Butyl propionate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to EPA OTS 798.4900 (Prenatal Developmental Toxicity Study) and in accordance with the Principles of Good Laboratory Practice (GLP)

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EPA OTS 798.4900 (Prenatal Developmental Toxicity Study)

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

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 propanoate

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

Details on test animals or test system and environmental conditions:

- Source: Charles River Laboratories Inc.
- Age at study initiation: approximately 10 weeks old at receipt and approximately 12 weeks when paired for breeding
- Weight at study initiation: minimum of 220 grams
- Housing: individually housed and the animals were paired for mating in the home cage of the male.
- Diet: ad libitum, Purina Certified Rodent Chow #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): 70.0 °F to 73.22 °F
- Humidity (%): 33.7-57.8%
- Air changes (per hr): 10 fresh air changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours light/dark cycle

Administration / exposure

Route of administration:

inhalation

Type of inhalation exposure (if applicable):

whole body

Vehicle:

unchanged (no vehicle)

Details on 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 (24 mm ID glass tube approximately 20 cm long) filled with 8 and 12-mm glass beads. The vaporization column was wrapped with a 206-watt flexible 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 FID and a 3396 Series II integrator
Detector: Flame ionization at 250 °C
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:

Target conentrations - 0, 500, 1000 and 2000 ppm
Daily nominal concentrations means for the entire study - 0, 542, 1159 and 2116 ppm
Mean analytical concentrations - 0, 495, 1011 and 2000 ppm

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage: 1:1 male:female
- Further matings after two unsuccessful attempts: no
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

10 consecutive days (gestation days 6-15)

Frequency of treatment:

6 hours/day

Duration of test:

April 23, 1996 - July 3, 1996

No. of animals per sex per dose:

24 Sprague Dawley rats/dose

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 concentrations of 500, 1000 and 2000 ppm were
selected for the definitive developmental toxicity study of n-Butyl Propionate in rats.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for moribundity and mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily prior to exposure, during the exposure period and one hour following completion of the exposure period.

BODY WEIGHT: Yes
- Time schedule for examinations: maternal body weights were recorded on gestation days 0, 6, 16 and 20

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

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: the thoracic, abdominal and pelvic cavities were opened by a ventral midline incision and the contents were examined

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
-Other: Uteri with no macroscopic evidence of nidation were excised, opened and subsequently placed in 10% ammonium sulfide solution for detection of early implantation loss.

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: all per litter

Statistics:

Standard statistical methods were employed

Indices:

Postimplantation loss on group mean litter and a proportional litter basis, and fetal devlopmental findings were summarized

Historical control data:

Yes

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
All maternal animals survived to the scheduled necropsy on gestation day 20. The predominant exposure-related clinical signs observed during exposure included dose-related incidences of slightly drooping eyelids and salivation in the 1000 and 2000 ppm groups. Sporadic occurrences of half-closed (and/or completely closed) eyelids and lacrimation also were observed in these animals during exposure. Wet tan or yellow matting on various body surfaces was noted primarily in the 2000 ppm group one hour following exposure on the first two days of test article administration. Other clinical signs noted in the treated groups were observed infrequently and/or at a similar frequency in the control group.

In the 500, 1000 and 2000 ppm groups, slightly reduced mean body weight gains (500 and 1000 ppm groups) or a mean body weight loss (2000 ppm group) occurred during gestation days 6-9. The differences from the control group were statistically significant p < 0.05 or p < 0.01). During the remainder of the exposure period (gestation days 9-12 and 12-16) and the post-exposure period (gestation days 16-20), mean body weight gains in the exposed groups were similar to the control group values. When the entire treatment period (gestation days 6-16) was evaluated, mean body weight gains in all of the exposed groups were slightly reduced relative to the control group value; the differences in the 1000 and 2000 ppm groups were statistically significant p < 0.01). Mean body weights in all of the exposed groups were slightly reduced from gestation days 7 through 20; the differences from the control group values were often statistically significant p < 0.05 or p < 0.01). Slight reductions in net body weight gain were observed in the 1000 and 2000 ppm groups relative to the control group value; the difference between the control and 2000 ppm groups was statistically significant p < 0.05). Mean gravid uterine weight and net body weight (the day 20 body weight minus the weight of the uterus and contents) were unaffected by treatment with the test article. Differences between the control and treated groups were slight and were not statistically significant.

