244.8	+
test item [nL/mL]	62.5	32.6	81.3	26.5	73.3	+
	250	17.3	52.7	9.1	222.1	+
	350	9.7	26.1	2.5	488.8	+
	400	8.7	28.9	2.6	405.1	+
	450	13.1	34.6	4.5	318.6	+
	500	3.9	10.0	1.2	618.4	+
	550	7.9	39.0	3.1	347.4	+

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative without metabolic activation
positive with metabolic activation

1,3-BDDMA was mutagenic with metabolic activation and not mutagenic without metabolic activation in the mouse lymphoma L5178Y test system under the experimental conditions described in this study. However, precipitation starting from 62.5 nL/mL and higher with metabolic activation and/or separation of the test substance from the culture medium to form suspended globules at 250 nL/mL was reported. It cannot be excluded that different local concentrations due to not completely dissolved test substance were leading to unreproducible results regarding cytotoxicity and mutant frequency. Thus, this test is considered not be not reliable.

Executive summary:

In a mammalian cell gene mutation assay equivalent to OECD guideline 476 (thymidine kinase (TK)), L5178Y mouse lymphoma cells cultured in vitro were exposed to 1,3-BDDMA in DMSO in the following concentrations:

 

Experiment 1

Without metabolic activation: 15.6, 31.3, 62.5, 125 and 250 nL/mL

With metabolic activation: 3.9, 31.3, 62.5, 125 and 250 nL/mL

Experiment 2

With metabolic activation: 250, 300, 400, 500, 600 nL/mL

Experiment 3

With metabolic activation: 400, 500, 600 nL/mL

Experiment 4

With metabolic activation: 400, 500, 600 nL/mL

Experiment 5

With metabolic activation: 62.5, 250, 350, 400, 450, 500, 550 nL/mL

 

1,3-BDDMA was tested up to cytotoxic concentrations. Positive controls induced the appropriate response.

No increase in mutant frequency was observed without metabolic activation. As in the first experiment with metabolic activation, no sufficient cytotoxicity was achieved, higher concentrations have been tested.

In the second experiment mutant frequencies were elevated in cultures exposed to 500 and 600 nL/mL, however mutant frequencies induced by the positive control in this experiment was low.

In the third experiment, mutant frequencies were elevated in cultures treated with 400, 500 and 600 nL/mL. As the increase was only small at 400 and 500 nL/mL, further tests have been conducted.

In the forth experiment mutant frequencies were elevated in cultures treated with 400, 500 and 600 nL/mL. All treatments were also highly toxic and resulted in depressed cloning efficiencies.

In the fifth experiment all concentrations starting from 62.5 nL/mL resulted in increased mutant frequencies. But concentrations of 250 nL/mL and higher were highly toxic.

There was evidence of induced mutant colonies over background with metabolic activation.

However, precipitation starting from 62.5 nL/mL and higher with metabolic activation and/or separation of the test substance from the culture medium to form suspended globules at 250 nL/mL was reported.

It cannot be excluded that different local concentrations due to not completely dissolved test substance were leading to unreproducible results regarding cytotoxicity and mutant frequency. Thus, this test is considered not be not reliable.

 

 

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