2-methylanthraquinone

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

secondary literature

Justification for type of information:

Data is from secondary literature.

Data source

Reference

Reference Type:

other: J check

Title:

Twenty- Eight Day Repeat Dose Oral Toxicity Test Of 2Amino 5Methylbenzenesulfonic Acid In Rats

Author:

Ito Yoshihiko ,Yuzuru Yamamoto,Yuuji Shimodaira,Hiroshi Akagi, Naemi Fukuda, Cleo Pan

Year:

2016

Bibliographic source:

J check, 2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: As mention below

Principles of method if other than guideline:

To evaluate the repeated dose toxicity of test substance in male and female Crj: CD (SD) rats.

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

4-aminotoluene-3-sulphonic acid

EC Number:

201-831-3

EC Name:

4-aminotoluene-3-sulphonic acid

Cas Number:

88-44-8

Molecular formula:

C7H9NO3S

IUPAC Name:

2-amino-5-methylbenzenesulfonic acid

Constituent 2

Reference substance name:

p-toluidine-m-sulfonic acid

IUPAC Name:

p-toluidine-m-sulfonic acid

Details on test material:

- Name of test material: 2-amino -5-methyl benzene sulfonic acid
- Molecular formula: C7H9NO3S
- Molecular weight: 187.2181g/mol
- Substance type: Organic
- Physical state: fine yellowish white powder
- Impurities (identity and concentrations): 99.96% pure
- Lot/batch No.: 4231

Test animals

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Source: Japan SD system was carried from Charles River (Ltd.) [Crj: CD (SD)]
- Age at study initiation: 5 week old
- Weight at study initiation: male -168-183 g, female 138 162 g
- Fasting period before study: No data available.
- Housing: Housed individually in wire mesh cages
- Diet (e.g. ad libitum): Solid feed [Nosan Co., lab MR stock] ad libitum
- Water (e.g. ad libitum): Water ad libitum
- Acclimation period: Male rat 8 days ,female rat 9 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 ℃,
- Humidity (%): 55± 10%
- Air changes (per hr): 10 or more times
- Photoperiod (hrs dark / hrs light): lighting 12 hours (at 6-18)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: Sesame oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Administration solution, which was prepared by suspending the pharmacopoeial sesame oil (Miyazawa Pharmaceutical), was sealed stored under the cold shielding until use. Test substance drug substance and administration solution of the test substance was confirmed to be stable.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Sesame oil
- Concentration in vehicle: 0,100 ,300 and 1000 mg/kg/bw/day
- Amount of vehicle (if gavage): 0.5 ml per body weight 100 g.
- Lot/batch no. (if required): No data available
- Purity: No data available

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

No data

Duration of treatment / exposure:

28 days

Frequency of treatment:

Once daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0,100 ,300 and 1000 mg/kg/bw/day
Basis:

No. of animals per sex per dose:

Total no of animals 72
0 mg/kg/bw/day- 6 male and 6 female
100 mg/kg/bw/day - 6 male and 6 female
300 mg/kg/bw/day - 6 male and 6 female
1000 mg/kg/bw/day - 6 male and 6 female

In 14 days recovery period
0 mg/kg/bw/day- 6 male and 6 female
1000 mg/kg/bw/day- 6 male and 6 female

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: A 14 days repeated oral toxicity Study was observed in 1 group 4 males and 4 females, and the 0,100,250,500,1000 and 2000 mg / kg / day dose was administered for 14 day daily by oral gavage. General state, body weight, food consumption, urinalysis, hematology testing, in the blood biochemical examination and organ weight, suggest change the toxicity of the test substance was observed. In the autopsy, the cecum of expansion was observed in male and female in all cases of 2000 mg / kg group. Therefore the final dose selected for this study was 100, 300 and 1000 mg/kg/bw/day.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily
- Cage side observations checked in table [No.?] were included.- General behaviour

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: Body weight was measured on day1 of administration (immediately before administration on the first day of administration), 3 days and thereafter twice a week, every 3 or 4 days, and on the day of slaughter.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data available.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): - No data available.

OPHTHALMOSCOPIC EXAMINATION: No data available.
- Time schedule for examinations:
- Dose groups that were examined:

HAEMATOLOGY: Yes
Blood collection was performed in the administration period and at the time of necropsy, the day after the end of the recovery period. Animals were excreted from 5 pm the day before blood collection and only water was fed. The collected blood was divided into three, a part of which was subjected to coagulation (prevention treatment with EDTA), and the number of red blood cells (electric resistance detection method), the number of red blood cells Hematocrit value (pulse detection method), average red blood cell volume, average red blood cell hemoglobin amount, mean red blood cell hemoglobin concentration (calculated values), white blood cell count and platelet count (both above, electric resistance detection Method), smear samples were prepared, and reticulocyte count (Brilliant cresyl blue staining) and leukocyte% (MayGiemsa staining) were measured. In addition, a part was treated with a 3.8% sodium citrate solution to obtain plasma, and the plasma prothrombin time (Quick single step method) and activated partial thromboplastin time (elastinic acid activity Method) was measured.

CLINICAL CHEMISTRY: Yes
Serum was separated from a part of the collected blood and analyzed for total protein (Biuret method), albumin (BCG), A / G (BCG) by a biochemical automated analyzer [JEOL Ltd., JCAVX1000 type cleaner] the ratio (calculated value), blood glucose, triglycerides, total cholesterol (or more, enzymatic method), total bilirubin (Jendrassik method), urea nitrogen (UreaseUV method), creatinine (Jaff method), GOT, GPT, γGTP ( or more Sodium, potassium and chlorine were added to an alkaline phosphatase (GSCC method), calcium (OCPC method) and inorganic phosphorus (enzymatic method) by an electrolyte automatic analyzer [Toa Denpa Kogyo Co., Ltd., NAKL1]
.
URINALYSIS: Yes
Male urine sample was received on 22 days of administration and 13 days after the administration, female’s urine sample was received on 26 or 27 days after administration and 12 days after administration finished. In order to collect the sample , rats were housed in a metabolism cage for about 3 hours .Urine sample was observed for appearance, specific gravity , pH, occult blood, proteins, sugar, ketone bodies, bilirubin, urobilinogen [all of these, Martistics, Miles • Sankyo Co., Ltd.] and sediment (stained with URICEL solution, Cambridge Chemical Products Co.) .

NEUROBEHAVIOURAL EXAMINATION: No data available.

OTHER: No data available

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Organ weight was observed for following organs- brain, heart, thymus, liver, kidney, spleen, adrenal glands, testis, epididymis and ovaries.

HISTOPATHOLOGY: Yes
Histopathological examination was performed by fixing the organs in 10% neutral phosphate buffered formalin solution.
In control group and 1000 mg / kg/day group, the brain, pituitary, eyeball, thyroid (including parathyroid gland), thymic gland, The pancreas, the testicle, the epididymis, the seminal vesicle, the prostate, the ovary, the uterus (cyst), the trachea, the lung, the liver, the kidney, the spleen, the adrenal gland, the stomach, the small intestine (duodenum jejunum ileal) , Vagina, bladder, lymph node (cervical lymph node / mesenteric lymph node), spinal cord (neck enlarged part • hip swollen part), sciatic nerve, bone marrow were examined. In addition, since thymus weight was changed in females of 100 mg / kg group, 100 and 300 mg / kg group was also examined for female thymus. In the examination, paraffin sections were prepared by a conventional method and subjected to microscopic examination with haematoxylineosin staining

Other examinations:

No data

Statistics:

Statistics was observed by Dunnett's multiple comparison tests in administration group while in recovery group it is measured by T-test and U-test.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

Mortality- Mortality was not observed at all dose level in treated group both in administration and recovery group. Clinical sign-No significant change were observed in the clinical sign at dose level of 100, 300 and 1000 mg/kg/bw/day in treated group compare to control both in administration and recovery group.

Mortality:

no mortality observed

Description (incidence):

Mortality- Mortality was not observed at all dose level in treated group both in administration and recovery group. Clinical sign-No significant change were observed in the clinical sign at dose level of 100, 300 and 1000 mg/kg/bw/day in treated group compare to control both in administration and recovery group.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weight- No significant change were observed in the body weight at dose level of 100, 300 and 1000 mg/kg/bw/day in treated group compare to control both in administration and recovery group.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption- No significant change were observed in the food consumption at dose level of 100, 300 and 1000 mg/kg/bw/day in treated group compare to control both in administration and recovery group.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not specified

Details on results:

Haematology: Significant decrease in white blood cell count was observed in males in the 1000 mg / kg treated group compare to control. Decrease in white blood cell count was mainly due to the decrease in lymphocytes in view of the percentage of white blood cells. There was no significant change in white blood cell count in the recovery group of 1000 mg / kg /day treated group.

Clinical chemistry: Significant decrease in total cholesterol was observed in male at dose level of 1000 mg / kg/day group. Significant increase in GPT was observed in females while significant decreases in blood glucose were observed in females in the 100 and 1000 mg / kg groups. Reduction in blood glucose was observed in the 1000 mg / kg/ day treated group compare to control group. In the 1000 mg / kg recovery group, such changes were not observed, and were recovered.

Urinanalysis: Significant increase in specific gravity and decrease in pH were observed in males at the dose level of 1000 mg/kg/day in treated group compare to control both in administration and recovery group.

Organ weights: Significant decrease in the absolute and relative weights of the thymus was found in females in the 100 mg / kg/day treated group compare to control. A significant increase in the relative weight of the spleen was observed in the females of each group (100, 300 and 1000 mg/kg/bw/day) treated with the test substance .But when dose and organ weight were correlated, No specific change was observed in result.

Gross pathology: Mild dilatation due to cecal contents retention was observed at dose level of 1000 mg / kg/day of treated group compare to control. There was no change in the cecum in the recovery group of 1000 mg / kg.

Histopathology: Mild changes in the lung, liver, pancreas, kidney and prostate were observed in the control group and 1000 mg / kg/day group, but findings were dose independent.

Effect levels

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw (total dose)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No significant change were observed in mortality, clinical sign, body weight food consumption, haematology , clinical chemistry and urine analysis and gross pathology and histopathology.

Remarks on result:

other: No toxic effect were observed

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Urinary examination of the male rats treated orally with2amino 5methyl benzene sulfonic acid in the 28 days repeat dose toxicity test

Sr no	Dose mg/kg/day	No of animals	Specific gravity	pH 8.0
Administration period
1	0	12	1.037±0.014	3
2	100	6	1.044±0.014	1
3	300	6	1.046±0.017	1
4	1000	12	1.057±0.019*	1**
Recovery period
1	0	6	1.045±0.015	2
2	1000	6	1.047±0.006	3

Significant difference from control group*P<0.05, **P<0.01   

 

Clinical chemistry examination of the male rats treated orally with2amino 5methyl benzene sulfonic acid in the 28 days repeat dose toxicity test

Dose level Mg/kg/day	0		100	300	1000	0		1000
		Administration period					Recovery period
No of animals	6		6	6	6	6		6
Total cholesterol	90±10		77±9	85±9	74±10*	101±14		91±10

Significant difference from control group*P<0.05

Clinical chemistry examination of the female rats treated orally with2amino 5methyl benzene sulfonic acid in the 28 days repeat dose toxicity test

Dose level Mg/kg/day	0		100	300	1000	0		1000
		Administration period					Recovery period
No of animals	6		6	6	6	6		6
GPT	24±4		27±2	24±5	32±5	25±4		26±6
Glucose	138±6		121±14	126±11	116±9**	137±15		130±11

Significant difference from control group*P<0.05, **P<0.01   

 

Hemotology examination of the male rats treated orally with2amino 5methyl benzene sulfonic acid in the 28 days repeat dose toxicity test

Dose level Mg/kg/day	0		100	300	1000	0		1000
		Administration period					Recovery period
No of animals	6		6	6	6	6		6
Leukocyte(10^2/mm^3)	76±16		67±9	73±26	49±11*	88±28		85±17

Significant difference from control group*P<0.05, **P<0.01   

Applicant's summary and conclusion

Conclusions:

The No Observed Adverse Effect Level (NOAEL) was considered to be 1000 mg/kg/day for test substance in male and female Crj: CD (SD) rats during 28 days repeated dose toxicity study.

Executive summary:

In 28 days repeated dose toxicity study for test substance was determined in in male and female Crj: CD (SD) rats when they were exposed in a concentration of 0,100,300 and 1000mg/ kg /day for 28 days by oral gavage . There was recovery period of 14 days in control and 1000 mg/kg/day group. No significant change were observed in mortality, clinical sign, body weight food consumption at dose level of100 ,300 and 1000 mg/kg/bw/day in treated group compare to control. Significant change were observed in thehaematology , clinical chemistry and urine analysis , organ weight and gross pathology and histopathology at dose level of 1000 mg/kg/day of treated group. These changes were not recovered during recovery period. No significant changes were observed at the dose level of 1000 mg/kg/day. Therefore the No Observed Adverse Effect Level (NOAEL) was considered to be 1000 mg/kg/day for test substance in male and female Crj: CD (SD) rats during 28 days repeated dose toxicity study.