Registration Dossier

Administrative data

Description of key information

There was no evidence of a tumorigenic response in male and female rats following lifetime exposure to up to 12000 ppm di (2-ethylhexyl) terephthalate in the diet. The substance is non carcinogenic.

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records
Reference
Endpoint:
carcinogenicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
104 weeks
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Reliable without restriction; study was conducted according to OPPTS 870.4200 guideline and GLPs.
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.4200 (Carcinogenicity)
Deviations:
no
GLP compliance:
yes
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals or test system and environmental conditions:
Test Animals:
-Source: Charles River, Inc., Wilmington, MA, USA
-Sex: male and female
-Age at receipt: approximately 26-30 days of age
-Age at study initiation: approximately 37-41 days of age
-Acclimation period: 12 days
-Weight at study initiation: Males: 89-127 g
Females: 81-110 g
-Housing: Singly housed in polypropylene cages with a stainless steel mesh floor (area was 889 cm2) suspended above absorbent paper. The cages constituting each group were blocked together by sex within the batteries. The batteries were rotated around the room at weekly intervals (cage positions within each group were not changed). Males and females were housed in separate rooms in the animal facility.
-Diet: Rat and Mouse No. 1 Maintenance Diet, Special Diets Services Ltd., Witham, Essex, England (ad libitum), except when urine was being collected and overnight before routine blood sampling.
-Water: Local municipality ad libitum via polycarbonate bottles fitted with sipper tubes, except when urine was being collected.
-Method of animal identification: Each animal was assigned a unique number and identified within the study by a tail tattoo. The animals were randomly assigned to four groups of 50 animals per sex. Each cage label was color-coded according to group and was numbered uniquely with cage and study number, as well as the identity of the occupant.
-Method of animal distribution: On arrival, the rats were removed from the transit boxes and allocated to study cages. Using the sequence of cages in the battery, one animal at a time was placed in each cage; this was repeated until each cage held the appropriate number of animals. Each sex was allocated separately.
-Enrichment: Each animal was provided with an Aspen chew block (B & K Universal Ltd., Grimston, Kingston-upon-Hull, East Yorkshire, England) for environmental enrichment from Week 16 to the end of the study.
-Preliminary screening: A routine health check was performed on the animals within 24 hours of arrival. Four animals per sex were selected at random, bled, euthanized and subjected to a routine macroscopic examination. No evidence of specific infectious disease was seen macroscopically, and the rats were considered acceptable for use on the study.

Environmental Conditions:
-Temperature: 19 to 23 °C
-Humidity: 40-70%
-Photoperiod: 12 hours continuous light and 12 hours continuous dark per 24 hours
-Air exchanges: Each animal room was kept at positive pressure with respect to the outside by its own supply of filtered fresh air, which was passed to atmosphere and not re-circulated.

In-Life Study Dates:
-Study Initiation Date: December 19, 2001
-Experimental Start Date: January 17, 2002
-Experimental Completion Date: October 18, 2004

Route of administration:
oral: feed
Vehicle:
other: Rat and Mouse No. 1 Maintenance Diet, Special Diets Services Ltd., Witham, Essex, England
Details on exposure:
The test substance was administered continuously via the diet at concentrations of 1500, 6000, or 12000 ppm for 104 weeks. Animals did not have access to mixed diet beyond the end of its shelf-life (as determined by a stability test). Control animals received untreated diet at the same frequency, and from the same batch of basal diet, as treated animals.

