Registration Dossier
Registration Dossier
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EC number: 219-674-4 | CAS number: 2495-37-6
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Bacterial reverse gene mutation assay according to OECD 471; not mutagenic
mammalian cell gene mutation assay according to OECD guideline 476, not mutagenic
mammalian cell micronucleus assay according to OECD guideline 478, not mutagenic
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2011-08-23 to 2011-10-25
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- GLP.
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- dated May 30, 2008
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- adopted 21 July 1997
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- certificate dated 30 March 2009
- 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): Benzyl methacrylate
- 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 containing Hank’s salts supplemented with 10% FBS, 5 µg/mL neomycin, 1% amphotericin B
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
- Other: each batch screened for spontaneous mutant frequency - Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- Sß mix
- Test concentrations with justification for top dose:
- range finding pre-experiment: 13.8 to 1760 mg/L (approx. 10 mM, the guideline limit concentration)
Doses applied:
Experiment I:
13.8, 27.5, 55, 110, 220, 330, 440 mg/L without S9 mix, exposure period 4 h; 13.8, 27.5, 55, 110 mg/L chosen for mutation rate analysis
55, 110, 220, 440, 880, 1760 mg/L with S9 mix, exposure period 4 h; 55, 110, 220, 440, 880 mg/L chosen for mutation rate analysis
Experiment II:
13.8, 27.5, 55, 110, 165, 220 mg/L without S9 mix, exposure period 24 h; 13.8, 27.5, 55, 110, 165 mg/L chosen for mutation rate analysis
27.5, 55, 110, 220, 440, 880 mg/L with S9 mix, exposure period 4 h; 27.5, 55, 110, 220, 440 mg/L chosen for mutation rate analysis - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO (final concentration in medium: 0.5%)
- Justification for choice of solvent/vehicle: solubility properties, relative non-toxicity to cell cultures - Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- other: (without metabolic activation)
- Positive controls:
- yes
- Positive control substance:
- 7,12-dimethylbenzanthracene
- other: (with metabolic activation)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration:
Experiment I: 4 h with and without metabolic activation
Experiment II: 4 h with metabolic activation, 24 h without metabolic activation
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 8 days
- Fixation time (start of exposure up to fixation or harvest of cells): 15 days
SELECTION AGENT (mutation assays): 11 mg/L 6-thioguanine (6-TG)
NUMBER OF REPLICATIONS: 2 replicates per experiment
DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
OTHER:
- colonies used to determine the cloning efficiency (survival) were fixed and stained approx. 7 days after treatment
- the colonies were stained with 10 % methylene blue in 0.01 % KOH solution; colonies with more than 50 cells were counted.
- Following the expression time of 7 days five cell culture flasks were seeded with about 3 - 5x10E05 cells each in medium containing 6-TG; 2 additional flasks were seeded with approx. 500 cells each in non-selective medium to determine the viability. - Evaluation criteria:
- The assay is considered acceptable if it meets the following criteria:
- the numbers of mutant colonies per 10E06 cells found in the solvent controls fall within the laboratory historical control data range
- the positive control substances must produce a significant increase in mutant colony frequencies
- the cloning efficiency (absolute value) of the solvent controls must exceed 50%
A positive response is described as follows:
- reproducible induction of a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment
- reproducible concentration-related increase of the mutation frequency; such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed - Statistics:
- A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated 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.
Biological and statistical significance were considered together. - Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: solvent control: 7.33; 1760 mg/L BNMA: 7.31 -> no relevant shift
- Effects of osmolality: solvent control: 365 mOsm; 1760 mg/L BNMA: 344 -> no relevant shift
- Water solubility:
Phase separation was observed in the first experiment at 220 mg/L and above with metabolic activation and at 110 mg/L and above without metabolic activation. In the second experiment phase separation was observed at 110 mg/L and above with and without metabolic activation.
RANGE-FINDING/SCREENING STUDIES: pre-test for cytotoxicity, cytotoxicity at 110 mg/L and above without metabolic activation, and 880 mg/L and above with metabolic activation
COMPARISON WITH HISTORICAL CONTROL DATA: mutant frequencies were within the historical range of solvent controls - Conclusions:
- Interpretation of results: negative
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. Therefore, BNMA is considered to be non-mutagenic in this HPRT assay. - Executive summary:
In a mammalian cell gene mutation assay according to OECD guideline 476, adopted 21 July 1997 (HPRT test), V79 cells cultured in vitro were exposed to BNMA (98.37% a.i.) at the following concentrations in the presence and absence of mammalian metabolic activation (S9 mix).
Experiment I:
Without metabolic activation: 0 (control), 13.8, 27.5, 55, 110 mg/L
With metabolic activation: 0 (control), 55, 110, 220, 440, 880 mg/L
Experiment II:
Without metabolic activation: 0 (control), 13.8, 27.5, 55, 110, 165 mg/L
With metabolic activation: 0 (control), 27.5, 55, 110, 220, 440 mg/L
BNMA was tested up to cytotoxic or phase-separation concentrations. The positive controls induced the appropriate response. There was no evidence of induced mutant colonies over background.
In this HPRT test, BNMA was found to be not mutagenic.
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 / micronucleus study
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2011-09-07 to 2011-12-01
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- GLP.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
- Version / remarks:
- adopted July 22, 2010
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- certificate dated 30 March 2009
- Type of assay:
- in vitro mammalian cell micronucleus test
- Specific details on test material used for the study:
- - Name of test material (as cited in study report): Benzyl methacrylate
- Target gene:
- n/a (micronucleus test)
- Species / strain / cell type:
- lymphocytes: human, healthy donors not receiving medication
- Details on mammalian cell type (if applicable):
- - Type and identity of media: Dulbecco's modified eagle medium/Ham's F12 medium, mixture 1:1, supplemented with 200 mM GlutaMax, penicillin/streptomycin (100 U/mL/100 µg/mL), phytohemagglutinin (PHA, 3 µg/mL), 10 % FBS (fetal bovine serum), 10 mM HEPES, heparin (125 U.S.P.-U/mL)
- Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix
- Test concentrations with justification for top dose:
- pre-test: 11.6 - 1789.0 mg/L of BNMA (approx. 10 mM)
concentrations evaluated:
Experiment I:
4 h treatment without metabolic activation: 62.3, 109.0, 190.7, 333.8 mg/L
4 h treatment with metabolic activation: 62.3, 1022.3, 1789.0 mg/L
Experiment IIA:
20 h treatment without metabolic activation: 62.3, 109.0, 190.7 mg/L
4 h treatment with metabolic activation: 109.0, 584.2, 1022.3, 1789.0 mg/L
Experiment IIB:
20 h treatment without metabolic activation: 100, 250, 300 mg/L
The dose calculations were adjusted to purity. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures. - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- 0.5% DMSO
- Positive controls:
- yes
- Positive control substance:
- mitomycin C
- other: Demecolcin (without metabolic activation)
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- other: (With metabolic activation)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration:
Experiment I:
4 hours pulse treatment (with and without metabolic activation), expression phase 16 hours, cytokinesis block 20 hours.
Experiment II:
4 hours pulse treatment (with metabolic activation), expression phase 16 hours, cytokinesis block 20 hours
20 hours continuous treatment (without metabolic activation), cytokinesis block 20 hours
- Expression time (cells in growth medium): 20 h (+ cytochalasin B)
- Fixation time (start of exposure up to fixation or harvest of cells): 40 h
SPINDLE INHIBITOR (cytogenetic assays): 4 mg/L cytochalasin B
STAIN (for cytogenetic assays): Giemsa
NUMBER OF REPLICATIONS: 2 per experiment
NUMBER OF CELLS EVALUATED: 1000 binucleate cells per culture were scored for cytogenetic damage
DETERMINATION OF CYTOTOXICITY
- Method: CBPI (Cytokinesis-block proliferation index) was determined in approximately 500 cells per culture and cytotoxicity is expressed as % cytostasis - Evaluation criteria:
- The micronucleus assay is considered acceptable if it meets the following criteria:
- The number of micronuclei found in the negative and solvent controls falls within the range of the laboratory historical control data
- The positive control substances should produce significant increases in the number of cells with micronuclei.
A test item can be classified as non-mutagenic if:
- the number of micronucleated cells in all evaluated dose groups is in the range of the laboratory historical control data and/or
-no statistically significant or concentration-related increase in the number of micronucleated cells is observed.
A test item can be classified as mutagenic if:
- the number of micronucleated cells is not in the range of the historical laboratory control data and
- either a concentration-related increase of micronucleated cells in three test groups or a statistically significant increase of the number of micronucleated cells is observed. - Statistics:
- Chi square test
- Key result
- Species / strain:
- lymphocytes: primary human
- 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: 7.4 – 7.6 for solvent control as well as test item in all experiments
- Effects of osmolality: 359 – 393 for solvent control, 348 – 387 for test item in all experiments -> no relevant influence
- Precipitation: No precipitation of the test item was observed up to highest concentration tested
- other confounding effects:
Phase separation:
- in Experiment I at 109.0 mg/L and above with and without S9 mix,
- in Experiment IIA at 109.0 mg/L and above without and at 190.7 mg/L and above with S9 mix
- in Experiment IIB at 150.0 mg/L and above without S9 mix
RANGE-FINDING/SCREENING STUDIES:
- Pre-test for cytotoxicity: concentrations 11.6, 20.3, 35.6, 62.3, 109.0, 190.7, 333.8, 584.2, 1022.3, 1789.0 mg/L with and without metabolic activation
- toxic effects were observed after 4 h treatment with 333.8 mg/L and above in the absence of S9 mix
- no cytotoxicity in the presence of S9 mix up to the highest concentration
COMPARISON WITH HISTORICAL CONTROL DATA:
- micronucleus rates of all evaluated concentrations without S9 mix were within the range of historical control data
- in Experiment I the micronucleus rate of the highest evaluated concentration (1789.0 mg/L) with S9 mix was above the range of historical control data; this finding was not reproducible in Experiment IIA
- micronucleus rates of all other cultures with metabolic activation were within the range of historical controls
ADDITIONAL INFORMATION ON CYTOTOXICITY:
- in Experiment I with S9 mix, no cytotoxicity was observed up to the highest applied concentration
- in Experiment I without S9 mix, cytotoxicity was observed at the highest evaluated concentration (333.8 mg/L; 77.8% cytostasis)
- in Experiment IIA with S9 mix, cytotoxicity was observed at the highest evaluated concentration (1789.0 mg/L; 59.4% cytostasis) - Conclusions:
- Interpretation of results: negative
In this test under the experimental conditions reported, the test item BNMA did not induce micronuclei in human lymphocytes in vitro when tested up to cytotoxic or the highest evaluable concentration. - Executive summary:
In a mammalian cell micronucleus assay according to OECD guideline 478 (adopted July 22, 2010), primary human lymphocyte cultures were exposed to BNMA (98.37%) in DMSO with and without metabolic activation (S9 mix).
The following concentrations were evaluated (dose calculations adjusted to purity):
experiment I:
4 h treatment without metabolic activation: 62.3, 109.0, 190.7, 333.8 mg/L
4 h treatment with metabolic activation: 62.3, 1022.3, 1789.0 mg/L
experiment IIA:
20 h treatment without metabolic activation: 62.3, 109.0, 190.7 mg/L
4 h treatment with metabolic activation: 109.0, 584.2, 1022.3, 1789.0 mg/L
experiment IIB:
20 h treatment without metabolic activation: 100, 250, 300 mg/L
BNMA was tested up to cytotoxic or the guideline limit concentration of 10 mM (corresponding to 1789.0 mg/L). Cytotoxic effects were observed after 4 h treatment with 333.8 mg/L and above in the absence of S9 mix; no cytotoxicity was observed in the presence of S9 mix up to the guideline limit concentration.
Without metabolic activation no biologically relevant increase in the number of cells carrying micronuclei was observed after treatment with the test item.
Cells treated with 1789.0 mg/L BNMA in the presence of S9 mix showed a statistically significant increase in micronucleated cells above the range of the historical solvent control data in the first experiment. However, this finding could not be confirmed in the second experiment. Therefore it can be considered to be of no biological relevance.
Positive controls induced the appropriate response.
