Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted as per OECD TG 422, EPA OPPTS 870.3650 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:
2005
Report Date:
2005

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: USEPA OPPTS 870.3650: Combined Repeated Dose Toxicity Study With the Reproduction/ Developmental Toxicity Screening Test
Deviations:
no
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): n-propyl propionate
- Physical state: clear liquid
- Analytical purity: 99.8 ± 0.06% as measured by gas chromatography with thermal conductivity (GC/TCD), and corrected for water content (100.0% area percent-0.21% water).
- Lot/batch No.: SG06551G01

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Inc. (Portage, Michigan)
- Age at study initiation: 8 weeks
- Fasting period before study: no
- Housing: one per cage in stainless steel cages
- Diet: LabDiet Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, Missouri) in meal form, ad libitum, except during exposure
- Water: municipal water was provided ad libitum, except during exposure
- Acclimation period: at least one week prior to the start of the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): range of 22 ± 1°C (with a maximum permissible excursion range of ± 3°C)
- Humidity (%): 40-70%
- Air changes (per hr): Room air was exchanged approximately 12-15 times/hour.
- Photoperiod (hrs dark / hrs light): 12- hour light/dark photocycle

Administration / exposure

Route of administration:
inhalation
Type of inhalation exposure (if applicable):
whole body
Vehicle:
clean air
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 4m3 whole-body exposure chambers under dynamic airflow conditions
- Method of holding animals in test chamber: individually housed
- Source and rate of air: room air
- Method of conditioning air: The various concentrations of n-propyl propionate were generated using the glass J tube method. Liquid test material was pumped into the glass J tube assembly and vaporized by heated nitrogen gas passing through the bead bed of the glass J-tube (approximately 20-40 liters per minute). Nitrogen was heated as needed with a flameless heat torch (FHT-4, Master Appliance Corporatio n, Racine, Wisconsin) to the minimum extent necessary to vaporize the test material. The generation system was electrically grounded and the J-tubes were changed as needed. The nitrogen gas and n-propyl propionate vapors were diluted and mixed with room air to achieve an appropriate total flow rate of 900 liters per minute at the desired concentration of n-propyl propionate vapors.
- Temperature, humidity, pressure in air chamber: The chamber temperature and relative humidity were controlled by a system designed to maintain values of approximately 22 ± 3°C and 30-70%, respectively. The chambers were operated at a slight negative pressure relative to the surrounding area.
- Air flow rate: appropriate total flow rate of 900 liters per minute
- Air change rate: 12-15 air changes/hour
- Treatment of exhaust air: All test chamber exhausts were passed through an activated charcoal bed to remove test material from the exhaust system

