tert-butyl methacrylate

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2006

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Principles of method if other than guideline:

Animals are treated with the test chemical once and samples of bone marrow are taken only 24 hours after treatment (In the preliminary study the bone marrow sampling times were set at about 24, 48 and 72 hours after the administrations); Tween® 80 was used as dispersant aid to prepare dosing solutions

GLP compliance:

not specified

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

tert-butyl methacrylate

EC Number:

209-548-7

EC Name:

tert-butyl methacrylate

Cas Number:

585-07-9

Molecular formula:

C8H14O2

IUPAC Name:

tert-butyl 2-methylprop-2-enoate

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (supplier): Office of Chemical Safety, Evaluation and Licensing Division, Pharmaceutical and Food Safety Bureau, Ministry of Health, Labour and Welfare
- Lot/batch number of test material: not specified
- Purity: 99.6%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Cool dark place (in a refrigerator, measured temperature range: 3-8°C).
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions and during storage: The test substance was analyzed by the test substance manufacturer on completion of the animal study, and was confirmed as stable. Stability and homogeneity were confirmed for 5 and 100 mg/ml test substance suspensions (vehicle: aqueous 0.5 w/v% CMC-Na solution with 0.1 % Tween® 80 added) that had been stored at room temperature for 24 hours.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final concentration of a dissolved solid: 25, 50 and 100 mg/ml

FORM AS APPLIED IN THE TEST
- The test substance was obtained as a homogenous suspension
- The volume administered to each animal was calculated based on the body weight on the day of administration

OTHER
Test solution of each concentration used in the main study was sampled, taking 10 ml from the upper, medium and lower layer, respectively; the concentration and homogeneity of these samples were confirmed. The results revealed that these concentrations were 95.2-98.4% of the nominal value of the respective concentrations, and the homogeneity (C.V.) was 0.1-1.3%. Thus there was no problem with concentration or homogeneity, as both were within 100 ± 10% of the nominal value.

Test animals

Species:

mouse

Strain:

other: CrlJ:CD1 (ICR) SPF

Details on species / strain selection:

Mice are widely used in micronucleus studies, and so mouse was selected as the animal species; the strain of mouse used in the present study was chosen because the characteristics of the strain are well known, and there is a lot of background data.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Atsugi Breeding Center, Charles River Laboratories Japan, Inc.
- Age at study initiation: 8 weeks
- Weight at study initiation: 31.9-35.7 g
- Assigned to test groups randomly: Animals that displayed no abnormality throughout the acclimatization and quarantine period were used. These animals were stratified according to body weight on the day of group allocation (administration day), and allocation was performed so that the mean body weights of the groups were as uniform as possible. Allocation was performed using a computer, by a combination of the block placement method and the random extraction method (the required groups were assembled by the block placement method, and the test groups and individual numbers therein were assigned randomly). Animals remaining after group allocation were euthanized under deep ether anesthesia on the study completion day.
- Fasting period before study: not specified
- Housing: individually
- Diet: Solid feed CRF-1, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature: 21-22°C
- Humidity: 47-54%
- Air changes: 10-15 times per hour
- Photoperiod: illumination 12 hours a day (07:00-19:00)

IN-LIFE DATES: not specified

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle 1: Sodium carboxymethyl cellulose (CMC-Na)
- Type and concentration of dispersant aid: 0.1 % Tween® 80
- Justification for choice of solvent/vehicle: Because the test substance is poorly soluble in water, the vehicle selected was aqueous 0.5 w/v% sodium carboxymethyl cellulose solution with 0.1 % Tween® 80 added, in which the present test substance can be obtained as a homogenous suspension.
- Concentration of test material in vehicle: 0.5 w/v% sodium carboxymethyl cellulose solution with 0.1 % Tween® 80
- Amount of vehicle: 20 ml/kg bw
- Lot/batch no.: Lot No.: 4Y01 (CMC-Na) and 7398H (Tween® 80)
- Purity: not specified

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- Treatment groups: For each concentration, the test substance was weighed, and thereto was added aqueous 0.5 w/v% CMC-Na solution with 0.1 % Tween® 80 added; the test substance was suspended therein using a homogenizer (high speed homogenizer Polytron Model K, Kinematica AG, for approx. 30 seconds, at setting 3), and the resulting suspension was made up to the specified volume. Any residual test solution was absorbed using a high-absorbency paper towel, and incinerated.
- Negative control group: The required amount of CMC-Na was weighed out and dissolved in water for injection [Lot No.: 6F74 to obtain 0.5 w/v% aqueous CMC-Na solution. Thereto was added Tween®80, to obtain aqueous 0.5 w/v% CMC-Na solution with 0.1 % Tween® 80 added.
-Positive control group: 0.1 mg/ml aqueous MMC solution was prepared at the time of use. Specifically, the contents of one vial of MMC (2 mg/vial) were dissolved in 5 ml water for injection, then 2 ml was collected and made up to 8 ml by adding 6 mL physiological saline.

