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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:
key study
Study period:
August 30, - December 10, 2010.
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Cross-reference
Reason / purpose:
reference to other assay used for intermediate effect derivation
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
October 2, 1991 - November, 1991.
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
- Source: Tama Chemical Industry Co., Ltd.
- Lot: 10705
- Purity of 99.7 %
Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Japan Co., Ltd.
- Age at study initiation: 5 weeks
- Weight at study initiation: 170-197 (males), 137-158 (females)
- Housing: 2 per cage (same sex), in polycarbonate cages (265 W x 426 D x 200 H mm: Tokiwa Scientific Instruments Co., Ltd.) .) laid with animal bedding (Beta chip: Nippon Charles River Co., Ltd.). The cage, feeder and water bottle were sterilised once per week.
- Diet: MF: Oriental Yeast Co., Ltd. ad libitum.
- Water: tap water, irradiated with ultraviolet light and filtered with a 5 µm filter, ad libitum.
- Acclimation period: 9 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-25ºC
- Humidity (%): 40-70%
- Air changes (per hr): 12 times/per hour
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark


IN-LIFE DATES: Obtained on October 2, 1991.
Route of administration:
oral: gavage
Vehicle:
olive oil
Remarks:
(Japanese Pharmacopoeia)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Olive oil (Japanese Pharmacopoeia) was added to the test substance, and the mixture was warmed at around 40 ºC and dissolved to a predetermined concentration;
the solution was prepared every 9 days, and it was kept in a cool dark place until immediately before administration.

VEHICLE
- Amount of vehicle (if gavage): the dosing volume was 5 ml/kg, based on the weight at the nearest measurement day.
- Purity: compliant with Japanese Pharmacopoeia standards.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stabilty of the solution (test item in olive oil) was analyzed, and it was confirmed that it was stable for 11 days after preparation, when stored in a cool dark place. No further details provided on the method. Accordingly, the test item solution was prepared every 9 days, and it was kept in a cool dark place until immediately before administration.
Duration of treatment / exposure:
28 days
Frequency of treatment:
Daily (once in the morning)
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
15 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:
6 males / 6 females per group in 15 mg/kg and 100 mg/kg groups.
12 males / 12 females per group in control and 500 mg/kg group.
Control animals:
yes
Details on study design:
- Dose selection rationale: Dose levels were selected based on a preliminary oral toxicity study. Repeated oral administration of the test substance to SD rats at doses of 125, 250 and 500 mg/kg for 7 days resulted in toxic signs such as salivation, loss of locomotor activity, weight gain suppression, liver weight gain at 500 mg/kg; at 125 and 250 mg/kg, toxic signs such as spontaneous motor activity decrease were also observed. Based on this study, the high dose of this study was 500 mg/kg, the middle dose was 100 mg/kg, and the low dose was 15 mg/kg. In addition to this, a control group (only vehicle) was provided.
- Administration period: 28-days in the three doses-group and control group
- Post-exposure recovery period: 14 days in 6 males / 6 females for control and high dose groups (satellite groups).
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS:
- Time schedule: daily for morbidity, mortality, behaviour (4 weeks for all groups, up to 42 days for both recovery groups).

DETAILED CLINICAL OBSERVATIONS
- Time schedule: detalied observations including palpation once a week (4 weeks for all groups, up to 42 days for both recovery groups).

BODY WEIGHT:
- Time schedule for examinations: at the beginning of treatment and once a week thereafter (4 weeks for all groups, up to 42 days for both recovery groups).

FOOD CONSUMPTION: Food consumption was measured, although it is not a 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 (4 weeks for all groups, up to 40 days for both recovery groups).

FOOD EFFICIENCY: No.

WATER CONSUMPTION AND COMPOUND INTAKE: Water consumption was measured, although it is not a drinking water study.
- Time schedule for examinations: the tare was measured once a week and compared (4 weeks for all groups, up to 42 days for both recovery groups).

OPHTHALMOSCOPIC EXAMINATION: No.

HAEMATOLOGY:
- Time schedule for collection of blood: suvirval animals at the time of each planned killing (28 or 42 days) were bled from posterior vena cava after anesthesia by pentobarbital sodium (Nembutal injection solution: Dynabot Corporation) intraperitoneally and examined on the following items: red blood and white blood cell count, platelet count, hemoglobin concentration, hematocrit value, leukocyte percentage, reticulocyte count, prothrombin time, activated partial thromboxane plastin time, mean red blood cell volume, mean red blood cell hemoglobin content and mean red blood cell hemoglobin concentration.

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: in the remaining blood used for hematalogical examination the following parameters were examined: total protein, albumin, A/G ratio, glucose, triglyceride, total cholesterol, urea nitrogen, creatinine, calcium, inorganic phosphorus, GOT (AST), GPT (ALT), γ-GTP, ALP, sodium, potassium, chloride.

URINALYSIS:
- Time schedule for collection of urine: fresh urine of the survival animals was collected before the end of the administration period (3 weeks) and on the end of th recovery period. The following parameters were determined: pH, protein, glucose, ketones, bilirubin, urobilinogen, ocult blood, specific gravity, sediment, urine volume, sodium, potassium and chlorine.

