Isobutyl acetate

Neurotoxicity

Description of key information

For isobutyl acetate, no data on neurotoxicity could be located.
To compensate for this lack of data, information resulting from isobutanol and n-butyl aceatate as supporting substances will be used as substitute.
In a valid subchronic neurotoxicity study with n-butyl acetate, transient narcotic effects (sedation) but no other neurotoxic effects were observed. For neurotoxicity a NOAEC of 500 ppm (ca. 2400 mg/m3) was derived.
In a valid subchronic (90 d) toxicity inhalation study with isobutanol, neurotoxicologic parameters (FOB, MA) were observed too. For neurotoxicity a NOAEC of 2500 ppm (ca. 7700 mg/m³) was determined.
In a valid subchronic (14 weeks) neurotoxicity inhalation study with isobutanol, a schedule controlled operant behavior test was conducted. From results, a NOAEC for neurotoxicity of 2500 ppm (ca. 7700 mg/m³) was derived.
The NOAEC for isobutyl acetate (conversion of mass concentratiaon/m³ using the respective molecular weight) is 2410 mg/m³

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

Reference

Endpoint:

neurotoxicity: sub-chronic inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 02 SEP 1994 to 29 JAN 1996

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study (EPA OTS 798.6050; 798.6200, 798.6500, 798.2450 ), but as read-across from supporting substance maximum reliablity is 2 Read-across hypothesis: for details please see read-across report in section 13.

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

EPA OTS 798.6050 (Neurotoxicity Screening Battery)

Qualifier:

according to guideline

Guideline:

EPA OTS 798.6500 (Schedule-Controlled Neurotoxicity Study)

Qualifier:

according to guideline

Guideline:

other: EPA OTS 798.6200 (motor activity)

Qualifier:

according to guideline

Guideline:

other: 40 CFR Part 798.2450

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

The study (Laboratory Project ID: 940305I5) consisted of two sets of animals
- Experiment No. 940305I5* (male rats restricted to 12 -14 g of fed per day, designated for schedule-controlled operant behaviour - SCOB) and
- Experiment No. 940305I6* (male and female ad libitum-fed rats designated for functional observational battery, motor activity, and neuropathology - FOB/MA/NP)

TEST ANIMALS
SCOB rats:
- Source: Charles River Hollister (Hollister, CA)
- Age at study initiation: 125 days (57 days at receipt)
- Weight at study initiation: 304 +/- 12 g (males)

FOB/MA/NP rats
Source: Charles River Kingston (Stone ridge, NY)
- Age at study initiation: 60 days (48 days at receipt)
- Weight at study initiation: 266 +/- 11 g (males); 205 +/- 10 g (females)

ALL ANIMALS
- Fasting period before study: no
- Housing: individually during non exposure periods; SCOB and FOB/MA/NP rats were housed in separate rooms
- Diet: a) Certified Rodent Diet (Agway Prolab RMH 3200, pellets), ad libitum except during exposure for the FOB/MA/NP group and b) Certified Rodent Diet (Agway Prolab RMH 3200, ground chow), 12-14 g/day beginning after release from quarantine for the SCOB group
- Water: ad libitum, except during exposure, SCOB sessions, and motor activity testing


ENVIRONMENTAL CONDITIONS
- Temperature: 67-75°F
- Humidity: 46-70%
- Photoperiod (hrs dark / hrs light): 12/12

Rooms for SCOB and motor activity testing:
- Temperature: 70-75°F
- Humidity: 37-70%

Study dates:
-Initiation date: 02 September 1994
- experimental start: 12 September 1994
- expserimental completion: 04 October 1995

Route of administration:

inhalation: vapour

Vehicle:

unchanged (no vehicle)

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 4200 L stainless-steel and glass inhalation chambers
- Method of holding animals in test chamber: cages
- System of generating vapour: test substance was metered into glass distillation columns packed with glass beads; filtered, compressed air was passed through the glass bead-packed columns to evaporate the test substance; distillation columns were heated to about 50°C to enhance vaporization; the resultant vapour was directed via glass tubing to a tee just upstream of the inhalation chamber where it was mixed with filtered, conditioned outside air
- Temperature, humidity in air chamber: 20.6-24.7°C; 36.7-68.7%
- Air flow rate: 836 to 965 Lpm
- Air change rate: 12 to 14 air changes per hour
- Method of particle size determination: Micro Laser Particle counter (µLPC-301, Particle Measuring Systems, Inc, Coulder, USA); indicating that an aerosol fo the test subsance was not present

TEST ATMOSPHERE
- Brief description of analytical method used: MIRAN IA infrared gas analyzer (Wilks Foxboro Analytical, South Norwalk, CT) set at a wavelength of 3.38 µM
- Samples taken from breathing zone: no; collection of chamber vapour samples

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

MIRAN IA infrared gas analyzer (Wilks Foxboro Analytical, South Norwalk, CT) set at a wavelength of 3.38 µM
- chamber vapour samples were continuously collected from each chamber throught TEFLON tubing (3/16" i.d.)
- valve position was pepriodically changed to sample from each chamber at least once each hour

Duration of treatment / exposure:

- 13 consecutive weeks for the SCOB animals
- 14 weeks for the FOB/MA/NP animals (an extra week of exposure was added, because these animals were not exposed on days FOB and MA testing were conducted)
- each animal received at least 65 exposures

Frequency of treatment:

6 hours per day, 5 days per week

Remarks:

Doses / Concentrations:
0, 500, 1500, 3000 ppm
Basis:
other: target concentration

No. of animals per sex per dose:

0 ppm: 25 males, 15 females
500 ppm: 20 males, 10 females
1500 ppm: 20 males, 10 females
3000 ppm: 25 males, 15 females

10 male and 10 female rats from each treatment group were designated for FOB/MA/NP; another 10 male rats from each treatment group were designated for SCOB; the control (0 ppm) and high concentration group (3000 ppm) contained an additional 5 male and 5 female rats designated for possible evaluation of recovery

Control animals:

yes, sham-exposed

Details on study design:

- Dose selection rationale: Range finding study: 2-Weeks repeated exposure in which animals were exposed to 0, 750, 1500 or 3000 ppm n-butyl acetate. The test substance produced concentration-related reductions in general activity levels during exposure periods. Animals appeared to acclimate to the 750 and 1500 ppm concentrations but not to 3000 ppm. Mean body weights for the female 1500 ppm animals and for the 3000 ppm male and female animals were lower than the control group on Days 7 and 14, but no statistically significant differences were noted. 3000 ppm was selected as an exposure concentration that would produce overt signs of toxicity, and 500 ppm was selected as an exposure concentration that was expected to have no effect. An exposure concentration of 1500 ppm was selected as the intermediate exposure concentration.

- the target concentration of the low dose group was set to 550 ppm at request of the sponsor to ensure that all animals were exposed to at least 500 ppm of the test substance

Observations and clinical examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily (prior to exposure, once per hour during exposure, 30 minutes to 1 hour after exposure)
- Cage side observations were included.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily


BODY WEIGHT: Yes
- Time schedule for examinations: FOB/MA/NP animals: weekly (prior to exposure) and on the days the FOBs were performed; SCOB animals: weekly during training and daily (except weekends) prior to SCOB testing


FOOD CONSUMPTION AND COMPOUND INTAKE
- not determined for ad libitium-fed animals
- SCOB animals were fed 12-14 grams chow/rat/day


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No



OPHTHALMOSCOPIC EXAMINATION: No

Specific biochemical examinations:

NEUROPATHY TARGET ESTERASE (NTE) ACTIVITY: No

CHOLINESTERASE ACTIVITY: No

Neurobehavioural examinations performed and frequency:

Time schedule for neurotoxicity examinations: see below


FUNCTIONAL OBSERVATIONAL BATTERY: Yes
- Parameters examined:
* severity and degree of lacrimation, salivation, and nasal discharge
* hair coat
* diarrhea
* excessive or diminished urination or defecation
* palpebral closure
* severity of convulsions and tremors
* ranking of reactivity
* alertness
* coordination of movement
* sensory function (vision and pain perception)
* pinna reflex
* righting reflex
* approach response
* touch response

