Registration Dossier

Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1991

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
- Source: Tama Chemical Industry Co., Ltd.
- Lot: 10705
- Purity of 99.7 %

Test animals

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.

Administration / exposure

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)
Doses / concentrationsopen allclose all
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).

Examinations

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.

Results and discussion

Results of examinations

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.

Effect levels

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

Target system / organ toxicity

open allclose all
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

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.