There was no evidence of micronucleated cells induced over background. Therefore, BNMA is considered to be non-clastogenic in this in vitro micronucleus test, when tested up to cytotoxic or the highest evaluable concentration.
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:
- key study
- Study period:
- 2006-12-20 to 2007-01-30
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study
- Remarks:
- Without detailed documentation. Non-GLP.
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- adopted 21 July 1997
- GLP compliance:
- no
- 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): Benzil methacrylic acid
- Target gene:
- his
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix
- Test concentrations with justification for top dose:
- - range finding test: 4.88, 19.5, 78.1, 313, 1250 and 5000 µg/plate
- official test: 9.77, 19.5, 39.1, 78.1, 156, 313 µg/plate without metabolic activation; 9.77, 19.5, 39.1, 78.1, 156, 313, 625, 1250 µg/plate with metabolic activation
- validation test: 9.77, 19.5, 39.1, 78.1, 156, 313 µg/plate with and without metabolic activation - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: no data - Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- other: 2-Aminoanthracene (without metabolic activation)
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- sodium azide
- other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (with metabolic activation)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: preincubation
DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h
NUMBER OF REPLICATIONS: 2
DETERMINATION OF CYTOTOXICITY
- Method: colony count; if the mutation factor is below ca. 0.5 compared to solvent control, the test item is considered cytotoxic - Evaluation criteria:
- A test item is considered mutagenic if:
- a clear and dose-related increase in revertant number occurs
- a biologically relevant positive response for at least one dose group occurs: in TA 100 and E. coli uvrA number of revertants at least twice as high as in solvent control; TA 98, TA 1535, TA 1537 at least three times higher number of revertants as in solvent control - Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- E. coli WP2
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: no data
- Precipitation: no sedimentation
RANGE-FINDING/SCREENING STUDIES:
Concentrations 1250 and 5000 µg/plate were toxic in all strains with and without metabolic activation
COMPARISON WITH HISTORICAL CONTROL DATA: not available - Conclusions:
- Interpretation of results: negative
In this reverse mutation assay in bacteria, BNMA induced no mutant colonies over background. BNMA was tested up the cytotoxic concentration of 313 µg/plate. - Executive summary:
In a reverse gene mutation assay in bacteria similar to OECD guideline 471(adopted 21 July 1997), S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 strains were exposed to BNMA (no information on purity) in DMSO at concentrations of 9.77, 19.5, 39.1, 78.1, 156 and 313 µg/plate in the presence and absence of mammalian metabolic activation. The test was performed as pre-incubation test with 20 minutes pre-incubation.
BNMA was tested up to cytotoxic concentrations.
The positive controls induced the appropriate responses in the corresponding strains.
There was no evidence of induced mutant colonies 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.
Referenceopen allclose all
Conc. [mg/L] |
S9 mix |
Rel. CE [%] |
Mutant colonies /10E06 cells |
Induction factor |
Rel. CE [%] |
Mutant colonies /10E06 cells |
Induction factor |
|
Experiment I, 4 h treatment |
Culture I |
Culture II |
||||||
Solvent control |
- |
100.0 |
16.9 |
1.0 |
100.0 |
18.3 |
1.0 |
|
P.C. (EMS) |
- |
97.9 |
132.8 |
7.0 |
102.5 |
147.1 |
8.0 |
|
Test item |
13.8 |
- |
99.3 |
8.1 |
0.4 |
104.0 |
20.9 |
1.1 |
Test item |
27.5 |
- |
99.9 |
10.4 |
0.6 |
104.3 |
9.7 |
0.5 |
Test item |
55 |
- |
97.9 |
12.4 |
0.7 |
111.0 |
14.5 |
0.8 |
Test item |
110 |
- |
89.5 |
8.0 |
0.4 |
72.8 |
10.4 |
0.6 |
Test item |
220 |
- |
0.0 |
not continued |
0.0 |
not continued |
||
Test item |
330 |
- |
0.0 |
not continued |
0.0 |
not continued |
||
Test item |
440 |
- |
0.0 |
not continued |
0.0 |
not continued |
||
Experiment I, 4 h treatment |
Culture I |
Culture II |
||||||
Solvent control |
+ |
100.0 |
29.1 |
1.0 |
100.0 |
14.6 |
1.0 |
|
P.C. (DMBA) |
+ |
45.5 |
983.2 |
33.7 |
60.0 |
675.1 |
46.1 |
|
Test item |
55 |
+ |
95.9 |
47.4 |
1.6 |
96.0 |
29.9 |
2.0 |
Test item |
110 |
+ |
97.3 |
21.0 |
0.7 |
99.5 |
26.1 |
1.8 |
Test item |
220 |
+ |
94.0 |
17.5 |
0.6 |
97.3 |
8.6 |
0.6 |
Test item |
440 |
+ |
90.9 |
10.1 |
0.3 |
94.5 |
16.9 |
1.2 |
Test item |
880 |
+ |
61.0 |
16.8 |
0.6 |
75.3 |
14.1 |
1.0 |
Test item |
1760 |
+ |
31.3 |
not continued |
55.2 |
not continued |
||
Experiment II, 24 h treatment |
Culture I |
Culture II |
||||||
Solvent control |
- |
100.0 |
4.6 |
1.0 |
100.0 |
20.4 |
1.0 |
|
P.C. (EMS) |
- |
99.4 |
136.3 |
29.7 |
87.6 |
153.1 |
7.5 |
|
Test item |
13.8 |
- |
95.3 |
5.6 |
1.2 |
84.6 |
10.1 |
0.5 |
Test item |
27.5 |
- |
102.8 |
6.1 |
1.3 |
79.6 |
8.8 |
0.4 |
Test item |
55 |
- |
99.0 |
8.4 |
1.8 |
82.3 |
7.7 |
0.4 |
Test item |
110 |
- |
84.0 |
7.5 |
1.6 |
79.9 |
18.6 |
0.9 |
Test item |
165 |
- |
40.2 |
13.8 |
3.0 |
55.9 |
6.8 |
0.3 |
Test item |
220 |
- |
5.2 |
not continued |
11.3 |
not continued |
||
Experiment II, 4 h treatment |
Culture I |
Culture II |
||||||
Solvent control |
+ |
100.0 |
7.0 |
1.0 |
100.0 |
3.6 |
1.0 |
|
P.C. (DMBA) |
+ |
48.5 |
487.9 |
70.1 |
38.2 |
444.0 |
125.1 |
|
Test item |
27.5 |
+ |
100.4 |
6.8 |
1.0 |
101.3 |
5.3 |
1.5 |
Test item |
55 |
+ |
97.8 |
12.6 |
1.8 |
99.5 |
6.8 |
1.9 |
Test item |
110 |
+ |
96.9 |
10.9 |
1.6 |
98.8 |
9.0 |
2.5 |
Test item |
220 |
+ |
100.0 |
11.0 |
1.6 |
102.2 |
7.0 |
2.0 |
Test item |
440 |
+ |
101.1 |
13.8 |
2.0 |
100.4 |
5.8 |
1.6 |
Test item |
880 |
+ |
71.6 |
not continued |
86.2 |
not continued |
||
P.C. = positive control
CE = cloning efficiency
Exp. |
Test item concentration [mg/L] |
Proliferation index CBPI |
Cytostasis [%] |
Micronucleated cells [%] |
