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Repeated dose toxicity: oral

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Administrative data

sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Japanese Ministry of Health Study - English translation

Data source

Reference Type:

Materials and methods

Test guideline
equivalent or similar to guideline
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
not specified
GLP compliance:
not specified
Limit test:

Test material

Constituent 1
Reference substance name:
Constituent 2
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Constituent 3
Reference substance name:
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): 2-tert.-butylphenol
- Physical state: liquid
- Analytical purity: 99.97%
- Purity test date: 2014-05-07
- Lot/batch No.: 1419
- Expiration date of the lot/batch: 05/2015
- Stability under test conditions: stable
- Storage condition of test material: under N2 in tightly closed container at a cool, well ventilated place
- Colour: clear

Test animals

Crj: CD(SD)
Details on test animals or test system and environmental conditions:
- Source: Charles River Laboratories Japan, Inc
- Age at study initiation: 5 weeks
- Weight at study initiation: 154 to 185 g for males and from 134 to 154 g for females
- Housing: polycarbonate cages with bedding for experimental animals (Beta Chip, Charles River Laboratories Japan, Inc.)
- Diet (ad libitum): pelleted diet for experimental animals (MF Oriental Yeast Co., Ltd.)
- Water (ad libitum): tap water which had been filtered through 5-μm filters and sterilized by ultraviolet light irradiation
- Acclimation period: 7 days

- Temperature (°C): 22 ± 2°C
- Humidity (%): 55 ± 15%,
- Air changes (per hr): 12 times per hour
- Photoperiod (hrs dark / hrs light):lighting for 12 hours/day (from 7:00 to 19:00)

Administration / exposure

Route of administration:
oral: gavage
olive oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: prepared once a week and preserved in a refrigerator until use for administration

- Justification for use and choice of vehicle (if other than water): olive oil
- Concentration in vehicle: The stability of the test article in the dosing solutions in a refrigerator for 8 days was verified before administration for the concentration range from 0.4 to 200 mg/mL.
- Amount of vehicle (if gavage): 10 ml/kg
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
The dosing solution for each dose group was analyzed at the time of first preparation to confirm that the concentration of the test article in each dosing solution was within ±10% of the set concentration.
Duration of treatment / exposure:
Oral gavage once daily in the morning
Frequency of treatment:
Single dose daily for 28 days
Doses / concentrations
Doses / Concentrations:
500, 100, 20, 4 mg/kg/d
actual ingested
No. of animals per sex per dose:
n=6 males, n=6 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: In a preliminary study, a single oral administration of the test article at dose levels of 500, 1000 or 2000 mg/kg was
associated with deaths of all females at 1000 and 2000 mg/kg and a marked decrease in locomotor activity in males in these groups.
In a 12-day repeated oral study, administration at 0, 100, 300 and 500 mg/kg was associated with ataxic gait in males and females in the 500 mg/kg group and females in the 300 mg/kg group, high values in the absolute and relative weights of the liver in males in the 500 mg/kg group and
high values in the relative weight of the liver and kidney in females in the 500 mg/kg group.

Based on these results, the dosing regimen was set at 500, 100, 20 and 4 mg/kg.
Positive control:


Observations and examinations performed and frequency:
All animals were observed for clinical signs every day.
Body weight and food consumption were measured on the starting day of administration, and then once a week thereafter.
For food consumption, the mean one-day food consumption per animal for each period was calculated.

Haematological: On Day 29 (the day following the final administration) and Day 43 (after the end of the recovery period), blood was collected via the caudal vena cava from all animals under non-fasting conditions under anesthesia by administration of thiopental sodium. The following measures were made: erythrocyte count (Sheath flow DC impedance detection method), hemoglobin concentration (SLS hemoglobin method), hematocrit value (Erythrocyte pulse wave high value detection method), platelet count (Sheath flow DC impedance detection method) and leukocyte count (RF/DC impedance detection method) using a multi-item automatic cytometer (NE-4500, Sysmex Corporation), reticulocyte percentage (Flow cytometry using argon razor) using an automatic reticulocyte analyzer (R-2000, Sysmex Corporation), prothrombin time (PT, Quick 1 stage test) and activated partial thromboplastin time (APTT, activated cephaloplastin method) by blood coagulation analyzer (KC10A, Amelung GmbH), and leukocyte percentage (Wright stained smear specimens) using a blood cell analyzer (MICROX HEG-70A, Omron Corporation).

Blood Chemistry: On Day 29 (the day following the final administration) and Day 43 (after the end of the recovery period), a part of the blood sample collected was allowed to stand at room temperature for approximately 30 minutes and centrifuged at 3000 rpm for 10 minutes to separate serum,
which was subjected to an automatic analyzer (Hitachi 736-10, Hitachi, Ltd.) to analyze ASAT (GOT, modified JSCC method), ALAT (GPT, modified JSCC method), γ-GT (modified SSCC method), ALP (modified JSCC method), total bilirubin (BOD method), urea nitrogen (Urease-GLDH method), creatinine (Jaffé method), glucose (GlcK-G6PDH method), total cholesterol (CES-CO-POD method), triglyceride (LPL-GK-G3PO-POD method), total protein
(Biuret method), albumin (BCG method), calcium (OCPC method), inorganic phosphorus (PNP-XOD-POD method), and sodium, potassium and
chloride (Ion selective electrode method).

Urinalysis: Fresh urine was collected from 6 males and 6 females from each group on Day 23 (final week of administration) and subjected to measurement of the following items: pH, protein, glucose, ketones, bilirubin, occult blood and urobilinogen (Test paper method, Multistix, Bayer-Sankyo Co., Ltd.) using a urine analyzer (Clinitek 100, Bayer-Sankyo Co., Ltd.).
Sacrifice and pathology:
On Day 29 (the day following the final administration) or Day 43 (after the end of the recovery period), after blood collection, all animals were
sacrificed by exsanguination via the abdominal aorta and necropsied. For all animals, the following organs were weighed: brain, heart, lung, liver,
kidneys, adrenals, thymus, spleen, testes, ovaries, uterus, epididymides, pituitary and thyroid. For all animals, the following organs/tissues were
brain, spinal cord, pituitary, eyeballs and Harderian glands, lymph nodes (mandibular / mesenteric), thymus, trachea, lungs and bronchus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, pancreas, thyroid and parathyroid glands, heart, liver, spleen, kidneys, adrenals, urinary bladder, testes, epididymides, seminal vesicles, ventral lobe of prostate, ovaries, uterus, femur and femoral bone marrow, femoral muscles and sciatic nerves.
Then, all organs/tissues were fixed and preserved in 10% neutral buffered formalin except the eyes and Harderian glands which were fixed in
Davidson’s solution and the testes and epididymides which were fixed in Bouin’s solution.

For all males and females in the control group and 500 mg/kg group at the end of the administration period, the following organs and macroscopic
lesions of all animals including those of the control group were subjected to preparation of hematoxylin and eosin (H&E) staining by an ordinary
method and examined microscopically: thymus, heart, liver, spleen, kidneys, adrenals, testes, epididymides, seminal vesicles, ovaries, brain, spinal
cord (cervical, thoracic and lumbar regions), sciatic nerve, and femoral muscles.
Quantitative data were tested for homogeneity of variance by the Bartlett test and homogeneous data were analyzed by one way analysis of variance while heterogeneous data were analyzed by the Kruskal-Wallis test. Significant differences between the groups were analyzed by the Dunnett method or a Dunnett-type multiple comparison. Urinalysis data and histopathological findings were subjected to chi-square test of a × b, and then differences between the control group and each dose group were analyzed by the Armitage chi-square test, if significant differences were observed. The level of significance was set at 5%.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
salivation, ataxic gait
mortality observed, treatment-related
Description (incidence):
salivation, ataxic gait
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
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:
effects observed, treatment-related
Description (incidence and severity):
males and females high dose; transient increased liver weights
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: Ataxic gait was observed in 9 males and all females in the 500 mg/kg group sporadically during the administration period. These changes were observed from Day 1, and they occurred after dosing but disappeared within 5 hours of dosing.
Salivation was observed in 6 males and 2 females in the 100 mg/kg group and all males and 11 females in the 500 mg/kg group. It was a transient
change which was observed within 30 minutes of dosing from Day 13 in males and Day 14 in females in the 100 mg/kg group and from Day 7 in
males and females in the 500 mg/kg group.

ORGAN WEIGHTS: In the measurement of organ weights, a high value in the relative weight of the liver was observed in males and females in the
500 mg/kg group at the end of the administration period. However, there were no statistically significant changes in the absolute weight of the liver and histopathological examination showed no changes that were related to the change in the relative weight.
These changes were no longer observed at the end of the recovery period.

Effect levels

open allclose all
Dose descriptor:
Effect level:
20 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: clinical signs - ataxic gait observed at high dose (500 mg/kg) and salivation (at 100 mg/kg)
Dose descriptor:
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: Salivation: a transient change was seen within 30 minutes of dosing. This is not considered adverse toxicity of the test material.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Based on the clinical signs of ataxic gait seen in males and females at 500 mg/kg/d and lack of adverse effects seen at the next lowest dose tested (100 mg/kg/d), the NOAEL is considered to be 100 mg/kg/day.
Executive summary:

In a study report provided by the Japanese Ministry of Health, a 28-day repeat dose study on o-tert butyl phenol (OTBP) was described, as similar to the principles of an OECD guideline 407 study. Crj:CD(SD)IGS rats (n=6 males and n=6 females per dose group) were administered with OTBP in olive oil, by oral gavage, at doses of 500, 100, 20 and 4 mg/kg. There were no changes that were thought to be caused by administration of the test article in the results of body weight measurement, food consumption measurement, hematological examination, blood chemistry examination, urinalysis, necropsy or histopathological examination.

Clinical signs of ataxic gait caused by administration of the test article were observed in both males and females in the 500 mg/kg group. Transient salivation within 30 minutes was observed as the only clinical sign in males and females in the 100 mg/kg group.

It is considered that the no observed adverse effect level (NOAEL) of o-tert-butylphenol is 100 mg/kg/day for both males and females under the conditions of this study.