TEST ATMOSPHERE
- Brief description of analytical method used: The chamber concentrations of n-propyl 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) at a wavelength determined pre-exposure. The chamber analytical concentrations were collected from the IR spectrometer, printed and stored using a CAMILE TGâ Data Acquisition and Control System (Camile Products, LLC, Indianapolis, Indiana). The IR spectrophotometer was calibrated and a standard curve was compiled prior to, at the midpoint, and at the end of the study using air standards prepared by vaporizing measured volumes of n-propyl propionate into Tedlar sample bags (Series 233, SKC, Eighty Four, Pennsylvania) along with the metered volumes of dry, compressed air. Analytical concentrations during the exposures were interpolated using the standard curve. The analytical system was checked prior to each exposure with a n-propyl propionate standard of known concentration. The CAMILE TGÒ Data Acquisition and Control System toggled the IR spectrophotometer between the chambers for concentration sampling. The nominal concentration of the test material in each chamber was calculated based on the amount of test material used each day and the total airflow through the chamber for each exposure period. Each of the chambers was checked pre-exposure to ensure that a uniform distribution of vapor was present in the breathing zone of the animals.
- Samples taken from breathing zone: yes
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 2 weeks
- Proof of pregnancy: [vaginal plug or sperm in vaginal smear] referred to as [day 0] of pregnancy
- After 14 days of unsuccessful pairing no replacement of first male by another male with proven fertility
- Further matings after two unsuccessful attempts: [no]
- After successful mating each pregnant female was caged: individual
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Nominal concentrations - 0, 50, 250, and 500 ppm
Mean chamber concentrations - 0, 50.8 ± 1.5, 253.3 ± 2.2, and 504.1 ± 7.0 ppm
Duration of treatment / exposure:
Males were exposed six hours per day for at least two weeks prior to mating and continued throughout mating until necropsy for a total of 37 days. Females were exposed six hours per day for 14 days pre-mating, continued throughout mating (two weeks) and gestation (three weeks through gestation day 20).
Frequency of treatment:
six hours per day, seven days/week
Details on study schedule:
Females were observed for signs of parturition beginning on or about GD 20. In so far as possible, parturition was observed for signs of difficulty or unusual duration. The day of parturition was recorded as the first day the presence of the litter was noted and was designated as LD 0. Litters were examined as soon as possible after delivery. The following information was recorded on each litter: the date of parturition, litter size on the day of parturition (LD 0), the number of live and dead pups on days 0, 1, and 4 postpartum, and the sex and the weight of each pup on LD 1 and 4. Any visible physical abnormalities or demeanor changes in the neonates were recorded as they were observed during the lactation period. Any pups found dead were sexed and examined grossly, if possible, for external and visceral defects and then were discarded.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 50, 250, and 500 ppm (nominal concentrations)
Basis:
other: 0, 50.8 ± 1.5, 253.3 ± 2.2, and 504.1 ± 7.0 ppm (analytical concentrations)
No. of animals per sex per dose:
12 male and 12 female Crl:CD(SD) rats/dose
Control animals:
yes, sham-exposed
Details on study design:
- Dose selection rationale: based on previous study (Supporting study (2)_Repeated dose toxicity: inhalation (14 days)_duPont_2000) and range finding study
- Rationale for animal assignment: random
Positive control:
not applicable

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: on all animals once daily throughout the study

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations (DCO) were conducted on all rats pre-exposure and weekly throughout the study. Mated females received DCO examinations on GD 0, 7, 14, and 20, and lactation day (LD) 3. The DCO was conducted at approximately the same time each examination day, according to an established format. The examination included cage-side, hand-held and open-field observations, which were recorded categorically or using explicitly defined scales (ranks).

BODY WEIGHT: Yes
- Time schedule for examinations: All rats were weighed at least once during the pre-exposure period and on the first day of exposure. Body weights for males were recorded weekly throughout the study. Females were weighed weekly during the pre-breeding period. During gestation, females were weighed on GD 0, 7, 14, and 20. Females that delivered litters were weighed on LD 1 and 4. Females that failed to mate or deliver a litter were not weighed during the gestation or lactation phases. Body weight gains were determined for the following intervals: GD 0-7, 7-14, 14-20, 0-20 and LD 1-4.

FOOD CONSUMPTION: Yes
- Feed consumption was determined pre-exposure and weekly during the two-week prebreeding period for all animals by weighing feed containers at the start and end of a measurement cycle. During breeding, feed consumption was not measured in males or females due to co- housing. Following breeding, feed consumption for males was not measured. For mated females, feed consumption was measured on GD 0, 7, 14 and 20. For females delivering litters, feed consumption was measured on LD 1 and 4. Feed consumption was not measured for females that failed to mate or deliver a litter.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: after last exposure for males and after lactation day 4 for females
- Anaesthetic used for blood collection: Yes - CO2
- Animals fasted: Yes
- How many animals: all animals, except from animals that were euthanized in a moribund condition prior to their scheduled necropsy
- Parameters examined: Blood samples were mixed with ethylenediamine-tetraacetic acid (EDTA), smears were prepared, stained with Wright-Giemsa stain, cover-slipped and filed for evaluation at the discretion of the pathologist. Hematologic parameters were assayed using a Advia 120 Hematology Analyzer (Bayer Corporation, Tarrytown, New York) - Hematocrit (HCT), Hemoglobin (HGB) concentration, Red blood cell (RBC) count, Total white blood cell (WBC) count, Differential WBC count, Platelet (PLAT) count, Reticulocyte (RET) count, RBC indices: Mean Corpuscular Hemoglobin (MCH), Mean Corpuscular Volume (MCV), Mean Corpuscular Hemoglobin Concentration (MCHC), Coagulation - Sample Preparation - Blood samples were collected in sodium citrate tubes, centrifuged and plasma collected and assayed using an ACL9000 (Instrumentation Laboratory, Lexington, Massachusetts). Assay - Prothrombin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after last exposure for males and after lactation day 4 for females
- Animals fasted: Yes
- How many animals: all animals
- Parameters examined: Blood samples were collected and serum was 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), Cholesterol (CHOL), Creatinine (CREA), Electrolytes (NA, K, PHOS, CL and CA), Glucose (GLU), Total bilirubin (TBIL), Total protein (TP), Urea nitrogen (UN)

