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Toxicological information

Toxicity to reproduction

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Administrative data

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
Cross-reference
Reason / purpose:
reference to same study
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).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999

Materials and methods

Test guideline
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

Test material

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

Test animals

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

Administration / exposure

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
Doses / concentrationsopen allclose all
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

Examinations

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

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

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 / performance (P0)

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.

Details on results (P0)

In the 20 mg/kg administration group, no effect of TSA administration was observed in any of the examinations.

Effect levels (P0)

open allclose all
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

Target system / organ toxicity (P0)

Critical effects observed:
not specified

Results: F1 generation

General toxicity (F1)

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

Effect levels (F1)

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

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

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

Any other information on results incl. tables

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; %           

Applicant's summary and conclusion

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.