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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
Study period:
07/03/2000 - 20/04/2000
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Cross-referenceopen allclose all
Reason / purpose:
reference to other assay used for intermediate effect derivation
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1998
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
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: Charles River Co., Ltd. Atsugi Rearing Center, Japan
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 8 weeks old
- Weight at study initiation: Males 333.6 g (± 8 g); Females 216.3 g (± 6 g)
- Housing: individually housed in metal cages (220 × 270 × 190 (m)), 2 animals/cage at the time of mating. Maternal animals after 14 days of pregnancy (sperm confirmation = pregnancy day 0) were housed in a breeding cage for rats (350 X 400 X 180 ffl), and pulp and paper chips (ALPHA-dri, Kado) were appropriately supplied as bedding.
- Diet: solid feed (CE-2, CLEA Japan, Ltd.), ad libitum
- Water: tap water (Hadano City Waterworks Bureau water supply), ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24 ± 1 (meas. 23.5-25.8)°C
- Humidity (%): 50 - 65 (meas. 52.0-66.5) %
- Air changes (per hr): 15 times/hour
- Photoperiod (hrs dark / hrs light): 12 hours (7 am to 7 pm) light / 12 hours dark

IN-LIFE DATES: Purchase date: January 14, 1998. Start of administration: January 21, 1998
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Test substance was administrated dissolved in an 0.5%(w/v) CMC solution; the preparation was stored under refrigeration and administered within 7 days after preparation.

VEHICLE
- Amount of vehicle (if gavage): 5 mL/kg
- Lot/batch no.: 6Z09 (JPA water for injection: Hikari Pharmaceutical, Lot. no.: 9707SA)
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:
Males: for 42 days; Females: from 14 days before mating to day 3 of lactation.
Frequency of treatment:
Daily (once in the morning, 9 am to 12 pm)
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
control (0.5% w/v CMC aqueous solution)
Dose / conc.:
20 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
No. of animals per sex per dose:
13 animals per sex per dose.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on the results of a preliminary toxicity test (A-97-029), at doses of 0 (0.5% CMC-Na, 5 mL/kg), 125, 250 and 500 mg/kg TSA. Since clear toxicity effects were observed at 500 mg/kg, this was selected as top dose. Using a factor of 5, the next doses were set at 100, 20 and
4 mg/kg bw/d.
- Rationale for animal assignment: random
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: once a week during the test period (male: administration 1, 8, 15, 22, 29, 36, 42 days, female: administration 1, 8, 15 days) and at necropsy day. Also, for mated females, body weights were measured on 0, 7, 14 and 20 days of pregnancy, and for females who gave birth, 0 and 4 days of lactation.

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: on the 42nd day of dosing
- Anaesthetic used for blood collection: Yes (pentobarbital)
- Animals fasted: Yes (18-24h)
- How many animals: all
- Parameters: Red blood cell count (RBC), White blood cell count (WBC), Hemoglobin level (Hb), Average red blood cell volume (MCV), Platelet count, Hematocrit (Ht), Mean Corpuscular Hemoglobin (MCH), Mean Corpuscular Hemoglobin concentration (MCHC), Prothrombin time (PT), Activated partial thromboplastin time (APTT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after blood collection for hematological examination, blood was collected using heparin as an anticoagulant, and blood plasma was separated.
- Animals fasted: Yes (18-24h)
- How many animals: all males
- Parameters: Total protein concentration; Albumin concentration; Total cholesterol concentration; triglyceride concentration; glucose concentration; urea nitrogen concentration (BUN); creatinine concentration; alkaline phosphatase activity (ALP); GOT activity; GPT activity; total bilirubin concentration; calcium concentration; Phosphorus concentration (inorg. Phos.); γ-GTP activity; A / G ratio.

URINALYSIS: Yes
- Time schedule for collection of urine: for males, on the 42nd day of dosing
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters: pH, occult blood, protein, sugar, ketone body, urobilinogen, bilirubin, sediment, color tone, turbidity, urine volume and specific gravity.

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 (all animals)
- Sacrifice, Male animals: All surviving animals as soon as possible after the last litters in each generation were produced.
- Sacrifice, Maternal animals: All surviving animals, after the last litter of each generation was weaned (day 4). Females whose copulation was confirmed but not delivered were sacrificed on the 25th day of gestation.
- Following blood collection, the axillary artery was cut and exsanguinated and sacrificed as needed, and organs and tissues were observed macroscopically. Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS: Yes
- In all males, the following tissues were prepared for microscopic examination and weighed, respectively: brain, heart, thymus, liver, kidney, spleen, adrenal gland, testis and epididymis; and the specific weight value (relative weight) was calculated. Among the fixed organs, liver, kidney, spleen, heart, bladder and thymus were examined in all treatment groups, and other organs were only observed in the control group and high dose groups.
- In females, histopathological examination was performed on the ovaries of infertile cases. In addition , brain, heart, thymus, liver, kidney, spleen, and adrenal gland weights were measured in all animals. Among the fixed organs, liver, kidney, spleen, heart, bladder and thymus were examined in all treatment groups, and other organs were only observed in the control group and high dose groups.
Statistics:
The copulation rate and the conception rate were subjected to χ2 test including Yates' correction. Histopathological findings were tested using Mann-Whitney's U test and for positive total scores, Fisher's exact probability one-sided test was used. Statistical analysis was not performed for general condition, autopsy findings and urinalysis data. The other data were tested for uniformity of each group's variance by Bartlett method, using the values obtained for each individual or the average value for each litter as one sample. Based on the results, one-way analysis of variance or Kruskal-Wallis rank test was performed, and when there is significance between the groups, either the Dunnett method or Scheffe's method is used to determine the difference between the control group and each test substance administration group. The significance level was 5% and 1%.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Decreased locomotor activity and appearance of prone position and salivation were observed in both sexes at 100 and 500 mg/kg bw.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Three (3/13) females died and two females were sacrificed in moribund condition during the pre-mating period at 500 mg/kg bw. No males died in any of the groups.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
A decrease in body weight gain was observed at the start of the study; low body weights were recorded in both sexes at 100 and 500 mg/kg bw.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Diminished food consumption was observed at the beginning of the study.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
In the hematological examination performed at necropsy, the average erythrocyte pigment amount increased in all TSA-administered groups.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Alkaline phosphatase activity decreased in all TSA administration groups, and total cholesterol concentration increased in the 100 mg/kg or more administration group. Also, in the 500 mg/kg administration group, the total protein concentration increased, and the A/G ratio, glucose concentration and triglyceride concentration decreased.
Urinalysis findings:
no effects observed
Description (incidence and severity):
Urinalysis conducted after 42 doses showed no abnormalities attributable to TSA administration.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
In the dose groups receiving 100 mg/kg test item or more, the specific body weights of the liver and kidney increased, and the heart, adrenal and testis weight ratios value also increased.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Darkening or swelling was observed in the liver and kidney in the 100 mg/kg or more administration groups.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Hypertrophy of the centrilobular hepatocytes with the cytoplasm having a ground glass appearance was observed in both sexes at 100 and 500 mg/kg bw in a dose-dependent manner. In addition, the incidence of fibrosis and cellular infiltration of the pericardium, and fibrosis and cellular infiltration of the capsule and atrophy of the thymus were significantly increased in females at 500 mg/kg bw. In the kidneys, eosinophilic body was observed in males of all treated groups, maybe due to the complex accumulation of this chemical with the male rat specific protein, alpha-2u-globulin.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Details on results:
- In the 20 mg/kg administration group, no effect of TSA administration was observed in any of the examinations.
Key result
Dose descriptor:
NOEL
Effect level:
20 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
histopathology: non-neoplastic
Critical effects observed:
not specified

Table 1. Body weights of male rats treated orally with o-TSA in the combined repeat dose and reproductive/developmental toxicity screening test.

Dose (mg/kg)

 

0

 

20

 

100

 

500

 

Days of administration

 

 

 

 

 

 

 

 

 

1            (Init, wt.)

333.1± 7.4

(13)

333.7 ± 82

(13)

333.7 ± 83

(13)

3317 ± 7.8

(13)

8

370.5

± 13.3

(13)

366.1 ± 117

(13)

351.2 ± 1.13"

(13)

348.9 ± 14.8**

(13)

15

405.2

± 19.0

(13)

402.3 ± 16.6

(13)

383.6 ± 183*

(13)

350.2 ± 21.0**

(13)

22

430.5

± 22.1

(13)

429.4 ± 1913

(13)

403.8 ± 22.0*

(13)

405.6 ± 216*

(13)

29

456.0

± 29.7

(13)

451.9 ± 214

(13)

435.1 ± 27.7

(13)

426.9 ± 25.2*

(13)

36

483.8

± 3.0 (13)

478.1 ± 24.0

(13)

460.1 ± 28.8

(13)

449.4 ± 26.3*

(13)

42

503.4

± 35.9 (13)

497.1 ± 25.8

(13)

479.6 ± 32.4

(13)

467.4 ± 26.9*

(13)

Values are expressed as mean±8 D. in grams.

 

 

 

 

 

 

 

 

 

Parenthesis indicates number of animals.

 

 

 

 

 

 

 

 

 

* :significant difference from control, p<0.05

 

 

 

 

 

 

 

 

 

**:significant difference from control, p<0.01

 

 

 

 

 

 

 

 

 

 

Table 2. Food consumption of male rats treated orally with o-TSA.

Dose (mg/kg)

 

0

 

20

 

 

10]

 

 

500

 

Days of administration

 

 

 

 

 

 

 

 

 

 

 

1 - 8

1990

± 17.8

(13)

194.3 ± 114

(13)

179.4

± 17,2**

(13)

166.5

± 12.0**

(13)

8 -15

196.1

± 18.0

(13)

1993 ± 12.6

(13)

181.6

± 14.6*

(13)

187.2

± 14.1

(13)

29 - 36

207.9

± 22.8

(13)

209.5 ± 13.7

(13)

204.0

± 15.8

(13)

201.5

± 14.5

(13)

36 - 42

181.8

± 21.4 (13)

182.9 ± 11.9

(13)

176.4

± 18.7

(13)

174.0

± 12.1

(13)

 

Table 3. Body weights of female rats treated orally wit h o-TSA.

 

Dose(mg/kg)

 

0

 

20

 

 

100

 

 

500

 

Days of administration

 

 

 

 

 

 

 

 

 

 

 

(Pre-mating period)

 

 

 

 

 

 

 

 

 

 

 

1            (Init. wt.)

216.3

± 6.2

(13)

216.4 ± 5.6

(13)

216.3

± 5.8

(13)

216.1

± 6.3  (13)

 

8

232.9

± 7.9

(13)

232.2 ± 7.7

(13)

226.5

± 9.1

(13)

215.4

± 17.1*(8)

15

251.3

± 15.6

(13)

250.9 ± 12.9

(13)

241.9

± 8.1

(13)

234.3

± 17.2   (8)

Days of pregnancy

 

 

 

 

 

 

 

 

 

 

 

0

255.2

± 15.9

(10)

255.6 ± 13.1

(13)

246.5

± 9.3

(12)

237.2

± 16.3   (7)

7

303.6

± 19.4

(10)

3006 ± 12.5

(13)

282.2

± 10.3*

(12)

278.4

± 23.5*(7)

14

341.1

± 25.3

(10)

340.9 ± 18.2

(13)

319.5

± 12.1

(12)

315.5

± 26.2   (7)

20

414.5

± 35.2

(10)

4099 ± 35.3

(13)

389.1

± 18.3

(32)

375.9

± 40.0   (7)

Days of lactation

 

 

 

 

 

 

 

 

 

 

 

0

313.1

±18.7

(10)

303.7 ± 21.3

(12)

296.2

±23.2

(12)

281.9

± 23.7   (7)

4

344.1

± 19.7

(10)

337.0 ± 196

(12)

317.6

± 24.8*

(12)

299.8

± 17.0** (7)

 

Table 4. Food consumption of female rats treated orally with o-TSA.

Dose (mg/kg)

 

0

 

20

 

 

100

 

 

500

 

Days of administration

 

 

 

 

 

 

 

 

 

 

 

(Pre-mating period}

 

 

 

 

 

 

 

 

 

 

 

1 - 8

136.9

± 11.5

(13)

137.3 ± 8.9

(13)

123.6

± 12.2

(13)

93.9

± 20.3**

(8)

8 - 15

146.1

± 13.6

(13)

151.3 ± 10.8

(13)

136.5

± 8.8

(13)

127.8

± 23.6 

(8)

Days of pregnancy

 

 

 

 

 

 

 

 

 

 

 

0 - 7

182.4

± 18.8

(10)

182.6 ± 11.0

(13)

162.2

± 10.9

(12)

148.2

± 24.9*

(7)

7 - 14

197.4

± 25.5

(10)

201.2 ± 16.1

(13)

181.3

± 13.8

(12)

178.1

± 20.6

(7)

11- 20

147.6

± 16.0

(10)

149.7 ± 98

(13)

140.3

± 13.2

(12)

130.5

± 20.3  

(7)

Days of lactation

 

 

 

 

 

 

 

 

 

 

 

0 - 4 

143.3

± 16.9

(10)

148.5 ±256

(12)

136.0 ± 19.4

(12)

117.6

± 14.0

(7)

Table 5. Hematological findings of male rats treated orally with o-TSA.

Dose (mg/kg)

0

20

100

330

Male Red blood cells

Count (x 10^4/mm3)

835 ± 29 (13)

830 ± 36

(13)

820 ± 3i

(13)

822 ± 37

(13)

Hemoglobin (g/d L)

14.9 ± 1.3 (13)

15.5 ± 0.6

(13)

1515 ± 015

(13)

155 ± 015

(13)

Hematocrit (%)

45.1 ± 3.9 (13)

4 6.5 ± 19

(13)

458 ± 1.6

(13)

45 1 ± 1.7

(13)

MCV (µm2)

54.3 ± 4.1 (13)

56.2 ± 16

(13)

550 ± 1.3

(13)

31 .9 ± 1.3

(13)

MCH (p g)

17.9 ± 1.4 (13)

18.7 ± 0.4*

(13)

189 ± 04**

(13)

189 ± 04**

(13)

MCHC (%)

32.9 ± 0.5 (13)

333 ± 0.6

(13)

33.9 ± 07**

(13)

34/1 ± 05**

(13)

White blood cells

Count (x 10^4/mm3)

69 ± 20 (13)

37 ± 11

(13)

8i ± I8

(13)

68 ± 15

(13)

Band neutrophil (%)

0 ± 0 (13)

0 ± 0

(13)

0 ± 0

(13)

0 ± 0

(13)

Se gmented neutrophil (%)

12 ± 8 (13)

15 ± 7

(13)

16 ± 7

(13)

15 ± 7

(13)

Eosinophil (%)

0 ± 1 (13)

1 ± 1

(13)

0 ± 1

(13)

1 ± 1

(13)

Basophil (%)

0 ± 0 (13)

0 ± 0

(13)

0 ± 0

(13)

0 ± 0

(13)

Monocyte (%)

1 ± 1 (13)

1 ± 1

(13)

1 ± 1

(13)

1 ± 1

(13)

Lymphocyte (%)

87 ± 9 (13)

83 ± 8

(13)

83 ± 7

(13)

83 ± 7

(13)

Platelet

Count (x 10^4/mm3)

96.5 ± 78 (13)

97.0 ± 7.9

(13)

9016 ± 59

(13)

101.7 ± 6.3*

(13)

 PT (sec)

29.9 ± 8.8 (13)

31.1 ± 7.2

(13)

22.7 ± 52*

(13)

21. 6 ± 53*

(13)

APTT (sec)

26.9 ± 2.5 (13)

280 ± 3.1

(13)

21.2 ± 3.3

(13)

21 .5 ± 2.3

(13)

Table 6. Blood chemical findings of male rats treated orally with o-TSA.

Dose (mg/kg)

0

 

20

100

 

330

Total protein (g/dL)

54 ± 0.2

(13)

53 ± 0.3

(13)

5.4 ± 0.2

(13)

5.6

 ± 0.2*

(13)

Albumin (g/dL)

3.0 ± 0.2

(13)

2.9 ± 0.2

(13)

3.0 ± 0.2

(13)

3.0

 ± 01

(13)

A/G

1.29 ± 0.14

(13)

1.19 ± 0.13

(13)

1.26 ± 0.13

(13)

1.15

 ± 0.10*

(13)

 

BUN (mg/dL)

17 ± 2

(13)

18 ± 4

(13)

19 ± 3

(13)

20

 ± 2

(13)

Creatinine (mg/d L)

0.7 ± 0.1

(13)

0.6 ± 0.0

(13)

0.7 ± 0.1

(13)

0.6

 ± 0.1

(13)

Glucose (mg/ dL)

144 ± 10

(13)

142 ± 13

(13)

135 ± 11

(13)

123

 ± 14**

(13)

Total cholesterol (mg/dL)

37 ± 6

(13)

39 ± 3

(13)

49 ± 13*

(13)

66

 ± 15**

(13)

Triglyceride (mg/dL)

45 ± 15

(13)

35 ± 14

(13)

39 ± 13

(13)

27

 ± 11**

(13)

Total bilirubin(mg/dL)

 0.08 ± 0.03

(13)

0.08 ± 0.01

(13)

0.09 ± 0.02

(13)

010

 ± 002

(13)

Na (mEq/L)

145.1 ± 0.8

(13)

145.1 ± 0.7

(13)

145.1 ± 0.9

(13)

145.0

 ± 1.0

(13)

K (mEg/L)

4.14 ± 0.15

(13)

4.13 ± 0.26

(13)

4.04 ± 0.16

(13)

3.99

 ± 021

(13)

Cl (mEq/L)

108.4 ± 0.8

(13)

108.9 ± 1.1

(13)

108.6 ± 10

(13)

107.5

 ± 1.1

(13)

Ca (mg/dL)

87 ± 0.2

(13)

85 ± 0.1

(13)

88 ± 0.1

(13)

8.9

 ± 03

(13)

Inorg. phos.(mg/dL)

6.5 ± 0.5

(13)

6.3 ± 0.4

(13)

6.0 ± 06

(13)

5.9

 ± 03

(13)

ALP (U/L)

261 ± 35

(13)

218 ± 40**

(13)

216 ± 34**

(13)

199

 ± 31**

(13)

GPT (U/L)

32 ± 7

(13)

32 ± 7

(13)

28 ± 3

(13)

38

 ± 15

(13)

GOT (U/L)

69 ± 14

(13)

68 ± 10

(13)

63 ± 7

(13)

71

 ± 23

(13)

y-GTP (U/L)

0 ± 0

(13)

0 ± 0

(13)

0 ± 0

(13)

0

 ± 1*

(13)

Table 7. Absolute and relative organ weights of treated rats

Dose (mg/kg)

 

0

20

100

300

MALE

 

 

 

 

Terminal body weight

(g)

469.6 ± 33.8 (13)

464.0 ± 24.8 (13)

443.9 ± 30.7

(13)

432.7 ± 26.4**

(13)

 

 

 

 

 

 

 

 

Brain

(g)

2.04 ± 0.06*(13)

2.01 ± 0.05(13)

2.00 ± 0.08

(13)

1.98 ± 0.09

(13)

 

 

0.44 ± 0.02

0.44 ± 0.03

0.13 ± 0.03

 

0.46 ± 0.03

 

Heart

(g)

1.33 ± 013 (13)

13.5 ± 0.13(13)

1.33 ± 0.09

(13)

1.31 ± 0.12

(13)

 

 

0.28 ± 0.02

0.29 ± 0.02

0.31 ± 0.02

 

030 ± 0.02*

 

Liver

(g)

12.27 ± 1.49 (13)

12.30 ± 1.15(13)

12.10 ± 125

(13)

14.37 ± 1.18**

(13)

 

 

2.61 ± 0.20

2.65 ± 0.14

2.79 ± 0.16

 

3.32 ± 0.17**

 

Kidneys

(g)

3.15 ± 0.21 (13)

3.24 ± 0.18 (13)

3.27 ± 0.35

(13)

3.51 ± 0.23**

(13)

 

 

0.67 ± 0.05

0.70 ± 0.03

0.74 ± 007**

 

082 ± 0.06**

 

Spleen

(g)

0.75 ± 0.09 (13)

0.79 ± 0.11 (3)

0.76 ± 010

(13)

0.75 ± 6.12

(13)

 

 

0.16 ± 0.02

0.17 ± 0.02

0.17 ± 0.02

 

0.11 ± 0.03

 

Thymus

(mg)

351.3 ± 86.3 (13)

319.8 ± 86.5(13)

3113 ± £2.5

(13)

278.6 ± 56.9

(13)

 

 

74.9 ± 18.3

68.9 ± 17.8

707 ± 20.1

 

64.2 ± 11.8

 

Adrenal glands

(mg)

54.3 ± 01 (13)

58.5 ± 11.1(13)

59.4 ± 8.7

(13)

62.2 ± 4.8

(13)

 

 

11.6 ± 1.8

12.6 ± 2.0

13.4 ± 16*

 

14.4 ± 1.1**

 

Testes

(g)

3.03 ± 073 (13)

3.40 ± 0.20(13}

3.36 ± 0.26

(13)

3.35 ± 0.21

(13)

 

 

0.65 ± 0.16

0.74 ± 0.06

0.76 ± 0.68*

 

0.78 ± 0.05**

 

Epididymides

(g)

1.09 ± 0.21 (13)

1.22 ± 0.09(13)

1.17 ± 0.09

(13)

1.04 ± 0.09

(13)

 

 

0.23 ± 0.05

0.26 ± 0.02

0.26 ± 0.66*

 

0.25 ± 0.03

 

FEMALE

 

 

 

 

 

 

 

Terminal body weight

(g)

344.1 ± 19.7

(10)

337.0 ± 19.6 (12)

317.6 ± 21.8*

(12)

299.8 ± 17.0**

(12)

 

 

 

 

 

 

 

 

 

Brain

(g)

1.94 ± 0.08

(10)

1.84 ± 0.07 (12)

1.37 ± 0.07

(12)

185 ± 0.10

(12)

 

 

0.56 ± 0.03

 

