2-methylpropan-1-ol

Endpoint summary

Description of key information

oral
90 d, rat, drinking water: NOAEL >= ca. 1450 mg/kg bw/ day (= 16000 ppm; no effects observed; OECD 408, GLP; BG Chemie 1990)
90 d, rat, gavage: NOAEL >= 1000 mg/kg bw/ day; NOEL  =316 mg/kg bw/day due to transient clinical signs and transient body weight gain reduction (GLP; US EPA 1985)
dermal
4-6 times 0.3 mL for 24 h within 7 d, rabbit, occlusive: no systemic toxicity studied; local: highly irritant (TSCATS OTS 0510692, 1986; Val. 4)
inhalation
90 d, rat, 6 h/d, 5 d/wk: NOAEL systemic >= ca. 7.5 mg/L/day (2500 ppm); NOEL systemic = ca. 3.0 mg/L/day due to slight hematologic effects with questionable biological significance (GLP, neurotoxicity guideline, CMA 1996a)
2-gen study/ca. 17 wks for the parental generation, 6 h/d, 7 d/wk: NOAEL systemic >= ca. 7.5 mg/L/day (2500 ppm, no effects observed; GLP, EPA OPPTS 870.3800; ACC 2003)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

GLP compliance:

yes

Specific details on test material used for the study:

- Name of test material (as cited in study report): 2-Methyl-1-propanol
- Physical state: liquid
- Analytical purity: 99.8 %

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Dr. Karl Thomae GmbH
- Age at study initiation: 42 days
- Weight at study initiation: mean males: 172g; mean females: 147 g
- Housing: individually
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 6 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24 °C
- Humidity (%): 30-70 %
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: drinking water

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was weighed for each particular test group and the specific quantity of drinking water (also weighed) added. To obtain a homogeneous solution of the test substance in the drinking water the mixture was then stirred for about 30 minutes using a magnetic stirrer.
The drinking water solutions were prepared twice a week.
The drinking bottles were filled using a semi-automatic metering device (Fortuna Optifix).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical investigations were carried out to characterize the stability and homogeneity of test substances in drinking water and to verify the target concentrations. The homogeneity of the test substances was guaranteed by the high degree of purity. The stability and the homogeneity of the test substances in the drinking water over a period of 6 days were analyzed. To check the correctness of the target concentration in drinking water, a sample of each concentration was taken for analysis by capillary gas chromatography at the beginning and at the end of the application period.

Duration of treatment / exposure:

90 days

Frequency of treatment:

continuous

Dose / conc.:

80 mg/kg bw/day (nominal)

Remarks:

in water

Dose / conc.:

340 mg/kg bw/day (nominal)

Remarks:

in water

Dose / conc.:

1 450 mg/kg bw/day (nominal)

Remarks:

in water

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
In a test study n which 2-Methyl-1-propanol and a similar substance in doses of 0 and 20000 (first 2 weeks) resp. 16000 ppm (next 2 weeks) were administered to each of 3 rats/sex the following observations, covering the entire study period, were made:
- No substantial differences as to the body weight gain and feed consumption of both sexes.
- The drinking water consumption of the males (20000 ppm) had increased by about 16% around the end of week 2 of the study; at the end of the test study, after the dose had been reduced to 16000 ppm, the increase recorded was even as high as about 43% compared to control.
- After a 14-day administration of 20000 ppm, the drinking water consumption of the females had decreased by about 16%; after the concentration had been changed to 16000 ppm, the situation at the end of the study was different, as the drinking water consumption was by about 15% higher than the control value.
- The gross-pathological examinations established no differences between the control animals and the rats which received the test substance.

On the basis of the data above and to guarantee a procedure parallel to the study with a similar substance the following doses with the factor 4 were chosen for the subchronic study with administration of the test substance via the drinking water:
1000 ppm: as the no adverse effect level to be expected
4000 ppm: as the dose with marginal toxic effects or as guarantee of a higher no adverse effect level
16000 ppm: as the dose with toxic effects to be expected

Positive control:

no data

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS/DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were examined at least once daily for clinical signs and mortality. The animals were subjected once a week to an additional exact clinical examination.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded at the beginning of the study and weekly throughout the study.

FOOD CONSUMPTION:
- Time schedule for examinations: Food consumption were determined once a week for a period of 7 days.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Water consumption was determined once a week for a period of 4 days. The mean daily intake of the test substances (in mg per kg body weight) was calculated at the intervals at which water consumption was determined.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Ophthalmological examinations were carried out prior to the beginning of treatment and at the termination of the studies using an ophthalmoscope.
- Dose groups that were examined: Control animals and high dose group

HAEMATOLOGY: Yes
- Time schedule for collection of blood: blood was taken on day 87 of the study
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals: all surviving animals per test group and sex
- Parameters examined: white blood cells, red blood cells, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelets, reticulocytes, differential blood count, prothrombin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: blood was taken on day 87 of the study
- Animals fasted: No
- How many animals: all surviving animals per test group and sex
- Parameters examined:sodium, potassium, chloride, inorganic phosphate, calcium, glucose, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, serum-gamma-glutamyltransferase, urea, albumin, blood creatinine, total bilirubin, total protein, globulins, triglycerides, cholesterol

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
For post-mortem examinations at the end of the 90-day treatment period, the animals were killed, necropsied and assessed by gross pathology. The weight of the anesthetized animals and the weights of their livers, kidneys, adrenal glands and testes were determined.
HISTOPATHOLOGY: Yes
Organs or tissues required by guidelines as well as all gross lesions were fixed in a 4% formaldehyde solution. Histological examination and assessment of the findings were carried out after histotechnical processing and staining with hematoxylin and eosin.

Statistics:

Mean values and standard deviations were calculated for body weight, food and water consumption, intake of the test substances, hematological and clinical chemistry parameters as well as for absolute and relative organ weights. The organ weights were statistically evaluated using the DUNNETT's test for comparison of the dose groups with the control groups. The analysis of variance (ANOVA) with subsequent DUNNETT's test was used to compare the body weights as well as the hematological and clinical biochemistry data of the dose groups with those of the control groups.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

All animals participating in the study were essentially free from clinical signs. Only one animal (4000 ppm) showed reddish smear on one eye and another animal (16000 ppm) were found to have an increased urine section, an increased drinking water consumption and at palpation apparently enlarged kidneys. These effects were assessed as being not substance-induced, i.e. incidental and spontaneous in nature.

Description (incidence):

After 42 days of the study one animal (0 ppm, male) was found dead in the cage. Otherwise no further animal died intercurrently during the entire study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The body weight gain of all dosed animals of both sexes was, within the biological limits, analogous to the controls.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

The males of test group 1 (1000 ppm) showed in the course of the second half of the study a marginal increase in the feed consumption values being significant only in some cases. The females of the same group showed throughout the study isolated cases where the feed consumption was slightly increased. Apart from these findings there were no effects on the feed consumption of the males and females of test groups 2 (4000 ppm) and 3 (16000 ppm).
In respect to the isolated occurrence and the lack of a dose-response relationship, the increase in the feed consumption is assessed as being incidental in nature.

