Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information
In vitro tests: Bacterial gene mutation assays: The test item did not show a mutagenic potential in an Ames test. Also the read-across substance n-butly methacrylate did not show a mutagenic potential in the Ames test. Gene mutation in mammalian cells: The read-across substance methyl methacrylate was tested in several mouse lymphoma assay and did not show a mutagenic potential. Chromosomal aberration assay in mammalian cells: The test substance caused a weak positive response in a mammalian chromosome aberration assay only at one single test item concentration in the absence of metabolic activation. Because only a weak increase in clastogenicity was observed for just one concentration and this result was not reproduced with any other test item concentration under similar conditions, the result of this test was regarded as negative. The read-across substance n-butly methacrylate did not cause a positive response in the mammalian chromosome aberration assay. In vivo test: The read-across substance n-BMA was tested negative in a mouse micronucleus test.
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:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study, GLP.
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OTS 798.5395 (In Vivo Mammalian Cytogenics Tests: Erythrocyte Micronucleus Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
Swiss
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Nossan s.r.l., Corezzana, Italy,
- Age at study initiation: 5-6 weeks
- Weight at study initiation: 26-34 grams for males and 22-27 grams for females
- Assigned to test groups randomly: no data
- Housing: 5/polycarbonate cage, by sexes
- Diet: Altromin MT diet
- Water: tap water
- Acclimation period: at least 5 days

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

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

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

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

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

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

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

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

Additional information

Additional information from genetic toxicity in vivo:

Reliable in vitro studies of the test substance t-butyl methacrylate (t-BMA) and of appropriate analogous substances were used to assess the potential of the t-BMA for gene mutations in bacteria and mammalian cells, cytogenicity in mammalian cells. Furthermore, an in vivo mouse micronucleous assay was conducted witth the analogue substance n-butyl methacrylate. A weight of evidence apporach was used to derive a conclusion of the in genetic toxicity potential of the test item assessed in vitro. Overall, based on the results obtained in the in vitro and in vivo studies the test item is not considered to be genotoxic/mutagenic or clastogenic.

 

In vitro assays

Bacterial gene mutation assay

In a non-GLP study according to Ames et al. (1975) the test substance t-butyl methacrylate was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several Salmonella typhimurium strains (TA 1535, TA 1537, TA 1538, TA 98and TA100) in a dose range from 40 µg to 2500 µg/plate with and without metabolic activation (Waegemaekers & Bensink, 1984). An increase in the number of his+ revertants was not observed either with or without metabolic activation. No bacteriotoxic effect was observed.

According to the results of the present study, the test substance t-butyl methacrylate is not mutagenic in the Salmonella typhimurium reverse mutation assay under the experimental conditions chosen here.

 

The potential of the read-across substance n-butyl methacrylate (n-BMA) to induce reverse mutation in Salmonella typhimurium (strains: TA 1535, TA 1537, TA 98, and TA 100) and in Escherichia coli WP2 uvrA was evaluated in accordance with the international guidelines (OECD 471, EU Method B13/14) in compliance with GLP Nakajima et al., 1998a).

n-Butyl methacrylate was tested in two independent experiments, with and without a metabolic activation system, both performed according to preincubation method. Bacteria were exposed to the test item at 7 or 8 dose-levels (three plates/dose-level) selected from a preliminary toxicity test: 9.77, 19.5, 39.1, 78.1, 156, 313 and 625 µg/plate without S9 and 9.77, 19.5, 39.1, 78.1, 156, 313, 625 and 1250 µg/plate with S9. After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored. n-butyl methacrylate did not induce any noteworthy increase in the number of revertants, both with and without S9 mix, in any of the four Salmonella typhimurium strains and in Escherichia coli WP2 uvrA.

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

 

Chromosomal aberration assay in mammalian cells

The test substance t-butyl methacrylate was tested in an in vitro chromosomal aberration test in Chinese hamster lung fibroblasts cells (CHL/IU) according to the OECD 473 guideline and GLP. The test was conducted with and without metabolic activation using the following test concentrations and exposure times:

without S9 mix (24 h treatment): 0, 100, 200, 400 μg/mL

without S9 mix (48 h treatment): 0, 50, 100, 200 μg/mL

without S9 mix (6 h pulse treatment): 0, 175, 350, 700 μg/mL

with S9 mix (6 h pulse treatment): 0, 188, 375, 750 μg/mL

The test item caused a weak positive response (clastogeniciy) in a mammalian chromosome aberration assay only at the highest test concentration tested with a 24 h exposure in the absence of metabolic activation (400 µg/mL). The remaining test concentrations, whether with or without metabolic activation, did not induce a positive response. Because only a weak increase in clastogenicity was observed for just one concentration and this result was not reproduced with any other test item concentration under similar conditions, the result of this test was regarded as negative. In addition a negative in vivo test is available ( see below).

 

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

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

 

Gene mutation assay in mammalian cells

As there are no data for in vitro gene mutation assays in mammalian cells for n-BMA, available data of the analogous substance methyl methacrylate (CAS 80-62-6) are used.

 

Three mouse lymphoma assays were described for methyl methacrylate.

 

According to Myhr et al. (1990) methyl methacrylate (MMA) was weakly positive with and without S-9 mix. With S-9 mix a dose-dependent increase in mutation frequencies was obtained for doses ranging from 250 nl/ml (doubling of control level, 72% relative total growth) to 1,500 nL/mL (more than 3-fold the control level, relative total growth 25%). Without S-9 mix the substance was positive in 1 out of 2 experiments for doses ranging from 500 to 1,000 nL/mL; 1,500 nl/ml led to total toxicity. In a second experiment a weak positive response was obtained at 1,500 nL/mL. Clear and reproducible increases in mutation frequencies were bound to high toxicity below 20% relative total growth.

Litton Bionetics (1981) reported on a mouse lymphoma assay which was weakly positive in presence and negative in absence of S-9 mix. Without S-9 mix doses up to 100 nl/ml were tested, higher doses led to total toxicity. With S-9 mix methyl methacrylate was positive in the dose range 100 nl/ml to 250 nl/ml, however, clear effects were observed only at doses with high toxicity below 20% relative growth.

 

In a third mouse lymphoma assay which was only run without S-9 mix, weak effects were obtained for doses producing high toxicity (Moore et al., 1988). According to the authors, 2000 µg/ml was positive in both experiments (92 and 98 mutants per 10e6survivors vs. 54 and 68 in the negative controls), relative survival was approximately 20% and 30%; in one experiment the highest dose of 2499 µg/ml induced 143 mutants at 10% relative survival; in the second experiment the highest dose of 3100 µg/ml induced 220 mutants with 11% relative survival. The vast majority of induced colonies were small ones (indicating that the genetic effect was derived from clastogenicity and not from gene mutations).

 

In several in vitro tests MMA induced an increase in gene mutations – associated with the induction of chromosomal aberrations in the same dose range. This potential seems to be limited to high doses with strong toxic effects.

 

In vivo assays

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


Justification for selection of genetic toxicity endpoint
In vivo study with structural analog substance.

Justification for classification or non-classification

Based on the results obtained in the in vitro and in vivo studies the test item is not considered to be genotoxic/mutagenic or clastogenic and thus is not to classified according to Directive 67/548/EEC and Regulation (EC) No 1272/2008.