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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1988
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study. Mortality attributed to gavage trauma but does not influence reliability of NOAEL.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1988
Report Date:
1988

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
EPA OTS 795.2600 (Subchronic Oral Toxicity Test)
Deviations:
not specified
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): ethyl acetate
- Analytical purity: 99.9%
- Lot/batch No.: 3299

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc, Portage, MI
- Age at study initiation: 43 days
- Weight at study initiation: 163 to 232 grams for males; 129 to 181 grams for females
- Housing: individually in stainless steel, wire-bottomed cages
- Diet (e.g. ad libitum): Purina Certified Rodent Chow #5002 ad libitum except prior to blood collection and scheduled sacrifice.
- Water (e.g. ad libitum): Filtered tap water
- Acclimation period: 14 days
- Unique identification: Yes, by metal ear tags


ENVIRONMENTAL CONDITIONS
- Temperature (°C): generally 68 - 78 degrees F
- Humidity (%): generally 30 - 70% relative humidity
- Photoperiod (hrs dark / hrs light): 12 hour light / dark cycle


IN-LIFE DATES: From: September 18, 1985 To: December 19, 1985

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
Animals assigned to the 300, 900, or 3,600 mg/kg test groups were administered either 3.0, 9.0, or 36% solutions of the test article, respectively, by oral gavage for either 90, 91, or 92 consecutive days at the rate of 10 ml/kg of body weight. Dosing volumes were adjusted weekly.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of solutions used for dosing revealed ethyl acetate concentrations within 7% of target for all samples analyzed. Analysis of dosing solutions stored under ambient conditions for up to 7 days revealed concentrations within 5% of target. The dosing solutions were considered stable for 7 days ambient storage.
Duration of treatment / exposure:
90 - 92 d
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
900 mg/kg bw/day (actual dose received)
Dose / conc.:
3 600 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
30
Control animals:
yes, concurrent vehicle
Details on study design:
Post-exposure period: none
Positive control:
No

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were observed for mortality, morbidity, and obvious abnormal clinical/overt toxic signs twice daily in the early morning and afternoon.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Animals were observed for detailed clinical observations after dosing on each day. Animals were also closely examined for signs of physical changes and palpated for tissue masses weekly.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were determined during quarantine for randomization, on the initial day of dosing, and weekly thereafter. Final body weights (fasted weights for scheduled sacrifice animals) were determined for all animals prior to necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Individual food consumption was determined weekly during the study.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: on all animals during quarantine and on all surviving animals prior to study termination.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: during quarantine, at interim sacrifice and at terminal sacrifice
- Anaesthetic used for blood collection: Yes
- Animals fasted: Yes
- How many animals: 10 males and 10 females per group
- Parameters examined: Total leukocyte count, erythrocyte count, hemoglobin, hematocrit, calculated cell indices (MCV,
MCH, and MCHC), platelet count, differential leukocyte count.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: during quarantine, at interim sacrifice and at terminal sacrifice
- Animals fasted: Yes
- How many animals: 10 males and 10 females per group
- Parameters examined: Sodium, potassium, chloride, calcium, glucose, urea nitrogen, creatinine, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, phosphorus (inorganic), total protein, albumin, globulin (calculated), albumin/globulin ratio (calculated), total bilirubin, cholesterol

URINALYSIS: Yes
- Time schedule for collection of urine: at interim sacrifice and at terminal sacrifice
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined: specific gravity, pH, protein, glucose, ketones, bilirubin, urobilinogen, microscopic examination
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- Scheduled sacrifices were done on study days 44 and 45 (interim sacrifice) and on study days 91,
92, and 93 (final sacrifice). Ten (10) males and 10 females of the vehicle control, 300 mg/kg, and 900 mg/kg groups and 9 males and 10 females of the 3,600 mg/kg group were sacrificed at the interim and all surviving animals were sacrificed at study termination. One male (AH0949) of the 3,600 mg/kg group, selected for interim sacrifice, was found dead on study day 43.

All external surfaces, orifices, and organs; the cranial cavity; carcass; external and cut surfaces of the brain; the abdominal, thoracic, and pelvic cavities and their viscera; and cervical tissues and organs were examined during necropsy.

HISTOPATHOLOGY: Yes
- All tissues and organs listed below from all animals found dead and from all vehicle control and high dose (3,600 mg/kg) animals sacri£iced at study termination were processed and examined histologically. The heart, kidneys, liver, and any gross changes and tissue masses of low dose (300 mg/kg) and mid-dose (900 mg/kg) animals found dead or sacrificed at study termination were also processed and examined histologically.
- Adrenals, lung and mainstem bronchi, aorta, lymph nodes (mandibular and mesenteric nodes), bone (femur), bone marrow (sternum), mammary gland, brain (sections of cerebrum, cerebellum, and brainstem) pituitary, pancreas, salivary gland (mandibular), esophagus, skeletal muscle with sciatic nerve, eyes and optic nerve, gonads (testes with epididymides or ovaries), uterus/cervix, corpus,
spinal cord (mid-thoracic), spleen, heart, stomach (cardiac, fundic, and pyloric portions), intestine (sections of cecum, colon, duodenum, ileum, jejunum, and rectum), thymus (or thymic remnant), thyroids (with parathyroids), kidneys, trachea, liver, urinary bladder, any gross changes of uncertain nature or tissue masses (including adjacent tissue).
Other examinations:
ORGAN WEIGHTS: Yes
- The adrenals, brain, gonads (testes with epididymides or ovaries), heart, kidneys, liver, and spleen of each final sacrifice animal. Paired organs were weighed separately. Organ to body and brain weight ratios were calculated. Final fasted body weights were used for organ to body weight ratio calculations.
Statistics:
Body weight, food consumption, appropriate clinical pathology, and organ weight data were analyzed by an analysis of variance with any significant differences examined further by either Tukey's or Scheffe's Test of Multiple Comparisons. Nonparametric data, such as organ weight ratios and differential leukocyte counts, were analyzed by Kruskal-Wallis' Test with any significant differences examined further by Kruskal-Wallis' Multiple Comparison Test. Statistically significant differences between the vehicle control and any test group(s), as determined by the multiple comparison test, were reported as statistically significant. The 0.05 and 0.01 levels of significance were used.

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):
effects observed, treatment-related
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
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
One male and one female animal in the 900 mg/kg group, and five males and two females in the 3600 mg/kg group were found dead during the study. Histopathology studies revealed evidence consistent with the diagnosis of pulmonary accident or gavaging trauma for several animals that were found dead. Antemortem observations of salivation, irregular breathing, and lethargy were seen in the 3,600 mg/kg male and female groups at notably increased incidence and frequency when compared to other test and vehicle control groups. Incidences of moist rales and vocalization were also notably increased in the 3,600 mg/kg male group. Incidences of the observations noted above in other groups and of other observations in all groups were few in number and were seen in non-treatment-related patterns.

BODY WEIGHT AND WEIGHT GAIN
Body weights of the 3,600 mg/kg male test group at weeks 1 through 6 and 10 through 13 and total weight changes were statistically lower than vehicle control group values. Body weights and weight changes of other male and all female test groups were comparable to vehicle controls.

FOOD CONSUMPTION
Food consumption of the 3,600 mg/kg male test group was significantly lower than vehicle control values at weeks 1, 2, 7, 10, and 11. Statistically lower food consumption values for the 900 mg/kg and 3,600 mg/kg female groups at week 10 were not considered to be toxicologically meaningful. All other test and vehicle control food consumption data were comparable.

OPHTHALMOSCOPIC EXAMINATION
Ophthalmology examinations done prior to final sacrifice revealed abnormalities for animals in all groups that were not considered to be treatment related.

HAEMATOLOGY, CLINICAL CHEMISTRY, URINALYSIS
Hematology, clinical chemistry, and urinalysis studies done at the interim (mid-point) and termination of the study revealed comparable test and vehicle control values. Statistical
differences were not considered to be clinically significant.

ORGAN WEIGHTS
Organ weight and ratio statistical analysis revealed significantly decreased kidney weights, kidney/brain weight ratios, liver weights and ratios (body and brain), spleen weights, and final body weights for the 3,600 mg/kg male group. Significantly decreased liver to brain weight ratios of the 3,600 mg/kg female group and significantly increased testes to body weight ratios of the 3,600 mg/kg male group were also observed.

GROSS PATHOLOGY
Mesenteric abnormalities (discolored, thickened, and nodular) were necropsy findings seen only in the 3,600 mg/kg group (one male/8 females). Incidences of liver discoloration (pale) in all male groups (test and control) were notably high. Other necropsy findings were few in number and seen in
non-treatment-related patterns.

HISTOPATHOLOGY
The test article, ethyl acetate, was not associated, under the conditions of this study, with any histopathologic alterations in rats during the test period.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
900 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects seen at this dose
Dose descriptor:
LOAEL
Effect level:
3 600 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
body weight and weight gain
food consumption and compound intake

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Mortality

The pathology report states: “Several of the animals found dead during the study had significant evidence consistent with a diagnosis of pulmonary accident or gavaging trauma.  These lesions included pulmonary edema and inflammatory changes.”  Animals with this diagnosis are not specifically identified in the report.  Lung changes were noted in all of the high dose animals that died.  While lung changes were not noted in the two mid-dose animals that died, both animals were noted in the pathology report to have “foreign material in the esophagus”. 

 

The pre-mortem clinical picture in the high dose animals that died does not appear to be distinguishable from animals that survived to the terminal sacrifice.  No abnormal clinical signs were observed in the two mid-dose animals that died.

 

Body Weights and Food Consumption

Reduced body weight gain and reduced food consumption were recorded in the high-dose male animals early in the study (Week 1).  Similar findings were not seen in the high-dose female animals, or in the mid-dose males.  A review of the individual animal data indicates that the body weight suppression occurred in nearly all of high-dose male animals.  It does not appear that exclusion of animals showing lung changes at the terminal sacrifice and / or animals that died during the study would materially alter this finding.  Rather it seems that the body weight suppression is more likely linked to reduced food consumption in the high-dose male group which began in Week 1.

 

Clinical Signs

Salivation, irregular breathing, and lethargy were observed at notably increased frequency in the high dose males and females.  Moist rales and vocalization were also observed at increased frequency in the high dose males.

 

Organ Weights

Organ weights differences noted in the high-dose male animals are considered to be a reflection of the suppressed body weight gain.

 

In summary, the mortality seen in this study can reasonably be considered not to be a test substance effect on the basis of the pathology findings and report indicating gavage trauma in these animals.  Clinical signs were seen at increased frequency in both the high dose male and the high dose female rats surviving the study.  Reduced body weight gain recorded in the high-dose male animals appears to be linked to the reduced food consumption seen in these animals, beginning early in the study.

Applicant's summary and conclusion

Executive summary:

The subchronic oral toxicity of ethyl acetate was evaluated in rats by US EPA. Groups of 30 male and 30 female rats received daily doses of 0, 300, 900 or 3600 mg/kg bw/day by gavage. Mortality in the mid- (1 male and 1 female) and high-dose (5 males and 2 females) groups are considered to be the result of gavage trauma. High dose male rats showed significantly depressed body weight gain and reduced food consumption. Salivation, irregular breathing, and lethargy were observed at notably increased frequency in the high dose male and female animals when compared to control animals.  Moist rales and vocalization were also observed at increased frequency in the high dose males. Based on these findings the subchronic oral NOAEL for ethyl acetate in rats is considered to be 900 mg/kg bw/day.