- Minimization of bias:
- Same technicians used throughout testing: Yes (one observer and one recorder; The same observer was used for all the FOB evaluations except for one day on which that individual was ill. On that day, the person who previously served as the recorder observed the animals with an additional person recording the observations)
- Technicians were blind to treatment status of animals: Yes

- Time schedule for examinations: see below
- Scoring criteria (if any): score 1 = normal; score 2 to 4 for different levels of behaviour
- Description of equipment where required: for quantitative assessment of forelimb and hindlimb strength grip strenght was performed using an apparatus equipped with a digital push-pull gauge (Model DFIS, John Chatillon & Sons)


MOTOR ACTIVITY: Yes
- Equipment: Automated cage rack photobeam activity system (PAS) using a Compaq 386SX computer. The system distinguishes and records two types of horizontal movement: 1) simple motor activiry (single beam break) and 2) ambulation (multiple beam breales pver tje 60 min time period). Motor activity (single beam breaks) was compiled every ten minutes for one hour. The total number of ambulations and total motor activty were calculated for the entire one-hour period.


SCHEDULE-CONTROLLED OPERANT BEHAVIOUR
(1) Overall testing design
- Number of animals: 10 males per dose
- Days of testing: see below

(2) Equipment used
- Type of equipment: operant chambers in isolation cubicles with a house light, three cue lights (over the lever), one lever (on the right side), a 2.5 kHz tone, and a food pellet dispenser (all: Coulbourn Instruments, Inc., Allentown, Pennsylvania, USA)
- Environmental conditions: test sessions were performed in a room separate from the housing room

(3) Procedures
- Animals were motivated to press a lever by by restricting their food and reinforcing lever presses with 45 mg food pellets. A multiple schedule of four fixed-ratio components (reinforcement after 20 responses; 4FR20) followed by two fixed-interval components (reinforcement after 120 sec; 2FI120) was used. The lights above the lever served as the cue for the FR component, and a 2.5 kHz tone at 70 +/- 2 dB served as the cue for the FI component.
- Animals acquired the behaviour over the course of several weeks prior to the start of the study using a weekly progression from continuous FR to FR5 (fixed ratio 5 lever presses) followed by FR10 (fixed ratio 10 lever presses) schedules, then adding a FI60 (fixed interval 60 seconds) component. The FR and FI components were then increased to the final multiple FR:FI schedule. Stable behaviour (coefficients of variation for FI index of curvature and FI response rate of < 20%) were demonstrated prior to the initiation of exposures.

(4) Testing
- Operant behaviour was measured for four consecutive days (Tuesday-Friday) prior to the first exposure to establish baseline response rates. EACHSCOB session consisted of four fixed-ratio (FR) sessions of 20 lever presses for each food pellet followed by two fixed interval (FI) sessions of 120 seconds for each food pellet (4 FR20:2FI120). Each animal had a designated chamber. Groups were evenly distributed across the test equipment.

(5) Calculation
Each FR and FI run during a day's session were combined and a daily average calculated for each animal. FR running rates, pause duration, FI response rates, and index of curvature values are presented as a mean for each animal basoed on values from Tuesday ghrough Friday of each week. Weekly means were then compared with the baseline value and the percent of baseline calculated.
The FR running rate is the number of lever presses per minute during the time interval from the first lever press to the 20th lever press. Lever presses which occurred during the first 250 msec were not counted (overflow responses). The post-reinforcement pause duration is the time interval from the reinforcement of the last FR run to the first lever press of the next FR run. If overflow responses occur during this time, the pause duration will be the interval from the overflow response to the first lever press of the next FR run.
The FI response rate is the number of lever presses per minute during the time interval from the start of the FI run until the reinforcement. The Index of curvature is a ratio of the cumulative lever presses in the last time segment of the run compared to the total cumulative responses.

Sacrifice and (histo)pathology:

- Time point of sacrifice: FOB/MA/NP group: week 14; SCOB group: week 13
- Number of animals sacrificed: FOB/MA/NP group: 15 or 10 per sex per group, but only 5 per sex per group underwent perfusion and neurohistopathology; SCOB group: 10 males (no perfusion and neurohistopathology)
- Procedures for perfusion: animals were anesthetized with sodium pentobarbitla containing heparin (10% v/v); perfused through the ascending aorta (4% paraformaldehyde followed by 5% glutaraldehyde, both in 0.1 M phosphate butter, pH 7.4, at 4°C); peripheral nerves from the left leg were fixed for an additional period of at least 2 hours in 5% glutaraldehyde and stored in sodium phosphate buffer for appr. 3 weeks until processed
- Number of animals perfused: 5 males and 5 females per group
- Tissues evaluated: brain (including the forebrain, cerebrum, nidbrain, cerebellum, pons, medulla oblongata), spinal cord swellings with dorsal and ventral roots (cervical and lumbar), dorsal root ganglia (cervical and lumbar), sciatic nerve (both hindlimbs at mid-thigh and sciatic notch), tibial nerve (both hindlimbs including branches to the calf musculature)
- Type of embedment and staining: brain and spinal cord: paraffin embedment, hematoxylin-eosin staining; others: embedded in glycol methacrylate, sectioned at 2 µm, stained with 1% toluidine blue

Positive control:

Historical positive control data from the test facility demonstrating the sensitivity of FOB, motor activityk, neuropathology, and SCOB have benn submitted to the Sponsor.

Statistics:

Mean values were calculated for analytical concentration, chamber temperature, chamber relative humidity, body weight. Body weight data were evaluated using the following statistical tests: Bartlett's test (p Continuous FOB data and behaviour scores, total motor activity values, and total ambulation values were analyzed using a repeated measures analysis of variance/multivariate analysis. Baseline (pre-exposure) values were subtracted from test-day (Weeks 4, 8, and 13) values to normalize the variance. Categorical data were analyzed using a two-way and multiway frequency table/log-linear model. Time points indicating significant changes were further analyzed using Fisher's Exact test. A probability of p SCOB data were analyzed as mean weekly percent of baseline values using ANOVA (p

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not specified

Clinical biochemistry findings:

not examined

Behaviour (functional findings):

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
- no spontaneous mortality
- one male control FOB/MA/NP animal was euthanatized and necropsied on Day 78 due to poor physical conditions and a body weight loss of 24% over a two week period

3000 ppm rats
- reduced activity levels of minor severity during exposure
- no evidence of a cumulative effect of exposure on the severity of reduced activity
- signs of sialorrhea, gasping, and red discoloration on the chin hair

1500 ppm rats
- reduced activity levels of minimal severity during exposure
- no evidence of a cumulative effect of exposure on the severity of reduced activity

500 ppm and control rats
- appeard normal during exposure

animals in all groups
- after exposure: porphyrin nasal discharges and dried porphyrin stains around the nose; these clinical signs were occasionally seen in the morning before exposure

BODY WEIGHT AND WEIGHT GAIN
3000 ppm ad libitum-fed rats:
- significantly (p - significantly (p - overall weight gains: 64% (males) or 59% (females) of those for the control group

1500 ppm ad libitum-fed rats:
- no differences in body weight of male rats in comparison to the control group
- significantly (p - significantly (p - overall weight gains: 82% (males) or 74% (females) of those for the control group

500 ppm ad libitum-fed rats:
- body weights and weight gains were comparable to the controls throughout the study

fed-restricted (SCOB) rat:
- no effects

NEUROBEHAVIOUR
- no relevant effects
- no evidence of neurotoxicity during FOB examinations
- minor changes in severity scores of isolated FOB parameterswere not considered to be toxicologically or neurobehariorally significant
- mean total motor activity for the 3000 ppm male group was significantly (p - no time-treatment interactions in total ambulations for male groups observed
- no significant motor activity differences for female rats
- no significant differences in SCOB at any concentrations

GROSS PATHOLOGY
- no effects

NEUROPATHOLOGY
- no effects

Dose descriptor:

NOAEC

Effect level:

500 ppm (nominal)

Based on:

test mat.

