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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:
90 days
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Reliable without restrictions; study was conducted in accordance with GLPs and under methods similar to those described in EPA guideline 799.9310 TSCA 90-Day Oral Toxicity Study in Rodents.

Data source

Referenceopen allclose all

Reference Type:
publication
Title:
Unnamed
Year:
1995
Reference Type:
study report
Title:
Unnamed
Year:
1985
Report Date:
1985

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
other: EPA guideline 799.9310 TSCA "90-Day Oral Toxicity Study in Rodents"
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
-Test substance: Di (2-ethylhexyl) terephthalate
-Synonyms: DEHT
-Supplier: Tennessee Eastman Company, Inc. Kingsport, TN
-Lot number: X-16765-22-1
-Physical Characteristics: Clear, viscous liquid
-Odor: Odorless
-Purity: 98.4-98.9% pure when determined prior to study initiation by gas chromatography, gas chromatography-mass spectroscopy, and high pressure liquid chromatography methodologies. The test substance was determined to be stable, since at study termination, the purity of di (2-ethylhexyl) terephthalate was determined to be 99.1%.
-Impurities: The major impurity, present at 0.6%, was identified as the mixed ester methyl 2-ethylhexyl terephthalate.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
Test animals:
-Strain: Sprague-Dawley (CRL: COBS® CD® (SD) BR)
-Source: Charles River Laboratories, Wilmington, MA, USA
-Sex: Male and Female
-Age at receipt: Males: approximately 4 weeks old; Females: approximately 6 weeks old
-Age at study initiation: approximately 6 (males) - 8 (females) weeks old
-Acclimation period: 2 weeks
-Weight at study receipt: Males: 250 ± 50 g
Females: 200 ± 50g
-Housing: Animals were housed 5 per cage in stainless steel wire-mesh cages; males and females were housed on separate racks.
-Diet: Purina Laboratory Rodent Chow 5002 (certified) ad libitum
-Water: Local municipality (Monroe County, NY) ad libitum
-Method of animal identification: Unique metal ear tag and a standardized ear punch
-Method of animal distribution: Computer-generated randomization procedure.
-Quality control animals: On the day of receipt, 3 animals/sex were randomly selected from each of six separate animal containers. The animals were euthanized and necropsied within 4 hours of arrival, and blood was collected for determination of complete blood counts, a serum chemical chemistry screen and a serology screen. All organs were examined grossly and nasal passages, salivary glands, trachea, liver and kidneys were processed for histolopathological examination.

Environmental Conditions:
-Temperature: 71-78 °F
-Humidity: 33-59%
-Three excursions outside the temperature and humidity limits were neither large nor long-standing, and were not considered to have affected the test animals adversely.
-Photoperiod: 12:12 light:dark
-Air exchange: The room that the animals were housed in was equipped with a laminar flow air system.

In-Life Study Dates:
-Study Initiation Date: August 10, 1984
-Experimental Start Date: September 5, 1984
-Experimental Completion Date: March 13, 1985


Administration / exposure

Route of administration:
oral: feed
Vehicle:
other: Purina Laboratory Rodent Chow 5002 (certified)
Details on oral exposure:
Rats (20 rats/sex/group) were exposed per os to di (2-ethylhexyl) terephthalate at dose concentrations of 0.1, 0.5, or 1.0% in a diet of Purina Laboratory Rodent Chow 5002 (certified) for 90 days. Control animals (20 rats/sex) were provided the standard rat chow without any added di (2-ethylhexyl) terephthalate.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of di (2-ethylhexyl) terephthalate in the diet:
The concentration of di (2-ethylhexyl) terephthalate in the test diets was determined by extraction with acetonitrile and subsequent analysis by HPLC.

Homogeneity of di (2-ethylhexyl) terephthalate concentrations in the diet:
All diets were tested for homogeneity by analyzing samples of the diet taken from the top, middle and bottom of the mixing container. In addition, following termination of use of each batch of diet, a small amount of the remaining mixture was re-analyzed to confirm stability. With a single exception, the diets proved to be homogeneous, with no more than 3-4% variation between the measured concentrations from the top to the bottom of a batch. The single non-homogeneous diet was remixed before it was used on study and reanalysis confirmed the homogeneity of the remixed diet.

Stability of di (2-ethylhexyl) terephthalate in the diet:
-Test diets containing either 0.1 or 1.0% di (2-ethylhexyl) terephthalate in the feed were prepared prior to study initiation and were analyzed once a week for 6 weeks. The analyses of these diets indicated that little degradation of di (2-ethylhexyl) terephthalate occurred with time, with all samples yielding equal to or greater than 90% of the target dose. Di (2-ethylhexyl) terephthalate was considered to be stable for six weeks when mixed with ground laboratory chow. The mean analyzed concentration of the 1.0, 0.5 and 0.1% diets was 0.983, 0.490, and 0.096%, respectively, at the time of mixing. Analyses of the same diets after use on the study revealed the concentrations to be 0.937, 0.460, and 0.091%. In agreement with the above analysis, the comparison of final versus initial concentration revealed a loss of only 8% or less in all diets, as final dietary concentrations of di (2-ethylhexyl) terephthalate, when measured in the experimental diets six weeks after mixing, averaged 92.3% of the original target dose.
-Dose level selection:
The high dose was set at 1% in order to provide a significant exposure to the test substance without markedly changing the nutritive characteristics of the diet, whereas the two other dose levels were chosen to represent 1/2 and 1/10 of the high dose. Previous experience at the testing facility with palatable compounds mixed into diets at a concentration of 1.0% resulted in a dose level of nearly 1000 mg/kg bw/day when offered to a growing animal.
Duration of treatment / exposure:
Approximately 90 days (ad libitum)
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0.1% (54 mg/kg bw/day male and 61 mg/kg bw/day female)
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
0.5% (277 mg/kg bw/day male and 309 mg/kg bw/day female)
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
1.0% (561 mg/kg bw/day male and 617 mg/kg bw/day female)
Basis:
nominal in diet
No. of animals per sex per dose:
20 per sex per dose
Details on study design:
Rats (20 rats/sex/group) were exposed per os to di (2-ethylhexyl) terephthalate at dose concentrations of 0.1, 0.5, and 1.0% in the diet for approximately 90 days. Control animals (20 rats/sex) were provided standard rat chow without the test substance. The following examinations/ measurements were conducted during the course of the study: clinical observations, body weights, food consumption, clinical chemistry, hematology, urinalysis, and hepatic peroxisome proliferation. At the end of the study, all animals were subjected to a gross necropsy, select organ weights were measured, and histopathology was performed.
Positive control:
For the peroxisome study, five male rats were randomly assigned to receive a positive control substance (2-ethylhexanol; known to cause liver enlargement and hepatic peroxisome proliferation) at a dose level of 1500 mg/kg bw/day by oral gavage. Due to excessive toxicity after three doses, the dose level was reduced to 1000 mg/kg bw/day starting on day 4. Dosing continued for a total of 3 consecutive weeks.

Examinations

Observations and examinations performed and frequency:
Examinations: Clinical examinations were conducted on Days 0, 3, 7, and once each week thereafter. Cage side observations were conducted each weekday afternoon and on mornings the examinations were not conducted. Cage side observation included, but was not limited to, examination of the hair, skin, eyes, motor activity, feces and urine.

Body Weights: Body weights were measured prior to study start, on Days 0, 3, 7, and weekly thereafter.

Feed Consumption: Feed consumption was determined on Days 3, 7 and twice weekly thereafter, except during Week 12 when it was determined only once.

Ophthalmic examinations: Ophthalmic examinations were performed on all animals prior to study start and at study termination.

Urinalysis: Ten animals were randomly selected for urine zinc analysis during Weeks 12 and 13. Selected animals were transferred to Nalgene metabolism cages for 24 hours for urine collection on two consecutive days. Urine volumes were measured and quantitation of zinc concentrations and calculation of total zinc excreted were determined.

Clinical Chemistry: Blood was collected from the posterior vena cava while the animals were under CO2 anesthesia at the time of necropsy. The following parameters were examined on blood from 10 animals/sex/dose level: Aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, urea nitrogen, glucose, creatinine, sorbitol dehydrogenase, gamma glutamyl transpeptidase, triglycerides, cholesterol, total protein, albumin, albumin/globulin ratio, and total bilirubin.

Hematology: Blood was collected from the posterior vena cava at the time of necropsy (see above), and the following parameters were examined on all 20 animals/sex/dose level: red blood cell count, hemoglobin concentration, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, prothrombin time, Heinz body count, white blood cell count, differential white blood cell count, nucleated red blood cell count, and red and white blood cell morphology.

Sacrifice and pathology:
Study Termination: Animals were necropsied on Days 91-94. All animals were fasted overnight prior to necropsy. On the day of necropsy, the animals were anesthetized with CO2 and blood was collected from the posterior vena cava for hematology and clinical chemistry analysis. The animals were euthanized by exsanguination and subjected to a complete necropsy.

Organ Weights: Liver, kidneys, heart, adrenal glands, spleen, testes, ovaries, and brain. Paired organs were weighed together.

Tissues Collected and fixed in 10% buffered formalin: Trachea, lungs, thymus, heart, aorta, tongue, salivary glands, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, pancreas, liver, kidneys, urinary bladder, adrenal glands, pituitary gland, thyroid glands, parathyroid glands, spleen, mesenteric lymph nodes, femoral bone marrow, quadriceps femoris muscle, skin, rib, femur, brain, cervical spinal cord, sciatic nerve, testes, epididymides, male accessory sex glands, male mammary gland, ovaries, uterus, vagina, fallopian tubes, female mammary glands and gross lesions. The eyes were collected and fixed in Zenker's (Russell's) fixative.

Histopathology: All collected tissues from the high-dose and control groups were examined microscopically. Target organs and gross lesions in other dose groups were also examined.
Other examinations:
Peroxisome Analysis: Five male rats from the treated and control groups were randomly selected for the peroxisome assay. Additionally, a group of five male rats treated with 1000 - 1500 mg 2-ethylhexanol/kg by gavage 5 days/week for the last 3 weeks of the study served as positive controls. The livers from these animals were removed at necropsy and fixed in 5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4. Samples were immersed in a peroxidase reaction medium containing 3,3’-diaminobenzidine. Following staining, the samples were rinsed in buffer and post-fixed in Dalton’s chrome-osmium solution, dehydrated, and embedded in Epon 812 epoxy resin. Samples were thin-sectioned using a microtome and examined using a Hitachi H600 electron microscope. One randomly chosen field from each of five blocks per rat was photographed, and morphometric analysis was accomplished using an Elographics digitizer attached to a PDP 11/44 computer. A computer program was used to calculate peroxisome area, peroxisome cell fraction, and the number of peroxisomes per 100 µm2 of hepatocyte cytoplasm.
Statistics:
All numerical data were evaluated using the following computerized statistical models: one-way analysis of variance (ANOVA), Bartlett's test, and Duncan's multiple range test. Additional procedures used in the peroxisome assay included the F-test and the Student's t-test.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
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:
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:
not examined
Details on results:
Mortality: No mortality was observed during the study.

Clinical abnormalities: Clinical abnormalities observed during the study were limited to transitory enlargement of the salivary glands, malocclusion of the incisors, and porphyrin nasal discharges. None of these abnormalities were considered treatment-related.

Body Weight Gains: Mean body weight gains were comparable among groups throughout the study.

Food Consumption: Mean feed consumption values were comparable among groups during the course of the study. Indications of slight decreases in food consumption were observed at two points for females during the study, but these were considered spurious and unrelated to the test material.

Male rats consumed an average of 26.85 grams of feed/day throughout the study, and females consumed an average of 19.13 grams/day. Initial daily consumption of the test material for animals consuming the 1.0% diet was 800 mg/kg/day in males and 750 mg/kg/day in females. The overall mean consumption over the study was 561 and 617 mg/kg/day in males and females, respectively, consuming the 1.0% diet. Effective daily doses were 277 and 309 mg/kg in the 0.5% males and females, and 54 and 61 mg/kg in the 0.1% males and females, respectively.

Ophthalmoscopic examination: During the acclimation period, four animals were found to have abnormalities of the eye and were eliminated from the animal pool. The remaining animals were in good general health. None had ophthalmologic abnormalities, and all were released to the study. The animals randomly assigned to the study were examined again for ophthalmologic irregularities during week 13, and none were found to have any abnormalities of the eye at the second examination.

Hematology: Mean hemoglobin, hematocrit, mean corpuscular hemoglobin (MCH), and mean corpuscular volume (MCV)values were significantly lower for male rats from the 1.0% dose group, though absolute reductions averaged only 4-5%. Mean MCH values were also lower for male rats from the 0.5% (2% lower) dose group. For female rats, mean MCV and MCH values were significantly lower for the 1.0 and 0.5% dose groups; reductions averaged 3%. Hemoglobin concentration was slightly and significantly decreased at 0.1% in the diet but this finding was considered spurious since the difference was not present in animals consuming 0.5% di (2-ethylhexyl) terephthalate in the diet.
Examination of red cell morphology in males revealed a number of variations in erythrocyte appearance in all groups which included: microcytosis, anisocytosis, poikilocytosis, and spherocytosis. Single animals had incidences of minimal macrocytosis (0.1% group) and minimal presence of Howell-Jolly bodies (0.5% group). Similar variations were observed in the females, though not as many animals were identified as having each abnormality. The observations made on the morphological characteristics of the red blood cells did not fit a recognizable pattern and were determined to be unrelated to di (2-ethylhexyl) terephthalate exposure and cell morphology observations were not corroborated by calculated numerical changes in hematocrit, hemoglobin concentration, or red cell indices.
No biologically significant differences were seen among males between treated and control animals in white blood cell count, differential white blood cell count, platelet count or prothrombin time. Statistically significant differences were observed in the percentage of polymorphonuclear leukocytes, lymphocytes, and eosinophils, when control animals were compared to animals fed diets containing 0.1 or 0.5% di (2-ethylhexyl) terephthalate. However, the differences were not dose related, as they were not observed in hematology data obtained from animals fed the 1.0% diets. No biologically or statistically significant differences in any of the observed parameters were observed in the females.
Since these changes in hematology were minimal in severity (less than 5%), not dose-dependent, and not accompanied by other abnormalities indicative of anemia, they were not considered to be biologically significant.

Clinical Chemistry: Changes in clinical chemistry parameters were limited to lower mean glucose values for female rats from the 0.1% dose group. However, since this change was not seen in any of the other dose groups, it was considered to be incidental. All other clinical chemistry values were comparable among groups.

Urinalysis: There were no treatment-related differences in urinary zinc concentrations.

Gross Pathology: No test substance-related abnormalities were observed during gross examination of the animals.

Organ Weights: In the males, absolute liver weight was increased by 9% and liver weights relative to body weights were increased 11% in animals consuming 1.0% di (2-ethylhexyl) terephthalate in the diet when compared to controls. These changes were not statistically significant. In contrast to the findings regarding absolute weight, the increase in relative liver weight was statistically significant. In females, the liver weights were similar to those observed in the males; the absolute liver weight was increased by 7% and the relative liver weight was increased by 9% in animals provided the 1.0% di (2-ethylhexyl) terephthalate diet. Similar to what was observed in males, the elevation in relative weights in females was statistically significant at the 1% level, but the absolute weight was not. No other mean absolute or relative (to body weight) organ weights were significantly altered in either sex. When organ weights were expressed relative to brain weights, no changes were present in either sex, though a trend was observed toward increasing relative liver to brain weights in both sexes at 1.0% in the diet. The differences did not reach statistical significance, but liver to brain weight ratios were increased 9 and 10% in the males and females, respectively, when compared to controls.

Histopathology: Histopathological examinations of organs from the control and high-dose groups as well as gross lesions from other groups did not reveal any treatment-related effects. The abnormalities observed in tissues from the animals were limited to lesions commonly observed in animals of this strain in the testing laboratory.

Peroxisome Assay: Based on the results of the peroxisome assay, there was no indication of peroxisome induction in animals from the 1.0% dose group. In contrast, the positive control caused a 28% increase in the liver peroxisome fraction and a 33% increase in peroxisome density.

Effect levels

open allclose all
Dose descriptor:
NOEL
Effect level:
277 other: mg/kg/day
Sex:
male
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOEL
Effect level:
309 other: mg/kg/day
Sex:
female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Quality control animals:

The profile of results on the quality control animals was similar to that routinely obtained from the vendor and correlated well with historical data obtained within the testing facility.

Applicant's summary and conclusion

Conclusions:
When di (2-ethylhexyl) terephthalate was administered to rats via the diet at concentrations of 0, 0.1, 0.5, and 1% for 90 days, the NOEL for systemic toxicity was determined to be 0.5% (equivalent to 277 and 309 mg/kg bw/day for males and females, respectively) based on minor effects on red blood cell formation, and enlargement of the liver in both sexes at a dose concentration of 1.0%.

There were no deaths, no functional changes in any organ system, and no significant adverse effects on clinical chemistry parameters, hematology or urinalysis during the course of the study. Although there was a statistically significant increase in relative liver weight to body weight ratios for both sexes at the 1% dose level, there was no corresponding organ damage seen at gross or microscopic examination, no evidence of liver dysfunction, and no effects on clinical chemistry parameters that would be suggestive of liver damage. Based on an absence of significant effects and clear NOEL values of 277 mg/kg bw/day and 309 mg/kg bw/day for males and females, respectively, following exposure in the diet for 90 days, di (2-ethylhexyl) terephthalate is not classified for “Specific Target Organ Toxicity – Repeated Exposure” according to GHS.

Executive summary:

In a subchronic dietary toxicity study, di (2-ethylhexyl) terephthalate was administered to 20 rats/sex/group at target concentrations of 0, 0.1, 0.5, and 1.0% continuously for 90 days. All animals survived and there were no treatment-related clinical signs. Toxicity related to the administration of di (2-ethylhexyl) terephthalate was limited to minor effects on red blood cell formation, and enlargement of the liver in both sexes at a dose concentration of 1.0%. There were no corresponding functional changes in the liver, no gross or microscopic liver changes, and no adverse effects on any clinical chemistry parameters that would indicate liver damage. All other findings in the study occurred in a non dose-dependent manner, were spurious in nature, or were biologically irrelevant and were not considered related to di (2-ethylhexyl) terephthalate consumption. Under the conditions of this study, the NOEL in male and female rats exposed via the diet to di (2-ethylhexyl) terephthalate was 277 and 309 mg/kg bw/day in males and females, respectively.