0.553 ± 0.03

0.39 ± 0.05

 

0.62 ± 0.05*

 

Heart

(g)

0.98 ± 0.07

(10)

0.95 ± 0.07 (12)

0.97 ± 010

(12)

0.93 ± 0.12

(12)

 

 

0.28 ± 0.02

 

0.28 ± 0.02

0.31 ± 0.02

 

0.31 ± 0.04

 

Liver

(g)

13.57 ± 0.99

(10)

13.96 ± 139 (12)

14.20 ± 1.71

(12)

15.12 ± 1.54

(12)

 

 

3.95 ± 0.30

 

4.11 ± 0.31

1.16 ± 0.31**

 

5.05 ± 0.40**

 

Kidneys

(g)

2.10 ± 0.20

(10)

2.08 ± 0.23 (12)

2.19 ± 0.15

(12)

2.19 ± 0.28

(12)

 

 

0.61 ± 0.06

 

0.62 ± 0.05

0.70 ± 0.10

 

0.73 ± 0.08*

 

Spleen

(g)

0.70 ± 0.15

(10)

0.66 ± 0.06 (12)

0.70 ± 0.13

(12)

0.65 ± 0.10

(12)

 

 

0.20 ± 0.04

 

0.20 ± 0.01

0.22 ± 0.03

 

0.21 ± 0.04

 

Thymus

(mg)

276.4 ± 51.0

(10)

219.3 ± 60.5 (12)

217.0 ± 97.4

(12)

188.8 ± 68.5

(12)

 

 

80.3 ± 13.7

 

65.3 ± 183

67.8 ± 28.6

 

62.1 ± 21.3

 

Adrenal glands

(mg)

74.6 ± 10.8

(10)

796.5 ± 9.3 (12)

75.2 ± 8.0

(12)

85.5 ± 16.4

(12)

 

 

21.7 ± 81

 

23.7 ± 3.5

23.8 ± 3.0

 

28.6 ± 57**

 

Values are expressed as mean ± SD. Parenthesis indicates number of animals.

a: ab solute weight; b: relative weight (g or mg per 100 g body weight)

*: significant difference from control. p<0.05; **:significant difference from control. p<0.01.

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

A Combined Repeated Dose Toxicity with Reproduction / Developmental Toxicity Screening Test was conducted on the test item, according to OECD Guideline 422, under GLP conditions. Based on the results of a preliminary acute oral toxicity test, 13 male and 13 female Sprague Dawley rats (Crj:CD (SD) IGS, SPF) were administered 0 (control), 20, 100 or 500 mg/kg bw/day test item in 0.5% CMC Na aqueous solution by gavage from 14 days before mating to 14 days after mating in males and from 14 days before mating to day 3 of lactation in females. Observations included general condition, clinical signs, body weights, food intake, haematology, clinical chemistry, urianalysis, evaluation of reproduction, necropsy, organ weights and histopathology. Three females died and two females were sacrificed in moribund condition during the pre-mating period at 500 mg/kg bw. Clinical signs such as decrease in locomotor activity and appearance of prone position and salivation, and significant lowering of body weights were observed in both sexes at 100 and 500 mg/kg bw. In the 500 mg/kg bw group, the delivery index, number of pups alive on day 0 of lactation and birth index were slightly decreased but not statistically significant. There was no effect of the test compound on the ovulation, copulation, fertility, delivery and lactation at dose levels up to 100 mg/kg bw. Significant reduction in body weights of pups was observed on days 0 and 4 in both sexes at 500 mg/kg bw. 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 considered to be 20 mg/kg/bw/day in rats (parental animals, both sexes).

Reason / purpose:
reference to other assay used for intermediate effect derivation
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1998
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Limit test:
no
Justification for study design:
SPECIFICATION OF STUDY DESIGN WITH JUSTIFICATIONS
- Premating exposure duration for parental (P0) animals: 2 weeks
- Basis for dose level selection: the dose of this study was set based on the results of a single-dose oral toxicity test of o-TSA in rats performed at Tano Research Center (study number: A-97-029: TSA at doses of 0 (0.5% CMC-Na, 5 mL/kg), 125, 250 and 500 mg/kg was administrated continuously to 5 8-week old rats per group for 14 days. In this study, both male and female rats showed clear toxicity effects, such as weight gain and food consumption suppression, occult blood positive reaction in urinalysis, increased liver and kidney weight. Based on these results, 500 mg/kg was set as the high dose. Using a factor of 5, the next doses were set at 100, 20 and 4 mg/kg bw/d.
- Route of administration: oral, gavage
Species:
rat
Strain:
Sprague-Dawley
Remarks:
(Crj: CD (IGS), SPF)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Atsugi Rearing Center, Japan
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (P) 8 wks; (F1) x wks
- Weight at study initiation: (P) Males:Males 333.6 g (± 8 g); Females 216.3 g (± 6 g)
- Housing: individually housed in metal cages (220 × 270 × 190 (m)), 2 animals/cage at the time of mating. Maternal animals after 14 days of pregnancy (sperm confirmation = day 0 of pregnancy) were housed in a breeding cage for rats (350 X 400 X 180 ffl), and pulp and paper chips (ALPHA-dri, Kado) were appropriately supplied as bedding.
- Diet: ad libitum solid feed (CE-2, CLEA Japan).
- Water: ad libitum tap water (Hadano City Water Bureau).
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24 ± 1 (meas. 23.5-25.8)°C
- Humidity (%): 50 - 65 (meas. 52.0-66.5) %
- Photoperiod (hrs dark / hrs light): 12 hrs light (7 am to 7 pm) / 12 hrs dark

IN-LIFE DATES: Purchase date: January 14, 1998. Start of administration: January 21, 1998
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5% w/v aquoeous solution
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Test substance was administrated dissolved in an 0.5% (w/v) CMC solution; the preparation was stored under refrigeration and administered within 7 days after preparation.

VEHICLE
- Amount of vehicle (if gavage): 5 mL/kg
- Lot/batch no.: 6Z09 (JPA water for injection: Hikari Pharmaceutical, Lot. no.: 9707SA)
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: up to 14 days
- Proof of pregnancy: sperm in vaginal smear, referred to as day [0] of pregnancy.
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
Males: for 42 days; Females: from 14 days before mating to day 3 of lactation.
Frequency of treatment:
Daily (9 am to 12 pm)
Details on study schedule:
- Age at mating of the mated animals in the study: 10 weeks
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
20 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
No. of animals per sex per dose:
13/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on the results of a preliminary toxicity test (A-97-029), at doses of 0 (0.5% CMC-Na, 5 mL/kg), 125, 250 and 500 mg/kg TSA. Since clear toxicity effects were observed at 500 mg/kg, this was selected as top dose. Using a factor of 5, the next doses were set at 100, 20 and 4 mg/kg bw/d.
- Rationale for animal assignment: random
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: once a week during the test period (male: administration 1, 8, 15, 22, 29, 36, 42 days, female: administration 1, 8, 15 days) and at necropsy day. Also, for mated females, body weights were measured on 0, 7, 14 and 20 days of pregnancy, and for females who gave birth, 0 and 4 days of lactation.

