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28 Day Oral Study, Imatanaka (1995)

Under the conditions of this study, the NOEL of the test material for rats in this study was considered to be 40 mg/kg/day since increase in total cholesterol, decreases in platelet and activated partial thromboplastin time were noted in the 200 mg/kg females.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31 January 1995 to 16 June 1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Version / remarks:
1981
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: '28-day Repeated Dose Toxicity Study in Mammalian Species" prescribed in "The Notification on Partial Revision of Testing Methods Relating to New Chemical Substances'
Version / remarks:
Notification No. 700 of Kanpogyo, No. 1039 of Yakuhatsu, and No. 1014 of 61 Kikyoku, Dec. 5, 1986
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 5 weeks old
- Weight at study initiation: 142.6 - 163.9 g for males and 120.1 - 136.5 g for females
- Fasting period before study:
- Housing: The animals were housed individually in a hanging stainless steel cage with wire-mesh floor (165 W x 300 D x 150 H mm). The trays were changed twice a week, cages once a week and racks once/two weeks.
- Diet: ad libitum
- Water: ad libitum
- The animals were quarantined and acclimatised and healthy animals with favourable weight gains were allocated to groups to ensure the homogeneity of the mean body weight using a body weight-stratified randomisation for the test.

DETAILS OF FOOD AND WATER QUALITY: The diet and housing materials were autoclaved at 121°C for 30 minutes prior to use. Analysis of contaminants in both the diet and drinking water confirmed that they would not affect the test system.

ENVIRONMENTAL CONDITIONS
- Temperature: 23 ± 2°C
- Humidity: 55 ± 10%
- Air changes: 10 - 15 air changes per hour
- Photoperiod: artificial light for 12 hours (between 7:00 and 19:00)
Route of administration:
oral: gavage
Details on route of administration:
Treatment by oral gavage was carried out daily using a Nelaton catheter (Terumo Corporation) and a syringe (Terumo Corporation) in the morning.
Vehicle:
other: Arabia gum (5% for final volume) mixed with purified water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
- The test material and Arabia gum (5% for final volume) were accurately weighed, and mixed with purified water to make 4 concentrations of 10.0, 2.0, 0.4 and 0.08 w/v%. These were prepared once a week.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- The stability and uniformity of the test material in the test concentration preparations were confirmed by on site laboratories.
Duration of treatment / exposure:
28 days of treatment followed by a 14 day recovery period
Frequency of treatment:
Once daily
Dose / conc.:
8 mg/kg bw/day (nominal)
Dose / conc.:
40 mg/kg bw/day (nominal)
Dose / conc.:
200 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
6 animals per sex per dose (plus additional 6 animals per sex at 1000 mg/kg for recovery period observations)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: A 14-day repeated-dose preliminary toxicity study was carried out at three doses of 50, 250 and 1,000 mg/kg. As a result, there were abnormalities in blood chemical examinations in the 250 and 1,000 mg/kg groups. In the main study the maximum dose was chosen at 1,000 mg/kg and three lower doses at 200, 40 and 8 mg/kg. Recovery groups were set at 1,000 mg/kg and the vehicle control groups.
Observations and examinations performed and frequency:
- The day of the start of dosing was defined as day 1, and the day before as day -1. The week of the start of dosing period was defined as week 1. Also, the next day of final dosing was defined as recovery day 1, and the week of the start of recovery period as recovery week 1.

CAGE SIDE OBSERVATIONS: Yes
- All animals were observed at least once per day.

BODY WEIGHT: Yes. All animals were weighed as follows:
- Before dosing: day -2 (at the time of grouping)
- During the dosing period: days 1 (at the start of dosing), 3, 5, 8, 10, 12, 15, 17, 19, 22, 24, 26 and 28
- During the recovery period: days 1 (at the start of the recovery period), 3, 5, 8, 10, 12 and 14
- In addition, immediately before necropsy, body weights were measured for calculation of relative organ weights.

FOOD CONSUMPTION: Yes. Food consumption was measured as follows:
- Before dosing: Once
- During the dosing and recovery periods: Twice a week

HAEMATOLOGY: Yes
- All animals were fasted overnight (16 - 20 hours) at the end of the dosing and recovery periods, and blood samples were taken via abdominal aorta from rats under ether anaesthesia. Sodium citrate was used as an anticoagulant for examinations of prothrombin time and activated partial thromboplastin time, and EDTA-2K was used for another parameters.
- The following parameters were examined for the blood and plasma samples obtained: Red blood cell count (RBC), White blood cell count (WBC), Haemoglobin concentration (Hb), Haematocrit value (Ht), Mean corpuscular volume (MCV), Mean corpuscular haemoglobin (MCH), Mean corpuscular haemoglobin concentration (MCHC), Platelet count, Reticulocytes count, Prothrombin time (PT), Activated partial thromboplastin time (APTT) and Differentiation of leukocytes (%): Band form neutrophils (N-Band), Segmental neutrophils (N-Seg), Eosinophils (Eosino), Basophils (Basa), Lymphocytes (Lymph) and Monocytes (Mono).

CLINICAL CHEMISTRY: Yes
- Sera were separated from the blood samples used in haematological examinations, and examined as follows: GOT, GPT, Alkaline phosphatase (ALP), Cholinesterase (ChE), γ-GTP, Total cholesterol (T-Cho), Triglyceride (TG), Glucose, Total protein (T-Protein), Albumin, A/G ratio, Blood urea nitrogen (BUN), Creatinine, Total bilirubin (T-Bil), Ca, IP, Na, K and Cl.

URINALYSIS: Yes
- Sixteen-hour urine samples were collected in individual metabolic cages, from all animals at day 28 and recovery day 14 and examined for volume, colour and additional items of pH, protein, ketone bodies, bilirubin, occult blood, glucose and urobilinogen, which were tested using a test paper (N-Multistix®, Miles·Sankyo).
Sacrifice and pathology:
GROSS PATHOLOGY: Yes all animals were necropsied in detail.
- The following organs were weighed wet in all animals: Brain, liver, spleen, kidneys, adrenal glands, testes (or ovaries)

HISTOPATHOLOGY: Yes
- The following organs and tissues from all animals were preserved in 10% formalin: Brain (cerebrum, cerebellum), hypophysis, eyeball, thyroid glands (with parathyroid glands), heart, lung, liver, kidneys, spleen, adrenal glands, stomach, intestine (duodenum to rectum), testes (or ovaries), urinary bladder, bone marrow (femur), gross legions
- Light microscopic examinations were performed on the following organs and tissues after paraffin embedding and sectioning followed by haematoxylin and eosin staining at Bio Pathology Institute Ltd

Dosing period
Vehicle control and 1,000 mg/kg groups: Liver, spleen, kidneys, heart, stomach, intestine ( duodenum, jejunum, ileum, cecurn, colon, rectum) and adrenal glands.
200 mg/kg group: Kidneys (male)

Recovery period
Vehicle control and 1,000 mg/kg groups: kidneys (male)

Gross lesions
Dosing period
Male: Vehicle control group (No. 1): Testes, 8 mg/kg group (No. 14): Eye ball and 200 mg/kg group (No. 30): Eyeball.
Female, 40 mg/kg group (No. 64): Kidneys
Statistics:
- Data regarding body weights, food consumption, haematological examination, blood chemical examination, urine volume and organ weights were analysed using Bartlett's test for homogeneity of variance. If the variances were homogeneous at a significance level of 5%, one way analysis of variance was performed. When there was a significant difference in this analysis, the difference between the vehicle control group and each of the treatment group was analysed by Dunnett's test (equal number of data) or Scheffe's test (unequal number of data).
- If the variances were not homogeneous in the Bartlett's test, Kruskal-Wallis's test was used. When there was a significant difference in this test, the difference between the vehicle control group and each of the treatment group was analysed by nonparametric Dunnett's test (equal number of data) or non-parametric Scheffe's test (unequal number of data).
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
During the dosing period
- Male: Scab formation in cervical region (1/6), dark reddish change of right eye ball (1/6), loss of left upper incisor (1/6) in the 8 mg/kg group, and soft faeces (1/6) in the 200 mg/kg group were noted.
- Female: No abnormalities were noted.

