Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial gene mutation assay

The potential of n-butyl methacrylate to induce reverse mutation in Salmonella typhimurium (strains: TA 1535, TA 1537, TA 98, and TA 100) and in Escherichia coli WP2 uvrA was evaluated in accordance with the international guidelines (OECD 471, Commission Directive No. B13/14) in compliance with the Principles of Good Laboratory Practice (Nakajima et al., 1998a). n-Butyl methacrylate was tested in two independent experiments, with and without a metabolic activation system, both performed according to preincubation method. Bacteria were exposed to the test item at 7 or 8 dose-levels (three plates/dose-level) selected from a preliminary toxicity test: 9.77, 19.5, 39.1, 78.1, 156, 313 and 625 µg/plate without S9 and 9.77, 19.5, 39.1, 78.1, 156, 313, 625 and 1250 µg/plate with S9. After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored. n-butyl methacrylate did not induce any noteworthy increase in the number of revertants, both with and without S9 mix, in any of the four Salmonella typhimurium strains and in Escherichia coli WP2 uvrA. Under these experimental conditions, n-butyl methacrylate did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium and Escherichia coli.

The potential of n-butyl methacrylate (n-BMA) to induce reverse mutation in Salmonella typhimurium (strains: TA 98, TA 100, TA 1535, TA 1537) was evaluated according to a protocol comparable to the OECD guidelines 471 (Zeiger et al., 1987). n-BMA was tested in single experiment, with and without a metabolic activation system, according to the preincubation method (20 min at 37 °C). Concentrations of n-BMA (0, 1, 3.3, 10, 33, 100, 333, 1000, 3333, and 10000 µg/plate.), overnight culture of S. typhimurium (0.05-0.10 ml) and S-9 mix or buffer were incubated without shaking for 20 minutes. The top agar was added and the contents of the tubes were mixed and poured onto the surfaces of Petri dishes. His+ (histidine dependent) colonies arising on plates were machine-counted after two days incubation at 37 °C. Testing was without metabolic activation, with 10% rat liver S-9, or with 10% hamster liver S-9. The positive control chemicals induced a significant increase of the revertant frequency in all tester strains, either with or without metabolic activation. n-BMA was negative, in the presence and absence of metabolic activation, in all tester strains.

 

Chromosomal aberration assay in mammalian cells

n-Butyl methacrylate (n-BMA) was tested in an in vitro chromosomal aberration test in Chinese hamster lung fibroblasts (CHL; Nakajima et al., 1998b). The study was performed following the OECD 473 guideline and GLP. The concentrations were set based on the results of previously conducted cell growth inhibition tests. The test was performed at concentrations of 178, 355, 710 and 1420 µg n-BMA/ml (≈10mM) with treatments of 6, 24 and 48 hours (continuous treatment method) without metabolic activation and at concentrations of 355, 710 and 1420 µg n-BMA/ml for 6 hours (short term treatment) with metabolic activation.

Both the positive control substances mitomycin C (MMC) in the continuous treatment method and cyclophosphamide (CP) in the short term treatment method induced a high frequency of chromosomal structural aberrations. n-BMA did not induce a higher frequency of chromosomal aberration for both the continuous treatment method and short term treatment method.

 

Gene mutation assay in mammalian cells

The study was performed to investigate the potential of n-BMA (n-Butyl methacrylate) to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster in an OECD guideline 476 according to GLP requirements (Envigo, 2016). The treatment period was 4 hours with and without metabolic activation. The maximum test item concentration of the pre-experiment (1420 µg/mL) was equal to a molar concentration of about 10 mM. The concentration range of the main experiment was limited by phase separation of the test item.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in the main experiment. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

In conclusion it can be stated that under the experimental conditions reported the test item n-butyl methacrylate (n-BMA) did not induce gene mutations at the HPRT locus in V79 cells.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine operon
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9: Rat liver, induced with phenobarbital and 5,6-benzoflavone 
Test concentrations with justification for top dose:
-S9 mix: 9.77, 19.5, 39.1, 78.1, 156, 313 and 625 µg/plate
+S9 mix: 9.77, 19.5, 39.1, 78.1, 156, 313, 625 and 1250 µg/plate
Vehicle / solvent:
DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: -S9 mix; 2-(2-Furyl)-3-(5-nitro-2-furyl) acrylamide (TA100, TA98, WP2 uvrA), Sodium azide (TA1535), 9-Aminoacridine (TA1537)  +S9 mix; 2-Aminoanthracene(all strains) 
Details on test system and experimental conditions:
100μl of the solvent used, the test substance solution and the positive control substance was placed in a capped tube, and then 500μl of 0.1M sodium phosphate buffer (pH7.4) for the direct method, or 500μl of the S9 mix for the metabolic activation method was added. Next, after adding 100μl of the pre-incubated test bacteria strain suspension, this was subject to 20 minutes of shaking incubation (pre-incubation) at 37°C using an incubator shaker. After incubation was completed, 2ml of top agar was added and the contents mixed. Then, the mixed solution was poured and evenly spread on the plate. After the layered top agar solidified, each plate was sealed with cellophane tape and incubated for 48 hours under conditions of 37°C using an incubator.
Three plates per dose were used. Also, to verify reproducibility, these tests were independently performed two times.
Evaluation criteria:
The number of reverse mutated colonies increased to nearly double that of the control solvent, and when the reproducibility and the test substance dose dependence was confirmed, it was deemed positive
Statistics:
A statistical analysis was not performed
Species / strain:
other: Salmonella typhimurium TA100, TA1535, TA98, TA1537, Escherichia coli WP2 uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
>= 156 µg/plate in the 5 strains without S9. >=313 µg/plate (TA100, Ta1535, TA98, TA1537) and >= 625 µg/plate (WP2 uvrA) with S9
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
This chemical was not mutagenic in Salmonella typhimurium TA100, TA1535, TA98, TA1537 and Escherichia coli WP2 uvrA, with or without an exogenous metabolic activation system. 
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

In a valid guideline study the test substance was not mutagenic in Salmonella typhimurium TA100, TA1535, TA98, TA1537 and Escherichia coli WP2 uvrA, with or without an exogenous metabolic activation system. 
Executive summary:

The potential of n-butyl methacrylate to induce reverse mutation in Salmonella typhimurium (strains: TA 1535, TA 1537, TA 98, and TA 100) and in Escherichia coli WP2 uvrA was evaluated in accordance with the international guidelines (OECD 471, Commission Directive No. B13/14) in compliance with the Principles of Good Laboratory Practice.

n-Butyl methacrylate was tested in two independent experiments, with and without a metabolic activation system, both performed according to preincubation method. Bacterias were exposed to the test item at 7 or 8 dose-levels (three plates/dose-level) selected from a preliminary toxicity test: 9.77, 19.5, 39.1, 78.1, 156, 313 and 625 µg/plate without S9 and 9.77, 19.5, 39.1, 78.1, 156, 313, 625 and 1250 µg/plate with S9. After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored.

n-butyl methacrylate did not induce any noteworthy increase in the number of revertants, both with and without S9 mix, in any of the four Salmonella typhimurium strains and in Escherichia coli WP2 uvrA.

Under these experimental conditions, n-butyl methacrylate did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium and Escherichia coli.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
July 26 - Sept 05, 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
adopted 28 July 2015
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
May 30, 2008
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Version / remarks:
August 1998
GLP compliance:
yes
Type of assay:
other: In Vitro Mammalian Cell Gene Mutation Test (HPRT)
Target gene:
HPRT
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: supplied by Laboratory for Mutagenicity Testing; Technical University, 64287 Darmstadt, Germany)

MEDIA USED
- Type and identity of media: MEM (minimal essential medium) containing Hank’s salts, neomycin (5 µg/mL), 10% FBS, and amphotericin B (1 %)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
44.8 - 1420 µg/mL
(without and without S9 mix: 44.8; 88.8; 177.5; 355.0; 710.0; 1420.0 µg/mL)
Highest dose corresponds to 10 mM as requested in OECD TG 476
Vehicle / solvent:
- Solvent used: DMSO
- Justification for choice of solvent: The solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures. The final concentration of ethanol in the culture medium did not exceed 0.5 % (v/v).
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
other: EMS; ethylmethane sulfonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in suspension

DURATION
- Exposure duration: 4 h
- Expression time (cells in growth medium): 7 d
- Selection time (if incubation with a selection agent): 8 d

SELECTION AGENT (mutation assays): 6-TG (6-thioguanine)

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 500

DETERMINATION OF CYTOTOXICITY
- Method: relative cloning efficiency
Evaluation criteria:
The gene mutation assay is considered acceptable if it meets the following criteria:
a) the mean values of the numbers of mutant colonies per 10e6 cells found in the solvent controls of both parallel cultures remain within the 95% confidence interval of the laboratory historical control data range.
b) the positive control substances should produce a significant increase in mutant colony frequencies and remain within the historical control range of positive controls.
c) the cloning efficiency II (absolute value) of the solvent controls must exceed 50 %.
Statistics:
A linear regression (least squares, calculated using a validated excel spreadsheet) was performed to assess a possible dose dependent increase of mutant frequencies. The numbers of mutant colonies generated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05.
A t-Test was performed using a validated test script of “R”, a language and environment for statistical computing and graphics, to evaluate an isolated increase of the mutation frequency at a test point exceeding the 95% confidence interval. Again a t-test is judged as significant if the p-value (probability value) is below 0.05.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
No relevant and reproducible increase in mutant colony numbers/10e6 cells was observed in the main experiment up to the maximum concentration.
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Relevant cytotoxic effects indicated by an adjusted cloning efficiency I below 50% occurred at 355.0 µg/mL without metabolic activation (in both cultures).
Vehicle controls validity:
valid
Remarks:
The viability (cloning efficiency II) of the solvent control of the second culture without metabolic activation did not exceed the lower limit of 50%. The data are valid however, as the solvent control of the parallel culture exceeded this limit.
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Remarks:
EMS (300 µg/mL) and DMBA (2.3 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.
Additional information on results:
The 95% confidence interval was exceeded at 44.8 µg/mL and 355.0 µg/mL in the first culture of the first experiment without metabolic activation (50.1 and 35.9 versus an upper limit of 29.7 mutant colonies/10e6 cells). The t-Test over both parallel cultures at both concentrations turned out significant. However, no increase of the mutation frequency above the 95% confidence interval was noted in the parallel culture and the increase observed in the first culture was not dose dependent as indicated by the lacking significance of the linear regression analysis. Consequently, the isolated increases of the mutation frequency described above were judged as biologically irrelevant as they were not reproduced in the parallel culture and not dose dependent.
A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in any of the experimental groups.

