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Neurotoxicity

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Description of key information

Inhalation
90 d, rat: NOAEC neurotoxicity >= 7.5 mg/L (2500 ppm;GLP, neurotoxicity guideline 82-7 F, CMA 1996a).
90 d, rat: NOAEC neurotoxicity/behaviour >= 7.5 mg/L (2500 ppm;GLP, neurotoxicity guideline 85 F, CMA 1996b).
Acute, rat: LOEC neurotoxicity = 4.5 mg/L (1500 ppm; slight hypoactivation during exposure; EPA guidelines 798.6050 & 789.6200; CMA 1994)

Key value for chemical safety assessment

Effect on neurotoxicity: via oral route

Endpoint conclusion
Endpoint conclusion:
no study available

Effect on neurotoxicity: via inhalation route

Link to relevant study records

Referenceopen allclose all

Endpoint:
neurotoxicity: sub-chronic inhalation
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:
other: Guideline: 82-7, Subdivision F (Neurotoxicity Screening Battery)
GLP compliance:
yes
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: Analyses were conducted using gas chromatography with a flame ionization detector. The material was assayed before and after the study by comparison with an independently obtained high purity standard that was stored in the dark and in a refrigerator.
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Raleigh
- Age at study initiation: 8 weeks
- Weight at study initiation: males: 285-365 g; females: 170-242 g
- Housing: individually
- Diet: ad libitum (no food during exposure)
- Water: ad libitum (no water during exposure)
- Acclimation period: 3 weeks (including quarantine and pretest evaluation periods)


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24 °C
- Humidity (%): 26-62 %
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
inhalation: vapour
Vehicle:
air
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Four 2000-liter stainless steel and glass Hazelton H-2000 chambers
- Method of holding animals in test chamber: Individual stainless steel wire mesh cages
Test atmospheres were generated by feeding isobutanol to a Laskin-type nebulizer mounted in the supply air inlet at top of the chamber. The test atmosphere was mixed with the filtered supply air as it entered the chamber. The chamber exposure concentration was controlled by regulating the rate at which isobutanol was delivered to the nebulizer using an adjustable-flow valveless pump.
Chamber airflow, temperature and % relative humidity were monitored continuously and recorded approximately every 30 minutes.
The chambers operated at airflow of approximately 500 liters/minute (15 air changes/hour).

TEST ATMOSPHERE
- Brief description of analytical method used: 18 test atmosphere samples were drawn, at approximately equal intervals, through a Fourier Transform infrared analyzer calibrated for isobutanol. Light from the desired wavenumber was absorbed by the test chemical, the absorption was detected by the instrument and output through a computer file.
- Samples taken from breathing zone: yes (In the animal breathing zone from a sampling line at the center of the animal area)

Nominal Concentration: Calculated once per exposure by determining the total amount (weight) of test material delivered to the chamber and dividing this amount by the total air volume passing through the chamber during the exposure.

Chamber Atmosphere Distributions: Distribution of the test material throughout the chambers was determined twice for each exposurechamber during the study period, by comparing the concentrations from six different points within the animal area of each chamber.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
18 test atmosphere samples were drawn, at approximately equal intervals, through a Fourier Transform infrared analyzer calibrated for isobutanol. Light from the desired wavenumber was absorbed by the test chemical, the absorption was detected by the instrument and output through a computer file.
Duration of treatment / exposure:
3 months (102 days)
Frequency of treatment:
6 hours/day, 5 days/week (total of 70-73 days during the study)
Dose / conc.:
250 ppm (nominal)
Dose / conc.:
1 000 ppm (nominal)
Dose / conc.:
2 500 ppm (nominal)
No. of animals per sex per dose:
controls and high dose: 20/sex
low and mid dose: 10/sex
Control animals:
yes
Observations and clinical 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.
Neurobehavioural examinations performed and frequency:
The behavioral tests consisted of a Functional Observational Battery (FOB) and a test for motor activity (MA). Both tests were conducted prior to initiation of exposure to the chemical and during the 4th, 8th, and l3th weeks of exposure. For each time period, behavioral tests were conducted over a 4-day period with 25 animals tested each day. Animals were distributed such that the time of testing for all periods was balanced across groups and devices. MA and FOB tests were conducted at approximately the same time of day throughout this study.

FUNCTIONAL OBSERVATIONAL BATTERY: Yes
An FOB was performed to detect gross functional deficits and to quantify behavioral effects. A single trained observer was used to conduct the FOB on all animals. Previous to the study, the observer demonstrated proficiency in conducting the FOB.
Animals were identified using codes that did not provide information on exposure concentration so that the observer was unaware of each animal's treatment. In addition, the observer could not associate any particular animal with others from the same group. Finally, the order in which the animals were tested was randomized so that there was no discernible pattern to the order in which animal from different exposure concentrations were tested.

LOCOMOTOR ACTIVITY: Yes
Motor activity was tested by placing individual animals into separate automated photobeam activity recording devices. Each monitoring session was 1 hour long. This length of time had been previously determined as being long enough for the activity of untreated rats to approach asymptotic levels for the last 20% of the session. Prior to each test session, a diagnostic test was performed to ensure proper photocell operation and equivalent functioning across devices. The 25 animals (5 - 8 per exposure concentration) tested each day were randomly distributed across the activity devices.
Sacrifice and (histo)pathology:
Gross Necropsv 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 groups
Extent 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 reproductive 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.

Histopathology of Nervous Tissues
Processing of Peripheral Nervous System Tissue: Peripheral nerves and ganglia were washed, dehydrated, embedded in glycol methacrylate, sectioned at approximately 2 microns, and stained with toluidine blue.
Processing of Central Nervous System Tissue: The brain and spinal cord were washed, dehydrated, embedded in paraffin, sectioned at approximately 5 microns, and stained with hematoxylin and eosin.
Tissues Examined: All retained nervous tissues from 5 control and 5 high exposure concentration animals of each sex were examined by light microscopy.

Histopathology of Animals Selected for Tissue and Blood Collection (Excluding Male Reproductive Tissues):
Processing of Tissues: Formalin fixed tissues, excluding testes and epididymides (see below), were washed, dehydrated, embedded in paraffin, sectioned at approximately 5 microns, and stained with hematoxylin and eosin.
Tissues Examined: All retained tissues from animals designated for tissue and blood collection (5/sex/group) were examined by light microscopy.

Histopathology of Male Reproductive Tissues and Andrological Measurements:
Processing of Frozen Tissues: Frozen testes were transported to RTI for andrological measurements.
Processing of Formalin Fixed Tissues: Male reproductive tissues (as inclicated above) were transported to RTI for processing. These tissues were subsequently examined by light microscopy at the EHL.
Other examinations:
Hematology Determinations:
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 evalluated: 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.

Blood Chemistry Determinations:
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.
Positive control:
Positive control data for the MA, FOB, and Neuropathology tests were collected prior to this study in accordance with EPA guidelines on Neurotoxicity Testing. The FOB positive control data provided evidence of the ability of the observational methods used by this laboratory to detect major neurotoxic endpoints including limb weakness (repeated exposure to acrylamide), tremor (p,p'-DDT), and autonomic signs (carbaryl).
The neuropathology positive control data demonstrated the sensitivity of the procedures used and ability of the pathologist to detect central nervous system pathology (trimethyltin) and peripheral nervous system pathology (acrylamide). The MA positive control data showed that the procedure for measuring motor activity is sensitive to chemically-induced increases (amphetamine) and decreases (chlorpromazine) in activity.
Statistics:
Weekly measurements of body weight (different from those measured as part of the FOB) and food consumption were analyzed by Dunnett's multiple comparison test (two-tailed).
EHL decision-tree analysis: Hematology data, climical chemistry data, terminal body weights, absolute organ weights, and organ/body weight and organ/brain weight ratios (not required by protocol) were evaluated by decision-tree statistical analyses which, depending on the results of tests for normality and homogeneity of varianees [Bartlett's Test], utilized either parametric [Dunnett's Test and Linear Regression] or nonparametric [Kruskal-Wallis, Jonckheere's and/or Mann-Whitney Tests] routines to detect differences and anailyze for trend.
Fisher's Exact Test (one-tailed): Incidence of microscopic lesions.
Grubbs' Test was used to detect outliers in the organ weight data.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
One female rat in the 2500 ppm group was sacrificed in a moribund condition after approximately two months on test. Upon microscopic examination, this rat was found to have lymphoblastic leukemia of the vertebral column and surrounding tissues. This condition was not attributed to exposure to the test material. The remaining rats survived to terminal sacrifice.

