4-tert-butyltoluene

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose 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:

other: GLP guideline study.

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Reference 2

Reference Type:

other: abstract

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Guideline for 28-Day Repeated Dose Toxicity Test in Mammalian Species (Chemical Substances Control Law of Japan)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

4-tert-butyltoluene

EC Number:

202-675-9

EC Name:

4-tert-butyltoluene

Cas Number:

98-51-1

Molecular formula:

C11H16

IUPAC Name:

1-tert-butyl-4-methylbenzene

Details on test material:

- Name of test material (as cited in study report): p-tert-butyltoluene; supplier: Fuso Chemical Co. Ltd., Osaka, Japan
- Physical state: colourless transparent liquid
- Analytical purity: 95.93%
- Impurities (identity and concentrations): no data
- Lot/batch No.: Lot No. 09006
- Storage condition of test material: room temperature; dark
No further data.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
male and female Crj:CD(SD)IGS, SPF rats
- Source: Charles River Laboratories, Inc
- Age at study initiation: 4 weeks upon arrival at the testing facility; 6 weeks at beginning of dosing
- Weight at study initiation: 161 - 188 g (males); 128 - 151 (females)
- Fasting period before study: no
- Housing: 5 per cage (during acclimatization), individually after assignment to experimental groups; stainless steel cages
- Diet: pellet diet (CRF-1; ORIENTAL YEAST Co., Ltd.), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 5 days quarantine, 7 days acclimatization

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 26 °C
- Humidity (%): 40 - 70 %
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: no data

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
p-tert-Butyltoluene was adjusted by dissolving and diluting it in corn oil. The amount of the test substance was calculated using its purity in preparing the test substance. It is approved that prepared solutions at 0.2, 2, 20, and 200 mg/mL can be kept stable if stored at room temperature under a shaded condition for 7 days. Therefore, the prepared solution at each concentration was stored at room temperature under a shaded condition, and was used within 7 days after preparation. Concentrations of the test substance in administration samples used on the day of start of administration and the day of the termination of administration were measured. The measurement result showed no problems with concentrations of the test substance.

VEHICLE: corn oil
- Justification for use and choice of vehicle (if other than water): no data
- Concentration in vehicle: 0.2, 2, 20, and 200 mg/mL
- Amount of vehicle (if gavage): 5 ml/kg bw

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily

Doses / concentrations

Remarks:

Doses / Concentrations:
1.5, 5, 15, 50 mg/kg bw/d
Basis:
actual ingested

No. of animals per sex per dose:

12 males and 12 females per group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The doses were determined according to the results of “Preliminary Reproduction Toxicity Screening Test of p-tert-Butyltoluene by Oral Administration in Rats" (dose levels: 0, 1.5, 5, 15, and 50 mg/kg; 12 males and 12 females in each group). There was one death in females of the 15 mg/kg group, and one death in males and 6 deaths in females of 50 mg/kg group. There were decreases in body weight in females of 5 mg/kg group and males and females of the 15 mg/kg and higher dose groups, and atrophy of the testis and epididymis in males of the 15 mg/kg and higher dose groups. Hence, the dose levels of this study were set at the highest 50, lower 15, 5, and 1.5 mg/kg by a common ratio of approximately 3. The control group was administered with the vehicle (corn oil) alone at the same volume.
- Rationale for animal assignment (if not random):
Grouping was carried out by random sampling after body weights were stratified by a computer so that the mean body weight and standard deviation are equivalent between each group.
- Rationale for selecting satellite groups:
12 male and 12 female animals were used in each group. Six animals of each sex were used for necropsy on the termination of administration period, and 6 animals of each sex were used for necropsy on the termination of recovery period.
- Post-exposure recovery period in satellite groups: 14 days
Animals were treated for 28 days and were sacrificed at day 29 or 43 after beginning of treatment.
- Section schedule rationale: random

Positive control:

no

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS and DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical signs and death were observed twice a day, before and after administration, during the administration period and once a day during the recovery period.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight was measured twice a week during both the administration and recovery periods.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: The food consumption was measured once a week during both the administration and recovery periods.

WATER CONSUMPTION: Yes
- Time schedule for examinations: The water consumption was measured once a week during both the administration and recovery periods.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the next day of the last administration and after the termination of recovery period
- Anaesthetic used for blood collection: Yes (sodium pentobarbital)
- Animals fasted: Yes
- How many animals: all
- Parameters checked in tables 5 and 6 (see attached study report) were examined.
Parameters examined: erythrocyte count (RBC), hemoglobin, hematocrit, platelet count, and leukocyte count (WBC), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), reticulocyte count, leukocyte percentage, prothrombine time (PT), activated partial thromboplastin time (APTT), plasma fibrinogen

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the next day of the last administration and after the termination of recovery period
- Animals fasted: Yes
- How many animals: all
- Parameters checked in tables 7 and 8 (see attached study report) were examined.
Parameters examined: AST, ALT, ALP, gamma-GTP, total protein, albumin A/G ratio, total bilirubin, BUN, creatinine, glucose, total cholesterol, triglycerides, sodium, potassium, chloride, calcium, inorganic phosphate.

URINALYSIS: Yes
- Time schedule for collection of urine: before the termination of administration period and before the termination of recovery period
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in tables 1-4 (see attached study report) were examined.
Urine was collected using diuresis cages before the termination of administration period and before the termination of recovery period. The following examinations were conducted for the urine collected in 3 hours (3-hour urine) under a condition that the animals were fasted and supplied with water, the urine collected in the subsequent 21 hours (21-hour urine) under a condition that the animals were supplied with food and water, and the urine that the above urines were totaled (24-hour urine).
Parameters examined: volume, specific gravity, colour, pH, protein, glucose, ketone body, bilirubin, occult blood, urobilinogen, sediments

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
The animals were sacrificed by exsanguination after collecting blood samples, and subsequently necropsy was conducted. The following organs were weighed: brain (cerebrum, cerebellum, and medulla oblongata), pituitary, thyroid, thymus, heart, liver, spleen, kidneys, adrenal glands, testes, epididymides, and ovaries (the pituitary and thyroid were weighed after fixed in 20% neutral buffer formalin for one night). These organs were fixed in 20% neutral buffer formalin together with the lungs, trachea, pancreas, salivary glands (sublingual gland and submandibular gland), esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, lymph nodes (mandibular and mesenteric), urinary bladder, seminal vesicles, prostate, uterus, vagina, parathyroid, spinal cord, sciatic nerve, eyeballs, harderian glands, sternum, femurs, and mammary glands. However, the testes and epididymides were fixed in Bouin’s solution from 2 to 3 hours and subsequently refixed in 90% alcohol. The eyeball was fixed in glutaraldehyde-formalin mixture for one night and subsequently refixed in 20% neutral buffer formalin.

HISTOPATHOLOGY: Yes
The investigators/authors produced HE-stained tissue sample of each organ and tissue of the animals of the control and 50 mg/kg groups that were necropsied on the termination of administration period and, conducted the histopathological examination. For the organs and tissues that the number of animals that showed abnormalities was different compared with the control group in the examination of the 50 mg/kg group, the examination was conducted in the same manner for the 1.5, 5, and 15 mg/kg groups on the termination of administration period, and for the control group and the 1.5, 5, 15, and 50 mg/kg groups on the termination of recovery period.

Statistics:

Statistical analyses were carried out between the control group and each administration group with a significance level of 5% as explained in the following.
The means and standard deviations were calculated for the body weight, food consumption, water consumption, urine volume, urine specific gravity, results on the hematological and blood chemical examinations, and absolute and relative organ weights for each group. Subsequently, Bartlett’s test was performed to assess the homogeneity of the variances. If the variances were homogenous, one-way ANOVA was conducted. Further, if there was statistical significance, Dunnett’s test was conducted. On the other hand, if the variance was not homogenous, one-way ANOVA by ranks (Kruskal-Wallis test) were conducted. If there was statistical significance, a Dunnett-type rank test was performed.
There was a toxicological effect in the 50 mg/kg group in the histopathological examination. The Dunnett-type rank test was conducted on the findings on the organs and tissues that the examination was conducted, for comparison between the control group and groups of the other dose levels.

Results and discussion

Results of examinations

Details on results:

CLINICAL SIGNS AND MORTALITY
1) Administration Period
There was no death or mobidity in males and females in any group.
In the observation of clinical signs, no abnormality was found in males and females of the control, 1.5, and 5 mg/kg groups. Transient salivation was found in males and females of the 15 and 50 mg/kg groups.
2) Recovery Period
There was no death or mobidity in males and females in any group.
In the observation of clinical signs, there was no abnormality in males and females in any group.

BODY WEIGHT AND WEIGHT GAIN
1) Administration Period (Figs. 1 and 2; see attached study report)
There was no significant difference in body weight at any day of measurement in males and females of the 1.5, 5, 15, and 50 mg/kg groups compared with the control group.
2) Recovery Period (Figs. 1 and 2; see attached study report)
There was no significant difference in body weight at any day of measurement in males and females of the 1.5, 5, 15, and 50 mg/kg groups compared with the control group.

FOOD CONSUMPTION
1) Administration Period (Figs. 3 and 4; see attached study report)
In males, there was no significant difference in food consumption at any day of measurement in the 1.5 and 5 mg/kg groups compared with the control group. There were significant decreases in food consumption at day 3 of the administration period in the 15 and 50 mg/kg groups compared with the control group.
In females, there was no significant difference in food consumption at any day of measurement in the 1.5 and 5 mg/kg groups compared with the control group. There was a significant decrease in food consumption at day 3 of the administration period in the 50 mg/kg group compared with the control group. There were significant increases in food consumption at day 17 of the administration period in the 15 and 50 mg/kg groups compared with the control group. However, these were transient changes, and were not considered to be a toxicological effect.
2) Recovery Period (Figs. 3 and 4; see attached study report)
There was no significant difference in food consumption at any day of measurement in males and females of the 1.5, 5, 15, and 50 mg/kg groups compared with the control group.
WATER CONSUMPTION
1) Administration Period (Figs. 5 and 6; see attached study report)
In males, there was no significant difference in water consumption at any day of measurement in the 1.5 and 5 mg/kg groups compared with the control group. There was a significant increase in water consumption at day 17 of the administration period in the 15 mg/kg group compared with the control group. There weresignificant increases in water consumption from day 3 to day 24 of the administration period in the 50 mg/kg group compared with the control group.
In females, there was no significant difference in water consumption at any day of measurement in the 1.5, 5, and 15 mg/kg groups compared with the control group. There was a significant increase in water consumption at day 10 of the administration period in the 50 mg/kg group compared with the control group.
2) Recovery Period (Figs. 5 and 6; see attached study report)
In males, there was no significant difference in water consumption at any day of measurement in the 1.5, 5, 15, and 50 mg/kg groups compared with the control group.
In females, there was no significant difference in water consumption at any day of measurement in the 1.5, 5, and 50 mg/kg groups compared with the control group. There was a significant decrease in water consumption at day 10 of the recovery period in the 15 mg/kg group compared with the control group. However, this change was not due to the dose, and therefore not considered to be a toxicological effect.

HAEMATOLOGY
1) On Termination of Administration Period (Table 5; see attached study report)
In males, there were no significant differences in any measurement subjects in the 1.5 mg/kg group compared with the control group. There were significant decreases in APTT and fibrinogen in the 5, 15, and 50 mg/kg groups compared with the control group. There were significant decreases in MCH in the 15 and 50 mg/kg groups compared with the control group. However, since there were no differences in erythrocyte count, hemoglobin concentration, and hematocrit value, it is not considered to be a toxicological effect.
In females, there were no significant differences in any measurement subjects in the 1.5 mg/kg group compared with the control group. There was a significant increase in fibrinogen in the 15 mg/kg group compared with the control group. There was a significant decrease in fibrinogen and a significant increase in PT in the 50 mg/kg group compared with the control group. Further, there was a significant decrease in platelet in the 5 mg/kg group compared with the control group. There were significant decreases in MCHC and platelet in the 15 mg/kg group compared with the control group. There were significant decreases in MCH and MCHC in the 50 mg/kg group compared with the control group. The significant differences in platelets found in the 5 and 15 mg/kg groups did not represent changes due to the administration since the differences from the control group were small and there were no significant differences in the high dose groups. Further, it is not considered that the significant decrease in MCHC found in the 15 mg/kg group and the significance decreases in MCH and MCHC found in the 50 mg/kg group are not toxicological effects since there were no differences in hemoglobin and hematocrit.
2) On Termination of Recovery Period (Table 6; see attached study report)
In males, there were no significant differences in any measurement subjects in the 1.5 and 5 mg/kg groups compared with the control group. There were significant decreases in erythrocyte count, hemoglobin concentration, and hematocrit value in the 15 and 50 mg/kg groups compared with the control group.
In females, there were no significant differences in any measurement subjects in the 1.5, 5, and 15 mg/kg groups compared with the control group. There were significant decreases in hemoglobin and hematocrit in the 50 mg/kg group compared with the control group.
CLINICAL CHEMISTRY
1) On Termination of Administration Period (Table 7; see attached study report)
In males, there were no significant differences in any measurement subjects in the 1.5 mg/kg group compared with the control group. There were significant decreases in total protein and triglyceride and significant increases in AST, blood urea nitrogen, and inorganic phosphorus in the 5 mg/kg group compared with the control group. There were significant decreases in total protein, albumin, and triglyceride and significant increases in AST, A/G, total bilirubin, blood urea nitrogen, and inorganic phosphorus in the 15 mg/kg group compared with the control group.
There were significant decreases in total protein, albumin, total cholesterol, triglyceride, and Na, and significant increases in AST, A/G, total bilirubin, blood urea nitrogen, creatinine, and inorganic phosphorus in the 50 mg/kg group compared with the control group. Further, there was a significant increase in ALT in the 5 mg/kg group. However, since the difference from the control group was small and there were no significant differences in the high dose groups, this was not considered to be a change due to the administration.
In females, there were no significant differences in any measurement subjects in the 1.5 and 5 mg/kg groups compared with the control group. There were significant decreases in total protein, albumin, total cholesterol, triglyceride, and Ca, and a significant increase in gamma-GTP in the 15 mg/kg group compared with the control group. There were significant decreases in total protein, albumin, triglyceride, K, and Ca, a decreasing tendency of total cholesterol, and significant increases in gamma-GTP and total bilirubin in the 50 mg/kg group compared with the control group.
2) On Termination of Recovery Period (Table 8; see attached study report)
In males, there were no significant differences in any measurement subjects in the 1.5, 5, 15, and 50 mg/kg groups compared with the control group.
In females, there were no significant differences in any measurement subjects in the 5, 15, and 50 mg/kg groups compared with the control group. There was a significant decrease in glucose in 1.5 mg/kg group compared with the control group. However, this did not represent a change due to the administration since the difference from the control group was small and there were no significant differences in the high dose groups.

URINALYSIS
1) Before Termination of Administration Period (Tables. 1 and 2; see attached study report)
In males, there were no significant differences in urine volume and urine specific gravity in the 1.5 and 5 mg/kg groups compared with the control group. There was a significant increase in urine volume in the 15 mg/kg group compared with the control group. There was a significant increase in urine volume and a significant decrease in urine specific gravity in the 50 mg/kg group compared with the control group. Colour, pH, protein, glucose, ketone body, bilirubin, occult blood, urobilinogen, and urine sediments were at almost the same level as the control group in the 1.5, 5, and 15 mg/kg groups. There were decreasing tendencies in pH and protein in the 50 mg/kg group compared with the control group.
In females, there were no significant differences in urine volume and urine specific gravity in the 1.5 and 5 mg/kg groups compared with the control group. There was a significant increase in urine volume in the 50 mg/kg group compared with the control group. There was a significant increase in urine specific gravity in the 15 mg/kg group compared with the control group. However, this change was not due to the dose. Colour, pH, protein, glucose, ketone body, bilirubin, occult blood, urobilinogen, and urine sediments were at almost the same level as the control group in the 1.5, 5, and 15 mg/kg groups. There was a decreasing tendency in pH in the 50 mg/kg group compared with the control group.
2) Before Termination of Recovery Period (Tables. 3 and 4; see attached study report)
In males, there were no significant differences in urine volume and urine specific gravity in the 1.5, 5, and 15 mg/kg groups compared with the control group. There was a significant increase in urine volume and a significant decrease in urine specific gravity in the 50 mg/kg group compared with the control group. Colour, pH, protein, glucose, ketone body, bilirubin, occult blood, urobilinogen, and urine sediments were at almost the same level as the control group in the 1.5, 5, 15, and 50 mg/kg groups.
In females, there were no significant differences in urine volume and urine specific gravity in the 1.5, 5, 15, and 50 mg/kg groups compared with the control group. Colour, pH, protein, glucose, ketone body, bilirubin, occult blood, urobilinogen, and urine sediments were at almost the same level as the control group in the 1.5, 5, 15, and 50 mg/kg groups.

ORGAN WEIGHTS and GROSS PATHOLOGY
1) On Termination of Administration Period (Table 9; see attached study report)
In males, there were no significant differences in absolute and relative weights of each organ in the 1.5 and 5 mg/kg groups compared with the control group. There was a significant increase in relative weight of the liver in the 15 mg/kg group compared with the control group. There were significant decreases in absolute weights of the testis and epididymis and relative weight of the testis and significant increases in absolute and relative weights of the liver in the 50 mg/kg group compared with the control group. Further, there was a significant decrease in absolute weight of the heart in the 50 mg/kg group compared with the control group. However, since there was no significant difference in relative weight, this was considered to be a change based on the difference in body weight, and that the change was not due to the administration.
In females, there was a significant decrease in body weight at the day of necropsy in the 50 mg/kg group compared with the control group. In the organ weight examination, there were no significant differences in absolute and relative weights of each organ in the 1.5 and 5 mg/kg groups compared with the control group. There were significant increases in absolute and relative weights of the liver in the 15 mg/kg group compared with the control group. There was a significant decrease in absolute weight of the ovary and significant increases in absolute weight of the liver and relative weights of the liver, kidney, and adrenal gland in the 50 mg/kg group compared with the control group. Further, there was a significant decrease in absolute weight of the thymus in the 50 mg/kg group compared with the control group. However, since there was no significant difference in relative weight, it was considered to be a change based on the difference in body weight, and that the change was not due to the administration.
2) On Termination of Recovery Period (Table 10; see attached study report)
In males, There were no significant differences in absolute and relative weights of each organ in the 1.5, 5, and 15 mg/kg groups compared with the control group. There were significant decreases in absolute and relative weights of the epididymis and decreasing tendencies of absolute and relative weight of the testis in the 50 mg/kg group compared with the control group.
In females, there were no significant differences in absolute and relative weights of each organ in the 1.5 and 5 mg/kg groups compared with the control group. There was a significant increase in relative weight of the liver in the 15 mg/kg group compared with the control group. There were significant increases in absolute and relative weights of the liver in the 50 mg/kg group compared with the control group.

HISTOPATHOLOGY
1) On Termination of Administration Period (Table 11; see attached study report)
(a) Male Rats
Liver: There was periportal hepatocyte hypertrophy in 4 animals in the 50 mg/kg group.
Testis: There was atrophy of the seminiferous tubules and hyperplasia of Leydig cells in 6 animals in the 50 mg/kg group.
Epididymis: There was a decrease in sperm count in the lumen of the ductus epididymis in 6 animals in the 50 mg/kg group.
Statistical significance was observed in periportal hepatocyte hypertrophy in the liver, atrophy of seminiferous tubules and hyperplasia of Leydig cells in the testis, the decrease in sperm count in the lumen of the ductus epididymis in the epididymis compared with the control group. The other observed changes are normally observed in the control group. Hence, those were considered accidental changes.
(b) Female Rats
Liver: There was periportal hepatocyte hypertrophy in one animal in the 50 mg/kg group.
The other observed changes are normally observed in the control group. Hence, those were considered accidental changes.
2) On Termination of Recovery Period (Table 12; see attached study report)
(a) Male Rats
Testis: There was atrophy of the seminiferous tubules in 6 animals in the 50 mg/kg group. There was atrophy of the seminiferous tubules in one animal of the 5 mg/kg group. However, this was considered an accidental change since there was no such change in the 15 mg/kg group.
Epididymis: There was a decrease in sperm count in the lumen of the ductus epididymis in 6 animals in the 50 mg/kg group.
Statistical significance was observed in atrophy of the seminiferous tubules in the testis and the decrease in sperm count in the lumen of the ductus epididymis in the epididymis in the 50 mg/kg group compared with the control group. The other observed changes are normally observed in the control group. Hence, those were considered accidental changes.
(b) Female Rats
There were microgranulomas and vacuolation of periportal hepatocytes in the liver. However, these changes are normally observed in the control group. Hence, these were considered accidental changes.

Effect levels

1

Dose descriptor:

NOEL

Effect level:

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

Sex:

male

Basis for effect level:

clinical biochemistry

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

urinalysis

2

Dose descriptor:

NOEL

Effect level:

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

Sex:

female

Basis for effect level:

clinical biochemistry

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

urinalysis

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

According to the authors, the NOELs for repeated dose toxicity are considered to be at 1.5 mg/kg bw/d for males and at 5 mg/kg bw/d for females.