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Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to OECD TG 412, EU Method B.8 and in accordance with the Principles of Good Laboratory Practice (GLP).
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:
no
Remarks:
The GLP status of the certificate of anaylsis for the analytical standard used for the determination of exposure chamber concentration could not be verified
Qualifier:
according to
Guideline:
EU Method B.8 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:
no
Remarks:
same as above
Principles of method if other than guideline:
not applicable
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): UCAR n-pentyl propionate
- Molecular formula: C8H16O2
- Molecular weight: 144.2
- Physical state: liquid
- Analytical purity: 99.973% ± 0.001% by gas chromatography
- Impurities (identity and concentrations): 273 ± 13 ppm water by Karl Fisher coulometric titration
- Lot/batch No.: lot # WL0901FTC1

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc.
- Age at study initiation: 8-9 weeks
- Housing: group housed (2-3 rats/cage) during acclimation and individually housed in stainless steel cages after randomization
- Diet (e.g. ad libitum): Animals were provided LabDiet Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, Missouri) in meal form, ad libitum, except during exposure
- Water (e.g. ad libitum): Municipal water was provided ad libitum
- Acclimation period: one week prior to start of the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 1°C
- Humidity (%): 40-70%
- Air changes (per hr): 12-15 times/hour
- Photoperiod (hrs dark / hrs light): 12-hour light/dark

Administration / exposure

Route of administration:
inhalation
Type of inhalation exposure:
whole body
Vehicle:
not specified
Remarks on MMAD:
MMAD / GSD: not applicable
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: The animals were exposed to filtered air or n-pentyl propionate vapors in 2 m3 stainless steel and glass Rochester-type whole body exposure chambers (1.3 meters x 1.2 meters wide x 1.2 meters deep with a pyramidal top and bottom).
- Method of holding animals in test chamber: Rats were singly-housed to minimize crowding during the exposure
- Source and rate of air: filtered air
- Chamber airflow was maintained at approximately 450 liters per minute. This flow rate was sufficient to provide the normal concentration of oxygen to the animals and 12-15 calculated air changes per hour. The chambers were operated at a slightly negative pressure, relative to the surrounding area. Rats were singly-housed to minimize crowding during the exposure. The time to equilibrium (T99) was 20.5 minutes.
The various concentrations of n-pentyl propionate were generated using the glass J-tube method. Liquid test material was pumped into the glass J-tube assemblies (1 per exposure chamber) and vaporized by the flow of nitrogen gas passing through the bead bed of the glass J-tube. The nitrogen was heated as needed with a flameless heat torch (FHT-4, Master Appliance Corporation, Racine, Wisconsin) to the minimum extent necessary to vaporize the test material. All chambers, including the 0 ppm (control) chamber received the same amount (35 liters per minute) of supplemental nitrogen (carrier gas). The minimum amount of nitrogen necessary to reach the desired chamber concentrations was used. The generation system was electrically grounded and the J-tubes were changed as needed. The vaporized test material and carrier gas were mixed and diluted with supply air to achieve a total flow of 450 liters per minute at the desired test chamber concentration.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The chamber concentrations of n-pentyl propionate, measured approximately in the center of the breathing zone of the animals, were determined at least once per hour with a Miran 1A infrared (IR) spectrophotometer (Foxboro/Wilks, South Norwalk, Connecticut). The chamber analytical concentrations were collected from IR, printed and stored using a CAMILE data acquisition and control system. The IR spectrophotometer was calibrated and a standard curve was compiled prior to and at the end of the study, with standards of vaporized test material generated dynamically in the chamber in which the animals were exposed to cover the range of exposure concentrations tested. The IRdetermined concentration during each exposure was interpolated from the standard curve. The IR system was checked prior to each exposure with a standard of known concentration. Output from the infrared spectrophotometer was monitored by the CAMILE TG Data Acquisition and Control System, displayed, logged to memory, and printed for inclusion in the study file.
To determine the actual concentrations of n-pentyl propionate within the exposure chambers a pump and tube sampling method was employed. A 780- mg silica gel tube (SKC Cat# 226-15) was connected directly to the exposure chamber and chamber atmosphere was drawn through for 10 minutes at ~40 mL/min. The tubes were desorbed in acetone and the extracts analyzed for n-pentyl propionate using gas chromatography with flame ionization detection (GC/FID). At least weekly, a sample from each exposure chamber was taken using the tube method described above. These samples served as exposure chamber reference standards and were used to derive a weekly correction factor that was applied to all IR-determined values collected that week (correction factor = mean weekly chamber concentration determined via the tube method/mean weekly chamber concentration based on the IR measurements).
Airflow through the chambers was determined by measuring the pressure drop across a calibrated orifice plate, and was maintained at approximately 450 liters per minute. Chamber airflow data were collected using Setra Differential Pressure Transducers (Setra Systems, Inc., Monterey, California). The signal from the pressure transducer was sent to the CAMILE TG Acquisition and Control System and recorded in liters per minute. The differential pressure transducer was calibrated with a gas meter (Singer Aluminum Diaphragm Meter, Model AL-1400, American Meter Division, Philadelphia, Pennsylvania) prior to the start of the study. Chamber temperature and relative humidity data were collected using Omega HX94C Probes (Omega Engineering Inc., Stamford, Connecticut) coupled to the CAMILE TG Data Acquisition and Control System. Calibration of the temperature and relative humidity sensors was conducted prior to the start of the study. The chamber temperature and relative humidity were controlled by a system designed to maintain values of approximately 22 ± 2 °C and 30 - 70%, respectively. Chamber temperature, relative humidity, and airflow data were automatically recorded from the CAMILE TG Data Acquisition and Control System once per hour for all chambers with the exception of the airflow for the control (0 ppm) chamber. The control (0 ppm) chamber was not hard-wired to CAMILE to allow automatic collection of airflow data. Therefore, for the control chamber, once per hour chamber airflow data were manually recorded from a calibrated magnehelic in the chamber exhaust stream.
Duration of treatment / exposure:
6 hours/day, 5 days/week for 4 consecutive weeks (20 exposures)
Frequency of treatment:
6 hours/day, 5 days/week for 4 consecutive weeks (20 exposures)
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 ppm (chamber concentration)
Basis:
other: 0 pppm (nominal and analytical concentration)
Remarks:
Doses / Concentrations:
100 ppm (chamber concentration)
Basis:
other: 141.0 ± 3.3 ppm (nominal concentration); 116.3 ± 4.5 ppm (analytical concentration)
Remarks:
Doses / Concentrations:
300 ppm (chamber concentration)
Basis:
other: 443.9 ± 43.3 ppm (nominal concentration); 350.0 ± 24.6 ppm (analytical concentration)
Remarks:
Doses / Concentrations:
1000 ppm (chamber concentration)
Basis:
other: 1462.2 ± 29.6 ppm (nominal concentration); 1375.0 ± 195.2 ppm (analytical concentration)
No. of animals per sex per dose:
5 males + 5 females/dose group
Control animals:
yes, sham-exposed
Details on study design:
- Dose selection rationale: based on studies conducted earlier
- Rationale for animal assignment: animals were stratified by body weight and then randomly assigned to treatment groups using a computer program designed.
- Rationale for selecting satellite groups: not applicable
- Post-exposure recovery period in satellite groups: not applicable
- Section schedule rationale: not applicable
Positive control:
not applicable