Remarks:

n-butyl acetate

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Remarks on result:

other:

Read-across justification: for details please see read-across report in section 13.

 

Target concentration (ppm)		0	500	1500	3000
Target concentration (mg/L)		0	2.41	7.23	14.46
Analyzed concentration (ppm)	Mean	0.0	547.5	1488.8	3009.7
	SD	0.0	15.7	44.7	63.0
Nominal concentration (ppm)	Mean	0.0	747.6	2508.6	3393.3
	SD	0.0	30.3	150.3	171.8

 

Analytical concentration: time weighted average concentrations (within 10% of the target concentrations)

Nominal concentration: calculated from the total amount of test substance (gas volume under normal conditions) divided by total chamber air flow

Conclusions:

Exposure of rats to n-butyl acetate vapour (1500 and 3000 ppm; 13 weeks, 6 h/day) resulted in acute, transient signs of reduced activity levels on a daily basis . There was no evidence of neurotoxicty based on FOB, motor activity, neuropathology, and SCOB endpoints. There was no evidence of a cumulative effect during the 13-week exposure. The no-observable adverse effect concentration (NOAEC) is 500 ppm (2.4 mg/L) in this study.

Executive summary:

A subchronic inhalation neurotoxicity study was performed in Sprague-Dawley rats (at least 20 males and 10 females per group). The study consisted of two sets of animals, male and female ad libitum-fed rats designated for functional observational battery, motor activity, and neuropathology endpoints (FOB/MA/NP) and male rats restricted to 12 -14 g of feed per day and which were designated for schedule-controlled operant behavior (SCOB). Both sets of animals were exposed to concentrations of 0, 500, 1500, or 3000 ppm n-butyl acetate for at least 65 exposures over 14 weeks (6 h/d). The target analytical concentration for the 500 ppm group was set to 550 ppm to guarantee that the animals were exposed to at least 500 ppm. FOB and MA were measured during weeks -1, 4, 8, and 13. SCOB-testing was performed daily prior to each exposure to maintain the operant behavior; the data from weeks -1, 4, 8, and 13 were evaluated for evidence of neurotoxicity.

No spontaneous mortality occurred during the study. Transient signs of sedation and hypoactivity were observed during exposure to the 1500 and 3000 ppm concentrations. The only signs of systemic toxicity were reduced body weights for the 3000 ppm groups and for the females at 1500 ppm.

FOB examinations did not reveal any evidence of neurotoxicity. No effect on motor activity was seen in males and females during weeks 4, 8, and 13, with the exception of a significantly higher activity of the 3000 ppm male group (p</= 0.05) during week 4. Moreover, no significant differences were seen in operant behaviour at any test concentrations.

Neurohistopathologic investigations of sections from the brain, spinal cord, dorsal and ventral spinal roots, dorsal root ganglia, sciatic nerve, and tibial nerve of animals in the control and 3000 ppm groups did not reveal any treatment-related effects.

Overall, exposure to n-butyl acetate vapour resulted in acute, transient signs of reduced activity levels on a daily basis at 1500 and 3000 ppm. There was no evidence of a cumulative effect on activity during the 13-week exposure. Additionally, there was no evidence of neurotoxicity based on FOB, motor activity, neuropathology, and SCOB endpoints up to 3000 ppm.

The no-observable adverse effect concentration (NOAEC) is 500 ppm (2.4 mg/L), based on the transient sedation and hypoactivity seen at 1500 and 3000 ppm (Bernard et al., 1996; David et al., 1998). The study is reliable with restrictions (RL2) as maximum reliability for read-across from supporting substance is 2.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

2 410 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

Results from subchronic neurotoxicity study with supporting substance (n-butyl acetate) in rats is of high reliability (Klimisch score 2) and is supported by findings from other studies.

Effect on neurotoxicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

For isobutyl acetate, no valid study concerning neurotoxicity could be identified. The study of Munch (1972) uses an unsuitable test system. Due to the serious methodological deficiencies, the study cannot be used for the evaluation of the neurotoxicity potential of isobutyl acetate.

