Registration Dossier

Diss Factsheets

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

Administrative data

Endpoint:
chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 452 (Chronic Toxicity Studies)
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Di-''isononyl'' phthalate
EC Number:
249-079-5
EC Name:
Di-''isononyl'' phthalate
Cas Number:
28553-12-0
Molecular formula:
C26H42O4
IUPAC Name:
bis(7-methyloctyl) phthalate
Constituent 2
Reference substance name:
DINP
IUPAC Name:
DINP
Details on test material:
- Name of test material (as cited in study report): Di(isononyl)phthalate
- Physical state: clear, colorless liquid
- Analytical purity: >99%
- Lot/batch No.: QCL 9004-273

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Portage, Michigan
- Age at study initiation: 6 weeks
- Weight at study initiation: males 96-134 g and females 82-109 g
- Housing: single
- Diet (e.g. ad libitum): Purina Certified Rodent Chow 5002
- Water (e.g. ad libitum): tap water
- Acclimation period: 2 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Humidity (%): 50±20
- Air changes (per hr): 10 or more
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: feed
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: For each dietary level, the test material was weighed on an appropriate balance and was placed into a pocket that was made in approximately 200 g of feed contained in a glass beaker. The test material/feed was mixed in a Waring blender for approximately 2 to 3 minutes to ensure an apparent homogeneous mixture. Based on dietary level, multiple premixes were required (approximately 30 g test material /premix). The premixes were added to approximately 5 kg of additional feed, mixed in a Hobart mixer for 15 minutes, then transferred to a Patterson-Kelley twin-shell mixer (fitted with an intensifier bar) that contained the remaining amount of basal feed. The diets were mixed for 1 minute/kg feed.


DIET PREPARATION
- Rate of preparation of diet (frequency): once a week
- Storage temperature of food: refrigerated (2-8°C)


Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Results of routine concentration analyses indicated that all formulations were within 15% of target.
Duration of treatment / exposure:
104 weeks for the test groups and 78 weeks for the recovery group (high dose level)
Frequency of treatment:
7 days per week
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
ca. 29.2, 88.3, 358.7, and 733.2 mg/kg bw/d for males and ca. 36.4, 108.6, 442.2, and 885.4 mg/kg bw/d for females
Basis:
actual ingested
Remarks:
Doses / Concentrations:
500, 1500, 6000, 12000 ppm
Basis:
nominal in diet
No. of animals per sex per dose:
500 and 1500 ppm dose group: 70 males and 70 females
0, 6000 and 12000 ppm dose group: 85 males and 85 females
Recovery (12000 ppm) group: 55 males and 55 females
Control animals:
yes, concurrent no treatment
Positive control:
[4-Chloro-6-(2,3-xylidino)- 2-pyrimidinylthio] acetic acid was used as positive control for Hepatocellular Proliferation and Biochemical Analyses. Fifteen males were administered the positive control substance for 13 weeks at dietary level of 1000 ppm.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily. In addition, detailed clinical observations by physical examination were performed once each week. Special attention was paid to mass development. The following information on each grossly visible or palpable mass was recorded: time of onset, location, size, appearance, progression.


BODY WEIGHT: Yes
- Time schedule for examinations: prior to treatment, and weekly for Weeks 1-17 and once every 4 weeks thereafter. An additional weight was obtained from all groups on the first day of Week 79.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined: Yes, weekly for Weeks 1-16 and once every 4 weeks thereafter. Food consumption was also determined at Week 78. When obvious spillage or wastage of food was recorded, the estimate of the food consumed by the animal was excluded from the group mean calculation for that particular interval.



HAEMATOLOGY: Yes
- Time schedule for collection of blood: During Weeks 26, 52, 78, and 104
- Animals fasted: Yes
- How many animals: 10 animals/sex/group
- Parameters checked: absolute reticulocyte count, mean cell hemoglobin, corrected leukocyte count, mean cell hemoglobin concentration, erythrocyte count mean cell volume, hematocrit, platelet count, hemoglobin, reticulocyte count, leukocyte differential and cellular morphology, myeloid/erythroid ratios (at necropsy), leukocyte count.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: During Weeks 26, 52, 78, and 104
- Animals fasted: Yes
- How many animals: 10 animals/sex/group
- Parameters checked: aspartate aminotransferase, alanine aminotransferase, gamma glutamyltransferase, creatinine, urea nitrogen, total bilirubin, sodium, chloride, calcium, potassium, inorganic phosphorus, glucose, total protein, albumin, globulin.

URINALYSIS: Yes
- Time schedule for collection of urine: During Weeks 26, 52, 78, and 104
- Metabolism cages used for collection of urine: Yes, urine collection racks
- Animals fasted: Yes
- Parameters checked: total volume, calcium, chloride, creatinine, creatinine clearance, osmolality, phosphorus, potassium, sodium, appearance/color, specific gravity (calculated), protein, glucose, ketones, bilirubin, Urinalysis, occult blood, microscopic sediment, pH, turbidity, urobilinogen


Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
All animals found dead or sacrificed in extremis (using sodium pentobarbital and exsanguination) during the study were subjected to a gross postmortem examination. Fifteen mice/sex/group in the control, and all test groups were sacrificed after 78 weeks of compound administration. Following at least 104 weeks of compound administration, the surviving mice were necropsied. At the interim and terminal sacrifices, the following organs (when present) were weighed: brain with stem, spleen, kidneys (paired weight), liver/gallbladder (drained), testes with epididymides (paired weight ), lung, uterus.
Five mice/sex/group in all test groups were designated for hepatocellular proliferation studies following 78 weeks and five mice/sex/group in all test groups and recovery group were designated for hepatocellular proliferation studies following 104 weeks. Before sacrifice, these animals were labelled with BrdUrd (20 mg/ml) for 72 hrs using ALZET osmotic pump. Tissues from liver (standard tissue samples from left and right median, and right anterior lobes) and duodenum from each animal designated for hepatocellular proliferation studies and were preserved in 10% neutral-buffered formalin. The remainder of the liver was immediately frozen at appoximately -70°C for biochemical analysis.

Other examinations:
Hepatocellular Proliferation and Biochemical Analyses: The frozen tissue samples from the three liver lobes (right and left lateral, and median), and the duodenum were evaluated for hepatocellular proliferation and analysis of protein concentration, cyanide-insensitive palmitoyl-CoA oxidation, and DNA concentration from five animals/sex from control, 12000 ppm and recovery group sacrificed during Weeks 1, 2, and 13, and from five animals/sex from control, 6000 and 12000 ppm groups sacrificed after 104 weeks of treatment.
Statistics:
Trend analysis of survival was evaluated at the 5.0% one-tailed probability level. Weekly body weights and food consumption and the following intervals for mean body weight change (Weeks 1-53 and 1-105) and total food consumption (Weeks 1-52 and 1-104), clinical pathology data (except hemolysis and cellular morphology gradings and routine urinalysis data), fasted terminal body weights, and organ weight data of the control group were compared statistically to the data from the same sex of the treated groups.

Results and discussion

Results of examinations

Details on results:
CLINICAL SIGNS AND MORTALITY: Survival at Week 104 in the males of the 12000 ppm dose group was significantly lower than the control group. Analysis of the females revealed no difference in survival between control and treated groups. No treatment related clinical signs were observed.


BODY WEIGHT AND WEIGHT GAIN: The body weight in the animals of the 12000 ppm and recovery groups was significantly lower during weeks 5-105 and 2-105, respectively.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): In females of the 12000 ppm and recovery groups, statistically significant lower mean food consumption values were noted most frequently. However, even though these females were fed the same dietary concentration (12000 ppm), significantly lower mean weekly food consumption values were noted at 18/31 measurement intervals for the 12000 ppm group, but only 9/31 intervals for the recovery group during Weeks 1-78. Therefore, the statistically significant intergroup differences in mean weekly and total food consumption were spurious findings. For Weeks 1-104, the average daily compound consumption (mg/kg bw/d) for the 500, 1500, 6000, 12000 ppm dietary levels was 29.2, 88.3, 358.7, and 733.2 mg/kg bw/d in the males and 36.4, 108.6, 442.2, and 885.4 mg/kg bw/d in the females, respectively. For Weeks 1-78 in the recovery group, the average daily consumed dose was 637.3 mg/kg/day in males, and 773.6 mg/kg/day in females.


HAEMATOLOGY: Significant changes in the hematology data included mild decreases in the erythrocyte mass (erythrocyte count, hemoglobin, and hematocrit) in the 12000 ppm and recovery groups at most intervals, however, the mean values for the recovery group rats were, in general, not significantly different from controls at Week 104. Hematopoietic neoplasia (leukemia) was observed in the peripheral blood smears from several animals of the 12000 ppm and recovery groups.


CLINICAL CHEMISTRY: Increases (often significant) were observed in the mean values for urea nitrogen, alanine aminotransferase, and aspartate aminotransferase primarily in the 12000 ppm and recovery groups throughout the study. The elevations in these analytes corresponded to the histologic findings noted in the kidneys and livers of the males of the 6000 ppm and recovery group, and animals (both sexes) of the 12000 ppm group.


URINALYSIS: The mean values for urine volume were significantly increased in the males of the 12000 ppm group at Week 104. The mean creatinine clearance rate was significantly increased in the females of the 12000 ppm group at Week 104. The change was of low magnitude and was not accompanied by a significant change in the mean value for urine volume, urine creatinine, or serum creatinine. Decreases were observed in the mean values for urine calcium, urine potassium, urine chloride, and urine osmolality in some treated groups during the study. The significant changes in the aforementioned urinary chemistry tests are of questionable toxicologic importance as they occurred inconsistently over time and did not correlate with concurrent serum chemistry changes.


ORGAN WEIGHTS: Liver organ weights (absolute and relative to body) for all interim sacrifices were significantly increased with dose in animals of the 6000 and 12000 ppm groups, compared to the control group. Spleen and kidney weights (absolute and relative to body) were also statistically increased in the 6000 and 12000 ppm groups when compared to the control group of animals sacrificed at Week 79. Absolute and relative weights for testis/epididymis or uterus in the 6000 and 12000 ppm groups were not statistically different compared to controls. In the animals sacrificed after at least 104 weeks of treatment, liver weights (absolute and relative to body or brain) were noted as statistically increased in males and females of the 6000 and 12000 ppm groups. Liver weights in animals of the recovery group were increased when compared to control but not significantly. Absolute and relative spleen and kidney weights in females of the 12000 ppm group were significantly increased. Statistically significant increases of spleen and kidney weights were observed in females of the 6000 ppm group. There were no statistically significant differences in absolute and relative weights for testis/epididymis or uterus in any of the treated groups compared to same sex in controls.


GROSS PATHOLOGY: In males and females of the 6000, 12000 ppm and recovery group, that died or were sacrificed in extremis during the study (unscheduled deaths), the incidence of the following necropsy findings was notably increased: enlarged spleen and granular/pitted/rough appearance of the liver, as well as dark area of the stomach, thoracic cavity fluid, small testis and small seminal vesicle, and uterine mass. At the Week 79 sacrifice, a liver mass was observed in each of two males of the 12000 ppm group; and various gross-pathologies of the liver (enlarged, irregular shape, granular/pitted/rough appearance and raised area) were observed in the females of the 12000 ppm group, when compared to the control group. Small testis and small seminal vesicle were not observed in any males killed from the control, 6000 and 12000 ppm group, and the incidence of uterus mass was 0/15, 2/15, and 1/15 in females of the control, 6000 and 12000 ppm group, respectively. At necropsy for study termination, the following abnormalities were observed to have a dose-related increase in the males and females of the 6000 and 12000 ppm groups: enlarged spleen, enlarged and/or granular/pitted/rough appearance of the liver (seen primarily in the males), dark kidney, dark area of the stomach, various uterine pathologies (cyst, mass, or thickened wall). The incidences for small testis and small seminal vesicle were similar across all groups.


