Butan-1-ol

Neurotoxicity

Description of key information

Experimental investigations of the neurotoxic potential of Butan-1-ol were predominantly carried out within the framework of subacute and subchronic as well as in reproduction toxicity and specific designed prenatal developmental toxicity studies. For the read across substance, n-Butyl acetate, which is rapidly hydrolyzed into Butan-1-ol, guideline-conform acute and subchronic toxicity studies are available.

 

The weight of evidence of all data indicated that there is no evidence that Butan-1-ol has to be considered as neurotoxic or developmental neurotoxicant as it did not lead to adverse and/or persistent damage to the CNS or peripheral nervous system. Exposure of Butan-1-ol led only to transient effects or impairment of neurological functions (drowsiness and dizziness) typically for short chain alcohols which are classified accordingly.

 

Oral

Subchronic repeated dose toxicity study, rat, NOEL transient neurological effects (ataxia, hypoactivity) = 125 mg/kg bw/d(US EPA 1986, RL 1)

 

Explorative screening study, single application, mice, NOAEL (rotarod performance) = 500 mg/kg bw (groups of 23–25 male Swiss-Cox mice (Maickel and Nash 1985, RL 2).

 

Inhalation

Behavioral peri-, postnatal developmental (neuro)toxicity study, rat, NOAEC for behavioral effects = 6000 ppm (18.5 mg/L, Nelson 1989b, RL 2)

 

n-Butyl acetate inhalation

Acute inhalation neurotoxicity study, rat, NOEC for motor activity and FOB = 1500 ppm (7.6 mg/L). For transient effects during exposure (reduced activity and reduced response to stimulus), a NOAEC was not obtained, the LOEC was 1500 ppm (7.2 mg/L, OPP/CMA 1994, RL 1)

 

Subchronic inhalation neurotoxicity study, rat, NOEC (neurotoxicity) = 3000 ppm (15.3 mg/L, OPP/CMA 1996, RL 1)

Key value for chemical safety assessment

Effect on neurotoxicity: via oral route

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

125 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Comparable to guideline study under GLP conditions

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:

weight of evidence

Study period:

From 02 SEP 1994 to 29 JAN 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline 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

Dose / conc.:

0 ppm

Remarks:

target concentration

Dose / conc.:

500 ppm

Remarks:

target concentration

Dose / conc.:

1 500 ppm

Remarks:

target concentration

Dose / conc.:

3 000 ppm

Remarks:

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

Key result

Dose descriptor:

NOEC

Remarks:

subchronic neurotoxicity

Effect level:

3 000 ppm (nominal)

Sex:

male/female

Basis for effect level:

other: No subchronic neurotoxicity was observed in this study

 

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

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

18 500 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

well documented study confirmed by other guideline study under GLP conditions on n-butyl acetate

Effect on neurotoxicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Oral

Within a subchronic repeated oral toxicity study, which covered clinical also possible neurological findings, 4 groups of male and female Sprague-Dawley rats (30/sex/group) were administered Butan-1-ol daily at doses of 0, 30, 125 or 500 mg/kg bw/d by gavage for either 6 or 13 weeks (US EPA 1986). No dose-related differences were observed between treatment or control rats in body or organ weight changes, food consumption or mortality, gross pathology, and histopathological and ophthalmic evaluations. Clinical signs in the form of ataxia and hypoactivity (lasting less than 1 h, a typical effect of alcohols) were observed 2 to 3 minutes after dosing in both sexes of the high-dose group (500 mg/kg bw/d) during the final 6 weeks of dosing. Such ataxia and hypoactivity are typically seen following high oral doses of alcohols. The rapid induction/remission of these effects and the reported increased incidence after the interim kill may be due to the fact that post-dose observations could be more quickly collected since fewer animals required dosing. Butan-1-ol was not expected to persist or accumulate over time. Finally, the NOEL with regards to transient neurological effects was 125 mg/kg bw/d (for more details on this study, please refer to the endpoints summary “repeated dose toxicity”).

