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Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to EPA OTS 798.2450 (90-Day Inhalation Toxicity) and in accordance with the Principles of Good Laboratory Practice (GLP)

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1997
Report date:
1997
Reference Type:
study report
Title:
Unnamed
Year:
1996
Report date:
1996
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report date:
1999

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
EPA OTS 798.2450 (90-Day Inhalation Toxicity)
Deviations:
no
Principles of method if other than guideline:
not applicable
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Butyl propionate
EC Number:
209-669-5
EC Name:
Butyl propionate
Cas Number:
590-01-2
Molecular formula:
C7H14O2
IUPAC Name:
butyl propionate
Details on test material:
- Name of test material (as cited in study report): n-butyl propionate
- Physical state: clear, colourless liquid
- Analytical purity: 99.61%
- Purity test date: 1995-12-21
- Lot/batch No.: 0379267-010
- Stability under test conditions: considered to be stable under the storage conditions
- Storage condition of test material: stored at room temperature

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 Laboratories Inc.
- Age at study initiation: 38 days upon receipt
- Housing: individually housed
- Diet: ad libitum, PMI Feeds, Inc., Certified Rodent LabDiet 5002 was provided ad libitum, except during the exposure periods and during the period of fasting prior to blood collection
- Water: ad libitum, tap water was provided ad libitum except during the exposure periods
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 72 ± 4°F (22 ± 2°C)
- Humidity (%): 30-70%
- Photoperiod (hrs dark / hrs light): 12 hours light/dark cycle

Administration / exposure

Route of administration:
inhalation
Type of inhalation exposure:
whole body
Vehicle:
clean air
Remarks on MMAD:
MMAD / GSD: not applicable
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Exposures were conducted in four 1.0 m3 stainless steel and glass whole body inhalation chambers
- Method of holding animals in test chamber: Animals were individually caged in stainless steel wire mesh caging during the exposures.
- Source and rate of air: chamber supply air provided from a HEPA and charcoal filtered, temperature and humidity controlled source. The cage batteries were rotated through various cage positions in the chambers to help ensure a similar exposure for all animals within each group over the duration of the xposure period.
- System of generating vapour: Test material was metered from a reservoir using positive displacement pumps at a known and constant rate to a glass vaporization column filled with 8 and 12-mm glass beads. The vaporization column was wrapped with a 206-watt heating tape operated from a temperature controller. Vaporization air was metered to the bottom of the column using a mass flow meter. The concentrated vapors were piped to the chamber inlet where the concentration was diluted to the target level by the chamber ventilation airflow.
- Temperature, humidity, pressure in air chamber: Temperature, relative humidity, chamber ventilation rate and negative pressure within the chambers were continually monitored and generally recorded every 35 minutes through an appropriate software. Chamber temperature and relative humidity were monitored by RTD and polymeric sensors. Airflow through the chamber was monitored by the pressure drop across a sharp-edge orifice plate as measured by differential pressure gauges (calibrated) and was set such that there were 12-15 air changes/hour.
- Air change rate: 12-15 air changes/hour
- Treatment of exhaust air: Treatment of exhaust air consisted of activated charcoal and a HEPA filtration.

TEST ATMOSPHERE
- Brief description of analytical method used: Actual exposure concentrations were measured using gas chromatographic (GC) methods. Sapmples of the exposure atmospheres from each chamber were automatically collected at approximately 35 minute intervals using a loop and computer-controlled gas sampling and multiposition valves. The instrument conditions were as follows -
Instrument: Hewlett Packard 5890 Series II with a 3396 Series II integrator
Detector: Flame ionization
Column: 10' x 1/8" stainless steel; 10% SP-1000 on 80/100 mesh Supelcoport
Gases: Carrier - Helium; Pressure - 60 psig, Flow rate - 30 ml/min
Temperature (°C) - Column 150, isothermal; Detector - 250
Injection volume: 0.25 ml
Retention time (min.): approximately 1.6
- Samples taken from breathing zone: yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
targeted exposure concentrations - 0, 250, 750 and 1500 ppm
measured exposure concentrations - 0, 247, 747 and 1521 ppm
Duration of treatment / exposure:
13 consecutive weeks
Frequency of treatment:
six hours per day, five days per week, for 13 consecutive weeks (minimum of 65 total exposures)
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
250 ppm (targeted exposure concentration)
Basis:
other: 247 ppm (analytical concentration)
Remarks:
Doses / Concentrations:
750 ppm (targeted exposure concentration)
Basis:
other: 747 ppm (analytical concentration)
Remarks:
Doses / Concentrations:
1500 ppm (targeted exposure concentration)
Basis:
other: 1521 ppm (analytical concentration)
No. of animals per sex per dose:
15 male + 15 female rats/group
Control animals:
yes, sham-exposed
Details on study design:
- Dose selection rationale: based on range finding study