NEUROBEHAVIOURAL EXAMINATION: No.
IMMUNOLOGY: No.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes.
Surviving animals, at the time of each planned killing, were exsanguined, sacrificied and necropsied. Dead animals were necropsied as soon as they were found. Brain, liver, kidney, adrenal gland, testis and ovary were weighed.

HISTOPATHOLOGY: Yes.
The following organs were collected, fixed and stored: Harder's glands, brain, pituitary, eyeball (including accessory gland), lung, stomach, thyroid (including parathyroid), heart, liver, spleen, kidney, adrenal gland, bladder, testis or ovary, bone marrow (femur). The testes suspected of being altered, heart, liver, kidney, spleen, adrenal glands and those duodena that had shown changes in the autopsy, were microscopical examined. All the organs that were found abnormal at necropsy were examined too. Examinations were made in all dosing groups.
Statistics:
The equidistance test by the Bartlett method was performed, one-way ANOVA was performed when the variance was uniform and Kruskal-Wallis test was performed when the variance was not uniform. Where the were significant differences between the groups, the method of Dunnett was applied. For uirne qualtitative test and urine sediments, Armitage's test was used.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Reduced lococmotor activity and salivation were observed in the 500 mg/kg group.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female of the 500 mg/kg group died on day 13 of the administration period and another died on day 4 of the recovery period. Animals showed crouching, prone position, ataxic gait, tiptoe gait, clonic convulsions and gasping before death.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight gain was reduced in the 500mg/kg males and females group. A recovery tendency was observed in both sexs during the recovery period.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Food consumption decreased in 500 mg/kg group (males and females) in the first week of administration, but no signifcant differences were observed after that.
Water consumption and compound intake (if drinking water study):
effects observed, non-treatment-related
Description (incidence and severity):
Water intake was increased in the 500 mg/kg (males and females).
Ophthalmological findings:
not specified
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Reduction of mean red blood cell hemoglobin level and reduction of prothrombin time was detected in males of the highest dose group. Shortening of activated partial thromboplastin time and increase of platelet count were observed in females (highest dose group).
No change was observed at the end of the recovery period.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
A decrease in alkaline phospatase was observed in the 500 mg/kg males group and an increase in potassium in the 500 mg/kg males and females group.
At the end of the recovery period, an increase in alkaline phosphatase was observed in males (500 mg/kg). An increase in inorganic phosphorus in males was observed too, but it was within the physiological variation range.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Urine volume tended to increase in both sexes although no significant differences was observed. Urinary pH was increased in the 500 mg/kg females group.
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Males showed an increase in the liver/body weight ratio while females showed an increase in both actual weight and liver/body weight ratio in the 500 mg/kg group. Females in the 500 mg/kg recovery group showed an increase in liver weight but the difference with the control group was reduced. The weight of adrenal glands in females was decreased but there were no differences in the adrenal weight/ body weight ratios.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
There was an elargement of the duodenum with thickening of the mucosa in both sexes in the 500 mg/kg group. This change was not observed in the recovery group.

Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
In the surviving animals, changes in the liver and duodenum of the 500 mg / kg group due to the administration of the test substance were observed in both sexes: enlarged hepatocytes around the Glissons capsules, eosinophilic hepatocyte cytoplasm and thickening of the duodenal mucosa.
In addition, fading foci in the kidney cortex, cyst formation, localized yellowing in the liver and brown / dark red spots in the lung were observed in a few cases, but it was considered to be a contingent lesion.
Details on results:
After the recovery period, histological changes of the liver and duodenum dissapeared and other charged parameters tended to show values almost within the normal range.
Key result
Dose descriptor:
NOEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
body weight and weight gain
clinical biochemistry
histopathology: non-neoplastic
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
500 mg/kg bw/day (nominal)
System:
gastrointestinal tract
Organ:
duodenum
Treatment related:
yes
Dose response relationship:
not specified
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
500 mg/kg bw/day (nominal)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
not specified

Table 3. Body weight (group mean values)

 

Days after commencement/cessation of treatment

Dose level (mg/kg)

 

0

7

14

21

28/0

35/7

42/14

MALE

 

0

Mean

SD

N

185

8.5

12

255

14.2

12

322

19.4

12

382

26.8

12

423

34.5

12

457

47.3

6

484

48.5

6

15

Mean

SD

N

187

6.7

6

255

11.3

6

319

19.1

6

377

27.0

6

415

37.8

6

-

-

100

Mean

SD

N

186

5.2

6

253

6.7

6

325

8.7

6

383

12.6

6

424

13.6

6

-

-

500

Mean

SD

N

187

8.4

12

239

15.9

12

300

22.5

12

351

23.9

12

368

24.3

12

427

23.5

6

468

28.1

6

FEMALES

 

0

Mean

SD

N

146

5.4

12

176

9.5

12

205

13.2

12

231

15.4

12

249

17.3

12

259

21.5

6

271

23.6

6

15

Mean

SD

N

145

6.6

6

181

8.7

6

207

10.8

6

233

16.0

6

245

13.5

6

-

-

100

Mean

SD

N

146

6.1

6

182

5.0

6

211

5.0

6

234

5.4

6

251

3.6

6

-

-

500

Mean

SD

N

147

4.9

12

170

8.5

12

196

10.3

11

216

13.2

11

225

26.6

11

250

21.5

4

268

15.1

4

Table 4. Organ weight and ratio organ weight/body weight (6 animals per group per dose, treatment groups)

