Butan-2-ol

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March 26, 1990 - Jun 29, 1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods

Justification for type of information:

Isopropanol (IPA) is considered as a suitable structural analogue to secondary butanol (sBA) because it meets the criteria for read-across/grouping, as outlined in Annex XI, Section 1.5 of the REACH regulation and the most recent ECHA Read-Across Assessment Framework (RAAF). The criteria include:

a. Common functional group - both IPA (C3) and sBA (C4) are secondary alcohols
b. Common precursors and/or likelihood of common breakdown products via physical and/or biological processes which result in structurally-similar degradation products - IPA and sBA are both converted via oxidation to the corresponding ketone (acetone for IPA and methyl ethyl ketone for sBA) and ultimately carbon dioxide.
c. A constant pattern in the changing of the potency of the properties across the group (i.e. of physico-chemical and/or biological properties). - IPA and sBA are considered to be toxicologically similar.

A detailed documentation of the read-across justification is attached below.

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1991

Report date:

1991

Reference 2

Reference Type:

publication

Title:

Isopropanol 13-Week Vapor Inhalation Study in Rats and Mice with Neurotoxicity Evaluation in Rats.

Author:

Burleigh-Flayer HD, Gill MW, Strother DE, Masten LW, McKee RH, Tyler TR, Gardiner T

Year:

1994

Bibliographic source:

Fundamental and Applied Toxicology 23: 421-428

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Deviations:

no

GLP compliance:

yes

Remarks:

The portions of this study performed at the Bushy Run Research Center were conducted in accordance with Good Laboratory Practice Regulations, Toxic Substances Control Act (TSCA), 40 CFR Part 792.

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Isopropanol

IUPAC Name:

Isopropanol

Constituent 2

Reference substance name:

Propan-2-ol

EC Number:

200-661-7

EC Name:

Propan-2-ol

Cas Number:

67-63-0

Molecular formula:

C3H8O

IUPAC Name:

propan-2-ol

Details on test material:

- Name of test material (as cited in study report): Isopropanol
- Physical state: colorless liquid
- Analytical purity: 99.9%
- Lot/batch No.: Drum Number T0058; Date 022690; Unit Storage Tank 1542
- Expiration date of the lot/batch: not reported
- Storage condition of test material: samples used for the study were transferred to and stored in 1-gallon glass bottles

Specific details on test material used for the study:

Test material purity - 99.93%

Test animals

Species:

other: rat and mouse

Strain:

other: Fischer 344 rats and CD®-1 mice

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Fischer 344 rats from Harlan Sprague-Dawley, Inc. (Indianapolis, IN); CD®-1 mice from Charles River Breeding Laboratories, Inc. (Portage, MI).
- Age at study initiation: rats: 8-12 weeks; mice: 8-10 weeks
- Weight at study initiation: male rats: 140-165 g; female rats: 112-130 g; male mice: 25-37 g; female mice: 19-26 g
- Fasting period before study: not reported
- Housing: individually in stainless steel, wire-mesh cages (15 cm x 22 cm x 18 cm for rats; 22.5 cm x 9.5 cm x 12.5 cm for mice); during exposure, animals were individually housed, separated by sex and exposure group, in stainless steel, wire-mesh cages (14.5 cm x 9.5 cm x 17.5 cm or 17.5 cm x 12 cm x 18 cm for rats, and 7.5 cm x 9.5 cm x 17.5 cm for mice).
- Diet (e.g. ad libitum): powdered food (Certified Rodent Chow 5002, Ralston Purina Co., St. Louis, MO) ad libitum
- Water (e.g. ad libitum): water (Municipal Authority of Westmoreland County, Greensburg, PA) ad libitum
- Acclimation period: animals were acclimated to the exposure chambers (air-only exposure) on 2 days prior to the initiation of the exposure regimen


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-24 °C
- Humidity (%): 52-56 %
- Air changes (per hr): 13.5 per hr (in inhalation chamber)
- Photoperiod (hrs dark / hrs light): 12 hrs dark/12 hrs light

Administration / exposure

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

air

Details on inhalation exposure:

Inhalation chamber description and operation: The inhalation chambers used in this study were constructed from stainless steel with glass windows for animal observation. The volume of each chamber was approximately 1330 liters, and the airflow was approximately 300 liters/minute (13.5 air changes per hour). Chamber temperature and relative humidity were recorded using a Fisherbrand® dial type thermometer (Fisher Scientific, Pittsburgh, PA) and an Airguide humidity indicator (Airguide Instrument Co., Chicago, IL). Temperature and relative humidity measurements were recorded at least 12 times per exposure.