URINALYSIS: Yes
- Time schedule for collection of urine: Urine samples were obtained from all males the week prior to the scheduled necropsy
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined: The following parameters were examined: Color, appearance, specific gravity (refractometer) and urine volume. Semiquantitative analyses (MultistixÒ Reagent Strips, Bayer Corporation, Elkhardt, Indiana on the Clinitek 200+) of: pH, Bilirubin, Glucose, Protein, Ketones, Blood, Urobilinogen and for microscopic examination - urine samples were also collected from each male by manual compression of the urinary bladder. The urine samples were pooled from each group, and the microsediments were characterized microscopically.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: The functional tests (sensory evaluation, rectal temperature, grip performance and motor activity) were conducted pre-exposure and during the last week of exposures. Postexposure testing in the females took place on LD 4.
- Dose groups that were examined: all groups
Oestrous cyclicity (parental animals):
not examined
Sperm parameters (parental animals):
not examined
Litter observations:
Females were observed for signs of parturition beginning on or about GD 20. In so far as possible, parturition was observed for signs of difficulty or unusual duration. The day of parturition was recorded as the first day the presence of the litter was noted and was designated as LD 0. Litters were examined as soon as possible after delivery. The following information was recorded on each litter: the date of parturition, litter size on the day of parturition (LD 0), the number of live and dead pups on days 0, 1, and 4 postpartum, and the sex and the weight of each pup on LD 1 and 4. Any visible physical abnormalities or demeanor changes in the neonates were recorded as they were observed during the lactation period. Any pups found dead were sexed and examined grossly, if possible, for external and visceral defects and then were discarded.
Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Postmortem examinations (offspring):
Litters were examined as soon as possible after delivery. The following information was recorded on each litter: the date of parturition, litter size on
the day of parturition (LD 0), the number of live and dead pups on days 0, 1, and 4 postpartum, and the sex and the weight of each pup on LD 1 and 4. Any visible physical abnormalities or demeanor changes in the neonates were recorded as they were observed during the lactation period.Any pups found dead were sexed and examined grossly, if possible, for external and visceral defects and then were discarded. All pups surviving to LD 4 were euthanized by oral administration of sodium pentobarbital solution, examined for gross external alterations, and then discarded
Statistics:
Standard statistical methods were employed
Reproductive indices:
Female mating index, Male mating index, female conception index, male conception index, female fertility index, male fertility index and gestation index, post implantation index
Offspring viability indices:
gestation survival index, day 1 and 4 pup survival index

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
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
Description (incidence and severity):
Male and female rats exposed to 50, 250 or 500 ppm n-propyl propionate had a degeneration of the olfactory epithelium of the nasal turbinates that was very slight in degree, focal or multifocal and unilateral or bilateral in distribution.
Other effects:
not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY - No treatment-related effects on behavior or demeanor were observed in any phase of the study at any exposure level. In addition, there were no treatment-related clinical observations or external morphological anomalies in the offspring. Male (2218) was euthanized on day 22 of test following an accidental injury to the tail and concern for humane treatment. All remaining animals survived until termination.

