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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial reverse gene mutation assays


- OECD 471, Salmonella reverse mutation test, plate incorp- and pre-incubation method; S. typh. TA98, TA100, TA1535, TA1537; E.coli WP2 uvr A; +/- metabolic activation: negative (ICCR 2020)


- Similar to OECD 471, Salmonella reverse mutation test, plate incorporation method; S. typh. TA98, TA100, TA1535, TA1537, TA1538; +/- metabolic activation: negative (Waegemaekers 1984)


 


Gene Mutation Data in Mammalian Cells


- HPRT Assay, OECD 476, V79 Chinese hamster cell line; Cytogenetic test with V79 Chinese hamster cells; +/- metabolic activation: negative (RTC 2000)


- HPRT Assay, OECD 476, V79 Chinese hamster cell line; Cytogenetic test with V79 Chinese hamster cells; +/- metabolic activation: positive (CCR 1995)


 


Chromosome Mutation Data


- OECD 473, In vitro chromo-some aberration assay; Cytogenetic test with V79 Chinese hamster cells

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1994-10-12 to 1994-10-28
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: Performed according older version of OECD 471, 5th strain is missing
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted 26 May 1983
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
29 December 1992
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Name of test material (as cited in study report): 1,4-Butanediol dimethacrylate
Target gene:
his-locus
Species / strain / cell type:
S. typhimurium, other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 1538
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
Experiment 1 (plate incorporation test): 33.3, 100.0, 333.3, 1000.0, 2500.0, 5000.0 µg/plate
Experiment 2 (pre-incubation test): 10.0, 33.3, 100.0, 333.3, 1000.0, 2500.0 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility properties + relative nontoxicity to the bacteria
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
other: 4-nitro-o-phenylene-diamine
Remarks:
without metabolic activation
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) and preincubation

DURATION
- Preincubation period: 60 minutes
- Exposure duration: at least 48 h

NUMBER OF EXPERIMENTS: 2 (one performed as pre-incubation assay, one as plate incorporation assay), 3 plates per concentration each

DETERMINATION OF CYTOTOXICITY
- Method: Toxicity of the test article may be evidenced by a reduction in the number of spontaneous revertants, a clearing of the bacterial background lawn, or by degree of survival of treated cultures.

OTHER:
automatic colony count with “AUTOCOUNT” (Artek systems, Biosys)
Evaluation criteria:
- corresponding background growth on negative control and test plates
- normal range of spontaneous revertants: TA1535 (10-29), TA 1537 (5-28), TA 1538 (12-37), TA 98 (15-57), TA 100 (77-189)
- test material is considered mutagenic if either a dose related and reproducible increase in revertant numbers or a significant and reproducible increase in revertant numbers for at least one test concentration in induced
- a significant increase in revertants means at least twice as high in TA 100 and a three times higher number in TA 1535, TA 1537, TA 1538, TA 89 compared to the negative control
Statistics:
no appropriate statistical method available
Species / strain:
S. typhimurium, other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
No substantial increases in revertant colony numbers of any of the five tester strains were observed following treatment with 1,4-Butanediol dimethacrylate at any dose level, either in the presence or absence of metabolie activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of significance.

TEST-SPECIFIC CONFOUNDING FACTORS
- no data

RANGE-FINDING/SCREENING STUDIES:
- performed with 3.3, 10.0, 33.3, 100.0, 333.3, 1000.0, 2500.0 and 5000.0 µg/plate in TA 98 and TA 100
- normal background growth was observed up to 5000 µg/plate

COMPARISON WITH HISTORICAL CONTROL DATA:
controls were within the range of historical control data

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- in the plate incorporation assay concentrations of 2500 µg/plate (without metabolic activation) and higher reduced the number of revertants in TA 1535, TA 1537 and TA 1538
- in the pre-incubation assay the number of revertants was reduced at concentrations of 1000 µg/plate (without metabolic activation) and higher in TA 1535 and TA 1537; at 2500 µg/plate (with metabolic activation) in TA 1537 and at 2500 µg/plate (without metabolic activation) in TA 1538
Conclusions:
Interpretation of results: negative

In this study 1,4-BDDMA did not induce mutant colonies over background and is therefore considered non-mutagenic.
Executive summary:

In a reverse gene mutation assay in bacteria according to OECD guideline 471, adopted 26 May 1983, strains TA 1535, TA 1537, TA 98, TA 100 and TA 1538 of S. typhimurium were exposed to 1,4-BDDMA in DMSO at concentrations of 33.3, 100.0, 333.3, 1000.0, 2500.0, 5000.0 µg/plate in a plate incorporation assay and 10.0, 33.3, 100.0, 333.3, 1000.0, 2500.0 µg/plate in a pre-incubation assay in the presence and absence of mammalian metabolic activation (S9 mix).

1,4-BDDMA was tested up to cytotoxic concentrations. There was no evidence of induced mutant colonies over background.

The positive controls induced the appropriate responses in the corresponding strains.

This study is classified as acceptable and satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

NOTE: Any of data in this dataset are disseminated by the European Union on a right-to-know basis and this is not a publication in the same sense as a book or an article in a journal. The right of ownership in any part of this information is reserved by the data owner(s). The use of this information for any other, e.g. commercial purpose is strictly reserved to the data owners and those persons or legal entities having paid the respective access fee for the intended purpose.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1983
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: Performed according older version of OECD 471, 5th strain is missing
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted 26 May 1983
Deviations:
no
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Name of test material (as cited in study report): 1,4-Butanediol dimethacrylate
Species / strain / cell type:
S. typhimurium, other: S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
40, 160, 625, 2500 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
other: 4-nitro-o-phenylenediamine
Remarks:
without metabolic activation
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
other: 2-aminoanthracene
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48-72 hours

NUMBER OF REPLICATIONS: 2, Compounds were once tested with phenobarbital-induced S9 mix, once with Aroclor-1254-induced
S9 mix and twice without any additional metabolizing system. All tests were performed on triplicate plates.

Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 1538 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Conclusions:
Interpretation of results:
negative

There was no evidence of induced mutant colonies over background in any of the tester strains exposed to 1,4-BDDMA up to 2500 µg/plate in the presence or absence of mammalian metabolic activation in this study.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1999-11-10 to 1999-12-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
adopted 21 July 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EEC Council Directive 92/69, part B
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
- Name of test material (as cited in study report): 1,4-Butanediol dimethacrylate
Target gene:
not applicable (chromosome aberration test)
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: Ham's F10 + 15% FCS
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: no
- other: generation time, plating efficiency checked
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
1250, 625, 313, 156, 78.1, 39.1, 19.5 and 9.77 mg/L
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: not given
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 h; second experiment: 20 h continuous exposure without S9 mix
- Expression time (cells in growth medium): 17 h (recovery period)
- Fixation time (start of exposure up to fixation or harvest of cells): 20 h

SELECTION AGENT (mutation assays):
SPINDLE INHIBITOR (cytogenetic assays): 0.2 µg/mL colcemid for the last 3 h of the treatment period
STAIN (for cytogenetic assays): 3% Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 100 from each culture, two cultures were prepared at each test point

DETERMINATION OF CYTOTOXICITY
- Method: cell count

OTHER EXAMINATIONS:
- Determination of polyploidy: yes, recorded, but not included in the count of eligible metaphases
- Determination of endoreplication: yes, recorded, but not included in the count of eligible metaphases

Evaluation criteria:
A substance is considered clastogenic if: - any dose level shows a statistically significant increase in aberration-bearing cells - the increase is over historical controls - the increase is present in both replicates
Statistics:
Fisher's exact test with solvent control as reference point
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:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no effects
- Effects of osmolality: no effects
- Precipitation: opacity occurred above 313 mg/L

COMPARISON WITH HISTORICAL CONTROL DATA:
- the number of cells with chromosome aberrations were in the range of the historical control data

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- in the first experiment in the absence of S9 mix cytotoxicity was observed at 625 mg/L and higher (number of viable cells reduced to 3 and 13% compared to solvent control); at 313 mg/L the number of viable cells was 61% compared to solvent control
- in the second experiment marked toxicity was observed at 313 mg/L and higher (cell counts < 22% compared to solvent control); at 156 mg/L the number of viable cells was reduced to 54% of the solvent control
- in the presence of S9 mix, no dose-related toxicity was observed
Remarks on result:
other: all strains/cell types tested

The test substance did not induce an increase in chromosomal aberrations at any test point selected for scoring in the absence or presence of S9 mix.

A slight, but not dose-related increase in endoreduplication, was observed in the presence of S9 mix.

Increases in gaps over the negative control value were observed in the 3 h treatment group (313 mg/L) without metabolic activation. This was seen in only one replicate and therefore not considered biologically relevant.


Significant increase in aberration-bearing cells was observed in the positive controls indicating the correct functioning of the test system.


3 h treatment, without S9 mix mg/L mean rel. Cell count % cells scored cells with aberrations (+gaps) cells with aberrations (-gaps) %CA







untreated
107 200 11 4 2.0
solvent
100 200 5 3 1.5
test substance 1250 13


–

625 3


–

313 (#) 61 200 15 5 2.5

156 (#) 93 200 8 4 2.0

78.1 (#) 79 200 12 6 3.0

39.1 87


–

19.5 101


–

9.77 94


–
Mitomycin C 0.3 75 100 72 68 68.0 ***







3 h treatment, with S9 mix mg/L mean rel. Cell count %


%CA







untreated
70 200 9 3 1.5
solvent
100 200 11 4 2.0
test substance 1250 (#) 89 200 16 6 3.0

625 (#) 69 200 14 8 4.0

313 (#) 103 200 19 7 3.5

156 86


–

78.1 68


–

39.1 86


–

19.5 107


–

9.77 69


–
Cyclophosphamide 15 88 100 62 56 56.0 ***







20 h treatment, without S9 mix mg/L mean rel. Cell count %


%CA







untreated
143 200 1 0 0.0
solvent
100 200 3 0 0.0
test substance 1250 0


–

625 3


–

313 22


–

156 (#) 54 200 6 2 1.0

78.1 (#) 112 200 3 1 0.5

39.1 (#) 116 200 7 1 0.5

19.5 120


–

9.77 94


–
Mitomycin C 0.3 55 150 62 62 41.3 ***


%CA = percentage of cells bearing aberrations (excl. gaps)

(#) used for scoring

* statistically significant at p< 0.05

** statistically significant at p< 0.01

*** statistically significant at p< 0.001

Conclusions:
Interpretation of results (migrated information):
negative

In this chromsome aberration test 1,4-BDDMA did not induce chromosomal aberrations in CHO cells after in vitro treatment in the presence and absence of S9 metabolic activation.
Executive summary:

In a mammalian cell chromosome aberration assay according to OECD guideline 473, adopted 21 July 1997, CHO cell cultures were exposed to 1,4-BDDMA (93.59% a.i.) in DMSO at concentrations of 0 (control), 1250, 625, 313, 156, 78.1, 39.1, 19.5 and 9.77 mg/L with and without metabolic activation (S9 mix). 1,4-Butanediol dimethacrylate was tested up to cytotoxic concentrations.

In the first experiment the cells were exposed to the test substance for 3 h with and without metabolic activation and in a second experiment for 20 h without metabolic activation.

