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Neurotoxicity

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Description of key information

n-Butyl acetate induced neurotoxicity (mainly sedatory effects) in rats exposed to 1500 ppm (7.2 mg/L) test item for a single 6 hour period or subchronically. Effects occurred only transiently during exposure, no effects were detectable shortly after stop of exposure. No neurotoxicity could be detected in rats exposed subchronically to 500 ppm (2.4 mg/L) n-butyl acetate, whereas transient sedation was seen at 1500 ppm and above.

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:
supporting study
Study period:
From 02 SEP 1994 to 29 JAN 1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (EPA OTS 798.6050; 798.6200, 798.6500, 798.2450 )
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)
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Remarks on result:
other:

 

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 without restrictions (RL1).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
2 410 mg/m³
Quality of whole database:
Results from reliable, GLP conform subchronic neurotoxicity study with rats is of high reliability (Klimisch score 1) and is supported by findings from other studies.

Effect on neurotoxicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In an acute inhalation neurotoxicity study Sprague-Dawley rats (10 per sex per group) were exposed to concentrations of 0, 1500, 3000 or 6000 ppm of n-butyl acetate for a single 6 h period followed by an observation period of 14 days. To assess the neurotoxicity of n-butyl acetate a motor activity test and a functional observation battery (FOB) were performed. Motor activity was measured over a 60 minutes period before exposure (day -6), immediately after exposure and on days 1, 7, and 14. FOB was conducted prior to exposure (day -3 to -6) and again after motor activity determination on day 0 as well as on days 7 and 14 (Bernard and David, 1994, corresponds to IUCLID study record OPP/CMA, 1994).

Animals of all dose groups showed reduced activity and reduced response to stimulus during exposure, additionally, hypoactivity, siallorhea, and occasionally tearing were observed. No clinical signs were noted at any time post-exposure. Immediately after exposure (day 0), total motor activity and total ambulation for the 3000 and 6000 ppm animals were significantly lower than for the control groups. No differences were noted on day 1, 7 and 14. On Day 0, the hair coat scores of the 6000 ppm group animals were significantly higher than that of the controls, indicating that the hair coat appeared slightly unkempt. In addition, forelimb grip strength of the female 3000 ppm group was significantly higher than the control. No differences were noted on Days 7 and 14. No mortality was observed during the study, no exposure-related changes were noted at gross pathology.

Based on the results of motor activity and FOB tests after exposure (transient effects for the 3000 ppm and 6000 ppm group) a NOAEC for changes that occurred after the animals were removed from the vapour was derived: NOAEC= 1500 ppm (7.2 mg/L). The LOAEC for transient sedative effects during exposure is at the same concentration level (1500 ppm).

Additionally, 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; corresponds to IUCLID study record OPP/CMA, 1996). The study is reliable without restrictions (RL1).

These findings were supported by three other studies: Groups of ten male Wistar rats were exposed for 4 hours to different concentrations of n-butyl acetate. Neurotoxic effects of the test item were measured in a hot plate behaviour test and a Rotarod Performance test. Exposure to the test item lead to an impaired Rotarod Performance (EC50: 8339 ppm (95% CI: 7399 -9622 ppm) and to a decreased sensitivity to pain (EC50: 4491 ppm (95% CI: 3578 -5643 ppm)) (Korsak and Rydzynski, 1994, RL2). Male mice were exposed for 20 minutes to n-butyl acetate vapour (static cell; 0, 1000, 2000, 4000, 8000 ppm). Locomotor activity was dose dependently affected, effects were statistically significant at 8000 ppm. Effects on FOB were also reported, statistical significance was reached at least at the highest exposure concentration, sometimes even at lower concentrations (Bowen and Balster, 1997, RL2). Mice were exposed for 10 (90 mg/L), 15 (80 mg/L), 17 (70 mg/L), 25 (60 mg/L), 31 (50 mg/L), 45 (40 mg/L), 215 (35 mg/L), 305 (30 mg/L), 360 min (20 and 28 mg/L) to different concentrations of n-butyl acetate. No neurotoxicity was observed at 20 mg/L for 360 min; unbalancing was detected at 28 mg/L for 360 min. At all other exposure regimes deep narcosis was observed (Flury and Wirth, 1933, RL3).

These data on acute and subchronic neurotoxicity testing reveal that n-butyl acetate acts as an neurotoxicant which induces acute and transient narcotic effects (NOAEC 500 ppm; 2.4 mg/L; LOAEC 1500 ppm; 7.2 mg/L). No tendency to accumulate and to increase the effects with ongoing exposure could be detected. Therefore, it is concluded that n-butyl acetate should be classified for specific target organ toxicity - single exposure category 3 (STOT SE 3) but no classification for specific target organ toxicity - repeated exposure is necessary.


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:
This study with the longest duration (subchronic) and the lowest NOAEC was chosen.

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

Exposure to n-butyl acetate vapour causes acute, transient signs of reduced activity at concentrations of 1500 and 3000 ppm, but not at 500 ppm. There is no evidence of a cumulative effect. Additionally, there is no evidence of neurotoxicity based on FOB, motor activity, neuropathology, and SCOB endpoints. The no-observable subchronic adverse effect level (NOAEC) is 500 ppm (2.4 mg/L).

n-Butyl acetate induces transient narcotic effects without any tendency to accumulate. Therefore, it is recommended to classify n-butyl acetate as specific target organ toxicity- single exposure category 3. No classification for specific target organ toxicity-repeated exposure is necessary.