Dose level (mg/kg)

 

Final body weight (g)

Brain

Brain/

body weight

Liver

Liver/

body weight

Kidneys

Kidneys/

body weight

Adrenals(x10-3)

Adrenals /

body weight

Testes /Ovaries

(x10-3)

Test-ovar/body weight

 

 

 

(g)

%

(g)

%

(g)

%

(g)

%

(g)/ (mg)

 

MALE

 

 

 

 

 

 

 

 

 

 

 

 

0

Mean

SD

430

31.3

2.00

31.3

0.47

0.029

18.06

2.234

4.19

0.249

3.06

0.297

0.71

0.054

58.3

0.276

13.7

1.58

3.03

0.276

0.71

0.055

15

Mean

SD

415

37.6

1.97

0.089

0.48

0.051

17.61

2.925

4.22

0.316

3.01

0.210

0.73

0.021

61.3

7.57

14.8

1.69

3.24

0.464

0.79

0.178

100

Mean

SD

424

13.8

2.04

0.037

0.48

0.024

18.20

1.611

4.29

0.272

3.08

0.225

0.73

0.055

57.4

7.75

13.5

1.49

3.10

0.092

0.73

0.044

500

Mean

SD

4386

28.3

1.99

0.029

0.52

0.043

19.58

1.886

5.07 *

0.166

3.01

0.229

0.79

0.068

57.2

9.92

14.8

1.83

3.06

0.178

0.80

0.064

FEMALE

 

 

 

 

 

 

 

 

 

 

 

 

0

Mean

SD

251

18.0

1.92

0.065

0.77

0.058

9.54

1.031

3.60

.0236

1.89

0.059

0.76

0.056

64.9

7.60

26.0

3.42

95.5

9.64

38.1

3.36

15

Mean

SD

246

13.5

1.88

0.053

0.77

0.051

9.13

0.811

3.70

0.194

1.85

0.126

0.75

0.046

68.5

6.76

28.0

3.88

95.7

13.69

38.9

4.75

100

Mean

SD

251

2.6

1.90

0.052

0.76

0.024

9.62

0.539

3.84

0.244

1.89

0.258

0.76

0.106

67.0

6.61

26.8

2.78

83.3

14.11

33.2

5.48

500

Mean

SD

231

17.3

1.84

0.054

0.80

0.062

12.61*

0.796

5.43 *

0.328

1.87

0.109

0.81

0.039

63.9

7.25

27.9

4.95

86.0

13.53

37.2

4.67

* Significantly different from control value p < 0.01

Table 5. Organ weight and ratio organ weight/body weight (6 animals per group per dose, recovery groups)

Dose level (mg/kg)

 

Final body weight

Brain

Brain/

body weight

Liver

Liver/

body weight

Kidneys

Kidneys/

body weight

Adrenals(x10-3)

Adrenals /

body weight

Testes /Ovaries

(x10-3)

Test-ovar /body weight

 

 

(g)

(g)

%

(g)

%

(g)

%

(g)

%

(g)/ (mg)

 

MALE

 

 

 

 

 

 

 

 

 

 

 

 

0

Mean

SD

485

47.8

2.10

0.049

0.44

0.039

20.80

4.627

4.27

0.524

3.57

0.314

0.74

0.023

63.4

9.21

13.2

2.29

3.36

0.272

0.70

0.081

500

Mean

SD

467

27.8

2.11

0.077

0.45

0.020

20.39

2.464

4.35

0.260

3.33

0.280

0.71

0.028

57.1

9.62

12.3

2.64

3.22

0.245

0.69

0.036

FEMALE

 

 

 

 

 

 

 

 

 

 

 

 

0

Mean

SD

271

23.7

1.9

0.061

0.73

0.059

10.20

1.640

3.75

0.284

19.99

0.166

0.74

0.056

76.7

5.68

28.6

4.34

110.2

15.88

40.7

5.62

500

Mean

SD

268

15.0

1.91

0.043

0.71

0.038

12.58

0.638*

4.69 *

0.080

1.97

0.137

0.74

0.045

66.4 *

6.44

24.9

3.65

113.1

11.99

42.2

3.15

* Significantly different from control value p < 0.01

Table 6. Total incidence macroscopic and microscopic findings

MACROSCOPIC FINDINGS

 

28 DAYS

RECOVERY

 

SEX

MALE

FEMALE

MALE

FEMALE

 

DOSE LEVEL (MG/KG)

0

15

100

500

0

15

100

500

0

500

0

500

ORGAN FINDINGS

NUMBER OF ANIMALS

6

6

6

6

6

6

6

6

6

6

6

4

Duodenum

enlargement

0

0

0

5

0

0

0

4

0

0

0

0

Kidneys

Focal discoloration

Cyst

 

0

0

 

1

0

 

0

0

 

0

0

 

0

0

 

0

0

 

0

1

 

0

0

 

0

0

 

0

0

 

0

0

 

0

0

Liver

Focal yellowish change

0

0

0

0

0

0

0

0

1

0

0

0

Lungs

Brownish/dark patch

0

0

0

0

0

0

1

0

0

1

0

0

MICROSCOPIC FINDINGS

 

28 DAYS

RECOVERY

 

SEX

MALE

FEMALE

MALE

FEMALE

 

DOSE LEVEL (MG/KG)

0

15

100

500

0

15

100

500

0

500

0

500

ORGAN FINDINGS

NUMBER OF ANIMALS

6

6

6

6

6

6

6

6

6

6

6

4

Liver

Enlarged/eosinophilic hepatocytes

Neutrophilic infiltration

Focal fatty change

 

0

 

0

 

0

 

0

 

0

 

0

 

0

 

0

 

0

 

5

 

0

 

0

 

0

 

0

 

0

 

0

 

0

 

0

 

0

 

0

 

0

 

6

 

1

 

0

 

0

 

0

 

1

 

0

 

0

 

0

 

0

 

0

 

0

 

0

 

0

 

0

Duodenum

thickening of mucosa

0

0

0

5

0

0

0

4

0

0

1/5

0

Heart

Focal myocardial degeneration

 

0

 

-

 

-

 

1

 

0

 

-

 

-

 

0

 

-

 

-

 

-

 

-

Kidneys

Basophilic change of tubular epithelium

Focal linphocytic in interstitium

Hyaline droplets in tubular epithelium

Cyst

 

3

 

1

0

 

0

 

 

0/1

 

0/1

1/1

 

0/1

 

 

-

 

 

2

 

0

0

 

0

 

 

1

 

1

0

 

0

 

-

 

0/1

 

0/1

0/1

 

1/1

 

2

 

2

0

 

0

 

-

-

-

-

Lungs

Focal hemorrhage into alveoli

0

0

0

0

0

0

1

0

0

1

0

0

- not examined

Conclusions:
The NOEL was determined to be 100 mg/kg bw/day in male and female rats.
Executive summary:

A 28-day repeated dose toxicity test was performed according toaccording to the Guidelines on Chemical Substitution Law (1986) (Guideline 28-Day Repeat Dose Toxicity Test of Chemical, Japan), similar to OECD 407 (GLP study). SD (Crj: CD) rats were treated with 0, 15, 100 and 500 mg/kg of the test substance by oral gavage for 28 days. 6 males and 6 females were selected for the groups of 15 and 100 mg/kg and 12 males/12 females for the control and 500 mg/kg groups. After the 28-days treatment, 6 males and 6 females for the control and 500 mg/kg groups were left to study their recovery over a period of 14 days. Animals of both sexes in the 500 mg/kg group showed reduced locomotor activity, salivation, inhibition of body weight gain, decreased food consumption and increased water intake. Two females in the 500 mg/kg dose group died, one during treatment and one during the recovery period. A decrease in alkaline phosphatase was also observed in males in the 500 mg / kg group. Pathological examination revealed increased relative liver weight, enlargement of the duodenum due to mucosal thickening and hepatocellular swelling in both sexes of the higher dose group. Histological changes in the liver and duodenum disappeared due to discontinuation of administration, and other changes also disappeared or showed a recovery tendency after the 14 days post-exposure. No effects were observed in the 100 and 15 mg/kg group in both males and females. Based on the above results, the NOEL under the test conditions was considered to be 100 mg/kg/day in both sexes.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
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 the 28-day repeated dose toxicity test (See 'Cross-reference'): In this test, animals were dosed with 15, 100 or 500 mg/kg test item for 28 days. In the high dose group, 2 females died, and reduced locomotor activity, supression of body weight gain, increase in liver weight, thickening of the duodenal mucosa, etc. were observed in the remaining animals; no effects were observed at 100 mg/kg bw. In the range finding study, toxicity symptoms such as reduced locomotor activity were also observed in animals dosed with 125 and 250 mg/kg test item for 7 days. Based on the available information, the high dose of this study was set at 250 mg/kg bw, and the spacing factor was 2.5, so the medium dose was 100 mg/kg bw and the low dose 40 mg/kg bw.
- Route of administration: oral, gavage

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:
- Purity: 99.1%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Refrigerated until use, storage under light shielding (measured temperature 3 to 7 ° C).
- Stability under test conditions: The stability of the test substance was confirmed at Hadano Laboratory before administration (August 30, 2010) and after the end of the administration period (December 10, 2010).

Test animals

Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl: CD (SD), SPF
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Atsugi Breeding Center (Japan).
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (P) 10 wks.
- Weight at study initiation: (P) Males: 354.0 to 436.0 g; Females: 215.5 to 280.2 g.
- Housing: individually housed, 2 animals per cage at the time of mating.
- Diet: ad libitum feed (CE-2, CLEA Japan).
- Water: ad libitum tap water.
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 25ºC
- Humidity (%): 40 - 75%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12h light/dark cycle.

IN-LIFE DATES: From: September 1, 2010 (62 males and 73 females, arrival date).

