Registration Dossier

Administrative data

Description of key information

Weight of evidence. Based on the available data, the test item has a NOAEL = 20 mg/kg bw/d in rats.

- Method according to OECD 422, GLP study. The NOAEL was determined to be 20 mg/kg bw/day in male and female rats.

- Method according to OECD 407, GLP study. The NOAEL for the subacute oral repeated dose toxicity was determined to be 20 mg/kg bw/day in male and female rats.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
07/03/2000 - 20/04/2000
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Reason / purpose:
reference to other assay used for intermediate effect derivation
Reason / purpose:
reference to other assay used for intermediate effect derivation
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crj:CD (SD) IGS, SPF
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Japan Charles River Co., Ltd.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 5 weeks old
- Weight at study initiation: Males 135.1 to 152.9 g (average 144 g) Females 114.1 to 136.6 g (average 124.8 g)
- Housing: wire mesh floor cage (220 W × 270 d × 190 mm)
- Diet: solid feed (CE-2, CLEA Japan, Ltd.), ad libitum
- Water: tap water (Kanno City Waterworks Bureau water supply), ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23-25​​°C
- Humidity (%): 50-65%
- Air changes (per hr): 15 times/hour
- Photoperiod (hrs dark / hrs light): 12 hours (7-19) light / 12 hours dark

IN-LIFE DATES: Purchase date: February 28, 2000 / Administration date: March 7, 2000
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS: The test substance was weighed for each dose, medium was added to give a predetermined concentration, and the sample was prepared by stirring and suspension. Since the stability of the sample for 8 days had been confirmed, it was prepared once a week and stored under airtight and refrigerated conditions until the time of use.

- VEHICLE : 0.5 % (w/v) CMC in water
- Amount of vehicle (if gavage): 5 ml/kg, the dose (mL) was calculated for each individual animal based on the recent body weight.
- Lot/batch no. (if required): carboxymethyl cellulose sodium (Japanese Pharmacopoeia CMC sodium, lot No. 6Z09, Maruishi Pharmaceutical Co., Ltd.); in water for injection (production number 9707SA, Hikari Pharmaceutical Co., Ltd.)
- Purity: compliant with Japanese Pharmacopoeia standards.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The dosed samples were checked for test item content and homogeneity after preparation. No further details provided.
Duration of treatment / exposure:
28 days (recovery 14 days)
Frequency of treatment:
Daily (once in the morning)
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
control (0.5% w/v CMC aqueous solution)
Dose / conc.:
4 mg/kg bw/day (nominal)
Dose / conc.:
20 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10 animals per sex per dose in each of the control and high dose groups, 5 animals per sex per group in the low and middle dose groups.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected based on the results of the repeated oral dose toxicity and reproductive developmental toxicity combined trial conducted previously (study No. A-97-029, test substance lot No. GC01, doses of 0, 20, 100 and 500 mg / kg). In this study, formation of eosinophilic bodies in the kidney and decreased alkaline phosphatase activity was observed in males of all test groups. In females, administration of 100 mg/kg or more caused deterioration of general conditions such as decreased locomotor activity, suppressed weight gain and food intake, and increased the relative weight of the liver, resulting in a centrilobular liver. Cell hypertrophy was observed. In addition, in the 500 mg/kg group, 5/13 animals died. From the above, 100 mg/kg, in which a clear change in toxicity was observed, was taken as the highest dose in this study, and divided by a common ratio of 5 to obtain the medium and low doses. In both sexes, the control group was administered 0.5% CMC Na aqueous solution (vehicle).
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily (before and after administration).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: detalied observations including palpation once a week

BODY WEIGHT: Yes
- Time schedule for examinations: twice a week, at the end of the administration period, at the end of the recovery period and on the day of necropsy.

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. Food was weighed at the beginning of the treatment and once a week thereafter, and the mean food intake per mouse per period was calculated.

HAEMATOLOGY: Yes
- Time schedule for collection of blood:
- Anaesthetic used for blood collection: Yes (pentobarbital)
- Animals fasted: Yes (18-24h)
- How many animals: all
- Parameters: Red blood cell count, average red blood cell volume, platelet count (electric resistance method), white blood cell count (flow cytometry / laser light scattering / electric resistance method) by an automatic blood analyzer (CELL-DYN3500SL, Dynabot, Inc.) Hematocrit, average red blood cell level and average white blood cell level were calculated (flow cytometry / laser light scattering method) and the amount of hemoglobin (absorbance method). In addition, for plasma: prothrombin time and active partial thromboplastin time (any time) using a fully automatic blood coagulation measurement device (CA-1000, Toa Medical Electronics Co., Ltd.).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after blood collection for haematology
- Animals fasted: Yes
- How many animals: all
- Parameters: Protein concentration (biuret method), albumin concentration (BCG method), total cholesterol concentration (COD / DAOS method), sucrose concentration (glucokinase G6 PDH method), urea nitrogen concentration (Urease Gr. DH method), creatinine concentration (Jaffe Rate Method), ALP activity (GSCC method), GOT activity (IFCC method), GPT activity (IFCC method), γ-GTP activity (γ-glutamyl-3-carboxy-4-nitroanilide substrate method), triglyceride concentration The A/G ratio was calculated by measuring the total bilirubin concentration (Jendrassik / Grof method), the inorganic phosphorus concentration (molybdic acid direct method) and the calcium concentration (OCPC method), GPO / DAOS method). Sodium, potassium and chlorine concentrations were measured by a fully automatic electrolyte analyzer (EA05, A & T Co., Ltd.).

