Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1991-10-22 to 1991-12-02
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Equivalent or similar to OECD 475 guideline, GLP study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report date:
1992

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
Deviations:
not specified
GLP compliance:
yes
Type of assay:
other: Mammalian bone marrow chromosome aberrration test

Test material

Constituent 1
Chemical structure
Reference substance name:
Allyl methacrylate
EC Number:
202-473-0
EC Name:
Allyl methacrylate
Cas Number:
96-05-9
Molecular formula:
C7H10O2
IUPAC Name:
prop-2-en-1-yl 2-methylprop-2-enoate
Test material form:
other: liquid

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Portage,MI., USA
- Age at study initiation: Adult (9 weeks)
- Weight at study initiation: males = 25.5 - 40.5 g, females = 21.3 - 31.1 g
- Assigned to test groups randomly: yes
- Fasting period before study:
- Housing: Animals were group-housed by sex up to five per cage.
- Diet: Purina Certified Laboratory Chow #5002 ad libitum
- Water: ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 72±6
- Humidity (%): 50±20
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle used: corn oil, water
- Justification for choice of solvent/vehicle: The solubility of the test article was evaluated in a previously conducted dose range finding assay. Based upon the solubility data from the dose range finding assay, the vehicle used to solubilize the test article for the bone marrow micronucleus assay was sterile deionized water for Trial 1. Due to the extreme variability in the target doses and the analytical verification of these concentrations, a Trial 2 was conducted using corn oil (Duke's Brand, LotIlOB2806:04) as the vehicle.
- Amount of vehicle: 10 mL/kg bw
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Trial 1:
The dosing solutions for the assay were prepared by making a 40.0 mg/mL stock for the high dose (400 mg/kg bw). This was achieved by adding 38.3 mL of sterile deionized water to 1600.3 mg of Allyl Methacrylate, for a final volume of 40.0 ml and a final concentration of 40.0 mg/mL. This was a translucent emulsion. Dilutions of this stock were prepared for the 200 and 100 mg/kg bw dose levels. All dosing stocks were homogenized in ablender, and were mixed continuously with a ma-gnetic stirrer during the dosing procedure.
Trial 2:
The dosing solutions for the assay were prepared by making a 30.0 mg/mL stock for the high dose (300 mg/kg). This was achieved by adding 37.47 mL of corn oil to 1154.2 mg of Allyl Methacrylate, for a final volume of 38.47 ml and a final concentration of 30.0 mg/mL. This was a clear, golden solution. Dilutions of this stock were prepared for the 150 and 75 mg/kg bw dose levels.
Duration of treatment / exposure:
24, 48 and 72 h
Frequency of treatment:
single treatment
Post exposure period:
no
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 100, 200 and 400 mg/kg bw
Basis:
actual ingested
Trial 1
Remarks:
Doses / Concentrations:
0, 75, 150, 300 mg/kg bw
Basis:
actual ingested
Trial 2
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Justification for choice of positive control(s): standard positive control substance
- Route of administration: oral, gavage
- Doses / concentrations: 80 mg/kg bw