The treated animals did not appear different from controls in terms of their general activity and posture in the inhalation chambers. Typically, animals are curled up and sleeping in the chambers during inhalation exposure and only limited clinical observations can be made. None of the visible rats exposed to isobutanol at any concentration were responsive to a light brush on the exterior of the exposure chamber at any point during the study. During the first week of exposure, a few rats of each sex in the 2500 ppm group were also unresponsive to a sharp tap on the chamber.
However, during the remainder of the study, all rats responded to the latter stimulus. All control rats responded to both stimuli throughout the study.
Responses to stimuli included raising the head, flinching (startle), and moving the ears and/or body. Thus, it can be concluded that animals exposed to all concentrations of isobutanol had a slight decrease in response to external stimuli. A few rats at the 2500 ppm concentration exhibited a more pronounced generalized depression of the central nervous system during the first week of exposure.

Immediately before and after exposure, there were no abnormal clinical signs that were regarded as treatment-related.

There were no abnormal clinical signs that were regarded as treatment related.
Several abnormalities were noted in the female rat that was sacrificed after about two months on test but these were considered to be secondary to that rat's leukemic disease state. One male rat in the 250 ppm group had bloodlike urinary color for a few days during the third month of the study. However, since remission of this effect occurred and there were no such findings in any rats from the 1000 or 2500 ppm groups, it was not attributed to isobutanol exposure. One female rat in the 250 ppm had a palpable mass which appeared near the end of the study. It, too, was considered to be unrelated to treatment.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no statistically significant differences from control values in body weights of rats of either sex. Cumulative body weight gain was similarly unaffected by exposure to the test material.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
There were sporadic statistically significant increases in food consumption in the male rats exposed to 1000 ppm isobutanol as compared to the male controls. The toxicological significance of these differences, however, is doubtful since there was no such effect at 2500 ppm and thus there was no dose response relationship. Additionally, no such effect occurred in female rats exposed to isobutanol at any exposure concentration.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No ocular abnormalities were noted during ophthalmic examinations.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were statistically significant increases in total erythrocyte count, hematocrit, and hemoglobin parameters in female rats in the 2500 ppm group as compared to the female controls. These changes may have been related to treatment, but their toxicological significance was not determined. There was a statistically significant increase in serum calcium levels in the 1000 ppm male rats but since the change did not occur in a dose related fashion, it was not considered to be related to isobutanol exposure.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
There were no statistically significant differences between treated and control groups for any behavioral parameter at any point in the study. There were a few incidental clinical observations occurring in single animals scattered among different exposure groups at various timepoints. In males, decreases in alertness and rearing occurred with increasing time on test for all treated and control groups. These decreases were equivalent across exposure groups. In summary, there were no behavioral changes observed that could be attributed to exposure to isobutanol.

In general, there was a decrease in activity level in both sexes in control and treated groups over time. However, motor activity was unaffected by exposure to isobutanol at any concentration.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no statistically significant differences in terminal body weights, absolute organ weights, organ to body weight ratios, or organ to brain weight ratios (nonperfused rats only) in any group or for any tissue. The female rat that was sacrificed in mid-study had abnormalities in a number of tissues but they were probably secondary to the disseminated leukemia and/or a generally compromised condition. One male rat, also in the 2500 ppm group, had abnormalities of tissues throughout the urinary tract (kidneys, ureters, and urinary bladder). One female rat from the 250 ppm group had a subcutaneous mass. Neither these nor the other scattered abnormalities that were noted were attributed to isobutanol exposure.

