Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Weight of evidence. Based on the available data, the test item has a NOAEL = 100 mg/kg bw/d in rats.

- Method according to OECD 422, GLP study. The NOAEL for reproductive/developmental toxicity of the test item in Sprague Dawley rats (m/f) was found to be 100 mg/kg bw/d.

- Method according to OECD 421, GLP study. The NOAEL for reproductive/developmental toxicity of the test item in Sprague Dawley rats (m/f) was found to be 100 mg/kg bw/d.

- Two-generation lifetime feeding study (no TG, no GLP, study well documented, acceptable for assessment). Decrease of litter size and pup body weight was observed at 250 mg/kg bw/day.

Link to relevant study records

Referenceopen allclose all

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.

Endpoint:
one-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1974
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Reason / purpose:
reference to same study
Qualifier:
no guideline followed
Principles of method if other than guideline:
- Principle of test: Two-generation lifetime feeding study
- Short description of test conditions: 50 male and 50 female weanling Sprague-Dawley rats were administered the test item at different doses in their diet. After 3 months on test, F0 rats were bred and 50 pups of each sex, from every group, were weaned onto the parental diets, which both generations received for their lifetime.
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratory, Canadian Breeding Farms, Montréal, Québec, Canada.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (P) 32 days; (F1) 0 wks
- Housing: The animals were housed individually, except during mating, in stainless-steel cages with screened floors. Cages were steam disinfected every 2 weeks and the "pull-papers" under the cages were changed daily.
- Diet: the basal diet was Master Laboratory Cubes (Master Feeds, Toronto, Ontario, Canada) to which 4% (w/w) corn oil had been added after the cubes were ground. Weighed amounts of the appropriate feed were distributed to the animals on a weekly basis in stainless steel feed cups.
- Water: drinking water was supplied by the Municipality of Ottawa, ad libitum, provided in a clear glass bottle, with a rubber stopper and stainless-steel nipple, which had been previously sterilized. The water bottles were changed 3 times/week.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 1°C
- Humidity (%): 50-55%
- Photoperiod (hrs dark / hrs light): 12 h light/dark cycle
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): the appropriate amount of test chemical was premixed with 500g of ground rat chow. This concentrate premix was added to an appropriate amount of basal diet plus corn oil and mixed thoroughly on the day prior to use.
- Storage temperature of food: room temperature, protected from light.
Details on mating procedure:
- M/F ratio per cage: one-to-one basis.
- After 3 months on test, the F0 rats were mated on a one-to-one basis; all litters were culled to 8 pups (4 males and 4 females) 4 days post partum in a random manner. The pups were weaned onto their parents' diet, and 50 males and 50 females from each group were randomly selected to constitute the second generation (F1). Thus, the pups were exposed to the test item in utero, during lactation via the dam's milk, and for the remainder of their life. The two generations remained on test for 127 (F1) and 142(F0) weeks.
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
F0: 142 weeks; F1: 127 weeks.
Frequency of treatment:
Daily
Details on study schedule:
- Age at mating of the mated animals in the study: 3 months (F0).
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
2.5 mg/kg bw/day (nominal)
Dose / conc.:
25 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
Remarks:
plus 1% NH4CI in drinking watter, which was added to prevent the formation of alkaline urine (associated with the production of bladder calculi and bladder tumors in a previous study).
No. of animals per sex per dose:
50
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: the doses were selected to be equivalent to the levels resulting from the dietary additions of Remsen-Falberg-produced saccharin used in previous studies (WARF, FDA).
- Rationale for animal assignment: Random.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

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, weekly
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION AND COMPOUND INTAKE: A limited water balance study was undertaken with the male and female F1 generation animals when they were 20 months of age. These consisted of determining the volume of water consumed and the volume of urine during a 24h period, by each of 10 males and 10 females from the following groups: control, 250 mg/kg o-TS and 250 mg/kg o-TS + 1% NH4Cl in water.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 0.1 - 0.125 ml were obtained from the tail aorta at 0, 4.5, 21, 44.5, 57, 65, 69, 96 and 98 weeks for F0; and at 4.5, 18.5, 32.5 and 45.5 weeks of age.
- Anaesthetic used for blood collection: Yes (ether)
- How many animals: 10 per group
- Parameters checked: erythrocyte enumeration (RBC), hematocrit (HTC), hemoglobin (Hb), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentratoin (MCHC), total leukocyte enumeration (WBC), differential leukocyte count including absolute values, and occasionally hematopoietic organs (bone marrow, lymph node, and spleen imprints) were examined in cases where hematologic involvement was observed in moribund animals.

URINALYSIS: Yes
- Time schedule for collection of urine: at 6 months intervals, at 9 AM and 2 PM (groups: for control, 250 mg/kg o-TS and 250 mg/kg o-TS + 1% NH4Cl in water)
- Metabolism cages used for collection of urine: No
- Parameters: pH, turbidity, Bililabstix determination (for presence of glucose, ketones, bilirubin, protein and blood), presence of sediment or exfoliated epithelial cells (microscopical examination), presence of parasites (Trichosomoides crassicauda).
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

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,

GROSS EXAMINATION OF DEAD PUPS:
[no / yes, for external and internal abnormalities; possible cause of death was/was not determined for pups born or found dead.]

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: no.

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals / Maternal animals: All surviving animals at the end of the study (142 weeks). Animals were anesthetized with ether and exsanguinated from the abdominal aorta.

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

HISTOPATHOLOGY / ORGAN WEIGHTS
Representative portions from all organs and all grossly abnormal areas of dermal, supportive or skeletal tissues were embedded in paraffin and sectioned at a thickness
of 4 μm, or directly by cryostat when appropriate. The tissues were stained with hemalum-phloxine -saffron (HPS, unless a special stain was indicated). In addition, slides of the bladder tumors were reviewed by a panel of pathologists not associated with the design/conduct of the study.
Postmortem examinations (offspring):
SACRIFICE
- Male animals / Maternal animals: All surviving animals at the end of the study (142 weeks). Animals were anesthetized with ether and exsanguinated from the abdominal aorta.