Test material formulation: The test substance was prepared for administration as a series of graded concentrations in the diet by dilution of an appropriate premix. An initial premix at a concentration of 100000 ppm was prepared. The required amount of test material was initially mixed with a similar weight of fine, sieved diet by gentle stirring. Additional quantities of coarse diet were then added to the premix and stirred until the final weight was achieved. This was then mixed using a Turbular mixer for 100 revolutions to ensure that the test material was dispersed in the diet. Aliquots of this premix were then diluted with coarse diet to produce the required concentrations (1500, 6000 and 12000 ppm). Each batch of treated diet was mixed for a further 100 revolutions. Batches of test diets were prepared weekly, issued in sealed metal containers and stored at ambient temperature.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Detailed records of compound usage were maintained. The amount of test substance necessary to prepare the formulations and the amount actually used were determined on each occasion. The difference between these amounts was checked before the formulations were dispensed. Before treatment commenced, the suitability of the proposed mixing procedure was determined, and specimen formulations were analyzed to assess the homogeneity and stability of the test substance in the diet. Samples of each formulation prepared for administration in Weeks 1, 13, 26, 52, 78 and 104 of treatment were analyzed for achieved concentration of the test substance. The mean concentrations of test material in all formulations were within 9% of intended, confirming accurate formulation. Homogeneity and stability of samples stored at ambient temperature for 35 days were confirmed at 1500 and 12000 ppm.
Duration of treatment / exposure:
104 weeks
Frequency of treatment:
Ad libitum in the diet for 104 weeks
Remarks:
Doses / Concentrations:
1500 ppm
Basis:
other: Based on feed consumption and analytical concentration, equivalent to 79 and 102 mg/kg/day in males and females, respectively
Remarks:
Doses / Concentrations:
6000 ppm
Basis:
other: Based on feed consumption and analytical concentration, equivalent to 324 and 418 mg/kg/day in males and females, respectively
Remarks:
Doses / Concentrations:
12000 ppm
Basis:
other: Based on feed consumption and analytical concentration, equivalent to 666 and 901 mg/kg/day in males and females, respectively
No. of animals per sex per dose:
50 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
The dietary concentrations used in the study (0, 1500, 6000 and 12000 ppm) were selected in conjunction with the Sponsor. Previous studies of ortho-phthalate ester plasticizers have reported liver tumors in F344 rats at 6000 and 12000 ppm in the diet. These two concentrations were chosen to compare the carcinogenic potential of the test substance to other phthalate ester plasticizers; the concentration of 1500 ppm was selected to provide a no-observed-effect level.

Four groups of 50 rats/sex/group received the test substance per os, via the diet, at concentrations of 0, 1500, 6000 or 12000 ppm. During the study, clinical observations, bodyweight, food consumption, ophthalmic examination, haematology, blood chemistry, urinalysis, organ weight, macropathology and histopathology investigations were undertaken at specified intervals.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes for mortality/morbidity or reaction to the test substance
-Time schedule: twice daily, 7 days per week. The observations were designed to identify any abnormalities in at least the following: skin and fur, eyes and mucous membranes, respiratory system, circulatory system, autonomic and central nervous system, somatomotor activity and behavior pattern. Any deviation from normal was recorded at the time in respect to nature and severity, date and time of onset, duration and progress of the observed condition (as appropriate).

DETAILED CLINICAL OBSERVATIONS: Yes
-Time schedule: Clinical observations were recorded weekly throughout the study. The detailed clinical observations included palpation of each animal to monitor general health, while particular attention was paid to superficial or palpable swellings, for which the location, size, consistency, time of first observation and subsequent history were recorded. Cages and cage-trays were inspected daily for loose feces.

BODY WEIGHT: Yes
-The weight of each rat was recorded on the first day of treatment (Week 0), at weekly intervals for the first 16 weeks of treatment, once every four weeks thereafter (to coincide with the end of a food consumption measurement period) and before necropsy. More frequent weighing was instituted for animals displaying any signs of illness, so that the progress of the observed condition could be monitored.

FOOD CONSUMPTION: Yes
-The weight of food supplied to each animal, that remaining and an estimate of any spilled was recorded each week for the first 16 weeks and every four weeks thereafter. Mean weekly food consumption per animal (g/rat/week) was calculated.

FOOD EFFICIENCY: Yes
Food conversion efficiency was calculated as body weight gain (g) / total food consumed (g/rat) x 100. The group mean achieved dosage for each sex (mg/kg/day) = food consumed (g/rat) x ppm test material/midweek body weight (g) x 7.

WATER CONSUMPTION: no data

OPTHALMOSCOPIC EXAMINATION: Yes
-During treatment Week 104, the eyes of all surviving animals were examined by means of a binocular indirect ophthalmoscope. The pupils of each animal were dilated using 0.5% tropicamide ophthalmic solution (Mydriacyl, Alcon Laboratories Ltd.) and examination included the adnexa, conjunctiva, cornea, sclera, anterior chamber, iris (pupil dilated), lens, vitreous and fundus.

HEMATOLOGY: Yes
-During weeks 52 and 78 of treatment, blood smears were prepared from samples obtained from the tail vein of all surviving animals (without overnight fasting or use of anesthetic). The smears were air-dried, fixed in methanol, stained using a Romanowsky stain, and examined for the following characteristics: differential leukocyte count and abnormalities of the blood film.

During Week 104, surviving animals were fasted overnight and anesthetized with isoflurane; blood samples were obtained from the retro-orbital sinus and collected into tubes containing EDTA. The blood was subsequently examined for hematocrit, hemoglobin concentration, red blood cell count, mean cell hemoglobin, mean cell hemoglobin concentration, mean cell volume, total white cell count, differential leukocyte count, neutrophils, lymphocytes, eosinophils, basophils, monocytes, large unstained cells and platelet count using a Technicon H1 hematology analyzer. Abnormal morphology was flagged by the analyzer and the most common morphological changes (anisocytosis, micro/macrocytosis and hypo/hyperchromasia) were recorded as follows: - = no abnormalities detected; + = slight; ++ = moderate; +++ = marked.

Blood films were also prepared for all samples and films containing blood flagged by the Technicon analyzer were stained with Romanowsky stain and examined for abnormalities by light microscopy. Confirmation or a written description from the blood film was made where appropriate. Further analysis included staining with brilliant cresyl blue and basic blue 24 to identify normal and abnormal reticulocytes (including those with Heinz bodies). Additional blood samples (0.5 mL) were taken and placed into collection tubes containing citrate and examined for prothrombin time and activated partial thromboplastin time using an ACL 1000 Analyzer with IL PT-Fibrinogen reagent or IL APTT reagent, respectively.

CLINICAL CHEMISTRY: Yes
-Additional blood samples were collected in tubes containing lithium heparin during study week 104 from all surviving animals. It is assumed (but not stated) that these samples were taken from the retro-orbital sinus after anesthesia. All tubes were mechanically agitated for at least two minutes; the sample was centrifuged at 3000 rpm for 10 minutes in order to separate the plasma, and assayed for: alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, total bilirubin, urea, creatinine, glucose, total cholesterol, triglycerides, sodium, potassium, chloride, calcium, inorganic phosphorus and total protein using a Hitachi 917 Clinical Chemistry Analyzer. Electrophoretic protein fractions albumin, alpha 1, alpha 2, beta and gamma globulin were analyzed with agarose gel, using a Beckman test kit, staining with Ponceau-S and scanning with a suitable densitometer. The albumin/globulin ratio (A/G Ratio) was calculated from total protein and albumin concentrations.

URINALYSIS: Yes
-Overnight urine samples were collected during study week 103 from all surviving animals. Animals were individually placed in a metabolism cage without food or water at approximately 16:00 hours, and urine was collected until approximately 08:30 hours the following day. The individual samples were examined for volume, visual appearance (brown, brown-yellow, dark orange, medium yellow, orange, pale yellow, cloudy with medium yellow color, or cloudy with pale yellow color), pH (using a Radiometer PHM 92 pH meter), specific gravity (with a Atago UR-1 digital refractometer), protein (with a Hitachi 917 Clinical Chemistry Analyzer), and glucose, ketones, bile pigments and heme pigments using Multistix (Bayer plc, Newbury, England).

A microscopic examination of the urine sediment also was performed. An aliquot of the urine sample was centrifuged, the deposit was stained with Kova stain, spread on a microscope slide, and the slide was examined for the presence of crystals, epithelial cells, leukocytes, erythrocytes, casts, spermatozoa and precursors and other abnormal components. The frequency of each finding was scored on a scale of 0 (none found) to 3 (many in all fields examined).

NEUROBEHAVIORAL EXAMINATION: no data
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
-Animals judged in extremis were euthanized during the study. Otherwise, animals were euthanized after blood collection at 104 weeks using CO2 asphyxiation, and a complete necropsy was performed including a full macroscopic examination of the tissues. All external features and orifices were examined visually. The cranial roof was removed to allow observation of the brain, pituitary gland and cranial nerves. After making a ventral midline incision, the neck and associated tissues and the thoracic, abdominal and pelvic cavities and their viscera were exposed and examined in situ, and any abnormal position, morphology or interaction was recorded.

Any abnormality in the appearance or size of any organ or tissue was recorded and photographs of unusual findings were taken at the discretion of the necropsy supervisor. The tissues saved for possible histopathological examination were: adrenals, aorta (thoracic), brain, cecum, colon, duodenum, epididymides, eyes, femur (one), head, heart, ileum, jejunum, kidneys, lachrymal glands, larynx, liver, lungs, lymph nodes (mandibular, mesenteric, regional to masses), mammary area (caudal), nose, esophagus, ovaries, pancreas, pharynx, pituitary, prostate, rectum, salivary gland (one), sciatic nerve (one), seminal vesicles, skeletal muscle (one thigh), skin, spinal cord, spleen, sternum, stomach, testes, thymus, thyroid (with parathyroids), trachea, urinary bladder, uterus (with cervix) and vagina; the tissues were preserved in 10% neutral buffered formalin except for the testes and epididymides which were fixed in Bouin's solution prior to transfer to 70% methyl alcohol. The eyes were fixed in Davidson's fluid. Samples of any abnormal tissues were also retained and processed for examination and in those cases where a lesion was not clearly delineated, contiguous tissue was fixed with the grossly affected region and sectioned as appropriate.

ORGAN WEIGHTS: Yes
-Adrenals, brain, epididymides, heart, kidneys, liver, ovaries, spleen, testes, and uterus with cervix were weighed, and bilateral organs were weighed together. Organs weights were adjusted for terminal body weight.

HISTOPATHOLOGY: Yes
-Tissue samples were dehydrated, embedded in paraffin wax, sectioned at approximately four to five micron thickness and stained with hematoxylin and eosin (except the testes, which were stained using a standard periodic acid/Schiff method). Sections of both bilateral organs were prepared. Microscopic examinations were conducted on all preserved tissues from the control and high dose animals that survived to study termination and for all animals euthanized or dying prematurely during the treatment period. The liver, kidney, nasal turbinates (females only), eyes, and grossly abnormal tissues were examined for all animals in the low- and mid-dose groups that survived to study termination. The pancreas and testes were examined for all mid-dose animals surviving to study termination. Samples of the head (apart from the nasal turbinates) and the remaining femur, salivary gland, sciatic nerve and skeletal muscle (thigh) were not examined. Findings were either reported as "present" or assigned a severity grade. If assigned a severity grade one of the following five grades was used - minimal, slight, moderate, marked or severe. A reviewing pathologist conducted a peer review of the microscopic findings.
Statistics:
Statistical analyses were used throughout the study. Means were calculated for body weights, food consumption, and survival in days. All other analyses were carried out using the individual animal as the basic experimental unit. For categorical data, including pathological findings, the proportion of animals was analysed with a Fisher’s Exact test for each treated group versus the control. For continuous data, Bartlett’s test was first applied to test the homogeneity of variance between the groups. For bodyweight gains and organ weights, if the Bartlett’s test was found to be statistically significant, a Behrens-Fisher test was used to perform pairwise comparisons; otherwise, a Dunnett’s test was used.

For clinical pathology data: if 75% of the data (across all groups) were the same value, then a frequency analysis was applied where the treatment groups were compared using a Mantel test for a trend in proportions and also pairwise Fisher's Exact tests for each dose group against the control. If Bartlett's test for variance homogeneity was not significant at 1%, parametric analysis was applied. If the F1 test for monotonicity of dose-response was not significant at the 1% level, Williams' test for a monotonic trend was applied. If the F1 test was significant, Dunnett's test was performed to determine if a dose-response existed. If Bartlett's test was significant at the 1% level, logarithmic and square-root transformations were tried. If Bartlett's test was still significant, the H1 test for monotonicity of dose-response was conducted and if it was not significant at the 1% level, a Shirley's test for a monotonic trend was applied. If the H1 test was significant, suggesting that the dose-response was not monotonic, Steel's test was performed instead.

Inter-group differences in mortality and tumor incidence were performed using the approach of Peto et al. Differences between control and treated groups were considered significant at p<0.05 or p<0.01.
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):
no effects observed
Food efficiency:
effects observed, treatment-related
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
effects observed, treatment-related
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
effects observed, treatment-related
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:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
MORTALITY: The survival rates of females receiving 0, 1500, 6000 and 12000 ppm were 56%, 62%, 68% and 72%, respectively. The survival rates of females receiving 6000 or 12000 ppm were slightly higher than control. The trend test in females was statistically significant (p = 0.048) when all groups were included, but not when the high dose was excluded (p = 0.136). None of the pairwise comparisons was statistically significant. There was no difference in survival between treated and untreated males (the survival rates of males treated with 0, 1500, 6000 or 12000 ppm were 58%, 52%, 58% and 52%, respectively).

CLINICAL SIGNS: The behavior of the animals was considered unaffected by treatment. There was a slightly higher incidence of perigenital staining in males exposed to 6000 or 12000 ppm (3 and 6) vs. control (0). Brown staining of the left forelimb was observed in 9 males exposed to 12000 ppm (vs. 0 in controls). Yellow perigenital staining was found in 16 males exposed to 12000 ppm (vs. 8 in controls). There was a slightly higher incidence of yellow staining of the ventral body surface (16 vs. 1) and perianal region (9 vs. 0) in females receiving 12000 ppm. There was also a slightly higher incidence of both (14 vs. 7) or right (14 vs. 2) opaque eyes for females receiving 12000 ppm, when compared to controls. However, the incidence of this finding in left eyes of females was similar to control (8 vs. 11, respectively). None of the clinical changes were considered toxicologically significant since the incidences were low and the findings were not observed in both sexes.

The total number of swellings palpated in-life was lower in males receiving 6000 (43) or 12000 ppm (42) vs. controls (55) and in females receiving 12000 ppm (7) than in controls (18). The number of animals with swellings also was lower in 12000 ppm females (6) than controls (15). The mean time of onset of the first palpable swelling in treated animals was similar to controls (59 - 70 weeks in males and 82 - 90 weeks in females).

BODY WEIGHT: Body weight gain of males and females receiving 12000 ppm was significantly lower than controls throughout the treatment period (overall bodyweight gain was 93 and 75% of the control value, respectively). The body weight gain of males and females receiving 6000 ppm was significantly lower than controls during the first year of the study (body weight gain to Week 52 was 97 and 96% of control value, respectively). At the end of the study, the overall bodyweight gain of these animals was similar to that of the controls. There was no effect of treatment on body weight gain at 1500 ppm at any time during the study.

FOOD CONSUMPTION: There was no effect of treatment on food consumption. Treated males and females consumed an average of 98 - 102% of the amount of food consumed by controls throughout the study.

FOOD CONVERSION EFFICIENCY: Over the first 16 weeks of the study, food conversion efficiency of high dose males and females (9.6 % and 5.3 %, respectively) was slightly (but not significantly) lower than controls (10.6% and 6.1%, respectively).

OPHTHALMIC EXAMINATION: The ophthalmoscopy examination during Week 104 revealed a higher incidence of fundic hyperreflection and conjunctival discharge for females (but not males) receiving 12000 ppm (44% and 58%), when compared to controls (28% and 31%, respectively). There was also a higher incidence of anterior subcapsular opacity in the lens of females (but not males) receiving 6000 (26%) or 12000 ppm (28%), when compared to controls (10%). There was no relationship between the animal position in the batteries and the ophthalmic lesions.

HEMATOLOGY: There was no effect of treatment on differential leukocyte numbers in blood smears prepared during Weeks 52 or 78 of treatment. Hematocrit and hemoglobin concentrations were significantly lower (p < 0.05) than control in females receiving 12000 ppm. Mean cell hemoglobin and mean cell volume were significantly lower than control (p < 0.01) in males and females receiving 6000 or 12000 ppm. At these concentrations there also was a significant increase in red blood cell count of males (p < 0.01). Prothrombin times were slightly longer (p < 0.05) and activated partial thromboplastin times slightly shorter (p < 0.01) than control for females receiving 12000 ppm. The differences were, however, small and were not considered to be toxicologically significant. There was also a trend towards high platelet count in females receiving 6000 (p < 0.05) or 12000 ppm (p < 0.01). A number of other inter-group differences attained statistical significance when compared with the controls, but these showed no dose-relationship, were seen in one sex only, and individual values were within normal background ranges at these laboratories. These differences were, therefore, not attributed to treatment. Such changes included the variation of reticulocyte count in males, which was attributed to high individual values for controls that increased the mean value for this group.

BLOOD CHEMISTRY: In Week 104, plasma urea concentrations were significantly higher (p < 0.01) and total cholesterol and triglyceride levels significantly lower than control for females receiving 12000 ppm. In females receiving 6000 ppm and in males and females receiving 12000 ppm there was a significant increase (p < 0.05) in plasma glucose concentration, though the increase was not dose-related in females. A number of other inter-group differences attained statistical significance (when compared with the respective controls), but these showed no dose-response relationship, were seen in one sex only, and individual values were within normal background ranges at these laboratories. These differences were, therefore, not attributed to treatment. Such variations included the intergroup differences for plasma electrolyte concentrations and plasma protein in females.

URINALYSIS: Urinalysis investigations in Week 103 indicated significantly lower volume and higher specific gravity in males receiving 6000 ppm and in males and females receiving 12000 ppm when compared to controls (all p < 0.01). In females receiving 12000 ppm there was a reduction of urinary protein (p < 0.05). In addition, the pH of urine produced by males or females receiving 6000 or 12000 ppm was lower than control values (p < 0.01). Compared to controls, fewer urine specimens of females receiving 6000 ppm (9/34 treated vs. 24/30 controls) and of males (2/28 treated vs. 9/29 control) and females (0/36 treated vs. 24/30 control) receiving 12000 ppm appeared cloudy. The significance of this finding was unclear.

ORGAN WEIGHTS: After 104 weeks of treatment, the relative liver and kidney weights to body weights of high dose females were significantly higher than control (p < 0.01 and 0.05, respectively). The relative liver weight of high dose males also was higher than control, but the difference was not statistically significant. Other inter-group differences in organ weights which attained statistical significance were either a result of high individual variability, or were considered to be secondary to the effects on body weight and not directly associated with treatment.

GROSS NECROPSY: There were no treatment-related gross findings in animals that died or were euthanized during the study. For animals euthanized after 104 weeks of treatment, there was a higher incidence of dark kidneys (9/36 treated vs. 1/27 controls) and opaque eyes (21/36 treated vs. 8/27 controls) in females which received 12000 ppm and a lower incidence of pale areas on the liver for females which received 6000 (19/34) or 12000 ppm (15/36), when compared to controls (25/27). The reason for the latter finding may be an abnormally high incidence of pale areas on the liver of female controls (only 8 control males had pale areas on the liver). Fluid distension was noted in the uteri of 7/36 high dose terminal females, compared to 0/27 controls (p < 0.05). When all females were taken into account (including decedents), the incidence of this lesion was not significantly different between high dose (9/50) and control (2/50) females.

HISTOPATHOLOGY, NON-NEOPLASTIC:
Treatment-related non-neoplastic findings were seen in the kidneys, liver, eyes and nasal turbinates. In high dose females (terminal and combined terminal and decedent), the incidence of chronic progressive nephropathy decreased significantly. The combined incidence of this lesion was 11/50 high dose females vs. 24/50 controls. There was no effect of treatment on the incidence of this lesion in males. A decreased incidence of mineralization of the pelvic/papillary epithelium also was seen in kidneys of treated males and females. This was statistically significant for males (terminal and combined) at 6000 or 12000 ppm, for males (decedent and combined) at 1500 ppm, for females (terminal and combined) at 12000 ppm and for females (decedent, terminal and combined) at 6000 ppm. The combined incidences of this lesion in males treated with 0, 1500, 6000 and 12000 ppm were 42/50, 30/50, 15/50 and 15/50, respectively. The corresponding incidences of this lesion in females were 40/50, 42/50, 25/50 and 24/50.

In the liver, a statistically significant decreased incidence of periportal hepatocyte vacuolization was seen at all dietary concentrations in males euthanized after 104 weeks of treatment (incidences at 0, 1500, 600 and 12000 ppm were 13/29, 4/26, 0/29 and 1/26, respectively) and all males (total incidences at 0, 1500, 600 and 12000 ppm were 14/50, 5/50, 0/50 and 2/50, respectively). This phenomenon was not seen in females or decedent males.

In the eyes, a statistically significantly increased incidence of loss of the outer nuclear layer of the retina was seen in females given 6000 or 12000 ppm (p < 0.01). This lesion occurred both unilaterally and bilaterally and was thought to be an exacerbation of an age-related finding. This lesion was noted in 27/50 and 40/50 mid and high dose females (respectively) that were killed or died during the treatment period (vs. 9/50 controls). Increased incidences of this lesion were not noted in low dose female animals or in males. High dose females that were killed or dying also had a significant (p < 0.05) increase in the incidence of vascular mural mineralization of the sclera (5/14 vs. 1/23 control). The incidence of this lesion also was increased in high dose terminal males but was not significant.

In the nasal turbinates, an increased incidence of prominent eosinophilic inclusions was seen in terminal and combined terminal and prematurely decedent females which received 6000 or 12000 ppm (36/50 total and 47/50 total, respectively, vs. 29/50 total controls). This was statistically significant for terminal and combined females at 12000 ppm. It is believed that this may have been an exacerbation of an age-related finding. This phenomenon was not seen in males at any of the dietary concentrations in this study or in females from the lower dosage group.

There were no other histopathological findings associated with treatment. There was a decreased incidence of lymphoid aggregates in the urinary bladder in males which received 12000 ppm (0/50 treated vs. 6/50 control), but this was only statistically significant when decedent and terminal were considered together. This may have been due to an abnormally high number of control males being affected (none of the control females were affected). There were no major factors determined to be contributory to death or euthanization during the study that were considered to be related to treatment.

HISTOPATHOLOGY, NEOPLASTIC:
There was no evidence of a treatment-related effect on the incidence of any tumor type for any group of rats. There were no statistically significant differences in incidences of specific tumors between treated and control groups, with the exception of an increased incidence of large granular cell lymphomas in low dose males (19/26 low dose treated vs. 13/29 control). The incidence of this lesion actually decreased with dose (16/29 mid dose and 8/26 high dose); therefore, it is not considered to be related to treatment.
Relevance of carcinogenic effects / potential:
Under conditions of this study, there was no evidence of a treatment-related effect on the incidence of any tumor type for any group of rats. Di (2-ethylhexyl) terephthalate is unlikely to pose a significant carcinogenic risk to humans exposed to low levels of this chemical.

Dose descriptor:
NOEL
Effect level:
1 500 ppm (analytical)
Sex:
male/female
Basis for effect level:
other: Toxicity was limited to low weight gain and food conversion efficiency in male and females receiving 6000 or 12000 ppm and ocular changes in females receiving 6000 or 12000 ppm.
Remarks on result:
other: Effect type: toxicity (migrated information)
Dose descriptor:
NOEL
Effect level:
12 000 ppm (analytical)
Sex:
male/female
Basis for effect level:
other: There was no effect on tumor incidence caused by the test substance at any dose level tested.
Remarks on result:
other: Effect type: carcinogenicity (migrated information)
Dose descriptor:
NOEL
Effect level:
12 000 ppm (analytical)
Sex:
male
Basis for effect level:
other: No adverse changes were observed in the testes during the study.
Remarks on result:
other:
Remarks:
Effect type: other: testicular toxicity (migrated information)
Dose descriptor:
NOEL
Effect level:
12 000 ppm (analytical)
Sex:
male/female
Basis for effect level:
other: There were increased liver weights in the study but no adverse histopathological findings and the increased liver weight was considered only an adaptive response to the test substance.
Remarks on result:
other:
Remarks:
Effect type: other: liver toxicity (migrated information)

Despite the urinalysis, biochemical, and gross pathological changes indicative of a kidney effect, histopathological examination did not identify any adverse effect of the test material on the kidney. The decreased incidences of chronic progressive nephropathy in treated females and mineralization of the pelvic/papillary epithelium in treated males and females were determined to be of no toxicological relevance.

 Since the magnitude of the red blood cell changes was small and did not elicit an adaptive response (there was no evidence for an increase in bone marrow activity, extramedullary hematopoeisis, congestion of the spleen, or an increase in bilirubin), it was thought that they were non-specific effects. The increase in liver weights in high dose females was thought to be adaptive, since it was not associated with any histopathological change. The changes in glucose, cholesterol and triglyceride were attributed to an alteration of hepatic metabolism. However, there was no evidence for disruption of hepatic fat metabolism, such as increased vacuolization. The reduced incidence of periportal hepatocyte vacuolization in treated males was not considered to be of toxicological significance.

Conclusions:
In a 2-year chronic toxicity study, di (2-ethylhexyl) terephthalate was administered to 50 F344 rats/sex/dose ad libitum via the diet, at dose concentrations of 0, 1500, 6000, or 12000 ppm for 104 weeks. Survival was slightly higher in di (2-ethylhexyl) terephthalate-treated female animals compared to controls but no changes in survival were observed in males. Final mean body weight was up to 25% less than controls in the high-dose group. There was no effect of treatment on food consumption. When all animals were included, there were no treatment-related effects on gross pathology and there were no effects on tumor incidence. Treatment-related non-neoplastic findings were seen in the kidneys, liver, eyes and nasal turbinates.

Based on the findings of the study, the no-observed-effect level (NOEL) for tumorogenicity was 12000 ppm (666 mg/kg bw/day in males and 901 mg/kg bw/day in females); the NOEL for chronic toxicity was 1500 ppm (79 mg/kg bw/day in males and 102 mg/kg bw/day in females) based on reduced weight gain and food conversion efficiency in males and females receiving 6000 or 12000 ppm and ocular changes in females receiving 6000 or 12000 ppm. While other phthalate esters have been reported to cause liver tumors at 6000 and 12000 ppm in the diet, no liver tumors and no significant non-neoplastic effects were observed in the livers of animals exposed to up to 12000 ppm di (2-ethylhexyl) terephthalate in the diet for up to 2 years. In addition, no adverse effects were observed in the testes which have been the site of toxicity following exposure to other phthalate esters such as DEHP.

Based on the findings of the study, di (2-ethylhexyl) terephthalate is not classified under GHS for “Carcinogenicity”. Based on the absence of adverse effects on any reproductive organ, di (2-ethylhexyl) terephthalate is not classified for “Reproductive Toxicity” according to GHS. Based on the absence of any significant target organ toxicity following chronic exposure in the diet at concentrations up to 12000 ppm, di (2-ethylhexyl) terephthalte is not classified for “Specific Target Organ Toxicity-Repeated Exposure” according to GHS.
Executive summary:

Three groups of 50 animals/sex/group were exposed to di (2-ethylhexyl) terephthalate per os, via the diet, at concentrations of 1500, 6000 or 12000 ppm for 2 years. A similarly constituted control group received the untreated diet for the same duration. There were no clinical signs of toxicity related to treatment. In males receiving 6000 or 12000 ppm, there was a reduction in the total number of palpable swellings, whereas in females receiving 12000 ppm there was both a reduction in the number of animals with swellings and the total number of swellings identified. Survival amongst females receiving 6000 or 12000 ppm was slightly higher than that of the controls, but there were no differences in survival in males. Body weight gain was statistically reduced in males and females receiving 12000 ppm throughout the treatment period and in males and females receiving 6000 ppm only during the first year of the study. Food consumption was not affected by treatment, but food conversion efficiency was initially slightly lower in the 12000 ppm animals.

  

Ocular changes were also noted at study termination in females consuming the 12000 and 6000 ppm diets. A number of minor hematological and clinical chemistry changes were noted in the 6000 and 12000 ppm groups including: lower haematocrit and haemoglobin concentration in females receiving 12000 ppm, lower mean cell haemoglobin and mean cell volume in animals receiving 6000 or 12000 ppm, and high erythrocyte counts in males receiving 6000 or 12000 ppm. Increased plasma urea, but lower total cholesterol and triglyceride concentrations were noted in females receiving 12000 ppm. Plasma glucose concentrations were elevated in 6000 ppm females and in both sexes at 12000 ppm. Urinary volume was decreased and specific gravity was increased in 6000 ppm males and in 12000 ppm males and females. Urinary protein was reduced in 12000 ppm females, and urinary pH was lower at 6000 and 12000 ppm. Females consuming 12000 ppm had increased relative (to body weight) liver and kidney weights; the12000 ppm group also had an increased incidence of dark kidneys and opaque eyes, whereas animals receiving 6000 or 12000 ppm had a low incidence of pale areas on the liver. There were no treatment-related macroscopic findings in males.

  

Non-neoplastic changes related to treatment were present in the kidneys, liver, eyes and nasal turbinates. These include a decreased incidence of chronic progressive nephropathy in the kidneys in the 6000 and 12000 ppm females, and a decreased incidence of mineralization of the pelvic/papillary epithelium in males and females at 6000 or 12000 ppm and in males at 1500 ppm. Periportal hepatocyte vacuolation was observed in the liver in all treated males, and there was an increased incidence of loss of the outer nuclear layer of the retina in females at 6000 or 12000 ppm. Prominent eosinophilic inclusions in the nasal turbinates were seen in females at 6000 or 12000 ppm. There was no effect of treatment upon tumor profile.

  

Under the conditions of the study, there was no effect upon tumor incidence and the no-observed-effect level (NOEL) for tumorogenicity was 12000 ppm (666 mg/kg bw/day in males and 901 mg/kg bw/day in females). The no-observed-effect level (NOEL) for chronic toxicity in the study was 1500 ppm (79 mg/kg bw/day in males and 102 mg/kg bw/day in females) based on toxicity related to low weight gain and food conversion efficiency in males and females receiving 6000 or 12000 ppm and ocular changes in females receiving 6000 or 12000 ppm. There were no adverse changes seen in the testes and the no-observed-effect level (NOEL) for testicular toxicity was 12000 ppm (666 mg/kg bw/day in males and 901 mg/kg bw/day in females). There were increased liver weights but no adverse histopathology and the no-observed-effect level (NOEL) for liver toxicity was 12000 ppm (666 mg/kg bw/day in males and 901 mg/kg bw/day in females).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
666 mg/kg bw/day
Study duration:
chronic
Species:
rat
Quality of whole database:
Reliable, OECD GLP study

Justification for classification or non-classification

There was no evidence of a tumorigenic response in male and female rats following lifetime exposure to up to 12000 ppm di (2-ethylhexyl) terephthalate in the diet. In addition, results were negative for induction of genetic damage in a series of in vitro mutagenicity studies as well as an in vivo study which examined gene expression in the testes of fetal rats following in utero exposure to di (2-ethylhexyl) terephthalate. Di (2-ethylhexyl) terephthalate was not previously classified under Directive 67/548/EEC, i.e., Annex I of the Dangerous Substances Directive for carcinogenicity. Based on a weight-of-the-evidence assessment, di (2-ethylhexyl) terephthalate is unlikely to pose a significant risk for the development of any type of cancer in humans exposed to this chemical and would not be classified for “Carcinogenicity” according to the UN Globally Harmonized System of Classification and Labeling (GHS) or the EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation (EC) no. 1272/2008. 

 

Additional information

The potential for di (2-ethylhexyl) terephthalate to cause cancer is well understood. In a 2-year study conducted according to EPA Guideline OPPTS 870.4200, groups of male and female Fischer 344 rats were provided 1500, 6000 or 12000 ppm of di (2-ethylhexyl) terephthalate ad libitum in the diet. At the highest concentration tested, this was equivalent to 666 and 901 mg/kg bw/day in males and females, respectively. These dose levels were chosen because previous studies of ortho-phthalate ester plasticizers have reported liver tumors in Fischer rats at concentrations of 6000 and 12000 ppm in the diet. Under the conditions of this assay, there was no statistically significant increase in any tumor type in either sex. Although relative liver weights in the 12000 ppm-dose groups were higher than controls, there were no significant non-neoplastic gross or microscopic changes observed in the livers of either sex.