Registration Dossier

Administrative data

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:
weight of evidence
Study period:
1989-10-10 to 1989-12-19
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was conducted under the regulations of GLP and the respective guideline. A read-across approach to the free acid of the test substance was used. For justification please refer to IUCLID section 13.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1990
Report Date:
1990

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: U.S. EPA FIFRA Guideline 84
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder

Test animals

Species:
mouse
Strain:
ICR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Sprague-Dawley, Inc., Frederick, MD.
- Assigned to test groups randomly: yes, under following basis: body weight
- Weight at study initiation: 28.6 - 39.1 grams and 20.0 - 24.3 grams for the male and female animals
- Housing: group-housed by sex up to five per cage
- Diet: Purina Certified Laboratory Chow* #5002 ad libitum
- Water: tab water ad libitum
- Acclimation period: at least 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
- Justification for choice of vehicle: Common vehicle
- Concentration of test material in vehicle: 400, 133.4, 40 mg/mL
- Amount of vehicle: 12.5 ml/kg
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Based upon the solubility data from the dose limit test, the vehicle used to solubilize the test article for the bone marrow micronucleus assay was corn oil. The dosing solutions for the assay were prepared by making a 400 mg/mL stock for the high dose (5000 mg/kg). Dilutions of this stock were prepared for the 1667 and 500 mg/kg dose levels.
Duration of treatment / exposure:
single treatment
Frequency of treatment:
Once
Post exposure period:
24, 48, 72 hours
Doses / concentrations
Remarks:
Doses / Concentrations:
5000, 1667, 500 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
5 animals per sex per dose
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide
- Route of administration: orally by gavage
- Dose: 80 mg/kg bw

Examinations

Tissues and cell types examined:
Bone marrow cells
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
The dose levels used in this assay were based upon the results of the previously conducted dose limit test in which there was no toxicity at 5000 mg/kg bw over a fourteen day observation period.

TREATMENT AND SAMPLING TIMES:
The test article dosed animals were euthanatized 24, 48 and 72 hours after administration of the test article. The positive and vehicle control animals were euthanatized 24 hours after the administration of the control articles.

DETAILS OF SLIDE PREPARATION:
At the appropriate harvest time, the animals were euthanatized with C02 and the adhering soft tissue and epiphyses of both tibiae were removed. The marrow was flushed into a centrifuge tube (one tube for each animal) with 3 mL fetal calf serum. Following centrifugation to pellet the tissue, most of the supernatant was drawn off, the cells were resuspended, and the suspension spread on slides and air-dried. The slides were then fixed in methanol, stained in May-Gruenwald solution followed by Giemsa, and rinsed in deionized water. After being air-dried, the slides were coverslipped using Depex mounting medium.

METHOD OF ANALYSIS:
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. 1000 PCEs per animal were scored. The frequency of micronucleated cells as 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%.
The frequency of PCEs versus NCEs was determined by scoring the number of NCEs observed in the optic fields while scoring the 1000 PCEs for micronuclei.
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 micronucieus; thus the occasional cell with more than one micronucieus was counted as one micro-nucleated 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:
Data were summarized to include tables indicating the individual animal results and in tables with animal results summarized by sex and dose groups at the different time points. The analysis of the data was performed using an Analysis of Variance (p<0.05) on the square root arcsine transformation (Sokal and Rohlf, 1981) which was performed on the proportion of cells with micronuclei per animal (square root arcsine proportion). Once the Analysis of Variance had been performed, Tukey's Studentized range test (HSD) with adjustment for multiple comparisons (p<0.05) was used at each harvest time to determine which dose groups, if any, were significantly different from the vehicle control. Analyses were performed separately for each harvest time and sex combination, and also at each harvest time for the sexes combined.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF DEFINITIVE STUDY
- Clinical signs of toxicity in test animals: All animals appeared normal after dosing and remained healthy until the appropriate harvest times.
- Induction of micronuclei: The test article induced no significant increases in micronucleated polychromatic erythrocytes over the levels observed in the vehicle controls in either sex or at any of the harvest times. The positive control, CP, induced significant increases in micronucleated PCEs in both sexes, with means and standard errors of 2.48% ± 0.54% and 1.28% ± 0.69% for the males and females, respectively.
- Ratio of PCE/NCE:
Vehicle control: Males: 0.57 ± 0.06; Females: 0.71 ± 0.09
Positive control: Males: 0.76 ± 0.16; Females: 0.69 ± 0.03
500 mg/kg bw (48h): Males: 0.60 ± 0.06; Females: 0.83 ± 0.16
1667 mg/kg bw (48h): Males: 0.45 ± 0.07; Females: 0.58 ± 0.07
5000 mg/kg bw (48h): Males: 0.49 ± 0.08; Females: 0.65 ± 0.04

Applicant's summary and conclusion