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once a day usually in the morning

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: on all animals pre-exposure and once per week throughout the study

BODY WEIGHT: Yes
- Time schedule for examinations: All rats were weighed pre-exposure and twice per week during the exposure period. Body weight gains were calculated

FOOD CONSUMPTION:
- Food consumption for each animal determined: Yes. Feed consumed was determined at least weekly for all animals.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at termination
- Anaesthetic used for blood collection: Yes (O2/CO2)
- Animals fasted: Yes
- How many animals: all animals
- Parameters examined: Hematologic parameters were assayed using the Bayer Advia 120 Hematology Analyzer (Siemens Medical Solutions Diagnostics, Tarrytown, New York). Hematocrit (HCT), Hemoglobin (HGB) concentration, Red blood cell (RBC) count, Total white blood cell (WBC) count, Differential WBC count, Platelet (PLT) count, Reticulocyte (RET) count, RBC indices: Mean Corpuscular Hemoglobin (MCH), Mean Corpuscular Volume (MCV), Mean Corpuscular Hemoglobin Concentration (MCHC) and Coagulation - Prothrombin time (Blood samples were collected in sodium citrate tubes, centrifuged, plasma collected, and assayed using the Instrumentation Laboratory ACL9000 Analyzer (Beckman Coulter, Inc., Miami, Florida). Blood samples for a complete blood count were mixed with ethylenediaminetetraacetic acid (EDTA). Blood smears were prepared, stained with Wright- Giemsa stain, cover-slipped and archived for potential future evaluation if warranted.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at termination
- Animals fasted: Yes
- How many animals: all animals
- Parameters examined: Blood samples were collected and sera were separated from cells as soon as possible. Serum parameters were measured using a Hitachi 912 Clinical Chemistry Analyzer (Roche Diagnostics, Indianapolis, Indiana). Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Gamma-glutamyl transpeptidase (GGT), Albumin (ALB), Albumin/Globulin Ratio (A/G) - calculated, Cholesterol (CHOL), Creatinine (CREA),
Electrolytes (NA, K, PHOS, CL and CA), Globulin (GLOB) - calculated, Glucose (GLUC), Total bilirubin (TBIL), Total protein (TP), Triglycerides (TRIG), Urea nitrogen (UN)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
OTHER: Organ weights (Absolute and relative)
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):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
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:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY - No treatment related clinical findings were noted in any of the animals and there were no incidences of mortality noted

BODY WEIGHT AND WEIGHT GAIN - Although no time-sex-dose interaction was identified, when the data for male and female rats were combined (time-dose interaction), rats exposed to 1375 ppm had body weights that were statistically identified as lower than their respective controls. There were no statistically-significant differences in the 116 or 350 ppm exposed groups when compared to controls.