For more details please see table 1 in section "Any other information on materials and methods incl. tables"

Storage temperature:
- Treatment groups: Prepared at time of use, and not stored.
- Negative control group: The vehicle was stored in a refrigerator (measured temperature for storage: preliminary study 3-5°C, main study 3-4°C), and used within 4 days after preparation (expiry: 8 days after preparation).
Positive control group: Prepared at time of use, and not stored.

Duration of treatment / exposure:

24 h

Frequency of treatment:

single administration via a flexible gastric tube

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

2 000 mg/kg bw (total dose)

Doses / concentrations 2

Dose / conc.:

1 000 mg/kg bw (total dose)

Doses / concentrations 3

Dose / conc.:

500 mg/kg bw (total dose)

No. of animals per sex per dose:

6/dose

Control animals:

yes, concurrent vehicle

Positive control(s):

mitomycin C (MMC)
- Justification for choice of positive control: Because MMC is widely used for micronucleus studies and there is a lot of background data; selected according to “toxicity study guidelines”.
- Route of administration: intraperitoneally administered once using a 25G injection needle
- Doses / concentrations: 1 mg/kg (dosage volume of 10 ml/kg bw)

Examinations

Tissues and cell types examined:

- Sample material: bone marrow cells from the bilateral femoral bones
- For each sample, the number of immature erythrocytes (polychromatic erythrocytes (PCE)), and the number of normochromatic erythrocytes, per 200 total erythrocytes were counted, and at the same time, the number of polychromatic erythrocytes having micronuclei (MNPCE) per 2000 PCE was counted.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
In the preliminary study, the maximum dose was set at 2000 mg/kg, which is the maximum dose as stipulated in the guidelines for toxicity tests; then doses of 1000, 500 and 250 mg/kg were set by dividing by a common ratio of 2. There there were no changes in general condition and no deaths. Accordingly, the doses in the main study were set as follows: the preliminary study maximum
dose of 2000 mg/kg was set as the high dose; then doses of 1000 and 500 mg/kg were set by dividing by common ratio of 2, giving 3 doses in total.

DETAILS OF SLIDE PREPARATION:
Samples for observing micronuclei were produced in accordance with the Schmid method (W. Schmid, Mutation Res. 31, 9-15 (1975), W. Schmid, “Chemical Mutagens,” Vol. 4, ed. by A. Hollaender, Plenum Press, N.Y., London, 1976, pp.76-78.). Specifically, at the specified time after administration, the respective mice were euthanized by cervical dislocation, and the bilateral femoral bones were extracted. Then, using a 1 ml disposable syringe and a 23G injection needle, bone marrow cells were washed out into a centrifuge tube using about 0.1-0.2 ml bovine fetal serum. Next, using the same syringe and injection needle,
the bone marrow cells and bovine fetal serum were mixed to loosen the cells, the mixture was centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, and the precipitate was mixed thoroughly in a mixer and then smeared
onto a slide glass (for the right and left femoral bones, one slide was prepared for each side, per animal). The smear samples were air-dried, fixed with methanol for 3 minutes, and then air-dried again. When preparing the smear samples, a corresponding table showing the animal numbers and sample numbers was compiled, and a label stating the respective test number, stage, sample number, type of test and preparation date was attached to each sample.

METHOD OF ANALYSIS:
In order to conduct the observation by a blinded method, one slide with excellent smearing was selected based on sample number. The selected bone marrow smear sample was then stained using acridine orange fluorescent stain and observed in accordance with the method of Hayashi et al. (M. Hayashi, T. Sofuni, M. Jr. Ishidate, Mutat. Res., 120, 241 (1983); Hayashi, M. “Micronucleus tests”, Scientist Press Co., Tokyo, 1991, pp. 44-55.). A small amount of 40 µg/mL aqueous acridine orange solution was dropped onto a cover glass, then the bone marrow smear sample was mounted thereon. The sample was observed at 600x total magnification using a fluorescent microscope equipped with excitation light of wavelength around 490 nm, and an observation filter permeable to ≥515 nm wavelengths.

Statistics:

For each sample, the number of MNPCE per 2000 PCE, the MNPCE frequency (%), the number of PCE per 200 total erythrocytes, and the PCE frequency (%) were calculated. In addition, the mean values and standard deviation for the number of MNPCE, the MNPCE frequency (%), the number of PCE and the PCE frequency (%) were calculated for each group, and the maximum and minimum values for each frequency (%) were also calculated. Furthermore, to assess the significance of the micronuclei frequencies in the main study, it was confirmed that the MNPCE frequencies in the negative control group and in the positive control group were within mean ± 3 S.D. of the background data at the present laboratories (this institute), and then the negative control group and test substance administration groups were compared, and subjected to a Kastenbaum and Bowman test (one-sided p≤0.05) based on binominal distribution, and a Cochran Armitage trend test (two-sided, p≤0.05, 0.01). For the PCE frequencies, homogeneity of variance (one-sided p<0.05) between the negative control group and each test substance administration group was examined using the F-test, and as the distributions were equal, the Student t-test (two-sided, p<0.05, 0.01) was performed.
Moreover, for the positive control group, the MNPCE frequency (%) was compared with the negative control group, and a Kastenbaum and Bowman test (one-sided p≤0.05) based on binominal distribution was performed. For the PCE frequencies, homogeneity of variance (onesided p<0.05) with regard to the negative control group was examined using the F-test, and because the distributions were equal, the Student t-test (two-sided, p<0.05, 0.01) was performed.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: the test substance was administered at 250, 500, 1000 and 2000 mg/kg
- Solubility: Because the test substance is poorly soluble in water, the vehicle selected was aqueous 0.5 w/v% sodium carboxymethyl cellulose solution with 0.1 % Tween® 80 added, in which the present test substance can be obtained as a homogenous suspension.
- Clinical signs of toxicity in test animals: No changes in general condition were observed in any test substance administration group.
- Evidence of cytotoxicity in tissue analysed: No dose-dependent increase in MNPCE frequency in any test substance administration group was observed, at any sampling time. The PCE frequency per 200 total erythrocytes in each test substance administration group revealed a decrease in immature erythrocytes in one male in the 2000 mg/kg group 72 hours after administration, but not other dose-dependent decrease was observed at any sampling time.
- Rationale for exposure: not specified
- Observation time points: 24, 48 and 72 hours after administration

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: In the test substance administration groups, the MNPCE frequency was 0.08 ± 0.06% in the 500 mg/kg group, 0.09 ± 0.04% in the 1000 mg/kg group and 0.13 ± 0.04% in the 2000 mg/kg group. Comparing these values with the negative control group, 0.15 ± 0.06%, revealed no statistically significant increase (p≤0.05) or dose-dependent increase (p≤0.05, 0.01) in any of the administration groups. For all test substance administration groups, there was no statistically significant decrease (p≤0.05, 0.01) in PCE frequency per 200 total erythrocytes when compared to the negative control group. Moreover, the MNPCE frequency in the positive control group was statistically significantly higher than in the negative control group. Furthermore, the MNPCE frequencies in the negative control group and the positive control group were within the range of mean ± 3 S.D. of the background data.
- Appropriateness of dose levels and route: Yes
- Statistical evaluation: Yes
- Other: Because there were no obvious differences between the sexes in terms of the appearance of toxicity, the main study was conducted using males only.

Applicant's summary and conclusion

Conclusions:

It was judged from the above results that, under the conditions of this study, the test substance does not induce chromosomal aberration in the bone marrow of Crlj:CD1(ICR) SPF mice.

Executive summary:

The test substance was assessed in a similar to OECD TG 474 study for its potential to induce chromosomal aberration in CrlJ:CD1 (ICR) SPF mice using the micronucleus test method. For this purpose, the test substance, suspended in aqueous 0.5 w/v% sodium carboxymethyl cellulose (CHC-Na) solution with 0.1 % Tween® 80 added, was administered once orally to male animals at dose levels of 500, 1000 and 2000 mg/kg in a volume of 20 ml/kg body weight in each case. The vehicle served as negative control while mitomycin C served as positive controls for clastogenicity and for spindle poison effects, respectively. Bone marrow smear samples were produced approximately 24 hours after administration. The results revealed no statistically significant increase in the frequency of immature erythrocytes with micronuclei in any test substance administration group, compared to the negative control group, and no dose-dependent increase was observed. Moreover, there was no statistically significant decrease in the frequency of immature erythrocytes in 200 whole erythrocytes in any test substance administration group, compared to the negative control group, and therefore the test substance was judged to have no inhibitory effect on the proliferation of bone marrow cells. The frequency of immature erythrocytes with micronuclei in the negative and positive control groups were within the range of mean ± 3S.D. of the background data of the present laboratories (present institute), and so the study was thought to have been conducted appropriately. From the above results, the test substance was judged not to induce chromosomal aberration in the bone marrow of CrlJ:CD1 (ICR) SPF mice under the conditions of the present study.