HISTOPATHOLOGY: NON-NEOPLASTIC: In the 6000 ppm dose group, there was no histopathological correlate to the increased liver weight. Livers from the rats killed at Week 79 and at study termination were remarkably free of treatment-related histologic findings, with no histologic evidence of hepatocellular enlargement. Treatment-related histopathologic lesions in the kidney at Week 79 and study termination included an increase in the incidence and severity of mineralization of the renal papilla in males, but not females, and increased incidence and severity of tubule cell pigment in both sexes. In the 12000 ppm group, histological findings in the livers of the males and females were diffuse hepatocellular enlargement (first detected at Week 2), increased cytoplasmic eosinophilia (first detected at Week 13) and increased pigment in Kupffer cell/bile canaliculi (first detected at Week 79). Treatment- related histopathologic lesions in the kidney at Week 79 and study termination, were an increase in the incidence and severity of mineralization of the renal papilla in males, but not females, and increased incidence and severity of tubule cell pigment in both sexes. In the recovery group, treatment-related histological effects in the liver also appeared to be reversible in that diffuse hepatocellular enlargement and increased cytoplasmic eosinophilia were not evident, and the incidence of pigment in Kupffer cell/bile canaliculi was comparable to the control group incidence. Kidney enlargement also appeared to be reversible in that mean kidney weights (absolute and relative) in the males and females were comparable to control values. However, mineralization of the renal papilla in males, and renal tubule cell pigment in both sexes, was only partially reversible with the incidence and severity approximating values in 6000 ppm group.


HISTOPATHOLOGY: NEOPLASTIC: In the 6000 ppm dose group, the incidence of mononuclear cell leukemia was 49% and 45% in males and females, respectively, compared to 34% and 26% in the control males and females, respectively. In the 12000 ppm group, increased incidence of neoplasms (first detected at Week 79) in liver was observed. Malignant tubule cell carcinoma was detected in kidneys of two males. The incidence of mononuclear cell leukemia was 46% and 46% in males and females, respectively, compared to 34% and 26% in the control males and females, respectively. In the recovery group, there was absence of an appreciable increase in liver neoplasms compared to the control group. Unlike the liver, treatment-related neoplasia in the kidney of males, and hematopoietic tissue of both sexes, was not reversible. No kidney neoplasms were detected in females, but malignant tubule cell carcinoma was found in four of the males, of which three were killed following the recovery phase. The incidence of mononuclear cell leukemia was 62% and 48% in males and females, respectively, compared to 34% and 26% in the control males and females, respectively.



OTHER FINDINGS: Cell proliferation: Significant increases in the labeling index were observed during Week 1 in the livers of male and female rats treated with the test material at all dose levels. The response was not observed at Week 2, 13, or 104. Large increases in the labeling index were observed in the positive control animals at Week 1, but questionable responses were observed at Weeks 2 and 13. No positive control animals were included for the Week 104 sacrifice.
Biochemical analyses: Increases in palmitoyl CoA oxidase, a monitor of the level of peroxisome proliferation were observed in rats of the 12000 ppm group at all timepoints. At Week 104, there was also an elevation in palmatoyl-CoA oxidase activity in female rats of the 6000 ppm group. Male rats treated at 6000 ppm did not have elevated enzyme levels.
DNA and Protein Analyses: Levels of DNA varied randomly among the groups and at the different timepoints. Statistical analysis confirmed the lack of a consistent pattern. Protein analysis also did not indicate a clear pattern in the 6000, 12000 ppm or positive control groups. Liver weights were significantly elevated in animals of the 12000 ppm group throughout the study, and also in the positive control animals included at 1, 2 and 13 weeks.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
ca. 88.3 mg/kg bw/day (actual dose received)
Sex:
male
Basis for effect level:
other: liver and kidney toxicity, mononuclear cell leukemia
Dose descriptor:
NOAEL
Effect level:
ca. 108.6 mg/kg bw/day (actual dose received)
Sex:
female
Basis for effect level:
other: liver and kidney toxicity, mononuclear cell leukemia

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion