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Repeated dose toxicity: oral

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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:
other: Data is from J check

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:
other:
Principles of method if other than guideline:
To evaluate the repeated dose toxicity of 2amino 5methyl benzene sulfonic acid in male and female Crj: CD (SD) rats.
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
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 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 720 mg/kg/bw/day- 6 male and 6 female100 mg/kg/bw/day - 6 male and 6 female300 mg/kg/bw/day - 6 male and 6 female1000 mg/kg/bw/day - 6 male and 6 femaleIn 14 days recovery period0 mg/kg/bw/day- 6 male and 6 female1000 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:
300 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.

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(102/mm3)

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 found to be 300 mg/kg/day for 2-amino-5-methyl benzene sulfonic acid 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 2-amino-5-methyl benzene sulfonic acidwas determined in in male and female Crj: CD (SD) rats when they were exposed in a concentration of0, 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 300 mg/kg/day. Therefore the No Observed Adverse Effect Level (NOAEL) was considered to be 300 mg/kg/day for 2-amino-5-methyl benzene sulfonic acid in male and female Crj: CD (SD) rats during 28 days repeated dose toxicity study.