The test substance did not induce an increase in chromosomal aberrations at any test point selected for scoring in the absence or presence of S9 mix.

A slight, but not dose-related increase in endoreduplication, was observed in the presence of S9 mix.

Increases in gaps over the negative control value were observed in the 3 h treatment group (313 mg/L) without metabolic activation. This was seen in only one replicate and therefore not considered biologically relevant.

Positive controls induced the appropriate response.

There was no evidence of chromosome aberrations induced over background.

NOTE: Any of data in this dataset are disseminated by the European Union on a right-to-know basis and this is not a publication in the same sense as a book or an article in a journal. The right of ownership in any part of this information is reserved by the data owner(s). The use of this information for any other, e.g. commercial purpose is strictly reserved to the data owners and those persons or legal entities having paid the respective access fee for the intended purpose.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1994-09-14 to 1994-11-18
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
29 December 1992
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
adopted 26 May 1983
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
- Name of test material (as cited in study report): 1,4-Butanediol dimethacrylate
Target gene:
not applicable (chromosome aberration test)
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM, supplemented with 10% FCS
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: no data
- other: determination of doubling time
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Experiment I (total 18 h):
without S9 mix: 10, 30, 60, 100, 150, 200 µg/mL
with S9 mix: 60, 100, 300, 600, 1000, 1200 µg/mL

Experiment II (total 18 h):
without S9 mix: 25, 50, 75, 100, 125, 150 µg/mL
with S9 mix: 200, 400, 600, 800, 1000, 1200 µg/mL

Experiment II (total 28 h):
without S9 mix: 75, 100, 125, 150 µg/mL
with S9 mix: 600, 800, 1000, 1200 µg/mL

The treatment interval was 4 h with metabolic activation, 18 h / 28 h without metabolic activation.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen according to its solubility properties and its non-toxicity to the cells. The final concentration of DMSO in the culture medium did not exceed 1% v/v.
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:
cyclophosphamide
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 h with metabolic activation; 18/28 h without metabolic activation
- Expression time (cells in growth medium): 18/28 h
- Fixation time (start of exposure up to fixation or harvest of cells): 18/28 h

SPINDLE INHIBITOR (cytogenetic assays): 0.2 µg/mL colcemid for the last 2.5 h before harvesting
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 100 per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
Evaluation criteria:
The chromosomal aberration assay is considered acceptable if it meets the following criteria:
a) The number of aberrations found in the negative and/or solvent controls falls within the range of historical laboratory control data: 0.00 % - 4.00 %.
b) The positive control substances should produce significant increases of the number of cells with structural chromosomal aberrations.

A test article is classified as mutagenic if it induces reproducibly either a significant concentration-related increase in the number of structural chromosomal aberrations or a significant and reproducible positive response for at least one of the test points.
A test article producing reproducibly neither a significant concentration-related increase in the number of structural chromosomal aberrations nor a significant and reproducibly positive response at any one of the test points is considered nonmutagenic in this system.
Statistics:
Chi-square test
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
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
- Effects of pH: no data
- Effects of osmolality: no data
- Precipitation: in a range from 600 to 1200 µg/mL in both experiments with S9 mix precipitation of the test article was observed

RANGE-FINDING/SCREENING STUDIES:
In a pre-test on toxicity (XTT-assay) with concentrations of 10, 30, 60, 100, 300, 600, 1000 and 1200 µg/mL in the absence and presence of metabolic activation cytotoxic effects were observed: without S9 mix at concentrations of 300 µg/mL and higher; with S9 mix at a concentration of 1200 µg/mL.

COMPARISON WITH HISTORICAL CONTROL DATA:
The number of cells with chromosome aberrations in the negative and solvent controls were within the range of historical laboratory control data

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In experiment I in the absence of metabolic activation slight reductions of the mitotic indices after treatment with 60 and 100 µg/mL were noted (relative mitotic index: 86.0 and 72.4 % compared to the corresponding solvent control). In the presence of S9 mix no reduction up to the highest tested concentration of 1000 µg/mL was observed.
In experiment II (18 h fixation interval) without S9 mix the mitotic indices were reduced at all tested concentration. With S9 mix slight reductions of mitotic index were observed at 600 and 800 µg/mL; higher concentrations were not evaluable due to cytotoxicity.
At a fixation interval of 28 h reduced mitotic indices were obtained in the absence of S9 mix at 75 µg/mL and in the presence of S9 mix at 1200 µg/mL.
Remarks on result:
other: all strains/cell types tested

mean % aberrant cells

concentration [µg/mL]

mitotic index [% control]

incl. gaps

excl. gaps

exchanges

experiment I; fixation interval 18 h; without metabolic activation

negative control

100

1.5

1

0.5

solvent control

100

1.5

0.5

0

positive control

86.7

24.5

20.5

8.5

test item

10

110.3

1.5

0

0

60

86

5.5

2

0

100

72.4

9

3.5

0

experiment I; fixation interval 18 h; with metabolic activation

negative control

100

2

1

0

solvent control

100

0.5

0.5

0

positive control

158.4

8.5

7

2.5

test item

100

110

1.5

1

0

600

103.3

2.5

2.5

0.5

1000

108.8

9

5.5

1

experiment II; fixation interval 18 h; without metabolic activation

negative control

100

1.5

1

0

solvent control

100

2.5

1

0

positive control

121.4

16.5

12

8

test item

25

64.6

5

2.5

0

75

70

8.5

5.5

0

100

77.9

12

8.5

0

experiment II; fixation interval 18 h; with metabolic activation

negative control

100

4

1.5

0

solvent control

100

6.5

3.5

0

positive control

86.2

13

10

3

test item

200

135

4.5

2.5

1

600

76.3

4.5

2

0.5

800

82.9

3.5

1.5

0

experiment II; fixation interval 28 h; without metabolic activation

solvent control

100

5

2.5

0

test item

75

64.3

15

10

2

experiment II; fixation interval 28 h; with metabolic activation

solvent control

100

2.5

1

0

test item

1200

65.2

4.5

2

0.5

In the presence of metabolic activation an enhanced chromosome aberration frequency was detected only after treatment with 1000 µg/mL in experiment I. This was not reproduced in experiment II due to higher toxicity.

Due to precipitation of the test item in the range of 600 to 1200 µg/mL in both experiments in the presence of S9 mix, it cannot be excluded that different local concentrations were leading to unreproducible results regarding cytotoxicity and chromosomal aberrations.

 

In the absence of S9 mix, chromosome aberration frequency (excl. gaps), was statistically significant increased in a concentration-dependent manner.

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

In this chromosome aberration test 1,4-BDDMA induced chromosomal aberrations in V79 cells after in vitro treatment in the absence of S9 metabolic activation, but not in the presence of metabolic activation.
Executive summary:

In a mammalian cell chromosome aberration assay according to OECD guideline 473, adopted 21 July 1997, Chinese hamster lung fibroblast (V79) cell cultures were exposed to 1,4-BDDMA (93.59% a.i.) in DMSO with and without metabolic activation (S9 mix) at the following concentrations:

 

Experiment I (total 18 h):

without S9 mix: 10, 30, 60, 100, 150, 200 µg/mL

with S9 mix: 60, 100, 300, 600, 1000, 1200 µg/mL

 

Experiment II (total 18 h):

without S9 mix: 25, 50, 75, 100, 125, 150 µg/mL

with S9 mix: 200, 400, 600, 800, 1000, 1200 µg/mL

 

Experiment II (total 28 h):

without S9 mix: 75, 100, 125, 150 µg/mL

with S9 mix: 600, 800, 1000, 1200 µg/mL

 

The treatment interval was 4 h with metabolic activation, 18 h / 28 h without metabolic activation. 1,4-BDDMA was tested up to cytotoxic or precipitating concentrations.

In the presence of metabolic activation an enhanced chromosome aberration frequency was detected only after treatment with 1000 µg/mL in the first experiment. However, this was not reproduced in the second experiment due to higher toxicity. Due to precipitation of the test item in the range of 600 to 1200 µg/mL in both experiments in the presence of S9 mix, it cannot be excluded that different local concentrations were leading to unreproducible results regarding cytotoxicity and chromosomal aberrations.

In the absence of S9 mix, chromosome aberration frequency (excl. gaps), was statistically significant increased in a concentration-dependent manner.

 

Positive controls induced the appropriate response.

There was evidence of chromosome aberrations induced over background in the absence of metabolic activation.

 

NOTE: Any of data in this dataset are disseminated by the European Union on a right-to-know basis and this is not a publication in the same sense as a book or an article in a journal. The right of ownership in any part of this information is reserved by the data owner(s). The use of this information for any other, e.g. commercial purpose is strictly reserved to the data owners and those persons or legal entities having paid the respective access fee for the intended purpose.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1994-09-14 to 1994-11-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
adopted 4 April 1984
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Specific details on test material used for the study:
- Name of test material (as cited in study report): 1,4-Butanediol dimethacrylate
Target gene:
HPRT
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM + 10% FCS
- 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:
S9 mix
Test concentrations with justification for top dose:
pretest for cytotoxicity
- 10, 30, 60, 100, 300, 600, 1000, 1200 mg/L with and without S9 mix

experiment 1
- without S9 mix: 10, 30, 60, 100, 150, 200 mg/L
- with S9 mix: 60, 100, 300, 600, 1000, 1200 mg/L

experiment 2
- without S9 mix: 10, 30, 60, 80, 100, 120 mg/L
- with S9 mix: 60, 100, 300, 600, 1000, 1200 mg/L
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: chosen due to solubility properties and non-toxicity to the cells
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:
7,12-dimethylbenzanthracene
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): 7 days
- Selection time (if incubation with a selection agent): 9 days
- Fixation time (start of exposure up to fixation or harvest of cells): 16 days

SELECTION AGENT (mutation assays): 6-Thioguanine

NUMBER OF REPLICATIONS: 2 per experimental point for cloning efficiency; 1 per experimental point for mutagenicity

DETERMINATION OF CYTOTOXICITY
- Method
- pretest for cytotoxicity, XTT-assay (metabolic activity) for dose range selection
- during main test: cloning efficiency

OTHER:
colonies were stained with methylene blue; colonies with > 50 cells were counted
Evaluation criteria:
Validity criteria:
- numbers of mutant colonies per 10E06 cells in negative and solvent controls are within the range of historical data (0 – 45 x10E06)
- positive controls induce a significant increase in mutant colony frequency
- cloning efficiency of negative and solvent controls >50%

Evaluation:
A test substance is classified positive if it induces either a concentration-related increase of mutant colony frequency or a reproducible positive response for one of the test points.
A response is significant if the mutation frequency is at least 3 times higher than the spontaneous mutation frequency (negative/solvent control).
Statistics:
adequate statistical method not available, since distribution of mutant cells does not follow known statistical methods
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
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
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: no data
- Precipitation:
- in experiment 1: at 200 mg/L without S9 mix and at 300 mg/L and higher with S9 mix
- in experiment 2: at 600 mg/L and higher with S9 mix

RANGE-FINDING/SCREENING STUDIES:
- toxicity was found at concentrations at 300 mg/L and higher without S9 mix

COMPARISON WITH HISTORICAL CONTROL DATA:
Controls were in the range of historical control data

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Experiment I: without metabolic activation showed toxic effects in the cloning efficiency (CE) after treatment with concentrations higher than 100 mg/L. The cell density at first subcultivation was cleary reduced at 100 mg/L. Higher concentrations than 100 mg/L could not be performed, since no cell growth occurred. In the presence of metabolic activation toxic effects were observed at concentrations higher than 600 mg/L in the cloning efficiency of the cells. The cell density at the first subcultivationc was slightly reduced at the highest investigated concentration (1200 mg/L).
In experiment II without metabolic activation toxic effects were observed at 120 mg/L only, where the cell growth at the first subcultivation was reduced to 56.6 % compared to the corresponding solvent control. With metabolic activation the cloning efficieney of the cells as weIl as the cell density at the first subcultivation were clearly reduced after treatment with 1000 and 1200 mg/L.

experiment 1

mg/L

S9 mix

CE% absolute

CE% relative

mutant colonies per 10E06 cells

mutation factor

negative control

0

-

66.9

100

16

solvent control (DMSO)

0

-

60.1

100

10.8

1.00

positive control (EMS)

600

-

57.7

86.3

428.3

39.66

1,4-BDDMA

10

-

56.5

93.9

3.4

0.31

30

-

59.6

99.1

5.7

0.53

60

-

53.9

89.7

4.3

0.40

100

-

56.6

94.1

0.6

0.06

150

-

36

59.8

n.c.

200

-

0

0

n.c.

negative control

0

+

55.1

100

11.5

0.82

solvent control (DMSO)

0

+

63.2

100

14.1

1.00

positive control (DMBA)

3.85

+

48.6

76.9

533.6

37.84

1,4-BDDMA

60

+

59.6

94.2

n.c.

100

+

58.8

93

n.c.

300

+

56.9

90.1

5.4

0.38

600

+

61.1

96.7

6.2

0.44

1000

+

19.5

30.8

14.5

1.03

1200

+

10.2

16.1

23.1

1.64

experiment 2

mg/L

S9 mix

CE% absolute

CE% relative

mutant colonies per 10E06 cells

mutation factor

negative control

0

-

56.8

100

16.6

solvent control (DMSO)

0

-

59.4

100

7.8

1.00

positive control (EMS)

600

-

55.7

98

780.2

100.03

1,4-BDDMA

10

-

65.2

109.9

23.7

3.04

30

-

61.5

103.7

n.c.

60

-

56.4

95.1

9.8

1.26

80

-

54.3

91.4

n.c.

100

-

56.1

94.6

13.4

1.72

120

-

54.2

91.2

6.9

0.88

negative control

0

+

62

100

3.9

0.45

solvent control (DMSO)

0

+

63.1

100

8.6

1.00

positive control (DMBA)

3.85

+

48.6

76.9

420.9

48.94

1,4-BDDMA

60

+

61.2

97

n.c.

100

+

64.4

101.9

n.c.

300

+

64

101.3

0.6

0.07

600

+

57.1

90.4

8.9

1.03

1000

+

10.2

16.2

5.9

0.69

1200

+

2.4

3.7

16.2

1.88

n.c. = culture not continued: concerning concentration range and toxicity 4 concentrations were selected to be evaluated at the end of the experiment

With metabolic activation a slight concentration-dependent increase in mutant colony numbers was observed from 300 mg/L up to 1200 mg/L. However, the results indicate only a minor increase in mutant factors (1.64 and 1.88 for 1200 mg/L in 1stand 2ndexperiment, respectively). They do not exceed the historical control data range. No threefold increase of mutation rates over the spontanous mutation rate is reached at any concentration of the test article. Thus, the results do not meet the criteria for mutagenicity.

The test article is therefore considered as non mutagenic in this HPRT assay.

Conclusions:
Interpretation of results:
negative

In this mutagenicity assay 1,4-BDDMA did not induce gene mutations over background at the HPRT locus in V79 cells.
Executive summary:

In a mammalian cell gene mutation assay according to OECD guideline 476, adopted 4 April 1984 (HPRT test), V79 cells cultured in vitro were exposed to 1,4-BDDMA , in DMSO at concentrations of 10, 30, 60, 80, 100, 120, 150, 200 mg/L in the presence and 60, 100, 300, 600, 1000, 1200 mg/L in the absence of mammalian metabolic activation (S9 mix) in two independent experiments.

1,4-BDDMA was tested up to cytotoxic concentrations. With metabolic activation a slight concentration-dependent increase in mutant colony numbers was observed from 300 mg/L up to 1200 mg/L. However, the results indicate only a minor increase in mutant factors (1.64 and 1.88 for 1200 mg/L in 1st and 2nd experiment, respectively) and they did not exceed the historical control data range. No threefold increase of mutation rates over the spontanous mutation rate is reached at any concentration of the test article. Thus, the results do not meet the criteria for mutagenicity.

The positive controls did induce the appropriate response.

The test article is therefore considered as non mutagenic in this HPRT assay.