Food consumption, evaluated as g/animal/day and g/kg/day, was slightly reduced in a dose-related manner in all of the treated groups relative to the control group values during gestation days 6-9, 9-12 and 12-16, and when the entire treatment period (gestation days 6-16) was evaluated. The differences from the control group values were generally statistically significant (p < 0.05 or p < 0.01). During the post-treatment period (gestation days 16-20), food consumption in the treated groups was similar to that in the control group; differences were slight and were not statistically significant. At the scheduled necropsy, no treatment-related findings were observed at any exposure level. Dark red lungs (all lobes) were observed in animal nos. 48997 and 49026 in the 1000 and 2000 ppm groups, respectively. In the 500 ppm group, animal no. 48965 had reddening of the cervical lymph nodes. No other remarkable internal findings were noted in animals in the treated groups.

Effect levels (maternal animals)

Dose descriptor:

LOAEL

Effect level:

500 ppm (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Intrauterine growth and survival were unaffected by treatment with the test article at exposure levels of 500, 1000 and 2000 ppm. Parameters evaluated included postimplantation loss, live litter size, fetal body weights, fetal sex ratios and the numbers of corpora lutea and implantation sites. Differences between the control and treated groups were slight and were not statistically significant.

The numbers of fetuses (litters) available for fetal morphological examination were 372(24), 3 18(22), 341 (24) and 353(24) in the control, 500, 1000 and 2000 ppm groups, respectively. The numbers of fetuses (litters) with malformations were 2(2), 0(0), 4(4) and l(1) in these same exposure groups, respectively.

External malformations were observed in 3 and 1 fetuses in the 1000 and 2000 ppm groups, respectively. Fetus nos. 49001-06 and 49030-06 in the 1000 and 2000 ppm groups, respectively, had vertebral agenesis. Fetus nos, 49017-01 and 49062-03 in the 1000 ppm group had microphthalrnia (unilateral or bilateral). No external developmental variations were observed in fetuses at any exposure level.

Soft tissue malformations were observed in two fetuses each in the control and 1000 ppm groups. Fetus no. 48983-06 in the control group and fetus no. 48997-05 in the 1000 ppm group had situs inversus. Fetus no. 48997-05 also had a heart and great vessel anomaly (the aortic arch was interrupted, the right carotid and subclavian arteries arose from the ascending and descending aorta, respectively, and the interventricular septum was absent) and lobular agenesis of the lungs (only one lobe was present on each side). Fetus no. 49001-06 in the 1000 ppm group had malpositioned testes and kidneys, fused kidneys and fused adrenals. Fetus no. 49032-02 in the control group had a heart and great vessel anomaly (situs inversus [heart]; the ascending aorta arose from the smaller ventricle on the left side, and the pulmonary trunk arose from the larger ventricle on the right side). Soft tissue developmental variations were not observed in fetuses at any exposure level.

Skeletal malformations were not observed in fetuses at any exposure level in this study. Skeletal developmental variations were observed in all of the exposure groups, including the control group. None of the occurrences were attributed to exposure to the test article. The incidence (percent per litter) of ossification of cervical centrum no. 1 was reduced in the 2000 ppm group relative to the control group value; the difference was statistically significant p < 0.05). However, because of the absence of other indications of developmental delay in these fetuses (e.g. reduced mean fetal body weight), the reduced incidence of ossification of cervical centrum no. 1 was not attributed to the test article. Statistically significant (p < 0.05 or p < 0.01) increases were observed in the incidence of reduced ossification of the 13th ribs in the 500, 1000 and 2000 ppm groups (numbers of litters and percent per litter). However, the incidences expressed as percent per litter (3.2, 6.6 and 5.1 percent per litter in the 500, 1000 and 2000 ppm groups, respectively) were well within the range in the WIL historical control data (0.0-11.5 percent per litter), and no dose-response relationship was apparent; therefore, the increases were not attributed to the test article. The only other statistically significant (p < 0.05) difference between the control and treated groups was an increase in the number of litters in the 1000 ppm group with unossified sternebra(e) no. 5 andlor 6. Because the incidence (3.7 percent per litter) was well within the range in the WIL historical control data (0.6-37.5 %), and because a similar increase was not observed in the 2000 ppm group, the difference was attributed to biological variation. Other skeletal developmental variations that occurred in the treated groups were observed infrequently and/or at similar frequencies in the control group.

