Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1991
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study without detailed documentation. The publication is sufficiently detailed to allow a scientific evaluation of the results

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report Date:
1991

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
- 1000 polychromatic erythrocytes were evaluated per animal, rather than 2000
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Isopropanol
- Physical state: Clear colorless liquid
- Analytical purity: 99.8%
- Lot/batch No.: TS-1621101,
- Storage condition of test material: Room temperature in the dark

Test animals

Species:
mouse
Strain:
ICR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: from Harlan Sprague-Dawley, Inc., Frederick, MD
- Age at study initiation: 8 to 11 weeks old
- Assigned to test groups randomly: yes, under following basis: by a computer generated randomization program
- Housing: 5 mice/cage
- Diet (e.g. ad libitum): Purina Certified Laboratory Chow #5002 ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: Quarantined for seven days before being placed on study

ENVIRONMENTAL CONDITIONS
- Temperature: Reported in the study to be 72 ± 6 ºF (approximately 22.2 ºC)
- Humidity (%): 50 ± 20%
- Photoperiod (hrs dark / hrs light): 12 hours: 12 hours

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: 0.9% sodium chloride
Details on exposure:
Thirty randomly assigned mice/group (15 males/15 females) were treated with isopropanol dissolved in 0.9% sodium chloride at dose levels of 0 (vehicle control), 350, 1173 and 3500 mg isopropanol/kg bw by intraperitoneal injection. Mice were observed for toxic symptoms and/or mortalities immediately after dosing and twice daily for the duration of the assay.
Duration of treatment / exposure:
Single exposure
Frequency of treatment:
Single exposure
Post exposure period:
None
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
350 mg/kg bw
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
1173 mg/kg bw
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
2500 mg/kg bw
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
3500 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
15/sex/dose
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Route of administration: oral (gavage)
- Doses / concentrations: 80 mg/kg bw

Examinations

Tissues and cell types examined:
Bone marrow was extracted from 10 mice/group (5 males/5 females) at 24, 48, and 72 hours after dosing. An additional group of 10 mice (5 males/5 females) was treated with 3500 mg/kg bw and held to ensure that 10 mice were available for bone marrow extraction at each interval. These mice were scheduled for use only if mortalities occurred in the primary dose group. As needed, mice were randomly selected from this secondary group for bone marrow extraction and unused mice were euthanized at the completion of the trial. Bone marrow for the positive control group was harvested at 24 hours. Terminal body weights were collected on all animals prior to euthanization for bone marrow harvest.
Details of tissue and slide preparation:
At the appropriate harvest time, the animals were euthanized with CO2 and the adhering soft tissue and epiphyses of both tibias 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 and stained in May-Gruenwald solution followed by Giemsa. After being air-dried, the slides were coverslipped using Depex mounting medium. The coded slides were then scored blind 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%. 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 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).
Statistics:
ANOVA followed by Tukey's

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
See remarks section.
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid

Any other information on results incl. tables

All animals in the 3500 mg/kg bw dose group became prostrate after dosing. Within 22 hours after dosing, 35 of the 40 animals dosed at 3500 mg/kg had expired. Due to this toxicity, the 3500 mg/kg bw dose level was eliminated from the study.

Immediately after dosing, the animals receiving 2500 mg/kg became prostrate. Approximately four hours after dosing, the animals were languid with squinted eyes. The following morning, approximately 23 hours after dosing, one male (#8371) was languid with squinted eyes. This animal also exhibited dyspnea approximately 30 hours after dosing. All other animals appeared normal; however, within 46 hours of dosing three animals (male #8371; female #’s 8319 and 8354) were found dead. Another female (#8369) expired approximately 53 hours after dosing and several other animals were languid. On the third morning, approximately 70 hours after dosing, two males (#’s 8326 and 8405) were found dead. All remaining test article dosed animals had rough haircoats and this condition remained at the 72 hour harvest time. A gross necropsy was performed on all animals which expired during the observation period. Male #8371 had a moderate amount of clear yellow fluid in the trachea and thoracic cavity and an irregular black stomach mucosa. Female #8369 also had a moderate amount of a clear orange fluid in the thoracic cavity. The other necropsied animals had moderate to significant distension of the stomachs or colons, two with an abnormally thin fluid content. No other abnormalities were noted in the animals examined. The terminal body weight range of the animals harvested in this trial of the micronucleus assay was 28.1 - 37.4 grams for males and 19.0 - 26.9 grams for females. Terminal body weight gains were significantly lower at the 48 and 72 hour sacrifice intervals in mice treated with 2,500 mg/kg bw compared to corresponding vehicle control body weight gains indicating that there was a test article related reduction in body weights.

The test article, isopropanol, 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 3.24% ± 0.84% and 1.22% ± 0.19% for the males and females, respectively. The vehicle and positive control rates of micronucleated PCEs were within the historical control values in this laboratory (TRIAL 1).

The test article, isopropanol, 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 1.56% ± 0.42% and 1.66% ± 0.39% for the males and females, respectively. The vehicle and positive control rates of micronucleated PCEs were within the historical control values in this laboratory (TRIAL 2).

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative