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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:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Referenceopen allclose all

Reference Type:
publication
Title:
Evaluation Of Subchronic Toxicity 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:
2001
Bibliographic source:
Fd. Chem. Toxicol. 39: 877-886, 2001
Reference Type:
secondary source
Title:
Evaluation Of Subchronic Toxicity 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:
2001
Bibliographic source:
Fd. Chem. Toxicol. 39: 877-886, 2001; cited in the OECD SIDS dossier 2008 of CAS 109-60-4, propyl acetate
Reference Type:
secondary source
Title:
Unnamed
Year:
1996

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EPA OTS 798.2450 (90-Day Inhalation Toxicity)
Deviations:
yes
Remarks:
tissues from the central and peripheral nervous systems were not examined histologically
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
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 and environmental conditions:
TEST ANIMALS
- Source: Charles River Kingston, USA
- Age at study initiation: ca. 60 d
- Mean weight at study initiation: 271 g (males); 215 g (females); The variability in body weight of individual animals in the selected population did not exceed 20% of the mean for each sex.
- Housing: individually
- Diet: Certified Rodent Diet ad libitum
- Water: filtered municipal tap water

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.
Chamber vapor concentrations were determined at least once each hour with a infrared gas analyzer set at a wavelength of 3.38 µm.
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
6 h
Frequency of treatment:
5d/week = 65 exposure days within 13 weeks
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 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:
15
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: 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:
OBSERVATIONS
Body weights and feed consumption were measured weekly prior to exposure. Animals were fasted the day prior to necropsy. Fasted body weights were measured after exsanguination, but prior to necropsy. This procedure is standard practice in our laboratory because it allows for blood collection from the posterior vena cava while the animal is under anesthesia, and reduces the variability in the ratio of body and organ weight due to differences in body fluids. Before and after exposure, each rat was removed from its cage and examined. Cageside observations were conducted once a day on weekends. Observations included, but were not limited to, examination of the hair, skin, eyes and mucous membranes, motor activity, feces, urine, respiratory system, circulatory system, autonomic nervous system, central nervous system, and behavior patterns.

HEMATOLOGY and CLINICAL CHEMISTRY
Animals were fasted beginning after their last exposure. The following day, animals were anesthetized with Metofane, and blood was collected from the posterior vena cava. The blood was placed into vacutainer tubes and allowed to clot for analyses of serum. Other tubes containing an anticoagulant were used for analyses of whole blood samples. Blood smears were also prepared for blood cell counts. Following blood collection, the animals were killed humanely by exsanguination under anesthesia. Animals were bled and euthanatized in random order based on a computer-generated list.
Whole blood samples were analyzed for red blood cell count, total white blood cell counts, hemoglobin, hematocrit and red blood cell indices using an hematology analyzer.
Prothrombin time was measured using a BBL Fibrosystems analyzer . Slides with blood smears were stained and examined for cellular morphology, differential white blood cell count and platelet count. Serum samples were analyzed for total protein, total bilirubin, calcium, phosphorous, urea nitrogen, creatinine, glucose, gamma-glutamyltransferase, aspartate aminotransferase, alanine aminotransferase, sorbitol dehydrogenase and alkaline phosphatase using a serum analyzer. Albumin concentration and isozyme profile were determined using a Gel Electrophoresis System. The albumin/globulin ratio was calculated from total protein and albumin concentrations. Serum sodium and potassium concentrations were determined using a Photometer and serum chloride concentration was measured using a Chloride Analyzer.

OPHTHALMOSCOPY
All rats were examined by a veterinarian for retinal and corneal lesions prior to the start of the study using a direct ophthalmoscope. During the last week of exposure, animals from the control and high-concentration groups were re-examined. Because no changes were detected in the eyes of the high-concentration animals, the animals from the low- and mid-concentration groups were not re-examined.
Sacrifice and pathology:
After 13 weeks of exposure, animals were fasted overnight. The following day, animals were anesthetized with Metofane, and blood was collected from the posterior vena cava. The animals were then exsanguinated and weighed. Wet weights of the liver, kidneys, testes or ovaries, spleen, adrenal glands, lungs and brain were recorded for all animals at necropsy. Paired organs were weighed together, except for the testes, which were weighed individually. All tissues listed in the US EPA Health Effects Testing Guideline for Inhalation Toxicity (40 CFR 798.2450) were collected and preserved in 10%
buffered formalin (pH 7.4). The capsule of the right testis was pricked with a 22-gauge needle and preserved in Millonig’s fixative (10% neutral buffered formalin with phosphate buffered saline, pH 7.4).
All tissues listed below were embedded in paraffin, sectioned at 5 mm, and stained with hematoxylin and eosin (H&E). The nasal passages were decalcified prior to being embedded and sectioned. The lungs were sectioned along a plane allowing visual examination of the major bronchi and bronchioles. The right testis was embedded in glycol methacrylate, sectioned, and stained with H&E and periodic Schiff’s reagent. All tissues except for the brain, spinal cord and peripheral nerve were examined microscopically from the control and high-concentration groups. Nervous tissue from animals exposed simultaneously were evaluated in the neurotoxicity study (David et al., 1998). The lungs, nasal passages, thymus (males only), stomach (females only) and gross lesions were examined from the mid- and lowconcentration groups.
Other examinations:
The left testis and left epididymis of each male rat were placed into individual bags and frozen at -25°C for sperm counts. Samples were shipped to Research Triangle Institute (Research Triangle Park, NC, USA) for analysis. The frozen tissues were weighed to assess the effect of freezing, and homogenized according to the procedure described in Fail et al. (1991). The number of elongated spermatids (testes) or spermatozoa (epididymis)
were counted. The results are expressed per gram tissue weight.
Statistics:
Body weight, feed consumption, serum chemistry, hematology, organ weight and sperm count data were evaluated using the following statistical tests: Bartlett’s test (P<=0.01), one-way analysis of variance (ANOVA) (P<=0.05), and Duncan’s multiple range test (P<=0.05) or Dunnett’s test to indicate statistical significance.
A probability of P<=0.05 (two-tailed) was used to determine significance. If the Bartlett’s test indicated unequal variances, the data were evaluated
using the Kruskal–Wallis H-test and the Mann–Whitney U-test.

Results and discussion

Results of examinations

Details on results:
MORTALITY
No mortality occurred in any of the treated groups during the study.

CLINICAL SIGNS
Animals exposed to 3000 ppm (14.100 mg/L) had reduced activity levels during exposure that were of generally minor severity. Signs of diarrhea and red discoloration on the chin hair were also observed. Animals exposed to 1500 ppm (7.050 mg/L) exhibited reduced activity levels during exposure that were of generally minimal severity. Reduced activity was defined as less movement and slower response to tapping on the chamber wall during exposure as compared to control animals. Control and 500 ppm (2.350 mg/L) animals appeared normal during exposure. After exposure, animals in all groups had porphyrin nasal discharges and dried porphyrin stains around the nose. These clinical signs were occasionally seen during the morning examination before exposure.

BODY WEIGHT And FOOD CONSUMPTION
Mean body weights for the 3000 ppm (14.100 mg/L) groups were significantly lower than the control group throughout the study. Overall weight gains for the 3000 ppm (14.100 mg/L) group were 62 and 78% of weight gains for the control group (males and females, respectively). Mean feed consumption for the 3000 (14.100 mg/L) ppm groups was significantly lower than for the control group throughout the study. Mean weekly feed consumption values for the 3000 ppm (14.100 mg/L) groups were 14-25% lower than the control group for male rats and were 6-16% lower than the control group for female rats. Mean body weights for the 1500 ppm (7.050 mg/L) groups were significantly lower than the control group at certain times during the study. Overall body weight gains for the 1500 ppm groups were 77 and 70% of the control group in males and females, repectively. Mean feed consumption values for the 1500 ppm (7.050 mg/L) groups were significantly lower than for the control group throughout the study. Mean weekly feed consumption values for the 1500 ppm (7.050 mg/L) groups were 4-17% lower than the control group for male rats and were 10-15% lower than
the control group for female rats. Mean body weights for the 500 ppm (2.350 mg/L) groups were comparable to the control group throughout the study. Overall weight gains were 90 and 107% of the control group in males and females, respectively. However, mean feed consumption values for the 500 ppm (2.350 mg/L) groups were significantly lower than for the control group on several days throughout the study. Mean weekly feed consumption values for the 500 ppm (2.350 mg/L) groups were 3-12% lower than the control group for male rats and were from 2% higher to 7% lower than the control group for female rats. No biologically significant differences in hematologic parameters were seen after 30 days or 90 days of exposure.
Mean terminal body weights were significantly lower for the 1500 and 3000 ppm (7.050 and 14.100 mg/L) male and female groups as compared with the control group.

CLINICAL CHEMISTRY
No biologically significant differences in serum chemistries were observed among groups.

OPHTHALMOSCOPY
No treatment-related ophthalmologic changes were observed.

ORGAN WEIGHTS
Organ weight changes independent of the body weight changes (noted above) were slight. These changes included lower spleen weights in the male 3000 ppm (14.100 mg/L) group, higher testes weights in the male 1500 and 3000 ppm (7.050 and 14.100 mg/L) groups, higher lung weights for the 3000 ppm (14.100 mg/L) male group, and higher adrenal gland weights for the 1500 ppm female (7.050 mg/L) and 3000 ppm (14.100 mg/L) male and female groups. Relative organ weights (to body weight) for these organs were not significantly different with the exception of the spleen to body weight ratio for the 3000 ppm male group, which was significantly lower as compared to male controls.

NECROPSY
Signs of necrosis of the olfactory epithelium observed in 1500 and 3000-ppm (7.050 and 14.100 mg/L) rats represent a localized, site-of-contact effect due to n-butyl acetate. Signs of irritation in the stomach were observed in females exposed to 3000 ppm. There were no other lesions observed microscopically that were considered to be compound-related.

SPERM COUNTS
There was no effect on either epididymidal or testicular sperm counts.

Effect levels

Dose descriptor:
NOAEL
Remarks:
local & systemic
Effect level:
2.35 mg/L air (analytical)
Sex:
male/female
Basis for effect level:
other: corresponding to 500 ppm; based on reduced body weight and transient CNS effects; signs of necropsy of the olfactory epithelium

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion