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Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: As a recent, guideline study reported in detail and carried out to GLP by a reputable laboratory, this is regarded as highly reliable without restrictions and the preferred study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EPA OTS 798.2450 (90-Day Inhalation Toxicity)
Deviations:
no
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): Ethyl acetate
- Molecular weight (if other than submission substance): 88.1
- Physical state: clear, colorless liquid
- Analytical Purity: 99.92% ± 0.001
- Supplier: Monsanto Chemical Company, Springfield, MA
- Stability under test conditions: Stable. The measured purity of samples analyzed before and after the exposure series was identical.

Test animals

Species:
rat
Strain:
other: Crl:CD®BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Raleigh, North Carolina.
- Age at study initiation: seven weeks old
- Weight at study initiation: 205 - 268g (males); 141 - 192g (females)
- Housing: During quarantine and the majority of the pretest rats were housed three per cage in suspended, stainless steel, wire-mesh cages. Upon grouping, rats were housed individually. During the test period, rats were housed singly.
- Diet: Purina® Certified Rodent Chow® #5002 was available ad libitum except during exposure and fasting periods prior to clinical laboratory evaluations.
- Water: tap water was available ad libitum
- Acclimation period: Rats were quarantined for 6 days and in pretest for approximately two weeks prior to test initiation.

ENVIRONMENTAL CONDITIONS
- Temperature: 23 ± 1°C
- Humidity: 50 ± 10%.
- Photoperiod: 12-hour light/12-hour dark cycle

Administration / exposure

Route of administration:
inhalation
Type of inhalation exposure:
whole body
Vehicle:
other: unchanged (no vehicle)
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Three 0.75 m3 and one 1.0 m3 (0 ppm) stainless steel or glass.
- Method of holding animals in test chamber: wire-mesh cages
- Temperature, humidity, pressure in air chamber: 23 ± 1°C, 50 ± 10%, oxygen concentration was targeted to at least 19%.
- Air flow rate: 190-220 L/min over the course of the study
- Air change rate: 12 air changes per hour
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The atmospheric concentration of ethyl acetate was determined by gas chromatography at approximately 30-minute intervals during each exposure.
Duration of treatment / exposure:
94 days
Frequency of treatment:
6 hours/day - 5 days/week (68 total exposures).
Doses / concentrationsopen allclose all
Dose / conc.:
350 ppm
Dose / conc.:
750 ppm
Dose / conc.:
1 500 ppm
No. of animals per sex per dose:
10
Control animals:
yes, sham-exposed

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily on non-exposure days (weekends and holidays), once every hour during exposure period
- Cage side observations checked included: Morbundity and abnormal behavior (non-exposure period); technicians judged whether the group of rats visible to him/her within a given exposure level appeared to display a normal, diminished, or excessive response to an alerting stimulus relative to rats in the 0 ppm (control) chamber (exposure period).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: regular intervals

BODY WEIGHT: Yes
- Time schedule for examinations: morning of the first exposure (test day 1), test days 2, 5, 8, 9, 12, and approximately weekly thereafter.

FOOD CONSUMPTION:
- Food consumption: Yes
- Time schedule for examinations: The amount of food consumed by each rat was determined for the intervals between successive body weight measurements on test days 1,2,5,8,9, 12, 19,26,33,40,47,54,61,68, 75, 82, 89, and 93. From these determinations, mean individual daily food consumption was calculated for each of the same intervals. The food consumption and corresponding body weight gain (loss) data were used to calculate food efficiency for each of the same intervals.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Both eyes of all rats were examined by focal illumination and indirect ophthalmoscopy. The eyes were examined in subdued light after mydriasis was produced with 1% Mydriacyl® solution. An examination was performed during the pretest period on all rats received for the study prior to selection and grouping, and on all surviving rats on test day 73.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: On test days 43 (males) and 44 (females), and 85 (males) and 86 (females)
- Anaesthetic used for blood collection: Yes
- Animals fasted: Yes, 14 hours
- Parameters examined: number RBC, WBC, PLAT, Hb, Ht, MCV, MCH, MCHC, Neut, Band, Lymph, Alym, Mono, Eosin, Baso. Absolute values for the various types of leukocytes were calculated from the leukocytic data. Blood cell counts, hemoglobin concentration, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration were determined on a Serono Baker 9000® hematology analyzer Differential cell counts and reticulocyte counts were determined on a Hematrak® Automated Differential System cell counter.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On test days 43 (males) and 44 (females), and 85 (males) and 86 (females)
- Animals fasted: Yes
- Parameters examined: ALP, ALT, AST, SDH, GLUCO, BUN, CALC, PHOS, BILRN, CHOL, CREAT, TRIG, TPROT, ALBMN, Na, K, CI. Clinical chemistry
parameters were measured on a Boehringer MannheimlHitachi 717 clinical chemistry analyzer using Boehringer Mannheim reagents. Serum globulin (GLOBN) concentration was calculated from the total protein and albumin concentrations.

URINALYSIS: Yes
- Time schedule for collection of urine: One day prior to the bleeding time, an overnight (approximately 14-hour) urine specimen was collected from each rat to measure VOL, OSMOL, UROBL, and pH; and to determine the presence of hemoglobin or occult blood (BLOOD), glucose, protein, bilirubin, and ketone (acetoacetic acid). Osmolality was determined on a Precision Systems Multi-Osmette™ model 2430 osmometer. Urine biochemical constituents were measured on a Clinitek® 200 urine chemistry analyzer using Ames Multistix® urine chemistry dipsticks. Urine appearance (color and transparency) was recorded and the sediment from each specimen was microscopically examined.

OTHER: SPERM ANALYSIS: On test days 93 or 94, all surviving male rats were analyzed for epididymal sperm count and concentration, testicular spermatid count and concentration, motility, and morphology.
Sacrifice and pathology:
All surviving rats were sacrificed by carbon dioxide anesthesia and exsanguination and necropsied after approximately 90 days on study. Due to technical error, rats were not fasted prior to necropsy. This protocol deviation was judged to have had no adverse effects on the validity of the study because animals in all groups were treated comparably and comparisons to control are valid.

GROSS PATHOLOGY: Yes, a complete gross examination was performed and liver, kidneys, lungs, heart, spleen, brain, adrenal glands, testis (right), and ovaries were removed and weighed.

HISTOPATHOLOGY: Yes, liver, kidneys, lungs, heart, skeletal muscle, spleen, aorta, brain (cerebrum, midbrain, cerebellum, medulla/pons), spinal cord (cervical, thoracic, lumbar), stomach, duodenum, jejunum, ileum, pancreas, cecum, colon, rectum, mesenteric lymph node, salivary glands, mandibular lymph node, harderian glands, exorbitallacrimal glands, thymus, adrenal glands, sciatic nerve, pituitary gland, thyroid gland, parathyroid glands, trachea, esophagus, pharynxllarynx, eyes, skin, mammary glands (female), ovaries, uterus, vagina, urinary bladder, prostate, seminal vesicles, testis (right), femur (including joint), sternum, bone marrow (femur, sternum), nose (four sections) and selected gross lesions.
- All tissues were fixed in 10% neutral buffered formalin except right testis and eyes, which were fixed in Bouin's solution. The lungs were inflated with 10% neutral buffered formalin after weighing.
- All tissues, including selected gross lesions, collected from rats in the high-concentration and control groups that were sacrificed at the end of the exposure period were. processed, embedded in paraffin, cut at a nominal thickness of 5 micrometers, stained with hematoxylin and eosin (H&E) and examined microscopically. Nose, pharynxllarynx, liver, kidneys, lungs, and selected gross lesions from rats in the low- and intermediate-concentration groups were also processed to slides and examined microscopically.
Statistics:
Means and standard deviations were used to summarize group data for each weighing date. Body weights and body weight gains were analyzed via univariate Analysis of Variance (ANOVA), with Dunnett's Test used to identify which treatment groups, if any, were significantly different from the control group. Separate analyses were performed on data for male and female rats. Significance levels were judged at p < 0.05. Data were evaluated with the individual rat as the unit of analysis.
Food consumption and food efficiency data were analyzed via univariate Analysis of Variance (ANOVA), with Dunnett's Test used to identify which treatment groups, if any, were significantly different from the control group. Separate analyses were performed on data for male and female rats. Significance levels were judged at p < 0.05. Data were evaluated with the individual rat as the unit of analysis.
All sperm parameters were statistically analyzed using Jonchkeere's test for trend. Significance levels were judged at p < 0.05.

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):
effects observed, treatment-related
Food efficiency:
effects observed, treatment-related
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:
no effects observed
Behaviour (functional findings):
effects observed, treatment-related
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
There were no test-substance related effects on mortality at any exposure concentration. One male in the 750 ppm group was sacrificed in extremis due to an injured leg. Necropsy revealed a fractured tibia. The cause of the injury could not be determined. There were no test-substance related adverse clinical signs collected at the time of weekly body weight determinations among either males or females at any exposure concentration.

BODY WEIGHT AND WEIGHT GAIN
Males: On test day 93 the 1500 ppm group expressed a 6.6% weight difference compared to controls. Body weight gain was significantly lower during test days 1-5, and body weight gain over the interval of test days 1-93 was 14% lower than the control value. There were no test substance related effects in body weight in the 750 ppm, but body weight gain was significantly reduced during the test day 1-2 interval. There were no effects on body weight gain in the 350 ppm group.
Females: Test substance-related decrements in body weight and body weight gain occurred in 750 and 1500 ppm females during the course of the study. The 750 ppm had significantly lower body weight for test days 61 and 75-89, and significantly lower body weight gain on test days 2-5 and 54-61. The 1500 ppm had significantly lower body weight for test days 40-93, and significantly lower body weight gain on test days 1-5 and 54-61. Mean body weight gain values over the interval of test days 1-93 for 750 and 1500 ppm groups were significantly lower (21% and 32%, respectively) than the control value. Since the body weight decrements exceeded 20% of the control value for body weight gain, they were considered to be toxicologically significant. There were no test substance-related or statistically significant differences in body·weight or body weight gain for the 350 ppm group.

FOOD CONSUMPTION
Males: 1500 ppm group food consumption values were slightly lower than control values, and the overall mean values over test days 1-93 was 7% lower than the control values, respectively. Instances of statistically lower food consumption for 1500 ppm group occurred over test days 1-5 and 54-61. There were no test substance-related effects on food consumption in the 750 ppm group or below. Although instances of higher and lower food consumption was observed during test days 1-93, they were not consistent over the duration of the study and were not considered to be toxicologically significant.
Females: Test substance-related decreases in food consumption was observed in the 750 or 1500 ppm groups. Food consumption in the 1500 ppm group was significantly lower for test days 1-5,9-47,54-68, and 75-89; and the overall mean food consumption for the 93-day period was 15% lower than the control value. Food consumption in the 750 ppm group was significantly lower for test days 9-12 and 40-47; and the overall mean food consumption for the 93-day period was 8% lower than the control value over the 93-day test period. There were no test substance-related effects on food consumption in the 350 ppm group exposed to ethyl acetate over a 93-day period.

FOOD EFFICIENCY
Males: 1500 ppm group food efficiency values were slightly lower than control values, and the overall mean values over test days 1-93 was 8% lower than the control values, respectively. Food efficiency was significantly lower during test days 1-2. An instance of significantly higher food efficiency for 1500 ppm group was also observed during test days 5-8. There were no test substance-related effects on food efficiency in the 750 ppm group or below. Although instances of higher and lower food efficiency was observed during test days 1-93, they were not consistent over the duration of the study and were not considered to be toxicologically significant.
Females: Test substance-related decreases in food efficiency was observed in the 750 or 1500 ppm groups. Food efficiency in the 1500 ppm group was significantly lower for test days 1-5, and 5461; while the overall mean food efficiency value was 21% lower than control for the interval of test days 1-93. Similarly, food efficiency in the 750 ppm group was significantly lower for test days 2-5 and 54-61, and the overall mean for efficiency was 14% lower than the control value. There were no test substance-related effects on food efficiency in the 350 ppm group exposed to ethyl acetate over a 93-day period.

OPTHALMOLOGY
There were no test-substance related effects on ophthalmologically visible structures of the eye,

HAEMATOLOGY
Males: the 1500 ppm group had mild, statistically significant decreases in the indicators of circulating erythrocyte mass (RBC, Hb, Ht) at the 90-day sampling time.
Female: Significantly decreased mean MCV in 1500ppm females at the 90-day sampling time was not considered to be biologically adverse because there were no relevant changes in the parameters which measure circulating erythrocyte mass (RBC, Hb, Ht). At the 45-day sampling time, significantly decreased mean WBC and lymphocyte counts in 750 ppm females and significantly decreased mean monocyte counts in all treated groups of females were not considered to be test substance related because the mean values did not exhibit a dose-response relationship.

CLINICAL CHEMISTRY
Exposure to ethyl acetate apparently caused decreases in serum triglyceride concentration in 1500 ppm males and females and in 750 ppm males. Also, serum total protein and albumin concentrations were mildly decreased in 1500 ppm females. Mean serum triglyceride concentration was statistically significantly decreased in 750 and 1500 ppm males at the 45-day sampling time and in 1500 females at both sampling times. Exposure to the ethyl acetate likely caused altered lipid metabolism, but the nature of altered lipid metabolism could not be elucidated from the data. Mean serum total protein and albumin concentrations were mildly decreased in 1500 ppm females at both sampling times. Although these statistically significant changes were mild, they indicated possible test substance related decreased albumin synthesis and they were considered to be biologically adverse.

URINALYSIS
Test substance related or biologically adverse urine analytical changes did not occur during this study.

NEUROBEHAVIOUR
Rats exposed to 750 or 1500 ppm had a diminished response to an alerting stimulus one or more times during exposure, beginning with the first exposure session. There was no evidence for development of cumulative toxicity since the rats exposed to 350 ppm, with one exception, consistently
demonstrated a normal response to the stimulus. On test day 5 a diminished response in the 350 ppm group was noted four hours after initiation of the daily exposure. However, when the stimulus was presented six hours after initiation of exposure, the 350 ppm rats exhibited a normal response on this day. Since this is a subjective assessment of the response of an entire group of animals, and since the responses to the stimulus were determined to be normal for the remainder of the exposure sessions, this single occurrence was not considered to be biologically relevant. The 750 and 1500 ppm rats generally continued to display a diminished response to the stimulus over the 68 exposures, with the exception of occasional instances in which a normal response to the stimulus was recorded. At the 90-day sampling time, significantly decreased mean neutrophil count in 350 ppm
females and significantly increased mean neutrophil count in 1500 ppm females were not considered to be test substance related because the mean values did not exhibit a doseresponse relationship. Furthermore, the magnitude of increased neutrophil count in 1500 ppm females was minuscule and biologically inconsequential and it was not accompanied by changes in total leukocyte count.

ORGAN WEIGHTS
There were no test substance related effects on organ weights of rats exposed to the test substance.

GROSS PATHOLOGY
There were no test Substance-related gross observations. All gross observations, except for the tibial fracture, were considered to be spontaneous lesions that occur sporadically in this strain and age of rat. The tibial fracture was considered to be an accidental injury.

HISTOPATHOLOGY: NON-NEOPLASTIC
The only primary test substance-related pathology finding in this study was degeneration of the olfactory mucosa in males and females at all test concentrations.

OTHER
Exposure up to 1500 ppm ethyl acetate had no effect on sperm number, motility or morphology.

Effect levels

open allclose all
Dose descriptor:
LOEC
Effect level:
350 ppm
Sex:
male/female
Basis for effect level:
other: nasal irritation.
Dose descriptor:
NOEC
Remarks:
systemic toxicity
Effect level:
350 ppm
Sex:
male/female
Basis for effect level:
other: all other acute effects, site of contact effects and systemic toxicity

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Microscopic lesions related to ethyl acetate exposure were limited to degeneration of the nasal olfactory mucosa.  The incidence of this lesion in the 350 ppm exposure group was 8 of 20 animals affected with a grade of “minimal” severity.  In the 750 and 1500 ppm groups, the incidence was 100% with a severity grade of “minimal” to “moderate” for the 750 ppm group and “minimal” to “severe” for the 1500 ppm group. 

Applicant's summary and conclusion

Conclusions:
Under the conditions of the study, the no-observed-effect concentration (NOEC) for systemic toxicity was 350 ppm; a NOEC was not demonstrated for respiratory irritation as nasal irritation was observed at all exposure concentrations in both males and females.
Executive summary:

In a guideline and GLP study, rats exposed to concentrations of 350, 750, or 1500 ppm (1.28, 2.75, 5.49 mg/L) in air for 94 days showed no significant toxic effects. Diminished response to delivery of an alerting stimuli was noted during exposure at the 750 and 1500 ppm levels. This transient diminished response was confined to the exposure period and was attributed to acute sedative properties of ethyl acetate. Other findings at the 750 and 1500 ppm levels were limited to reduced food consumption and body weight gain, and lower levels of serum triglycerides. No other changes were seen. Some evidence of nasal mucosa degeneration was seen at 350 ppm.

Synopsis

The NOAEL for systemic toxicity in this study is considered to be 350 ppm (1.28 mg/L) and the LOAEL 750ppm (2.75 mg/L)

The LOAEL for respiratory irritant effects (nasal toxicity) is 350 ppm (1.28 mg/L).