- A Combined 2-Week Range-Finding Inhalation Toxicity and Developmental Toxicity Study of n-Butyl Propionate in Rats; Report no. 228009 and Company Study no. - HET K-012981-008 listed under references: A range-finding study of n-Butyl Propionate in rats was conducted to determine exposure concentrations for subsequent 13-week and developmental toxicity studies. The range-finding study consisted of two phases: a subchronic toxicity phase and a developmental toxicity phase. For the subchronic toxicity phase, the test article was administered via whole-body inhalation to four groups, each comprised of five male and five female Sprague-Dawley Crl:CD BR rats, for six hours per day, five days per week, for two consecutive weeks. For the developmental toxicity phase, the test article was administered via whole-body inhalation to four groups of 12 bred female rats each for six hours per day for 10 consecutive days (gestation days 6-15). Target exposure concentrations were 250, 500, 2500 and 4000 ppm (parts per million). For each phase, a concurrent control group of identical design was exposed to clean, filtered air on a comparable regimen. Throughout the study period, all rats were observed daily for appearance and behavior prior to exposure, for clinical signs during and within approximately one hour after exposure, and for mortality and moribundity. Body weights and food consumption were recorded at appropriate intervals. For the subchronic toxicity phase, necropsies were performed on all animals, and selected organs were weighed. A microscopic examination was performed on nasal tissues from all animals. For the developmental toxicity phase, a laparotomy and macroscopic examination were performed on each animal on gestation day 20. The uteri and ovaries were examined, and numbers of fetuses, early and late resorptions, total implantations and corpora lutea were recorded. Mean gravid uterine weights and net body weight changes were recorded for each group.
Test article exposure had no adverse effect at any exposure level on survival, organ weight data (subchronic toxicity phase), intrauterine survival (developmental toxicity phase) or at the macroscopic examination. Test article-related clinical signs were noted in the 2500 and 4000 ppm groups and consisted primarily of drooping eyelids and salivation during exposure, and red or brown material or staining around the nose and/or mouth one hour following exposure. In both phases, body weight gain and food consumption were inhibited in the 2500 and 4000 ppm groups generally throughout the treatment period. Mean gravid uterine weights, net body weights and net body weight gains in these groups were also reduced in the developmental toxicity phase. Exposure-related lesions were noted at the microscopic examination of nasal tissues in the subchronic toxicity phase. These consisted of cytoplasmic vacuolation, necrosis and/or atrophy of the olfactory epithelium (with or without dilatation of Bowman's glands) in nasal sections III, IV, V and VI of the 2500 and 4000 ppm groups. Based on the results of this study, target exposure concentrations of 250, 750 and 1500 ppm were selected for the 13-week toxicity study with n-Butyl Propionate in rats.
- Pathology Advisory Group Review of the Histopathology of the Nasal Olfactory Mucosa from selected inhalation toxicity studies conducted with volatile chemicals - Study no. HET K-002610-015 - listed under references: Histopathology of the nasal olfactory mucosa from selected animals from the range finding study (above) were evaluated by an independent pathology Advisory Group. Nasal sections were examined from one male (48530) and one female (48559) control rat. Nasal sections were also examined from one male (48521) and one female (48542) rat in Group 4 (2500 ppm) and one male (48523) and one female rat (48563) in Group 5 (4000 ppm) which had changes reported to involve the olfactory mucosa.
The primary alterations were observed in the olfactory epithelium lining the dorsal medial meatus of Level II but also extended posteriorly to involve Levels III and IV. The changes were centrally located with extension laterally. Changes were also observed on the tip and medial surface of the dorsal scroll of the third ethmoturbinate.
The changes were characterized by degeneration of the mucosal epithelium with loss of sensory and sustentacular cells. Areas of regeneration were present along with the degenerative lesions in some portions of the olfactory mucosa. In more severely affected areas, the degeneration of the sensory and sustentacular cells was accompanied by atrophy or loss of the nerve bundles in the lamina propria.

- Rationale for selecting satellite groups: for reversal groups
- Post-exposure recovery period in satellite groups: 8 weeks
Positive control:
not applicable

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: midppoint during exposure and once daily in the morning, detailed physical examinations were conducted weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION:
- Food consumption for each animal determined weekly and mean daily diet consumption calculated as g/animal/day: Yes

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to exposure and during study week 12. All ocular examinations were conducted using an indirect opthalmoscope and/or a slit lamp or other suitable equipment, preceded by pupillary dilation with an appropriate mydriatic agent.
- Dose groups that were examined: all animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: during study week 4 and study week 14
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes
- How many animals: all animals
- Parameters examined: total leukocyte count, erythrocyte count, hemoglobin, hematocrit, MCV, MCHC, MCH, platelet count, prothrombin time, APTT and differential leucocyte count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: during study week 4 and study week 14
- Animals fasted: Yes
- How many animals: all animals
- Parameters examined: albumin, total protein, globulin, A?G ratio, total bilirubin, urea nitrogen, creatinine, ALP, ASAT, ALAT, GGT, glucose, total cholesterol, calcium, chloride, phosphorous, potassium and sodium

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
ORGAN WEIGHTS: Yes (arenals, brain, kidneys, liver, lungs (prior to inflation with fixative), ovaries and testes.
Other examinations:
None
Statistics:
Standard statistical methods were employed

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY - No treatment related clinical findings were noted during the exposure or recovery periods. Other findings in the treated groups (salivation, swollen ears, scabbing, rales, red material on various body surfaces, etc.) were seen at a similar incidence in the control group or were observed infrequently, typically in single animals. One male animal from the 250 ppm group was found dead during exposure on day 41 and the cause of death was a marked upper and lower urinary tract infection which was not considered to be treatment related. All other animals survived to the scheduled necropsies.

BODY WEIGHT AND WEIGHT GAIN - Exposure related effects on body weight data were limited to the male animals of 1500 ppm group. Mean body weight and weight gains were reduced in the 1500 ppm group males generally throughout the exposure period. During the recovery period, mean body weight gains in the 1500 ppm group males were similar to greater than the control group values. The female animals from the 1500 ppm dose groups revealed slight, transient reductions in mean body weight during the initial weeks of exposure (upto week 3), subsequently no other effects were noted during the experimental period. No exposure related effects on body weights and weight gain were noted in animals of the 250 and 750 ppm dose group.

FOOD CONSUMPTION - Statistically significant reductions in feed consumption were noted in the 1500 ppm group males throughout the exposure period, while a recovery in the feed consumtion was noted during the recovery period. No other reamekable differences were apparent in food consumption during the exposure or recovery periods.

OPHTHALMOSCOPIC EXAMINATION - Opthalmic examinations revealed no changes attributable to n-butyl propionate exposure.

HAEMATOLOGY - Hematology parameters were unaffected by exposure to n-butyl propionate, however some statistically significant differences were noted in MCHC and MCH values between the control and treated dose groups. In the absence of any treatment related trend for these parameters, these changes were not considered to be treatment related.

CLINICAL CHEMISTRY - No exposure related changes were noted in serum chemistry parameters at the study week 4 and 13 evaluations, however some statistically significant differences were noted in ALAT, GGT, phosphorous and potassium levels. In the absence of any treatment related trend for these parameters, these changes were not considered to be treatment related.

ORGAN WEIGHTS - No exposure related changes were apparent in organ weight at the scheduled necropsies. Certain changes such as statistically significant reduced mean absolute liver weight and increased mean relative brain and testes weight noted in the 1500 ppm male dose group were considered to be not treatment related as a similar trend was not noted in the female animals of the same group. These changes were considered secondary to the reduced final body weight in this group.

GROSS PATHOLOGY - No treatment related gross pathological changes were noted in the male animal from the 250 ppm dose group found dead on day 41 and all animals terminated at the scheduled necropsy. Incidental findings such as dilated renal pelvis, white areas on various organs, splenic cysts, reddened and/or enalrged lymph nodes were noted sporadically in animals across all the dose groups.

HISTOPATHOLOGY: No exposure related histopathological findings were observed in the 250 ppm group male that was found dead on day 41. Microscopic evaluation at the study week 13 primary necropsy revealed exposure related findings in the olfactory epithelium (vacuolation, cell necrosis and/or atrophy) of the 750 and 1500 ppm groups. Vacuolation of the olfactory epithelium was noted in all animals in the 750 and 1500 ppm groups. Necrosis of the olfactory epithelium was observed in all animals in the 750 ppm group and 19/20 animals in the 1500 ppm group. Atrophy of the olfactory epithelium was noted in 4/20 animals in the 750 ppm group and 6/20 animals in the 1500 ppm group. The changes were found in the nasal sections taken at levels 3, 4, 5 and 6; however, the degenerative changes were most pronounced and consistently present at levels 3 and 4. Lesions found at levels 5 and 6 were typically less prominent than those at levels 3 & 4. The affected areas (the dorsal meatus, dorsal medial meatus and/or tips of the ethmoid turbinates) consisted of relatively small portions of the olfactory mucosal surface overall. No other exposure related lesions were noted at the study week 13 primary necropsy. Although vacuolation of the olfactory epithelium was noted in the control and 250 ppm groups, no differences were noted in the severity of findings between these groups. Therefore, this finding was not considered to be exposure related in the 250 ppm group. Other findings in the treated groups (myocardial inflammation, mineralization of the pulmonary vessels, hepatocellular cytoplasmic vacuolization, etc.) were present at a similar incidence in the control group or were considered to be common background lesions in rats.
At study week 21 recovery evaluation, microscopic examination of nasal tissues suggested substantial to complete recovery from the effects of n-butyl propionate. The olfactory epithelia in the treated groups were of normal height and cellularity, suggesting replacement of cells lost as a result of necrosis. Minimal amounts of necrosis of the olfactory epithelium were noted in 4/10 animals in the 1500 ppm group and minimal to mild degrees of vacuolation of the olfactory epithelium were noted in 2/5 and 3/5 males in the 250 and 1500 ppm groups, respectively. However, these changes were probably representative of histologic artifact due to the minimal degree and small number of cells involved (necrosis) or due to the lack of similar findings in the 750 ppm group (vacuolation). Other findings in the treated groups (dacryosolenitis, cytoplasmic hyalinization, squamous metaplasia, etc.) were seen at a similar incidence in the control grooup or were noted in single animals.