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

effects observed, non-treatment-related

Description (incidence and severity):

Throughout the course of the study, an increase in the drinking water consumption of males and females of test groups 1 (1000 ppm), 2 (4000 ppm) and 3 (16000 ppm) was observed sporadically. It varied within the test groups, showed no clear dose-response relationship and was only evident in few animals over the whole study period.
In respect to this, the sporadically increased drinking water consumption was assessed as being not substance induced.
The amount of test substance intake (in mg) consumed each day by the animals per kilogram body weight was calculated at the times at which the drinking water consumption was also determined.
males (1000 ppm): ca. 75 mg/kg bw/day; females (1000 ppm): ca. 91 mg/kg bw/day
males (4000 ppm): ca. 300 mg/kg bw/day; females (4000 ppm): ca. 385 mg/kg bw/day
males (16000 ppm): ca. 1251 mg/kg bw/day; females (16000 ppm): ca. 1657 mg/kg bw/day

Ophthalmological findings:

no effects observed

Description (incidence and severity):

The ophthalmological examinations carried out before the beginning of administration and towards the end of the study using a hand-held slit lamp revealed no substance induced impairment of the refracting media.
All examinations revealed no differences between treated and untreated animals.

Haematological findings:

no effects observed

Description (incidence and severity):

The oral administration of 2-Methyl-1-propanol in doses of 1000; 4000 and 16000 ppm via the drinking water for 3 months to male and female rats caused no changes related to the test substance either in the clinicochemical or in the hematological examinations.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

The mean absolute and relative testes weights of the animals of dose group 3 (16000 ppm) were slightly decreased when compared with the corresponding control group and they were below the mean weights of 13 historical control studies. However, this weight decrease was not statistically significant. The individual absolute and relative testes weights of the two animals with gross lesions were far below those of all other treated and untreated animals of the study.The mean absolute and relative testes weights of the remaining 8 animals of dose group 3 (16000 ppm) were comparable to the controls and to those of the historical control data of 14 comparable studies.

Description (incidence and severity):

The gross pathology revealed that 2 animals in group 3 (16000 ppm) had small testes, and this was marked and moderate for the animals respectively.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

The histopathology of these testes revealed diffuse tubular degeneration and diffuse hyperplasia of Leydig's cells. Both these changes were moderate and slight in the animals respectively. The testis of 1 control animal was found to have slight focal tubular degeneration. It appears unlikely that this is an effect of the test substance, especially since the testis weight s of the animals in group 3 (16000 ppm) were not statistically significantly different from those in the control group.

No other alterations of the testicles were noted in any of the other treated or control animals histopathologically.

A minimal increase in extramedullary hematopoiesis was noticed in the spleen of 5 male control animals and 4 males in group 3 (16000 ppm). The same was found in 1 female control animal and 5 females in group 3 (16000 ppm). However, this cluster of findings in the females in group 3 is not regarded as related to the test substance, especially since there was no such clustering in the males in group 3 and there were no corresponding changes found in the hematology.

1 female in group 3 (16000 ppm) had very high kidney weights (10.95 g). The gross pathology of both kidneys revealed several cysts with a diameter up to 15 mm. The histopathology of these kidneys showed extreme dilation of the renal pelvis. These changes are an incidental finding, and an effect of the test substance can be ruled out.

All the other histopathological changes are regarded as unrelated to the test substance.

1 male control animal died prematurely. Marked central necrosis 41 was found in the liver of this animal.

Key result

Dose descriptor:

NOAEL

Effect level:

> 1 450 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: original value: 16000 ppm; no effects observed

Key result

Critical effects observed:

no

With respect to the results obtained, it appears rather unlikely that the testicular findings of two animals of dose group 3 (16000 ppm) represent an effect of the test substance. The spontaneous origin of the observed tubular degeneration is supported by the fact that focal tubular degeneration was also noted in one control male whereas no precursor lesions were observed in any of the other treated animals of the high dose group. Moreover, neither the reduced absolute or relative testes weights of this group were statistically significantly altered nor did the mean values of the remaining grossly and histopathologically unaffected 8 animals of this group show any biologically relevant deviation from the controls.

Reference 2

Endpoint:

sub-chronic toxicity: oral

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Principles of method if other than guideline:

The purpose of this study was to evaluate the toxicity of isobutyl alcohol in a rat subchronic toxicity (90 days) study. In order to assess the toxicologic potential after both 1 month and 3 months of dosing, an interim sacrifice at the 1 month interval was included.

GLP compliance:

yes

Specific details on test material used for the study:

- Name of test material (as cited in study report): Isobutyl alcohol

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc.
- Age at study initiation: 22-23 days upon receipt
- Weight at study initiation: 45-55 g upon receipt
- Housing: individually
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/- 2 °C
- Humidity (%): 43 +/- 5 %
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Fresh solutions of the compound (in deionized water) were prepared weekly and dosed orally at a volume of 10 ml/kg.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of each dose concentration were saved during weeks 1, 3, 8, 9 and 13 and taken to the Muskegon County Wastewater Treatment Facility for chemical analysis at the laboratory under the direction of Dr . Avi Joshi, Physical Chemist. Duplicate samples were sent to Mr . John Maney, ERCO, A Division of Enseco, Inc ., 185 Alewife Brook Parkway, Cambridge, Massachusetts for referee analysis. No further information were given in the report.

Duration of treatment / exposure:

90 days

Frequency of treatment:

daily

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Dose / conc.:

316 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

30 (plus an additonal control group with 10 animals/sex)

Control animals:

yes

Positive control:

no data

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS/DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: The rats were observed at least twice daily 7 days a week for mortality and clinical effects.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded weekly.


FOOD CONSUMPTION:
- Time schedule for examinations: Food consumptions were recorded weekly


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: All rats received an ophthalmoscopic examination during the pretreatment period and week 13 by a veterinary ophthalmologist. Ophthalmologic examinations were conducted on all rats in a darkened room with an indirect ophthalmoscope. Six rats that had an eye lesion during the pretreatment period were not included in the study.
- Dose groups that were examined: all animals


HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were collected prior to initiation of dosing from the 10 male and 10 female rats in the additional control group, from all surviving rats scheduled for the interim sacrifice during week 4 or 5 and from the first 10 (numerically) surviving males and first ten surviving females from each group at the final sacrifice (week 13 or 14)
- Anaesthetic used for blood collection: Yes (CO2, the animals were sacrificed)
- Animals fasted: No data
- How many animals: 10/sex/dose
- Parameters examined: hemoglobin (HGB) hematocrit (PCV), erythrocyte count (RBC), mean cell volume (MCV), mean cell hemoglobin (MCH), mean cell hemoglobin concentration (MCHC) , total and differential leucocyte counts (WBC), estimated platelet count (PLT).