 

In an explorative screening study, groups of 23–25 male Swiss-Cox mice received a single oral Butan-1-ol application of 500, 1000, or 2000 mg/kg bw/d and body temperature (via rectal thermometer) and rotorod performance in groups of five mice each was assessed at 10, 20, 40, 80, and 120 minutes after treatment. Blood was collected for analysis of Butan-1-ol at the same time points. Treatment resulted in dose-dependent decreases in body temperature that persisted through 40 minutes after exposure in all groups and through 80 minutes in the mid- and high-dose groups. Exposure of 1000 and 2000 mg/kg bw resulted in dose-related decreases in rotorod performance (65 and 25% of baseline, respectively, 10 minutes after exposure, based on visual inspection of data presented graphically). Rotorod performance gradually improved over time until it was 100% of baseline by 80 minutes (at 1000 mg/kg bw) or 120 minutes (at 2000 mg/kg bw) after dosing. Exposure at 500 mg/kg did not affect rotorod performance (Maickel and Nash 1985).

Rats exposed to 4000 ppm butan-1 -ol (6 h/day x 5 days, N=10/group) had no impairment of hearing when tested using reflex modification audiometry (Crofton et al., 1994, see specific investigations: other studies).

 

Inhalation

Butan-1-ol was investigated for its possible behavioral developmental neurotoxicity in a scientifically reliable study in rats following in utero or paternal inhalation exposure. Groups of 15 pregnant female Sprague-Dawley rats were exposed to 0, 3000, or 6000 ppm (0, 9.2 mg/L or 18.5 mg/L) for 7 hours/day on GDs 1 – 19. Groups of 18 male Sprague-Dawley rats were exposed to the same concentrations for 7 hours/day for 6 weeks and then mated to non-exposed females. On the day of birth (PND 0), offspring were culled to four males and four females per litter and fostered to untreated controls. A limited number of neurobehavioral and neurochemical parameters were affected in neonates of mothers exposed but these effects were noted in the presence of maternal toxicity. Finally, no patterns of results characteristic of specific developmental neurotoxicity were seen. The NOAEL was 6000 ppm (18.5 mg/L), the highest concentration tested (Nelson 1989b, for more details on this study, please refer to the endpoints summary “toxicity to reproduction”).

Within an explorative subchronic inhalation study, groups of 12 male Wistar rats each were exposed to 50 or 100 ppm vapour of Butan-1-ol for 6h/d and 5d/wk (Korsak et al. 1994). Within this study, neurological effects were assessed by means of the animals performance on a rotarod and measurement of the level of analgesia determined by hot plate avoidance behavior. However, relevant elements of a standard study for repeated dose toxicity or neurotoxicity were missing. Especially no histopathological evaluation of tissues was performed. A decreased performance on the rotarod was observed at both concentrations. However, the data were only reported in graphical form. There was no effect regarding sensitivity of thermal response at both concentrations. Although the rotarod results may indicate a disturbed co-ordination performance, which is a frequent finding of an unspecific alcohol impact on the CNS, the finding is not considered as an indication for neurotoxicity in the sense of irreversible effects on the CNS or peripheral nervous system. Finally, due to major deficiencies this study was disregarded.

 

Neurotoxicity studies with the read across substance n-Butyl acetate

In an acute inhalation neurotoxicity study (OPP/CMA 1994) male and female Sprague-Dawley rats were randomly distributed into 4 groups of 10 per sex per group which were further separated into 4 replicates of 5 animals per group per replicate. Two replicates consisted of male rats and the other two replicates consisted of female rats. Each replicate was composed of groups which were exposed to concentrations of 0, 1500, 3000, or 6000 ppm of n-butyl acetate (99% pure) for a single 6-hour period. The replicates were exposed on consecutive days to allow time for post-exposure motor activity measurement and a functional observational battery (FOB). The time-weighted average analytical concentrations were generally within 10% of the target concentrations.

Animals near the chamber windows were observed every 30 minutes during exposure. No unusual clinical conditions were observed in control animals. Beginning immediately after onset of the exposure period and continuing until the end of the exposure period, treated groups had minimal reduced activity (hypoactivity) and minimal reduced responses to extrachamber stimulation (tapping on the outside wall of the inhalation chamber). At 6000 ppm, the severity of hypoactivity was minor to moderate. At 3000 ppm, the severity of hypoactivity in female rats was minor, while male 3000 ppm rats were characterized as having minimal hypoactivity. Only minimal hypoactivity was observed at 1500 ppm. Sialorrhea was also observed in treated male rats, but only occasionally in treated female rats. Tearing was also noted occasionally in treated female rats. No deaths were noted during exposure and no clinical conditions were noted at any time post-exposure.