Exposure period 4 hrs without S9 mix |
||||
I |
Negative control |
1.96 |
|
0.25 |
|
Solvent control |
1.79 |
|
0.30 |
|
Positive control |
1.97 |
n.c. |
3.30S |
|
62.3 |
1.61 |
22.8 |
0.35 |
|
109.0 |
1.77 |
2.5 |
0.50 |
|
190.7 |
1.74 |
6.1 |
0.45 |
|
333.8 |
1.18 |
77.8 |
0.45 |
Exposure period 20 hrs without S9 mix |
||||
IIA |
Negative control |
1.76 |
|
0.40 |
|
Solvent control |
1.82 |
|
0.35 |
|
Positive control |
1.70 |
7.8 |
2.40S |
|
62.3 |
1.83 |
n.c. |
0.35 |
|
109.0 |
1.81 |
1.6 |
0.35 |
|
190.7 |
1.75 |
8.3 |
0.45 |
IIB |
Negative control |
2.12 |
|
0.35 |
|
Solvent control |
1.99 |
|
0.25 |
|
Positive control |
1.79 |
29.5 |
4.10S |
|
100.0 |
1.97 |
2.4 |
0.35 |
|
250.0 |
1.85 |
13.9 |
0.50 |
|
300.0 |
1.76 |
23.5 |
0.50 |
Exp. |
Test item concentration [mg/L] |
Proliferation index CBPI |
Cytostasis [%] |
Micronucleated cells [%] |
Exposure period 4 hrs with S9 mix |
||||
I |
Negative control |
1.61 |
|
0.40 |
|
Solvent control |
2.01 |
|
0.70 |
|
Positive control |
1.30 |
50.8 |
3.30S |
|
62.3 |
1.98 |
3.07 |
0.45 |
|
1022.3 |
1.86 |
15.3 |
0.45 |
|
1789.0 |
1.67 |
33.9 |
2.10S |
IIA |
Negative control |
2.03 |
|
0.65 |
|
Solvent control |
1.64 |
|
1.05 |
|
Positive control |
1.64 |
37.4 |
3.60S |
|
109.3 |
1.67 |
n.c. |
0.40 |
|
584.2 |
1.65 |
n.c. |
0.65 |
|
1405.7 |
1.39 |
38.4 |
0.45 |
|
1789.0 |
1.26 |
59.4 |
0.90 |
S The number of micronucleated cells is statistically significantly higher than corresponding control values
n.c. Not calculated as the CBPI is equal or higher than the solvent control
Without S9 mix no biologically relevant increase in the number of cells carrying micronuclei was observed after treatment with the test item.
The micronucleus rates of cells after treatment with the test item (0.35 - 0.50 % micronucleated cells) were slightly above the range of the solvent control values (0.25 - 0.40 % micronucleated cells) but within the range of the laboratory historical control data (0.1 - 1.4% micronucleated cells).
With S9 mix in Experiment I the highest evaluated concentration (1789.0 mg/L) showed a statistically significant increase in micronucleated cells (2.1% micronucleated cells) above the range of the historical solvent control data (0.25 - 1.65 % micronucleated cells). However, in Experiment IIA this finding could not be confirmed. Therefore it can be considered to be of no biological relevance.
Official test results |
TA100 |
TA1535 |
E. coli uvrA |
TA 98 |
TA 1537 |
|||||
without S9 |
mean |
MF |
mean |
MF |
mean |
MF |
mean |
MF |
mean |
MF |
negative control |
105.5 |
1.0 |
13 |
1.0 |
15.5 |
1.0 |
14 |
1.0 |
7 |
1.0 |
9.77 |
99.5 |
0.9 |
12.5 |
1.0 |
28.5 |
1.8 |
15 |
1.1 |
10.5 |
1.5 |
19.5 |
106 |
1.0 |
14 |
1.1 |
18.5 |
1.2 |
18 |
1.3 |
7 |
1.0 |
39.1 |
93.5 |
0.9 |
10 |
0.8 |
20.5 |
1.3 |
16.5 |
1.2 |
3.5 |
0.5 |
78.1 |
100 |
0.9 |
10 |
0.8 |
28 |
1.8 |
13.5 |
1.0 |
3 |
0.4 |
156 |
76.5 |
0.7 |
6.5 |
0.5 |
17 |
1.1 |
7 |
0.5 |
3 |
0.4 |
313 |
0 |
0.0 |
0 |
0.0 |
19.5 |
1.3 |
4 |
0.3 |
3.5 |
0.5 |
positive control |
678.5 |
6.4 |
453.5 |
34.9 |
187.5 |