There were scattered occurrences of microscopic abnormalities in other tissues but since they occurred in small numbers of animals and/or did not exhibit a dose-response relationship, they were not considered to be related to treatment. The subcutaneous mass in the female rat from the 250 ppm group was diagnosed microscopically as a fibroadenoma. The renal mass in the male rat from the 2500 ppm group was found to be non-neoplastic.
Description (incidence and severity):
Sporadic instances of nerve fiber degeneration were observed in sections of nerves and spinal cord in control and high-dose animals. They were extremely mild and represent expected occurrences of focal spontaneous changes.
Key result
Dose descriptor:
NOAEL
Remarks:
Neurotoxicity
Effect level:
>= 7.5 mg/L air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: original data: 2500 ppm; no relevant effects observed
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
>= 7.5 mg/L air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: original data: 2500 ppm; light 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
Sex:
male/female
Basis for effect level:
other: ca. 1000 ppm

There were no behavioral or morphological effects indicative of a persistent or progressive action of isobutanol on the nervous system at exposure concentrations up to 2500 ppm. However, a slight, transient decrease in response to external stimuli was observed during exposures to all concentrations. This effect is likely an acute effect of exposure to isobutanol and is consistent with known effects of solvents which cause a general and non-specific depression of the nervous system.

Endpoint:
neurotoxicity: sub-chronic inhalation
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:
other: Guideline: 85, Subdivision F - Schedule-Controlled Operant Behavior
GLP compliance:
yes
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 %
Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Raleigh
- Age at study initiation: 16 weeks old
- Weight at study initiation: 275.5 - 306.6 g
- Housing: individually
- Diet: 11-14 g per day and 45-mg food pellets as reinforcement during SCOB sessions, no food during exposure
- Water: ad libitum; no water during exposure ore SCOB sessions
- Acclimation period: ca. 11 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-23 °C
- Humidity (%): 26-62 %
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
inhalation: vapour
Vehicle:
air
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Four 2000-liter stainless steel and glass Hazelton H-2000 chambers
- Method of holding animals in test chamber: Individual stainless steel wire mesh cages
Test atmospheres were generated by feeding isobutanol to a Laskin-type nebulizer mounted in the supply air inlet at top of the chamber. The test atmosphere was mixed with the filtered supply air as it entered the chamber. The chamber exposure concentration was controlled by regulating the rate at which isobutanol was delivered to the nebulizer using an adjustable-flow valveless pump.
Chamber airflow, temperature and % relative humidity were monitored continuously and recorded approximately every 30 minutes.
The chambers operated at an airflow of approximately 500 liters/minute (15 air changes/hour).

TEST ATMOSPHERE
- Brief description of analytical method used: 18 test atmosphere samples were drawn, at approximately equal intervals, through a Fourier Transform infrared analyzer calibrated for isobutanol. Light from the desired wavenumber was absorbed by the test chemical, the absorption was detected by the instrument and output through a computer file.
- Samples taken from breathing zone: yes (In the animal breathing zone from a sampling line at the center of the animal area)

Nominal Concentration: Calculated once per exposure by determining the total amount (weight) of test material delivered to the chamber and dividing this amount by the total air volume passing through the chamber during the exposure.

Chamber Atmosphere Distributions: Distribution of the test material throughout the chambers was determined twice for each exposure chamber during the study period, by comparing the concentrations from six different points within the animal area of each chamber.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
18 test atmosphere samples were drawn, at approximately equal intervals, through a Fourier Transform infrared analyzer calibrated for isobutanol. Light from the desired wavenumber was absorbed by the test chemical, the absorption was detected by the instrument and output through a computer file.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
6 hours/day, 5 days/week (65 exposures)
Dose / conc.:
250 ppm (nominal)
Dose / conc.:
1 000 ppm (nominal)
Dose / conc.:
2 500 ppm (nominal)
No. of animals per sex per dose:
10 males/dose
Control animals:
yes
Observations and clinical examinations performed and frequency:
CAGE SIDE OBSERVATIONS/DETAILED CLINICAL OBSERVATIONS: Yes
- 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: Yes
- Time schedule for examinations: Once weekly, prior to exposure and SCOB testing (Body weights were also taken on days that the SCOB dependent variables were analyzed, pretest and weeks 4, 8 and 13).