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were collected prior to initiation of dosing from the 10 male and 10 female rats in the additional control group, from all surviving rats scheduled for the interim sacrifice during week 4 or 5 and from the first 10 (numerically) surviving males and
first ten surviving females from each group at the final sacrifice (week 13 or 14)
- Animals fasted: No data
- How many animals: 10/sex/dose
- Parameters examined: alkaline phosphatase (Alk phos), blood urea nitrogen (BUN), glutamate pyruvate transaminase (SGPT), glutamate oxaloacetate transaminase (SGOT) , glucose (Gluc), total protein (TP) , albumin (Alb) , A/G ratio (calculated), globulin (calculated), total bilirubin (Tot. bili.), sodium (Na), pottssium (K), chloride (Cl) Calcium (Ca), creatinine, inorganic phosphate (phos.), total carbon dioxide (TCO2), total serum cholesterol (Chol).


URINALYSIS: Yes / No / No data
- Time schedule for collection of urine: Urine samples were collected prior to initiation of dosing from the 10 male and 10 female rats in the additional control group, from all surviving rats scheduled for the interim sacrifice during week 4 or 5 and from the first 10 (numerically) surviving males and first ten surviving females from each group at the final sacrifice (week 13 or 14)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No data
- Parameters examined: pH, specific gravity, glucose, protein, ketones, bilirubin, urobilinogen, microscopy of sediment

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
A necropsy was performed on the surviving rats of the first 10 males and 10 females from each dose group on day 29 or 30 of the study. On days 92 or 93 the remaining rats were sacrificed. Rats found dead were necropsied immediately after they were found. At the interim and final sacrifices, rats were anesthetized with CO2 and exsanguinated by cardiac puncture. The cranial, thoracic and peritoneal cavities were opened and the contents examined macroscopically. Organs and tissues were removed from each animal and preserved. Lungs of rats at the scheduled sacrifice were inflated with formalin via the trachea. Eyes with attached optic nerve from all rats killed at the interim and final sacrifices were preserved in a modified Zenker's fixative. The testes with attached epididymides of all male rats were preserved in Bouin's fixative. All other tissues were preserved in 10% neutral-buffered formalin. Feet were preserved with the tissues for positive identification of the rat. At the interim and final sacrifices, the following organs were weighed: brain, heart, liver, spleen, kidneys, testes with epididymides, and ovaries. Adrenals and thyroids with parathyroids were weighed at the final sacrifice. For paired organs, the organ weight was the combined weight of right and left members of the pair. Organ/body weight ratios were determined for each tissue. No organ weights were taken on rats found dead.

HISTOPATHOLOGY: Yes
The full tissue microscopic examination was done on the control and high-dose rats and on those dying on study or sacrificed in extremis. Also, livers, hearts, and kidneys of low- and mid-dose rats, and all gross lesions seen at necropsy were examined microscopically.
Tissues examined:
Heart and attached aorta (a longitudinal section); thymus; lung (sections from the left and caudal lobes); trachea (a cross-section); esophagus (a cross-section); stomach (a section from the nonglandular esophageal areathrough the area of the cardiac glands into the area of the fundic glands and another section from the duodenum through the pyloric sphincter into the area of the pyloric glands); salivary gland (sections of the sublingual and mandibular glands); small intestines (a separate cross-section of the duodenum, jejunum and ileum); colon (a cross-section); liver (sections from the left and right lobes); pancreas (a separate section in addition to sections commonly attached to the viscera); spleen (a cross-section); mesenteric lymph node; kidney (a cross-section of the right kidney); urinary bladder (an entire cross-section); adrenal (section through the cortex and medulla of one adrenal); pituitary; eye (with attached optic nerve); thyroids and parathyroids; thoracic spinal cord (a cross-section); lumbar spinal cord (a cross-section); brain (three sections including front cortex and basal ganglia, parietal cortex and thalamus and cerebellum and pons); femur with marrow; testis and epididymis (a cross-section of each); ovary; uterus (a cross-section of one uterine horn); cervix (a longitudinal section with uterine horns); skin; mammary gland; skeletal muscle (thigh); sciatic nerve; tissue masses and all other gross lesions.

As tissues were trimmed, the presence or absence of tissues and lesions was noted. The tissues were placed in Tissue Tek cassettes that were labeled with study number, rat number and cassette number. They were then processed on a Fisher Scientific Histomatic, or an AO TP/8000. After processing, the tissues were embedded in paraffin using a Tissue Tek embedding system. They were sectioned at 5-6 microns, mounted on numbered slides and stained with hematoxylin and eosin.

Statistics:

The body weight, food consumption, clinicopathologic, and organ weight data were tested for homogeneity of variance by Bartlett's method. If the data were found to be homogeneous, differences between control and treatment means were tested for statistical significance by the method of Dunnett. If the data were found not to be homogeneous, the method of Gill (modified Dunnett's) was employed.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Treatment related clinical signs were restricted to the high-dose group and included hypoactivity, ataxia, and salivation. Labored respiration and rales appeared to be related to treatment during week 1 but they were generally observed thereafter as agonal signs, along with prostration, hypothermia, and emaciation. Hypoactivity was the most frequently observed clinical sign. It occurred in every rat in the 1000 mg/kg/day dose group during week 1. However, by week 4, hypoactivity was seen in only 16 of 55 rats at the 1000 mg/kg/day dose and it occurred only sporadically thereafter. Onset was generally within a few minutes after dosing and duration was approximately ten minutes. Ataxia occurred at a low incidence throughout the study. The onset and duration were similar to that of hypoactivity. Salivation also occurred soon after dosing for a short period of time. It is frequently present in studies where a high concentration of a dosing solution is administered by gavage and is usually a local effect of the compound. During week 13, a control male injured its nasal region on a cage or feeder. This resulted in a swollen nasal region, labored respiration, a urine wet abdomen, and hypoactivity. Another control male developed a corneal opacity during week 2 and this remained throughout the study. The lacrimation, hypoactivity, labored respiration, and/or emaciation present in a control male during week 1 and in a female in the 100 mg/kglday dose group during weeks 1 and 2 were seen prior to the non-treatment related death of these rats.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

The mortality rate was 1/60, 1/60, 2/60, and 11/60 for the control, 100, 316, and 1000 mg/kg/day dose groups, respectively. The mortality data will be evaluated in the discussion section in relation to the gross necropsy and histologic findings.

Description (incidence and severity):

Compound administration had only a minor effect on body weight gain and food consumption. The only effect on weight gain was during week 1 when the males of the 1000 mg/kg/day dose group gained 18 % less than the controls. For the first 2 weeks of the study, food consumption averages of the 1000 mg/kg/day dose group males and females were significantly lower than controls. Thereafter, food consumption for the 1000 mg/kg/day females was generally similar to the controls but food consumption for the 1000 mg/kg/day males tended to be slightly lower than the controls.
In general, group mean body weight and food consumption values for the 100 and 316 mg/kg/day groups were similar to the controls. During week 2, body weight gain of females in the 316 mg/kg/day dose group was significantly higher than the control group and the food consumption of the females in the 100 and 316 mg/kg/day dose groups during week 4 was significantly higher than those of the controls. However, these statistically significant differences were small , occurred only at these times, and are considered not to be of toxicologic significance.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No treatment-related effect was found. The pathology observed was considered to be within normal limits for the age, sexes, and strain.

Description (incidence and severity):

There was no definite treatment- related effect on clinical pathologic parameters. However, the possibility of treatment-related effects was suggested by differences between control and treatment group mean serum potassium, albumin, and total protein concentrations. Group mean serum potassium concentration for males and females in the 1000 mg/kg/day group was 11-15% lower than the control group means at the interim evaluation, but group mean serum potassium concentrations were similar for treated and control groups at the final clinical pathologic evaluation.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Group mean albumin (and hence, total protein) concentrations were slightly lower than control group means for females of the 316 mg/kg/day dose group (7 %) and females of the 1000 mg/kg/day dose group (9 %) at the final evaluation. This appeared to be a result of higher than expected values in the control group rather than abnormally low values in the treated groups. At the interim evaluation (days 29 and 30), albumin and total protein concentrations were similar for control and treated groups of males and females. At the final evaluation (days 92 and 93) group mean albumin concentration was 3-5 % higher than at the interim evaluation for control and treated males and 5-8% in treated females, while the albumin concentration in control females increased 15 %. A similar situution existed for the total protein averages. Other statistically significant differences between control and treated groups were present, but they were small, occurred at one dose level, in one sex, and at one evaluation only. Thus, they were considered not to be treatment-related.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related effect on organ weight values was observed. No trends were apparent and no statistically significant differences in absolute organ weight values were present at the interim or the final sacrifices. A few statistically significant differences between control and treated group mean relative organ weight values were noted. However, these differences were small (8-17 %), occurred in only one sex, and at one dose level, and can be explained by the slight differences in terminal body weights.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The most frequently observed lesions among the rats that were found dead were of the thoracic cavity. White or tan material in the thoracic cavity and/or adhesions between the organs and tissues of the thoracic cavity (eg., heart, lungs, diaphragm, rib cage) were observed in two rats (a control group male and a 100 mg/kg/day female). In addition an esophageal tear was visable grossly in the female.
One rat at the 1000 mg/kg/day dose also had a torn esophagus, plus had a clear fluid in the thoracic cavity. These lesions indicated that the esophagi had been ruptured during dosing. Dark areas in the lungs were present in one female from the 316 mg/kg/day group and in two males and a female from the 1000 mg/kg/day dose group. Other lesions present in the rats found dead and in rats necropsied at the interim-sacrifice were lesions commonly seen in laboratory rats. At the final sacrifice, lung lesions and enlarged and or dark lymph nodes were the most frequently observed lesion. Their distribution between the control and treatment groups was not indicative of a treatment related effect. The enlarged uterine horns appeared to be a reflection of the estrual cycle.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related lesion was seen from the rats at the interim or final sacrifice or from the rats found dead during the study. Lesions were seen in the tissues from the thoracic cavity of all 15 rats which were found dead. Tracheal inflammation and/or necrosis were seen in 8 rats-3 males and 5 females of the 1000 mg/kg/day dose group, and tracheal inflammation without necrosis was seen in 2 females of the 316 mg/kg/day dose group. The lesions were characterized by necrosis of the tracheal epithelium and inflammatory exudate consisting of mixed inflammatory cells and in some cases a prominent fibrinopurulent exudate. Also, hemorrhage, pleuritis, bronchitis, bronchopneumonia, congestion, and/or edema were seen in the lungs of these rats. Pyknosis or hemorrhage was seen in the thymus of 4 of these rats and inflammation was seen in the heart of 1 rat. These lesions were considered to be due to aspiration of the compound and/or its vapors at the time of dosing and were considered not to be directly related to treatment.
Four rats were determined to have died from a perforation of the esophagus (incurred during dosing). Lesions related to the cause of death seen in the thoracic cavity of these rats were: perforation and/or inflammation of the esophagus and/or the lungs, edema of the lungs, presence of food particles and bacteria in the lungs and/or thoracic cavity, pyknosis and/or inflammation of the thymus, and pericarditis and bacteria in the heart.
These lesions were considered not to be directly related to treatment. The cause of death of one rat from the 1000 mg/kg/day dose group that was found dead could not be ascertained but edema was present in the lung. Fifteen rats from the final sacrifice had pneumonia which did not affect their clinical health and was more common in controls than in treated rats. Agonal hemorrhages (red spots grossly) were seen in the lungs of eleven rats from the final sacrifice. These are related to the euthanasia and are of no clinical or toxicological significance.
One rat in the 1000 mg/kg/day dose group had fibrinopurulent tracheitis. It is ascribed to a regurgitation and aspiration of the isobutyl alcohol. The chronic focal myositis in 2 males of the 1000 mg/kg/day dose group at the final sacrifice probably is due to the dosing process.
All other lesions seen at the interim and final sacrifices were of low incidence and severity, common in these laboratory animals, and unrelated to the treatment.

Key result

Dose descriptor:

NOEL

Effect level:

316 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Clinical signs and effects on body weight and food consumption were transient and were considered as adaptive and therefore as not adverse.

The mortality rate was 1/60, 1/60, 2/60, and 11/60 for the control 100, 316, and 1000 mg/kg/day dose groups, respectively. These rates indicate a treatment-related increase in mortality at the 1000 mg/kg/day dose level. However, histological evaluation revealed that four of the fifteen rats died of an esophageal rupture and another ten apparently died from aspiration of the compound and/or its vapors into the trachea. Thus, these deaths were not due to a systemic toxicity of orally administered isobutyl alcohol.

During dosing on the first day, the dosing solution was observed coming up into the mouths of some of the rats. In addition, two rats at the 316 mg/kg/day dose level and one at the 1000 mg/kg/day dose level died shortly after dosing. On day 1, an 18 gauge, 3 inch, ball tip (214 mm) metal dosing cannula had been used for compound administration.

On day 2, a number 8 French rubber catheter was placed over the metal cannula.

This change increased the diameter of the dosing device and markedly decreased the incidence of compound flowing from the stomach anteriorly around the dosing device and into the oral cavity. However, it may have increased esophageal injury as three of the four rats (one control, one low-, and one high-dose) that died of esophageal perforation did so early in the study (days 3, 4, and 8).

This is a well recognized entity and is not uncommon early in a study, since the esophagus is smaller at a younger age. The cause of death of another ten rats was acute tracheitis (or bronchial hemorrhage), which was apparently due to the regurgitation and subsequent aspiration of the isobutyl alcohol and/or its vapors into the trachea (and probably the nasal cavity). The acute lesions of the trachea (inflammation and/or necrosis) seen in the histological examination support this theory. Five of these ten rats died in the first week of the study, so, the large diameter dosing cannula alleviated, but did not completely prevent, the problem.

Two alterations occurred in clinical pathologic parameters that suggested a treatment related effect because they were present in both sexes of the high-dose group or in one sex at the mid and high-dose group. At the interim (day 29 and 30) evaluation, serum potassium concentrations in the high-dose males and females were slightly (11-15%) lower than control averages. At the final (day 92 and 93) evaluation serum potassium averages were similar for control and treated groups. Thus, even if the lower potassium concentrations were real, they were small and transitory.

Group mean albumin (and hence, total protein) in females of the 316 and 1000 mg/kg/day dose groups were 7% and 9% lower (respectively)than the control average group means at the final evaluation only.