 

As substitute, data for n-butyl acetate and isobutanol will be used (for details on read-across hypothesis and justification please see read-across report in section 13).

Supporting substance: n-butyl acetate

A subchronic inhalation neurotoxicity study was performed inSprague-Dawley rats (at least 20 males and 10 females per group). The study consisted of two sets of animals, male and female ad libitum-fed rats designated for functional observational battery, motor activity, and neuropathology endpoints (FOB/MA/NP) and male rats restricted to 12 -14 g of feed per day and which were designated for schedule-controlled operant behavior (SCOB). Both sets of animals were exposed to concentrations of 0, 500, 1500, or 3000 ppm n-butyl acetate for at least 65 exposures over 14 weeks (6 h/d, 5 d/w). FOB and MA were measured during weeks -1, 4, 8, and 13. SCOB-testing was performed daily prior to each exposure to maintain the operant behavior; the data from weeks -1, 4, 8, and 13 were evaluated for evidence of neurotoxicity.

No spontaneous mortality occurred during the study. Transient signs of sedation and hypoactivity were observed during exposure to the 1500 and 3000 ppm concentrations. The only signs of systemic toxicity were reduced body weights for the 3000 ppm groups and for the females at 1500 ppm. FOB examinations did not reveal any evidence of neurotoxicity. No effect on motor activity was seen in males and females during weeks 4, 8, and 13, with the exception of a significantly higher activity of the 3000 ppm male group (p</= 0.05) during week 4. Moreover, no significant differences were seen in operant behaviour at any test concentrations. Neurohistopathologic investigations of sections from the brain, spinal cord, dorsal and ventral spinal roots, dorsal root ganglia, sciatic nerve, and tibial nerve of animals in the control and 3000 ppm groups did not reveal any treatment-related effects.

Overall, exposure to n-butyl acetate vapour resulted in acute, transient signs of reduced activity levels on a daily basis at 1500 and 3000 ppm. There was no evidence of a cumulative effect on activity during the 13-week exposure. Additionally, there was no evidence of neurotoxicity based on FOB, motor activity, neuropathology, and SCOB endpoints up to 3000 ppm. The no-observable adverse effect concentration (NOAEC) is 500 ppm (2.4 mg/L), based on the transient sedation and hypoactivity seen at 1500 and 3000 ppm (Bernard et al., 1996; David et al., 1998; corresponds to IUCLID study record OPP/CMA, 1996). The study is reliable with restrictions (RL2; maximum reliability adjustable for read-across).

Supporting substance: isobutanol

 

Three studies relating to neurotoxicity with isobutanol as test substance have been located. Two are assigned a high reliability (subchronic inhalation studies: Li/Monsanto 1999 FOB/MA and Li/Monsanto SCOB). They are chosen as key studies. The third study is only available as a short abstract (acute inhalation study: Monsanto 1994).

 

In the two subchronic studies, a common NOAEC of 2500 ppm (7.700 mg/m³) has been determined. In the acute neurotoxicity study, a vapor concentration of 1500 ppm (4620 mg/m³) was considered to be the LOEC.

 

Monsanto 1994

 

The LOEC derived in the acute neurotoxicity study is based on minimal effects (hypoactivity) seen only during exposure in the 1500 ppm group (Monsanto 1994, RL4). In higher dose groups (3000 and 6000 ppm), the also rapidly reversible general depression of the central nervous system was much more pronounced.

 

Li/Monsanto 1999 FOB/MA

 

 In the subchronic (14 weeks) inhalation combined repeated dose/neurotoxicity study, four exposure levels were tested (0, 250, 1000, and 2500 ppm; isobutanol purity > 99%; exposure for 6 hours per day on 5 days per week). Neurotoxic effects in male rats were monitored by behavioral tests (functional observational battery test and automated test of motor activity).

 

A minimal general effect (depression) on the central nervous system was observed at all exposure levels (up to 2500 ppm), but only during exposure. Response to external stimuli on the exposure chamber was slightly reduced at all exposure concentrations. Immediately after exposure no functional or morphological signs of neurotoxicity could be detected in the treated groups any more. No significant, treatment-related differences between controls and exposure groups were determined for all neurological parameters examined. The slight transient reduction in responsiveness to external stimuli is likely an acute effect of isobutanol exposure but is no indication of a specific, persistent or progressive action of isobutanol on the nervous system.

On this basis a NOAEC for neurotoxicity of 2500 ppm (7.7 mg/L, highest dose administered) was set (Li/Monsanto 1999 FOB/MA).

 

Li/Monsanto 1999 SCOB

 

The second subchronic neurotoxicity inhalation study was conducted simultaneously. Exposure and treatment of the test animals was the same as above. Test object was to perform a schedule-controlled operant behavior (SCOB) study.

 

Rats were trained to perform a multiple fixed ratio 20 (FR20), fixed interval 120 second (FI120s) schedule of food reinforcement. Food was restricted so that their body weights were maintained approx. between 300 - 350 g. After performance of rats was stable, the animals were exposed to test substance. On each exposure day, rats were required to perform the multiple schedule of reinforcement for 47 minutes approx. the same time each day just prior to exposure.

  SCOB dependent variables were analyzed for four consecutive days during the pre-exposure phase of testing (baseline), and during week 4, 8 and 13 of the study.

  Isobutanol (purity > 99%) concentrations up to 2500 ppm did not cause any effects on performance on the multiple 4FR20 - 2FI120 sec schedule of food reinforcement. All concentrations caused slight reduction in responsiveness to external stimuli during exposure. However, immediately after exposure, this effect disappeared and there was no difference between control and treated animals. The effect observed is discussed to be a transient depressive effect on the central nervous system caused by the acute exposure to isobutanol.

  On basis of the results of this study, a NOAEC for neurotoxicity of 2500 ppm (7.7 mg/L, highest dose administered) can be deduced

Deduction of the NOAEC for isobutyl acetate

The NOAEC of isobutyl acetate will be calculated on basis of the NOAEC of n-butyl acetate (Bernard et al., 1998; David et al., 1998) using the mass concentration/m³ and the respective molecular weights (116.16 and 116.16)

The deduced NOAEC (neurotoxicity) for isobutyl acetate is 2.4 mg/L (500 ppm).

Justification for selection of effect on neurotoxicity via oral route endpoint:
Inhalation exposure is the most relevant route of exposure based on the vapour pressure of the substance. Adequate studies using this route of exposure are available.

Justification for selection of effect on neurotoxicity via inhalation route endpoint:
Reliable results from read-across substance n-butyl acetate available

Justification for selection of effect on neurotoxicity via dermal route endpoint:
Inhalation exposure is the most relevant route of exposure based on the vapour pressure of the substance. Adequate studies using this route of exposure are available.

Justification for classification or non-classification

In a neurotoxicity study with the supporting substance n-butyl acetate (structural isomer) the only neurotoxic effects observed were transient signs of sedation and hypoactivity at concentration of 1500 ppm or above. No effects on FOB/MA/NP or SCOB were observed at any test concentration (500 ppm or above). These findings are supported by studies with the supporting substance isobutanol (immediate metabolite of submission susbtance). Isobutanol causes acute, transient signs of reduced activity at concentrations of 250 ppm and higher. Effects are reported to be minimal up to concentrations of 1500 pmm and are more pronounded only at 3000 or 6000 ppm (but still reversible). There is no evidence of a cumulative effect. Additionally, there is no evidence of neurotoxicity based on FOB, motor activity, neuropathology, and SCOB endpoints. Due to the very slight effects on neurotoxicity at 250 ppm isobutanol the NOAEC for neurotoxicity of isobutyl acetate was determined to be 500 ppm (corresponding to 2.4 mg/L of isobutyl acetate).

Based on these effects (induction of transient narcotic effects without any tendency to accumulate) it is recommended to classify isobutyl acetate as specific target organ toxicity- single exposure category 3. No classification for specific target organ toxicity-repeated exposure is necessary.