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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:
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

Constituent 1
Chemical structure
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:
butane-1,3-diyl bis(2-methylacrylate)
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
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation
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 resultsopen allclose all
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
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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