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Toxicological information

Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study

Data source

Referenceopen allclose all

Reference Type:
publication
Title:
Evaluation Of Subchronic Neurotoxicity Of n-Butyl Acetate Vapor.
Author:
David, R.M., Tyler, T. R., Ouellette, R., Faber, W.D., Banton, M. I., Garman, R.H., Gill, M.W. and O’Donoghue, J.L
Year:
1998
Bibliographic source:
Neurotoxicology 19: 809-822, 1998
Reference Type:
publication
Title:
Evaluation Of Subchronic Neurotoxicity Of n-Butyl Acetate Vapor.
Author:
David, R.M., Tyler, T. R., Ouellette, R., Faber, W.D., Banton, M. I., Garman, R.H., Gill, M.W. and O’Donoghue, J.L
Year:
1998
Bibliographic source:
Neurotoxicology 19: 809-822, 1998; cited in the OECD SIDS dossier 2008 of CAS 109-60-4, propyl acetate

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: US EPA Pesticide Assessment Guidelines, Subdivision F, Hazard Evaluation; Human and Domestic Animals, addendum 10, Series 81, 82 and 83; EPA 540/09-91- 123
Deviations:
yes
Remarks:
Additional modifications utilized to accommodate the logistics of exposure and evaluation. Each neurotoxicity endpoint was designed to evaluate a different aspect of potential neurotoxicity (Tilson, 1987; Moser and MacPhail, 1990).
Principles of method if other than guideline:
Further references:
Moser, V.C. and MacPhail, R.C. 1990. Comparative sensitivity of neurobehavioral tests for chemical screening. Neurotoxicology 11: 285-291.
Tilson, H.A. 1987. Behavioral indices of neurotoxicity: What can be measured? Neurotoxicol. Teratol. 9: 427-443.
Tilson, H.A. 1993. Neurobehavioral methods used in neurotoxicological research. Toxicol. Lett. 68: 231-240.
GLP compliance:
not specified
Limit test:
no

Test material

Constituent 1
Reference substance name:
N-butyl acetate
EC Number:
204-658-1
EC Name:
N-butyl acetate
Cas Number:
123-86-4
IUPAC Name:
butyl acetate
Details on test material:
- Name of test material (as cited in study report): nBA
- Analytical purity: 99.9% by GC

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Hollister/ Kingston, USA
- Age and mean weight at study initiation: Male Sprague-Dawley rats designated for schedule controlled operant behavior (food-restricted animals) testing were received from Charles River Hollister (Hollister, California) and were 125 days of age at the initiation of exposures . Because these animals were foodrestricted for several weeks, they weighed 304 ± 12 g (mean ± SD) . Male and female Sprague-Dawley rats designated for the other neurotoxicity endpoints (ad libitum-fed animals) were received from Charles River Kingston and were 60 days of age and weighed 266 ± 11 g (males) or 205 ± 10 g (females) at the initiation of exposures. The variability in body weight of individual animals in the selected population did not exceed 20% of the mean for each sex.
- Housing: individually in stainless-steel, wire-mesh cages
- Diet and water: Ad libitum-fed animals were maintained on Certified Rodent Diet whereas food-restricted animals were fed daily amounts of 13 grams of Certified Rodent Diet. Water was available ad libititum from an automatic watering system except during operant behavior sessions, motor activity testing, and exposures.

ENVIRONMENTAL CONDITIONS
not reported

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
air
Details on inhalation exposure:
The test atmosphere was generated by metering the test substance into glass distillation columns packed with glass beads. Filtered, compressed air was passed through the glass bead-packed columns to evaporate the test substance. The distillation columns were heated to approximately 50°C to enhance vaporization. The oxygen content of the chamber exposure atmosphere was at least 19.0%. The total airflow was set at 12–14 air changes per h. The temperature and humidity were maintained at 20.6–24.7°C and 36.7–68.7%, respectively.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chamber vapor concentrations were determined at least once each hour with a infrared gas analyzer set at a wavelength of 3.38 µm.
Duration of treatment / exposure:
6 h
Frequency of treatment:
5d/week = 65 exposure days within 13 weeks
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
ca. 2.35, 7.05 and 14.1 mg/L
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
500, 1500 and 3000 ppm
Basis:
analytical conc.
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
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 food-restricted male (SD) rats which were designated for schedule-controlled operant behavior (SCOB).

Animals were distributed into 4 groups of 30 animals each. Ten males and 10 females from each treatment group were designated for FOB/MA/NP testing while another 10 males from each treatment group were utilized for SCOB testing. Control (0 ppm) and high-concentration groups contained an additional 10 animals (5 males and 5 females) for possible post-exposure/recovery evaluation. Exposures were conducted 6 hr/day, 5 days/wk for 65 exposures over 13 consecutive weeks for food-restricted animals assigned to the SCOB group. Ad-libitum-fed animals assigned to the FOB/MA/NP group were exposed for 65 exposures over 14 weeks; the extra week of exposures was required because animals were not exposed on days when the FOB and MA tests were conducted.

Concentrations were selected based on the results of a two-week repeated exposure study in which animals were exposed to 0, 750, 1500, or 3000 ppm nBA. A concentration of 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 .

Examinations

Observations and examinations performed and frequency:
CLINICAL OBSERVATIONS
Animals were observed each hour during exposure. Tapping sounds were made on the outside of the chamber to assess the animals' response to stimulation. Before and after exposure, each animal was handled and examined in detail for clinical signs of toxicity. On weekends, cageside observations were conducted.

BODY WEIGHT
For ad libitum-fed animals, body weights were measured weekly (prior to exposure) and on days the functional observational battery tests were performed. For food-restricted animals, body weights were measured daily prior to each schedule-controlled operant behavior session.
Sacrifice and pathology:

At the end of the exposure period, five ad libitum fed animals per sex were randomly selected from each group for perfusion and neurohistopathology evaluation. The remaining ad libitum-fed animals and all the food restricted animals were humanely killed, and the carcasses were discarded. Animals designated for neuropathologic examination were anesthetized with sodium pentobarbital containing heparin (10% by volume) and perfused through the ascending aorta. The perfusates were 4% paraformaldehyde followed by 5% glutaraldehyde, both in 0.1 M phosphate buffer, pH 7.4, at VC. The following tissues were collected from all perfused animals: brain (including the forebrain, cerebrum, midbrain, 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); and tibial nerve (both hindlimbs including branches to the calf musculature).
The 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 until processed. After processing was completed, the remaining peripheral nerve tissue from the left leg was stored in neutralbuffered 10% formalin.
Brain and spinal cord sections from the high concentration and control groups were processed by routine neuropathological techniques for paraffin embedment, hematoxylin-eosin staining, and light microscopic examination. The left sciatic and tibial nerves, dorsal and ventral roots, and the dorsal root ganglia from the high concentration and control groups were processed and embedded in glycol methacrylate, sectioned at 2 µm, and stained with 1% toluidine blue.
Other examinations:
Motor Activity Measurement
Motor activity for ad libitum-fed animals was measured from Tuesday through Friday of the week prior to study start (Week-1), and during Weeks 4, 8, and 13 of the study. Horizontal motor activity was measured by counting photobeam breaks at 10 minute intervals for a total of 60 minutes using an automated cage rack photobeam activity system. Following motor activity measurements, the animals were placed into their home cages for at least 30 minutes prior to conducting the functional observational battery. Ad libitum-fed animals were divided into 1 of 4 replicate groups containing 5 to 7 animals from each treatment group per sex. One replicate was tested each day at the same time of day (early morning). Animals were not exposed on the days during which motor activity was measured.

Functional Observational Battery
A functional observational battery was conducted using ad libitum-fed animals during the week prior to study start (Week -1), and during Weeks 4, 8, and 13 of the study. Animals were evaluated in random order with both observer and recorder blind to treatment. The numbers of vocalizations, urinations, and defecations during a 3-minute period of open field activity were recorded. The animals were observed, and their behavior or condition was scored based on the system described by O'Donoghue (1996: Clinical neurologic indices of toxicity in animals. Environ Health Perspect 1996; 104 (Suppl 2): 323-33 0). A quantitative assessment of forelimb and hindlimb strength grip strength was performed with an apparatus equipped with a digital push-pull gauge. The rat was placed on a rectangular screen and then lifted upwardly until it released its grip. The grip strength test was repeated for a total of three readings. The mean of the three readings was reported as the grip strength measurement. A quantitative assessment of foot splay was performed by releasing a rat 32 cm above a bench in a horizontal position and allowing it to drop on the. Foot splay was determined by measuring the distance between marks caused by ink placed on the outside digit of each hindfoot with a water soluble marker. Animals were divided into the same replicates used for motor activity measurements, but observed at least 30 minutes after returning to their home cage. Animals were not exposed on the days during which an FOB was conducted.

Schedule-Controlled Operant Behavior
The equipment consisted of operant chambers (Model E10-09) 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. Test sessions were conducted in a room separate from the housing room. Bedding material was placed in trays under the operant chambers and was changed daily. Food pellet dispensers were cleaned weekly. Operant chambers were cleaned weekly with an Alconox solution after which the chambers were wiped down with 70% isopropyl alcohol. The chambers were vacant and open for at least 24 hours after cleaning. Animals were motivated to press a lever by restricting their food to -13 grams/day and reinforcing lever presses with 45-mg food pellets. A multiple schedule of four fixed-ratio components followed by two fixed-interval components 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. The sessions were 47 minutes long and were conducted once daily, 5 days per week at the same time each day 1-2 h prior to exposure. The animals acquired the behavior 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 F160 (fixed interval 60 seconds) component. The FR and FI components were then increased to the final multiple FR:FI schedule. Stable behavior (coefficients of variation for Fl index of curvature and Fl response rate of <20%) were demonstrated prior to the initiation of exposures.
Operant behavior was measured for four consecutive days (Tuesday-Friday) prior to the first exposure to establish baseline response rates. Animals were divided into two replicates (20 per replicate) evenly distributed across exposure groups and equipment. Parameters calculated were FR run rate, post-reinforcement pause duration, F1 response rate, and index of curvature. The FR run rate is defined as the number of lever presses per minute during the time interval from the first lever press to the 20th lever press. Lever presses that occurred during the first 250 msec were not counted (overflow responses). The post-reinforcement pause duration is defined as the time interval from the reinforcement of the last FR run to the next lever press. If overflow responses occurred during this time, the pause duration was the interval from the last overflow response to the first lever press of the next FR run. The FI response rate is defined as the number of lever presses per minute during the time interval from the start of the F1 run until the reinforcement. The index of curvature is the ratio of the area under the response-time curve within each of the 5 bins to the area under the hypothetical straight line of constant-rate responses. Weekly means of FR run rates, pause duration, FI response rates, and FI index of curvature values were compared with the baseline values, and the percent of baseline was calculated for each animal and group. The data from Weeks 4, 8, and 13 were evaluated for evidence of neurotoxicity. Data were also collected for two weeks following the cessation of exposure.
Statistics:
Body weight data were evaluated using the following statistical tests: Bartlett’s test (p<=0.01) to test for equality of variances, one-way analysis of variance (ANOVA, p<=0.05), and Duncan’s multiple range test (p<=0.05) for statistical significance. Continuous data from FOB and MA testing were analyzed by repeated measures of analysis of variance (SAS Institute, Cary, NC, USA). Baseline (pre-exposure) values were subtracted from test-day (weeks 4, 8, 13) values to normalize the animal to animal variance in absolute scores. Time points indicating significant changes were further analyzed by a Fisher’s Exact Test. A probability of p<0.05 (two tailed) was used to determine significance which was not corrected for the number of comparisons conducted. If the Bartlett’s test demonstrated unequal variances, then the data were evaluated with a Kruskal-Wallis H-test and Mann-Whitney U-test.
SCOB data were analyzed as mean weekly percent of baseline values using ANOVA (p<0.05) and Dunnett’s t-test (p<0.05) to indicate statistical significance, and the absolute values were analyzed for trends using a multivariate, repeated-measures analysis of variance. SCOB data for weeks 13 and 15 were compared using a paired Student’s t-test and a probability of p<0.05 (two-tailed) was used to indicate significance.

Results and discussion

Results of examinations

Details on results:
MORTALITY
No spontaneous mortality occurred during the study, but one ad libitum-fed control male was euthanized in a moribund condition due to poor physical condition and excessive weight loss.

CLINICAL SIGNS
Animals exposed to 3000 ppm (ca. 14.1 mg/L) exhibited less movement, decreased alertness, and slower response to tapping on the exposure chamber wall (reduced reactivity) during exposure compared with activity levels exhibited by control animals. Animals exposed to 1500 ppm ( ca. 7.05 mg/L) appeared normal for the first 5 hours of Day 0 (the first day of exposure) and the first hour or two of Days 1 and 2, and thereafter exhibited reduced reactivity of generally minimal severity for the remainder of the daily exposure periods. Control and 500 ppm animals appeared normal during exposure. There was no apparent difference in the clinical conditions of ad libitum-fed and food-restricted animals. There were no signs of neurobehavioral effects or systemic toxicity immediately after exposure (30-60 minutes following the cessation of exposure).

