2-tert-butylphenol

Endpoint summary

Description of key information

An OECD guideline 408 90 day repeated dose study on o-tert-butylphenol. In addition, OECD guideline 407 28 day study on o-tert butyl phenol and an OECD gudieline 422 study on read across analogues o-sec butyl phenol, both from the Japanese Ministry of Health.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Japanese Ministry of Health Study - English translation

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

not specified

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- 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

ENVIRONMENTAL CONDITIONS
- 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)

Route of administration:

oral: gavage

Vehicle:

olive oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: prepared once a week and preserved in a refrigerator until use for administration

VEHICLE
- 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:

yes

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

Remarks:

Doses / Concentrations:
500, 100, 20, 4 mg/kg/d
Basis:
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:

None

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
collected:
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.

Statistics:

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%.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

salivation, ataxic gait

Mortality:

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.

Dose descriptor:

NOEL

Effect level:

20 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: clinical signs - ataxic gait observed at high dose (500 mg/kg) and salivation (at 100 mg/kg)

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

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.

Critical effects observed:

not specified

Conclusions:

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Klimisch 2. The study has been performed to OECD guideline 407 principles, but GLP compliance is not stated. Well documented.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In a 28-day study, reported by the Japanese Ministry of Health, to a protocol equivalent to OECD 407 guidelines,Sprague-Dawley strain (Crj:CD(IGS), SPF) rats (n=6 males, n =6 females per dose group) were dosed by oral gavage with o-tert butyl phenol dissolved in olive oil at doses of (0, 4, 20, 100 and 500 mg/kg). Clinical effects (ataxic gait and transient salivation (<30 minutes after dosing)) were observed in the high dose group (500 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, haematological examination, blood chemistry examination, urinalysis, necropsy or histopathological examination.No adverse effects were seen at 100 mg/kg or less.

The subchronic oral toxicity (OECD 408) of o-t-Butylphenol was determined by administering daily in powdered rodent diet to Sprague Dawley rats for 13 consecutive weeks at approximate dose levels of 20, 100 and 500 mg/kg/day and recovery from any treatment-related effect during a period of 4 weeks.

No mortality occurred and no treatment-related clinical signs were observed during the study. No signs of toxic or neurotoxic effects, which could be considered adverse, were seen during the in life phase of the study. The only effects observed (a slight increase of white blood cells in some males dosed at 100 mg/kg/day and in a number of animals of both sexes receiving 500 mg/kg/day; a reduction of prothrombin time in treated animals; and statistically significant fluctuations of some clinical chemistry parameters) during treatment were no longer observed at the end of recovery. Due to the low severity, the complete reversibility and/or the absence of dose-relation, all the changes were not considered adverse. The absolute and/or relative weights of the liver and kidneys showed slight increases which, due to the low magnitude, complete reversibility and the absence of a support from the histopathological examination, were not considered adverse.

In conclusion, no treatment-related changes, which could be considered adverse, were observed in male and female rats following dosing with o-t-Butylphenol, when administered in powdered rodent diet for 13 consecutive weeks at the approximate dosages of 20, 100 and 500 mg/kg/day (corresponding to mean achieved dose levels of 20, 104 and 498 mg/kg/day for the males and 20, 104 and 496 mg/kg/day for the females).

Therefore, it can be concluded that the No Observed Adverse Effect Level (NOAEL) for this study was 500 mg/kg/day.

For completeness and comparison, data are also included for a combined OECD guideline 422 repeat-dose and reproductive screening study, as a supporting study on a read across analogue o-sec butyl phenol dissolved in corn oil at doses of 0, 12, 60 and 300 mg/kg (n=13 males and n -13 females) administered by oral gavage to Sprague Dawley rats.

Similar clinical observations of transient salivation, ataxic gait and decreased activity were seen at 300 mg/kg. Transient salivation and decreased activity was seen at 60 mg/kg (males only). The NOEL in this study was 60 mg/kg (females) and 12 mg/kg (males).

 

It is concluded that the NOAEL from the 28-day key repeat dose study on o-tert butyl phenol at 100 mg/kg should be used for DNEL derivation.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
The only OECD guideline 407 study on o-tert butyl phenol, provided by the Japanese Ministry of Health

Justification for classification or non-classification

Adverse effects have been reported at 500 mg/kg/d in a 28d oral toxicity study in rodents. The NOAEL is 100 mg/kg/d. As the effects are neither severe nor organ specific a classification is not warranted.