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Toxicological information

Acute Toxicity: oral

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Administrative data

Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Not specified
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed in accordance with OECD and Japanese testing guidelines in compliance with GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Report date:
1996

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 401 (Acute Oral Toxicity)
GLP compliance:
yes
Test type:
standard acute method
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
6-tert-butyl-2,4-xylenol
EC Number:
217-533-1
EC Name:
6-tert-butyl-2,4-xylenol
Cas Number:
1879-09-0
Molecular formula:
C12H18O
IUPAC Name:
2-tert-butyl-4,6-dimethylphenol
Test material form:
liquid: viscous
Details on test material:
Name of test substance: 6-tert-butyl-2,4-xylenol
CAS No.: 1879-09-0
Code No.: B0903
Purity: 98.5%
Storage conditions: Room temperature, in the dark
Chemical name: 6-tert-butyl-2,4-xylenol
Molecular formula: (CH3)3 CC6H2 (CH3)2OH
Molecular weight: 178.27
Physical state: Liquid
Color: Very pale yellow, transparent
Coagulation temperature: 21.5 °C
Solubility: Insoluble in water
Specific gravity (d20/20): 0.9612
Refractive index (n20/D): 1.5186
Precaution for handling: Wear appropriate eye protection, protective gloves and protective mask during handling. Wash the hands and gargle sufficiently after handling.
Storage of test substance and disposal of the residue: After the end of administration, about 2 g of test substance was stored in BioSafety Research Center, and the remainder was discarded.

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
Test animals: Thirty males and 30 females of Crj:CD (SD) strain rats [SPF] were purchased from Japan Charles River Co. Ltd., (Atsugi-shi, Kanagawa) at the age of 5 weeks old and used in the study.
After inspection, the animals were acclimatized to the test environment and the administration of test substance was started at the age of 6 weeks old.
The animals were stratified by the body weights, and allocated to each test group by randomization procedure.
The animals were identified by the marking method with saturated picric acid in ethanol and attaching the labels specified with animal identification number (Animal ID-No.). The body weights at the start of administration were 145-164 g in male and 116-145 g in female.

Reason for test system selection: The species was selected considering the resistance to infectious diseases, genetic stability and others.

Animal husbandry: The animals were housed in an animal room (W 3.6 x D 10.0 x H 2.5m, 90 m3), the target environmental conditions were 23 ± 2°C of temperature, 55 ± 10% of relative humidity, 20 times per hour of ventilation frequency and 12-hour lighting (150-300 lux, lighting at 7: 00 a.m. and turn off at 7:00 p.m.).
Five animals were housed in each metal-mesh cage (W 21.5 x D 27.5 x H 19.5 cm, space: 11529 cm3). Cages were set on a water-flush breeding instrument (W 745.0 x D 50.0 x H 182.0 cm) supplied by Tokyo Giken Service Co. Ltd (Fuchu-shi, Tokyo).
Cages and food supplier were exchanged once a week.
The food used was solid food MF supplied by Oriental Yeast Co., Ltd. (Chuo-ku, Tokyo). The food was given to the animals ad libitum.
In addition, the animals were allowed free access to drinking water supplied by automatic water supply nozzles.
There were no environmental deviations which might influence the reliability of the study data during the duration of observation including the acclimatization period.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
Reasons for the dose selection: In the preliminary study 500, 1000 and 2000 mg/kg were orally administered to 3 males and 3 females each. As a result, 1 male and 1 female in 1000 mg/kg group died, and all animals in 2000 mg/kg group died. No animal died in 500 mg/kg group.
From the above results, the five dose levels were selected in the main study based on the geometric ratio of 1.25 as 819-2000 mg/kg in male and 655-1600 mg/kg in female.

Preparation of dosing solution: Appropriate amount of the test substance was weighed with an electronic balance and dissolved in corn oil (Nacalai Tesque Inc., Kyoto-shi, Kyoto). The concentration of solutions were 13.1, 16.4, 20.5, 25.6, 32.0 and 40.0 (w/v)% respectively for 655, 819, 1024, 1280, 1600 and 2000 mg/kg groups.
Doses:
Dose levels were 819, 1024, 1280, 1600 and 2000 mg/kg in male, 655, 819, 1024, 1280 and 1600 mg/kg in female.
No. of animals per sex per dose:
5 male and 5 female animals per dose group.
Control animals:
no
Details on study design:
Administration procedure: The administration route of the test substance was determined as oral route. The test substance dissolved in corn oil was administered by gavage using a steel cannula into the stomach of animals which were starved for 16 hours before the administration.
The volume to administer to each animal was determined as 0.5 mL per 100 g of body weight.
Food was given to the animals at approximately 3 hours after the administration of test substance.

Observations: Observations of toxic signs and mortality were conducted in the interval of an hour until 6 hours after the administration, and then, twice a day, in the morning and afternoon (morning only on holidays) until 14 days after the administration. Toxic signs observed were recorded in the sheets for observation findings.

Body weight: Animals were weighed immediately before the administration and at 7 and 14 days after the administration.
In addition, dead animals were weighed at the discovery of death.

Pathological examination
Macroscopic observation: Dead animals during the observation period were autopsied on the discovery, and survived animals were euthanized by exsanguination under ether anesthesia and autopsied on the day of observation end. Macroscopic abnormalities were recorded on the sheet for the record of macroscopic findings.

Histopathological examination: The organs or tissues with abnormalities were preserved in 10% neutral buffered formalin, and parts from them were processed for histopathological examination. Histological slides were prepared and examined under microscope, and the pathological findings were recorded for the type and severity of changes.

Disposal of remained animals: Remained animals were euthanized by carbon dioxide.
Statistics:
Calculation method of 50% lethal dose (LD50): LD50 was calculated by the method of Litchfield-Wilcoxon method (1949) from the mortalities at 14 days after the administration.

Results and discussion

Preliminary study:
In the preliminary study 500, 1000 and 2000 mg/kg were orally administered to 3 males and 3 females each. As a result, 1 male and 1 female in 1000 mg/kg group died, and all animals in 2000 mg/kg group died. No animal died in 500 mg/kg group.
Effect levelsopen allclose all
Sex:
male
Dose descriptor:
LD50
Effect level:
910 mg/kg bw
Based on:
test mat.
95% CL:
> 570 - < 1 452
Sex:
female
Dose descriptor:
LD50
Effect level:
972 mg/kg bw
Based on:
test mat.
95% CL:
> 814 - < 1 162
Mortality:
Mortalities in the male groups administered with 819, 1024, 1280, 1600 and 2000 mg/kg were 0, 60, 60, 80 and 100%, respectively. In female, the mortalities in the groups of 655, 819, 1024, 1280 and 1600 mg/kg were 0, 20, 60, 100 and 100%, respectively.
Clinical signs:
Decrease in locomotive activity was seen at 1 hour until 5 days after the administration. In addition, prone position and hunchback position were observed from 1 hour until 4 days after the administration, and abnormal gait was seen from 6 hours until 3 days after the administration. Hypothermia was observed from 6 hours until 4 days after the administration. In died animals lateral position was seen from 5 hours after the administration and brownish urine was observed from 2 days after the administration both in males and females.
Body weight:
The body weights of dead animals were decreased compared to those weighed before the administration both in male and female. On the other hand, the body weights of survived animals were normally increased on measurement days 7 and 14 after the administration both in male and female.
Gross pathology:
Macroscopic findings: In dead animals, black patch in the thymus, retention of brownish urine in the urinary bladder, and black contents and white patch in the small intestine were observed both in male and female. In addition, hydrothorax, black patch in the urinary bladder, white patch in the forestomach, black patch in the glandular stomach, white contents in the small and large intestine and brown contents in the small intestine in female animal were observed.
In the survived animals, adhesion with the forestomach and the diaphragm was seen both in male and female at the end of observation period. In addition, yellow patch in the liver, adhesion with the forestomach and the liver, adhesion with the liver and the diaphragm or the retro-peritoneum, adhesion with the liver and the spleen, adhesion with the spleen and the kidney and thickening of the forestomach were seen in male, atrophy of the spleen was seen in female.

Histopathology findings: In the survived male animals at the end of observation period, severe ulceration and granulomatous inflammation, and hyperplasia of the squamous epithelium and granulomatous inflammation were observed in the forestomach, and granulomatous inflammation was seen in the liver.
Other findings:
Detailed clinical signs
Male:
In 819 mg/kg group, decrease in locomotive activity was seen in 2 rats out of 5 from 1 hour after the administration, and in all rats from 2 hours to 3 days after the administration. Prone position was seen in 1 rat from 1 hour and 3 rats from 4 hours, hunchback position was seen in 1 rat from 5 hours after the administration and these signs lasted until 6-24 hours after the administration. In addition, abnormal gait appeared in 2 rats at 6 hours and later after the administration and lasted until 2 days after the administration. These signs were not severe and no death was observed.
In 1024 mg/kg group, decrease in locomotive activity was seen in all of 5 rats from 1 hour after the administration, and the sign lasted until 3 days after the administration in survived animals. Prone position was observed in 4 rats from 4 hours after the administration and it lasted until 3 days after the administration when the animals died, and hunchback position was observed in 2 rats from 5 hours after the administration and the sign lasted until 2 days after the administration. In addition, abnormal gait was observed in 2 rats at 6 hours and later after the administration and it lasted until 2 days after the administration, and lateral position was seen in 1 rat at 6 hours and later and lasted until the death of this animal. In addition to these changes, hypothermia was seen in 1 rat at 6 hours and later after the administration, it was observed in 2 rats out of 4 on day 3. Brownish urine was observed in 1 rat out of 4 at 3 days after the administration. Death was observed during 6-24 hours after the administration and 2 rats on day 3.
In 1280 mg/kg group, decrease in locomotive activity was observed in all of 5 rats from 1 hour after the administration and this sign in survived animals lasted until 5 days after the administration. Prone position was observed in 1 rat from 2 hours after the administration, in all animals during 4-6 hours after the administration and lasted until 3 days after the administration. Abnormal gait was seen in 1 rat during 6-24 hours after the administration. In addition, lateral position and hunchback position were seen in 1 and 2 rats, respectively at 6 hours and later after the administration, and these signs lasted until 2 or 3 days after the administration when the animals died. Hypothermia was seen in 2 rats at 6 hours and later and in 3 rats on day 2, and it lasted until 3 days after the administration. Furthermore, brownish urine was seen in 2 out of 4 rats at 3 days after the administration. One rat died on 2 days and 2 rats died on 3 days after the administration.
In 1600 mg/kg group, decrease in locomotive activity was seen in all 5 rats at 1 hour and lasted until 4 days after the administration in survived animals. Prone position was seen in 1 rat at 2 hours and in 3 rats at 4 hours and later and lasted until 6 hours after the administration. Hunchback position was seen in 1 rat at 4 hours after the administration, and lasted until 3 days after the administration, lateral position was observed in 1 rat from 5 hours after the administration and in 4 rats at 6 hours and later, and lasted until 2 days after the administration when the animals died. In addition, abnormal gait was observed in 1 rat at 6 hours and later after the administration, lasted until 3 days after the administration, and hypothermia was observed in 4 rats at 6 hours and later, lasted until the animals died. Four animals died on 2 days after the administration.
In 2000 mg/kg group, decrease in locomotive activity was observed in all 5 rats from 1 hour after the administration and lasted for 6-24 hours or until day 4 when the animals died. Prone position appeared from 4 hour after the administration in 4 rats, and lasted until 2 days after the administration. Hunchback position was seen from 5 hours in 1 rat, lasted during 6-24 hours after the administration, and abnormal gait was observed in 2 rats from 6 hours and later, lasted until 2 days after the administration. Lateral position was seen in 1 rat from 6 hours and later and in 1 survived rat during 3 and 4 days after the administration. In addition, hypothermia was observed in 2 rats at 6 hours and later after the administration and lasted until the animals died. All of the animals showing these signs died from 6 hours to 4 days after the administration.
Female:
In 655 mg/kg group, decrease in locomotive activity was seen 2 out of 5 rats from 3 hours and in 3 rats from 4 hours to 3 days after the administration. Prone position was observed in 2 rats from 5 hours after the administration and lasted until 6-24 hours. In addition, abnormal gait was observed in 1 rat from 6 hours and in 3 rats at 2 days after the administration. These symptoms were not severe and no death was observed.
In 819 mg/kg group, decrease in locomotive activity was seen in 4 rats out of 5 from 1 hour after the administration and in 5 rats from 2 hours to 2 days after the administration, and lasted until 3 days after the administration in survived animals. Prone position was seen in 1 rat from 4 hours after the administration and lasted until 6 hours after the administration. Abnormal gait and hunchback position were observed in 2 animals from 6 hours and later and lasted until 2 days after the administration. In addition, lateral position and hypothermia were seen in 1 animal from 6 hours after the administration and lasted until 2 days after the administration when the animal died. One death was noted on 2 days after the administration.
In 1024 mg/kg group, decrease in locomotive activity was seen in all 5 rats from 1 hour and lasted until 3 days after the administration in survived animals. Prone position was seen in 1 rat from 2 hours and in 4 rats from 4-6 hours after the administration. Abnormal gait was observed in 1 rat during 6-24 hours after the administration. In addition, lateral position and hypothermia were seen in 3 rats from 6 hours and lasted until 2 days after the administration when the animals died.
Hunchback position was seen in 1 rat from 6 hours and lasted until 2 day after the administration. Furthermore, brownish urine was noted in 1 rat on 2 days after the administration. 3 animals died on 2 days after the administration.
In 1280 mg/kg group, decrease in locomotive activity was observed in all 5 rats from 1 hour and lasted during 6-24 hours or until 2 days after the administration when the animals died. Prone position was seen in 3 rats from 4 hours after the administration and lasted until the animals died. Hunchback position was seen in 1 rat from 5 hours and lasted until 6 hours after the administration. In addition, lateral position was seen in 1 rat from 6 hours after the administration and in 4 rats at 6 hours and later, hypothermia was seen in 4 rats at 6 hours and later after the administration and these symptoms lasted until the animals died. Furthermore, brownish urine was seen in 1 rat out of 4 at 2 days after the administration. All animals which showed these symptoms died from 6 hours to 2 days after the administration.
In 1600 mg/kg group, decrease in locomotive activity was seen in all 5 rats from 1 hour after the administration, and lasted for 6-24 hours or until 2 days after the administration when the animals died. Prone position was seen in 2 rats from 2 hours and in 4 rats from 4-6 hours, and lasted until 6-24 hours after the administration. Hunchback position was seen in 1 rat from 4 hours after the administration, and lasted until the animal died. In addition, lateral position and hypothermia were seen in 3 rats from 6 hours after the administration, and lasted until the animals died. All animals which showed these symptoms died from 6 hours to 2 days after the administration.

Any other information on results incl. tables

Table 1. Mortality

Sex

Group

Dose level (mg/kg)

Number of animals

Number of deaths on the day

Mortality (%)

1

2

3

4

5

6

7

8

9

10

11

12

13

14

Male

1

819

5

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

2

1024

5

1

0

2

0

0

0

0

0

0

0

0

0

0

0

60

3

1280

5

0

1

2

0

0

0

0

0

0

0

0

0

0

0

60

4

1600

5

0

4

0

0

0

0

0

0

0

0

0

0

0

0

80

5

2000

5

2

2

0

1

0

0

0

0

0

0

0

0

0

0

100

Female

6

655

5

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

7

819

5

0

1

0

0

0

0

0

0

0

0

0

0

0

0

20

8

1024

5

0

3

0

0

0

0

0

0

0

0

0

0

0

0

60

9

1280

5

1

4

0

0

0

0

0

0

0

0

0

0

0

0

100

10

1600

5

2

3

0

0

0

0

0

0

0

0

0

0

0

0

100

                               LD50 (mg/kg)    95% Confidence limit

Male                     910                        (570 – 1452)

Female                972                        (814 – 1162)

Applicant's summary and conclusion

Interpretation of results:
harmful
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
LD50 calculated from the mortalities was 910 mg/kg (95% confidence limit: 570-1452 mg/kg) in male, and 972 mg/kg (95% confidence limit: 814-1162 mg/kg) in female.
On the basis of the results noted in the study, classification under the Dangerous Substance Directive (67/548/EEC) and the CLP Regulation (EC No 1272/2008 is as follows:

R22: Harmful if swallowed
H302: Harmful if swallowed
Executive summary:

Acute oral toxicity study of 6-tert-butyl-2,4-xylenol was conducted using 5 male and 5 female CD (SD) rats per each group.

6-tert-butyl-2,4-xylinol was dissolved in corn oil. Single oral administration was made at the dose levels of 819, 1024, 1280, 1600 and 2000 mg/kg for male, 655, 819, 1024, 1280 and 1600 mg/kg for female. During the observation period of 14 days, mortality, toxic signs, and the time of toxic sign appearance were observed. The results of the observations were summarized as follows.

 

LD50: Death of animals was seen at and above dose levels of 1024 mg/kg in male, and 819 mg/kg in female. All male animals treated with 2000 mg/kg died and all females treated with 1280 mg/kg died. LD50was 910 mg/kg (95% confidence limit: 570 to 1452 mg/kg) in male, 972 mg/kg (95% confidence limit: 814 to 1162 mg/kg) in female.

 

Clinical observations: Decrease in locomotive activity was seen at 1 hour until 5 days after the administration. In addition, prone position and hunchback position were observed from 1 hour until 4 days after the administration, and abnormal gait was seen from 6 hours until 3 days after the administration. Hypothermia was observed from 6 hours until 4 days after the administration. In died animals lateral position was seen from 5 hours after the administration and brownish urine was observed from 2 days after the administration both in males and females.

 

Body weight: Body weights of survived animals both in male and female were normally increased until the end of the observation period.

 

Pathological examination

Macroscopic findings: In dead animals, hydrothorax, black patch in the thymus, black patch and retention of brownish urine in the urinary bladder, white patch in the forestomach, black patch in the glandular stomach, white, brown or black contents in the small intestine, white patch in the small intestine and white contents in the large intestine were observed. In the animals survived until the end of observation period, yellow patch in the liver, adhesion with the liver and the spleen, diaphragm, or retro-peritoneum, atrophy of the spleen, adhesion with the spleen and the kidney, thickening of the forestomach, and adhesion with the forestomach and the liver or the diaphragm were observed.

 Histological findings: In the survived male animals at the end of the observation period, severe ulceration with granulomatous inflammation, and hyperplasia of the stratified squamous epithelium with granulomatous inflammation in the forestomach, and granulomatous inflammation in the liver were observed.

 

On the basis of the results noted in the study, classification under the Dangerous Substance Directive (67/548/EEC) and the CLP Regulation (EC No 1272/2008 is as follows:

R22: Harmful if swallowed

H302: Harmful if swallowed