Target concentrations and exposure regimen: Animals were acclimated to the exposure chambers (air-only exposure) for 2 days prior to the initiation of the exposure regimen. Target isopropanol vapor concentrations of 0 (control), 100, 500, 1500, and 5000 ppm were selected for this study. The rats (55 days of age) and mice (56 days of age) were exposed for 6 hours per day, 5 days per week for 13 weeks. During the 14th week, male and female rats (excluding those animals designated for neuroanatomic pathology evaluation) received 2 and 3 consecutive days of exposure, respectively. The 10 female rats of the 500, 1500, and 5000 ppm group designated for neuroanatomic pathology evaluation were exposed for 1 day during the 14th week; the male rats of the 500, 1500, and 5000 ppm group designated for neuroanatomic pathology evaluation were not exposed during the 14th week. Male and female mice received 4 and 5 consecutive days of exposure during the 14th week, respectively. The 6-hour exposure interval was defined as the time when the vapor generation system was turned on and subsequently turned off. Control (air-only exposure) animals were handled in an identical manner as the isopropanol-exposed animals.

The cage placement pattern was changed weekly in a predetermined manner within each chamber to compensate for any possible, but undetected, variations in chamber exposure conditions.

Isopropanol vapor generation: Liquid isopropanol was metered from a piston pump (Fluid Metering, Inc., Oyster Bay, NY) into a glass evaporator similar in design to that described by Snellings and Dodd (1990). Piston pumps were equipped with a 1/8'' piston (G-6 FMI pump), 1/4" piston (C-6 FMI pump), 3/8" piston (C-6 FMI pump) and 1/4" piston (G-20 FMI pump) for the 100, 500, 1500 and 5000 ppm exposure chambers, respectively. The temperature in the evaporator was maintained at a level sufficient to vaporize the test substance. Evaporator temperatures were measured without test substance on Days 4, 32, and 63 following the 6-hour exposure period. The probe was positioned on the glass column at the second spiral from the top of the evaporator using a type K thermocouple attached to a series 400A Digital Trendicator (Doric Scientific, San Diego, CA).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chamber concentrations of isopropanol vapor were analyzed by flame ionization gas chromatography. At least 2 samples per hour were obtained from each exposure chamber and also the control chamber. The nominal concentration was calculated daily by dividing the total amount of vapor delivered to the chamber by the total airflow. See Table 1 attached for data on the mean (± SD) for analytical concentrations, nominal concentrations and analytical-to-nominal concentration ratios.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

6 hours per day, 5 days per week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 ppm

Remarks:

Nominal concentration

Doses / concentrations 2

Dose / conc.:

500 ppm

Remarks:

Nominal concentration

Doses / concentrations 3

Dose / conc.:

1 500 ppm

Remarks:

Nominal concentration

Doses / concentrations 4

Dose / conc.:

5 000 ppm

Remarks:

Nominal concentration

No. of animals per sex per dose:

All exposure groups (except for the 100 ppm group) consisted of 25 rats/sex. Ten rats per sex were assigned to the 100 ppm group. Ten mice per sex were assigned to each exposure group.

Control animals:

yes, sham-exposed

Details on study design:

- Dose selection rationale: not reported
- Rationale for animal assignment (if not random): animals were assigned to the 4 exposure groups and one air-exposed control group using a computer-based randomization program
- Rationale for selecting satellite groups: not applicable
- Post-exposure recovery period in satellite groups: not applicable
- Section schedule rationale (if not random): not reported

Positive control:

Not applicable

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: during exposures, observations were recorded on a group basis. Preceding and following each exposure, animals were examined individually, and observations were recorded for each animal exhibiting overt clinical signs. At the time of body weight collection and just preceding sacrifice, detailed observations were performed on all animals. On nonexposure days, the animals were observed once a day for overt clinical signs and mortality.
- Cage side observations checked were included: tables 2 and 3 (attached) present summaries of the clinical signs observed following exposures for male and female rats, respectively. Corresponding data for mice are presented in Tables 4 and 5. The clinical signs observed during exposures were not tabulated.