Fetal external, soft tissue and/or skeletal malformations were observed in 2(2), 0(0), 4(4) and l(1) fetuses (litters) in the control, 500, 1000 and 2000 ppm groups, respectively, and were considered to be spontaneous in origin. Differences between the control and treated groups in the total numbers of malformations were not statistically significant. Statistically significant (p < 0.05) differences in the incidences of various skeletal developmental variations were noted between the control and treated groups. However, in all cases, the incidences were within the range in the WIL historical control data, were not observed when the data were expressed on a percent per litter basis andlor were not observed to occur in a dose-related manner. Therefore, the increased incidences were attributed to biological variation. Other developmental variations noted in the treated groups were observed infrequently and/or at similar frequencies in the control group.

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

2 000 ppm

Based on:

test mat.

Basis for effect level:

other: No treatment-related developmental toxic effects were noted for the three tested exposure concentrations.

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:

Based on effects on body weight and food consumption observed at exposure concentrations, a NOAEL (no observed adverse effect level) for maternal toxicity was not determined. The exposure concentration of 500 ppm was considered to be the LOAEL (lowest observed adverse effect level) for maternal toxicity. Alternatively, no treatment-related developmental toxic effects were noted for the three tested exposure concentrations. Therefore, the highest exposure 2000 ppm, was considered to be the NOAEL for developmental toxicity.

Executive summary:

The potential maternal toxicity and development toxicity of n-Butyl Propionate were evaluated in this study. Three groups of 24 bred Sprague-Dawley Crl:CD BR rats were exposed to the test article by whole body inhalation for a 6 hour period each day for 10 consecutive days (gestation days 6-15) Target concentrations were 500, 1000 and 2000 ppm (parts per million.) The actual mean measured exposure concentrations were observed daily for appearance and behavior prior to exposure, for clinical signs during and within approximately one hour after completion of exposure and for mortality and moribundity. Body weights and food consumption were recorded at appropriate intervals. On gestation day 20, a laparohysterectomy was performed on all animals. The uteri and ovaries were examined and the numbers of fetuses, early and late resorptions, total implantations and corpora lute were recorded. Mean gravid uterine weights and net body weight changes were calculated for each group. The fetuses were weighed, sexed and examined for external, soft tissue skeletal malformations and variations.

 

All maternal animals survived to the scheduled necropsy on gestation day 20. The predominant treatment related clinical signs observed were dose-related incidences of slightly dropping eyelids and salivation, noted during exposure in the 1000 and 2000 ppm groups. Body weight gain and food consumption were inhibited in the 500, 1000, 2000 ppm groups during the first three days of exposure. Food consumption remained reduced in all of the treated groups throughout the remainder of the exposure period. No treatment-related necropsy findings were noted at any exposure level on gestation day 20.

 

Intrauterine growth and survival were unaffected by exposure to the test article at levels of 500, 1000 and 2000 ppm. The malformations and developmental variations observed in this study occurred infrequently, at similar frequencies in the control group or at frequencies that were within the range of the WIL historical control data, and were not considered to be related to test article exposure.

 

Based on effects on body weight and food consumption observed at exposure concentrations, a NOAEL (no observed adverse effect level) for maternal toxicity was not determined. The exposure concentration of 500 ppm was considered to be the LOAEL (lowest observed adverse effect level) for maternal toxicity. Alternatively, no treatment-related developmental toxic effects were noted for the three tested exposure concentrations. Therefore, the highest exposure 2000 ppm, was considered to be the NOAEL for developmental toxicity.