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Acute Toxicity: oral

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acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
December 22, 1992 to January 21, 1993
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guidelineopen allclose all
according to
EPA OTS 798.1175 (Acute Oral Toxicity)
according to
OECD Guideline 401 (Acute Oral Toxicity)
GLP compliance:
Test type:
standard acute method
Limit test:

Test material

Specific details on test material used for the study:
A of isobutanol, Lot No. TS3370114 was used. The test substance was a colorless, transparent, low viscosity liquid.

NMR spectral data and mass spectral fragmentation data show that this sample is isobutanol. Sample purity, measured by capillary GC, is = 99.9%.

Test animals

Details on test animals and environmental conditions:
Male and female Sprague Dawley* albino rats were received from Harlan Sprague Dawley, Inc. (Indianapolis, IN). The strain and species were selected because of their availability and existing historical data. Male rats were ordered to be approximately 195 to 215 g (designated by the supplier to be approximately 7 to 8 weeks of age). Female rats were ordered to be approximately 220 to 240 g (approximately 11 to 12 weeks of age). The females were ordered to be nulliparous and nonpregnant. Occasionally, male and female rats that were ordered in at 6 weeks of age (initially received for other acute testing) were used as long as weight requirements were met on the day of dosing.

Once a month, 5 rats/sex received for acute testing and housed in Room 109 were subjected to a quality control evaluation, including gross pathology, parasitology and viral serology testing. Periodically, a Clinical Veterinarian examined the rats housed in Room 109 for any signs of health deficiencies. All animals were assigned a unique number and identified by cage cards. Animals were also identified by an ear tagging procedure during the week of receipt.

The animals were separated by se (up to 5/cage) in stainless steel, wire mesh cages (approximately 23.5 x 40.0 x 18.0 cm.). DACBQ (Deotized Animal Cage Board; Shepherd Specialty Papers, Inc.) was placed under each cage and changed regularly. An automatic timer was set to provide fluorescent lighting for a 12-hour photoperiod (approximately 0500 to 1700 hours for the light phase). Temperature and relative humidity were recorded (Cole-Parmer HygrothermographQ Seven-Day Continuous Recorder, Model No. 8368-00, Cole-Parmer Instrument Co., Chicago, IL). Temperature was routinely maintained at 66-77OF during the test period; relative humidity was routinely maintained at 40-70%. Any minor exceptions to these specified ranges can be found in the raw data.

Tap water (Municipal Authority of Westmoreland County, Greensburg, PA) was available ad libitum except during dosing and was delivered by an automatic watering system with demand control valves mounted on each rack. Water analyses were provided by the supplier, Halliburton NUS Environmental Laboratories, Materials Engineering & Testing Company, and Lancaster Laboratories, Inc. at regular intervals. EPA standards for maximum levels of contaminants were not exceeded. Pelleted, certified AGWAY@ PROLAB@ Animal Diet Rat, Mouse, Hamster 3000 (Agway Inc,) was available ad libitum. For the peroral test, the feed was removed the day before dose administration. After dosing, feed was again made available ad libitum. Analyses for chemical composition and possible contaminants of each feed lot were performed by Agway Inc. and the results are included in BRRC files. Feed and water analysis reports were reviewed by the Study Director as they were available.

Animal Acclimation
The animals were acclimated for at least 5 days before dosing. Detailed clinical observations were conducted twice, at the time of receipt and during animal identification and/or dosing. Cage-side observations and mortality checks were conducted at least once daily. Animals considered unacceptable for the study, based on the clinical signs or body weights (rabbits), were rejected for use on this study.

Study Organization
The animals were weighed and inspected for health on the day of the test. Only those exhibiting a healthy state were used. Healthy animals appeared lert, active and well groomed, with no evidence of discharge, diarrhea, breathing difficulties or locomotor abnormalities. A BRRC veterinarian was available for consultation regarding any animal health concerns. Animals were randomly assigned to cages and were designated for dosing according to need and availability.

Rat body weights were within 20% of the group mean for each sex. For the peroral test, the body weight range on the day of dosing was 281 to 292 g for males and 210 to 259 g for females (including those used for preliminary testing). A total of 3 male and 20 female rats were used for the definitive peroral test.

Administration / exposure

Route of administration:
oral: gavage
other: 0.25% w/v aqueous methyl cellulose solution
Details on oral exposure:
Each dosing mixture was prepared just prior to administration by diluting the appropriate amount of isobutanol with 0.25% w/v aqueous methyl cellulose solution (CAS No. for water is 7732-18-5; CAS No. for methyl cellulose, Sigma Chemical, is 9004-67-5). All resulting emulsions were mixed for approximately 15 to 30 minutes on a magnetic stirrer. Doses were administered by stomach intubation through a commercial 16-gauge (3-inch) ba11-end stainless steel needle attached to a disposable syringe. The exact amounts of test substance and emulsion given to each rat were recorded on the raw data form.

1000, 2000, 2830 and 4000 mg/kg (females)
2830 mg/kg (males)
No. of animals per sex per dose:
5 females/dose level
3 males at 2830 mg/kg
Control animals:
not specified
Details on study design:
The rats were fasted overnight before dosing. Five female rats were included on each of several dose levels in order to determine an LD50. Three male rats were included on an intermediate dose level for comparison. An additional 2 female rats were used for preliminary peroral toxicity testing. For individual animals, the dosing volume was adjusted according to body weight. Dosed rats were observed frequently for signs of toxicity on the first day of the test and twice a day thereafter (except on weekends or holidays when they were examined for death alone). Weights were recorded on the day of dosing and at 7 and 14 days after dosing or at death.

After 14 days, all survivors were sacrificed by CO2 overdose. Necropsies were performed on all animals that died or were sacrificed. Unless tissues were judged to be excessively autolyzed, the following tissues were collected from selected animals and retained in 10% neutral buffered formalin: kidneys, urinary bladder, liver, sciatic nerve, stomach, intestines and spleen. Lungs were also saved because of possible lung damage, based on clinical signs.

During the acute peroral toxicity test, several animals (including survivors) had varying amounts of blood present in the urine. Therefore, the Sponsor requested that histology evaluation be performed on all saved kidney and urinary bladder tissues. One female rat appeared to be pregnant at necropsy and the uterus was saved in order to verify this condition (since the animals are ordered to be nonpregnant).
An LD50 was calculated for female rats, based on the 14-day observation period. It was calculated by the moving average method (Thompson, 1947). An estimate of the slope was made by the formula developed by Weil (1983).

Thompson, W. R. (1947). Use of moving averages and interpolation to estimate medium-effective dose. Bacteriological Rev. 11, 115-145.

Weil, C. S. (1983). Economical LD50 and slope determination. Drug and Chemical Toxicology 6(6), 595-603.

Results and discussion

Preliminary study:
Not applicable.
Effect levelsopen allclose all
Key result
Dose descriptor:
Effect level:
3 350 mg/kg bw
Based on:
test mat.
95% CL:
2 860 - 3 920
Key result
Dose descriptor:
Effect level:
> 2 830 mg/kg bw
Based on:
test mat.
In preliminary testing, 1 female rat was dosed with 2000 mg/kg of isobutanol and 1 female rat was dosed with 8000 mg/kg (20% w/v emulsions in 0.25% aqueous methyl cellulose solution). The rat receiving 8000 mg/kg died. In the definitive test, the peroral LDs0 for female rats dosed with the test substance (emulsions in 0.25% aqueous methyl cellulose solution) was 3350 mg/kg. None of 3 male rats died after receiving peroral doses of 2830 mg/kg of isobutanol (a comparison dose that produced 0 of 5 female deaths), although signs were apparent.

Deaths occurred within 2 hours to 1 day. Survivors recovered within 0.5 hour to 6 days.
Clinical signs:
Signs of toxicity included sluggishness, unsteady gait, lacrimation, piloerection, slow breathing, prostration and a trace to large amount of blood -
in urine (positive by HEMASTIX" Reagent Strips).
Body weight:
Several females exhibited a slight weight loss within 7 to 14 days.
Gross pathology:
Necropsy of animals that died revealed discolored and/or mottled lungs (bright to dark red), tan to dark maroon and/or mottled livers (in 2), discolored stomachs (gray and/or yellow), 1 liquid-filled stomach, dark red and/or gray areas on the intestines, red to brown kidneys (in 1) and a large amount of blood in the urine of 1 (positive by HEMASTIX" Reagent Strips). There were no gross lesions apparent in any survivor at necropsy. One female survivor dosed with 2830 mg/kg of isobutanol appeared pregnant at necropsy (determined to be a pseudopregnancy during microscopic evaluation).

The kidneys and urinary bladders from 1 or 2 rats from each dose group (except 1000 mg/kg) were saved and examined microscopically. The only kidney lesions evident were single instances of tubular proteinosis, tubular basophilia, mineralization and congestion which were not considered to be attributable to the test substance. There were no lesions observed in the urinary bladders. In the uterus of the 1 female rat (2830 mg/kg) that appeared pregnant at necropsy, deciduoma of pseudopregnancy were apparent. This condition is somewhat unusual for animals of this age group and was, therefore, possibly related to the treatment.
Other findings:
No additional information available.

Any other information on results incl. tables

No additional information available.

Applicant's summary and conclusion

Interpretation of results:
Category 5 based on GHS criteria
The acute oral toxicity was examined. The LD50 in female rats was 3350 mg/kg and in male rats was >2830 mg/kg. Kidney lesions were apparent microscopically but were not considered to be treatment-related.
Executive summary:

The acute oral toxicity of isobutanol was examined. The LD50 in female rats was 3350 mg/kg and in male rats was >2830 mg/kg. Kidney lesions were apparent microscopically but were not considered to be treatment-related.