Main experiment - Summary of Results

 

 

 

 

relative

relative

rel.adjusted

mutant

95%

relative

relative

rel.adjusted

mutant

95%

 

conc.

 

S9

cloning

cell

cloning

colonies/

confidence

cloning

cell

cloning

colonies/

confidence

 

µg/mL

PS

mix

efficiencyI

density

efficiencyI

16cells

0

interval

efficiencyI

density

efficiencyI

16cells

0

interval

 

 

 

 

%

%

%

 

 

%

%

%

 

 

Column

1

2

3

4

5

6

7

8

9

10

11

12

13

Experiment I/ 4h treatment

 

 

 

culture I

culture II

Solvent control with DMSO

 

 

-

100.0

100.0

100.0

15.1

0.2-29.7

100.0

100.0

100.0

11.0

0.2-29.7

Positive control (EMS)

300.0

 

-

85.0

86.1

73.2

296.8

0.2-29.7

86.3

103.6

89.4

263.3

0.2-29.7

Test item

44.8

 

-

92.0

82.3

75.7

50.1

0.2-29.7

91.6

118.1

108.2

28.0

0.2-29.7

Test item

88.8

 

-

108.2

88.3

95.5

13.8

0.2-29.7

100.5

108.8

109.4

21.3

0.2-29.7

Test item

177.5

 

-

94.4

88.4

83.5

8.9

0.2-29.7

94.3

79.8

75.2

25.3

0.2-29.7

Test item

355.0

PS

-

62.0

56.0

34.7

35.9

0.2-29.7

53.9

76.5

41.3

17.9

0.2-29.7

Test item

710.0

PS

-

#

7.2

#

 culture was not continued #

Test item

1420.0

PS

-

culture was not continued #

culture was not continued #

Solvent control with DMSO

 

 

+

100.0

100.0

100.0

9.5

0.6-28.7

100.0

100.0

100.0

21.9

0.6-28.7

Positive control (DMBA)

2.3

 

+

84.2

90.6

76.3

157.1

0.6-28.7

56.9

86.9

49.4

178.8

0.6-28.7

Test item

44.8

 

+

94.1

108.3

101.9

13.4

0.6-28.7

81.0

84.6

68.5

13.3

0.6-28.7

Test item

88.8

 

+

99.5

95.7

95.2

12.8

0.6-28.7

93.9

78.5

73.7

26.8

0.6-28.7

Test item

177.5

 

+

87.7

97.3

85.3

26.6

0.6-28.7

104.0

82.5

85.8

9.7

0.6-28.7

Test item

355.0

PS

+

89.2

92.4

82.4

26.4

0.6-28.7

102.7

101.5

104.2

12.9

0.6-28.7

Test item

710.0

PS

+

87.5

93.9

82.1

8.3

0.6-28.7

81.0

72.4

58.6

27.1

0.6-28.7

Test item

1420.0

PS

+

86.8

81.8

71.0

##

76.8

84.7

65.1

##

PS = Phase separation at the beginning and at the end of treatment

# culture was not continued due to exceedingly severe cytotoxic effects

## culture was not continued to avoid analysis of too many insoluble concentrations

Conclusions:
Under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, n-BMA (n-Butyl methacrylate) is considered to be non-mutagenic in this HPRT assay.
Executive summary:

The study was performed to investigate the potential of n-BMA (n-Butyl methacrylate) to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster in an OECD guideline 476 according to GLP requirements (Envigo 2016).

The treatment period was 4 hours with and without metabolic activation.

The maximum test item concentration of the pre-experiment (1420 µg/mL) was equal to a molar concentration of about 10 mM. The concentration range of the main experiment was limited by phase separation of the test item.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in the main experiment.

Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
other: Chinese hamster lung fibroblasts (CHL)
Metabolic activation:
with and without
Metabolic activation system:
S9 : Rat liver, induced with phenobarbital and 5,6-benzoflavone 
Test concentrations with justification for top dose:
-S9 mix(continuous exposure): 0, 178, 355, 710, 1420 ug/mL
-S9 mix(short-term exposure): 0, 178, 355, 710, 1420 ug/mL
+S9 mix(short-term exposure): 0, 355, 710, 1420 ug/mL
Vehicle / solvent:
DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: -S9 mix, 1-methyl-3-nitro-1-nitrosoguanidine +S9 mix, Benzo[a]pyrene 
Details on test system and experimental conditions:
PRELIMINARY TESTS:
a. Test Doses
In the preliminary tests (5 doses of 4.05, 13.5, 45.0, 150 and 500 μg/plate: ratio: 10/3), a clear cell growth inhibition action was not observed. Using these results as a reference, 6 doses containing a concentration equivalent to 10mM (ratio: 2) were set. The range of test doses in each test is shown below. Two wells per dose were used.
Test Number of Doses Test Doses (μg/ml)
24 hours of continuous treatment method 6 110-~1420
48 hours of continuous treatment method 6 110-~1420
Short term treatment method –S9 treatment 6 110-~1420
Short term treatment method +S9 treatment 6 110-~1420

b. Continuous Treatment Method
In a cell incubation flask (incubation area 25cm2), 5ml of a cell suspension (4x10e4 cells) prepared in 8x10e3 cells/ml using an incubation solution was used for the 24 hour treatment and 5ml of a cell suspension (2x10e4 cells) prepared in 4x10e3 cells/ml was used for the 48 hour treatment. After incubating for 3 days, 25 μl of the solvent used (hereafter, the solvent) or the test substance solution was added, and incubation was continued for an additional 24 or 48 hours to determine the cell survival rate (ratio to the control solvent).

c. Short Term Treatment Method
5ml of a cell suspension (4x10e4 cells) prepared in 8x10e3 cells/ml using an incubation solution was incubated in a cell incubation flask. After incubating for 3 days, in the case of –S9 treatment, 2ml of the incubation solution was removed and 15 μl of the solvent or test substance solution was added while in the case of +S9 treatment, 2.5ml of the incubation solution was removed and 500 μl of the S9 mix and 15 μl of the solvent or test substance solution was added. Each was incubated for 6 hours. After removing the incubation solution from each flask, the cells were washed using Dulbecco’s phosphate buffered saline (Life Technologies Co.). 3ml of fresh incubation solution was added and incubation continued for an additional 18 hours to determine the cell survival rate.

d. Calculation of the 50% Cell Growth Inhibition Concentration
The incubation solution was removed from each flask in the cell growth inhibition test and the cells were washed once using saline solution. 10% neutral buffered formalin was added and after fixing the cells for approximately 10 minutes, a 0.1% crystal violet solution was used to dye this for 10 minutes. After rinsing each flask, they were completely dried.
10ml of a dye elution (30% ethanol, 1% acetate solution) was added to each flask and after leaving alone for 5 minutes, the absorbance at 580nm was measured using a spectrophotometer. With the control solvent group, the ratio to the absorbance (=cell survival rate) is determined for each dose group, and then the Probit model was used to calculate the 50% cell growth inhibiting concentration. The calculation used the 5 points of 184~1420 μg/ml (24 hour continuous treatment method), the 6 points of 110~1420 μg/ml (48 hour continuous treatment method) and the 6 points of 110~1420 μg/ml (short term treatment method –S9 treatment).

MAIN TEST:
a. Test Doses
Based on the cell growth inhibition test results, the 4~5 doses (ratio 2: refer to the chart below) for each test were used to set the dose for this particular test. For the short term treatment method, doses equivalent to 10mM were set as the maximum dose.
Two flasks per dose were utilized.
Test Test Doses (μg/ml)
24 hour continuous treatment method (88.8), 178, 355, 710, (1420)
48 hour continuous treatment method (88.8), 178, 355, 710, (1420)
short term treatment method –S9 treatment 178, 355, 710, (1420)
short term treatment method +S9 treatment (178), 355, 710, 1420
Doses in brackets were not observed with a microscope.

b. Continuous Treatment Method
In a cell incubation flask (incubation area 25cm²), 5ml of a cell suspension (4x10e4 cells) prepared in 8x103 cells/ml using an incubation solution was used for the 24 hour treatment and 5ml of a cell suspension (2x10e4 cells) prepared in 4x10e3 cells/ml was used for the 48 hour treatment. After incubating for 3 days, 25 μl of the solvent, test substance solution and 500 μl of the positive control substance solution was added, and incubation was continued for an additional 24 or 48 hours to produce the chromosome samples.

c. Short Term Treatment Method
5ml of a cell suspension (4x10e4 cells) prepared in 8x10e3 cells/ml using an incubation solution was incubated in a cell incubation flask. After incubating for 3 days, in the case of –S9 treatment, 2ml of the incubation solution was removed and 15 μl of the solvent, test substance solution and 300 μl of the positive control substance solution was added while in the case of +S9 treatment, 2.5ml of the incubation solution was removed and 500 μl of the S9 mix and 15 μl of the solvent and test substance solution and 300 μl of the positive control substance solution was added. Each was incubated for 6 hours. After removing the incubation solution from each flask, the cells were washed using Dulbecco’s phosphate buffered saline. 3ml of fresh incubation solution was added and incubation continued for an additional 18 hours to produce the chromosome samples.

d. Slide Production
A final concentration of 0.2 μg/ml of a colcemid was added two hours before producing the chromosome samples and cell division was stopped mid-cycle. Next, after moving all of the incubation solution to a centrifugal tube, a 0.25% trypsin solution was used to separate the cells from the flask, which were added to the incubation solution in the centrifugal tube. The incubation solution was removed by centrifugal separation for five minutes at 1000rpm and while maintaining the temperature at 37°C, approximately 5ml of a 75mM gallium chloride solution was added to the cell suspension and 20 minutes of hypotonic treatment was conducted at 37°C. After removing the hypotonic solution by centrifugal separation, the cells were fixed by a fixative (methanol-acetate: 3:1) chilled to 4°C. After replacing the fixative 3 times, a suitable amount of new fixative was added to make a cell suspension, and then after degreasing cleansing was completed, it was dripped onto a glass slide 1~2 drops at a time. The sample on the slide was completely dried and then using a 1/100M sodium-phosphate buffer (pH 6.8), dyed with a diluted 1.2% Giemsa stain for 12 minutes. After lightly rinsing the slide with water, it was dried.

e. Observation of the Chromosomes
100 mid-phase images per flask, or 200 per dose were observed under a microscope (x600) and based on the changes in chromosome form, the structural abberations were classified into gaps (gap), chromatid breaks (ctb), chromosome breaks (csb), chromatid exchange (cte), chromosome exchange (cse) and other (oth). At the same time, the appearance rate for polyploidy cells was recorded. There were un-stained regions on the chromatids or chromosomes, so if the cross-section image of the chromosome was confirmed and the un-stained regions were the same or greater than the chromatid width, and if they hadn’t shifted from the original position, they were calculated as a gap. A distinction was made between cells only containing gaps (+gap), and those without any (-gap), and the appearance frequency of the chromosomal aberrations is displayed.
After all of the samples were coded, they were observed with the masking method.





b. Continuous Treatment Method
In a cell incubation flask (incubation area 25cm²), 5ml of a cell suspension (4x104 cells) prepared in 8x10e3 cells/ml using an incubation solution was used for the 24 hour treatment and 5ml of a cell suspension (2x10e4 cells) prepared in 4x10e3 cells/ml was used for the 48 hour treatment. After incubating for 3 days, 25 μl of the solvent, test substance solution and 500 μl of the positive control substance solution was added, and incubation was continued for an additional 24 or 48 hours to produce the chromosome samples.

c. Short Term Treatment Method
5ml of a cell suspension (4x10e4 cells) prepared in 8x10e3 cells/ml using an incubation solution was incubated in a cell incubation flask. After incubating for 3 days, in the case of –S9 treatment, 2ml of the incubation solution was removed and 15 μl of the solvent, test substance solution and 300 μl of the positive control substance solution was added while in the case of +S9 treatment, 2.5ml of the incubation solution was removed and 500 μl of the S9 mix and 15 μl of the solvent and test substance solution and 300 μl of the positive control substance solution was added. Each was incubated for 6 hours. After removing the incubation solution from each flask, the cells were washed using Dulbecco’s phosphate buffered saline (Life Technologies Co.). 3ml of fresh incubation solution was added and incubation continued for an additional 18 hours to produce the chromosome samples.



DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other:


OTHER EXAMINATIONS:
- Determination of polyploidy:
- Determination of endoreplication:
- Other:


OTHER:
Evaluation criteria:
Cells with structural abberations in each group as well as the polyploid cell appearance frequency were determined using the standards shown below, and if there was a reappearance or test substance dose dependency, it was deemed positive. The final evaluation was performed if cells containing gaps were included.

Less than 5% Negative (-)
5% ~ 10% Suspected positive (±)
More than 10% Positive (+)
Statistics:
Determinations using statistical methods were not conducted.
Species / strain:
other: Chinese hamster lung fibroblasts (CHL)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: CE50 % for cell growth inhibition: continuous exposure; 532 µg/ml(24h), 386 µg/ml(48h), short-term exposure; 547 µg/ml(-S9)
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
other: Chinese hamster lung fibroblasts (CHL)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
CE50 >1420µg/ml (10 mM)
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
CYTOTOXICITY ASSAY (Table 1 and 2)
The 50% cell growth inhibiting concentration was 532 μg/ml for 24 hour continuous treatment, 386 μg/ml for 48 hour continuous treatment, 547 μg/ml for short term treatment method –S9 treatment and greater than 10mM for +S9 treatment (or 1420 μg/ml). At all dose levels, a white precipitate was created when the test substance was added but it disappeared after agitation. Furthermore, at doses greater than 511 μg/ml, oily droplets of the test substance were observed on the surface of the incubation solution but no changes of note were observed at the completion of incubation.