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: All rats and mice were weighed on Week 0 (prior to the start of exposures). These values were used as the preexposure reference weights and were subtracted from each subsequent weight to determine the change in body weight. Animals were weighed weekly during the study, at neurobehavioral evaluations, and immediately preceding sacrifice.

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Weekly. For rats, food consumption was determined over a 7-day period. For mice, food consumption was measured over a 6-day period rather than a 7-day period due to excessive food spillage during the first 24 hours after the containers were filled with fresh food. Mice and rats which caused excessive food spillage were excluded from the weekly statistical evaluation.

FOOD EFFICIENCY: No data
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION: Yes
- Time schedule for examinations: Weekly. For rats, water consumption was determined over a 7-day period. Water consumption for mice was measured over a 7-day period (except for the final collection period when it was determined over a 6-day period). Mice and rats which caused excessive water spillage were excluded from the weekly statistical evaluation.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to the first exposure and during Week 12
- Dose groups that were examined: all rats and mice

HAEMATOLOGY: Yes
- Time schedule for collection of blood: During the 6th week of the study in 10 rats/sex/group. At sacrifice in 10 rats/sex/group and 10 mice/sex/group.
- Anaesthetic used for blood collection: Yes (methoxyflurane)
- Animals fasted: Yes
- How many animals: 10 rats/sex/group during the 6th week; 10 rats/sex/group and 10 mice/sex/group at sacrifice
- Parameters checked in table 6 (attached) were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at sacrifice
- Animals fasted: Yes
- How many animals: 10 rats/sex/group and 10 mice/sex/group
- Parameters checked in table 6 (attached) were examined.

URINALYSIS: No
- Time schedule for collection of urine: not applicable
- Metabolism cages used for collection of urine: not applicable
- Animals fasted: not applicable

NEUROBEHAVIOURAL EXAMINATION: Yes (rats only)
- Time schedule for examinations: 10/15 rats selected were evaluated with the functional observational battery (FOB) prior to the first exposure and on the weekend following Weeks 1, 2, 4, 9, and 13. Motor activity evaluations were conducted on all 15 rats per sex selected from the 0, 500, 1500, and 5000 ppm groups prior to the first exposure and on the weekend following Weeks 4, 9, and 13.
- Dose groups that were examined: 15 rats per sex from each group
- Battery of functions tested: motor activity and Functional Observational Battery (described in more detail below)

Sacrifice and pathology:

All rats (excluding those designated for neuroanatomic pathology evaluation) and all surviving mice were killed by exsanguination via the brachial blood vessels following anesthesia with methoxyflurane. A complete necropsy examination was performed on each of these animals. Complete necropsy examinations were also performed on the two female mice which died prior to the study termination. Selected tissues, listed below, were fixed in 10% neutral buffered formalin.

Fixed tissues - Larynx, Spleen, Liver, Testes, Lungs, Thymus, Heart, Trachea, Ovaries, Pancreas, Stomach, Uterus, Thyroid/parathyroid, Aorta, Salivary glands, Duodenum, Gall bladder, Jejunum, Urinary bladder, Ileum, Mammary gland, Cecum, Thigh muscle, Colon, Exorbital lachrymal glands, Rectum, Epididymis, Prostate, Adrenals, Brain, Eyes, Cross lesions, Kidneys, Nasal turbinates, Pituitary, Sternum with bone marrow, Skin (flank), Esophagus, Lymph node (submandibular), Peripheral nerve (sciatic), Zymbal glands, Seminal vesicles, Femur (including articular, surface)
Spinal cord (1 section from each of the cervical, thoracic, and lumbar regions).

Microscopic evaluations were performed on all tissues from the 10 rats/sex and 10 mice/sex of the control and high concentration groups. In addition, microscopic evaluations of the lungs and liver from 10 rats/sex and 10 mice/sex as well as the kidneys of 10 rats/sex of the 100, 500, and 1500 ppm groups were performed. The details of the necropsy procedures and histopathologic evaluations are included in Appendix 6.
The animals designated for neuroanatomic pathology evaluation were anesthetized with sodium pentobarbital and fixed by intracardiac perfusion, first with a phosphate buffered solution of 52 methanol-free EM grade formaldehyde followed by a phosphate solution of 52 glutaraldehyde. The brain, spinal cord, and peripheral nerves were removed and immersion-fixed in methanol-free EM grade formalin for light microscopic examination or in glutaraldehyde for possible electron microscopic examination. Light microscopic examinations were performed on the following tissues, from 6 rats/sex/group.