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Twice (prior to assignment on study and 3 days after the last exposure)
- Dose groups that were examined: All rats at both time points
Neurobehavioural examinations performed and frequency:
SCHEDULE-CONTROLLED OPERANT BEHAVIOR (SCOB)
Apparatus:
Twenty operant chambers (Model E10-09, Coulbourn Instruments Inc., Allentown, PA) enclosed in ventilated and sound- and light-attenuated chambers were used. These chambers were connected to Compaq Prolinea 4/25S personal computers via a Coulbourn L91-12 LabLinc Test Cage Port and E91-12 Test Cage Interface). Each computer operated a maximum of 8 chambers. Up to twenty-four operant chambers were used during the training sessions.
Reinforcement consisted of one 45 mg food pellet. Each operant chamber was equipped with a food pellet dispenser, a recessed pellet trough with a light that was activated concomitantly with the delivery of a. food pellet, one lever mounted to the right of the food trough, a panel of three mullticolored cue lights positioned directly above the lever, a 2.9 KHz Sonalert tone generator (Coulbourn Instruments, Model E12-02), and a houselight. The houselight was turned on when the session began, remained on throughout the entire 47 minute session, and was turned off at the end of the session. Discriminative stimuli for the multiple schedule of reinforcement was either illumination of three multicolored lights above the lever (FR2O) or presentation of a 70 db, 2.9 KHz tone (FIl2Os). Background noise at approximately 66 decibels was provided by a white noise generation system (Lafayette Instrument Co., Lafayette, IN). All equipment and computer software was subjected to validation procedures. In addition, a diagnostic test was performed on each chamber prior to each test session to verify that the equipment, as described, above was functioning properly.

Training:
Animals arrived at the laboratory at approximately 5 weeks of age. Animals were quarantined for 14 days. On the seventh day of quarantine animals were placed on a restricted diet. Training began after the animals had been on the restricted diet for one week. Animals were restricted to a diet of 11 - 14 g/day, 7 days/week for the first three weeks of the training period. Following this, at an age of approximately 9 weeks, the animals were fed 12 g/day, 7 days/week, except during training weeks 4 through 7, when the animals were fed 14 g/day on weekends. This feeding schedule was targeted at achieving and maintaining body weights close to 300-350 grams. It should be noted that the diets were adjusted for all animals in a similar manner and were not adjusted on an individual basis. Rats were trained on a series of schedules 5 days a week, whenever possible. Initially, rats were trained to press the lever for food reinforcement on a concurrent Fixed Ratio 1-Fixed Time 1 min schedule. In other words, each rat automatically received reinforcement every time it pressed the lever and whenever a 1 minute period elapsed since the last lever press. After the rat learned to press the lever for food reinforcement, the schedule was shifted to a Fixed Ratio (FR) 3, followed successively by a FR5, FR 10, 4 FR 20 - 2 Fixed Interval (FI) 60 sec schedule, and finally a 4FR20-2FI120s schedule. The criteria used to shift an animal from one FR schedule to the next schedule were that the animal must receive 60 to 100 reinforcements in a session, on each schedule of reinforcement. Once the animals were performing on the 4FR60-2FI60s schedule, an animal advanced to the 4FR20-2FI120s schedule after responding on the lever 1000 times during a single session. Most of the rats reached the final schedule after 3 weeks of training. Once the final schedule was reached, it took approximately 6 more weeks before performance became stable. Animals were trained for 9 weeks before the pretest baseline measurements were made.

SCOB Behavior:
Forty male rats that were determined to have stable performance (defined in the Baseline Performance section of Results) an the 4FR20-2FI120s schedule of food reinforcement were randomly distributed among 4 groups (10 rats/group). In this schedule of reinforcement, four consecutive fixed ratio 20 (4FR20) schedules alternated with two consecutive- fixed interval 120 second (2FI120s) schedules. Under the FR20 schedule every 20th response (lever press) was succeeded by the reinforcement (food pellet). Under the FI120s schedule the first response after 120 seconds have elapsed resulted in the delivery of the reinforcement. This alternating sequence was repeated until the 47 minutes elapsed. The three-light cue panel was illuminated throughout the FR sequence. A tone cue was activated throughout the FI schedules. The houselight remained on throughout the entire 47 minute session.
Performance was maintained by having each rat perform under the schedule of reinforcement for one session per day five days per week.
Sessions for each animal started at approximately the same time each day.
Baseline behavior for each rat was determined by averaging the performance of that rat for Tuesday, Wednesday, Thursday and Friday of the week prior to initiation of exposures. Behavior for each week of exposure was determined in a similar manner. Animals were tested prior to exposure during the exposure phase of the study.

Data Acquisition and Analysis:
Schedule contingencies and data collection were controlled by computer (IBM-AT compatible computer with L2T2 Operant Control Software, Coulbourn Instruments Inc., Allentown, PA, Model D91-12, version 3.3). Coulbourn's SCOB Data Analysis and Reporting Software (Model D91-12A, version 1.0) was used to determine session values for a number of dependent variables for each rat. The dependent variables were FR running rate, length of FR post-reinforcement pause, FI- response rate, and FI index of curvature.
Sacrifice and (histo)pathology:
At the end of the experiment all the animals were sacrificed by C02 asphyxiation. Only one animal was given a gross necropsy because of the clinical observations of swollen genitalia during the last three weeks of the exposure period.
Positive control:
Positive control data for the SCOB test was collected prior to this study in accordance with EPA guidelines on Neurotoxicity Testing. The SCOB positive control data showed that the procedure for measuring SCOB is sensitive to chemically-induced increases and decreases in performance.
Statistics:
Only the data collected during the pretest week and the 4th, 8th and l3th week of exposure was statistically analyzed. An individual weekly mean value was determined for each dependent variable by averaging the daily session value for each rat over four consecutive days (Tuesday through Friday) during the test weeks (pretest, weeks 4, 8, and 13). These individual weekly mean values were analyzed across time and exposure concentration by repeated measures analysis of covariance (REPANOVA) using the baseline values as the covariates. The dependent variables were analyzed for homogeneity of variance by using the Levine's Test. The Dunnett's multiple comparison test (two-tailed) was used to compare treated groups with control using the unconverted dependent variables as well as the dependent variables expressed as percent of pretest.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
During Exposure:
The treated animals did not appear different from controls in terms of their general activity and posture in the inhalation chambers. Typically, animals are curled up and sleeping in the chambers during inhalation exposure and only limited clinical observations can be made. Both treated and control animals responded to finger taps on the side of the chamber wall. Responses included raising the head, flinching (startle), and movements of the ears and body. A difference between treated and control animals could be detected in their response to light finger brushes along the side of the chambers. All the control animals responded to these external stimuli, whereas the treated animals from all dose groups did not generally respond to these stimuli. Thus, it can be concluded that during exposure all treated animals had a slight decrease in response to external stimuli.
Immediately before and after exposure:
Immediately before and after exposure, as the animals were removed from the exposure cages and returned to their home cages, there were no abnormal clinical signs that were regarded as treatment-related. There was no obvious difference in responsiveness detected between control and exposed animals Weekly detailed clinical observations:
Singular incidences of skin abrasion and swollen genitalia were noted that are not attributed to treatment with isobutanol. There were no other clinical observations or palpable masses that were observed in these animals.
Mortality:
no mortality observed
Description (incidence):
All animals survived until the completion of the study.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
There were no statistically significant differences from control in body weight except at the mid exposure concentration during exposure week 2 (a decrease of 8 grams from control) and the high exposure concentration during exposure week 6 (an increase of 12 grams from control). There were statistically significant differences in cumulative weight gain at the high exposure concentration during exposure week 4 (study day 29) and continuing until the start of exposure week 9 (study day 63). These differences were increases above the control group that ranged from approximately 6 to 14 grams cumulative weight gain. The gain differences are relatively small compared to the mean body weights and the cumulative gains at the high exposure concentration returned to control levels before the end of the study. These results are not considered to be biologically significant.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related ocular abnormalities in all the animals after 3 months of exposure to isobutanol.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Baseline SCOB Performance:
All rats exhibited fairly stable baseline performance prior to exposure to isobutanol. Stable performance was operationally defined as performance in which the standard deviation of the mean FR response rate and of the mean FI IOC is less than 20% for most of the rats over 4 consecutive days (Tuesday-Thursday). The group mean coefficient of variation ranged from 5 to 7% for the FR running rate and from 5 to 11% for the baseline IOC.
Group mean values for FR rate ranged from 166 to 182 presses/minute. Group mean values for FI rate ranged from 57 to 74 presses/minute. Thus, there was a good separation in performance rate on the FR and FI schedules with group mean ratios of FR to FI rate ranging from 2.8 to 3.8.
The FI schedule produced a typical pattern of performance characterized by little or no responding during the early portions of each interval. The baseline IOC values (group means ranging from .44 to .49) reflected this positive acceleration of responding within the interval.

Effect of Isobutanol on SCOB performance:
The interaction term and dose term for the repeated measures analysis of covariance was not statistically significant for FR rate, FR pause, FI rate, or FI index of curvature. There were no significant differenees between control and treated groups during pretest, week 4, 8, or 13 for any of the dependent variables expressed as raw values or converted as percent of pretest.
FI rate decreased over time for all four groups. This was accompanied by a marginal increase in index of curvature. No remarkable changes could be noted in FR rate or FR pause.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
One male animal from the 1000 ppm group was given a gross necropsy because of the clinical observation that it had swollen testes during the last 3 weeks of the study. Necropsy findings for this animal included an enlarged epididymis, testes with abnormal color, soft consistency and multiple discolored foci and a subcutis mass in the left scrotal region. The mass was soft and filled with light green exudate. This incidental finding is not considered related to exposure to isobutanol.
Key result
Dose descriptor:
NOAEL
Remarks:
Neurotoxicity
Effect level:
>= 7.5 mg/L air
Based on:
test mat.
Sex:
male
Basis for effect level:
other: original value: 2500 ppm; no relevant effects observed
Remarks on result:
other:
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
>= 7.5 mg/L air
Based on:
test mat.
Sex:
male
Basis for effect level:
other: original value: 2500 ppm; no relevant effects observed
Remarks on result:
other:

Under the conditions of this study, there were no effects on performance under a 4 FR 20 - 2 FI 120 second schedule of food reinforcement after subchronic exposure to isobutanol at concentrations up to 2500 ppm (ca. 7.5 mg/liter).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
7.5
Study duration:
subchronic
Species:
rat

Effect on neurotoxicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Inhalation

Two GLP conform 13-week inhalation studies were performed with Sprague-Dawley rats exposed to 0, 250, 1,000 or 2,500 ppm (ca. 0, 0.75, 3.0, 7.5 mg/L), 6 hours/day, 5 days/week either following U.S. neurotoxicity guideline 82 -7, subdivision F (CMA 1996a) or following U.S. neurotoxicity guideline 85, subdivision F (CMA 1996b). These studies included expanded neurotoxicity endpoints (functional observational battery, motor activity, scheduled-control operant behavior, and neuropathology endpoints) as well as the standard parameters for subchronic studies. Intensive investigations of testicular parameters (homogenization-resistant spermatid head counts) were collected at necropsy. The highest exposure concentration (2500 ppm; 7.5 mg/L) did not have any adverse effects demonstrating a persistent or progressive effect of isobutanol on the central or peripheral nervous system in both studies. A slight reduction in responsiveness to external stimuli was noted during exposure in all treatment groups. Slight increases (9%) in red blood cell parameters (count, hematocrit, hemoglobin) were noted in female rats exposed to 2500 ppm (7.72 mg/L) in the first study but the slight nature of these findings made them of questionable biological significance. There were no changes in any other parameters in both studies. Based on the results of both studies, the NOAEL for neurotoxicity (and repeated dose toxicity) was at least 7.5 mg/L (2500 ppm).

An acute inhalation neurotoxicity study was performed with rats following GLP requirements and EPA guidelines 798.6050 & 789.6200 (CMA 1994). 10 male and 10 female rats were exposed to 0, 1500, 3000 and 6000 ppm isobutanol (corresponding to ca. 4.5, 9.0 and 18.0 mg/L). Neurobehaviour tests (functional observational battery and motor activity) were conducted prior to exposure (pretest), immediately after exposure (day 0), the day after exposure (day 1), and 7 and 14 days after dosing. 3000 and 6000 ppm vapours of isobutanol caused rapidly reversible narcosis, 1500 ppm caused slight hypoactivation in rats during, but not after exposure. No treatment-related effects were observed in gross necropsy. Therefore, only a LOEL of 1500 ppm (ca. 4.5 mg/L) was determined.

Justification for classification or non-classification

Effects indicative for CNS depression were observed after single and repeated application (see also chapters Acute Toxicity and Repeated Dose Toxicity). Therefore, the substance has to be classified with R67 according to 67/548/EEC critera and STOT single exposure, Cat. 3 (for narcotic effects) according to 1272/2008/EC (CLP) criteria, respectively.