Effect levels

open allclose all
Dose descriptor:
NOEL
Effect level:
247 ppm (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: based on olfactory epithelium damage
Dose descriptor:
NOEL
Remarks:
systemic toxicity
Effect level:
747 ppm (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: based on decreased body weight gain at the highest test group

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:
In conclusion, exposure of rats to n-butyl propionate for 13 weeks resulted in reduced body weight gains and food consumption in the 1500 ppm group males essentially throughout the exposure period, slight and transient reductions in body weight gain in the 1500 ppm group females and histopathologic lesions of the nasal tissues in the 750 and 1500 ppm group animals. Based on the data obtained during the 8-week recovery (nonexposure) period, the effects on body weight gain and food consumption were completely reversible. The histopathological lesions showed substantial to complete reversibility at the end of the 8-week recovery period. Based on the exposure related lesions observed in the nasal tissues at the study week 13 primary necropsy, the no-observable effect level (NOEL) for olfactory epithelium damage was considered to be 247 ppm. The NOEL for systemic toxicity, based on decreased body weight gain was considered to be 747 ppm.
Executive summary:

In this 13 week study, n-butyl propionate was administered via whole-body inhalation to three groups, each comprise of 15 male and 15 female Sprague-Dawley Crl:CR BR rats, for a period of six hours per day, five days per week, for 13 consecutive weeks (minimum of 65 total exposures). The targeted exposure concentrations were 250, 750 and 1500 ppm (parts per million). The test atmosphere concentrations were monitored by a gas chromatographic method and were found to be 247, 747 and 1521 ppm. A concurrent control group of identical design received only filtered air, on a comparable regimen. The animals were observed for clinical signs and effects on body weight, food consumption and clinical pathology parameters. After 13 weeks of exposure, five rats/sex/group entered an eight week post-exposure observation period, after which they were euthanized; necropsies were performed, and selected organs were weighed. The remaining rats in each group were euthanized immediately following the exposure period and necropsied. A microscopic examination was conducted on selected tissues from all groups.

There were no exposure-related deaths during the study. No nBP-related clinical findings were noted during the exposure or recovery periods. Hematology and serum chemistry parameters were unaffected at any exposure level of nBP. Ophthalmologic examination revealed no test article-related effects. There were no exposure-related gross lesions at either necropsy. Organ weights were not affected by the test article.

Mean body weight gains and food consumption were reduced generally throughout the exposure period (study weeks 0-1 to 12-13) for males exposed to 1521 ppm. (At study week 12-13, mean body weight gain and food consumption in the 1521 ppm group males were decreased by 20% and 15%, respectively, when compared to the control group.) Food consumption and body weight gains in males exposed to 1521 ppm were similar to the control group during the recovery period. Females exposed to 1521 ppm experienced slight, transient reductions in body weight gain during study weeks 0-1 and 2-3.

Test article-related microscopic findings were limited to the nasal cavity. Both males and females exposed to 747 and 1521 ppm exhibited vacuolation, cell necrosis and/or atrophy of the olfactory epithelium. Substantial to complete recovery of the nasal tissues was observed at the study week 21 recovery evaluation. Olfactory epithelia in exposed animals after the recovery period were of normal height and cellularity, suggesting replacement of cells lost as a result of necrosis. Similar findings in the olfactory epithelium lining were also noted in animals of the dose range finding study and this was also substantiated by an independent Pathology Advisory Group.

Based on the exposure-related lesions observed in the nasal tissues at the primary necropsy after 13 weeks of exposure, the no observable effect level (NOEL) for olfactory epithelium damage was considered to be 247 ppm and, based on decreased body weight gain, the NOEL for systemic toxicity was considered to be 747 ppm.