NOTE: Any of data in this dataset are disseminated by the European Union on a right-to-know basis and this is not a publication in the same sense as a book or an article in a journal. The right of ownership in any part of this information is reserved by the data owner(s). The use of this information for any other, e.g. commercial purpose is strictly reserved to the data owners and those persons or legal entities having paid the respective access fee for the intended purpose.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start date: 16 October 2020 - Experimental completion date: 04 November 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
OECD 471, GLP
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
Ninth Addendum to OECD Guidelines for Testing of Chemicals, Section 4, No. 471: Bacterial Reverse Mutation Test, corrected June 26, 2020
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
Commission Regulation (EC) No. 440/2008 B13/14, dated 30-05-2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
TEST MATERIAL:
- Name of Test material: 1,4-Butanediol dimethacrylate (1,4-BDDMA)
- physical state: liquid, colorless
Target gene:
his locus
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:
Phenobarbital/ beta-naphthoflavone induced rat liver S9 Mix
Test concentrations with justification for top dose:
Experiment I (plate incorporation test): 3/ 10/ 33/ 100/ 333/ 1000/ 2500/ 5000 µg/plate
Experiment II (pre-incubation test): 33/ 100/ 333/ 1000/ 2500/ 5000 µg/plate
Vehicle / solvent:
On the day of experiment, the test item was dissolved in DMSO (purity >99%). The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria. All formulations were prepared freshly before treatment and used within two hours of preparation.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 2-aminoanthracene: with metabolic activation: all strains; 4-nitro-o-phenylene-diamine: without metabolic activation: TA 1537 and TA 98
Details on test system and experimental conditions:
METHOD OF APPLICATION:
in agar (plate incorporation) and pre-incubation

DURATION:
- Pre-incubation period:
- Exposure duration:

NUMBER OF EXPERIMENTS:

DETERMINATION OF CYTOTOXICITY:
- Method: Toxicity of the test item my be evidenced by a reduction in the number of spontaneous revertants, a clearing of the bacterial background lawn, or by degree of survival of tre

OTHER:
automatic colony count with
Evaluation criteria:
The Salmonella typhimurium and Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:

- regular background growth in the negative and solvent control;
- the spontaneous reversion rates in the negative and solvent control are in the range of our historical data;
- the positive control substances should produce an increase above the threshold of twofold (strains TA 98, TA 100, and WP2 uvrA) or threefold (strains TA 1535 and TA 1357) the colony count of the corrresponding solvent control;
- a minimum of five analysable dose levels should be present with at least three dose levels showing no signs of toxic effects, evident as a reduction in the number of revertants below the indication factor of 0.5.
Statistics:
no appropriate statistical method available
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Cytotoxicity observed in Experiment II without metabolic activation at 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Cytotoxicity observed in Experiment I with metabolic activation at 5000 µg/plate and in Experiment II without metabolic activation above 2500 µg/plate and with metabolic activation at 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
1,4-BDDMA is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.
Executive summary:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base par changes or frameshifts in the genome of the strains used.

Therefore, the substance 1,4-BDDMA is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

 

NOTE: Any of data in this dataset are disseminated by the European Union on a right-to-know basis and this is not a publication in the same sense as a book or an article in a journal. The right of ownership in any part of this information is reserved by the data owner(s). The use of this information for any other, e.g. commercial purpose is strictly reserved to the data owners and those persons or legal entities having paid the respective access fee for the intended purpose.

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

Genetic toxicity in vivo

Description of key information

Chromosome Mutation Data

- OECD 474, Mouse bone marrow micro­nucleus test, oral gavage up to 2000 mg/kg: negative (BSL, 1999)

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1998-03-25 to 1998-05-03
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
draft version 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
29 December 1992
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
micronucleus assay
Specific details on test material used for the study:
- Name of test material (as cited in study report): 1,4-Butanediol dimethacrylate
Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann, Borchen, Germany
- Age at study initiation: no data
- Weight at study initiation: 26 – 36 g
- Assigned to test groups randomly: yes
- Fasting period before study: no data
- Housing: 5 animals of identical sex per cage
- Diet (e.g. ad libitum): pellet standard diet (Altromin)
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25°C
- Humidity (%): 55+/-10°C
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light):12/12

IN-LIFE DATES: no data
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: cotton seed oil
- Justification for choice of solvent/vehicle: relatively nontoxic to the animals
- Concentration of test material in vehicle:
- Amount of vehicle (if gavage or dermal): 16.7 mL/kg bw
- Lot/batch no. (if required): 27H0534
- Purity: no data
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
dilution in cotton seed oil; single standard volume of 16.7 mL/kg bw
Duration of treatment / exposure:
The animals received the test item once. Sampling of the bone marrow was carried out on animals 24 and 48 h after treatment.
Frequency of treatment:
single dose
Post exposure period:
The animals were sacrificed 24 and 48 hours after treatment.
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Remarks:
24 h interval
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
24 h interval
Dose / conc.:
2 000 mg/kg bw/day (actual dose received)
Remarks:
24 h interval
Dose / conc.:
2 000 mg/kg bw/day (actual dose received)
Remarks:
48 h interval
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Justification for choice of positive control(s): appropriate reference mutagen
- Route of administration: single administration i.p.
- Doses / concentrations: 30 mg/kg bw in 0.9% NaCl (10 mL/kg bw)
Tissues and cell types examined:
bone marrow erythrocytes (polychromatic erythrocytes)
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
dose selection based on acute toxicity pre-test: at 2000 mg/kg bw no toxic signs were found two lower dose groups (200 and 1000 mg/kg bw) were selected for 24 h treatment interval

DETAILS OF SLIDE PREPARATION:
- removal of femura
- bone marrow was flushed out with 1 mL FCS followed by resuspension of cells and centrifugation at 1500 rpm for 10 min
- cells were resuspended in FCS and smeared on slides
- air drying of slides, staining with May-Grünwald/Giemsa
- at least one slide prepared from each bone marrow sample

METHOD OF ANALYSIS:
- 2000 polychromatic erythrocytes were analysed per animal for micronuclei
- ratio between polychromatic erythrocytes (PCE) and normochromatic erythrocytes (NCE) was determined to assess cytotoxicity (expressed as NCE per 1000 PCE)
- analysis carried out with coded slides
Evaluation criteria:
A test item is classified mutagenic if it induces either a statistically significant dose related increase in the number of micronucleated PCE or a reproducible statistically significant positive result for at least one test point.
Statistics:
non-parametric Mann-Whitney test
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg/kg bw
- Solubility: no data
- Clinical signs of toxicity in test animals: after 1 hour reduced spontaneous activity
- Other: only pre-test for toxicity, no further analysis

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): no induction of micronuclei over background (see table); the mean values of micronuclei observed after treatment with the test item (0.11% to 0.15%) were in the same range as the negative control groups (0.10% to 0.13%)
- Ratio of PCE/NCE (for Micronucleus assay): slight effect on PCE/NCE ration by treatment with test item at a concentration of 2000 mg/kg bw at 24 and 48 h indicating slight cytotoxic properties
- Statistical evaluation: no significance found for 1000 mg/kg bw (24 h), 2000 mg/kg bw (24 h), 2000 mg/kg bw (48 h); lowest dose of 200 mg/kg bw (24 h) not tested for significance, as values were equal to or lower than negative control

group sex mean MN/2000 PCE %MN relative to N.C. PCE/NCE
N.C. m 2 0.1 1.0 1000/808.2
P.C. m 34.6 1.73 17.3 1000/1045.6
test item 2000mg/kg, 24 h m 2.6 0.13 1.3 1000/995.2
test item 2000mg/kg, 48 h m 3 0.15 1.5 1000/1011
test item 1000mg/kg, 24 h m 2.4 0.12 1.2 1000/868
test item 200mg/kg, 24 h m 2.2 0.11 1.1 1000/820.2
N.C. f 2.6 0.13 1.0 1000/844.2
P.C. f 38.8 1.94 14.9 1000/1055.8
test item 2000mg/kg, 24 h f 2.4 0.12 0.9 1000/972.8
test item 2000mg/kg, 48 h f 2.6 0.13 1.0 1000/1016.8
test item 1000mg/kg, 24 h f 2.8 0.14 1.1 1000/884.4
test item 200mg/kg, 24 h f 2.4 0.12 0.9 1000/828.6

N.C. = negative control

P.C. = positive control

m = male

f = female

Conclusions:
Interpretation of results (migrated information): negative
In this study under the experimental conditions reported, 1,4-BDDMA did not induce micronuclei over background as determined by the micronucleus test in bone marrow cells of the mouse.
Executive summary:

In a NMRI mouse bone marrow micronucleus assay according to OECD guideline 474, draft version 1997, 5 animals per sex per dose were treated by oral gavage (single dose) with 1,4-BDDMA (93.9% a.i.) at doses of 0, 200, 1000 and 2000 mg/kg bw. Bone marrow cells were harvested at 24 h (200, 1000 and 2000 mg/kg dose groups) and 48 h (2000 mg/kg dose group) post-treatment. The vehicle was cotton seed oil.

There were no signs of toxicity during the study based on mortality or clinical signs. A slight effect on the ratio of polychromatic to normochromatic erythrocytes was observed in the 2000 mg/kg dose group 24 and 48 h post-treatment indicating a possible weak toxic effect to the bone marrow. There was no significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any treatment time. 1,4-BDDMA was tested at an adequate dose evel which included the guideline limit dose of 2000 mg/kg bw. The positive control induced the appropriate response.

NOTE: Any of data in this dataset are disseminated by the European Union on a right-to-know basis and this is not a publication in the same sense as a book or an article in a journal. The right of ownership in any part of this information is reserved by the data owner(s). The use of this information for any other, e.g. commercial purpose is strictly reserved to the data owners and those persons or legal entities having paid the respective access fee for the intended purpose.

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

Additional information

For the assessment of the mutagenic potential of 1,4-BDDMA several studies are available: two reverse gene mutation assays in bacteria (Ames test), a mammalian cell gene mutation assay (HPRT test), two mammalian cell chromosome aberration assays and an in vivo micronucleus test.


 


Bacterial reverse gene mutation assays


In a guideline study according OECD 471 (corrected June 2020) S. typhimurum TA 1535, TA 1537, TA 98, TA 100 and E.coli WP2 uvr A were exposed to 1,4-BDDMA in DMSO at concentrations of 3/10/33/100/333/1000/2500/5000 µg/plate in a pre-incubation and a plate-incorporation assay with and without metabolic activation (S9 mix).


1,4-BDDMA was tested up to cytotoxic concentrations. There was no evidence of induced mutant colonies over background.


The positive controls induced the appropriate responses in the corresponding strains.


This study is considered reliable without restrictions.


 


In a supporting study, S. typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 1538 of S. typhimurium were exposed to 1,4-BDDMA in DMSO at concentrations of 33.3, 100.0, 333.3, 1000.0, 2500.0, 5000.0 µg/plate in a plate incorporation assay and 10.0, 33.3, 100.0, 333.3, 1000.0, 2500.0 µg/plate in a pre-incubation assay in the presence and absence of mammalian metabolic activation (S9 mix).


1,4-BDDMA was tested up to cytotoxic concentrations. There was no evidence of induced mutant colonies over background.


The positive controls induced the appropriate responses in the corresponding strains.


 


In a supporting study, S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100 were exposed to 1,4-BDDMA. There was no evidence of induced mutant colonies over background in any of the tester strains exposed to 1,4-BDDMA up to 2500 µg/plate in the presence or absence of mammalian metabolic activation in this study.


 


 

Mammalian cell gene mutation assay


In a mammalian cell gene mutation assay according to OECD guideline 467, adopted 4 April 1984 (HPRT test), V79 cells cultured in vitro were exposed to 1,4-BDDMA, in DMSO at concentrations of 10, 30, 60, 80, 100, 120, 150, 200 mg/L in the presence and 60, 100, 300, 600, 1000, 1200 mg/L in the absence of mammalian metabolic activation (S9 mix) in two independent experiments.


1,4-BDDMA was tested up to cytotoxic concentrations. With metabolic activation a slight concentration-dependent increase in mutant colony numbers was observed from 300 mg/L up to 1200 mg/L. However, the results indicate only a minor increase in mutant factors (1.64 and 1.88 for 1200 mg/L in 1st and 2nd experiment, respectively) and they did not exceed the historical control data range. No threefold increase of mutation rates over the spontaneous mutation rate is reached at any concentration of the test article. Thus, the results do not meet the criteria for mutagenicity. The positive controls did induce the appropriate response. The test article is considered as non mutagenic in this HPRT assay.


This study is considered reliable without restrictions.


Mammalian cell chromosome aberration assay


In a mammalian cell chromosome aberration assay according to OECD guideline 473, adopted 21 July 1997, CHO cell cultures were exposed to 1,4-BDDMA in DMSO at concentrations of 0 (control), 1250, 625, 313, 156, 78.1, 39.1, 19.5 and 9.77 mg/L with and without metabolic activation (S9 mix). 1,4-Butanediol dimethacrylate was tested up to cytotoxic concentrations.


In the first experiment the cells were exposed to the test substance for 3 h with and without metabolic activation and in a second experiment for 20 h without metabolic activation.


The test substance did not induce an increase in chromosomal aberrations at any test point selected for scoring in the absence or presence of S9 mix.


A slight, but not dose-related increase in endore duplication, was observed in the presence of S9 mix. Increases in gaps over the negative control value were observed in the 3 h treatment group (313 mg/L) without metabolic activation. This was seen in only one replicate and therefore not considered biologically relevant. Positive controls induced the appropriate response.


There was no evidence of chromosome aberrations induced over background.


This study is considered reliable without restrictions.


 


In a mammalian cell chromosome aberration assay according to OECD guideline 473, adopted 21 July 1997, Chinese hamster lung fibroblast (V79) cell cultures were exposed to 1,4-BDDMA (93.59% a.i.) in DMSO with and without metabolic activation (S9 mix) at the following concentrations:


Experiment I (total 18 h):


without S9 mix: 10, 30, 60, 100, 150, 200 µg/mL


with S9 mix: 60, 100, 300, 600, 1000, 1200 µg/mL


Experiment II (total 18 h):


without S9 mix: 25, 50, 75, 100, 125, 150 µg/mL


with S9 mix: 200, 400, 600, 800, 1000, 1200 µg/mL


Experiment II (total 28 h):


without S9 mix: 75, 100, 125, 150 µg/mL


with S9 mix: 600, 800, 1000, 1200 µg/mL


The treatment interval was 4 h with metabolic activation, 18 h / 28 h without metabolic activation. 1,4-BDDMA was tested up to cytotoxic or precipitating concentrations.


In the presence of metabolic activation an enhanced chromosome aberration frequency was detected only after treatment with 1000 µg/mL in the first experiment. However, this was not reproduced in the second experiment due to higher toxicity. Due to precipitation of the test item in the range of 600 to 1200 µg/mL in both experiments in the presence of S9 mix, it cannot be excluded that different local concentrations were leading to unreproducible results regarding cytotoxicity and chromosomal aberrations.


In the absence of S9 mix, chromosome aberration frequency (excl. gaps), was statistically significant increased in a concentration-dependent manner. Positive controls induced the appropriate response. There was evidence of chromosome aberrations induced over background in the absence of metabolic activation. No increase in chromosome aberration frequency was observed in the presence of metabolic activation.


This study is considered reliable with restrictions.


 


Due to the positive result obtained in the OECD 473 study, an OECD 474 was performed to clarify the positive result. 


 


In vivo micronucleus assay


In a NMRI mouse bone marrow micronucleus assay according to OECD guideline 474, draft version 1997, 5 animals per sex per dose were treated by oral gavage (single dose) with 1,4-BDDMA at doses of 0, 200, 1000 and 2000 mg/kg bw. Bone marrow cells were harvested at 24 h (200, 1000 and 2000 mg/kg dose groups) and 48 h (2000 mg/kg dose group) post-treatment. The vehicle was cotton seed oil. 1,4-BDDMA was tested at an adequate dose level which included the guideline limit dose of 2000 mg/kg bw.


The positive control induced the appropriate response.


There were no signs of toxicity during the study based on mortality or clinical signs. A slight effect on the ratio of polychromatic to normochromatic erythrocytes was observed in the 2000 mg/kg dose group 24 and 48 h post-treatment indicating a possible weak toxic effect to the bone marrow. There was no significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any treatment time.


This study is considered reliable without restrictions.


 


Although there is one mammalian cell chromosome aberration assay, which showed evidence of chromosome aberrations induced over background in the absence of metabolic activation, no increase in chromosome aberration frequency was observed in the presence of metabolic activation. Moreover, an in vivo micronucleus assay was negative for clastogenicity outweighing the result from the in vitro assay.


 


Thus, based on the available information, 1,4-BDDMA is not genotoxic. There are no data gaps for the endpoint genotoxicity. No human information is available for this endpoint. However, there is no reason to believe that these results would not be applicable to humans.

Justification for selection of genetic toxicity endpoint
No single key study has been selected, since all available studies were negative or are considered in a weight of evidence approach.


 


Compliance to REACh requirements


The in vitro mutagenity test requirements are all covered with an Ames test, two chromosome aberration tests, and a HPRT assay. The in vivo mutagenity test requirement is covered by an in vitro Micronucleus test. All mentioned studies are reliable (Reliability 1 or 2) and were performed with the substance itself. 





 





 

Justification for classification or non-classification

Based on the available data, 1,4-BDDMA does not need to be classified for mutagenicity according to the criteria given in regulation (EC) 1272/2008 or the former European directive on classification and labelling 67/548/EEC. Thus, no labelling is required.