BODY WEIGHT and FOOD CONSUMPTION
Mean body weights for the 3000 ppm groups were generally 15-19% lower than the mean body weight of the control group and were significantly lower than the control group beginning on Day 7 for male rats and Day 14 for female rats. Overall weight gains for the 3000 ppm group were 64 and 59% of weight gains for the control group (males and females, respectively). Overall, mean body weights for the 1500 ppm group were 9% lower than the mean body weights of the control group. However, mean body weights for the female 1500 ppm group were significantly lower than the control group on Days 42, 49, 70, 77, 84, 91, and 98. Mean body weights for the 500 ppm group were comparable to the control group throughout the study, and no statistically significant differences were noted.
No differences in mean body weight of food restricted animals were noted among groups.

HISTOPATHOLOGY
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.

Effect levels

Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
ca. 2.35 mg/L air (analytical)
Sex:
male/female
Basis for effect level:
other: corresponding to 500 ppm; based on reduced body weight and acute, transient signs of reduced activity levels

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Functional Observational

In male rats, significant time-treatment interactions were found in the severity scores of nasal discharge (a possible autonomic parameter). Pre-exposure baseline severity values of nasal discharge were substantially higher for the control group than for the 500 ppm group. As a result, mean severity scores of nasal discharge during Weeks 4 and 8, which were corrected for baseline, were significantly higher in the 500 ppm group than in the control group. This finding was not considered to be toxicologically or neurobehaviorally significant or related to exposure to butyl acetate. No other time-treatment interactions were observed. In female rats, no time-treatment interactions were observed.

 

Motor Activity

In general, the mean total motor activity counts and total ambulations for all male groups were higher during Week 4 than at other times although only slightly higher (11% for motor activity counts). Repeated-measures analysis indicated a time-treatment interaction in total motor activity counts with the baseline-corrected mean total motor activity for the 3000 ppm male group significantly higher than for the control group at 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. Given the slight difference between the counts for the 3000 ppm and control groups, and the lack of a difference at Weeks 8 and 13, the significance of the finding at Week 4 is questionable. In addition, graphic representation of motor activity during the 60 minute measurement period indicates that the activity was similar for all groups during Weeks 4 and 13. In contrast to total motor activity counts, no time-treatment interactions were observed in total ambulations for male groups. For female groups, the mean total motor activity counts and total ambulations were also higher during Week 4 than at other times, but there were no statistically significant differences. Mean total motor activity counts for female rats were also closer to baseline values during Weeks 8 and 13.

 

Schedule-Controlled Operant Behavior

Baseline schedule-con trolled operant behavior values were comparable among groups prior to the initiation of exposure, and no statistically significant difference in any schedule-controlled operant behavior parameter was noted among groups prior to exposure or at any of the four specified time periods. In general, FR run rates increased 20-37% over the course of the study and appeared to be unaffected by exposure to the test substance. Run rates increased in each group approximately the same amount relative to baseline. Changes in pause duration were variable over the course of the study, with the control and 3000 ppm groups having longer durations relative to baseline, and the 500 and 1500 ppm groups having shorter durations relative to baseline. These differences in pause duration among groups appear to reflect the number of animals in each group with pause durations of less than or greater than 6.0 seconds. In general, pause times of less than 6 .0 seconds at baseline became longer over the course of the study, whereas pause times of greater than 6.0 seconds became shorter over the course of the study. These trends appeared to be unaffected by exposure to the test substance. Under the FI schedule, the activity of the control group remained relatively constant throughout the study. Mean overall response rates for the control group were within 10% of baseline during Weeks 4,13, and 13; by contrast, Fl response rates for the treated groups decreased over the course of the study to 70% of baseline. Decreases in mean response rates were first observed in the 3000 ppm group at Week 4, but by Week 13, all treated groups had lower mean response rates relative to baseline. Index of curvature values reflect the FI response rates in that index of curvature values for the control group remained near baseline throughout the study, and index of curvature for the treated groups increased. There were no apparent concentration-related differences among groups. There were no significant concentration-time interactions in any SCOB parameters, and there were no significant changes in SCOB parameters following cessation of exposure relative to the last week of exposure.

Applicant's summary and conclusion