MAIN STUDY:
1) 24 Hour Continuous Treatment Method (Table 3)
The chromosomal aberrations and polyploidy cell appearance frequency in the butyl methacrylate treatment group were identical to that of the solvent control. Furthermore, at the maximum dose of 1420 μg/ml, observable division mid-phase images were almost undetectable.
On the other hand, many chromosomal aberrations were observed in cells treated with the positive control substance MMC, and the appearance frequency at +gap was 71.0%.

2) 48 Hour Continuous Treatment Method (Table 4)
The chromosomal aberrations and polyploidy cell appearance frequency in the test substance treatment group were identical to that of the solvent control. Furthermore, at the maximum dose of 1420 μg/ml, only a few living cells were observed, while at 710 μg/ml, there was a dramatic reduction in observable division mid-phase images.
In the positive control, chromosomal aberration cells had a 60.5% appearance in +gap.

3) Short Term Treatment Method –S9 Treatment (Table 5)
The chromosomal aberrations and polyploidy cell appearance frequency in the test substance treatment group were identical to that of the solvent control. Furthermore, at the maximum dose of 1420 μg/ml, observable division mid-phase images were almost undetectable while at 710 μg/ml, there was a slight decrease.
On the other hand, since metabolic activation was not performed with the group treated with CP, clear induction of chromosomal aberration was not confirmed.

4) Short Term Treatment Method +S9 Treatment (Table 6)
The chromosomal aberrations and polyploidy cell appearance frequency in the test substance treatment group were identical to that of the solvent control. Furthermore, marked cell toxicity was not observed in any of the groups.
On the other hand, cells treated with the positive control substance CP required for metabolic activation exhibited a large amount of aberration, structural aberration was confirmed in 67.5% of the cells with +gap.
In all of the test doses, the incubation solution created a white precipitate when the test substance was added but it disappeared after agitation. Also, at doses greater than 710 μg/ml, oily droplets of the test substance were observed on the surface of the incubation solution but no changes of note were observed at the completion of incubation.
Conclusions:
Interpretation of results (migrated information):
negative

In a valid guideline study, this chemical did not induce structural chromosomal aberrations and/or polyploidy in CHL cells, with or without an exogenous metabolic activation system.
Executive summary:

n-Butyl methacrylate (nBMA) was tested in an in vitro chromosomal aberration test in Chinese hamster lung fibroblasts (CHL). The study was performed following the OECD 473 guideline and GLP. The concentrations were set based on the results of previously conducted cell growth inhibition tests. The test was performed at concentrations of 178, 355, 710 and 1420 µg nBMA/ml (≈10mM) with treatments of 6, 24 and 48 hours (continuous treatment method) without metabolic activation and at concentrations of 355, 710 and 1420 µg nBMA/ml for 6 hours (short term treatment) with metabolic activation.

Both the positive control substances mitomycin C (MMC) in the continuous treatment method and cyclophosphamid (CP) in the short term treatment method induced a high frequency of chromosomal structural aberrations. nBMA did not induce a higher frequency of chromosomal aberration for both the continuous treatment method and short term treatment method.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The ability of n-butyl methacrylate to cause chromosomal damage in vivo was investigated in a micronucleus OECD 474 test (RTC, 1999). Dose-levels for treatment were selected on the basis of a preliminary toxicity test. Male and female Swiss CD-1 mice were dosed once intraperitoneally with vehicle only, corn oil, 2000, 1000 and 500 mg/kg bw n-butyl methacrylate and the positive control Mitomycin-C. Five animals per sex from each group were sacrificed at the 24 hour sampling time. The additional animals were sacrificed at the 48 hour sampling time. Following treatment with n-butyl methacrylate, no statistically significant increase in the incidence of micronucleated PCE's over the control value was observed at any dose-level. Slight increases in the ratio of mature to polychromatic erythrocytes, compared to the vehicle control, were seen at the 48 hour sampling time for both male and female animals from the high-dose group, indicating that the test substance exerted a mild toxic effect on the bone marrow cells. Following treatment with the positive control Mitomycin-C, statistically significant increases in the incidence of micronucleated PCE's over the control values were seen in the positive control group indicating the correct functioning of the test system. It is concluded that n-butyl methacrylate administered intraperitoneally at dose-levels of 2000, 1000 and 500 mg/kg bodyweight to both male and female animals, does not induce micronuclei in the polychromatic erythrocytes of treated mice, under the reported experimental conditions.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OTS 798.5395 (In Vivo Mammalian Cytogenics Tests: Erythrocyte Micronucleus Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
Swiss
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Nossan s.r.l., Corezzana, Italy,
- Age at study initiation: 5-6 weeks
- Weight at study initiation: 26-34 grams for males and 22-27 grams for females
- Assigned to test groups randomly: no data
- Housing: 5/polycarbonate cage, by sexes
- Diet (e.g. ad libitum): Altromin MT diet
- Water (e.g. ad libitum): tap water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 55 +/- 10
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: solubility
- Concentration of test material in vehicle: no data
Duration of treatment / exposure:
Single administration
Post exposure period:
24 and 48 hour sampling time
Remarks:
Doses / Concentrations:
0 (vehicle), 500, 1000 and 2000 mg/kg
Basis:

