Ethyl 3-ethoxypropionate

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Not applicable

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-study according to OECD guideline 413.

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 Type:

study report

Title:

Unnamed

Year:

1986

Report date:

1986

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Principles of method if other than guideline:

This study was conducted by procedures comparable to the Toxic Substances Control Act Test Guidelines (Environmental Protection Agency,
September 27, 1985) and; Guidelines for Testing of Chemicals: TG-413, Subchronic Inhalation Toxicity: 90-Day Study (Organization for Economic
Cooperation and Development, May, 1981) and; Annex V: Sub-Chronic 90-Day Repeated Dose Inhalation Study (European Economic Community, Notification of New Substances Regulations, 1982).

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Ethyl 3-ethoxypropionate

EC Number:

212-112-9

EC Name:

Ethyl 3-ethoxypropionate

Cas Number:

763-69-9

Molecular formula:

C7H14O3

IUPAC Name:

ethyl 3-ethoxypropanoate

Details on test material:

- Name of test material (as cited in study report): Ethyl 3-ethoxypropionate; EEP
- Physical state: liquid
- Analytical purity: >99% by GC/FID
- Purity test date: 15 May 1985; 29 July 1985; 23 September 1985
- Lot/batch No.: X-18626-184-6 & X-18626-184-7
- Stability under test conditions: Yes
- Storage condition of test material: Stored in and inhalation chamber maintained at room temperature and slightly negative pressure.

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 Breeding Laboratory, Wilmington, MA.
- Age at study initiation: approximately eight weeks old
- Weight at study initiation: 260 to 314 gm (males) and 179 to 232 gm (females)
- Fasting period before study: Pelleted was available during nonexposure perlods only.
- Housing: Animals were group housed, five per cage, in stainless steel suspended cages during the isolation period, but subsequently singly housed in multicompartmented stainless steel mesh cages designed for Inhalation studies. Males and females in each group were housed on opposite sides of the same rack.
- Diet: Feed (Certified Purina Laboratory Rodent Chow 5002 pelleted) was available during nonexposure perlods only.
- Water: ad libitum
- Acclimation period: approximately 20 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 64-71
- Humidity (%): 41-58
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 (6:00 am to 6:00 pm)

Administration / exposure

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

other: unchanged (no vehicle)

Remarks on MMAD:

MMAD / GSD: not applicable

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION/EXPOSURE CONDITIONS
- Exposures were conducted fn 4.2 m3 stainless steel and glass inhalation chambers. Chambers were maintained under negative pressure
(approximately 0.5" H2O) and at 13 alr changes per hour.