Tissues for light microscopic examinations - Forebrain, center of the cerebrum, center of the midbrain, cerebellum and pons, medulla oblongata, tibial nerve (below the knee), proximal sciatic nerve (mid-thigh and sciatic notch), spinal cord (cervical and lumbar), dorsal root ganglia, gasserian ganglia, dorsal and ventral root fibers, common peroneal nerve (below the knee) and sural (fibular) nerve (below the knee).

Other examinations:

Organ Weights: The brain, liver, lungs, kidneys, adrenals, testes (males), and ovaries (females) from all surviving animals (except animals designated for neuroanatomic pathology evaluation) were weighed at sacrifice. Organ weights were recorded as absolute weights and relative weights (as a percentage of both body and brain weight). For rats designated for neuroanatomic pathology evaluation, the brain was weighed and measured (length and width).

Hematology and serum clinical chemistry: During the sixth week of the study, hematologic evaluations were performed on blood samples collected from 10 rats/sex/group. At sacrifice, hematologic and serum clinical chemistry evaluations were performed on blood samples collected from 10 rats/sex/group and 10 mice/sex/group. Blood was obtained from the orbital sinuses of methoxyflurane-anesthetized animals. Food was removed from the animal cages prior to the start of the blood collection period (approximately 0.5 to 2 hours), but water was supplied ad libitum. The details of the materials and methods for hematologic and serum clinical chemistry evaluations are presented in Appendix 5. Table 6 provides a list of the analyses performed.

Statistics:

The data for continuous, parametric variables were intercompared for the exposure and control groups by use of Levene's test for homogeneity of variances, by analysis of variance, and by t-tests. The t-tests were used, if the analysis of variance was significant, to delineate which groups differed from the control group. If Levene's test indicated homogeneous variances, the groups were compared by an analysis of variance for equal variances followed, when appropriate, by pooled variance t-tests. If Levene's test indicated heterogeneous variances, the groups were compared by an analysis of variance for unequal variance followed, when appropriate, by separate variance t-tests. Frequency data, such as microscopic diagnoses, were compared using Fisher's exact test. All statistical tests, except the frequency comparisons, were performed using BMDP Statistical Software (Dixon, 1985). The frequency data tests are described in Biometry (Sokal and Rohlf, 1969). The probability value of p < 0.05 (two-tailed) was used as the critical level of significance for all tests.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

A summary of clinical signs observed for male/female rats are provided in attached tables 2 and 3. Corresponding data in mice are provided in tables 4 and 5.