BODY WEIGHT AND WEIGHT GAIN - There were no effects on male body weights at any exposure concentration at any time. The body weights of females in the 250 and 500 ppm group were slightly lower (4-7%) than those of controls during the pre-mating phase, corresponding with decreased feed consumption in these animals during this phase of the study. Body weights of the latter females were also decreased slightly during the first two weeks of gestation. These lower body weights were statistically identified on pre-mating day 14 and gestation days 7 and 14 in the 500 ppm females, but only on pre- mating day 14 in the 250 ppm females. Slight, non-statistically significant decreases (3-6%) in body weights of the 250 and 500 ppm females were also evident on lactation day 1 and 4. Consistent with the very slight nature of the body weight effects in the 250 and 500 ppm group females, there were no statistically significant effects on gestation or lactation body weight gains in these groups.

FOOD CONSUMPTION - There were no treatment-related effects on male feed consumption at any time in any of the exposure groups. During the pre-mating phase, mean feed consumption of females in the 250 and 500 ppm groups was slightly lower (7-8%) than that of control females (Text Table 2). Although only the value for the 500 ppm group females on days 7-14 was statistically identified, these slight decreases in food consumption correlated with slightly decreased body weights during the same period and, thus, were considered treatment related. There were no statistically significant effects on food consumption during gestation or lactation in the 250 or 500 pm group females, nor at any time in the 50 ppm group females.

HAEMATOLOGY - There were no treatment related hematologic effects at any exposure level.

CLINICAL CHEMISTRY - There were no treatment-related changes for males and females at any exposure level. Males exposed to 500 ppm had a statistically identified increase in aspartate aminotransferase activity that was slightly outside of the historical control range for our laboratory. A similar increase in this enzyme did not occur in males at lower dose levels or females at any dose level. This slightly higher enzyme activity was interpreted not to be toxicologically significant due to the absence of microscopic necrosis in the heart, liver and/or skeletal muscle and the fact that significant increases in aspartate aminotransferase activity are normally associated with 2- to 3-fold increases in activity, rather than the minor increase observed in this study.

URINALYSIS - There were no treatment-related changes at any exposure level.

NEUROBEHAVIOUR - There were no treatment-related changes at any exposure level.

ORGAN WEIGHTS - There were no treatment-related alterations in the final body weights and organ weights of males at any exposure level. However, the final body weights of females of the 500 ppm group were significantly lower (8%) than those of controls. Males exposed to 50 or 500 ppm had statistically identified increases in absolute and relative adrenal weights. These adrenal weight differences were interpreted not to be treatment related because the weights were within the historical control range, there was no clear dose-response relationship, there was a lack of a similar response in the females, and there was no histopathologic correlate to the decreased adrenal weights.

GROSS PATHOLOGY - Five females exposed to 500 ppm had a pale focus involving the right medial lobe of the liver, compared to an incidence of zero in the control females. This alteration is commonly observed in rats in this specific location, consists of a very focal vacuolation of hepatocytes and is also known as tension lipidosis. This alteration was interpreted to be a spontaneous change that has no toxicologic significance, and was not related to exposure to n-propyl propionate. There were no treatment-related gross pathologic observations. One male (2218) exposed to 50 ppm was euthanized (Day 22) due to a traumatic injury to the tail.

HISTOPATHOLOGY - Male and female rats exposed to 50, 250 or 500 ppm n-propyl propionate had a degeneration of the olfactory epithelium of the nasal turbinates that was very slight in degree, focal or multifocal and unilateral or bilateral in distribution. The incidence of olfactory degeneration increased with exposure concentration. These degenerative changes were primarily observed in Level 2 of the nasal turbinates and affected the medial and lateral aspects of the dorsal portion of the nasoturbinates turbinates and infrequently the maxilloturbinates (Level 1) and ethmoturbinates (Level 3 and 4). Females exposed to 500 ppm also had very slight degenerative effects involving olfactory epithelium in Level 1. Very slight, focal or multifocal, squamous metaplasia of olfactory or respiratory epithelium was also noted in two males exposed to 500 ppm, two females exposed to 50 ppm and two females exposed to 250 ppm. These effects were interpreted to be primary treatment-related effects that were attributed to n-propyl propionate.
Males exposed to 500 ppm and females exposed to ³ 50 ppm had a higher incidence of adipose tissue atrophy than the controls. The toxicologic significance of these observations was questionable given that they were graded as very slight or slight, and they were not associated with any marked changes in body weights.

REPRODUCTIVE EFFECTS:
There were no treatment-related effects at any exposure level on reproductive indices, time to mating, gestation length, post-implantation loss, pup survival, or pup sex ratio. Although there was a statistically identified decrease in postnatal day (PND) 4 survival, this was not considered to be toxicologically significant as the values were well within the laboratory’s historical control range (Text Table 7) and the statistical significance was largely influenced by the very high value of 100% in the control group. In fact, only four pups died during the early postnatal period, which is not unexpected for this sample size.In addition, there were no statistically identified differences on litter size, PND 1 survival, or other reproductive end points that would be indicative of a reproductive effect.

Effect levels (P0)

open allclose all
Dose descriptor:
NOEC
Effect level:
500 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on no reproductive effects and no effects on neurological function observed at 500 ppm
Dose descriptor:
LOEC
Effect level:
50 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

There were no treatment-related effects at any exposure level on reproductive indices, time to mating, gestation length, post-implantation loss, pup survival, or pup sex ratio. Although there was a statistically identified decrease in postnatal day (PND) 4 survival, this was not considered to be toxicologically significant as the values were well within the laboratory’s historical control range and the statistical significance was largely influenced by the very high value of 100% in the control group. In fact, only four pups died during the early postnatal period, which is not unexpected for this sample size.In addition, there were no statistically identified differences on litter size, PND 1 survival, or other reproductive end points that would be indicative of a reproductive effect. There were no treatment related effects on litter or pup body weights at any exposure level tested.

Effect levels (F1)

Dose descriptor:
NOEC
Generation:
F1
Effect level:
500 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: There were no treatment-related clinical observations or external morphological anomalies in the offspring. There were no treatmentrelated effects on litter or pup body weights at any exposure level tested.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:
Exposure to n-propyl propionate resulted in slight reductions in food consumption and body weight throughout the study in females exposed to 250 and 500 ppm. Treatmentrelated histopathologic effects were also evident in the nasal tissues of males and females exposed to all test concentrations. The nasal tissue effects consisted of very slight degeneration of the olfactory epithelium. In addition males exposed to 500 ppm and females exposed to >= 50 ppm had a higher incidence of adipose tissue atrophy than the controls. This was interpreted to have questionable significance. No treatment-related effects were seen in reproductive performance, pup survival and growth, neurologic function, clinical chemistry, or hematology. Therefore, the no-observed-effect concentration (NOEC) for general toxicity could not be determined for male and female rats. The NOEC for reproductive effects and neurological function was 500 ppm, the highest concentration tested.
Executive summary:

This study evaluated n-propyl propionate in the OECD 422 study design. Groups of 12 male and 12 female Crl:CD(SD) rats were whole-body exposed to target concentrations of 0, 50, 250 or 500 ppm vaporized n-propyl propionate for six hourslday, seven dayslweek. Female rats were exposed daily for two weeks prior to breeding, through breeding (up to two weeks), and continuing through gestation day 20. Females were necropsied on post-partum day 5. The males were exposed for two weeks prior to breeding and continuing through breeding (two weeks) until necropsy (test day 38). Effects on reproductive and neurological function as well as general toxicity were evaluated. In addition, post mortem examinations included a gross necropsy of the adults with collection of organ weights and extensive histopathologic examination of tissues. Litter size, pup survival, sex, body weight, and the presence of gross external abnormalities were also assessed.

Exposure to n-propyl propionate resulted in slight reductions in food consumption and body weight throughout the study in females exposed to 250 and 500 ppm. Treatment-related histopathologic effects were also evident in the nasal tissues of males and females exposed to all test concentrations. The nasal tissue effects consisted of very slight degeneration of the olfactory epithelium. In addition males exposed to 500 ppm and females exposed to >= 50 ppm had a higher incidence of adipose tissue atrophy than the controls. This was interpreted to have questionable significance. No treatment-related effects were seen in reproductive performance, pup survival and growth, neurologic function, clinical chemistry, or hematology parameters.

Therefore, the no-observed-effect concentration (NOEC) for general toxicity could not be determined for male and female rats. The NOEC for reproductive effects and neurological function was 500 ppm, the highest concentration tested.