-Vapors were generated by metering the test material dropwise into a heated glass bead-packed column supplied with metered dried oil-free
compressed air. Controls were treated identically to the test groups, except that the generator was maintained at room temperature and exposure was to filtered air only. Chamber concentrations were determined at least once per hour by a MIRAN® I A infrared analyzer equipped for automated sampling and analysis. In addition, temperature and humidity was determined hourly and nominal chamber concentrations calculated daily. Twice daily
(morning and afternoon of each exposure) concentration of background nongaseous material was measured in the high concentration chamber
relative to the control chamber in order to insure that exposures were to vapor and not aerosol. Animal cage assignments were rotated daily within
their respective chambers to minimize potential positional effects. Distributfon of test material was determined initially by measurement from numerous positions throughout the chamber and was compared to a fixed reference position. Subsequently, chamber concentration was monitored continuously and recorded about once per hour from the fixed reference position.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Test Atmosphere Generation: Liquid EEP was metered ( Lab Pump Model RPSY, Fluid Metering, Inc., Oyster Bay, NY) from a graduated reservoir into a thermoregulated (Thermowatch®, IZR, Cheltenham, PA) glass distilling column (7/8" x 15", Ace Glass Inc., Vineland, NJ). The liquid trickled over the surface of tightly packed glass beads (4 mm diameter) within the column. Metered compressed air (approximately 24, 33, 42, and 59 L/min or the 0, 250, 500, and 1000 ppm chambers, respectively) was passed through an indicating silica gel drier and an oil filter (Deltech Engineering Inc., New Castle, DE) entering the column and passing over the surface of the glass beads. Vapor temperature was measured in a round bottom flask as it exhausted the distilling column. The vapor temperature was maintained (mean ± standard deviation) at 81 ± 9, 92 ±8, and 81 ± 8 ºC for the 250, 500, and 1000 ppm concentration chambers, respectively. Together, the vapor temperature, liquid delivery rate, and compressed airflow rate were adjusted as necessary to produce the desired chamber target concentration. The resultant vapor was directed via glass tubing to a tee just upstream of the inhalation chamber where it was mixed with dilution air to produce a total chamber airflow rate of approximately 889 L/min (313 air changes per hour).
Vapor Analysis: Chamber vapor concentrations were monitored with a multipositional air sampling and analysis system. The system consisted of a Miran® IA infrared gas analyzer (Wilks Foxboro Analytical, South Norwalk, CT), and a four-port multipositional environmental sampling valve (Valco Instruments, Houston, TX). Chamber vapor samples were continuously collected, from a fixed reference position within each chamber, through the four port valve using Teflon® tubing (3/16" ID). The voltage output of a sampled atmosphere was integrated over a one minute time slice following a five minute flushing period. A Perkin-Elmer SIGMA 15 mlcroprocessor calculated the vapor concentration by linear interpolation between selected calibration data points. An AATS program called "MIRAN", was used by the SIGMA 15 microprocessor for automated storage and retrieval of calibration data. Vapor concentration is calculated by simple linear interpolation between specific data points on the calibration curve. Then, the multipositional environmental sampling valve was automatically advanced to the next valve position, and the procedure repeated until termination of the daily exposure.
The nomlnal concentration was calculated by dividing the amount of EEP vaporized from the graduated reservoir (determined volumetrically) by the total chamber air flow using the formula: C (ppm) = V1 xp/V2 x24.45/MW where: V1 = amount vaporized (mL); p = EEP density = 0.950 g/cc; V2 = total daily chamber air flow = 319.9 m3; 24.45 = molar volume at 1 atm and 25 ºC; MW = EEP molecular weight = 146.2

Total chamber flow was monitored via a Magnehelic®gauge (Dwyer Instruments, Inc., Michigan City, IN) which measured the differential pressure across an orifice located in the chamber's exhaust duct. The chamber flow corresponding to the gauge value was determined and recorded from a differential pressure versus chamber flow calibration curve. Each chamber was maintained at a negative pressure of 0.5 inch of water.

Duration of treatment / exposure:

six hours per day

Frequency of treatment:

Five days per week, including holidays for a total of 65-67 exposures spanning 13 weeks.

Doses / concentrations

Remarks:

Doses / Concentrations:
250, 500 or 1000 ppm
Basis:
nominal conc.

No. of animals per sex per dose:

15

Control animals:

yes, concurrent no treatment

Details on study design:

Inhalation exposure was chosen because it is a possible route of human exposure to EEP.

Positive control:

Not applicable

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Each rat was removed from its cage before and after each exposure for examination.

BODY WEIGHT: Yes
- Time schedule for examinations: Day 0 of the study and then weekly thereafter. Terminal body weights were determined at time of necropsy in
those animals surviving to the end of the study.

FOOD CONSUMPTION:
- Not measured

FOOD EFFICIENCY:
- Not measured

WATER CONSUMPTION:
- Not examined

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Prior to study initiation and prior to study termination
- Dose groups that were examined: All

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the time of necropsy
- Anaesthetic used for blood collection: Yes (CO2 anesthesia)
- Animals fasted: Yes
- How many animals: All rats
- Parameters examined: hemoglobin concentration, hematocrit, red blood cell count, white blood cell count, differential white blood cell count, platelet count, red blood cell indices, and examination of blood smears for cellular morphology.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the time of necropsy
- Animals fasted: Yes
- How many animals: All rats
- Parameters examined: aspartate aminotransferase (AST), alanine aminotransferase IALT), sorbitol dehydrogenase (SDH), alkaline phosphatase (AP), urea nitrogen (UN), glucose, and creatinine.

URINALYSIS: Not examined

NEUROBEHAVIOURAL EXAMINATION: Not examined

ORGAN WEIGHTS: Yes
-spleen; heart; testes; kidneys; thymus; brain; ovaries

Sacrifice and pathology:

GROSS PATHOLOGY: Yes. Animals were selected for necropsy using a stratiffed random pattern which resulted in equal numbers of animals of each sex from each exposure level being autopsied on each day.
HISTOPATHOLOGY: Yes. The following tissues were collected in 10% buffered formalin: nasal passages, trachea, lungs, heart, tongue, aorta, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, pancreas, liver, salivary glands, kidneys, urinary bladder, pituitary gland, adrenal glands, thyroid glands, parathyroid glands, thymus, spleen, mesenterlc lymph nodes, bone marrow (femoral), brain, sciatic nerve, testes, epididymides, male accessory sex glands, ovaries, vagina, uterus, Fallopian tubes, rfb, and gross lesions.

Other examinations:

Not applicable

Statistics:

All numerical data was evaluated using the following computer generated statistical tests: one-way analysis of variance (ANOVA), Bartlett's test, and Duncan's multiple range test using a P value of ≤0.05 to indicate statistical significance.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

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:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
There was one spontaneous death during the study. A female exposed to 1000 ppm died an Day 54. Substantial autolysis had occurred and no cause of death was determined. Clinical signs of irritation in males were lacrimation, sialorrhea, red or brown discoloration of facial hair, and an unkempt haircoat. These signs were seen either before or after exposure on one or several occasions. The severity of these signs was generally minimal and reflect a slight irritant property of EEP. Lacrimation was seen in two animals exposed to 250 ppm, one animal exposed to 500 ppm and four animals exposed to 1000 ppm. Sialorrhea was seen in five animals exposed to 1000 ppm. Discoloration of facial hair was seen in three animals exposed to 500 ppm and 13 animals exposed to 1000 ppm and may be secondary to lacrimation. One animal exposed to 1000 ppm had an unkempt haircoat.
Alopecia seen in two animals exposed to 250 ppm and one animal exposed to 500 ppm may not have been compound-related but may have been exacerbated by the presence of EEP. Corneal opacity was seen in one animal in the control group and is occasionally seen in this strain of laboratory rat.
Slight lethargy was seen in males during the first and second exposure to 1000 ppm. The animals appeared normal during all subsequent exposures.
No clinical signs of toxicity were seen during exposure to 500 and 250 ppm.

Clinical signs of irritation in females were lacrimation, sialorrhea, and red or brown hair discoloration of the face, neck, and abdomen. These signs were seen either before or after exposure on one or several occasions. The severity of these signs was generally mlnimal. Lacrimation was seen in two animals exposed to 1000 ppm. Sialorrhea was seen in one animal exposed to 250 ppm and seven animals exposed to 1000 ppm. Hair discoloration was seen in two control animals, four animals exposed to 250 ppm, 10 animals exposed to 500 ppm, and 10 animals exposed to 1000 ppm. Alopecia seen in one animal exposed to 250 ppm, two animals exposed to 500 ppm, and eight animals exposed to 1000 ppm may not have been compound-related but may have been exacerbated by the presence of EEP. Hyperemla of the ears was found in one control animal. Slight lethargy was seen during the first and second exposure to 1000 ppn. The animals appeared normal during all subsequent exposures. No clinical signs of toxicity were observed during exposure to 500 or 250 ppm.

BODY WEIGHT AND WEIGHT GAIN
A concentration dependent decrease in body weight gain was seen in males. This difference was significant at each weighing period to the end of the study in the 1000 ppm group beginning in Week 2 and in the 500 ppm group beginning Week 5 (except for Week 7). Although slightly decreased, the weight gain of the 250 ppm group never differed statistically from control values. By Week 13 (Day 89) body weights were 15 , 10, and 5% less than control values for the 1000, 500, and 250 ppm groups, respectively. A very slight decrease in mean body weight gain, which was not concentration dependent was seen in females exposed to all concentrations of EEP. The decrease in mean body weight gain was statistically significant for the 1000 ppm exposure group in Weeks 9 and 13, for the 500 ppm exposure group in Weeks 2, 9, 12, and 13, and for the 250 ppm exposure group in Week 2. By Week 13, body weights were 7, 9, and 5% less than control values for the 1000, 500, and 250 ppm groups, respectively. Weight at all other time periods for males and females were comparable to control values at each exposure concentration.

FOOD CONSUMPTION
Not measured

FOOD EFFICIENCY
Not measured

WATER CONSUMPTION
Not measured

OPHTHALMOSCOPIC EXAMINATION
No effects

HAEMATOLOGY
A very slight concentration dependent increase in lymphocytes was seen in females exposed to EEP. The increase was statistically significant in the high concentration group only. All other hematology determlnations in females as well as all hematology determinations in males were comparable to control values.

CLINICAL CHEMISTRY
There was a slight but statistically significant decrease in serum glucose ana a slight but statistically significant increase in creatinine in males exposed to 1000 ppm. All other clinical chemistry values for males were comparable to controls.

In females, AST was statistically significantly reduced in all exposure groups. However, unusually high values in at least two of the control animals may account for this difference. A statistically significant difference was also seen in ALT concentration in females exposed to 250 and 500 ppm. The 1000 ppm exposure group was comparable to control values. Again, high concentrations of ALT were seen in control animal. There was an exposure dependent increase in alkaline phosphatase concentration which was statistically signiffcant in the 500 and 1000 ppm exposure groups. A slight decrease in urea nitrogen values was seen in all treated groups. Although statistically significant the decreases were not concentration dependent and were extremely small. Creatinine concentration in females exposed to 1000 ppm was slightly, but significantly increased over control values. SDH values were decreased in a non-concentration dependent manner compared to controls. Although each was significantly different statistically from controls, values for two control animals were extremely large and may account for the significance.

TERMINAL BODY WEIGHTS and ORGAN WEIGHTS
There was a concentration dependent decrease in terminal body weights of males. The decrease was statistically significant in the 1000 and 500 ppm groups. In males, absolute liver and kidney welghts of all groups were comparable to control values. Relative liver weights were statistically increased slightly in the 1000 ppm group. There was a statistically significant increase in relative kidney weights in all groups exposed to EEP. Absolute testes weights were comparable to control values while relative weights of testes in all exposure groups showed a concentration dependent increase which was statistically different from controls. The results suggest that body weight loss in males may not have been sufficient to cause weight retardation of liver, kidneys, or testes. Absolute heart weights were decreased in an exposure dependent manner in all groups but were statistically significant in the 1000 and 500 ppm groups only. The relative heart weights were normal compared to controls. Absolute brain welghts were very slightly decreased statistically in the 1000 and 500 ppm groups, while the relative brain weights in these two groups were very slightly increased. The magnitude of the heart and brain weight losses were not unexpected relative to that of the body weight loss observed. Absolute and relative spleen and thymus weights of all expoaure groups were comparable to control values.

Terminal body weights of females were statistically significantly decreased in all exposure groups. Absolute liver weights were significantly increased in the 1000 ppm group while relative weights were statistically increased in the 1000 and 500 ppm groups. Absolute and relative kidney weights were increased in a concentration dependent: manner. The absolute increases were statistically significant in the 1000 ppm group whlle the relative increases were significant at all exposure concentrations. Relative brain weights were statistically elevated in the 500 and 1000 ppm exposure groups. Absolute brain weights were comparable to control values. The results suggest that body weight loss in females may not have been sufficient to cause weight retardation of liver, kidneys, or brain. Absolute and relative spleen, thymus, and heart weights of all exposure groups were comparable to control values.

GROSS PATHOLOGY
Seven males showed one of the following gross Lesions after exposure to 1000 ppm EEP: moderate adipose tissue atrophy (2/15), moderate thymic atrophy (3/15), or minimal and minor hemorrhage of the glandular mucosa of the stomach (2/15). Minimal hemorrhage of the glandular mucosa of the stomach was also seen in one animal exposed to 500 ppm. There were no compound-related gross changes in males exposed to 250 ppm. Three females exposed to 1000 pprn showed one of the following gross lesions: minimal hemorrhage of the glandular mucosa of the stomach, moderate thymic atrophy, and moderate adipose tissue atrophy. There were no compound-related gross changes observed in females exposed to 500 or 250 ppm.

HISTOPATHOLOGY: NON-NEOPLASTIC
Histologic examination of males revealed minimal thymic atrophy in one animal exposed to 1000 ppm. There were no compound-related histopathologic changes detected In the groups exposed to 500 or 250 ppm. Histologic exanation of females exposed to 1000 ppm revealed minimal thymic atrophy in one female and serous atrophy of adipose tissue in another. There were no compound-related histologic changes detected in the groups exposed to 500 or 250 ppm.
One rat died during the study (a female exposed to 1000 ppm for 54 days). The stomach was full of feed, suggesting a sudden death. Because of an advanced autalysls, no cause of death could be established.

OTHER FINDINGS
EXPOSURE CONDITIONS
The overall mean exposure concentrations and standard deviations were 996±61, 510±23, and 251±10 ppm comparing favorably to the targeted (1000, 500, and 250 ppm) and nominal (1119 ±57, 629 ±15, and 290±9 ppm) concentrations. A drop in the 1000 ppm chamber concentration on day 14 was due a generator pump malfunction which was promptly adjusted. Overall mean chamber temperature and relative humidity varied from 22 to 23ºC and 50 to 56%, respectively. Particle count data indicated that an aerosol of the test material was not present.

Effect levels

1

Dose descriptor:

NOEC

Effect level:

250 ppm

Sex:

male/female

Basis for effect level:

other: No-observed effects at 250 ppm

2

Dose descriptor:

NOAEC

Effect level:

500 ppm

Sex:

male/female

Basis for effect level:

other: 10% decrease in bodyweight

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Not applicable

Applicant's summary and conclusion

Conclusions:

The no-observed effect level for toxicity is 250 ppm EEP and the no-observed adverse effect level is 500 ppmwith the principal change being weight gain depression following exposure to 500 and 1000 ppm EEP for 13 weeks.

Executive summary:

Groups of 15 male and 15 female rats were exposed to target vapor concentrations of 1000, 500, 250, or 0 ppm ethyl 3-ethoxypropionate (EEP) 6h/day, 5 days/week, and received 65-67 exposures over 13 weeks. Effects related to exposure were lethargy during exposure to 1000 ppm, decreased body weight gain and clinical signs of irritation such as sialorrhea and lacrimation at 1000 and 500 ppm. Differences in hematology, clinical chemistry, terminal body and organ weight values were seen, but the effects were small, the severity of the effects were slight and they appeared to be secondary to the weight gain depression. The effects seen with EEP were of a non-specific nature. One female exposed to 1000 ppm died on Day 54 of the study. No cause of death was determined.

The no-observed effect level for toxicity is 250 ppm EEP and the no-observed adverse effect level is 500 ppm with the principal change being weight gain depression following exposure to 500 and 1000 ppm EEP for 13 weeks.