During exposures, ataxia, narcosis, hypoactivity, and a lack of a startle reflex were observed in some rats and mice of the 5000 ppm group; narcosis was not observed in rats after Week 2 of the study. During exposures to 1500 ppm, narcosis, ataxia, and hypoactivity were observed in some mice while only hypoactivity was observed in rats. No clinical signs were noted during exposures for either rats or mice of the 100 and 500 ppm groups. Clinical signs observed following exposures included a markedly increased incidence of swollen periocular tissue in rats of the 5000 ppm group (females only) and an increased incidence of perinasal encrustation in male rats of the 500, 1500, and 5000 ppm groups. Additionally, ataxia and/or hypoactivity were observed in one male rat, three female mice, and one male mouse of the 5000 ppm group immediately following exposure. Paresis was noted in one female rat of the 5000 ppm group from Week 8 to the termination of the study, although this was not believed to be a result of exposure. No exposurerelated clinical signs were observed following exposures in male or female rats of the 100 ppm group; no exposure-related clinical signs were observed following exposures for male or female mice of the 100, 500, and 1500 ppm
groups.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Two female mice died during the study as a result of caging accidents.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Summaries of absolute body weights and body weight changes for rats and mice are provided in attached Table 7. Absolute body weight and/or body weight gains for the male and female rats of the 5000 ppm group were statistically significantly lower at the end of Week 1; the percent decrease in body weight gain of the 5000 ppm group was 16% and 48% for male and female rats, respectively. Decreased body weight and body weight gain were also observed in the female rats of the 1500 ppm group at the end of Week 1; the percent decrease in body weight gain was 17%. An increased body weight and/or body weight gain were observed for the 100 and 500 ppm group of male rats at the end of the first week. However, these increases were considered to be spurious because of the slightly greater mean body weight of these two groups prior to the start of the study and the lack of effects following this time point. Decreased body weight and/or body weight gain observed in rats during Week 1 were not present during the second week of the study. In contrast to the initial decrease, absolute body weight and body weight gains were increased (usually statistically significantly) beginning at approximately Week 5 in both male and female rats of the 1500 and 5000 ppm groups. At the end of Week 13, the percent increases in body weight gain were 12% and 16% for the male and female rats of the 5000 ppm group, respectively, and 7% and 8% for the male and female rats of the 1500 ppm group, respectively. Statistically significantly increased body weight and body weight gain (starting at approximately Week 3) were also observed in female mice of the 5000 ppm group. The percent increases in body weight and body weight gain at
the end of the study were 13% and 71%, respectively. Occasional statistically significant increases observed for females of the 100 and 500 ppm groups were not considered to be related to the isopropanol exposures, since these increases were inconsistent and were not concentration related. There were no exposure-related effects on body weight or body weight gain of male mice during the study.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Summaries of food consumption data for male/female rats and mice are provided in attached Table 8. A statistically significant decrease in food consumption was observed in female rats of the 5000 ppm group at the end of Week 1. In contrast to the initial decrease, statistically significant increases in food consumption were observed for both sexes of rats of the 5000 ppm group beginning at Week 4 or 5. Occasional statistically significant increases observed for male or female rats in other exposure groups were not considered to be related to the exposure considering the magnitude of the changes, the lack of a concentration-response relationship, and lack of consistency. There were no exposure-related effects on food consumption in male or female mice of any exposure group.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

Summaries of water consumption data for male/female rats and mice are provided in attached Table 9. Increased water consumption was observed (beginning at approximately Week 2) in both male and female rats of the 1500 and 5000 ppm groups. Increased water consumption was also observed for male mice of the 1500 and 5000 ppm group during Weeks 1 and 2. Water consumption was increased for female mice of the 5000 ppm group throughout the study. Other statistically significant changes in food or water consumption noted during the study were not considered to be related to the exposure since they were inconsistent and not concentration dependent.

Ophthalmological findings:

effects observed, treatment-related

Description (incidence and severity):

During Week 12, conjunctivitis was observed in one female rat of the 5000 ppm group and blepharospasm (squinting) was observed in one male mouse of the 5000 ppm group. In addition, conjunctivitis and swollen eyelids were observed in one male mouse of the 1500 ppm group. These findings may be indicative of chemical irritation.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Summaries of hematological findings for male/female rats and mice are provided in attached Table 10. Decreased total erythrocytes, hemoglobin, hematocrit, and platelet counts were observed at Week 6 in both sexes of rats of the 5000 ppm group. In addition, increased MCV and MCH were noted at this time point for male rats. Lymphocytes were also increased at Week 6 for females of the 5000 ppm group. At Week 6 in the 1500 ppm group, decreased platelet counts were observed for male rats, and decreased total erythrocytes were noted for female rats. These
results at Week 6 are indicative of a slight anemia which was not sufficient to stimulate an increase in reticulocytes but did increase production of immature cells, as indicated by the increases in MCV and MCH in male rats. Certain hematologic effects seen in male and female rats of the 5000 ppm group at Week 6, such as decreased total erythrocytes, hemoglobin, and hematocrit, were no longer present at Week 14. However, at Week 14, MCV and MCH were still increased in male rats of the 5000 ppm group. In addition, increased MCV was also observed for female rats of the 5000 ppm group. In contrast to the decrease observed at Week 6, platelet counts were increased in the 1500 and 5000 ppm groups of male rats. These results indicate that the slight anemia present at Week 6 was no longer present at Week 1.