No. of animals per sex per dose:
Each group consisted of five male and five female animals with the exception of the control and high-dose groups, which included an additional five animals of each sex per group.
Control animals:
yes, concurrent vehicle
Positive control(s):
Mitomycin C
- Route of administration: intraperitoneal
- Doses / concentrations: 2.0 mg/kg bw
Tissues and cell types examined:
5 animals/sex/group were sacrificed at the 24-hour sampling time. The  additional animals were sacrificed at the 48-hour sampling time. At least  2000 polychromatic cells per animal were examined for the presence of micronuclei. The ratio of mature to polychromatic erythrocytes was also  determined.  
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
A preliminary toxicity study was conducted in which groups of two male and two female mice were dosed once (i.p. injection) with n-butyl  methacrylate at 2000, 1500 and 1000 mg/kg. Clinical signs were observed  in all animals on the day of dosing, but all animals recovered by the  following day. No bone marrow cytotoxicity, as measured by increases in  the NCE/PCE ratio, was observed. The doses for the definitive study were  selected based on this preliminary toxicity study. 

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Five animals per sex from each group were sacrificed at the 24 hour sampling time. The additional animals were sacrificed at the 48 hour sampling time.

DETAILS OF SLIDE PREPARATION:
The femurs were removed and bone marrow cells obtained by flushing with foetal calf serum. The cells were centrifuged and a concentrated suspension prepared to make smears on slides. These slides were air-dried and then stained with May-Gruenwald and Giemsa, and mounted with Eukitt. Three slides were made from each animal.

METHOD OF ANALYSIS:
The slides were randomly coded by a person not involved in the subsequent microscope scoring. The slides were examined under low power (x 16 objective) and one slide from each animal was selected according to staining and quality of smears. At least 2000 polychromatic cells per animal were examined for the presence of micronuclei at high power (x 100 objective, oil immersion). At the same time the numbers of normal and micronucleated normochromatic erythrocytes were also recorded.
Evaluation criteria:
The test substance is considered to induce micronuclei if a statistically significant increase in the micronucleus incidence in polychromatic erythrocytes (at P<0.05) is observed in any treatment group, in the pooled data for both sexes, or for either sex considered separately.
Statistics:
Only counts obtained from polychromatic cells were subjected to statistical analysis. Using the original observations (and not the micronucleus frequencies per 1000 cells), a modified Chi-squared calculation was employed to compare treated and control groups. The degree of heterogeneity within each group was first calculated and where this was significant it was taken into account in the comparison between groups. Variance ratios or Chi-squared values are taken to show the significance of any difference between each treated group and the controls.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Remarks:
but tested up to the limit dose
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
No statistically significant increase in the incidence of micronucleated PCE's over the control value was observed at any dose-level. Slight increases in the ratio of mature to polychromatic erythrocytes, compared to the vehicle control, were seen at the 48 hour sampling time for both male and female animals from the high-dose group.

Following are the results for the male and female animals combined:

                        Incidence of         NCE/PCE
            Dose        micronucleated        Mean
Treatment   Level              PCE              Ratio
           (mg/kg)        Mean      S.E.
-----------------------------------------------------------        
24 hr. Sampling time
-----------------------------------------------------------        
Vehicle (corn oil)
            10           0.5      0.2         1.12
-----------------------------------------------------------
           (ml/kg)
n-butyl methacrylate
           500           0.6      0.2         1.19
n-butyl methacrylate        
          1000           0.9      0.2         0.93
n-butyl methacrylate
          2000           0.6      0.2         1.15
-----------------------------------------------------------
Mitomycin-C  2.0        33.6***   6.5         1.32
-----------------------------------------------------------
48 hr. Sampling time
-----------------------------------------------------------        Vehicle (corn  oil)
           10            0.8      0.3         1.10
-----------------------------------------------------------
          (ml/kg)
n-butyl methacrylate
         2000            1.2      0.3         1.48
-----------------------------------------------------------
*** Incidence significantly greater than control value at p < 0.0001

Conclusions:
Interpretation of results (migrated information): negative
In a valid guideline study, no statistically significant increase in the incidence of micronucleated PCE's over the control value was observed at any dose-level. Slight increases in the ratio of mature to polychromatic erythrocytes, compared to the vehicle control, were seen   at the 48 hour sampling time for both male and female animals from the  high-dose group. The substance was negative for genotoxicity in this test.
Executive summary:
The ability of n-butyl methacrylate to cause chromosomal damagein vivowas investigated in a micronucleus OECD 474 test. Dose-levels for treatment were selected on the basis of a preliminary toxicity test. Male and female Swiss CD-1 mice were dosed once intraperitoneally with vehicle only, corn oil, 2000, 1000 and 500 mg/kg bw n-butyl methacrylate and the positive control Mitomycin-C. Five animals per sex from each group were sacrificed at the 24 hour sampling time. The additional animals were sacrificed at the 48 hour sampling time. Following treatment with n-butyl methacrylate, no statistically significant increase in the incidence of micronucleated PCE's over the control value was observed at any dose-level. Slight increases in the ratio of mature to polychromatic erythrocytes, compared to the vehicle control, were seen at the 48 hour sampling time for both male and female animals from the high-dose group, indicating that the test substance exerted a mild toxic effect on the bone marrow cells. Following treatment with the positive control Mitomycin-C, statistically significant increases in the incidence of micronucleated PCE's over the control values were seen in the positive control group indicating the correct functioning of the test system. It is concluded that n-butyl methacrylate administered intraperitoneally at dose-levels of 2000, 1000 and 500 mg/kg bodyweight to both male and female animals, does not induce micronuclei in the polychromatic erythrocytes of treated mice, under the reported experimental conditions.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Data availability

For n-BMA gene mutation data in bacteria and mammalian cells and chromosome mutation data in mammalian cells are available as in vitro data. In addition, a micronucleus test in vivo is available for n-BMA. Data from the category Lower Alkyl (C1-C8) Methacrylates support the findings in n-BMA.

Data from analogous substances and metabolites

Analogous substances

Reliable in vitro gene mutation assays in mammalian cells, test method OECD Guideline 476 (In vitro Mammalian Cell Gene Mutation Test), are available for methyl methacrylate (CAS 80-62-6); ethyl methacrylate (CAS 97-63-2) and 2-ethyl-hexyl methacrylate (CAS 688-84-6).In general, methyl methacrylate and ethyl methacrylate were positive in high and mainly toxic concentrations in several mouse lymphoma assays (small colony mutants, indicating that the genetic effect was derived from clastogenicity and not from gene mutations). 2-ethylhexyl methacrylate did not induce gene mutations at the HPRT locus in V79 cells (this study is attached as read-across information).[KK1] 

Further support for the absence of genotoxic potential in vivo can be gained by read-across from MMA (CAS 80-62-6), referring to a dominant lethal test in CD-1 mice (Anderson and Hodge, 1976). In this study groups of 20 male CD-1 mice were exposed via inhalation to MMA at 100, 1000, or 9000 ppm (416, 4160 and 37440 mg/m³) for 6 h/day for 5 days. Each male was subsequently mated with 2 different unexposed female mice weekly over a period of 8 weeks. MMA did not induce dominant lethal mutations as indicated by no adverse effect on total implants and early or late post-implantation death in the offspring of treated males compared to controls (this study is attached as read-across information).

 

Metabolites

It has been established that the lower alkyl methacrylates have a common mode of chemical reactivity via the C=C double bond and Michael addition and as such this lends them the potential to be chemically reactive towards macromolecules such as protein and DNA though a mechanism of electrophilic attack, albeit with a low reactivity (see ch. 5.1.3 toxicokinetics; Schwöbel et al., 2010, Cronin, 2012, 2015). It has also been established that the primary metabolic pathway of the esters is fast hydrolysis by ubiquitous carboxylesterases in the human body with a half-life in the order of minutes (Jones 2002). The resultant acid (methacrylic acid) and alcohol metabolites are non genotoxic.

The EU ESR on MAA (methacrylic acid, CAS 79-41-4) concluded: “Methacrylic acid is negative in a bacterial gene mutation test. Further testing on methacrylic acid is lacking. However, taking into consideration the data on the structurally related substance methyl methacrylate - which indicate that this substance does not express a genotoxic potential in vivo - there is no need for further testing.”

None of the lower alkyl alcohol metabolites are regarded as mutagenic, including n-butanol.

 

Summary                                  

Overall, n-BMA is negative in gene mutation tests in in bacteria and mammalian cells. n-BMA did not induce chromosome aberrations in vitro or in vivo and this, also, is supported by analogous substances from the lower alkyl methacrylates category. In conclusion, n-BMA is regarded as non-genotoxic.


Short description of key information:
Gene mutation in bacteria
Negative S. typhimurium TA 1535, TA 1537, TA 98, and TA 100, and E. coli WP2 uvrA, with and without metabolic activation (OECD 471) (Nakajima, 1998a)
Gene mutation in mammalian cells
Negative HPRT test in V79 cells, OECD 476, (Envigo, 2016)
Cytogenicity in mammalian cells
Negative Chromosomal aberration test in CHL cells, with and without metabolic activation, OECD 473, (Nakajima, 1998b)

In vivo
Negative Micronucleus assay, mouse bone marrow, OECD 474 (RTC, 1999)
Negative Dominant lethal assay in the mouse (by read-across from methyl methacrylate) (Anderson and Hodge, 1996)

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

According to the available data and the CLP criteria for classification as germ cell mutagens, no classification is warranted for n-butyl methacrylate.