tert-pentylbenzene

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

No data

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

tert-pentylbenzene

EC Number:

218-076-0

EC Name:

tert-pentylbenzene

Cas Number:

2049-95-8

Molecular formula:

C11H16

IUPAC Name:

(2-methylbutan-2-yl)benzene

Details on test material:

- Substance type: Technical product.
- Analytical purity: 97.4%
- Impurities: Cumene: 0.7%, Heptylbenzene (C13H18): 0.6%, Nonylbenzene (C15H22): 0.6%
- Lot/batch No.: Tank BV1112 du 18 avril 2007.

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: no data
- Expiration date of the lot/batch: no data
- Purity: 99.5%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Sealed in a cool, dark place (Measured temperature range: 1°C-8°C)
- Stability under test conditions: stability during experiments was confirmed.
- Solubility and stability of the test substance in the solvent/vehicle: Insoluble in water. Easily soluble in ethanol, ether.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: no data

Test animals

Species:

rat

Strain:

Crj: CD(SD)

Details on species / strain selection:

Because the rat is the animal species that is commonly used in toxicity studies and the researchers have sufficient experience in handling rats at this laboratory, this animal strain was selected.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan Co., Ltd. (795 Shimo-furusawa, Atsugi-shi, Kanagawa ken)
- Age at study initiation: Forty-two four-week-old male and female rats each.
- Weight at study initiation: from 68 to 82 g (females) and from 68 to 93 g (males).
- Fasting period before study: no data
- Housing: individually
- Diet (e.g. ad libitum): ad libitum .
- Water (e.g. ad libitum): From the automatic water feeder, the animals were allowed to drink, ad libitum, tap water supplied by City of Sapporo.
- Acclimation period: 7 days under quarantine

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): 10-15 times/hour
- Photoperiod (hrs dark / hrs light): 12 hours of illumination (8:00-20:00, artificial lighting).


IN-LIFE DATES: No data

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
VEHICLE
- Justification for use and choice of vehicle (if other than water): No data
- Concentration in vehicle: , 0, 100, 300 or 1000 mg/kg bw/day
- Amount of vehicle (if gavage): 5 mL/kg bw
- Lot/batch no. (if required): V5R8265, Nacalai tesque
- Purity: No data
- Analytical verification of doses or concentrations: Yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Prepration of sample solution was conducted once each, and injection into HPLC system was conducted once each.

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily

Doses / concentrations

Remarks:

0, 100, 300 and 1000 mg/kg bw/day (mg/kg bw/day (nominal))

No. of animals per sex per dose:

6 males and 6 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Post-exposure period: yes. 14 days (0 and 1000 mg/kg bw/day)
- Dose selection rationale:
According to the results of the 14-day repeated oral dose study in which tert-pentylbenzene dissolved in corn oil was given to three male and female rats each in four dose groups (30, 100, 300 and 1000 mg/kg), the male animals of the dose groups treated at doses exceeding 300 mg/kg and one female animal of 1000 mg/kg dose group showed brown urine discoloration, and the male and female animals of the 1000 mg/kg dose group showed salivation and a trend of slightly high values of absolute and relative weight of the liver. Accordingly, the dose of 1000 mg/kg was regarded as the highest dose, thereafter the doses were divided by an approximate common ratio of three and the doses of 300 mg/kg and 100 mg/kg were regarded as the medium dose and the low dose respectively. Two dose groups, the control group and the 1000 mg/kg dose group, were used in the recovery study to examine whether recovery could beachieved by setting 14-day without treatment.

Positive control:

Not used.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: all the animals were observed in detail with respect to performance status before initiation of administration, Day 7, 14, 21 and 28 and Recovery Day 7 and 14.

BODY WEIGHT: Yes
- Time schedule for examinations: On Day 1, 7, 14, 21 and 28 (before administration), Recovery Day 7 and 14 and the day of autopsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal: On Day 1, 7, 14, 21 and 28 (before administration) and Recovery Day 7 and 14 (before administration).

FOOD EFFICIENCY: no.


WATER CONSUMPTION AND COMPOUND INTAKE: No


OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: twice
- Anaesthetic used for blood collection: Yes (no data on identity)
- Animals fasted: Yes
- How many animals: all tested animals.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: twice
- Animals fasted: Yes
- Anaesthetic used for blood collection: Yes (no data on identity)
- How many animals: all tested animals

URINALYSIS: Yes
- Time schedule for collection of blood: twice
- Animals fasted: Yes
- How many animals: all tested animals.

NEUROBEHAVIOURAL EXAMINATION: yes (week 4 and recovery week 2).

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
brain (cerebrum, cerebellum, medulla), pituitary, spinal cord, thymus, thyroid gland, parathyroid, adrenal gland, spleen, heart, tongue, esophagus, stomach, liver, pancreas, duodenum, jejunum, ileum (including Peyer’s patches), appendix, colon, rectum, mesenteric lymph node, submandibular lymph node, trachea, lung, kidney, bladder, testis, epididymis, prostate gland, seminal vesicle (including the coagulating gland), ovary, uterus, vagina, eyeball, harderian gland, femur (including bone marrow, right), sciatic nerve, injured skin of the neck detected as macroscopic abnormality.

HISTOPATHOLOGY: Yes

Other examinations:

Measurement of organ weight:
Organs: brain, pituitary, thyroid gland, adrenal gland, spleen, heart, liver, kidney, thymus, testis, epididymis, prostate gland, seminal vesicle (including the coagulating gland), ovary, uterus.

Statistics:

For grip strength, motor activity, body weight, weight gain and the rate of weight gain, feed intake, urine volume, and the results of hematological examination, blood chemistry test and measurement of absolute and relative organ weight, the means and standard deviations were calculated, and equality of variance was analyzed by Bartlett’s test. In the case of equal variance (p>0.05), one-way analysis of variance was used, while, in the case of unequal variance (p ≤0.05), Kruskal-Wallis test was used for further analysis. If one-way analysis of variance showed significant difference (p ≤0.10), Dunnett’s test was used to make a comparison with the control group. If Kruskal-Wallis test showed significant difference (p ≤0.10), Mann-Whitney U test was used to make a comparison with the control group.
Kruskal-Wallis test was used to analyze the items of detailed observation of performance status, the observation items of functional examinations, qualitative items of urinalysis and data on the urine specific gravity, and, in the case of presence of significant difference (p ≤0.10), Mann-Whitney U test was used to make a comparison with the control group.
A level of significance of 5% was used to perform a comparative test with the control group. For the statistical methods, the method for display was shown at the top of INDIVIDUAL DATA.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

1- Administration period
- In the control group, both male and female animals had no abnormality.
- In the 100 mg/kg dose group, one male animal had head injury on Day 24 or later. No abnormality was observed in the female animals.
- In the 300 mg/kg dose group, both male and female animals had no abnormality.
- In the 1000 mg/kg dose group, one male and three females occasionally had salivation, while one male and two females occasionally had contamination of the hair around the external urethral opening.
2- Recovery period
No abnormality was observed in both male and females of the control group and the 1000 mg/kg dose group.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

1- Administration period
Both male and female animals of the treatment groups showed no significant difference in mean body weight when compared with those of the control group although significantly low values of weight gain and rate of weight gain were recognized in the male animals of the 1000 mg/kg dose group.
2- Recovery period
Both male and female animals of the 1000 mg/kg dose group showed no significant difference in mean body weight when compared with those of the control group although significantly high values of rate of weight gain were recognized in the male animals.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

- Administration period:
There was no significant difference between the male and female animals of the treatment groups and those of the control group.
- Recovery period:
There was no significant difference between the male and female animals of the 1000 mg/kg dose group and those of the control group.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

1- Administration period
- In the 100 mg/kg dose group, the male animals showed significantly low RBC count, significant reduction of activated thromboplastin time, and significant changes in differential WBC count such as significantly high percentage of segmented neutrophils and significantly low percentage of lymphocytes. The female animals showed significantly low mean RBC hemoglobin value, significantly low platelet count, and significantly high WBC count.
- In the 300 mg/kg dose group, both male and female animals showed no significant changes.
- In the 1000 mg/kg dose group, female animals showed significant prolongation of APTT. No significant change was observed in the male animals
2- Recovery period:
In the 1000 mg/kg dose group, the male animals showed significantly low mean RBC hemoglobin values, and the female animals showed the significantly low percentage of monocytes (WBC differential count), when compared with those of the control group.

These effects were unlikely to be of toxicological significance because of absence of dose response relationship.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

1- Administration period effects observed.
- In the 100 mg/kg dose group, female animals showed significantly high γ–GTP values and significantly low K values when compared with the control group. No significant change was observed in the male animals.
- In the 300 mg/kg dose group, male animals showed significantly high TP and albumin values. No significant change was observed in the female animals.
- In the 1000 mg/kg dose group, both male and female animals showed significantly high TP values, while the female animals showed significantly high albumin values and the male animals showed a trend of high albumin values. Moreover, the male animals showed significantly high γ–GTP values and significantly low Cl values, while the female animals showed significantly high Ca values.
2- Recovery period
- In the 1000 mg/kg dose group, the male animals showed significantly high glucose values when compared with the control group. No significant change was observed in the female animals.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

1- Administration period
- In the 100 mg/kg dose group, both male and female animals showed no significant change when compared with the control group.
- Although a male animal (1/6) and a female animal (1/6) of the 300 mg/kg dose discharged light yellow/yellow urine and showed slightly brown urine discoloration, these findings did not indicate significant change. Moreover, some female animals showed significantly high values of urine specific gravity.
- Ten male animals and two female animals of the 1000 mg/kg dose group discharged light yellow/yellow urine and showed slightly brown urine discoloration, and the number of male animals with this symptom increased significantly.
2- Recovery period
The female animals of the 1000 mg/kg dose group showed significantly higher values of urine specific gravity when compared with the animals of the control group. No significant change was observed in the male animals.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

1- Administration period
There was no significant difference in the results of observation items between male and female animals of the treatment groups and those of the control group throughout the examinations, and no neurobehavioral disorders including sedation, excitement and behavioral disorder were recognized.
2- Recovery period
There was no significant difference in the results of observation items between male and female animals of the 1000 mg/kg dose group and those of the control group throughout the examinations, and no neurobehavioral disorders including sedation, excitement and behavioral disorder were recognized.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

1- Administration period RECOVERY
- In the 100 mg/kg dose group, both male and female animals showed no significant change when compared with the control group.
- The significantly high values of relative kidney weight and a trend of high values of absolute kidney weight were observed in the male animals of the 300 mg/kg dose group. No significant change was observed in the female animals.
- In the 1000 mg/kg dose group, the significantly high values of relative liver weight were observed in the male and female animals, while a trend of high values of absolute liver weight was observed in the male animals and the significantly high values of absolute liver weight were observed in the female animals. The significantly high values of absolute and relative kidney weight were observed in the male animals. Moreover, the significantly low values of absolute ovary weight were observed in the female animals.
2- Recovery period
In the 1000 mg/kg dose group, the male animals had significantly low values of absolute epididymis weight when compared with those of the control group. No significant change was observed in the female animals.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

1- Administration period
- No abnormal finding was obtained from both male and female animals of the control group.
- In the 100 mg/kg dose group, a male animal had injury on the skin of the neck. No abnormal finding was obtained from the female animals.
- No abnormal finding was obtained from the male and female animals of the 300 mg/kg and 1000 mg/kg dose groups.
2- Recovery period
- In the control group, a female animal had black patches over the mucosa of the glandular stomach, and a different female animal had adhesion of the spleen to the parietal peritoneum. No abnormal finding was obtained from the male animals.
- Enlargement of ventricle of the brain was observed in one female animal of the 1000 mg/kg dose group. No abnormal finding was obtained from the male animals.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

1- Administration period
* Liver: In the 1000 mg/kg dose group, a female animal had mild centrilobular hypertrophy of hepatocytes although no such a change was observed in the male animals.
According to the results of comparison between the control group and the treatment groups, other changes characterized by increased number of affected animals or higher grades were not observed in the male and female animals of the treatment groups.
* Kidney:
- In five male animals of the 100 mg/kg dose group, mild hyaline droplets and eosin bodies appeared in the proximal tubular epithelium.
- In the 300 mg/kg dose group, mild hyaline droplets appeared in the proximal tubular epithelium in five male animals, moderate hyaline droplets in the proximal tubular epithelium in one male animal, and mild eosin bodies in the proximal tubular epithelium in all the animals. In the 1000 mg/kg dose group, mild hyaline droplets appeared in the proximal tubular epithelium in two male animals, moderate hyaline droplets in the proximal tubular epithelium in four male animals, and mild eosin bodies in the proximal tubular epithelium in all the animals. These animals showed the positive reaction in α2u-globulin stain. According to the results of comparison between the control group and the treatment groups, other changes characterized by increased number of affected animals or higher grades were not observed in the male and female animals of the treatment groups.
* Other organs/tissues: According to the results of comparison between the control group and the treatment groups, the changes characterized by increased number of affected animals or higher grades were not observed in the male and female animals of the treatment groups.
2- Recovery period
* Liver: The male and female animals of the 1000 mg/kg dose group showed no changes characterized by increased number of affected animals or higher grades when compared with the control group.
* Kidney: Five of six male animals of the 1000 mg/kg dose group had mild regeneration of proximal tubular epithelium in the kidney and the incidence was higher in the 1000 mg/kg dose group than in the control group. The female animals showed no changes characterized by increased number of affected animals or higher grades when compared with the control group.
* Other organs/tissues: Both male and female animals of the 1000 mg/kg dose group showed no changes characterized by increased number of affected animals or higher grades when compared with the control group.

Histopathological findings: neoplastic:

not examined

Effect levels

1

Key result

Dose descriptor:

NOEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Remarks on result:

other: based on the high values of TP, albumin and kidney weight at 300 mg/kg bw/day. In the 1000 mg/kg dose group,and increased liver weight was observed.

2

Key result

Dose descriptor:

NOEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Remarks on result:

other: based on the high values of TP and albumin, significant prolongation of APTT, high Ca value, mild centrilobular hypertrophy of hepatocytes in the liver and increased liver weight observed at 1000 mg/kg bw/day.

3

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: The histopathological effects on kidneys, which were found for males at 300 mg/kg bw/day, are considered not relevant for humans. The other effects observed in males at 300 mg/kg bw were considered as not adverse.

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

Table 1/Absolute and relative liver weights of female rats in 28 days study:

 

Group	Number of animals	Body weight (g)	%
Control	6	6.680	3.22
100 mg/kg bw	6	7.200	3.287
300 mg/kg bw	6	6.853	3.438
1000 mg/kg bw	6	8.100++	4.052**
Recovery control	6	6.732	2.948
Recovery 1000 mg/Kg bw	6	6.320	2.977

Table 1/Absolute and relative liver weights of male rats in 28 days study:

Group	Number of animals	Body weight (g)	%
Control	6	11.333	3.245
100 mg/kg bw	6	11.365	3.247
300 mg/kg bw	6	11.473	3.335
1000 mg/kg bw	6	12.592	3.837**
Recovery control	6	12.778	3.090
Recovery 1000 mg/Kg bw	6	12.548	3.220

 **: Significantly different from the control group at p < = 0.01 (Dunnett's prosedure)

++: Significantly different from the control group at p <= 0.01 (Mann-Whitney's U-test).

 

Applicant's summary and conclusion

Conclusions:

Under the test conditions of this study:
- The NOEL is 100 mg/kg bw/day for males based on the high values of TP, albumin and kidney weight at 300 mg/kg bw/day. In the 1000 mg/kg dose group, the male animals showed the low values of weight gain, rate of weight gain and Cl, high γ-GTP value and increased liver weight (these effects are not adverse and reversible after 14 days of recovery).
- The NOEL is 300 mg/kg bw/day for females based on the high values of TP and albumin, significant prolongation of APTT, high Ca value, mild centrilobular hypertrophy of hepatocytes in the liver and increased liver weight observed at 1000 mg/kg bw/day (these effects are not adverse and reversible after 14 days of recovery).
- The NOAEL is 300 mg/kg bw/day for male and female rats. The histopathological effects on kidneys, which were found for males at 300 mg/kg bw/day, are considered not relevant for humans. The accumulation of protein (hyaline) droplets in the kidney can also explain the increased absolute kidney weight of the males (12 %). The biochemical findings showed a statistically significant increase of total protein (TP) (4,6 %) and albumin (5,1 %) for the male rats but this increase is not very high and was observed for male rats only.

Executive summary:

The toxicity of tert-pentylbenzene was investigated (OECD 407, GLP), when given by oral route to the Sprague Dawley rat for a 4- week period followed by a recovery period of 2 weeks from the last administration. Males and females were dosed at 0, 100, 300 and 1000 mg/kg bw/day.

There were no significant clinical findings of toxicity and no mortality. The male animals of the 1000 mg/kg dose group showed the significantly reductions of body weight and rate of weight gain during the administration period. During the recovery period, however, the significantly high values of rate of weight gain were observed. Both male and female animals of the treatment groups showed no change in food intake associated with administration of the test substance during the administration and recovery periods. Hematological examination showed significant prolongation of activated partial thromboplastin time (APTT) in the female animals of 1000 mg/kg dose group. A complete recovery was evident after the end of recovery. Blood chemistry test showed significantly high values of total protein (TP) and albumin in the male animals of 300 mg/kg dose group. In the 1000 mg/kg dose group, both male and female animals showed significantly high values or a trend of high values of TP and albumin, male animals showed significantly highγ-GTP values and significantly low Cl values, and female animals showed significantly high Ca values. A complete recovery was evident after the end of recovery. In urinalysis, urine discoloration was observed but did not suggest any adverse effect of the test substance on the kidney but indicated that the test substance was eliminated in the urine.

 At post mortem examination, the male animals of 300 and 1000 mg/kg dose groups showed significantly increase of kidney weight recorded both at absolute and relative kidney weight. The male and females of 1000 mg/kg dose group showed significantly increase of liver weight recorded both at absolute and relative liver weight. A complete recovery was evident after the end of recovery.

At microscopic level, mild centrilobular hypertrophy of hepatocytes in the liver of the females (1/6) of 1000 mg/kg dose group was observed. A complete recovery was observed.

In the male animals of the dose groups treated at doses exceeding 100 mg/kg, hyaline droplets and eosin bodies appeared in the proximal tubular epithelium in the kidney (α2u-globulin stain positive) and a dose-dependent grade increase was noticed although recovery was complete.

 

As mentioned above, as a change related to administration of the test substance, in the male animals of the dose groups treated at doses exceeding 100 mg/kg, mild hyaline droplets and eosin bodies appeared in the proximal tubular epithelium (α2u-globulin stain positive). It has been known that α2u-globulin, which is characteristic of male rats, cannot be extrapolated to humans.

In the dose groups treated at the doses exceeding 300 mg/kg, the male animals showed the high values of TP, albumin and kidney weight. In the 1000 mg/kg dose group, the male animals showed the low values of weight gain, rate of weight gain and Cl, high γ-GTP value and increased liver weight. The female animals showed the high values of TP and albumin, significant prolongation of APTT, high Ca value, mild centrilobular hypertrophy of hepatocytes in the liver and increased liver weight. All these effects were reversible after 14 days of recovery.

Consequently, the NOELs of tert-pentylbenzene under the present study conditions were 100 mg/kg/day in male animals and 300 mg/kg/day in female animals and the NOAEL is 300 mg/kg bw/day for male and female rats. Therefore, no classification is required according to CLP regulation (1272/2008).