Increased hemoglobin, hematocrit, MCV, and MCH were noted for female mice of the 5000 ppm group. There were no exposure-related changes in hematologic parameters for male mice.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

There were no exposure-related changes in serum clinical chemistry parameters for male or female rats at Week 14. Increased total protein, albumin, globulin, total bilirubin, direct bilirubin, and inorganic phosphorus along with decreased serum chloride were observed for female mice of the 5000 ppm group at Week 14 (Table 11). These changes may be a result of dehydration. There were no exposure-related changes in serum clinical chemistry parameters for male mice.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Exposure of rats to isopropanol did not result in changes in the functional observational battery performed following Weeks 1, 2, 4, 9, and 13 of exposure. An increase in motor activity was observed in female rats of the 5000 ppm group following Weeks 9 and 13. The percent increase in motor activity for female rats was 57% and 26% for Weeks 9 and 13, respectively.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The only organ in which possible exposure-related effects occurred was the liver . An increased relative liver weight, as a percentage of body weight, was observed for both sexes of rats and female mice of the 5000 ppm group. Absolute and relative liver weights were also significantly increased in female mice of the 100 and 1500 ppm groups; however, these changes were not considered to be exposure related based on the magnitude of the changes and the lack of a concentration-response relationship. Significant increases in other organ weights were a result of the increased mean body weight of the 1500 and 5000 ppm groups.

Organ weight data are provided in attached Table 12.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No significant differences in microscopic lesion frequencies were noted in the rats as a result of isopropanol vapor exposure. Eosinophilic hyaline droplets (which were best demonstrated with the Mallory Heidenhain stain) vere present in greater numbers and were generally of larger size within the proximal tubular epithelial cells of the kidneys of male rats in all of the isopropanol vapor-exposed groups (in comparison to the kidneys of control rats). While the degree of hyaline droplet formation was greatest for the 5000 ppm group rats, no apparent differences in the grades for this finding vere noted between the other male rat exposure groups. Hyaline droplets vere not noted in the kidney sections of any of the female rats. Although not significant, there was a numerical increase in mixed inflammatory cell nodules within the bone marrow (sternum and femur) of female rats exposed to 5000 ppm isopropanol (viz. 4 of 10 vs. 0 of 10 controls). These foci vere comprised of small aggregates of histiocytic cells and occasional other inflammatory cells and were graded, in three of the rats, as being minimal to mild in nature.

One 5000 ppm female rat, which had not been randomly selected for microscopic evaluation, developed posterior paresis during the study. Sections of the spinal cord, peripheral nerves, and skeletal muscles from the hind legs were processed for microscopic evaluation and were stained with H&E. This rat had a malignant oligodendroglia which involved the lumbar spinal cord. This is a relatively uncommon tumor in the F-344 rat and is particularly rare in such a young animal. Mineralization of the pulmonary vessels was significantly elevated in the 1500 ppm group females but not in the 5000 ppm group females. All the 1500 ppm group female rats with this finding were graded as having minimal degrees of mineralization. Pulmonary vessel mineralization is characterized microscopically, by small foci of mineral deposition within the walls of medium-sized pulmonary vessels. This represents a common but variable lesion in the F-344 rat and the increased frequency in the 1500 ppm group females is not considered to be treatment related. Numerous rats on this study (both male and female) had varying degrees of pulmonary inflammation classified in the specified microscopic diagnoses tables as "perivascular infiltrates", "pneumonitis", or "pneumonitis, interstitial". The etiology of these lesions is unknown, but they are not considered to be treatment related.

Neuropathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Upon neuropathologic examination, a minimal degree of myelin degeneration was seen in the sciatic or tibial/peroneal/sural nerves of a few rats (including controls). Since this finding was observed in only a few fibers of each affected nerve, the degeneration was believed to be spontaneous "background" myelin degeneration and not related to the exposures.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Upon histologic examination, the only exposure-related changes observed were the presence of increased numbers and sizes of hyaline droplets within the kidneys of male rats. These differences were not clearly concentration-related, although this microscopic change was most pronounced in the high concentration group. Hyaline droplets were not observed within the kidneys of female rats. Other microscopic changes noted included a malignant oligodendroglioma of the spinal cord in one female rat (paresis was observed in this animal during the study) and a thyroid adenoma in one female mouse of the 5000 ppm group; there was no evidence that either of these lesions was related to the isopropanol exposures. Foci of mixed inflammatory cell infiltration in the bone marrow were also observed in female rats. However, it was uncertain whether this finding was related to the exposures.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not specified

Details on results:

Exposure of rats and mice to isopropanol vapor for thirteen weeks produced signs of narcosis but no mortality. The narcotic effects of isopropanol were noted typically only during exposures at 1500 and 5000 ppm in this study. Narcosis, ataxia, lack of a startle reflex, or hypoactivity was observed during exposures in some rats and mice of the 5000 ppm group and in some mice of the 1500 ppm group. Narcosis was not observed during exposures in rats of the 5000 ppm group following Week 2, suggesting some adaptation to isopropanol. Tolerance to the narcotic effects of isopropanol has been previously reported. Clinical signs observed following exposures included swollen periocular tissue and perinasal encrustation at concentrations as low as 500 ppm which may have been due to the irritating effects of isopropanol; however, due to the lack of microscopic changes in the eyes and nasal cavities, the toxicologic significance of these clinical findings is uncertain. Neurobehavioral evaluations indicated no changes in the functional observational battery for either sex and no alterations in motor activity for males. An increase in motor activity for female rats of the 5000 ppm group was observed at Weeks 9 and 13. A possible hypothesis, based upon results observed with ethanol, is that the increase in motor activity is a result of withdrawal following exposure.

Transient decreases in body weight and body weight gain were observed during the first week of the study in male and female rats of the 5000 ppm group and female rats of the 1500 ppm group. Following this time point, increased body weight and body weight gain were observed for both of these exposure groups. Increased body weight and body weight gain were also observed in female mice of the 5000 ppm group. There were no exposure-related effects on body weight noted for male mice. Increases or decreases in food and water consumption generally corresponded to the changes in body weight. Changes in hematologic parameters noted in rats exposed to 5000 ppm isopropanol vapor indicated a slight anemia was present at Week 6 but not at Week 14. There were no changes in serum clinical chemistry parameters for male or female rats. Changes in hematologic and serum clinical chemistry parameters of female mice of the 5000 ppm group suggested a dehydration effect at Week 14. No exposure-related alterations in hematologic or serum clinical chemistry parameters were observed for male mice.

The only organ weight effect noted was an increase in liver weight in both sexes of rats and female mice of the 5000 ppm group. However, due to the lack of any microscopic changes observed in the liver, the significance of this effect is unclear. No gross lesions that could be attributed to isopropanol exposures were observed. Increased numbers and sizes of hyaline droplets within the kidneys of male rats exposed to isopropanol were the only microscopic changes noted. These differences were not clearly concentration related, although this microscopic change was most pronounced in the high concentration group. This kidney change, believed to be a result of the accumulation of a2u globulin in the proximal tubule, was observed previously in an isopropanol nine-day inhalation study (Burleigh-Flayer et al., 1990) and in various studies with hydrocarbon petroleum distillates, branched chain ketones, and other hydrocarbon solvents. The biological significance of this change is unknown since it has been observed only in male rats and has not been seen in female rats or other species, including man.

There were no microscopic lesions in female rats or in mice of either sex that could be attributed to the isopropanol exposures. In addition, there were no exposure-related microscopic changes noted within the central or peripheral nervous system during the neuropathologic evaluation.

Effect levels

Dose descriptor:

NOAEC

Effect level:

> 5 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No evidence of systemic effects and/or persistent neurological effects in the functional observation battery studies.

Target system / organ toxicity

Critical effects observed:

yes

Lowest effective dose / conc.:

100 ppm

System:

other: Excretory

Organ:

kidney

Treatment related:

yes

Dose response relationship:

no

Relevant for humans:

no

Any other information on results incl. tables

SUMMARY

Four groups, each containing 25 Fischer 344 rats per sex and 10 CD®-1 mice per sex, were exposed for six hours per day, five days per week, for thirteen weeks to isopropanol vapor (CAS No. 67-63-0). Target concentrations of isopropanol were 0 (control), 500, 1500, and 5000 ppm. An additional group of 10 Fischer 344 rats per sex and 10 CD®-1 mice per sex were exposed to a target concentration of 100 pprn with the same exposure regimen. Monitors for toxic effect included clinical observations, functional observational battery and motor activity (rats only), ophthalmic examination, body and organ weights, food and water consumption, hematologic and serum clinical chemistry evaluations, and macroscopic and microscopic evaluations. Detailed microscopic evaluations were performed on the nervous system of selected rats following perfusion fixation of tissues.