Examinations

Tissues and cell types examined:
Bone marrow cells
Details of tissue and slide preparation:
At the appropriate harvest time, the animals were euthanatized with CO2 and the adhering soft tissue and epiphyses of both tibiae were removed. The marrow was aspirated from the bone and mixed in a syringe with about 0.1 mL fetal calf serum to form a suspension. The cells were then placed on slides and air-dried, fixed in methanol, and stained in May-Grunwald solution followed by Giemsa. The air-dried slides were coverslippes using Depex mounting medium.
The coded slides were then scored for micronuclei and the polychromatic (PCE) to normochromatic (NCE) cell ratio. Standard forms were used to record these data. One thousand PCEs per animal were scored. The frequency of micronucleated cells was expressed as percent micronucleated cells based on the total PCEs present in the scored optic field. The normal frequency of micronuclei in this mouse strain is about 0.0-0.4%. Necropsy was conducted on all dead animals. The % PCEs was determined by scoring the number of PCEs and NCEs observed in the optic fields while scoring the first 1000 erythrocytes.
Evaluation criteria:
The criteria for the identification of micronuclei were those of Schmid (1976). Micronuclei were darkly stained and generally round, although almond and ring-shaped micronuclei occasionally occur. Micronuclei had sharp borders and were generally between 1/20 and 1/5 the size of the PCE. The unit of scoring was the micronucleated cell, not the micronucleus; thus the occasional cell with more than one micronucleus was counted as one micronucleated PCE, not two (or more) micronuclei. The staining procedure permitted the differentiation by color of PCEs and NCEs (bluish-grey and red, respectively).
The criteria for determining a positive response involved a statistically significant dose-related increase in micronucleated PCEs, or the detection of a reproducible and statistically significant positive response for at least one dose level. A test article that induced neither a statistically significant dose response nor a statistically significant and reproducible increase at one dose level was considered negative. In either case, the final decision was based on scientific judgment.
Statistics:
The raw data on the counts of MN-PCE for each animal was first transformed by adding 1 to each count and then taking the natural log of the adjusted number. The transformed MNPCE data on percent PCE were analyzed separately by threeway analysis of variance (sex, dose, and time) assuming the three-way interaction to be zero. From these initial analyses, the two-way interactions were reviewed for significance. Depending on these reviews, the data were analyzed by either one-, two-, or three-way analysis of variance looking at main effects only. Pairwise comparisons of treated vs. control groups were done, if necessary, by Dunnett's t-tests, one-sided (upper) for MN-PCE and two sided for percent PCE. Linear dase-related trend tests were performed if any of the pairwise comparisons yielded significant differences. All tests were conducted at an alpha level of 0.01.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Trial 1
All animals were observed immediately after dosing and periodically throughout the duration of the assay for toxic symptoms and/or mortalities. All animals in the vehicle and positive contral groups appeared normal after dosing and remained healthy until the appropriate harvest times. All test article dosed groups appeared normal immediately after dosing. However, one male from the 400 mg/kg bw dose group was found dead approximately 21 hours after dosing. This animal had no gross abnormalities in a necropsy examination. All remaining test article dosed animals appeared normal and remained healthy until the appropriate harvest times. Due to the differences in the concentrations of test material in the dosing solutions and the target concentrations, water was deemed an inapprapriate solvent, and the assay was aborted at this stage and no slide analysis was performed.
Trial 2
All animals were observed immediately after dosing and periodically throughout the duration,:of the assay for toxic symptoms and/or mortalities. All animals in the vehicle and positive control groups appeared normal after dosing and remained healthy until the appropriate harvest times. All test article dosed groups appeared normal immediately after dosing.
Approximately 20 hours after dosing, a total of 9 males and 1 female from the 300 mg/kg bw dose group were found dead. An additional 5 males at the 300 mg/kg bw dose group appeared languid. The following morning, 4 males at the 300 mg/kg bw dose group had expired. All the remaining test article dosed animals appeared normal after dosing and remained healthy until the appropriate harvest times.
Necropsy examination revealed that 11 of the dead animals had no observable lesions. A dark red stomach mucosa was observed in two animals. All lobes of the lungs were bright red in one animal.
The test article, Allyl Methacrylate, induced no significant increases in micronucleated polychromatic erythrocytes over the levels observed in the vehicle controls. The positive control, CP, induced significant increases in micronucleated PCEs compared to the vehicle control animals (about 0.01). The percent PCE value of allyl methacrylate-treated groups were not significantly different from the vehicle controls except at the 75 mg/kg bw body weight treatment level, where the analysis indicated a significant difference (about 0.01). There was no significant difference in percent PCE value between positive and negative control groups.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
The test material, allyl methacrylate, did not induce a significant increase in micronuclei in bone marrow polychromatic erythrocytes under
the conditions of this assay and is considered negative in the mouse bone marrow micronucleus test.