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: No / No data

URIANALYSIS: Yes
- Time schedule for collection of urine: for males, on the 42nd day of dosing
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the 42nd day of dosing
- Anaesthetic used for blood collection: Yes (pentobarbital)
- Animals fasted: Yes (18-24h)
- How many animals: all males
- Parameters: Red blood cell count (RBC), White blood cell count (WBC), Hemoglobin level (Hb), Average red blood cell volume (MCV), Platelet count, Hematocrit (Ht), Mean Corpuscular Hemoglobin (MCH), Mean Corpuscular Hemoglobin concentration (MCHC), Prothrombin time (PT), Activated partial thromboplastin time (APTT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after blood collection for hematological examination, blood was collected using heparin as an anticoagulant, blood plasma was separated, and the following items were examined.
- Animals fasted: Yes (18-24h)
- How many animals: all males
- Parameters: Total protein concentration; Albumin concentration; Total cholesterol concentration; triglyceride concentration; glucose concentration; urea nitrogen concentration (BUN); creatinine concentration; alkaline phosphatase activity (ALP); GOT activity; GPT activity; total bilirubin concentration; calcium concentration; Phosphorus concentration (inorg. Phos.); γ-GTP activity; A / G ratio.

OTHER: Mating rate [(number of mating animals / number of mating animals) x 100] for each group, Conception rate [(pregnant animal number / copulation animal number) x 100], number of days from the cohabitation start date to the mating confirmation date, and number of estrus which returned during that time were calculated.
Oestrous cyclicity (parental animals):
No
Sperm parameters (parental animals):
No
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, weights, litter weight.

GROSS EXAMINATION OF DEAD PUPS: yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: no.

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals as soon as possible after the last litters in each generation were produced.
- Maternal animals: All surviving animals, after the last litter of each generation was weaned (day 4). The females whose copulation was confirmed but not delivered were sacrificed on the 25th day of gestation.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.
- Necropsy was performed on all animals. The ovaries and uterus were removed in all cases of pregnancy and infertility, the number of implantations was counted for the uterus, and animals with implantation were regarded as pregnancy cases. The ovaries were counted under a stereomicroscope and the number of corpus luteum was fixed and stored in Bouin's solution.

HISTOPATHOLOGY / ORGAN WEIGHTS
- In all males, the following tissues were prepared for microscopic examination and weighed, respectively: brain, heart, thymus, liver, kidney, spleen, adrenal gland, testis and epididymis; and the specific weight value (relative weight) was calculated. Among the fixed organs, liver, kidney, spleen, heart, bladder and thymus were examined in all treatment groups, and other organs were only observed in the control group and high dose groups.
- In females, histopathological examination was performed on the ovaries of infertile cases. In addition, brain, heart, thymus, liver, kidney, spleen, and adrenal gland weights were measured in all animals. Among the fixed organs, liver, kidney, spleen, heart, bladder and thymus were examined in all treatment groups, and other organs were only observed in the control group and high dose groups.
Postmortem examinations (offspring):
SACRIFICE
- All F1 offspring were sacrificed at 4 days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGTHS: not examined.
Statistics:
The copulation rate and the conception rate were subjected to χ2 test including Yates' correction. Histopathological findings were tested using Mann-Whitney's U test and for positive total scores, Fisher's exact probability one-sided test was used. Statistical analysis was not performed for general condition, autopsy findings and urinalysis data. The other data were tested for uniformity of each group's variance by Bartlett method, using the values ​​obtained for each individual or the average value for each litter as one sample. Based on the results, one-way analysis of variance or Kruskal-Wallis rank test was performed, and when there is significance between the groups, either the Dunnett method or Scheffe's method is used to determine the difference between the control group and each test substance administration group. The significance level was 5% and 1%.
Reproductive indices:
Fertility indices:
- Mating period: maximum 14 days. Confirmation of mating completion was done by checking for the existence of sperm in vaginal plug and vaginal semen = day 0 of pregnancy.
- Time until mating,
- Mating rate [(number of copulated animals / number of animals kept in couples)×100],
- Conception rate [(number of pregnant animals / number of copulated animals)×100],
- Gestation period: number of days from day 0 of pregnancy to the day of delivery,
- Birth rate [(number of females with live pups / number of pregnant females)×100].
Offspring viability indices:
- Viability rate [(number of live pups /number of pups born)×100] on day 1
- Survival rate [(number of live pups on day 4 / number of live pups on day 1)×100] on day 4
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Decreased locomotor activity and appearance of prone position and salivation were observed in both sexes at 100 and 500 mg/kg bw.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Three (3/13) females died and two females were sacrificed in moribund condition during the pre-mating period at 500 mg/kg bw. No males died in any of the groups.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
A decrease in body weight gain was observed at the start of the study; low body weights were recorded in both sexes at 100 and 500 mg/kg bw.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Diminished food consumption was observed at the beginning of the study.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
In the hematological examination performed at necropsy, the average erythrocyte pigment amount increased in all TSA-administered groups,
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Alkaline phosphatase activity decreased in all TSA administration groups, and total cholesterol concentration increased in the 100 mg/kg or more administration group. Also, in the 500 mg/kg administration group, the total protein concentration increased, and the A/G ratio, glucose concentration and triglyceride concentration decreased.
Urinalysis findings:
no effects observed
Description (incidence and severity):
Urinalysis conducted after 42 doses showed no abnormalities attributable to TSA administration.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
In the dose groups receiving 100 mg/kg test item or more, the specific body weights of the liver and kidney increased, and the heart, adrenal and testis weight ratios value also increased.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Darkening or swelling was observed in the liver and kidney in the 100 mg/kg or more administration groups.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Hypertrophy of the centrilobular hepatocytes with the cytoplasm having a ground glass appearance was observed in both sexes at 100 and 500 mg/kg bw in a dose-dependent manner. In addition, the incidence of fibrosis and cellular infiltration of the pericardium, and fibrosis and cellular infiltration of the capsule and atrophy of the thymus were significantly increased in females at 500 mg/kg bw. In the kidneys, eosinophilic body was observed in males of all treated groups, maybe due to the complex accumulation of this chemical with the male rat specific protein, alpha-2u-globulin.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
TSA up to 500 mg/kg showed no effect on mating, ovulation and conception. In addition, there were no abnormalities in labor and feeding caused by TSA administration.
In the 20 mg/kg administration group, no effect of TSA administration was observed in any of the examinations.
Key result
Dose descriptor:
NOAEL
Effect level:
20 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
Critical effects observed:
not specified
Clinical signs:
not specified
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
No effects of TSA administration were observed on the survival, sex ratio and body weight of offspring at doses of 100 mg/kg or less, and no morphological abnormalities attributable to TSA administration were observed. In the 500 mg/kg dose group, but the number of survivors at 0 and 4 days of lactation and the rate of parturition tended to decrease.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In the 500 mg/kg dose group, a decrease was observed in the weight of male and female survivors at 0 and 4 days of lactation.
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Histopathological findings:
not specified
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
body weight and weight gain
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
500 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects occurring together with other toxic effects, but not as a secondary non-specific consequence of other toxic effects
Dose response relationship:
not specified
Relevant for humans:
not specified

Table 1. Body weights of male rats treated orally with o-TSA in the combined repeat dose and reprcductive/developmental toxicity screening test.

Dose (mg/kg)

 

0

 

20

 

100

 

500

 

Days of administration

 

 

 

 

 

 

 

 

 

1            (Init, wt.)