During the recovery period
- No abnormalities were noted in both sexes.
Mortality:
no mortality observed
Description (incidence):
There were no deaths on account of administration of the test material.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
During the dosing period
- No abnormalities were noted in both sexes.

During the recovery period
- No abnormalities were noted in both sexes.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
During the dosing period
- No abnormalities were noted in both sexes.

During the recovery period
- No abnormalities were noted in both sexes.
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):
At the end of the dosing period
- Male: No abnormalities were noted.
- Female: Decreases in platelet count and activated partial thromboplastin time were noted in the 200 and 1000 mg/kg groups.

At the end of the recovery period
- Male: No abnormalities were noted.
- Female: Decrease in mean corpuscular haemoglobin concentration was noted in the 1000 mg/kg group.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
At the end of the dosing period
- Male: Increase in total cholesterol and decrease in alkaline phosphatase were noted in the 1000 mg/kg group.
- Female: Increase in total cholesterol in 200 and 1000 mg/kg groups, and decrease in GOT in the 8 and 40 mg/kg groups, and decrease in GPT in the 40 mg/kg group were noted.

At the end of the recovery period
- No abnormalities were noted in both sexes.
Urinalysis findings:
no effects observed
Description (incidence and severity):
Since over 40 mL of urine volume was noted in the 200 mg/kg males (Nos. 25, 26) and considered to be mixed with drinking water, these data were not used in a statistical analysis.

At the end of the dosing period
- No abnormalities were noted in both sexes.

At the end of the recovery period
- No abnormalities were noted in both sexes.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
At the end of the dosing period
- No abnormalities were noted in both sexes.

At the end of the recovery period
- Male: No abnormalities were noted.
- Female: Decrease in relative spleen weight was noted in the 1000 mg/kg group.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
At the end of the dosing period
- Male: Small testis (1/6) in the vehicle control group, and loss of teeth (1/6) and cloudy reddish change of eye ball (1/6) in the 8 mg/kg group, and reddish change of eye ball (1/6) in the 200 mg/kg group, and pelvic dilatation in kidney (2/6) in the 1,000 mg/kg group were noted.
- Female: Pelvic dilatation in kidney (1/6) were noted in the 40 mg/kg group.

At the end of the recovery period
- No abnormalities were noted in both sexes.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
At the end of the dosing period
- Male: Necrosis of hepatocytes with cell infiltration (liver, 1/6) and decreased spermatogenesis (testis, ± , 1/1) in the vehicle control group, and degeneration of lens fibres (eye ball, 1/1) and hypoplasia of retina (eye ball, 1/1) in the 8 mg/kg group, and erythrocytes in chamber (eye ball, 1/1) in the 8 and 200 mg/kg groups, and pelvic dilatation (kidney, 2/6) in the 1000 mg/kg group were noted.
- Female: Cyst formation (kidney, 1/6) in the vehicle control group, and dilatation of tubules with cell infiltration (kidney, 1/1), pelvic dilatation (kidney, 1/1) in the 40 mg/kg group, and cyst formation (kidney, 1/6) in the 1000 mg/kg group were noted.