12.1 |
520 |
37.1 |
194.5 |
27.8 |
with S9 |
||||||||||
negative control |
111.5 |
1.0 |
10 |
1.0 |
26 |
1.0 |
25.5 |
1.0 |
12 |
1.0 |
9.77 |
23.5 |
0.9 |
||||||||
19.5 |
25.5 |
1.0 |
||||||||
39.1 |
130.5 |
1.2 |
9 |
0.9 |
31 |
1.2 |
29 |
1.1 |
9.5 |
0.8 |
78.1 |
102.5 |
0.9 |
11 |
1.1 |
28.5 |
1.1 |
25 |
1.0 |
8.5 |
0.7 |
156 |
118 |
1.1 |
6 |
0.6 |
30.5 |
1.2 |
18.5 |
0.7 |
18 |
1.5 |
313 |
90 |
0.8 |
5.5 |
0.6 |
22 |
0.8 |
25 |
1.0 |
21 |
1.8 |
625 |
74 |
0.7 |
5 |
0.5 |
25.5 |
1.0 |
|
|
6.5 |
0.5 |
1250 |
53 |
0.5 |
3 |
0.3 |
11.5 |
0.4 |
|
|
8.5 |
0.7 |
positive control |
1310.5 |
11.8 |
276 |
27.6 |
981 |
37.7 |
425.5 |
16.7 |
132 |
11.0 |
Validation test results |
TA100 |
TA1535 |
E. coli uvrA |
TA 98 |
TA 1537 |
|||||
without S9 |
mean |
MF |
mean |
MF |
mean |
MF |
mean |
MF |
mean |
MF |
negative control |
108.5 |
1.0 |
10.5 |
1.0 |
22 |
1.0 |
11 |
1.0 |
8 |
1.0 |
9.77 |
113 |
1.0 |
7 |
0.7 |
21 |
1.0 |
13.5 |
1.2 |
6.5 |
0.8 |
19.5 |
102.5 |
0.9 |
8 |
0.8 |
20 |
0.9 |
12.5 |
1.1 |
4 |
0.5 |
39.1 |
99.5 |
0.9 |
11 |
1.0 |
15.5 |
0.7 |
11.5 |
1.0 |
5.5 |
0.7 |
78.1 |
114 |
1.1 |
5 |
0.5 |
22.5 |
1.0 |
14 |
1.3 |
5.5 |
0.7 |
156 |
88.5 |
0.8 |
4 |
0.4 |
17 |
0.8 |
12.5 |
1.1 |
4 |
0.5 |
313 |
78 |
0.7 |
1 |
0.1 |
12 |
0.5 |
12 |
1.1 |
5.5 |
0.7 |
positive control |
694 |
6.4 |
370.5 |
35.3 |
206 |
9.4 |
375.5 |
34.1 |
214 |
26.8 |
with S9 |
|
|||||||||
negative control |
30 |
1.0 |
||||||||
9.77 |
30 |
1.0 |
||||||||
19.5 |
21.5 |
0.7 |
||||||||
39.1 |
25.5 |
0.9 |
||||||||
78.1 |
29.5 |
1.0 |
||||||||
156 |
20 |
0.7 |
||||||||
313 |
22 |
0.7 |
||||||||
positive control |
|
|
|
|
|
|
426.5 |
14.2 |
|
|
MF = mutation factor
empty cells: test not performed
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
For the assessment of the mutagenic potential of BNMA several reliable (RL1 -2), relevant and adequate studies are available: a reverse gene mutation assay in bacteria (Ames test), a mammalian cell gene mutation assay (HPRT test), an in vitro mammalian cell micronucleus assay, and as supporting studies an SOS/umu-test in Salmonella typhimurium TA 1535/pSK1002 and a mammalian cell DNA synthesis inhibition test in HeLa S3 cells.
Bacterial reverse gene mutation assay
In a reverse gene mutation assay in bacteria similar to OECD guideline 471 (adopted 21 July 1997), S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 strains were exposed to BNMA (no information on purity) in DMSO at concentrations of 9.77, 19.5, 39.1, 78.1, 156 and 313 µg/plate in the presence and absence of mammalian metabolic activation. The test was performed as pre-incubation test with 20 minutes pre-incubation. BNMA was tested up to cytotoxic concentrations. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.
Mammalian cell gene mutation
In a mammalian cell gene mutation assay according to OECD guideline 476, adopted 21 July 1997 (HPRT test), V79 cells cultured in vitro were exposed to BNMA (98.37% a.i.) at the following concentrations in the presence and absence of mammalian metabolic activation (S9 mix).
Experiment I:
Without metabolic activation: 0 (control), 13.8, 27.5, 55, 110 mg/L
With metabolic activation: 0 (control), 55, 110, 220, 440, 880 mg/L
Experiment II:
Without metabolic activation: 0 (control), 13.8, 27.5, 55, 110, 165 mg/L
With metabolic activation: 0 (control), 27.5, 55, 110, 220, 440 mg/L
BNMA was tested up to cytotoxic or phase-separation concentrations. The positive controls induced the appropriate response. There was no evidence of induced mutant colonies over background. In this HPRT test, BNMA was found to be not mutagenic.
In vitro micronucleus assay
In a mammalian cell micronucleus assay according to OECD guideline 478 (adopted July 22, 2010), primary human lymphocyte cultures were exposed to BNMA (98.37%) in DMSO with and without metabolic activation (S9 mix).
The following concentrations were evaluated (dose calculations adjusted to purity):
experiment I:
4 h treatment without metabolic activation: 62.3, 109.0, 190.7, 333.8 mg/L
4 h treatment with metabolic activation: 62.3, 1022.3, 1789.0 mg/L
experiment IIA:
20 h treatment without metabolic activation: 62.3, 109.0, 190.7 mg/L
4 h treatment with metabolic activation: 109.0, 584.2, 1022.3, 1789.0 mg/L
experiment IIB:
20 h treatment without metabolic activation: 100, 250, 300 mg/L
BNMA was tested up to cytotoxic or the guideline limit concentration of 10 mM (corresponding to 1789.0 mg/L). Cytotoxic effects were observed after 4 h treatment with 333.8 mg/L and above in the absence of S9 mix; no cytotoxicity was observed in the presence of S9 mix up to the guideline limit concentration.
Without metabolic activation no biologically relevant increase in the number of cells carrying micronuclei was observed after treatment with the test item. Cells treated with 1789.0 mg/L BNMA in the presence of S9 mix showed a statistically significant increase in micronucleated cells above the range of the historical solvent control data in the first experiment. However, this finding could not be confirmed in the second experiment. Therefore it can be considered to be of no biological relevance. Positive controls induced the appropriate response. There was no evidence of micronucleated cells induced over background. Therefore, BNMA is considered to be non-clastogenic in this in vitro micronucleus test, when tested up to cytotoxic or the highest evaluable concentration.
Supporting studies
In anSOS/umu-test Salmonella typhimurium TA 1535/pSK1002 was exposed to BNMA (>98%) in DMSO at concentrations of 0 (control), 0.3, 0.6, 1.3, 2.5, 5, 10 mmol/L corresponding to 52.8, 105.6, 228.8, 440, 880 and 1760 mg/L in the absence of metabolic activation.
BNMA was not mutagenic up to a concentration of 440 mg/L. At concentrations of 880 mg/L and higher the test substance was cytotoxic, thus no assessment for mutagenicity was possible for those concentrations. The positive control did induce the appropriate response.
In a mammalian cell DNA synthesis inhibition test, HeLa S3 cells culturedin vitrowere exposed to BNMA (>98%) in DMSO at concentrations of 0 (control), 0.3, 0.6, 1.3, 2.5, 5, 10 mmol/L corresponding to 52.8, 105.6, 228.8, 440, 880 and 1760 mg/L in the absence of metabolic activation. The positive control did induce the appropriate response. There was no evidence of genotoxicity for all concentrations tested.
Based on the available reliable, relevant and adequate data, there was no evidence of genotoxicity for BNMA. 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 for genotoxicity.
Short description of key information:
BNMA is not genotoxic based on the following results:
- not mutagenic in the Salmonella typhimurium reverse mutation assay
(OECD guideline 471; non-GLP)
- not mutagenic in the mammalian cell gene mutation assay (OECD
guideline 476; HPRT test, V79 cells; GLP)
- not clastogenic in an in vitro micronucleus test (OECD guideline 487;
human lymphocytes; GLP)
- negative in SOS/umu test and mammalian cell DNA synthesis inhibition
test (no guideline, non-GLP)
Endpoint Conclusion: No adverse effect observed (negative)
Justification for classification or non-classification
Based on the available data, BNMA does not need to be classified for mutagenicity according to the criteria given in regulation (EC) 1272/2008. Thus, no labelling is required.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