333.1± 7.4

(13)

333.7 ± 82

(13)

333.7 ± 83

(13)

3317 ± 7.8

(13)

8

370.5

± 13.3

(13)

366.1 ± 117

(13)

351.2 ± 1.13"

(13)

348.9 ± 14.8**

(13)

15

405.2

± 19.0

(13)

402.3 ± 16.6

(13)

383.6 ± 183*

(13)

350.2 ± 21.0**

(13)

22

430.5

± 22.1

(13)

429.4 ± 1913

(13)

403.8 ± 22.0*

(13)

405.6 ± 216*

(13)

29

456.0

± 29.7

(13)

451.9 ± 214

(13)

435.1 ± 27.7

(13)

426.9 ± 25.2*

(13)

36

483.8

± 3.0 (13)

478.1 ± 24.0

(13)

460.1 ± 28.8

(13)

449.4 ± 26.3*

(13)

42

503.4

± 35.9 (13)

497.1 ± 25.8

(13)

479.6 ± 32.4

(13)

467.4 ± 26.9*

(13)

Values are expressed as mean±8 D. in grams.

 

 

 

 

 

 

 

 

 

Parenthesis indicates number of animals.

 

 

 

 

 

 

 

 

 

* :significant difference from control, p<0.05

 

 

 

 

 

 

 

 

 

**:significant difference from control, p<0.01

 

 

 

 

 

 

 

 

 

 

Table 2. Food consumption of male rats treated orally with o-TSA in the combined repeat dose and reproductive/developmental toxicity screening test

Dose (mg/kg)

 

0

 

20

 

 

10]

 

 

500

 

Days of administration

 

 

 

 

 

 

 

 

 

 

 

1 - 8

1990

± 17.8

(13)

194.3 ± 114

(13)

179.4

± 17,2**

(13)

166.5

± 12.0**

(13)

8 -15

196.1

± 18.0

(13)

1993 ± 12.6

(13)

181.6

± 14.6*

(13)

187.2

± 14.1

(13)

29 - 36

207.9

± 22.8

(13)

209.5 ± 13.7

(13)

204.0

± 15.8

(13)

201.5

± 14.5

(13)

36 - 42

181.8

± 21.4 (13)

182.9 ± 11.9

(13)

176.4

± 18.7

(13)

174.0

± 12.1

(13)

 

Table 3. Body weights of female rats treated orally wit h o-TSA in the combined repeat dose and reproductive/developmental toxicity screening test

 

Dose(mg/kg)

 

0

 

20

 

 

100

 

 

500

 

Days of administration

 

 

 

 

 

 

 

 

 

 

 

(Pre-mating period)

 

 

 

 

 

 

 

 

 

 

 

1            (Init. wt.)

216.3

± 6.2

(13)

216.4 ± 5.6

(13)

216.3

± 5.8

(13)

216.1

± 6.3  (13)

 

8

232.9

± 7.9

(13)

232.2 ± 7.7

(13)

226.5

± 9.1

(13)

215.4

± 17.1*(8)

15

251.3

± 15.6

(13)

250.9 ± 12.9

(13)

241.9

± 8.1

(13)

234.3

± 17.2   (8)

Days of pregnancy

 

 

 

 

 

 

 

 

 

 

 

0

255.2

± 15.9

(10)

255.6 ± 13.1

(13)

246.5

± 9.3

(12)

237.2

± 16.3   (7)

7

303.6

± 19.4

(10)

3006 ± 12.5

(13)

282.2

± 10.3*

(12)

278.4

± 23.5*(7)

14

341.1

± 25.3

(10)

340.9 ± 18.2

(13)

319.5

± 12.1

(12)

315.5

± 26.2   (7)

20

414.5

± 35.2

(10)

4099 ± 35.3

(13)

389.1

± 18.3

(32)

375.9

± 40.0   (7)

Days of lactation

 

 

 

 

 

 

 

 

 

 

 

0

313.1

±18.7

(10)

303.7 ± 21.3

(12)

296.2

±23.2

(12)

281.9

± 23.7   (7)

4

344.1

± 19.7

(10)

337.0 ± 196

(12)

317.6

± 24.8*

(12)

299.8

± 17.0** (7)

 

Table 4. Food consumption of female rats treated orally with o-TSA in the combined repeatdose and reproductive/developmental toxicity screening test

Dose (mg/kg)

 

0

 

20

 

 

100

 

 

500

 

Days of administration

 

 

 

 

 

 

 

 

 

 

 

(Pre-mating period}

 

 

 

 

 

 

 

 

 

 

 

1 - 8

136.9

± 11.5

(13)

137.3 ± 8.9

(13)

123.6

± 12.2

(13)

93.9

± 20.3**

(8)

8 - 15

146.1

± 13.6

(13)

151.3 ± 10.8

(13)

136.5

± 8.8

(13)

127.8

± 23.6 

(8)

Days of pregnancy

 

 

 

 

 

 

 

 

 

 

 

0 - 7

182.4

± 18.8

(10)

182.6 ± 11.0

(13)

162.2

± 10.9

(12)

148.2

± 24.9*

(7)

7 - 14

197.4

± 25.5

(10)

201.2 ± 16.1

(13)

181.3

± 13.8

(12)

178.1

± 20.6

(7)

11- 20

147.6

± 16.0

(10)

149.7 ± 98

(13)

140.3

± 13.2

(12)

130.5

± 20.3  

(7)

Days of lactation

 

 

 

 

 

 

 

 

 

 

 

0 - 4 

143.3

± 16.9

(10)

148.5 ±256

(12)

136.0 ± 19.4

(12)

117.6

± 14.0

(7)

Table 5. Hematological findings of male rats treated orally with o-TSA in the combined repeat dose and reproductive/developmental toxicity screening test

Dose (mg/kg)

0

20

100

330

Male Red blood cells

Count (x 10^4/mm3)

835 ± 29 (13)

830 ± 36

(13)

820 ± 3i

(13)

822 ± 37

(13)

Hemoglobin (g/d L)

14.9 ± 1.3 (13)

15.5 ± 0.6

(13)

1515 ± 015

(13)

155 ± 015

(13)

Hematocrit (%)

45.1 ± 3.9 (13)

4 6.5 ± 19

(13)

458 ± 1.6

(13)

45 1 ± 1.7

(13)

MCV (µm2)

54.3 ± 4.1 (13)

56.2 ± 16

(13)

550 ± 1.3

(13)

31 .9 ± 1.3

(13)

MCH (p g)

17.9 ± 1.4 (13)

18.7 ± 0.4*

(13)

189 ± 04**

(13)

189 ± 04**

(13)

MCHC (%)

32.9 ± 0.5 (13)

333 ± 0.6

(13)

33.9 ± 07**

(13)

34/1 ± 05**

(13)

White blood cells

Count (x 10^4/mm3)

69 ± 20 (13)

37 ± 11

(13)

8i ± I8

(13)

68 ± 15

(13)

Band neutrophil (%)

0 ± 0 (13)

0 ± 0

(13)

0 ± 0

(13)

0 ± 0

(13)

Se gmented neutrophil (%)

12 ± 8 (13)

15 ± 7

(13)

16 ± 7

(13)

15 ± 7

(13)

Eosinophil (%)

0 ± 1 (13)

1 ± 1

(13)

0 ± 1

(13)

1 ± 1

(13)

Basophil (%)

0 ± 0 (13)

0 ± 0

(13)

0 ± 0

(13)

0 ± 0

(13)

Monocyte (%)

1 ± 1 (13)

1 ± 1

(13)

1 ± 1

(13)

1 ± 1

(13)

Lymphocyte (%)

87 ± 9 (13)

83 ± 8

(13)

83 ± 7

(13)

83 ± 7

(13)

Platelet

Count (x 10^4/mm3)

96.5 ± 78 (13)

97.0 ± 7.9

(13)

9016 ± 59

(13)

101.7 ± 6.3*

(13)

 PT (sec)