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
olive oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The medium was warmed at 40ºC or less, and the test item was weighed and added to the solution, at a concentration of 5% (w/v). Two further solutions were prepared at concentrations of 2 and 0.8 % (w/v). These solutions were stored in a refrigerated space (3 - 10ºC) and shielded from light, and used within 7 days after preparation (storage: from September 14th to October 30th, 2010). The doses were administered by gavage.

VEHICLE
- Justification for use and choice of vehicle (if other than water): the test item was found to be moisture sensitive; the stability of the test item in olive oil was confirmed by analysis.
- Concentration in vehicle: 5, 2 and 0.8% (w/v)
- Amount of vehicle (if gavage): 5 mL/kg
- Purity: according to Japanese Pharmacopoeia.
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: 2 weeks
- Proof of pregnancy: vaginal plug and sperm in vaginal smear, referred to as [day 0] of pregnancy.
- Further matings after two unsuccessful attempts: no.
- After successful mating each pregnant female was caged (how): females were individually housed in a plastic breeding cage for rats (350 w × 400 d × 180 h mm) from 18th day of pregnancy to 4th day of nursing, and a paper pulp chips (Pepper Clean, Japan SLC) were used as bedding. No anomalies in the breeding environment were observed during the period, the measured value of the temperature in the animal room was 23.5 to 24.0ºC, the measured value of humidity was 52.0 to 70.0%. In addition, it was confirmed that the analysis results of the feed, drinking water and bedding that were supplied were within the tolerance limits described in the standard operating procedure manual.
- Any other deviations from standard protocol: no.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test item content was measured by a validated analytical method after preparation of the dosing solution; the average content was found to be 90 - 110%, and the variation between measurements was within the acceptable range (average value ± 10%). No further details on analytical method provided (Annex D-1, D-2 not included).
Duration of treatment / exposure:
Males were treated from 2 weeks before mating until the day before necropsy (42 administrations in total). Females were treated from 2 weeks before mating until delivery.
Frequency of treatment:
daily.
Details on study schedule:
- Animals arrival date: September 1, 2010
- Quarantine end date: September 14, 2010
- Dosing prior to mating: 2 weeks
- Mating: up to 2 weeks
- Age at mating of the mated animals in the study: [12] weeks
- End of administration period: December 10, 2010
Doses / concentrationsopen allclose all
Dose / conc.:
40 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
No. of animals per sex per dose:
13 male and 13 female per group.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: the dose of this study was set based on the results of the 28-day repeated dose toxicity test (See 'Cross-reference'): In this test, animals were dosed with 15, 100 or 500 mg/kg test item for 28 days. In the high dose group, 2 females died, and reduced locomotor activity, supression of body weight gain, increase in liver weight, thickening of the duodenal mucosa, etc. were observed in the remaining animals; no effects were observed at 100 mg/kg bw. In the range finding study, toxicity symptoms such as reduced locomotor activity were also observed in animals dosed with 125 and 250 mg/kg test item for 7 days. Based on the available information, the high dose of this study was set at 250 mg/kg bw, and the spacing factor was 2.5, so the medium dose was 100 mg/kg bw and the low dose 40 mg/kg bw.
Positive control:
Not required.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily before and after administration during administration and once daily during other rearing periods.

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes
- Time schedule for examinations: Male animals were weighed on the day 1 (administration start date), 7, 14, 21, 28, 35, 42 days and on the autopsy day. Female animals were weighed on days 1, 7 and 14 of administration, pregnancy 0 (mating confirmation date), 7, 14, 20 days, day of delivery, 4 days after and on the autopsy day. If any animals died, they were weighed at the time of death.

FOOD CONSUMPTION AND COMPOUND INTAKE (not a feeding study):
- Male animals were measured for food intake on days 1 to 2, 7 to 8, 14 to 15, 29 to 30, 35 to 36, and 41 to 42.
- Female animals were measured for food intake on days 1 to 2, 7 to 8, 14 to 15 of administration; days 0 to 1, 7 to 8, 14 to 15, 20 to 21 of gestation and days 3 to 4 of rearing.
Oestrous cyclicity (parental animals):
For each group, vaginal smear specimens were prepared every day after the start of administration following the sexual cycle observation up to the grouping day, and the sex cycle was observed until the copulation was confirmed. In addition, the average number of days from estrous period to estrous period was calculated for each individual.
Sperm parameters (parental animals):
Parameters examined in [P] male parental generations:
testis weight, epididymis weight, sperm count in testes, sperm count in epididymides.
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.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: Not specified.

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: Not specified.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals as soon as possible after the last litters were produced.
- Maternal animals: All surviving animals after the last litter of each generation was weaned (day 4).

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.
- In males: liver, duodenum, prostate, seminal vesicle (including coagulated gland), mammary gland.
- In females: liver, duodenum, ovary, uterus, vagina, mammary gland. If the female or all babies died, also: heart, brain, kidney, lung. In females that had copulated but did not deliver or that died before delivery, liver and ovarian weights were measured.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated were prepared for microscopic examination and weighed:
- In males, histopathological examination was performed on the liver, duodenum, testis, epididymis (0.1 M phosphate buffered 10% formalin solution).
- In females, histopathological examination was performed on the liver, duodenum and ovaries. In females that had copulated but did not deliver or that died before delivery, the number of corpus luteum was counted under a stereoscopic microscope and the implantation rate [(number of implantation / number of pregnant corpus luteum) X 100,%] was determined. In females that died or when all babies died, histopathological examinations also included: heart, brain, kidney, lung.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at [4] days of age.
- These animals were subjected to postmortem examinations as follows: all pups were subject to macroscopic examination, and any organ presenting abnormalities was fixed in 0.1 M phosphate buffered 10% formalin solution and stored.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.
Statistics:
Fisher's exact test of significance (5%) was carried out on the frequency, the mating rate, the conception rate and the morphological abnormality frequency of the babies.From the histopathological findings of the treated groups, the graded pathological tissue findings were determined by Marm-Whitney's U test and the total value of positive groups was determined by Fisher's one-sided probability test. Significant difference test with the group was conducted (significance: 5%).
For the other data, a value obtained for each individual or an average value for each litter was taken. First, the uniformity of variance of each group was tested (significance: 5%) by Bartlett's method. When the variance was uniform, a one-way analysis of variance (significance level: 5%) was performed, and when significance was found between groups, multiple comparisons were performed by the Dmrnett method (significance Level: 5%). Finally, Kruskal-Wallis's rank test (significance level: 5%) was performed when the variance was 0 in any group and when the variance was not uniform, significance was observed between the groups In the case of multiple comparison by Dunnett type test method (significance level: 5%).
Reproductive indices:
- Copulation index = [Number of copulated pairs / number of mated pairs (%)]
- Fertility index = [number of fertile males / copulation index x 100 (%)]
- Implantation index = [Number of implantation scars / number of corpora lutea x 100 (%)]
Offspring viability indices:
- Gestation index = [Number of dams with live offspring / number of pregnant dams x 100 (%)]
- Sex ratio = [Number of male offspring / (number of male + female offspring)]
- Delivery index = [Number of offspring at birth / number of implantation scars x 100 (%)]
- Birth index = [Number of live offspring at birth / number of implantation scars x 100 (%)]
- Live birth index = [Number of live offspring at birth / number of offspring at birth x 100 (%)]
- Viability index = [Number of live offspring 21 days after birth / number of live offspring after culling x 100 (%)]

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Mortality:
mortality observed, treatment-related
Description (incidence):
Females: one animal in the 100 mg/kg group died at perinatal stage, five in the 250 mg/kg group (table 2).
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In the 250 mg/kg administration group, suppression of increase in body weight was observed in both males and females. No significant differences with the control were observed at any other dose.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In the 250 mg/kg administration group, female food intake decreased. No effects were observed at 100 mg/kg or less.
Food efficiency:
not specified
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
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The liver weight of the 250 mg / kg administration group showed an increasing trend in both males and females. There was no significant difference between the treated group and the control group in absolute weight and relative weight of testis, epididymis, prostate (vaginal), seminal vesicle and ovary.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
- Males: miniaturisation of the testis at 100 and 250 mg/kg; thickening of the intestinal wall of the duodenum.
- Females: In the 250 mg/kg group, thickening of the intestinal wall of the duodenum was seen in 1 animal, darkening of the lungs in 3 animals, and emphysema in 1 animal, spleen miniaturisation in all animals, dark spots in the stomach mucosa in 4 animals, and red urine in 2 animals. In females that died during pregnancy or parturition, the fetuses remained in the uterus.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histopathological examination of the liver revealed bile duct expansion and diffuse Kupffer cell proliferation around the Gleason sheath in males and females at 250 mg / kg administration group. On histopathological examination of the duodenum, thickening of the mucosa was observed in males and females at the dose of 250 mg / kg. Degeneration / necrosis of the proximal renal tubular epithelium in the kidney cortex was observed in females whose whole litter died by nursing day 4.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
The female sex cycle, mating rate and conception rate, number of corpus luteums of pregnant animals, implantation number, implantation rate and pregnancy period were not affected by administration of test substance.
The estrous regression days in the 250 mg/kg group increased, but it had been observed before administration that the same group's estrus regression days tended to be extended.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
testis, epididymis, prostate and seminal vesicle weights were not affected by the administration of the test substance.
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
Failure of labor condition was observed in the group administered 100 and 250 mg / kg, and the birth rate decreased: the number of births and the number of babies born decreased, the live birth rate and the birth rate decreased. No effects on delivery rate, neonatal survival rate or sex ratio.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
body weight and weight gain
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Effect level:
40 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There was no effect of administration of the test substance on the sex ratio, the form and weight of the newborn infant.
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
The birth rate, fertility rate and neonatal survival rate decreased in the 250 mg/kg administration group.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There was no effect of administration of the test substance on the sex ratio, the form and weight of the newborn infant.
Food efficiency:
not specified
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
Other effects:
no effects observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

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:
clinical signs
mortality

Overall reproductive toxicity

Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
100 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

Any other information on results incl. tables

Table 2. Mortality in parental animals.

Animal number

Dose

(mg/kg)

Death period

Internal uterine findings

at necropsy

F03002

100

During labor

8 fetus remains

F04001

250

Nursing 3rd

No abnormality

F04004

250

Pregnancy 23rd

15 fetus remains

F04006

250

During labor

2 fetus remains

F04008

250

During labor

10 fetus remains

F04009

250

During labor

1 fetus remains

Table 3. Body weights of male rats.

Group

 

Olive oil (vehicle)

DCC

40mg/kg

DCC

100mg/kg

DCC

250mg/kg

Number of males

 

13

13

13

13

Days of administration

1

397.4

±

15.0

402.5

±

14.7

397.0

±

10.8

400.3

±

14.7

 

7

429.3

±

19.5

427.2

±

22.0

421.2

±

14.8

420.7

±

16.6

 

14

459.0

±

24.9

455.4

±

24.8

449.9

±

20.0

445.0

±

20.3

 

21

481.7

±

24.1

479.8

±

26.1

470.2

±

22.0

460.9

±

23.6

 

28

510.7

±

26.4

502.4

±

26.5

494.1

±

24.1

485.5

±

29.5

 

35

533.7

±

30.6

526.6

±

26.3

516.2

±

28.0

506.9

±

32.2

 

42

554.0

±

31.8

547.2

±

30.3

535.6

±

31.0

525.5

±

35.5

Each value shows mean (g)±S.D.

Significantly different from the control group (*: P<0.05, **: P<0.01).

Table 4. Body weights of female rats.  

Group

 

Olive oil (vehicle)

DCC 40 mg/kg

DCC 100 mg/kg

DCC 250 mg/kg

Number of females

 

13

13

13

13

Days of administration

 

 

 

 

 

 

1

247.3 ± 11.9

248.1 ± 9.4

246.1 ± 12.2

250.2 ± 11.6

 

7

258.6 ± 12.3

258.9 ± 10.3

259.7 ± 12.0

257.5 ± 13.9

 

14

270.9 ± 15.9

270.9 ± 15.9

266.1 ± 16.7

260.7 ± 16.2

Number of dams

 

13

12

12

12

Days of pregnancy

0

280.9 ± 12.6

279.7 ± 14.4

276.9 ± 19.4

267.5 ± 15.3

 

7

314.9 ± 17.1

309.7 ± 17.0

308.1 ± 21.4

293.9 ± 17.5 *

 

14

351.6 ± 17.8

342.1 ± 18.4

347.7 ± 25.8

326.4 ± 18.5 **

 

20

432.6 ± 25.8

415.0 ± 20.8

423.5 ± 30.0

392.3 ± 22.9 **

Number of dams

 

13

12

11

10

Days of lactation

0

323.8 ± 24.3

315.8 ± 34.0

307.7 ± 34.2

291.5 ± 36.1

 

4

339.6 ± 26.7 (12)

335.3 ± 20.8 (10)

331.2 ± 26.9 (10)

325.2 ± 13.4 (4)

Each value shows mean (g) ± S.D. Significantly different from the control group (*: P<0.05, **: P<0.01). Figures in parentheses indicate number of dams.

 

Table 5. Organ weights of male rats

Group

 

Olive oil (vehicle)

DCC 40 mg/kg

DCC 100 mg/kg

DCC 250 mg/kg

Number of males

 

13

13

13

13

Body weight

(g)

528.4 ± 33.5

521.6 ± 28.4

507.6 ± 28.9

491.7 ± 33.8

Liver

(mg)

14917.5 ± 1803.5

14677.0 ± 1145.0

15266.9 ± 2121.6

15699.2 ± 1366.2

 

(mg/g)

28.160 ± 2.004

28.149 ± 1.780

29.991 ± 2.914

31.930 ± 1.807**

Testes

(mg)

3257.5 ± 563.1

3441.9 ± 243.3

3293.5 ± 419.8

3387.7 ± 532.2

 

(mg/g)

6.209 ± 1.230

6.618 ± 0.603

6.512 ± 0.912

6.922 ± 1.228

Epididymides

(mg)

1192.3 ± 194.7

1301.6 ± 102.0

1198.8 ± 88.7

1266.9 ± 158.8

 

(mg/g)

2.268 ± 0.406

2.501 ± 0.223

2.368 ± 0.207

2.588 ± 0.377

Prostate, ventral

(mg)

626.5 ± 101.8

637.6 ± 162.5

638.5 ± 130.1

650.3 ± 104.6

 

(mg/g)

1.194 ± 0.229

1.226 ± 0.313

1.256 ± 0.246

1.331 ± 0.245

Seminal vesicles

(mg)

1907.7 ± 430.4

1669.7 ± 338.9

1837.3 ± 216.5

1752.7 ± 126.9

 

(mg/g)

3.614 ± 0.781

3.205 ± 0.638

3.617 ± 0.336

3.570 ± 0.214

Each value shows mean±S.D. Significantly different from the control group (*: P<0.05, **: P<0.01).

Table 6. Organ weights of female rats

Group

 

Olive oil (vehicle)

DCC 40 mg/kg

DCC 100 mg/kg

DCC 250 mg/kg

Number of females

 

12

10

10

4

Body weight

(g)

307.7±25.5

306.0±20.4

297.5±23.7

289.6±20.6

Liver

(mg)

10146.4±740.0

10223.8±555.2

15266.9±2121.6

15699.2±1366.2

 

(mg/g)

33.120±3.014

33.501±2.342

34.760±2.501

35.956±4.321

Ovaries

(mg)

105.5±9.7

102.9±11.6

109.0±12.9

97.6±9.8

 

(mg/g)

0.344±0.039

0.336±0. 032

0.367±0.044

0.338±0.033

Table 7. Reproductive performance.

Group

Olive oil (vehicle)

DCC 40 mg/kg

DCC 100 mg/kg

DCC 250 mg/kg

Number of pairs

13

13

13

13

Number of copulated pairs

13

13

13

13

Copulation index

100

100

100

100

Number of fertile males

13

12

12

12

Fertility index

100

92.3

92.3

92.3

Pairing days until copulation

2.6±1.3 (13)

3.2±1.0 (13)

2.8±1.2 (13)

2.6± 1.0(13)

Table 8. Reproduction/developmental toxicity.

Group

Olive oil (vehicle)

DCC 40 m/kg

DCC100mg/kg

DCC250mg/kg

Number of dams

13

12

11

 

8

 

Gestation length (days)

Mean ± S.D. per dam

Number of corpora lutea

22.5 ± 0.5

22.3 ± 0.5

22.5 ± 0.5

 

22.5 ± 0.5

 

Total

216

187

202

 

193

 

Mean ± S.D. per dam

Number of implantation scars

16.6 ± 2,3

15.6 ± 1.4

16.8 ± 1.8

(12)

16.1 ± 1.9

(12)

Total

198

173

190

 

175

 

Mean ± S.D. per dam

15.2 ± 2.0

14.4 ± 1.4

15.8 ± 1.9

(12)

14.6 ± 5.3

(12)

Implantation index (%)

92.2 ± 8.7

92.7 ± 7.1

94.3 ± 7.6

(12)

90.7 ± 19.4

(12)

Gestation index (%)

100.0

100.0

91.7

 

66.7

 

Number of offspring at birth

 

 

 

 

 

 

Total

184

164

161

 

101

 

Mean ± S.D. per dam

Number of live offspring at birth

14.2 ± 2.8

13.7 ± 1.4

14.6 ± 1.4

 

12.6 ± 4.3

 

Male

87

69

78

 

39

 

Female

92

90

73

 

39

 

Total

179

159

151

 

78

 

Mean ± S.D. per dam

Sex ratio

13.8 ± 2.6

13.3 ± 1.4

13.7 ± 2.1

 

9.8 ± 5.7

 

Mean±S.D. per dam

Number of dead offspring

0.49 ± 0.06

0.43 ± 0.10

0.51 ± 0.12

 

0.40 ± 0.28

 

Total

5

5

10

 

23

 

Mean ±S.D. per dam

Delivery index

0.4 ± 0.9

0.4 ± 0.7

1.9 ± 3.9

 

2.9 ± 4.5

 

Mean%±S.D. per dam

Birth index

92.1 ± 8.9

94.8 ± 5.5

95.3 ± 6.6

 

90.6 ± 12.4

 

Mean% ±S.D. per dam

Live birth index

89.8 ± 9.5

910 ± 5.6

81.9 ± 28.3

 

72.2 ± 35.0

 

Mean% ±S.D. per dam

97.6 ± 5.4

97.1 ± 4.6

93.9 ± 12.1

 

77.6 ± 33.2

 

Number of offspring on day 4

 

 

 

 

 

 

Male

78

58

72

 

28

 

Female

Sex ratio

83

74

63

 

17

 

Mean ±S.D. per dam

Viability index

0.49 ± 0.06 (12)

0.44 ± 0.09 (10)

0.53 ± 0.11

(10)

0.62 ± 0.09

(4)

Mean% ±S.D. per dam

90.9 ± 27.4

83.3 ± 38.9

86.2 ± 31.7

 

57.1 ± 53.5

(7)

Number of external abnormalities 0

0

0

0

 

0

 

Mean% ± S.D. per dam

0

0

0

 

0

 


Applicant's summary and conclusion

Conclusions:
The NOAEL (based on toxicity) for both parent animals and first generation male/female Sprague Dawley rats was found to be 100 mg/kg bw; the NOAEL (based on reproductive ability) for parent male/female Sprague Dawley rats was found to be 40 mg/kg bw.
Executive summary:

A Reproduction / Developmental Toxicity Screening Test was conducted on the test item, according to OECD Guideline 421 (GLP study). 13 male and 13 female Sprague Dawley rats per group were exposed to 0 (control), 40, 100 or 250 mg/kg bw of test item by oral gavage, based on the results of a 28 -day repeated dose toxicity test performed previously. Males were dosed up to the day of conception and females were dosed throughout the study. Observations included clinical observations, body weight measurements, food intake, ostreus cycles, mating procedures, behavioural studies, evaluation of reproduction, weight of organs, and histopathological examinations. Toxicity signs were observed at the highest dose (lower body weight, enlarged liver, thickening of duodenum walls), and effects on reproduction were observed at the mid and high doses (lower birth rate, maternal deaths). Based on the available information, the NOAEL for general toxicity was set at 100 mg/kg bw, both for P0 and F1 generations, and the NOAEL for reproductive toxicity was set at 40 mg/kg bw.