URINALYSIS: Yes
- Time schedule for collection of urine: In all groups, animals were stored in metabolic cages and collected at week 4 and the end of the recovery period, and collected at about 4 and 24 hours.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Not specified
- Parameters: pH, occult blood, protein, sugar, ketone body, urobilinogen, bilirubin, the sediment by light microscope, color tone and turbidity by visual examination, urine volume (weight was measured with a balance and divided by specific gravity) and specific gravity (weight per unit volume) were checked using 24-hour urine.

FOOD EFFICIENCY: No / Not specified
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No / Not specified
OPHTHALMOSCOPIC EXAMINATION: No / Not specified
NEUROBEHAVIOURAL EXAMINATION: No / Not specified
IMMUNOLOGY: No / Not specified
Sacrifice and pathology:
GROSS PATHOLOGY: Yes.
Following blood collection, the axillary artery was cut and exsanguinated and sacrificed as needed, and organs and tissues were observed macroscopically. In addition, the weight of each animal's brain, heart, liver, kidney, thymus, spleen, adrenal gland, testis, epididymis, ovaries, thyroid and pituitary are measured, and each organ weight is divided by the weight of the autopsy day to calculate relative weights.

HISTOPATHOLOGY: Yes.
The brain, pituitary, spinal cord, eye, thyroid, parathyroid, heart, trachea, bronchi, lung, liver, kidney, thymus, spleen, adrenal, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, prostate, seminal vesicle, ovary, uterus, ankle, mammary gland, bladder, mandibular lymph node, mesenteric lymph node, skeletal muscle (lower leg), sciatic nerve, femoral bone marrow, pancreas, submandibular gland, sublingual gland, tongue, esophagus, aorta, Harder's gland, skin and lesions were fixed in 0.1 mol/L phosphate buffered 10% formalin solution, and testis and epididymis in Bouin's solution. For all lesions observed at the end of administration period, the sections were paraffin-embedded, sliced, and hematoxylin-eosin stained specimens were prepared. Thereafter, specimens of the control group and the high dose group (lesions were all groups) were examined histologically using a light microscope, and then the kidney and spleen were also examined histologically.
Statistics:
Body weight, food consumption, urinalysis excluding semi-quantitative examination, and values ​​of hematological examination and biochemical examination for routine autopsy cases and organ weights were determined as the mean and standard deviation for each group. In addition, when there are three or more test groups, multiple comparisons are performed using the variance uniformity test according to Bartlett's method, one-way analysis of variance, Kruskal-Wallis rank test, and Dunnett to Dunnett-type test method. In the case of 2 groups, F-test was performed, and a test of significance was performed using Student's t-test or Aspin-Welch's t-test. In addition, for semi-quantitative test results of urine, χ2 test using a 2 × 2 contingency table was performed. The graded data of the histopathological examination findings were the Mann-Whitney U test (two-sided test), and the total value of the positive grade was the Fisher's exact one-sided test. The significance test between the control group and the test substance administration group was 5% in all cases.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Swelling and decreased activity were observed 15 minutes after administration in the 100 mg/kg group of males and females, and in 1 male, a prone position was also observed. Fluctuation, decreased activity, and prone posture were also found in the combined test of repeated toxicity and reproductive toxicity of this substance.
In the 100 mg/kg group, salivation was observed from day 7 of administration onwards for males, and on day 9 onwards for females. No salivation was observed immediately after administration, and no gastrointestinal changes often observed when administering irritating substances, suggesting that it was not due to physical effects. Possibley related changes such as miosis were not observed. Activity was decreased from day 9 (males) / 10 (females) after administration and throughout the administration period.
No other changes were observed in other groups, neither during the administration period nor during the recovery period.
Mortality:
no mortality observed
Description (incidence):
There were no deaths during the administration period or recovery period.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Females in the 100 mg/kg group showed significantly lower values ​​compared to the control group at the time of measurement from day 4 to day 11 of the recovery test. No other significant differences in body weight were observed between the test substance administration groups and the control.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food intake was significantly reduced in females in the 100 mg/kg group in the third week of administration and in the males in the first week of the recovery test compared with the control group. However, no simultaneous effect on body weight was observed. No other significant differences were observed between the test substance administration groups and the control.
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):
At the end of the administration period, there was no significant change between the control group and the test substance administration group in any item. At the end of the recovery test period, the number of red blood cells decreased significantly, and the average red blood cell volume and the average red blood cell pigment amount increased significantly in the 100 mg/kg dose group of males. Histopathological examination of the spleen was performed for all male and female cases in the study, but no differences were observed in the degree of findings among each group. Based on this and the previous combined study, the change was considered non-treatment-related. No other significant changes were observed.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
At the end of the administration period, urea nitrogen concentration was increased in males in the 20 mg/kg dose group and in females in the 100 mg/kg dose group. At the end of the recovery test period, urea nitrogen concentration was significantly increased in males at 100 mg/kg, and creatinine concentration and GOT activity were significantly decreased. In females of the same group, albumin concentration, total cholesterol concentration, triglyceride concentration and calcium concentration decreased significantly. The change in urea nitrogen concentration was not observed in the previous combined study, in which a higher dose was administered for a long time, and it was not dose-dependent in males. In addition, no other changes in clinical biochemistry or any related changes were observed, so it was judged that the change was not attributable to administration of the test substance.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
In females in the 100 mg/kg dose group, the pH tended to be high in the urine examination at the end of the dosing period, but it was unclear whether the change was attributable to the test substance administration. No other significant differences were observed between the test substance administration groups and the control.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Significant increase in the relative weight of the male spleen in the 100 mg/kg dose group. No other significant effects were observed.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
At the end of the administration period, miniaturization of the testis, epididymis, seminal vesicles and prostate was observed in 1 male in the 4 mg/kg administration group, but no change was observed in both male and female groups. At the end of the recovery test period, miniaturization of the thymus was observed in 1 male in the 100 mg/kg group. No significant effects were observed.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
The eosinophilic body of the tubular epithelium tended to increase in the 100 mg/kg group in both autopsy cases at the end of the administration period and recovery period: At the end of the administration period, males in the 100 mg/kg group tended to increase the eosinophilic body of the tubular epithelium, but there was no statistical difference. At the end of the recovery test period, as in the end of the administration period, an increase in eosinophilic body in the tubular epithelium was observed in the 100 mg/kg dose group. There was no difference in the frequency and degree of occurrence. There was no histological change in the male thymus of the 100 mg/kg dose group, which was visually reduced in size, and no similar changes were found in the autopsy cases at the end of the administration period. Therefore, the changes was judged to be accidental.
In the case of 1 male in the 4 mg/kg administration group, degeneration of seminiferous cells and sperm cells was observed in seminiferous tubules, the number of spermatozoa in the epididymal lumen decreased and cell debris was observed, but no changes were seen in the seminal vesicles and prostate. These changes were not dose-dependent and were not observed at highest doses in the previous combined toxicity study, so they were judged to be accidental.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
20 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
Critical effects observed:
not specified

Table 1 - Urinalysis of rats treated orally with o-TSA in 28-days repeat dose toxicity test

Dose (mg/kg)

On day 23 of the administration period

On day 9 of the recovery period

0

4

20

100

0

100

Male

Number of animals

10

5

5

10

5

5

Volume (mL/24 hr) (a)

17.6 ± 8.2

20.6 ± 2.9

17.2 ± 6.3

17.2 ± 3.1

16.0 ± 5.0

16.6 ± 4.8

Specific gravity (a)

1.045 ± 0.015

1.038 ± 0.007

1.037 ± 0.009

1.042 ± 0.011

1.016 ± 0.015

1.052 ± 0.003

pH

6.5

0

0

0

0

1

0

 

7.0

3

1

1

1

2

1

 

7.5

6

3

1

4

2

1

 

8.0

1

0

2

2

0

1

 

8.5

0

1

1

2

0

0

 

≥9.0

0

0

0

1

0

2

Protein (b)

±

3

2

3

4

0

1

 

+

6

3

2

6

4

4

 

0

0

0

0

1

0

Ketone (c)

±

4

4

3

1

1

3

 

+

2

0

1

0

3

1

 

0

0

0

0

1

0

Occult blood (c)

±

0

0

0

0

0

0

 

+

0

0

0

0

0

1

Urobilinogen (d)

±

10

5

5

10

5

5

Microscopic examination of urinary sediment

Crystal (e)

±

9

4

5

10

5

5

 

 

 

 

 

 

 

 

Female

Number of animals

10

5

5

10

5

5

Volume (mL/24 hr) (a)

14.3 ± 4.2

13.0 ± 1.7

11.9 ± 3.4

14.6 ± 3.5

22.4 ± 5.3

14.3 ± 6.6

Specific gravity (a)

1.036 ± 0.013

1.037 ± 0.011

1.025 ± 0.008

1.027 ± 0.012

1.030 ± 0.004

1.014 ± 0.020

pH

6.5

3

1

0

0

1

1

 

7.0

2

2

0

0

1

1

 

7.5

2

2

3

3

3

1

 

8.0

2

0

0

4

0

0

 

8.5

1

0

1

1

0

1

 

≥9.0

0

0

1

2

0

1

Protein (b)

±

2

0

0

0

0

0

 

+

1

0

0

0

0

1

Ketone (c)

±

0

0

0

0

0

0

 

+

0

0

0

0

0

0

Occult blood (c)

±

0

0

0

0

0

0

 

+

0

0

0

0

0

0

Urobilinogen (d)

±

10

5

5

10

5

5

Microscopic examination of urinary sediment

Crystal (e)

±

8

3

5

10

4

4

a)Values represent mean ±S.D. b) ±:trace,+:30 mg/dL, ≠:100 mg/dL, c) ±: trace, +:slight, ±: trace, d)±:0.2 E.U./dL, e) ±: a few

Table 2. Hematological examination of rats treated orally with o-TSA in 28 days repeat dose toxicity test

Dose (mg/kg)

End of the administration period

End of recovery period

0

4

20

100

0

100

Male

Number of animals

5

5

5

5

5

5

 

 

 

 

 

 

 

RBC(x10^4/µL)

705 ± 57

732 ± 20

715 ± 40

716 ± 23

793 ± 32

745 ± 32*

Hemoglobin (g/dL)

14.3 ± 1.0

14.9 ± 0.4

14.2 ± 0.5

14.4 ± 0.4

15.0 ± 0.6

14.7 ± 0.6

Hematocrit (%)

42.7 ± 2.8

44.5 ± 1.3

42.3 ± 1.5

43.0 ± 1.2

44.6 ± 1.9

43.4 ± 2.1

MCV (fL)

60.6 ± 1.8

60.8 ± 0.5

59.2 ± 3.0

60.2 ± 2.2

56.3 ± 1.2

58.3 ± 1.3*

MCH (pg)

20.3 ± 0.4

20.4 ± 0.2

19.9 ± 1.1

20.1 ± 0.7

19.0 ± 0.4

19.7 ± 0.4*

MCHC (g/dL)

33.6 ± 0.3

33.5 ± 0.2

33.5 ± 0.3

33.4 ± 0.2

33.7 ± 0.2

33.8 ± 0.4

Platelet (x10^4/µL)

93.5 ± 11.5

107.5 ± 7.0

96.3 ± 12.1

109.0 ± 12.0

105.3 ± 8.7

99.2 ± 10.8

PT (sec)

16.0 ± 1.9

16.3 ± 2.2

16.2 ± 4.1

15.4 ± 3.6

17.9 ± 3.5

14.0 ± 1.2

APTT (sec)

20.2 ± 1.6

18.9 ± 1.4

19.3 ± 2.9

18.3 ± 4.3

20.2 ± 0.8

19.6 ± 1.1

WBC (x100/µL)

52.5 ± 15.1

67.6 ± 24.0

59.5 ± 16.8

58.9 ± 13.9

73.3 ± 23.1

60.3 ± 20.9

Differential leukocyte counts (%)

 

 

 

 

 

 

Neutrophil

12 ± 4

9 ± 5

8 ± 3

11 ± 3

11 ± 3

14 ± 5

Eosinophil

1 ± 0

1 ± 1

1 ± 0

1 ± 0

1 ± 1

2 ± 1

Basophil

0 ± 0

0 ± 0

1 ± 2

0 ± 0

0 ± 0

0 ± 0

Monocyte

4 ± 2

6 ± 4

8 ± 8

3 ± 1

4 ± 2

4 ± 2

Lymphocyte

83 ± 5

84 ± 6

82 ± 11

84 ± 2

83 ± 4

81 ± 6

 

 

 

 

 

 

 

Female

Number of animals

5

5

5

5

5

5

 

 

 

 

 

 

 

RBC(x10^4/µL)

689 ± 60

705 ± 32

737 ± 70

707 ± 66

726 ± 33

726 ± 40

Hemoglobin (g/dL)

14.2 ± 08

14.2 ± 0.5

14.5 ± 1.2

14.2 ± 1.3

14.2 ± 0.5

14.1 ± 0.4

Hematocrit (%)

41.9 ± 2.7

41.8 ± 1.2

43.1 ± 3.8

42.2 ± 4.0

41.7 ± 1.6

41.5 ± 1.0

MCV (fL)

60.9 ± 1.8

59.4 ± 2.0

58.5 ± 1.3

59.7 ± 1.3

57.5 ± 0.7

57.3 ± 1.9

MCH (pg)

20.6 ± 07

20.1 ± 0.4

19.7 ± 0.6

20.1 ± 0.4

19.5 ± 0.4

19.4 ± 0.6

MCHC (g/dL)

33.9 ± 0.1

33.9 ± 0.6

33.7 ± 0.3

33.8 ± 0.2

33.9 ± 0.4

33.9 ± 0.3

Platelet (x10^4/µL)

102.0 ± 7.5

104.2 ± 8.2

96.1 ± 12.9

98.1 ± 14.3

100.5 ± 10.5

109.7 ± 17.2

PT (sec)

12.1 ± 0.4

12.7 ± 0.7

12.3 ± 0.4

12.6 ± 0.5

11.7 ± 0.2

12.1 ± 0.7

APTT (sec)

15.8 ± 0.6

17.2 ± 0.8

15.3 ± 1.8

16.2 ± 0.9

15.7 ± 2.6

15.0 ± 1.5

WBC (x100/µL)

36.0 ± 2.3

31.2 ± 7.9

38.7 ± 14.7

32.7 ± 9.5

36.9 ± 16.2

27.5 ± 8.3

Differential leukocyte

counts (%)

 

 

 

 

 

 

Neutrophil

10 ± 3

13 ± 7

12 ± 5

10 ± 2

14 ± 5

11 ± 3

Eosinophil

2 ± 1

2 ± 1

1 ± 1

2 ±1

2 ± 1

2 ± 1

Basophil

0 ± 1

0 ± 1

0 ± 0

0 ± 0

0 ± 0

0 ± 0

Monocyte

8 ± 7

9 ± 7

8 ± 10

6 ± 3

4 ± 1

5 ± 2

Lymphocyte

81 ± 9

75 ±13

78 ± 14

82 ± 3

80 ± 6

81 ± 5

Values represent mean ± S.D.

*: Significant difference from 0 mg/kg, p < 0.05

Table 3. Blood chemical examination of rats treated orally with o-TSA in28 days repeat dose toxicity test

Dose (mg/kg)

End of the administration period

End of recovery period

0

4

20

100

0

100

Male

Number of animals

5

5

5

5

5

5

 

 

 

 

 

 

 

Total protein (g/dL)

5.2 ± 0.4

5.3 ± 0.4

5.1 ± 0.2

5.2 ± 0.4

5.4 ± 0.0

5.2 ± 0.3

Albumin (g/dL)

3.2 ± 0.3

3.2 ± 0.2

3.1 ± 0.2

3.1 ± 0.3

3.1 ± 0.1

3.0 ± 0.2

A/G

1.56 ± 0.15

1.60 ± 0.16

1.46 ± 0.14

1.46 ± 0.16

1.38 ± 0.11

1.40 ± 0.13

Glucose (mg/dL)

135 ± 24

118 ± 13

132 ± 19

125 ± 8

148 ± 24

128 ± 18

Total cholesterol (mg/dL)

35 ± 8

42 ± 3

35 ± 3

37 ± 6

36 ± 5

43 ± 7

Triglyceride (mg/dL)

37 ± 9

37 ± 12

31 ± 12

36 ± 11

44 ± 20

42 ± 19

BUN (mg/dL)

16 ± 2

17 ± 3

20 ± 2*

16 ± 2

19 ± 2

21 ± 1*

Creatinine (mg/dL)

0.7 ± 0.1

0.6 ± 0.0

0.6 ± 0.1

0.6 ± 0.1

0.6 ± 0.1

0.6 ± 0.1*

Total bilirubin (mg/dL)

0.10 ± 0.03

0.08 ± 0.02

0.08 ± 0.02

0.07 ± 0.02

0.08 ± 0.03

0.12 ± 0.03

Inorg. phos. (mg/dL)

8.8 ± 2.0

9.4 ± 1.6

9.5 ± 1.5

7.4 ± 0.6

6.6 ± 0.6

6.4 ± 0.4

Ca (mg/dL)

9.0 ± 0.3

9.1 ± 0.4

9.1 ± 0.4

9.3 ± 0.2

8.5 ± 0.2

8.6 ± 0.3

Na (mEq/L)

146.2 ± 1.3

147.5 ± 1.3

146.1 ± 0.6

147.0 ± 0.3

146.1 ± 1.2

145.9 ± 0.8

K (mEq/L)

5.25 ± 1.57

5.00 ± 2.28

4.87 ± 1.53

3.68 ± 0.52

4.02 ± 0.26

3.76 ± 0.29

Cl (mEq/L)

107.1 ± 1.9

107.1 ± 3.1

106.2 ± 1.4

107.4 ± 2.0

107.7 ± 1.1

107.6 ± 2.1

ALP (U/L)

474 ± 95

472 ± 39

464 ± 74

447 ± 85

260 ± 79

295 ± 33

GPT (U/L)

35 ± 11

35 ± 8

31 ± 3

30 ± 5

31 ± 5

27 ± 2

GOT (U/L)

81 ± 26

81 ± 19

76 ± 12

69 ± 8

74 ± 8

63 ± 4*

γ-GPT (U/L)

0 ± 0

1 ± 1

0 ± 1

1 ± 1

0 ± 0

0 ± 0

 

 

 

 

 

 

 

Female

Number of animals

5

5

5

5

5

5

 

 

 

 

 

 

 

Total protein (g/dL)

5.2 ± 0.2

5.1± 0.2

5.2 ± 0.3

5.1 ± 0.5

5.4 ± 0.3

5.0 ± 0.3

Albumin (g/dL)

3.3 ± 0.2

3.1 ± 0.3

3.2 ± 0.2

3.0 ± 0.6

3.4 ± 0.3

3.0 ± 0.2*

A/G

1.80 ± 0.16

1.53 ± 0.23

1.62 ± 0.11

1.51 ± 0.43

1.64 ± 0.18

1.47 ± 0.18

Glucose (mg/dL)

105 ± 18

107 ± 8

105 ± 13

106 ± 16

113 ± 10

120 ± 14

Total cholesterol (mg/dL)

39 ± 8

42 ± 7

46 ± 9

51 ± 4

48 ± 9

36 ± 7*

Triglyceride (mg/dL)

26 ± 10

23 ± 9

23 ± 11

26 ± 10

30 ± 2

21 ± 2**

BUN (mg/dL)

16 ± 1

19 ± 2

20 ± 4

22 ± 3

25 ± 3

21 ± 3

Creatinine (mg/dL)

0.6 ± 0.1

0.6 ± 0.1

0.6 ± 0.3

0.7 ± 0.1

0.6 ± 0.0

0.5 ± 0.1

Total bilirubin (mg/dL)

0.08 ± 0.02

0.11 ± 0.02

0.11 ± 0.02

0.10 ± 0.03

0.12 ± 0.02

0.10 ± 0.03

Inorg. phos. (mg/dL)

8.0 ± 1.9

7.4 ± 0.9

9.0 ± 0.5

7.9 ± 0.3

5.9 ± 0.5

6.4 ± 0.5

Ca (mg/dL)

8.9 ± 0.3

8.8 ± 0.3

9.0 ± 0.4

9.0 ± 0.5

8.7 ± 0.2

8.1 ± 0.5*

Na (mEq/L)

145.4 ± 2.1

146.2 ± 0.9

146.1 ± 1.4

146.1 ± 1.2

145.1 ± 1.0

146.1 ± 1.2

K (mEq/L)

5.61 ± 2.84

4.47 ± 1.39

4.99 ± 2.16

5.02 ± 1.67

4.02 ± 0.39

3.87 ± 0.34

Cl (mEq/L)

108.9 ± 2.1

109.3 ± 0.7

108.9 ± 1.7

109.1 ± 2.9

108.9 ± 1.2

110.2 ± 0.5

ALP (U/L)

315 ± 62

288 ± 62

271 ± 91

322 ± 51

196 ± 48

216 ± 26

GPT (U/L)

25 ± 2

29 ± 7

25 ± 9

28 ± 7

25 ± 4

24 ± 8

GOT (U/L)

69 ± 6

73 ± 9

73 ± 12

68 ± 11

63 ± 8

62 ± 17

GTP (U/L)

1 ± 1

1 ± 1

0 ± 1

0 ± 1

1 ± 0

0 ± 0

Values represent mean ± S.D.

*: Significant difference from 0 mg/kg, p<0.05

**: Significant difference from 0 mg/kg, p<0.01

 

Table 4. Organ weights of rats treated orally with o-toluenesulfonamide in28 days repeat dose toxicity test

Dose (mg/kg)

End of the administration period

End of recovery period

0

4

20

100

0

100

Male

Number of animals

5

5

5

5

5

5

 

 

 

 

 

 

 

Body weight (g)

302.7 ± 16.1

318.8 ± 18.7

3158.8 ± 25.2

318.6 ± 20.5

392.5 ± 32.1

371.2 ± 40.2

Absolute organ weights (mg)

 

 

 

 

 

 

Brain

1873.9 ± 72.7

1859.3 ± 71.0

1795.8 ± 63.8

1873.3 ± 57.6

1906.0 ± 96.1

1926.4 ± 51.1

Pituitary gland

8.9 ± 1.4

9.5 ± 0.8

9.8 ± 0.6

10.2 ± 0.3

11.2 ± 0.7

10.1 ± 2.0

Thymus

595.7 ± 85.7

473.4 ± 64.2

594.3 ± 167.9

532.7 ± 103.1

599.8 ± 104.6

511.2 ± 120.2

Heart

1014.0 ± 75.9

1114.4 ± 89.0

1085.1 ± 98.7

1097.9 ± 85.9

1280.5 ± 135.0

1127.0 ± 93.9

Liver

9662.9 ± 876.8

10330.2 ± 1234.0

9774.7 ± 1273.5

10301.8 ± 1119.2

11803.5 ± 1613.7

11086.0 ± 2337.5

Kidneys

2380.0 ± 221.2

2453.1 ± 164.8

2451.5 ± 161.5

2609.2 ± 160.4

2800.1 ± 148.5

2545.1 ± 336.6

Spleen

661.5 ± 69.6

684.2 ± 52.7

727.4 ± 53.2

687.1 ± 158.3

692.5 ± 110.2

838.0 ± 98.3

Thyroid gland

12.9 ± 2.8

15.2 ± 3.5

13.9 ± 1.6

16.0 ± 5.1

17.5 ± 1.6

18.9 ± 4.6

Adrenal glands

47.8 ± 4.1

43.5 ± 5.8

48.5 ± 6.0

48.3 ± 4.8

49.3 ± 6.6

53.8 ± 8.2

Testes

2797.0 ± 117.9

2615.2 ± 561.3

2776.9 ± 168.5

2841.3 ± 97.2

2960.9 ± 47.7

2936.6 ± 75.2

Epididymides

662.6 ± 55.2

624.8 ± 104.3

680.2 ± 65.6

652.9 ± 16.4

922.5 ± 47.3

894.8 ± 54.1

 

 

 

 

 

 

 

Relative organ weight (mg/g)

 

 

 

 

 

 

Brain

6.212 ± 0.524

5.846 ± 0.374

5.708 ± 0.379

5.891 ± 0.233

4.874 ± 0.343

5.238 ± 0.581

Pituitary gland

0.030 ± 0.004

0.030 ± 0.001

0.031 ± 0.002

0.032 ± 0.003

0.029 ± 0.002

0.027 ± 0.004

Thymus

1.977 ± 0.336

1.484 ± 0.167

1.873 ± 0.445

1.676 ± 0.323

1.546 ± 0.367

1.372 ± 0.274

Heart

3.350 ± 0.184

3.504 ± 0.340

3.434 ± 0.076

3.452 ± 0.265

3.261 ± 0.190

3.014 ± 0.119

Liver

31.922 ± 2.352

32.359 ± 2.766

30.862 ± 1.707

32.271 ± 1.721

29.982 ± 1.931

29.629 ± 3.332

Kidneys

7.870 ± 0.688

7.694 ± 0.228

7.780 ± 0.473

8.198 ± 0.371

7.152 ± 0.348

6.858 ± 0.556

Spleen

2.185 ± 0.193

2.153 ± 0.218

2.313 ± 0.226

2.159 ± 0.492

1.773 ± 0.305

2.263 ± 0.192*

Thyroid gland

0.043 ± 0.008

0.048 ± 0.011

0.044 ± 0.006

0.050 ± 0.013

0.045 ± 0.006

0.050 ± 0.007

Adrenal glands

0.158 ± 0.007

0.136 ± 0.013

0.155 ± 0.023

0.152 ± 0.014

0.125 ± 0.012

0.147 ± 0.030

Testes

9.256 ± 0.516

8.172 ± 1.594

8.807 ± 0.314

8.958 ± 0.785

7.589 ± 0.691

7.981 ± 0.806

Epididymides

2.190 ± 0.152

1.957 ± 0.297

2.152 ± 0.054

2.054 ± 0.107

2.369 ± 0.300

2.432 ± 0.291

Female

Number of animals

5

5

5

5

5

5

 

 

 

 

 

 

 

Body weight (g)

200.8 ± 11.1

201.7 ± 14.8

186.7 ± 21.8

193.2 ± 15.0

233.6 ± 20.6

208.3 ± 14.8

Absolute organ weights (mg)

 

 

 

 

 

 

Brain

1738.9 ± 47.0

1743.3 ± 87.4

1717.2 ± 89.6

1704.0 ± 71.0

1787.6 ± 48.9

1774.3 ± 22.1

Pituitary gland

12.5 ± 1.9

12.1 ± 0.5

12.1 ± 1.2

11.8 ± 1.6

13.7 ± 1.9

13.1 ± 1.1

Thymus

495.9 ± 103.7

481.9 ± 82.6

439.7 ± 106.6

404.8 ± 107.4

457.1 ± 94.2

395.8 ± 79.7

Heart

736.4 ± 77.2

746.3 ± 53.1

673.9 ± 68.4

690.0 ± 58.8

794.1 ± 60.0

765.0 ± 50.2

Liver

6600.5 ± 950.1

6268.1 ± 297.0

6073.5 ± 830.0

6407.4 ± 394.0

6723.6 ± 719.0

6093.4 ± 621.2

Kidneys

1591.9 ± 102.0

1710.1 ± 185.5

1619.1 ± 128.9

1729.6 ± 193.7

1768.3 ± 185.5

1673.3 ± 88.4

Spleen

514.5 ± 68.9

499.6 ± 110.9

450.6 ± 61.1

522.1 ± 42.7

765.3 ± 404.8

426.5 ± 54.0

Thyroid gland

14.7 ± 2.7

12.4 ± 2.2

12.4 ± 1.1

12.2 ± 3.2

18.6 ± 3.1

18.4 ± 5.8

Adrenal glands

63.8 ± 1.7

61.6 ± 7.2

60.6 ± 3.3

60.9 ± 7.4

62.7 ± 12.7

61.8 ± 10.4

Ovaries

86.7 ± 11.0

84.0 ± 6.8

78.1 ± 9.2

80.4 ± 13.6

81.4 ± 22.4

81.3 ± 16.2

 

 

 

 

 

 

 

Relative organ weight (mg/g)

 

 

 

 

 

 

Brain

8.680 ± 0.545

8.670 ± 0.614

9.275 ± 0.913

8.851 ± 0.554

7.701 ± 0.736

8.548 ± 0.510

Pituitary gland

0.062 ± 0.006

0.060 ± 0.004

0.065 ± 0.009

0.061 ± 0.007

0.059 ± 0.007

0.063 ± 0.003

Thymus

2.466 ± 0.457

2.382 ± 0.305

2.332 ± 0.329

2.075 ± 0.407

1.960 ± 0.397

1.901 ± 0.377

Heart

3.667 ± 0.322

3.705 ± 0.225

3.627 ± 0.336

3.574 ± 0.181

3.409 ± 0.271

3.680 ± 0.236

Liver

32.909 ± 4.835

31.132 ± 1.225

32.554 ± 2.995

33.280 ± 2.662

28.759 ± 1.342

29.218 ± 1.290

Kidneys

7.929 ± 0.307

8.472 ± 0.611

8.723 ± 0.732

8.975 ± 1.036

7.574 ± 0.529

8.015 ± 0.251

Spleen

2.568 ± 0.373

2.464 ± 0.444

2.410 ± 0.085

2.716 ± 0.300

3.253 ± 1.598

2.017 ± 0.201

Thyroid gland

0.074 ± 0.016

0.062 ± 0.012

0.067 ± 0.008

0.063 ± 0.014

0.080 ± 0.014

0.087 ± 0.022

Adrenal glands

0.319 ± 0.025

0.306 ± 0.033

0.327 ± 0.036

0.315 ± 0.021

0.269 ± 0.050

0.297 ± 0.050

Ovaries

0.434 ± 0.071

0.419 ± 0.051

0.421 ± 0.057

0.420 ± 0.087

0.345 ± 0.074

0.389 ± 0.060

Values represent mean ± S.D., *: significant difference from 0 mg/kg. p<0.05

Table 5. Histopathological findings of rats treated orally with o-TSA in 28 days repeat dose toxicity test