FOOD CONSUMPTION - Statistically identified lower mean feed consumption values were observed on test days 1-5 in male rats exposed to 350 and 1375 ppm when compared to their respective controls. This decrease in feed consumption was transient resolving by the second week of the study and correlated with
lower body weight gains in these groups. There were no significant differences in the amount of feed consumed for any other groups when compared to their respective controls.

HAEMATOLOGY - Males and females exposed to 116 ppm, 350 ppm or 1375 ppm had no treatment-related effect when compared to their respective controls

CLINICAL CHEMISTRY - There were no treatment related changes in any of the clinical chemistry parameters for males and females at any exposure level. However, a very slight increase in the mean chloride content was observed in females at 116 ppm. This alteration was interpreted to be unrelated to treatment because of the lack of a dose-response relationship

ORGAN WEIGHTS - There were no statistically identified or treatment-related alterations in any of the organ weights of any exposure group

GROSS PATHOLOGY - There were no treatment-related gross pathologic observations.

HISTOPATHOLOGY - Histopathologic changes attributed to repeated inhalation exposure to n-pentyl propionate were present in sections II and III of the nasal passage. The treatment-related lesions were slight, bilateral, multifocal olfactory epithelial degeneration of the the dorsal and dorso-medial meatus of all males and females exposed to 1375 ppm. The degeneration was characterized by thinning, disarray and atrophy of the olfactory epithelial cells. Individual necrotic and sloughed epithelial cells accompanied the degenerative change. Similar changes were also present in three females exposed to 350 ppm. Very slight, unilateral, focal olfactory epithelial degeneration occurred in one male and one female, exposed to 350 ppm.
Slight, unilateral, multifocal, olfactory epithelial degeneration with minimal neutrophilic inflammation was present in the nasoturbinate of section II and III of one control male, one low-dose (116 ppm) male and in one mid-dose (350 ppm) female. These unilateral changes in the control, low-and mid-dose rats were considered unrelated when compared to the anatomic distribution and the histopathology of the treatment-related change (slight, bilateral, multifocal olfactory epithelial degeneration of the the dorsal and dorso-medial meatus).

Effect levels

Dose descriptor:
NOEC
Remarks:
Crl:CD (SD) rats
Effect level:
116 ppm (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: based on overall effects and the presene of treatment related histopathologic changes in the nasal airways of male and female rats exposed to 350 and 1375 ppm n-pentyl propionate, equivalent to 686 mg/m3

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:
Based on the presence of treatment-related histopathologic alterations in the nasal passage of male and female rats exposed to 350 and 1375 ppm n-pentyl propionate, the no-observed-effect concentration (NOEC) for male and female Crl:CD(SD) rats repeatedly exposed to n-pentyl propionate for four weeks (20 exposures) was 116 ppm.
Executive summary:

Five male and five female Crl:CD(SD) rats per group were whole-body exposed to 0, 116, 350, or 1375 ppm (parts per million) (0, 686, 2065, or 8111 mg/m3, respectively) of n-pentyl propionate (6 hours/day, 5 days/week, 4 consecutive weeks) for 20 exposures to evaluate the potential for respiratory tract and systemic toxicity. Exposures occurred under dynamic airflow conditions. In-life observations, feed consumption, body weights, hematology, clinical chemistry, coagulation, and organ weights were evaluated. In addition, a gross necropsy was conducted followed by an extensive histopathological examination of tissues.

There were no treatment-related clinical observations, nor effects in hematology, prothrombin time, or clinical chemistry parameters. There were no treatment related gross pathologic observations. Although no time-sex-dose interaction was identified, when the data for male and female rats were combined (time-dose interaction), rats exposed to 1375 ppm had body weights that were statistically identified as lower than their respective controls. Statistically identified lower mean feed consumption values were observed on test days 1-5 in male rats exposed to 350 and 1375 ppm when compared to their respective controls. This decrease in feed consumption was transient resolving by the second week of the study and correlated with lower body weight gains seen in these groups.

Histopathologic changes attributed to repeated inhalation exposure to n-pentyl propionate were present in the nasal passage. The treatment-related lesions were slight, bilateral, multifocal olfactory epithelial degeneration of the dorsal and dorso-medial meatus of all males and females exposed to 1375 ppm. The degeneration was characterized by thinning, disarray and atrophy of the olfactory epithelial cells. Individual necrotic and sloughed epithelial cells accompanied the degenerative change. Similar changes were also present in three females exposed to 350 ppm. Very slight, unilateral, focal olfactory epithelial degeneration occurred in one male and one female, exposed to 350 ppm.

Based on the presence of treatment-related histopathologic alterations in the nasal airways of male and female rats exposed to 350 and 1375 ppm n-pentyl propionate, the no-observed-effect concentration (NOEC) for male and female Crl:CD(SD) rats repeatedly exposed to n-pentyl propionate for four weeks (20 exposures) was 116 ppm.