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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study followed guidelines similar to OECD TG 407 and appears to be well conducted permitting a meaningful evaluation of study results
Justification for type of information:
See attached document with the justification for the category/read-across approach.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
not specified
Principles of method if other than guideline:
not applicable
GLP compliance:
not specified
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Dibutyl adipate
EC Number:
203-350-4
EC Name:
Dibutyl adipate
Cas Number:
105-99-7
Molecular formula:
C14H26O4
IUPAC Name:
dibutyl adipate
Details on test material:
- Name of test material (as cited in study report): Dibutyl Adipate (DBA)
- Molecular weight (if other than submission substance): 258.40
- Physical state: colourless, transparent liquid
- Analytical purity: 99.8%
- Expiration date of the lot/batch: 3 months after receipt (stable at ordinary temperature).
- Stability under test conditions: In a stability test of dibutyl adipate in the solvent (DMSO) conducted by the Laboratory of Analytical Chemistry, Hatano Research Institute, dibutyl adipate was stable for 4 hours at a concentration range of 2.3-520 mg/ml
- Storage condition of test material: Light-resistant container at room temperature

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratries Japan Inc.
- Age at study initiation: 5 weeks
- Weight at study initiation: 131-155 grams for males and 121-134 grams for females. Individual body weights were confirmed to be within ±20% of the mean body weight for each sex of each group.

- Housing: Two animals/sex/cage
- Diet (e.g. ad libitum): pelleted diet ad libitum - (MF, Oriental Yeast Co., Ltd.) in which contaminants such as pesticide residue were below the concentrations prescribed in the SOP of the testing facility and feed was changed once weekly.
- Water (e.g. ad libitum): The animals were allowed free access to the tap water which was passed through a 5 μm filter and irradiated with ultraviolet light. Drinking water was changed once weekly. The quality of water is examined periodically in accordance with the Water Supply Act in Japan and was confirmed to be within the ranges prescribed as per the Ministry of Health and Welfare Ordinance No. 56.
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-25°C
- Humidity (%): 40-70 %
- Air changes (per hr): 12 fresh air changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours light/dark cycle

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
olive oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The test substance was dissolved in olive oil (Lot No. 3525, Maruishi Pharmaceutical Co., Ltd.) to yield the specified concentrations. The dosing formulations were prepared once weekly and stored under refrigeration until just prior to dosing. A stability test of the dosing formulations was conducted by the supplier of the test substance, Daihachi Chemical Industry Co., Ltd., and the dosing formulations have been confirmed to be stable for 10 days after preparation when stored in a dark cold place.
The dosing formulations were administered via oral gavage using syringes fitted with gastric intubation tubes.
The dose volume for each animal was set at 0.5 ml/100 g of body weight and the dosing volume for each animal was calculated based on the most recent body weight measurement.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The lot of the test substance used for the study was analyzed and confirmed to be stable by the sponsor prior to initiation and after termination of dosing.
Duration of treatment / exposure:
28 days
Frequency of treatment:
Dosing was conducted in the morning once daily for 28 days.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 mg/kg
Basis:
other: oral by gavage
Remarks:
Doses / Concentrations:
20 mg/kg
Basis:
other: oral by gavage
Remarks:
Doses / Concentrations:
140 mg/kg
Basis:
other: oral by gavage
Remarks:
Doses / Concentrations:
1000 mg/kg
Basis:
other: oral by gavage
No. of animals per sex per dose:
6 male + 6 female/group (main groups)
6 male + 6 female/group (reversal groups - control and high dose)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: During a preliminary 8-day repeated dose study conducted at dose levels of 0, 100, 300 and 1000 mg/kg using 3 animals/sex, no death occurred and no changes attributable to the test substance were noted in the clinical observations, body weights or gross pathology. Therefore, 1000 mg/kg, the upper limit stipulated in the Guidelines, was selected as the highest dose level for this study and then 140 and 20 mg/kg were selected as the mid and low dose levels, respectively. A control group receiving the solvent alone was included in this study
- Rationale for animal assignment: The animals were distributed into groups to nearly equalize the body weights in each group 1 day prior to initiation of dosing using a stratified randomization procedure based on the body weights.
- Post-exposure recovery period in satellite groups: control and high dose recovery groups
Positive control:
not applicable

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- All animals were observed daily for mortality and the appearance/behavior, etc. In addition, more detailed observations, including palpation, were conducted once weekly.

BODY WEIGHT: Yes
- All animals were weighed on the first day of dosing and once weekly thereafter using an electronic balance

FOOD CONSUMPTION: Yes
- Weights of feed, including the feeders, were measured for all cages on the first day of dosing and once weekly thereafter to calculate mean daily food consumption in each animal based on the weekly food consumption. An electronic balance (EB-5000, Shimadzu Corporation) was used for measurement.

OPHTHALMOSCOPIC EXAMINATION:No

HAEMATOLOGY: Yes
- Blood samples were collected from the posterior vena cava of all nonfasting animals which were anesthetized with thiopental sodium (RAVONAL, Tanabe Seiyaku Co., Ltd.) by intraperitoneal injection and necropsied at each scheduled interval. A 3.13% sodium citrate aqueous solution was used for prothrombin time and activated partial thromboplastin time and EDTA-2K was used for the other parameters as anticoagulants. The following parameters were analyzed –
Red blood cell count (RBC), White blood cell count (WBC), Platelet count, Hemoglobin concentration, Hematocrit, Differential count of leukocytes, Reticulocyte count, Prothrombin time, Activated partial thromboplastin time, Mean corpuscular volume, Mean corpuscular hemoglobin, Mean corpuscular hemoglobin concentration on an automated hematology analyzer (NE-4500, Toa Medical Electronics Co., Ltd.), Blood cell analyzer (MICROX HEG-70A, OMRON Tateisi Electronics Co., Ltd.), an automated reticulocyte counter (R-2000, Toa Medical Electronics Co., Ltd.) and an automated coagulation analyzer (KC-10A, Amelung)

CLINICAL CHEMISTRY: Yes
- The remaining portion of the blood samples used for hematology were allowed to stand for approximately 30 minutes at room temperature and centrifuged at 3000 rpm for 10 minutes. Serum samples obtained under non-fasting conditions were analysed for - total protein, albumin, A/G ratio, glucose, triglycerides, total cholestrol, urea nitrogen, creatinine, calcium, inrganic phosphorous, GOT (AST), GPT (ALT), gamma-GTP, ALP, sodium, potassium and chloride on a clinical analyzer (Hitachi 736-10)

URINALYSIS: Yes
- Fresh urine was collected from all animals 4 days prior to the terminal necropsy and analysed for pH, occult blood, protein, glucose, ketone bodies, bilirubin and urobilinogen on a urine anlyser (Clinitek 10)
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- All animals for necropsy at each scheduled interval were sacrificed by exsanguination from the abdominal aorta after blood sampling and observed for the presence/absence of lesions macroscopically

HISTOPATHOLOGY: Yes
- The following organs were removed from all animals and fixed and preserved in 10% neutral phosphate buffered formalin, with the exception that the eyes and Harderian glands were fixed and preserved in Davidson’s solution. Brain, pituitary, eyes with Harderian glands, lungs, stomach, thyroids with parathyroids, heart, liver, spleen, kidneys, adrenals, urinary bladder, testes or ovaries and femoral bone marrow The heart, liver, kidneys, adrenals and spleen from males and females necropsied at termination of dosing in the control and 1000 mg/kg groups were routinely embedded in paraffin, thin-sectioned and stained with hematoxylin and eosin. The specimens were examined for the presence/absence of histological lesions by light microscopy.
Other examinations:
The following organs from all animals necropsied at each scheduled interval were weighed using an electronic even balance (ED-H60, Shimadzu Corporation). In addition, relative organ weights were calculated based on the terminal body weights. Brain, liver, kidneys, adrenals and testes or ovaries
Statistics:
Quantitative data were analyzed for statistical significance using a multiple comparison test between the control and each test substance treated group. The data were analyzed for homogeneity of variance using Bartlett’s test. When the variance was homogeneous, one-way analysis of variance was conducted. When significant differences were noted between the groups, mean group values were compared using Dunnett’s or Scheffe’s test depending on whether the number of animals in each group was equal or not. When the variance was heterogeneous based on Bartlett’s test, Kruskal-Wallis H-test was conducted. When significant differences were noted between the groups, a rank sum test of the Dunnett type or Scheffe type was conducted depending on whether the number of animals in each group was equal or not.
Data obtained in the urinary qualitative tests were analyzed using Armitage’s χ2 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:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY - No mortality was observed and during the dosing period, salivation was observed transiently for the males and females in the 1000 mg/kg group, this was not observed after cessation of dosing.

BODY WEIGHT AND WEIGHT GAIN - Body weight changes in males and females in each treated group were similar to those in the control group.

FOOD CONSUMPTION - Food consumption of males and females in each treated group was comparable to those in the control group

HAEMATOLOGY - Shortening of APTT was noted in males in the 20 mg/kg group at termination of the dosing period and decreased MCHC and increased white blood cell counts were noted in males in the 1000 mg/kg group at termination of the recovery period. These were considered to be incidental changes within the ranges of physiological variation since they were not dose-dependent or were minimal changes.

CLINICAL CHEMISTRY - No appreciable changes were noted in any group.

URINALYSIS - Acidic pH changes were noted in females in the 20 mg/kg group prior to termination of dosing. However, this was considered to be an incidental change since it was not dose-dependent and was a minimal change.

ORGAN WEIGHTS - A decrease in the wet kidney weight was noted in males in the 140 mg/kg group at termination of the dosing period. However, this was considered to be an incidental change within a range of physiological variation since it was not dose-dependent and was a minimal change.

GROSS PATHOLOGY - No gross pathological lesions attributable to treatment with the test substance were observed in any treated group. Incidental lesions of a white nodule (1 cm in diameter) in the spleen and focal hemorrhage in the lungs were observed in 1 animal each.

HISTOPATHOLOGY: NON-NEOPLASTIC - No histopathological lesions attributable to treatment with the test substance were
observed in any group.
Diffuse fatty changes and microgranuloma in the liver and basophilic changes of the tubular epithelium, a hyaline cast and hyaline droplets in the tubular epithelium of the kidneys were sporadically observed in the control or 1000 mg/kg group. These were spontaneous lesions occasionally observed and no dose-relationship was noted in the incidences.

Effect levels

Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: based on overall effects - No changes attributable to treatment with the test substance were noted in the clinical observations, body weights, food consumption, hematology, clinical chemistry, urinalysis and pathology.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:
Under the conditions of the study, the no observed effect level (NOEL) of dibutyl adipate was considered to be 1000 mg/kg for males and females.
Executive summary:

Dibutyl adipate was administered to male and female SD rats for 28 days. A 14-day recovery period was set for the control and high dose groups. Dibutyl adipate was dissolved in olive oil and administered via oral gavage using gastric intubation tubes. Three dose levels were selected at 20, 140 and 1000 mg/kg and the control animals received olive oil alone. The dose volume was 0.5 ml per 100 g of body weight in all groups.

No death occurred throughout the observation period. No changes attributable to treatment with the test substance were noted in the clinical observations, body weights, food consumption, hematology, clinical chemistry, urinalysis or pathology.

Based on these results, the no observed effect level (NOEL) of dibutyl adipate is considered to be 1000 mg/kg for males and females under conditions of this study.