This appeared to be an artefact produced by a larger than expected age related increase in the group mean albumin level of the control females. Moreover, if a toxicologically significant alteration in kidney excretion or liver synthesis of albumin (protein) existed, histological lesions would be expected in these organs. Histopathologic evaluation of the kidneys and livers did not reveal a treatment related lesion. Therefore, the alterations in albumin and total protein did not appear to be treatment-related.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 450 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

repeated dose toxicity: inhalation

Remarks:

other: reproduction

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3800 (Reproduction and Fertility Effects)

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

- Name of test material (as cited in study report): Isobutanol
- Physical state: liquid
- Analytical purity: 99.9 %
- Stability under test conditions: the test substance was proven stable under the storage conditions
- Storage condition of test material: room temperature

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Inc.
- Age at study initiation: (P) ca. 7 wks; (F1) ca. 4 wks
- Weight at study initiation: (P) Males: 236-350 g; Females: 159-213 g; (F1) Males: post natal day 32: means: 84-97 g; Females: post natal day 32: means: 78-88 g
- Housing: individually
- Diet: ad libitum (no food during exposure)
- Water: ad libitum (no water during exposure)
- Acclimation period: 21 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/-3 °C
- Humidity (%): 30-70 %
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

air

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
Exposures were conducted in four 2.0 m3 stainless steel and glass whole-body inhalation chambers. One chamber was dedicated for each group for the duration of the study. Chamber supply air was provided from a HEPA- and charcoal-filtered, temperature- and humidity-controlled source. Treatment of exhaust air consisted of charcoal- and HEPA filtration.

The generation system was operated as follows: Vapors of the test article were generated using a heated bead column. The test article was introduced to the top of the column, while nitrogen entering the bottom of the column served as the carrier gas. For Groups 2 and 3 the column was 2.4 cm in diameter (ID), 40 cm long filled with 2, 3 and 4 mm beads. For Group 4, the column was 5 cm in diameter (ID) and 68 cm long filled with 3, 6, 8 and 12-mm beads. The columns were wrapped with heating tapes (Omega Engineering). Temperatures were set to approximately 100°C for chambers 2 and 3, and approximately 170-185ºC for chamber 4 using an Omega CN370 temperature controller. The chamber 4 temperature was significantly higher due to the need to maintain an internal temperature of at least 70ºC to ensure vaporization of the test article. The test article was vaporized as it dripped from 1/16-inch Teflon tubing onto the glass beads contained within each heated column.
The test article was metered from an amber glass reservoir to the column using an FMI pump (Fluid Metering, Inc., Oyster Bay, New York). Calibrated FMI pumps included 2 model no. QG-6 pumps with a 1/4-inch piston for chamber 2, and a 3/8-inch piston for chamber 3 and a model no. QG-20 pump with a 1/4-inch piston for chamber 4.
Vaporization nitrogen was delivered from the facility nitrogen generation system and was controlled using calibrated flowmeters (Gilmont Instruments, Barrington, Illinois). The vaporization nitrogen carried the isobutanol vapor through Teflon delivery lines (3/8-inch O.D. tubing for chambers 2 and 3, 1-inch O.D. tubing for chamber 4) to the chamber inlet where the concentration was reduced to the desired level with chamber ventilation air. The animal exposure was initiated by switching the FMI pumps and the compressed nitrogen on simultaneously.

TEST ATMOSPHERE
- Brief description of analytical method used: Exposure concentrations within each chamber were measured 9 to 10 times (approximately every 35 minutes) during each daily exposure period by a validated gas chromatographic method. At least one standard was analyzed each day prior to exposure to confirm gas chromatographic calibration. Chamber temperature, relative humidity, ventilation rate, and negative pressure within the chambers were monitored continuously and were recorded approximately every 35 minutes. Oxygen content within the chamber was measured during the pre-study method development phase. Nominal chamber concentrations were determined daily. Total air volume was calculated by multiplying mean chamber ventilation rate (in liters per minute) by the exposure duration (in minutes). Test atmosphere homogeneity was demonstrated during pre-study method development. There were no detectable aerosols at any evaluation interval.
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Exposure concentrations within each chamber were measured 9 to 10 times (approximately every 35 minutes) during each daily exposure period by a validated gas chromatographic method.

Duration of treatment / exposure:

for the F0/P generation: approx. 17 weeks (beginning at least 70 days before mating until postweaning)

Frequency of treatment:

daily for 6 hours per day

Dose / conc.:

500 ppm

Dose / conc.:

1 000 ppm

Dose / conc.:

2 500 ppm

No. of animals per sex per dose:

30

Control animals:

yes

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS/DETAILED CLINICAL OBSERVATIONS
Detailed physical examinations were recorded weekly for all parental animals throughout the study period. All animals were observed twice daily for moribundity and mortality; in addition, the animals were observed for appearance, behavior and pharmacotoxic signs within one hour after completion of exposure. Females expected to deliver were also observed twice daily during the period of expected parturition and at parturition for dystocia (prolonged labor, delayed labor) or other difficulties.

BODY WEIGHT
Time schedule for examinations: Individual F0 and F1 male body weights were recorded on study days 0, 1, 4 and 7, weekly thereafter throughout the study, and prior to the scheduled necropsy. Individual F0 and F1 female body weights were recorded on study days 0, 1, 4 and 7 and weekly thereafter until evidence of copulation was observed. Mean weekly body weights and body weight changes are presented for each interval. Once evidence of mating was observed, female body weights were recorded on gestation days 0, 4, 7, 11, 14 and 20 and on lactation days 1, 4, 7, 14 and 21; for the F0 females, body weights were also recorded on lactation day 28. Body weight changes are presented for each of these intervals. After weaning (lactation day 28), weekly body weights were recorded for these F0 females until the scheduled necropsy.

FOOD CONSUMPTION
Individual F0 and F1 male and female food consumption was measured on study days 0, 1, 4 and 7 and weekly thereafter until pairing. Food intake was not recorded during the mating period. Male food consumption was measured after mating on a weekly basis until the scheduled necropsy. Female food consumption was recorded on gestation days 0, 4, 7, 11, 14 and 20 and lactation days 1, 4, 7, 14 and 21; for the F0 females, food consumption was also recorded on lactation day 28. For the F0 generation, the last scheduled interval for weekly recording of food consumption was study day 126. Since final body weights and clinical observations were recorded on the scheduled necropsy days (after study day 126), food consumption was manually recorded at that time. These data are not presented in the report tables, but will be maintained in the raw data. Food consumption was calculated and reported as g/animal/day and g/kg/day for the corresponding body weight change intervals. Food efficiency (body weight gained as a percentage of food consumed) was also calculated and reported for these intervals.

Sacrifice and pathology:

SACRIFICE
- Male animals: All surviving adults were euthanized following the selection of the F1 generation and completion of a detailed clinical observation.
- Maternal animals: All surviving adults were euthanized following the selection of the F1 generation and completion of a detailed clinical observation.

GROSS NECROPSY
The necropsy included examination of the external surface, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, and the thoracic, abdominal and pelvic cavities including viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
organs collected: Adrenals (2); Aorta; Bone with marrow (sternebrae); Brain (forebrain, midbrain, hindbrain); Coagulating gland; Eyes with optic nerve (2); Gastrointestinal tract (Esophagus, Stomach, Duodenum, Jejunum, Ileum, Cecum, Colon, Rectum); Heart; Kidneys (2); Liver (sections of two lobes); Lungs (including bronchi, fixed by inflation with fixative); Lymph node (mesenteric); Ovaries and oviducts (2); Pancreas; Peripheral nerve (sciatic); Pituitary; Prostate; Salivary gland [submandibular (2)]; Seminal vesicles (2); Skeletal muscle (rectus femoris); Skin with mammary gland; Spinal cord (cervical); Spleen; Testes with epididymidesa (the right testis and epididymis were fixed in Bouin's solution) and vas deferens; Thymus; Thyroids [with parathyroids, if present (2)]; Trachea; Urinary bladder; Uterus with cervix and vagina; All gross lesions
Organ weights: Adrenals; Brain; Epididymides (total and caudal; these paired organs were weighed seperately); Kidneys; Liver; Ovaries; Pituitary; Prostate; Seminal vesicles with coagulating glands (with accessory fluids), Spleen, Testes (these paired organs were weighed seperately), Thymus gland; Uterus with oviducts and cervix
Organs examined: Adrenal glands: cortex and medulla; Brain; Cervix; Epididymis (right): caput, corpus and cauda; Kidneys; Liver; Ovaries; Pituitary, Prostate; Seminal vesivles with coagulatin glands (with accessory fluids); Spleen; Testis (right); Thymus, Uterus (with oviducts); Vagina; All gross (internal) lesions

Statistics:

see chapter Toxicity on Reproduction

Clinical signs:

no effects observed

Description (incidence and severity):

PARENTAL ANIMALS
After 91 days of exposure to the test article, female no. 74805 in the 500 ppm group had pale eyes and ears and appeared to be having difficulty during parturition (gestation day 23) and was euthanized in extremis on lactation day 0. This female delivered 12 pups and had four pups retained in utero. On the day of euthanasia, this female had pale ears and eyes and red discharge from the vagina. Microscopically, the cause of death for this female was determined to be severe acute renal tubular necrosis and moderate acute hepatic necrosis. Therefore, this death was not attributed to the test article; no evidence of dystocia was observed in females in the 1000 or 2500 ppm groups. All other animals survived to the scheduled necropsy.
There were no test article-related clinical observations at the weekly detailed physical examinations or one hour following exposure. The response to novel stimulus was similar in all groups, including the control group.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

PARENTAL ANIMALS
Mean F0 body weights, body weight gains and cumulative body weight gains in the 500,
1000 and 2500 ppm groups were unaffected by exposure to the test article during the premating period and one week following weaning (females) and throughout the study (males). The only statistically significant (p<0.05) difference from the control group was
a decreased mean body weight gain in the 1000 ppm group males during study days 84-91. A similar reduction was not observed in the 2500 ppm group; therefore, this transient decrease was not attributed to the test article. No test article-related effects on maternal body weights or body weight gains were observed in the test article-exposed groups during the F0 gestation period. Differences from the control group were slight and were not statistically significant.
No test article-related effects on F0 lactation body weights or body weight gains were observed in the test article-exposed groups. The only statistically significant (p<0.01) differences from the control group were a mean body weight loss of 33 grams in the 500 ppm group compared to a loss of 53 grams in the control group during lactation days 21-28, resulting in a statistically significant (p<0.05 or p<0.01) increase in mean body weight on lactation day 28 and in mean body weight gain during the entire lactation period (days 1-28). Since the control group lost more weight than the 500 ppm group, this difference was not considered to be an adverse change.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

PARENTAL ANIMALS
Food consumption, evaluated as g/animal/day and g/kg/day, and food efficiency in the 500, 1000 and 2500 ppm groups were unaffected by exposure to the test article during the pre-mating period and one week following weaning (females) and throughout the study (males). Occasional statistically significant (p<0.05 or p<0.01) reductions in food consumption (primarily g/kg/day) were observed in the 2500 ppm group males compared to the control group values during study days 14-21 through 112-119. Food consumption (g/animal/day) in these males was generally not affected during these intervals. Mean body weights and food efficiency in the 2500 ppm group males and females were similar to or greater than the control values. Therefore, these sporadic reductions were not attributed to exposure to the test article. The only other statistically significant differences (p<0.05 or p<0.01) from the control group were a decrease in food consumption (g/animal/day) in the 2500 ppm group males during study days 28-35, an increase in food consumption (g/kg/day) during day 0-1 in the 1000 ppm group males and a decrease in food efficiency in the 1000 ppm group males during study days 1-4. Similar differences were not observed in the 2500 ppm group males during this interval; therefore, no relationship to exposure was evident.
F0 maternal food consumption and food efficiency during gestation were unaffected by test article exposure in the 500, 1000 and 2500 ppm groups. The only statistically significant difference from the control group was a slight reduction in food consumption (g/kg/day) in the 2500 ppm group when the entire gestation period (days 0-20) was evaluated. Food efficiency and gestation body weight gain in this group was unaffected during this interval. Therefore, the reduction was not attributed to test article exposure.
F0 maternal food consumption and food efficiency in the test article-exposed groups were similar to the control group values throughout lactation. The only statistically significant (p<0.01) differences were a smaller decrement in food efficiency in the 500 ppm group during lactation days 21-28 than the control group and an increase in food efficiency in this group during the entire lactation period (days 1-28). Because similar effects were not observed at higher exposure levels, these differences were not considered test article related.

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

PARENTAL ANIMALS
No test article-related effects on organ weights were observed in the 500, 1000 and 2500 ppm group males or females. The only statistically significant (p<0.05 or p<0.01) differences from the control group were increases in mean absolute prostate weights in the 1000 ppm group males and mean relative prostate weights in the 500 and 1000 ppm group males and a decrease in mean absolute pituitary weight in the 500 ppm group females. Because similar effects were not observed in the 2500 ppm group males and females, these changes were not attributed to the test article.

Gross pathological findings:

no effects observed

Description (incidence and severity):

PARENTAL ANIMALS
Female no. 74805 in the 500 ppm group was euthanized on lactation day 0 due to difficulties during parturition. This female delivered 12 pups and had four fetuses with no apparent malformations retained in utero. This animal had dark red contents in the ileum and jejunum and a pale pituitary gland. Similar findings were not observed in females at the scheduled necropsy in the higher exposure groups. Therefore, these findings were not attributed to the test article.
At the scheduled necropsy, no exposure-related trends were observed in the macroscopic findings noted in the test article-exposed groups. Findings were observed similarly in control group animals, were noted infrequently and/or did not occur in an exposure-related manner. The mean number of implantation sites and the mean number of unaccounted sites in the F0 test article-exposed females were similar to the control group values; no statistically significant differences were observed.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

PARENTAL ANIMALS
Female no. 74805 was euthanized in extremis during parturition. Microscopic findings for this female included severe acute renal tubular necrosis, moderate acute hepatic necrosis, lymphoid depletion of the spleen and lymphoid necrosis of the thymus. The cause of the moribund condition of this female was renal and liver necrosis with multiple organ failure.
At the scheduled necropsy, no test article-related microscopic findings were observed including for animals that failed to breed or produce a litter. The only statistically significant (p<0.05) differences from the control group values were an increase in the incidence of hydronephrosis in the 500 ppm group females and a decrease in the incidence of dilatation of the uterine lumen in the 2500 ppm group females. A slightly increased incidence (not statistically significant) of basophilic tubules was observed in the kidneys of the 2500 ppm group males; however, the severity of this lesion was minimal, and a similar increase was not observed in the 2500 ppm group females. Therefore, this common, spontaneous alteration was not considered to be test article-related. Other microscopic findings were typical of spontaneous conditions in young rats.