Motor activity was measured over a 60 minute period prior to exposure on Day -6 at the same approximate time of day as the scheduled measurement post-exposure. Motor activity was also measured on Day 0 (day of exposure) immediately after the exhausting of the chamber (- 30 min), on Day 1 approximately 26 hrs after the initiation of exposure, and on Days 7 and 14. Mean total motor activity and total ambulations on Day 0 (post-exposure) by the 3000 and 6000 ppm male and female groups were significantly (p <= 0.05) lower than by the control groups. No differences were noted on Days 1, 7, or 14. Analysis of motor activity at each 10 minute interval indicated some differences between treated and control groups, but there was no overall effect on activity during exploratory behavior or habituation periods. Small differences which were observed between the control and the 1500 ppm groups at specific 10 minute intervals were not considered to be toxicologically significant.

A functional observational battery was performed on all animals the Friday prior to the exposure (Study Day -3 to Day -6), and again after motor activity determination on Day 0. FOB examinations were also performed on Days 7 and 14. On Day 0, the hair coat scores of the 6000 ppm male and female groups were significantly higher than the control groups, indicating that the hair coat appeared slightly unkempt. In addition, forelimb grip strength for the female 3000 ppm group was significantly higher on Day 0 than for the control group. No differences were noted on Days 7 and 14.

Individual animal body weights were measured on Day 0 prior to exposure, and on Days 7 and 14 prior to FOB examinations. The male 6000 ppm rats had significantly (p <= 0.05) lower mean body weights on Days 7 and 14 than did the control group. Male 1500 ppm rats also had significantly (p <= 0.05) lower mean body weights on Day 7, but not at any other time. The differences in mean body weight between treated and control groups were less than 10%. No differences were noted among female rats or between male 3000 ppm rats and the control group.

No treatment-related gross lesions were noted at necropsy.

The results of this study indicate that concentrations of 1500, 3000, and 6000 ppm reduce activity and response to stimulus during exposure. Thus, a no-observed effect level (NOEL) during exposure could not be established. Immediately after exposure, transient decreases in motor activity occurred in groups exposed to concentrations of 3000 and 6000 ppm. However, these changes were not observed during the FOB examination after motor activity on Day 0 or on the day after exposure. The 1500 ppm concentration was considered to be the no-observed effect level (NOEL) for changes that occurred after the animals were removed from the vapor.

 

Subsequently, a reliable subchronic neurotoxicity study was performed according to US EPA Pesticide Assessment Guidelines (OPP/CMA 297761P, 1996). The study consisted of two sets of animals, male and female ad libitum-fed Sprague-Dawley (SD) rats designated for functional observational battery, motor activity, and neuropathology endpoints (FOB/MA/NP) and male (SD) 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 of n-butyl acetate for at least 65 exposures over 14 weeks. The animals were exposed in 4200 L glass and stainless steel chambers for 6 hours per day. Vapors of the test substance were generated by metering the liquid test substance through heated glass distillation columns packed with glass beads. The time-weighted average analytical concentrations were within 10% of the target concentrations. The target analytical concentration for the 500 ppm group was increased to 550 ppm after consultation with the Sponsor because determination of chamber atmosphere homogeneity showed that the variation in actual exposure concentration at various locations in the chamber was on average 13 % lower than the reference point. Nominal concentrations were generally 13-70% higher than the analytical concentrations. The daily mean temperatures and relative humidity inside the chambers during exposure were 21.9 - 23.0°C and 46.9 -53.7%, respectively.

No spontaneous mortality occurred during the study, however, one male control FOB/MA/NP animal (Rat 502) was euthanatized and necropsied on Day 78 due to poor physical condition and a body weight loss of 24 % over a two week period.

Animals were observed for signs of toxicity prior to exposure, once per hour during exposure, and 30 minutes to one hour after exposure. Animals exposed to 3000 ppm had reduced activity levels which were of minor severity during exposure. There was 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 were also observed. Animals exposed to 1500 ppm exhibited reduced activity of generally minimal severity. There was no evidence of a cumulative effect of exposure on the severity of reduced activity. Control and 500 ppm animals appeared normal during exposure. There were no other apparent differences in the clinical condition of FOB/MA/NP and SCOB animals. After exposure, animals in all groups had porphyrin nasal discharges and dried porphyrin stains around the nose. These clinical signs were occasionally seen in the morning before exposure.

Mean body weights for the 3000 ppm ad libitum-fed group were significantly (p <= 0.05) lower than the control group for male rats on Days 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84, 91, and 98, and for female rats on Days 14, 21, 28, 35, 42, 56, 63, 70, 77, 84, 91, 98. Mean weight gains for the 3000 ppm ad libitum-fed male group during Weeks 1-3, 5-7, 11, and 14 were significantly (p <= 0.05) lower than for the control group. Mean weight gains for the 3000 ppm ad libitum-fed female group during Weeks 1-3, and 6 were significantly (p <= 0.05) lower than for the control group. Overall weight gains for the 3000 ppm groups (males and females, respectively) were 64 and 59 % of those for the control groups. No differences in body weight were seen for the 1500 ppm ad libitum-fed male group. Mean body weights for the female 1500 ppm ad libitum-fed group were significantly (p <= 0.05) lower than the control group during Weeks 6, 7, 10, 11, 12, 13, and 14. Mean weight gains for the 1500 ppm male ad libitum-fed group were significantly (p <= 0.05) lower than for the control groups during Weeks 9 and 14, while mean weight gains for the 1500 ppm ad libitum-fed female group were significantly (p <= 0.05) lower than for the control groups during Weeks 6 and 11. Overall weight gains for the 1500 ppm groups (males and females, respectively) were 82 and 74% of those for the control groups. Mean body weights and weight gains for the 500 ppm ad libitum-fed groups were comparable to the control groups throughout the study. No differences in body weight were noted among the male SCOB rats.

Neurotoxicity was evaluated in ad libitum-fed animals using an FOB and quantitative measurement of motor activity during Weeks -1, 4, 8, and 13, and neuropathology at termination. SCOB testing occurred daily in feed-restricted male rats during Weeks 1-13 of exposure and Weeks 14 and 15 following the cessation of exposure. No evidence of neurotoxicity was seen during the FOB examinations. Minor changes in severity scores of isolated FOB parameters were noted, but these were not considered to be toxicologically or neurobehaviorally significant. Mean total motor activity for the 3000 ppm male group was significantly (p <= 0.05) higher than for the control group during Week 4. Mean total motor activity counts for all male groups were closer to baseline values during Weeks 8 and 13 and no significant differences were observed among groups. No time-treatment interactions were observed in total ambulations for male groups. No significant motor activity differences were present for female rats. No significant differences were seen in SCOB at any test vapor concentration. No treatment-related changes were detected during gross necropsy examinations of male or female FOB/MA/NP rats exposed to the test substance. Microscopic evaluations of sections from the brain, spinal cord (cervical and lumbar regions), dorsal and ventral spinal roots, dorsal root ganglia, sciatic nerve, and tibial nerve of animals in the control and 3000 ppm groups did not indicate any treatment-related effects.

This study fulfills the requirements of the U.S. Environmental Protection Agency, Neurotoxicity Pesticide Assessment Guidelines, Subdivision F, Hazard Evaluation: Human and Domestic Animals, addendum 10, Series 81, 82, and 83 (1991) as specified in the Enforceable Consent Agreement of the Testing Consent Order. Exposures to n-butyl acetate vapors resulted in acute, transient signs of reduced activity levels on a daily basis at 1500 and 3000 ppm, but no evidence of a cumulative effect on activity during the 13-week exposure. In addition, there was no evidence of neurotoxicity based on FOB, motor activity, neuropathology, and SCOB endpoints. Therefore, the no-observable effect level (NOEL) for subchronic neurotoxicity for this study is 3000 ppm based on the lack of cumulative neurotoxicity following repeated exposure. 

N-Butyl acetate was tested for acute neurobehavioral effects in mice using locomotor activity and a functional observational battery. 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). The effects described in this study are general impairments of neurological and behavioral functions (drowsiness and dizziness) and which are typically seen with these type of substances.

Assessment of developmental neurotoxicity:

Recently an overview on Butan-1-ol induced developmental neurotoxicity and the potential mechanisms related to these effects was published (Bale AS and Lee JS, 2016). For Butan-1-ol in total five individual paper were mentioned, which are also part of this dossier, namely McLaughlin et al., 1964, Sitarek et al., 1994, Ema et al., 2005, Nelson et al., 1989a, Nelson et al., 1989b). Based on their evaluation, the authors stated that notable signs of neurotoxicity and developmental neurotoxicity have been observed in some studies where laboratory animals (rodents) were gestationally exposed and that mechanistic data supported these observations. Based on the very thorough assessment of the aforementioned studies in this CSR (see endpoint summaries “Toxicity to reproduction” and “Neurotoxicity”, we can’t follow the authors conclusion that butan-1-ol is a neuro-toxicant or induces developmental neurotoxicity.

Overview of the key findings by Bale and Lee vs. the assessment in this CSR:

	Assessment of the effects observed in the five papers cites by Bale and Lee:
	Bale AS and Lee JS (2016)	This CSR (2017):
McLaughlin et al., 1964	Increased incidence of corneal opacity (cataracts) and nerve damage at 320 and 480 mg/kg. No hatched eggs at 640 mg/kg.	No validated or appropriate exposure route, especially not for a severe irritant that causes serious damage to eyes. An in vitro method (Hen's Egg Test Chorioallantoic Membrane (HET-CAM)) has been established to test for severe ocular irritants and as butan-1-ol causes serious damage to eyes (Eye Cat. 1, H318) the observed effects in this test (corneal opacity and nerve damage) can be clearly associated to the local irritant effects of butan-1-ol. The test is considered invalid.
Sitarek et al., 1994	Increased litter incidence of dilation of the lateral and/or third ventricle and subarachnoid space of the brain in pups gestationally exposed to 300 mg/kg-day.	Study lacks historical control data which makes the interpretation of observed effects difficult. Most of the reported“congenital defects” are listed as variations or delayed development in commonly used historical databases, lack a dose response relationship and do not adversely affect survival or health. Thus a clear indication for developmental or developmental neurotoxicity can’t be deducted from this study.
Ema et al., 2005	No significant observations were noted in fetal brains, up to 5 g/kg-day, although developmental toxicity was noted.	Developmental effects at 5 g/kg/day were associated with clear maternal toxicity and are not considered as an independent effect. No significant or relevant observations were noted in fetal brains.
Nelson et al., 1989a	Enlarged brain ventricles observed in exposed fetuses but not significantly increased from control.	Developmental toxicity of Butan-1-ol appeared to be low and did not indicate selective foetal effects as it occurred only in the presence of maternal toxicity. No adverse or statistically significant effectswere noted in fetal brains.
Nelson et al., 1989b	No neurobehavioral effects in offspring, regardless of whether mothers or fathers exposed. Significantly higher levels of serotonin and dopamine in several brain regions (e.g. brain stem, midbrain).	No neurobehavioral effects in offspring, regardless of whether mothers or fathers exposed. The changes between control and high-dose neurotransmitter mean levels (serotonin and dopamine) observed in this study are within normal biological variance and are thus not considered adverse.
Overall interpretation of data:	Butan-1-ol induces developmental neurotoxicity	Overall (weight of evidence) there is no indication that butan-1-ol induces developmental neurotoxicity

 

 

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The weight of evidence of all data are considered as reliable and suitable for classification purposes under Regulation 1272/2008. There is no evidence that Butan-1-ol has to be considered as neurotoxicant or developmental neurotoxicant as it did not lead to adverse and/or persistent damage of the CNS or peripheral nervous system. Exposure of Butan-1-ol led only transiently to minor behavioural effects or impairment of neurological functions (drowsiness and dizziness) which are classified accordingly (STOT SE 3, H336). Those observations typically occur after exposure to alcohols.

As a result the substance is not considered to be classified for neurotoxicity under Regulation (EC) No 1272/2008, as amended for the eighth time in Regulation (EU) No 2016/218.