Mean isopropanol analytical concentrations of 100, 506, 1508 and 5008 ppm were measured. No exposure-related mortalities occurred during the study, although two mice died due to incidental causes. Clinical signs observed in some of the rats and mice during exposures at 5000 ppm included ataxia, narcosis, lack of a startle reflex, and hypoactivity. During exposures to 1500 ppm, narcosis, ataxia, and hypoactivity were observed in some mice, while only hypoactivity was observed in rats. The clinical signs noted during exposures were either seldom observed or completely absent following exposures. Clinical signs that were observed following exposures in rats of the 5000 ppm group included swollen periocular tissue (females only), perinasal encrustation (males only), ataxia (one male), and paresis (one female). Perinasal encrustation (males only) was also observed in rats of the 500 and 1500 ppm groups. Hypoactivity (one male) and ataxia (three females) were observed following exposures in mice of the 5000 ppm group. Neurobehavioral evaluations indicated no changes in the functional observational battery; however, increased motor activity for female rats in the 5000 ppm group was noted at Weeks 9 and 13.

Decreases in absolute body weight and body weight gain were observed in rats of the 5000 ppm group at the end of the first week of exposure. Absolute body weight and body weight gain were also slightly decreased in the 1500 ppm group of female rats. The decreases in body weight and body weight gain observed during the first week of exposure were, however, transient. Increased body weight and/or body weight gain were observed in male and female rats of the 1500 and 5000 ppm groups during the remaining weeks of the study. No exposure-related effects on body weight were noted in male mice; however, increased body weight and body weight gain were observed in female mice of the 5000 ppm group starting at approximately Week 3. Increases or decreases in food and water consumption generally corresponded to changes in body weight and body weight gain.

Upon hematologic evaluation at Week 6, decreases in total erythrocytes, hemoglobin, hematocrit, and platelet counts were observed in both sexes of rats of the 5000 ppm group. In addition, increases in mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) were observed in males of the 5000 ppm group. Increased MCV and/or MCH were also noted in both sexes of rats of the 5000 ppm group at Week 14. These changes indicated a slight anemia was present at Week 6 but not at Week 14. Another hematologic effect observed in male rats included increased platelet counts in the 1500 and 5000 ppm groups at Week 14. There were no exposure-related changes in serum clinical chemistry parameters for male and female rats. Various changes in hematologic and serum clinical chemistry parameters observed in female mice of the 5000 ppm group indicated a possible slight dehydration effect; there were no exposure-related changes in hematology or serum clinical chemistry for male mice.

The only organ weight effect noted was an increased relative liver weight in both sexes of rats and female mice of the 5000 ppm group. At necropsy, there were no gross lesions determined to be exposure related. Histologic examination revealed hyaline droplets within the kidneys of all male rats including controls. However, the size and frequency of the hyaline droplets were increased for the exposure groups, albeit not in a concentration-related manner. There were no microscopic lesions in female rats or in mice of either sex that could be attributed to the isopropanol exposures. Neuropathologic examination revealed no exposure-related lesions in the central or peripheral nervous system of exposed rats.

Applicant's summary and conclusion

Executive summary:

Repeated exposure to IPA for 98 days produced toxic effects only at the highest concentration (5000 ppm) and a kidney change of unknown biological signifiance. Clinical signs of toxicity on the central nervous system (including ataxia, narcosis, lack of a startle reflex, and/or hypoactivity) are acute effects and not relevant for limit value determination for repeated dose systemic effects. Decreases in absolute body weight and body weight gain, and changes in hematology parameters in animals exposed to 1500 and 5000 ppm of isopropanol, increased relative liver weight in male and female rats exposed to 5000 ppm, as well as increased motor activity for female rats in the 5000 ppm group have been observed.

Clinical signs of toxicity on the central nervous system (including ataxia, narcosis, lack of a startle reflex, and/or hypoactivity) during exposures to 1500 ppm in some mice are acute effects and not relevant for limit value determination for repeated dose systemic effects. In mice exposed to 1500 and 5000 ppm of isopropanol, increased body weight and body weight gain and in female mice of the 5000 ppm group are observed. Various changes in hematologic and serum clinical chemistry parameters are observed in female mice of the 5000 ppm group, and increased relative liver weight in female mice of the 5000 ppm group.