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

HISTOPATHOLOGY / ORGAN WEIGHTS
Representative portions from all organs and all grossly abnormal areas of dermal, supportive or skeletal tissues were embedded in paraffin and sectioned at a thickness
of 4 μm, or directly by cryostat when appropriate. The tissues were stained with hemalum-phloxine -saffron (HPS, unless a special stain was indicated). In addition, slides of the bladder tumors were reviewed by a panel of pathologists not associated with the design/conduct of the study.
Statistics:
Unless otherwise noted, the observed results for each test diet were compared to those for the control diet separately for each sex and time of observation, limiting the overall significance to 5% for each sex and time of observation, using the Bonferroni inequality. t-tests were used for continuous data a nd Fisher's test for quantal data.
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
The F0 generation animals were kept on test for 142 weeks, at which time less than 3 % of the initial animals were alive. The time-to-death was normally distributed in the F0 generation and was not affected by treatment.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The growth curves for the F0 males and females receiving o-TSA at 250 mg/kg or 250 mg/kg with 1% NH4Cl in the drinking water were significantly different (p < 0.05) from control animals.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Animals consuming o-TSA at 250 mg/kg or 250 mg/kg with 1% NH4Cl in the drinking water consumed less feed.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
not examined
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
Statistical analysis of urinary pH values for male rats during the 18th month on test revealed that only the urine from the group of males receiving o-TSA at 250 mg/kg with NH4Cl in the drinking water significantly different when compared to males in other treatment groups; it was more acidic.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
At necropsy, the most common lesions found for both F0 and F1 animals were pituitary and subcutaneous tumors. Grossly visible bladder stones were observed in 3 animals during the study. Microscopic examination revealed the following: epithelial hyperplasia in an F0 control animals, diffuse papillomatosis involving most of the bladder wall in an F0 animal receiving 2.5 mg/kg o-TS, no pathological changes in an F1 animal receiving 2.5 mg/kg o-TS. 3 F0 females from the control, 2.5 mg/kg o-TS and 250 mg/kg o-TS groups had grossly visible kidney stones, but none had urinary bladder tumors.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related effects associated with longevity.
Histopathological findings: neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related effects associated with longevity. Urinary bladder tumours, all of which were benign, were observed in one male each of the 0, 2.5 and 250 mg/kg b.w. group and in one female of the 2.5 mg/kg b.w. group in the first generation, and 2 females of the 2.5 mg/kg b.w. group in the second generation. However, the incidence was neither statistically significant nor dose-related. No other dose-related tumours were observed in any groups.
Other effects:
no effects observed
Reproductive function: oestrous cycle:
not specified
Description (incidence and severity):
No significant differences from the control group were observed for any of the reproductive parameters (no further details)
Reproductive function: sperm measures:
not specified
Description (incidence and severity):
No significant differences from the control group were observed for any of the reproductive parameters (no further details)
Reproductive performance:
not specified
Description (incidence and severity):
No significant differences from the control group were observed for any of the reproductive parameters (no further details). However, a statistically significant decrease in the litter size of the 250 mg o-TS/kg group was observed.
Key result
Dose descriptor:
LOAEL
Effect level:
250 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
Critical effects observed:
no
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
There was a statistically significant decrease in the litter size of the 250 mg o-TS/kg group.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
When the average pup body weight at 4 days postpartum was adjusted for litter size, there was a significantly lower average body weight at 4 days postpartum in both groups receiving 250 mg/kg o-TS. No other treatment-related effect was evident.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
not examined
Urinalysis findings:
effects observed, non-treatment-related
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, non-treatment-related
Histopathological findings:
effects observed, non-treatment-related
Other effects:
no effects observed
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
25 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
body weight and weight gain
Critical effects observed:
no
Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
250 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. Prenatal data for rats fed diets containing o-TS.  

o-TS Dose

(mg/kg bw/d)

No.of litters

Average litter size (day 1)

Average litter size (day 4)

Average body weight (day 4) (g)

0 (Control)

41

10.80

10.46

11.56

2.5

40

11.78

11.08

11.07

25

41

10.56

10.22

11

250

49

9.38*

9.04*

9.26**

250 + NH4Cl

33

9.7

9.12

9.49**

*: statistically significant (p<0.05); **: statistically significant (p<0.05) when the average pup weight was statistically adjusted to an average litter size of 11 pups based on an analysis of covariance with the covariate litter size.

Conclusions:
The NOAEL for reproductive/developmental toxicity is considered to be 25 mg/kg bw/day under test conditions.
Executive summary:

In a two generation lifetime feeding study (no TG, no GLP, study well documented, acceptable for assessment), SD rats were given o-TSA in the diet at 0 (control), 2.5, 25 or 250 mg/kg bw/day. At 90 days after onset of the administration, the rats were mated on a one-to-one basis in the same group and their pups after weaning were also given this chemical at the same dose as their parents. All diets were prepared on a weekly basis. The first generation (F0) was treated from 32 days of age to 142 weeks, and the second (F1) generation was treated from 21 days of age to 127 weeks. The time-to-death for parental animals was not affected by treatment. The average body weight for parental animals were significantly lower in the 250 mg/kg group than that in the control group, but there is no information on the period showing this result. There was a statistically significant decrease in the litter size at postpartum day 1 and 4 in the 250 mg/kg bw group. When the average pup body weight at day 4 after birth was adjusted for litter size, there was a significantly lower average body weight at 250 mg/kg bw/day. Based on the available data, the NOAEL for reproductive/developmental toxicity is considered to be 25 mg/kg bw/day.

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the available data (NOAEL for reproductive/developmental toxicity in rats = 100 mg/kg bw/d), the test item is not classified for reproductive toxicity according to CLP, Regulation (EC) No. 1272/2008.