29.9 ± 8.8 (13)

31.1 ± 7.2

(13)

22.7 ± 52*

(13)

21. 6 ± 53*

(13)

APTT (sec)

26.9 ± 2.5 (13)

280 ± 3.1

(13)

21.2 ± 3.3

(13)

21 .5 ± 2.3

(13)

Table 6. Blood chemical findings of male rats treated orally with o-TSA in the combined repeat dose and reproductive/developmental toxicity screening test

Dose (mg/kg)

0

 

20

100

 

330

Total protein (g/dL)

54 ± 0.2

(13)

53 ± 0.3

(13)

5.4 ± 0.2

(13)

5.6

 ± 0.2*

(13)

Albumin (g/dL)

3.0 ± 0.2

(13)

2.9 ± 0.2

(13)

3.0 ± 0.2

(13)

3.0

 ± 01

(13)

A/G

1.29 ± 0.14

(13)

1.19 ± 0.13

(13)

1.26 ± 0.13

(13)

1.15

 ± 0.10*

(13)

 

BUN (mg/dL)

17 ± 2

(13)

18 ± 4

(13)

19 ± 3

(13)

20

 ± 2

(13)

Creatinine (mg/d L)

0.7 ± 0.1

(13)

0.6 ± 0.0

(13)

0.7 ± 0.1

(13)

0.6

 ± 0.1

(13)

Glucose (mg/ dL)

144 ± 10

(13)

142 ± 13

(13)

135 ± 11

(13)

123

 ± 14**

(13)

Total cholesterol (mg/dL)

37 ± 6

(13)

39 ± 3

(13)

49 ± 13*

(13)

66

 ± 15**

(13)

Triglyceride (mg/dL)

45 ± 15

(13)

35 ± 14

(13)

39 ± 13

(13)

27

 ± 11**

(13)

Total bilirubin(mg/dL)

 0.08 ± 0.03

(13)

0.08 ± 0.01

(13)

0.09 ± 0.02

(13)

010

 ± 002

(13)

Na (mEq/L)

145.1 ± 0.8

(13)

145.1 ± 0.7

(13)

145.1 ± 0.9

(13)

145.0

 ± 1.0

(13)

K (mEg/L)

4.14 ± 0.15

(13)

4.13 ± 0.26

(13)

4.04 ± 0.16

(13)

3.99

 ± 021

(13)

Cl (mEq/L)

108.4 ± 0.8

(13)

108.9 ± 1.1

(13)

108.6 ± 10

(13)

107.5

 ± 1.1

(13)

Ca (mg/dL)

87 ± 0.2

(13)

85 ± 0.1

(13)

88 ± 0.1

(13)

8.9

 ± 03

(13)

Inorg. phos.(mg/dL)

6.5 ± 0.5

(13)

6.3 ± 0.4

(13)

6.0 ± 06

(13)

5.9

 ± 03

(13)

ALP (U/L)

261 ± 35

(13)

218 ± 40**

(13)

216 ± 34**

(13)

199

 ± 31**

(13)

GPT (U/L)

32 ± 7

(13)

32 ± 7

(13)

28 ± 3

(13)

38

 ± 15

(13)

GOT (U/L)

69 ± 14

(13)

68 ± 10

(13)

63 ± 7

(13)

71

 ± 23

(13)

y-GTP (U/L)

0 ± 0

(13)

0 ± 0

(13)

0 ± 0

(13)

0

 ± 1*

(13)

Table 7. Absolute and relative organ weights of treated rats

Dose (mg/kg)

 

0

20

100

300

MALE

 

 

 

 

Terminal body weight

(g)

469.6 ± 33.8 (13)

464.0 ± 24.8 (13)

443.9 ± 30.7

(13)

432.7 ± 26.4**

(13)

 

 

 

 

 

 

 

 

Brain

(g)

2.04 ± 0.06*(13)

2.01 ± 0.05(13)

2.00 ± 0.08

(13)

1.98 ± 0.09

(13)

 

 

0.44 ± 0.02

0.44 ± 0.03

0.13 ± 0.03

 

0.46 ± 0.03

 

Heart

(g)

1.33 ± 013 (13)

13.5 ± 0.13(13)

1.33 ± 0.09

(13)

1.31 ± 0.12

(13)

 

 

0.28 ± 0.02

0.29 ± 0.02

0.31 ± 0.02

 

030 ± 0.02*

 

Liver

(g)

12.27 ± 1.49 (13)

12.30 ± 1.15(13)

12.10 ± 125

(13)

14.37 ± 1.18**

(13)

 

 

2.61 ± 0.20

2.65 ± 0.14

2.79 ± 0.16

 

3.32 ± 0.17**

 

Kidneys

(g)

3.15 ± 0.21 (13)

3.24 ± 0.18 (13)

3.27 ± 0.35

(13)

3.51 ± 0.23**

(13)

 

 

0.67 ± 0.05

0.70 ± 0.03

0.74 ± 007**

 

082 ± 0.06**

 

Spleen

(g)

0.75 ± 0.09 (13)

0.79 ± 0.11 (3)

0.76 ± 010

(13)

0.75 ± 6.12

(13)

 

 

0.16 ± 0.02

0.17 ± 0.02

0.17 ± 0.02

 

0.11 ± 0.03

 

Thymus

(mg)

351.3 ± 86.3 (13)

319.8 ± 86.5(13)

3113 ± £2.5

(13)

278.6 ± 56.9

(13)

 

 

74.9 ± 18.3

68.9 ± 17.8

707 ± 20.1

 

64.2 ± 11.8

 

Adrenal glands

(mg)

54.3 ± 01 (13)

58.5 ± 11.1(13)

59.4 ± 8.7

(13)

62.2 ± 4.8

(13)

 

 

11.6 ± 1.8

12.6 ± 2.0

13.4 ± 16*

 

14.4 ± 1.1**

 

Testes

(g)

3.03 ± 073 (13)

3.40 ± 0.20(13}

3.36 ± 0.26

(13)

3.35 ± 0.21

(13)

 

 

0.65 ± 0.16

0.74 ± 0.06

0.76 ± 0.68*

 

0.78 ± 0.05**

 

Epididymides

(g)

1.09 ± 0.21 (13)

1.22 ± 0.09(13)

1.17 ± 0.09

(13)

1.04 ± 0.09

(13)

 

 

0.23 ± 0.05

0.26 ± 0.02

0.26 ± 0.66*

 

0.25 ± 0.03

 

FEMALE

 

 

 

 

 

 

 

Terminal body weight

(g)

344.1 ± 19.7

(10)

337.0 ± 19.6 (12)

317.6 ± 21.8*

(12)

299.8 ± 17.0**

(12)

 

 

 

 

 

 

 

 

 

Brain

(g)

1.94 ± 0.08

(10)

1.84 ± 0.07 (12)

1.37 ± 0.07

(12)

185 ± 0.10

(12)

 

 

0.56 ± 0.03

 

0.553 ± 0.03

0.39 ± 0.05

 

0.62 ± 0.05*

 

Heart

(g)

0.98 ± 0.07

(10)

0.95 ± 0.07 (12)

0.97 ± 010

(12)

0.93 ± 0.12

(12)

 

 

0.28 ± 0.02

 

0.28 ± 0.02

0.31 ± 0.02

 

0.31 ± 0.04

 

Liver

(g)

13.57 ± 0.99

(10)

13.96 ± 139 (12)

14.20 ± 1.71

(12)

15.12 ± 1.54

(12)

 

 

3.95 ± 0.30

 

4.11 ± 0.31

1.16 ± 0.31**

 

5.05 ± 0.40**

 

Kidneys

(g)

2.10 ± 0.20

(10)

2.08 ± 0.23 (12)

2.19 ± 0.15

(12)

2.19 ± 0.28

(12)

 

 

0.61 ± 0.06

 

0.62 ± 0.05

0.70 ± 0.10

 

0.73 ± 0.08*

 

Spleen

(g)

0.70 ± 0.15

(10)

0.66 ± 0.06 (12)

0.70 ± 0.13

(12)

0.65 ± 0.10

(12)

 

 

0.20 ± 0.04

 

0.20 ± 0.01

0.22 ± 0.03

 

0.21 ± 0.04

 

Thymus

(mg)

276.4 ± 51.0

(10)

219.3 ± 60.5 (12)

217.0 ± 97.4

(12)

188.8 ± 68.5

(12)

 

 

80.3 ± 13.7

 

65.3 ± 183

67.8 ± 28.6

 

62.1 ± 21.3

 

Adrenal glands

(mg)

74.6 ± 10.8

(10)

796.5 ± 9.3 (12)

75.2 ± 8.0

(12)

85.5 ± 16.4

(12)

 

 

21.7 ± 81

 

23.7 ± 3.5

23.8 ± 3.0

 

28.6 ± 57**

 

Values are expressed as mean ± SD. Parenthesis indicates number of animals.

a: ab solute weight; b: relative weight (g or mg per 100 g body weight)

*: significant difference from control. p<0.05; **:significant difference from control. p<0.01.

Table 8. Summary of reproductive performance in parental rats

Dose (mg/kg)

0

20

100

500

Number of mated pairs

13

13

13

8

Number of copulated pairs

13

13

13

8

Copulation index M

100

100

100

100

Number of pregnant animals

10

13

12

7

Fertility index

76.9

100

92.3

87.5

Pairing days until copulation

  Mean ± SD

2.5 ± 1.3

2.9 ± 1.8

2.7 ± 1.8

3.0 ± 2.2

Frequency of vaginal estrus

  Mean ± SD

11 ± 0.3

11 ± 0.3

10 ± 0.0

1.0 ± 0.0

A) Copulation index=(Number of copulated pairs/Number of mated pairs)X100;%

B) Fertility index=(Number at pregnant animals/Number of copulated pairs)X100;%

Table 9. Summary of development up of pups

Dose (mg/kg)

0

20

100

500

Number of pregnant females

10

13

12

7

Number of pregnant females

 with pups alive

10

12

12

7

Gestation index

100

02.3

100

100

Gestation length in days

22.6 ± 0.5 (10)

22.6 ± 0.5 (12)

22.6 ± 0.5 (12)

23.0 ± 0.0 (7)

Number of corpora lutea

16.7 ± 3.4 (10)

16.3 ± 3.3 (13)

17.8 ± 2.9 (12)

17.9 ± 4.4 (7)

Number of implantation sites

15.0 ± 2.7 (10)

150 ± 4.4 (13)

153 ± 2.9 (12)

136 ± 3.4 (7)

Implantation index

90.8 ± 105 (10)

87.5 ± 22.1 (13)

86.8 ± 12.4 (12)

87.7 ± 5.0 (7)

Day 0 of lactation

Number of pups born

13.6 ± 2.1 (10)

13.7 ± 4.3 (13)

1-1.0 ± 3.3 (12)

12.4 ± 4.8 (7)

Delivery index 01

91.2 ± 7.9 (10)

81.9 ± 26.7 (13)

915 ± 13.0 (12)

76.4 ± 20.6 (7)

Number of pups alive

13.5 ± 2.1 (10)

13.8 ± 4.3 (13)

13.9 ± 13 (12)

11.4 ± 4.0 (7)

Birth index

93.5 ± 7.9 (10)

84.5 ± 26.9 (13)

91.0 ± 13.0 (12)

73.0 ± 1&7 (7)

Live birth index

80.3 ± 2.1 (10

99.4 ± 2.0 (12)

99.4 ± 19 (12)

93.8 ± 86 (7)

Pup weight in grams

- Male

7.4 ± 1.0 (10)

7.1 ± 0.7 (12)

6.8 ± 08 (12)

6.2 ± 0.7* (7)

- Female

7.0 ± 0.7 (10)

6.7 ± 0.7 (12)

6.4 ± 06 (12)

38 ± 0.7** (7)

Sex ratio *

0.87 ± 0.34 (10)

1.43 ± 1.03 (12)

116 ± 082 (12)

1.38 ± 1.23 (7)

Day 4 of lactation

 Number of pups alive

13.3 ± 1.9 (10)

1-1.8 ± 1-1 (12)

13.5 ± 10 (12)

11.1 ± 3.8 (7)

 Viability index **

96.8 ± 4.0 (10)

100.0 ± 00 (12)

97.5- ± 4.0 (12)

97.6 ± 4.4 (7)

 Pup weight in grams

 - Male

12.0 ± 1.3 (10)

11.5 ± 0.9 (12)

11.0 ± 1.5 (12)

9.4 ± 2.4* (7)

 - Female

11.6 ± 1.1 (10)

10.9 ± 1.0 (12)

10.4 ± 1.4 (12)

8.7 ± 2.4** (7)

Values are expressed as mean ± SD.                                                                      

Parenthesis indicates the number of litters evaluated.                                 

A) Gestation index= (Number of pregnant females with pups alive/Number of pregnant females)x100; %

B) implantation index= (Number of implantation sites/Number of corpora lutea) x100; %

O) Delivery index= (Number of pups born/Number of implantation sites) x100; %           

Conclusions:
Based on the available data, under the test conditions, the NOAEL for repeated dose toxicity was 20 mg/kg bw/day in parental animals (m/f), whereas for reproductive and developmental toxicity it was 500 mg/kg bw/day and 100 mg/kg bw/day for offspring.
Executive summary:

A Combined Repeated Dose Toxicity with Reproduction / Developmental Toxicity Screening Test was conducted on the test item, according to OECD Guideline 422 (GLP study). Based on the results of a preliminary acute oral toxicity test, 13 male and 13 female Sprague Dawley rats per group were exposed via gavage to 0 (control), 20, 100 and 500 mg/kg bw from 14 days before mating to 14 days after mating in males and from 14 days before mating to day 3 of lactation in females. Observations included clinical observations, body weight measurements, food intake, mating procedures, evaluation of reproduction, weight of organs, and histopathological examinations. Three females died and two females were sacrificed in moribund condition during the pre-mating period at 500 mg/kg bw. Clinical signs such as decrease in locomotor activity and appearance of prone position and salivation, and significant lowering of body weights were observed in both sexes at 100 and 500 mg/kg bw In the 500 mg/kg bw group, the delivery index, number of pups alive on day 0 of lactation and birth index were slightly decreased but not statistically significant. There was no effect of the test compound on the ovulation, copulation, fertility, delivery and lactation at dose levels up to 100 mg/kg bw Significant reduction in body weights of pups was observed on days 0 and 4 in both sexes at 500 mg/kg bw.

Based on the available data, under the test conditions, the NOAEL for repeated dose toxicity was 20 mg/kg bw/day in parental animals (m/f), whereas for reproductive and developmental toxicity it was 500 mg/kg bw/day and 100 mg/kg bw/day for offspring.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Test animals

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

Administration / exposure

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)
Doses / concentrationsopen allclose all
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).

Examinations

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.

Results and discussion

Results of examinations

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

Effect levels

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

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.