At the end of the recovery period
- Male: Cyst formation (kidney, 1/6) was noted in the vehicle control group.
- Female: not examined.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Details on results:
DISCUSSION
The oral toxicity of the test material was examined in rats given daily doses of 8, 40, 200 and 1,000 mg/kg/day for 28 days, followed by the 14-day recovery period.
There were no deaths on account of administration of the test material.
No abnormalities were noted in general conditions, body weights and food consumption during the dosing period, and urinalysis, organ weights, necropsy and histopathological examinations at the end of the dosing period.
In haematological examinations, decreases in platelet and activated partial thromboplastin time in the 200 and 1,000 mg/kg females were noted.
In blood chemical examinations, increase in total cholesterol in the 200 and 1,000 mg/kg females and the 1,000 mg/kg males were noted. Although there were no another related changes, these were considered to be related to treatment since similar changes were noted in a 14-day repeated-dose preliminary toxicity study.
The main effects of the test material were noted in the coagulation system and cholesterol metabolism; however, these were very slight and there were no organic changes.
In histopathological examinations, pelvic dilatation of kidney was noted in 1,000 mg/kg males; however, this change was not considered to be treatment related since pelvic dilatation was observed in one kidney and such change is common in rats in this strain.
In the recovery test, no abnormalities were noted. Although decreases in relative spleen weight and mean corpuscular haemoglobin concentration were noted in 1000 mg/kg females these were considered to be unrelated to treatment since no other findings were associated with them, and they were not noted at the end of the dosing period.
The other statistically significant differences, and changes in general conditions, necropsy and histopathological examinations were not considered related to administration of the test material since there was no dose relationship and they were noted in the vehicle control group.
In conclusion, the NOEL of the test material for rats in this study was considered to be 40 mg/kg/day since increase in total cholesterol, decreases in platelet and activated partial thromboplastin time were noted in the 200 mg/kg females.
Key result
Dose descriptor:
NOEL
Effect level:
40 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
clinical biochemistry
haematology
Critical effects observed:
not specified

Table 1: Summary of Significant Haematology Parameters

Sex

Experimental Group (mg/kg/day)

Mean corpuscular haemoglobin concentration (MCHC) (%)

Platelet Count (x10^4/mm^3)

Activated partial thromboplastin time (APTT) (sec)

Male

Vehicle

35.0

121.2

24.0

8

34.8

118.2

25.4

40

35.2

129.0

25.9

200

34.8

125.4

24.0

1000

34.8

133.0

23.1

Recovery Vehicle

34.6

110.5

28.2

Recovery 1000

34.3

115.8

29.8

Female

Vehicle

36.0

144.1

22.0

8

36.1

129.4

20.6

40

35.5

128.1

21.4

200

35.7

119.9**

18.4**

1000

35.9

119.7**

19.5*

Recovery Vehicle

36.1

123.7

22.3

Recovery 1000

35.2*

121.6

20.6

* Significantly different from vehicle control at P < 0.05

** Significantly different from vehicle control at P < 0.01

 

 

Table 2: Summary of Significant Blood Chemistry Parameters

Sex

Experimental Group (mg/kg/day)

GOT (IU/I)

GPT (IU/I)

Alkaline phosphatase (ALP) (IU/I)

Total cholesterol (T-Cho) (mg/dL)

Male

Vehicle

79

29

613

54

8

79

26

469

49

40

78

28

425

54

200

78

27

458

61

1000

79

28

375**

43

Recovery Vehicle

70

29

374

46

Recovery 1000

77

28

376

40

Female

Vehicle

95

23

319

51

8

70**

22

268

55

40

74*

19*

266

53

200

77

22

249

65*

1000

86

19

243

65*

Recovery Vehicle

77

23

224

55

Recovery 1000

74

21

207

53

* Significantly different from vehicle control at P < 0.05

** Significantly different from vehicle control at P < 0.01

Conclusions:
Under the conditions of this study, the NOEL of the test material for rats in this study was considered to be 40 mg/kg/day since increase in total cholesterol, decreases in platelet and activated partial thromboplastin time were noted in the 200 mg/kg females.
Executive summary:

The repeated dose toxicity of the test material was investigated in accordance with the standardised guidelines OECD 407 and "The Notification on Partial Revision of Testing Methods Relating to New Chemical Substances" (Notification No. 700 of Kanpogyo, No. 1039 of Yakuhatsu, and No. 1014 of 61 Kikyoku, Dec. 5, 1986. The testing was performed under GLP conditions.

A 28-day repeated-dose oral toxicity study of the test material followed by a 14-day recovery test was conducted in male and female Crj: CD (SD) rats (6/sex/ group), 5 weeks of age at the start of dosing. The highest dose was set at 1000 mg/kg/day, and 3 lower doses at 200, 40 and 8 mg/kg/day. Recovery groups were separately provided for vehicle control and 1000 mg/kg groups.

There were no deaths on account of administration of the test material. No abnormalities were noted in general conditions, body weights and food consumption during the dosing period, and urinalysis, organ weights, necropsy and histopathological examinations at the end of the dosing period.

In haematological examinations, decreases in platelet and activated partial thromboplastin time in the 200 and 1000 mg/kg females were noted. In blood chemical examinations, increase in total cholesterol in the 200 and 1,000 mg/kg females and the 1,000 mg/kg males were noted. Although there were no another related changes, these were considered to be related to treatment since similar changes were noted in a 14-day repeated-dose preliminary toxicity study.

The main effects of the test material were noted in the coagulation system and cholesterol metabolism; however, these were very slight and there were no organic changes.

In histopathological examinations, pelvic dilatation of kidney was noted in 1000 mg/kg males; however, this change was not considered to be treatment related since pelvic dilatation was observed in one kidney and such change is common in rats in this strain.

In the recovery test, no abnormalities were noted. Although decreases in relative spleen weight and mean corpuscular haemoglobin concentration were noted in 1000 mg/kg females these were considered to be unrelated to treatment since no other findings were associated with them, and they were not noted at the end of the dosing period. The other statistically significant differences, and changes in general conditions, necropsy and histopathological examinations were not considered related to administration of the test material since there was no dose relationship and they were noted in the vehicle control group.

Under the conditions of this study, the NOEL of the test material for rats in this study was considered to be 40 mg/kg/day since increase in total cholesterol, decreases in platelet and activated partial thromboplastin time were noted in the 200 mg/kg females.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
40 mg/kg bw/day
Study duration:
subacute
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

28 Day Oral Study, Imatanaka (1995)

The repeated dose toxicity of the test material was investigated in accordance with the standardised guidelines OECD 407 and "The Notification on Partial Revision of Testing Methods Relating to New Chemical Substances" (Notification No. 700 of Kanpogyo, No. 1039 of Yakuhatsu, and No. 1014 of 61 Kikyoku, Dec. 5, 1986. The testing was performed under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

A 28-day repeated-dose oral toxicity study of the test material followed by a 14-day recovery test was conducted in male and female Crj: CD (SD) rats (6/sex/ group), 5 weeks of age at the start of dosing. The highest dose was set at 1000 mg/kg/day, and 3 lower doses at 200, 40 and 8 mg/kg/day. Recovery groups were separately provided for vehicle control and 1000 mg/kg groups.

There were no deaths on account of administration of the test material. No abnormalities were noted in general conditions, body weights and food consumption during the dosing period, and urinalysis, organ weights, necropsy and histopathological examinations at the end of the dosing period.

In haematological examinations, decreases in platelet and activated partial thromboplastin time in the 200 and 1,000 mg/kg females were noted. In blood chemical examinations, increase in total cholesterol in the 200 and 1,000 mg/kg females and the 1,000 mg/kg males were noted. Although there were no another related changes, these were considered to be related to treatment since similar changes were noted in a 14-day repeated-dose preliminary toxicity study.

The main effects of the test material were noted in the coagulation system and cholesterol metabolism; however, these were very slight and there were no organic changes.

In histopathological examinations, pelvic dilatation of kidney was noted in 1,000 mg/kg males; however, this change was not considered to be treatment related since pelvic dilatation was observed in one kidney and such change is common in rats in this strain.

In the recovery test, no abnormalities were noted. Although decreases in relative spleen weight and mean corpuscular haemoglobin concentration were noted in 1000 mg/kg females these were considered to be unrelated to treatment since no other findings were associated with them, and they were not noted at the end of the dosing period. The other statistically significant differences, and changes in general conditions, necropsy and histopathological examinations were not considered related to administration of the test material since there was no dose relationship and they were noted in the vehicle control group.

Under the conditions of this study, the NOEL of the test material for rats in this study was considered to be 40 mg/kg/day since increase in total cholesterol, decreases in platelet and activated partial thromboplastin time were noted in the 200 mg/kg females.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to repeated dose toxicty.