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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from August 6th 2000 to the March 17th 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD GLP and Japanese GLP study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 452 (Chronic Toxicity Studies)
Deviations:
yes
Remarks:
: no ophtalomologic examination, no urinalysis, no neurobehavioural examination, no food consumption monitoring.
GLP compliance:
yes
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Di(2-ethylhexyl)phthalate or DEHP
- Physical state: liquid
- Analytical purity: 99.6% for the both lots, FGG01 and FGJ01
- Lot/batch No.: FGG01 and FGJ01
- Storage: The test article was stored in a well-closed bottle protected from light at room temperature (Actual temperature: 11.0 to 28.0 C).
- Stability under test conditions: The test article was confirmed to be stable during the experimental period by the analytical report obtained from the supplier, Tokyo Chemical Co. Ltd. (Tokyo, Japan).
- Source: Tokyo Chemical Co., Ltd. (Tokyo, Japan)

Test animals

Species:
primate
Strain:
Marmoset
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Clea Japan Inc. (Tokyo, Japan)
- Age at study initiation: 90-110 days old (two female exceeded the range: administration started at 115 or 112 day old).
- Weight at study initiation: Males: 95-180 g and Females: 116-188 g
- Fasting period before study: no
- Housing: One animal per cage. Stainless-steel cages (300Wx600Dx650H mm, CLEA JAPAN Inc., Tokyo, Japan). The cages were washed with tap water once a day.
- Diet (e.g. ad libitum): CMS-1 and CMS-1M (CLEA JAPAN Inc., Tokyo, Japan), a solid chow for new-world primate. Ascorbic acid, powdered milk and water were added. An 80 g/head/day of the mixture of main diet and additives (200g of water and 1g of ascorbic acid for 1000g of pellet diet) prepared everyday was given every morning and replaced the next morning. Powdered milk (1 to 2g) was given to the animal that lacks appetite for the main diet.
- Water (e.g. ad libitum): All animals were provided access to water ad libitum through a nozzle of the water delivery system (Tokiwa Kagaku, Tokyo, Japan). Tap water filtered using a 5-μm filter and irradiated with ultraviolet rays was used.
- Acclimation period: quarantine+ 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Actual value: 22.4-28.9 (Target value: 26.0, permissible range: 23.0-29.0)
- Humidity (%): Actual value: 32.5-100.0 (Target value: 55.0%, permissible range: 35.0-75.0%)
- Air changes (per hr): 6-25 time/hours (all fresh filtered air)
- Photoperiod (hrs dark / hrs light): 12 hours (light: 08:00 to 20:00)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
Oral administration by gavage with a gastric tube (nasal feeding catheter) and glass syringe.
Dose volume was set at 5 mL/kg weight, and that of individual animal was calculated based on the most recently body measured weight.

The dosing suspension was administered once a day, 7 days per week for 65 weeks. Administration was discontinued for a while, when it is diagnosed that the animal could not tolerate the administration operation, by the veterinary observation. The dosing frequency for each animal, however, was more than 93.4%
of predetermined times of administration.

VEHICLE
- Lot/batch no. (if required): Lot No. 9J2240, OH2195, 1A2136, 1B2144, and 1I2140, Junsei Chemical Co., Ltd.)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Preparation method and frequency:
The test substance was mixed with the vehicle, corn oil (Junsei Chemical Co., Ltd., Tokyo, Japan), containing 20, 100, and 500 mg/mL. The solutions were prepared on a weekly basis, and were stored in a refrigerator (target range: 2° to 15 C, actual range: 1.5° to 16.0 C) until dosing. They were used within 10 days.

Confirmation of the stability:
The formulation was confirmed to be stable in the former study (study no.: 4L209, Mitsubishi Chemical Safety Institute Ltd.) at 20 and 500 mg/mL for 10 days when stored in a refrigerator.

Confirmation of the concentration:
At the first preparation, the dose solutions were analyzed to confirm each concentration. As a result, concentrations of the test substance in the vehicle were 102.0%, 102.0%, and 102.0% to the indicated concentrations, 20, 100, and 500 mg/mL, respectively.
Duration of treatment / exposure:
65 Weeks
Frequency of treatment:
once daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 500, 2500 mg/kg
Basis:
actual ingested
No. of animals per sex per dose:
74 males and 56 females in total (9 males and 6 females per dose)
Control animals:
yes, concurrent vehicle
Details on study design:
- Group assignment:
Animals of the main group (group 1) were treated as described above and were sacrificed by exsanguination from the abdominal aorta and vein under pentobarbital anesthesia, and subjected to necropsy.
A satellite group (group 2) was formed (with 3, 4, and 3 males treated at 0, 100, 500 and 2500 mg/kg respectively). Animals in Group 2 were perfused with glutaraldehyde solution systematically just before exsanguination.
- Dose selection rationale: Three dose levels (100, 500, and 2500 mg/kg) except the control (0 mg/kg) were set by a common ratio of 5. The high doses were the maximum tolerated level and the maximum technically-possible level according to a preliminary study (Study No. B000495). The control group was given the vehicle alone.
Positive control:
none

Examinations

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the scheduled sacrifice, blood samples were obtained from the femoral vein of all animals in Group 1 and 2. Using a portion of the blood samples, the following items were routinely determined by an auto analyzer. EDTA (dipotassium-salt) was used as an anticoagulant.
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: all
- Parameters examined:
Red blood cell counts (Isovolumetrically sphered two optical cytometric laser FCM)
Hemoglobin concentration (Modified cyanmethemoglobin method)
Hematocrit (Isovolumetrically sphered two optical cytometric laser FCM)
Mean corpuscular volume (MCV) (Calculated from the erythrocyte count and hematocrit)
Mean corpuscular hemoglobin (MCH) (Calculated from erythrocyte count and hemoglobin conc.)
Mean corpuscular hemoglobin concentration (MCHC) (Calculated from hemoglobin concentration and hematocrit)
Reticulocyte ratio (RNA-stained laser FCM)
Platelet count (Isovolumetrically sphered two optical cytometric laser FCM)
White blood cell count (Peroxidase cytometric FCM and Baso/lobularity laser FCM)
White blood cell count-differential (Counted after Wright’s stain)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Another portion of the blood sample obtained at the scheduled sacrifice was collected in heparinized (lithium-salt) tubes. Then the plasma was separated by centrifugation and routinely examined for the following items.
- Animals fasted: No data
- How many animals: all
- Parameters examined:
ASAT: UV-rate method (JSCC modified method)
ALAT: UV-rate method (JSCC modified method)
Gamma-glutamyl transpeptidase (G-GT): gamma-glutamyl-p-nitroanilide substrate method (SSCC modified method)
Lactate dehydrogenase (LDH): UV-rate method (modified JSCC method)
Alkaline phosphatase (ALP): P-nitrophenylphosphate substrate method (JSCC modified method)
Total bilirubin: Enzymatic method (BOD method)
Urea nitrogen: Enzymatic-UV method (urease-LEDH method)
Creatinine: Enzymatic method (creatine kinase method)
Glucose: Enzymatic-UV method (GlcK-G6PDH method)
Total cholesterol: Enzymatic method (CO-HDAOS method)
Free cholesterol: Enzymatic method (CO-POD method)
Triglyceride: Enzymatic method (GPO-HDAOS method)
Phospholipid: Enzymatic method (COD-DAOS method)
Total protein: Biuret method
Albumin: BCG method
A/G ratio: Calculated from total protein and albumin
Calcium (Ca): OCPC method
Inorganic phosphate (IP): UV method (PNP-XOD-POD method)
Sodium (Na): Ion selective electrodes method
Potassium (K): Ion selective electrodes method
Chloride (Cl): Ion selective electrodes method

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER: Hormonal analysis:
Prior to and 13, 26, 39, 52, and 65 weeks after commencement of the treatment, blood samples were obtained from the femoral vein of all animals in Group 1 and collected in heparinized (lithium-salt) tubes. Then the plasma was separated by centrifugation and examined for the following items. The plasma samples were stored at -80 C (acceptable range: below -70 C) until analysis.
Analysis of the following items was contracted to Panapharm laboratories Inc. (Kumamoto, Japan). Peptide hormones such as Follicle-stimulating hormone and luteinizing hormone were undetectable due to lack of cross-reactivity.
Testosterone: Radioimmunoassay
Estradiol (E2): Radioimmunoassay
Triiodothyronine (T3) and thyroxine (T4): Radioimmunoassay
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
On the day after final dosing, all animals were sacrificed by exsanguination from the abdominal aorta and vein under pentobarbital anesthesia, and subjected to necropsy.
Group 1: Animals were necropsied routinely.
Group 2: The animals were perfused with glutaraldehyde solution before necropsy (electronic microscopy on testis).

Light microscopic examination:
Light microscopic examination was performed on the sections of the following organs from all animals in Groups 1 and 2 (except testes from animals in Group 2). The sections were routinely stained with hematoxylin and eosin. All organs were weighed and the ratio of organ weight to body weight were then calculated (Pituitary, Thyroid, Pancreas, Liver, Adrenal, Kidney, Spleen, Testis, Epididymis, Prostrate, Seminal vesicle, Ovary, Uterus)

Histochemical examination of the testes and ovaries:
A 3beta-hydroxysteroid dehydrogenase (3beta-HSD) activity in the testes or ovaries from all animals in Groups 1 (females) and 2 (males) was examined histo-chemically.
Regarding the testis, the activity of 3beta-HSD in Leydig cell was classified in 4 grades on 100 fields of view, and then the mean positive area in each grade was calculated. Regarding the ovary, the ratio of positive area against whole area was measured using an image analyzer.

Electron microscopic examination of the testis:
Electron microscopic examination was performed on the thin sections double-stained with uranyl acetate and lead citrate using a electron microscope. Special attention was paid, among others, on Sertoli cell organelles like rough endoplasmic reticulum (rER) near junction, ribosome or Leydig cell for any change in organelle peroxisome.

Testicular component and enzyme activities
Examinations: Sperm count, Sorbitol dehydrogenase (SDH), gamma-glutamyl transpeptidase (G-GT), Glutathione content, Glutathione S-transferase (GST), Glutathione peroxidase (GSH-Px), Zinc (Zn) content.

Hepatic enzyme analysis:
A portion of the liver from all animals in Group 1 was used for measurements of cyanide-insensitive palmitoyl co-A beta-oxidation (FAOS), Carnitine acetyltransferase (CAT), Carnitine palmitoyltransferase (CPT), Cytochrome P-450 content, Testosterone 6beta-hydroxylation Lauric acid gamma- and gamma1-hydroxylation, Glutathione S-transferase (GST), Glutathione, Glutathione peroxidase (GSH-Px) enzyme activities or contents.
Statistics:
The metric data were first analyzed by Bartlett's test. When the group variances are determined to be homogeneous, all groups were compared by analysis of variance (ANOVA).
Dunnett's multiple range test (for an equal number of animals in each group) or Scheffe's test (for unequal numbers of animals in each group) were used when inter-group differences were found to be significant. When the result of Bartlett's test indicated heterogeneous group variances, all groups were compared by the Kruskal-Wallis test and Dunnett's rank sum test (for an equal number of animals in each group) or Scheffe's test (for unequal numbers of animals in each group).

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):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
Mortality
All of deaths or moribund sacrifices were considered not to be specific to the test-substance treatment. Dead or sacrificed animals were replaced (except for one male receiving the vehicle).

Clinical signs and body weight
There were no treatment-related abnormalities in any group.
Loose stool, reddish stool, or diarrhea was occasionally observed especially in the early stage of the treatment period. However, there was no difference in its incidence among all groups.
Dose related change in the body weight was not evident, except the wasting animals that showed marked decrease in body weight.

Hematology
There were no treatment-related abnormalities in any group.

Blood chemistry
There were no treatment-related abnormalities in any group.

Hormonal analysis
There were no treatment-related abnormalities in any group.
During the treatment period, all males experienced testosterone surge, and the testosterone levels in all treated groups were similar to that of the control group.

Organ weight
There were no treatment-related abnormalities in any group.
A statistically significant high value was observed in female reproductive organs such as ovary and uterus in the 500- and 2500-mg/kg groups. However, no histological abnormality was observed.
One animal in each DEHP-treatment group of Group 1 showed low weights in the testis, epididymis, seminal vesicle, and prostate. These animals usually showed low body weight and such animal as showing low body weight and small size in above organs also existed in the control animals of Group 2.

Sperm count
There were no treatment-related abnormalities in any group.
In the growing testis, low value of sperm count was observed as expected. The occurrence of the growing animal is not dose dependent. To examine the trend of administration effect on sperm count, inclusion of exceptional incidence was avoided.

Testicular enzyme activities and component
There were no treatment-related abnormalities in any group.

Hepatic enzyme activities and component
P450 content tended to increase with dose-dependent manner in general with statistical significance in females at 500 and 2500 mg/kg. With regard to specific CYPs, CYP3A and 2E, these were related to testosterone 6 beta-hydration and lauric acid gamma-1 hydration and CYP4A that is related to lauric acid gamma hydration, no marked or specific increase was noticed. Total GSH content also tended to decrease with dose-dependent manner in general. However, these changes were considered to be minimal. On the other hand, there were no changes in the peroxisomal enzyme activities such as CAT, CPT, and FAOS.

Necropsy finding
There were no treatment-related abnormalities of the testis in any group. One or 2 males in each group showed small size of genital organs such as testis, epididymis, seminal vesicle, prostate. However, this change was considered to not be a primary effect of the test substance but due to low body weight.
Large size of the ovary was observed in 2 females each receiving 500 and 2500 mg/kg of DEHP, no abnormality, however, was observed histopathologically. Dark-brownish change of the liver was observed in 1 male receiving 2500 mg/kg of DEHP and may be treatment related. However, no change was evidenced in the histopathological examination.

Histological findings
There were no treatment-related abnormalities in the organs examined from animals in any group.
Testis, epididymis, seminal vesicle, and prostate with small size due to low body weight did not mature satisfactorily (on growing). However, degenerative change ascribed to the DEHP-treatment was not observed in the above growing testis. In the ovary with large size in 2 females each receiving 500 and 2500 mg/kg of DEHP, a development of the corpus luteum was noticed, however such corpus luteum is usually observed in more mature females.

Histological findings (3beta-HSD)
There were no treatment-related abnormalities in the testis or ovary from animals in any group.
Lydig cells in the testis from the animals treated with DEHP were strongly positive to 3beta-HSD staining as well as that in control animals. However, in the growing testis, 3beta-HSD activity was undetectable as expected. The corpus luteum in the ovary was well stained by 3beta-HSD immunostaining, so the large variation in the positive area in the ovary, which increased in a dose dependent manner, depends on the development of the corpus luteum.

Electron microscopic findings
There were no treatment-related abnormalities in the Lydig cells, Sertoli cell, or spermatic cells in the testis from animals in any group. In this examination, the observation was focused especially on Sertoli cell. However, vacuolative change of rER (rough endoplasmic reticulum) near the tight junction or destruction of the desmosome that were observed in rodents were not observed in marmosets.

Effect levels

Dose descriptor:
NOAEL
Effect level:
2 500 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: No toxicologically relevant effect

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
DEHP was administered orally to juvenile marmoset at dosage levels of 0, 100, 500, and 2500 mg/kg/day for 65 weeks. The testicular effect that is well known in rodents were not observed despite extensive examination.
Executive summary:

DEHP was administered by oral gavage at doses of 0, 100, 500, and 2500 mg/kg for 65 weeks to juvenile common marmosets (about 3 months old) of both sexes, and its toxicity was assessed. An extensive and intensive investigation focused on testicular morphology, and function was realised.

Treatment-related change in the body weight was not evident. Treatment-related changes were not observed in general except for adaptive liver changes. During the treatment period, all males experienced a surge in testosterone, and the testosterone levels in all treated groups were similar to that of the control group. For the testis, electron microscopic examination was additionally applied, however, this revealed no treatment-related abnormalities. Histochemical examination after 3beta-hydroxysteroid dehydrogenase (3beta-HSD) staining did not reveal any alteration in steroid synthesis.

Consumption of peroxide scavenger like GSH, GST, or GSH-Px in the testis was not noticed, which suggests that the peroxysomal enzyme may not be operating in this organ. For functional examination, sperm count was conducted to show no treatment related effect in numerical changes.

The liver weight and its body weight ratio were not affected. P450 content tended to increase with dose-dependent manner in general, and that was considered to be adaptive change to the DEHP-exposure. However, with regard to specific CYP, CYP3A and 2E these are related to testosterone-6-beta-hydroxylation and lauric acid gamma-1 hydroxylation, respectively, and CYP4A related to lauric acid  hydroxylation, no marked increases were noticed. That showed induction of non-PP dependent oxidation and proved absorption of the test substance.

In the toxicological study, despite the high dose of 2500 mg/kg/day and adoptive liver change proving absorption, no testicular change was morphologically or functionally noticed in the extensive examinations.

In conclusion, when DEHP was administered orally to juvenile marmoset at dosage levels of 100, 500, and 2500 mg/kg/day for 65 weeks, the testicular effect that is well known in rodents were not observed despite extensive examination.