Sex

Male

Female

 

End of the

administration period

End of the

recovery period

End of the

administration period

End of the

recovery period

Dose (mg/kg)

0

4

20

100

0

100

0

4

20

100

0

100

Number of animals examined

(Kidney)

 

5

5

5

5

5

5

5

0

0

5

0

0

Eosinophilic body

±

1

1

1

0

2

1

0

 

 

0

 

 

 

+

0

0

0

3

0

3

0

 

 

0

 

 

 

0

0

0

1

1

0

0

 

 

0

 

 

 

Total

1

1

1

4

3

4

0

 

 

0

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Basophilic tubule,

±

3

1

3

4

1

3

4

 

 

3

 

 

cortex

Total

3

1

3

4

1

3

4

 

 

3

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Mineralization

±

0

0

0

0

0

0

3

 

 

3

 

 

 

+

0

0

0

0

0

0

0

 

 

1

 

 

 

Total

0

0

0

0

0

0

3

 

 

4

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Fibrosis, subcapsule,

±

0

0

0

0

0

0

1

 

 

0

 

 

focal

Total

0

0

0

0

0

0

1

 

 

0

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Cyst, cortico-medullary

±

0

0

0

0

0

1

0

 

 

0

 

 

junction

Total

0

0

0

0

0

1

0

 

 

0

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Cast, hyalin, cortex

±

0

0

0

0

0

1

0

 

 

0

 

 

 

Total

0

0

0

0

0

1

0

 

 

0

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Fibrosis, subcapsule, focal

±

0

0

0

0

1

0

0

 

 

0

 

 

 

Total

0

0

0

0

1

0

0

 

 

0

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Number of animals examined

(Spleen)

 

5

5

5

5

5

5

5

5

5

5

5

5

Haematopoiesis

±

3

2

2

1

2

3

5

5

5

3

4

5

extramedullary

+

2

3

3

4

3

2

0

0

0

2

1

0

 

Total

5

5

5

5

5

5

5

5

5

5

5

5

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Deposit, pigment,

±

0

0

0

0

4

4

0

0

0

0

5

5

brown

Total

0

0

0

0

4

4

0

0

0

0

5

5

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Number of animals examined

(Liver)

 

5

0

0

5

0

0

5

0

0

5

0

0

Fatty change, periportal

±

4

 

 

3

 

 

2

 

 

4

 

 

 

+

1

 

 

1

 

 

3

 

 

1

 

 

 

Total

5

 

 

4

 

 

5

 

 

5

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Vacuolization, cytoplasmic,

±

1

 

 

0

 

 

0

 

 

0

 

 

hepatocyte, centrilobular

+

1

 

 

0

 

 

1

 

 

0

 

 

 

Total

2

 

 

0

 

 

1

 

 

0

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Microgranuloma

±

0

 

 

0

 

 

1

 

 

1

 

 

 

Total

0

 

 

0

 

 

1

 

 

1

 

 

Grade of histopathological finding: ±: very slight, +: slight, ≠: moderate, ≠≠: severe, total: total of positive grade

Conclusions:
The NOAEL of the test item in male and female rats was determined to be 20 mg/kg bw/day.
Executive summary:

A 28-day repeated dose toxicity test was performed according to the Guidelines on Chemical Substitution Law (1986) (Guideline 28-Day Repeat Dose Toxicity Test of Chemical, Japan) and OECD 407, under GLP conditions.Based on the results of the repeated oral dose toxicity and reproductive developmental toxicity combined trial conducted previously 100 mg/kg was taken as the highest dose (as a clear change in toxicity had been observed), and divided by 5 to obtain the medium and low doses. Sprague-Dawley rats (Crj:CD (SD) IGS, SPF) were administered 0 (control), 4, 20 or 100 mg/kg bw/day test item in 0.5% CMC Na aqueous solution by gavage for 28 days and observed for further 14 days of recovery period. 10 animals per sex per dose were selected for the control and highest dose group, and 5 for the other groups. Observations included general condition, clinical signs, body weights, food consumption, haematology, clinical chemistry, urianalysis, necropsy and histopathology. No mortality was observed throughout the study. Animals of both sexes in the 100 mg/kg group showed salivation and decreased activity; some other effects observed like inhibition of body weight gain, and decreased food consumption were deemed non-treatment related. In the histopathological examination, there was a tendency of increased eosinophilic body of the tubular epithelium in males in the 100 mg/kg group, in autopsy cases at the end of the administration period and after the recovery period. No significant treatment-related effects were observed in the other groups related to control. Based on the above results, the NOAEL under test conditions was determined to be 20 mg/kg/bw/day in both sexes.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
20 mg/kg bw/day
Study duration:
subacute
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Based on the available data (NOEL = 20 mg/kg bw/d in rats), the test item is not classified for repeated dose toxicity according to CLP, Regulation (EC) No. 1272/2008.