Histopathological findings: neoplastic:

no effects observed

Key result

Dose descriptor:

NOAEL

Remarks:

parental, systemic

Effect level:

>= 7.5 mg/L air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: original value: 2500 ppm; no effects observed

Key result

Critical effects observed:

no

Reference 2

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: Guideline: 82-7, Subdivision F (Neurotoxicity Screening Battery)

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

see chapter Neurotoxicity

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

see chapter Neurotoxicity

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

air

Details on inhalation exposure:

see chapter Neurotoxicity

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

see chapter Neurotoxicity

Duration of treatment / exposure:

3 months (102 days

Frequency of treatment:

6 hours/day, 5 days/week

Dose / conc.:

250 ppm (analytical)

Dose / conc.:

1 000 ppm (analytical)

Dose / conc.:

2 500 ppm (analytical)

No. of animals per sex per dose:

controls and high dose: 20/sex
low and mid dose: 10/sex

Control animals:

yes

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS/DETAILED CLINICAL OBSERVATIONS
- Checks for Mortality, Moribundity and Noteworthy Signs of Toxicity: Twice daily (AM and PM)
- Observations During Exposures: Rats were observed for signs of toxicity once during the last hour of each exposure. This included brushing and tapping the exposure chambers' exteriors.
- Observations After Exposure: Animals were observed for abnormal clinical signs as they were removed from their exposure cages and returned to their home cages.
- Detailed Observations for Signs of Toxicity (including palpation for masses): Once weekly

BODY WEIGHT
Time schedule for examinations: Once weekly, (Body weights were also taken as part of the FOB on a monthily basis).

FOOD CONSUMPTION
-Time schedule: Once weekly

OPHTHALMOSCOPIC EXAMINATION
- Time schedule for examinations: Twice (prior to assignment on study and during the l4th week of the exposure)
- Dose groups that were examined: All rats were examined prior to assignment to study. The control and 2500 ppm groups were examined during the l4th week of exposure.

HAEMATOLOGY
At terminal necropsy, blood was drawn from five rats/sex/group for hematology and blood chemistry determinations. Whole blood treated with anticoagulant (EDTA pretreated cominercial tubes) was processed on a Technicon H*1E System blood cell analyzer using the manufacturer's methods. The following parameters were evaluated: total erythrocyte count (ABC), total leukocyte count (WVBC), hematocrit (HCT), level of hemoglobin (HGB), platelets, red blood cell indices [mean corpuseular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC)], activated partial thromboplastin time (APTT), and leukocyte differential. In addition, reticulocytes were evaluated using the Retic-COUNT system in a flow cytometer utilizing EDTA sample Retic-COUNT reagent.

CLINICAL CHEMISTRY
Blood urea nitrogen (BUN), creatinine, glucose, total protein, albumin, globulin (calculated), glutamic pyruvic transaminase (SGPT/ALT), alkaline phosphatase, gamma glutamyl transpeptidase, glutamic oxaloacetic transaminase (SGOT/AST), creatine phosphokinase, total and direct bilirubin, cholesterol, sodium, potassium, calcium, chloride and phosphorus were determined from serum collected after centrifugation of samples submitted in commercial clot tubes and assayed by standard manufacturers' methodology on a Hitachi 717 clinical analyzer.

Sacrifice and pathology:

Gross Necropsy of Animals Selected for Neuropathologic Evaluation:
Occurrence: At study termination (approximately 3 months)
Animals Examined: Five rats/sex/concentration were randomly selected to be perfused in preparation for neuropathologic evaluation. One rat that was sacrificed in a moribund state was similarly perfused. Since this rat was scheduled for perfusion at terminal sacrifice, it was replaced by another rat from the same group at that time.
Extent of Examination: These rats were not given a complete necropsy. However, abnormalities observed during perfusion or tissue collection were documented.
Perfusion and Fixation: Heparin sulfate was injected intraperitoneally approximately 10 minutes prior to necropsy. The rats were next anesthetized by inhalation of halothane or intraperitoneal injection of sodium pentobarbital (65 mg/ml). The thoracic cavity was then opened and whole body perfusion-fixation was accomplished by intracardial infusion of sodium nitrite followed by 4% formaldehyde/1.5% glutaraldehyde.
Organs Weighed: Testes and epididymides of male animals
Tissues Retained: Brain (including olfactory bulbs, forebrain, cerebrum, cerebellum, midbrain, pons, and medulla oblongata), spinal cord (cervical, thoracic, and lumbar segments) dorsal and ventral spinal nerve roots with dorsal root ganglia (C3-C6, L1-L4), Gasserian ganglion, sciatic, tibial, and sural nerves, testes, and epididymides. Except for the right testis, tissues were stored in perfusion fixative. The right testis was stored in Millonig's neutral buffered formalin and shipped overnight to Research Triangle Institute for processing.

Necropsy of Animals Selected for Tissue and Blood Collection:
Occurrence: At study termination (3 months)
Method of Euthanasia: Carbon dioxide anesthesia followed by exsanguination
Animals Examined: Five rats/sex/group
Extent of Examination: External surface and internal cavities. Organs were examined in place and then removed. Hollow organs were opened and examined.
Organs Weighed: Brain, lungs, liver-, kidneys, adrenals, testes (separately) and epididymides (separately) Tissues Retained: Adrenals, brain, epididymides (separately), eyes, gross lesions with possible histopathological correlates, heart, kidneys, liver, lungs, nose (3 sections), ovaries, skin, spleen, testes (separately), uterus and vagina
Fixatives: Eyes: 5 % buffered neutral formalin/0.5% glutaraldehyde
Remaining Tissues (except testes and left epididym'is): 10% buffered neutral formalin
Right Testis: Millonig's neutral buffered formalin
Left'Testis and Epididymis: Dry ice
Disposition of male reproductive tissues: The frozen left testis and epididymis were shipped to RTI for andrological measurements. The right testis was stored in Millonig's neutral buffered formalin and shipped to RTI for tissue processing. The right epididymis was stored in 10% neutral buffered formalin.

Necropsy of Remaining Male Animals Not Selected for Neuropathology:
Occurrence: At study termination (3 months)
Method of Euthanasia: Carbon dioxide anesthesia followed by exsanguination
Animals Examined: Ten male rats from each of the control and 2500 ppm grouExtent of Examination: External surface and internal cavities. Organs were examined in place and then removed. Hollow organs were opened and examined.
Organs Weighed: Testes (separately) and epididymides (separately)
Fixatives: Left testis and epididymis: 10% buffered neutral formalin
Right Testis and Epididymis: Dry ice
Disposition of male reproduetive tissues: The frozen right testis and epididymis were shipped to RTI for andrological evaluations. The left testis and epididymis were stored in 10% neutral buffered formalin.

Other examinations:

see chapter Neurotoxicity

Statistics:

see chapter Neurotoxicity

Details on results:

see chapter Neurotoxicity

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

>= 7.5 mg/L air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: original value: 2500 ppm; no adverse effects observed (Slight increases (9%) in red blood cell parameters in females of the high dose group were not taken into account due to unknown biological relevance)

Key result

Dose descriptor:

NOEL

Remarks:

systemic

Effect level:

ca. 3 mg/L air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: original value: 1000 ppm; no effects observed

Key result

Critical effects observed:

no

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

7 500 mg/m³

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

secondary literature

Principles of method if other than guideline:

Rabbits were exposed to isobutanol for 7 d to determine the skin irritation potential after repeated dermal exposition.

GLP compliance:

not specified

Specific details on test material used for the study:

Isobutanol

Species:

rabbit

Strain:

New Zealand White

Sex:

not specified

Type of coverage:

occlusive

Duration of treatment / exposure:

24 h per application

Frequency of treatment:

4-6 x 24 hr

Dose / conc.:

0.3 other: mL undiluted test compound

No. of animals per sex per dose:

no data

Control animals:

no

Details on study design:

Post-exposure period: 72 hr

Key result

Dose descriptor:

other: local toxicity

Basis for effect level:

other: highly irritant after repeated exposure under occlusive conditions; no systemic toxicity reported

Remarks on result:

not measured/tested

Four to 6 times repeated 24-hr occlusive exposure to 0.3 ml isobutanol caused severe edema and erythema with eschar formation, which persisted throughout the 72-hr post-observation period. Isobutanol was therefore judged to be "highly irritant”.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

secondary literature

Principles of method if other than guideline:

Rabbits were exposed to isobutanol for 7 d to determine the skin irritation potential after repeated dermal exposition.

GLP compliance:

not specified

Specific details on test material used for the study:

Isobutanol

Species:

rabbit

Strain:

New Zealand White

Sex:

not specified

Type of coverage:

occlusive

Duration of treatment / exposure:

24 h per application

Frequency of treatment:

4-6 x 24 hr

Dose / conc.:

0.3 other: mL undiluted test compound

No. of animals per sex per dose:

no data

Control animals:

no

Details on study design:

Post-exposure period: 72 hr

Key result

Dose descriptor:

other: local toxicity

Basis for effect level:

other: highly irritant after repeated exposure under occlusive conditions; no systemic toxicity reported

Remarks on result:

not measured/tested

Four to 6 times repeated 24-hr occlusive exposure to 0.3 ml isobutanol caused severe edema and erythema with eschar formation, which persisted throughout the 72-hr post-observation period. Isobutanol was therefore judged to be "highly irritant”.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Species:

rabbit

Additional information

There are reliable data available to assess the toxicity of isobutanol after repeated oral and inhalative exposure. The data for repeated dermal administration are very limited.

 

oral

In an oral subchronic study according to OECD guideline 408 and GLP, the test substance was administered via the drinking water at doses of 0, 1000, 4000, or 16,000 ppm (ca. 0, 80, 340, or 1450 mg/kg bw/day) to groups of ten male and female rats for 90 days, no significant differences were noted in the exposed animals compared to the controls (Schilling BG Chemie 1990). Testicular findings in two male animals of the high dose group were judged as incidental and not related to the test substance exposure. The NOAEL was 16,000 ppm (ca. 1450 mg/kg bw/day).

Another subchronic study according to GLP requirements was provided by US EPA (1985). Groups of thirty male and female rats received the test substance via oral intubation with dose levels of 0, 100, 316, or 1000 mg/kg bw/day for 29/30 days (interim sacrifice) or 92/93 days (final sacrifice) without recovery period. Clinical signs related to treatment with 1000 mg/kg dose level included hypoactivity, ataxia, and salivation. Clinical signs of hypoactivity and ataxia were resolved by the 4th week of the study. Slight decreases in feed consumption and body weight gains were noted in the first two weeks and were restricted to the 1000 mg/kg/day group. There were no changes in organ weights or gross or histopathology at any exposure level. Therefore, the LOAEL was 1000 mg/kg bw/ day and the NOEL 316 mg/kg bw/day.

Other available studies did not report results on systemic toxicity in adequate manner (Hilbom et al. 1974).

 

dermal

There is only a skin irritation study with a few repeated applications (4-6 times) available (TSCATS OTS 0510692 by Eastman Kodak Corp., 1986; Val. 4). Besides of the limited relevance due to the short study period, only local effects were studied. Here, isobutanol was judged as highly irritant to rabbit skin after 4-6 applications (each for 24 h) of 0.3 mL undiluted test substance under harsh occlusive conditions.

 

inhalation

A 13-week inhalation study was performed with Sprague-Dawley following GLP and a neurotoxicity guideline requirements (CMA 1996, see chapter Neurotoxicity). Rats were exposed to 0, 250, 1,000 or 2,500 ppm (ca. 0, 0.75, 3.0, 7.5 mg/L) for 6 h/day on 5 d/week. Clinical signs, body weight, necropsy, ophthalmology, haematology and clinical chemistry was observed/ studied in at least five of 10 (control and high dosage: 20) animals. There was only a slight (but statistically significant) increase in red blood cell counts, hematocrit, and hemoglobin parameters in the 2500 ppm female rats observed when compared to the control female rats. Although these effects were considered related, they are of unknown questionable biological significance and were considered as not relevant for the derivation of the systemic NOAEL. Therefore, the NOAEL systemic is 7.5 mg/L/ day and the NOEL is 3.0 mg/L/ day.

 

Additional information is provided by a GLP conform 2-generation study according to EPA OPPTS 870.3800 (ACC 2003, see also chapter Toxicity on reproduction). Here, groups of 30 male and 30 female Sprague-Dawley rats were exposed by inhalation (6 hours/day, seven days/week) to 0, 500, 1000, or 2500 ppm (ca. 1.5, 3.0, or 7.5 mg/L) of the test substance for two generations. Animals of the F0 generation were exposed for ca. 17 weeks (at least 70 days before mating until post-weaning). Exposure up to 2500 ppm (ca. 7.5 mg/L) of the test substance did not cause any systemic toxicity in the F0 generation. Thus, the NOAEL is considered to be ca. 7.5 mg/L (2500 ppm).

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
Acutally, two inhalation studies, the one selected above and the neurotoxicity study should be selected as key studies for this endpoint. Both assess different aspects, but result in the same NOAEC, i.e., no adverse effects observed at the highest dose tested. Blood, and neurological parameters were assessed only in the neurotoxicity study, while a detailed histopathology was only performed in the two generation study.

Justification for classification or non-classification

No adverse systemic effects were observed in any of the available studies . Consequently, there is no need for classification of effects according to 1272/2008/EC (CLP) requirements due to repeated exposure to the test substance.