Reaction mass of 2-ethyl-4-methyl-1H-imidazole-1-propiononitrile and 2-ethyl-5-methyl-1H-imidazole-1-propiononitrile

Endpoint summary

Description of key information

Key information:

• OECD TG407; 28 day repeated dose toxicity; Hoshuyama al. 2018

 

An OECD TG 407 study was conducted in order to examine the test substances' potential to toxicity following a sub-acute oral dosing regime. The results indicated treatment-related adverse effects in the liver, with secondary effects on the thyroid, and a potential effect on the nervous system. A NOAEL of 50 mg/kg bw/day was established.

 

The CLP guidance value range for STOT RE classification into category 1 is ≤ 30mg/kg and category 2 is ≤ 300mg/kg bw/day. Effects considered to be adverse and related to the test substance were reported at doses of 150mg/kg bw/day and above, therefore the effects are within the value range for its classification as STOT RE Category 2 (liver, thyroid).

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

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:

20th March 2018 - 30th July 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: 28-day Repeated Dose Toxicity Study in Mammalian Species

Version / remarks:

Stipulated in the 'Testing Methods for New Chemical Substances' (March 31, 2011, No. 0331-8, Pharmaceutical food safety bureau, Ministry of Health, Labour and Welfare, Japan; March 20, 2011 No. 6, Manufacturing Industries Bureau, Ministry of Economy, Trade and Industry, Japan; No. 110331010, Environmental Health Department, Ministry of the Environment, Japan).

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

3rd October 2008

GLP compliance:

yes

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Avoid direct sunlight, high temperatures and humidity. The test substance was placed in an air tight container and stored at room temperature. Temperature was stated as 10 - 30˚C.
- Stability under test conditions: Stable
- Solubility and stability of the test substance in the vehicle: water soluble >10g/L (visual)

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test material was melted in a water bath at about 40˚C
- Final dilution of a dissolved solid: Dose volume of 10 mL/kg at concentration range of 0.5 -4.5 w/v%.

Species:

rat

Strain:

other: Crl:CD (SD)

Details on species / strain selection:

Crl:CD(SD) rats were used as this strain is an established laboratory animal and is widely used in general toxicity studies. Additionally the laboratory has historical control data for the strain in question.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Japan Hino Breeding Center.
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: 5 weeks
- Weight at study initiation: Body weight ranges of 131.1 to 153.6 g for males and 119.9 to 140.2g for females
- Housing: The animals were housed in hanging stainless steel cages with wire floors and under-trays. the temperature was maintained at 21 to 25˚C, relative humidity of 40 to 70%, 10 to 15 air changes per hour with 12 hours light per day. The animals were acclimatised and quarantined for 6 days under group housing of five or less animals per cage in each sex. Additionally, the animals were acclimatised until seven days after the receipt(day one before the start of dosing). During the dosing period animals were grouped as per Table 1.
- Diet: The animals had access to a pelleted diet (MF, lot number 171221 and 180131 Oriental Yeast) ad libitum
- Water (e.g. ad libitum): The animals had ad libitum supply to drinking water via an automatic watering system. The drinking water had been maintained at 3 to 5ppm of chloric level prepared by adding sodium hypochlorite (Purelox) to Hita city-supply water.
- Acclimation period: The animals were acclimatised for 7 days under group housing of five or less animals per cage in each sex.

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 25
- Humidity (%): 40 - 70
- Air changes (per hr):10 - 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

other: Purified water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): Purified water used therefore no justification required
- Concentration in vehicle: 0.500, 1.5, 4.5 w/v%
- Amount of vehicle (if gavage):
- Lot/batch no. (if required): Lot No. PC171219
- Purity: Not stated

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Immediately after the first preparation, the concentrations of the 4.50, 1.50 and 0.500 w/v% dose formulations were analysed by HPLC in the testing facility. Consequently the actual concentrations of thr 4.50, 1.50 and 0.500 w/v% dose formualtions were 99.8, 96.7 and 96.4% compare to their nominal concentrations. Thus all concentrations of the dose formulations were confirmed to be within the acceptable range (100 +/- 10).

Duration of treatment / exposure:

28 days

Frequency of treatment:

once daily

Dose / conc.:

0 other: mg/kg/day

Remarks:

Vehicle control recovery group - Concentration of dosing formulation (w/v%) - 0 w/v%

Dose / conc.:

450 other: mg/kg/day

Remarks:

Recovery Group. concentration of dosing formulation (w/v%) - 4.5 w/v%

Dose / conc.:

0 other: mg/kg/day

Remarks:

Concentration of dosing formulation (w/v%) - 0 w/v%

Dose / conc.:

50 other: mg/kg/day

Remarks:

Concentration of dosing formulation (w/v%) - 0.500 w/v%

Dose / conc.:

150 other: mg/kg/day

Remarks:

Concentration of dosing formulation (w/v%) - 1.50 w/v%

Dose / conc.:

450 other: mg/kg/day

Remarks:

Concentration of dosing formulation (w/v%) - 4.5 w/v%

No. of animals per sex per dose:

5 animals per sex per dose and recovery groups, see table 1.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
A range finding study was performed, in this study three male and three female Crl:CD (SD) rats in each group (ages 5 weeks) were treated with the test substance dissolved in purified water at 0,100, 250, 500 and 1000 mg/kg/day by gavage for 7 days. General clinical observations and measurements of body weight were carried out during the dosing period. On the next day of the final dosing, necropsy and organ weight measurements were performed. As a result, decreasing spontaneous locomotion, decreased respiratory rate, incomplete eyelid opening etc were observed in the 1000 mg/kg groups and one male and one female were dead on day 2. although no animals were dead in the 500 mg/kg groups decreased spontaneous locomotion and suppression of body weight gain were observed in males with relative weight and enlargement of the liver in males and females of the was observed. Enlargement of the liver in males of the 250 mg/kg group was also observed. It was therefore predicted that severe toxicities like death or moribundity would be observed if treated at a dose of greater than 500 mg/kg/day for 28 days. Therefore, the high dose was set at 450mg/kg/day and lower dose of 150 and 50 mg/kg/day.

- Rationale for animal assignment (if not random): Random

The animals of the recovery groups were reared for 14 days without dosing after the dosing period. the initiation day and week of recovery were designated day 1 of recovery and week 1 of recovery, respectively.

- Fasting period before blood sampling for clinical biochemistry:
All animals were fasted overnight (16 to 20 hours).

Observations and examinations performed and frequency:

General Clinical Observations:
During the dosing period, the animals were observed thrice daily: before dosing, within one hour after dosing and 2 to 6 hours after dosing. During the recovery period, the clinical observation including mortality was performed once a day.

Detailed clinical observations were performed for the following table in all animals once before dosing period and once weekly thereafter. After the initiation of dosing, the animals were examined in a blind test basis, with the random numbering and observation labels without identifying the dose levels.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before dosing period and then once weekly.
After the initiation of dosing, the animals were examined in a blind test bias, with random numbering and observation labels without identifying the dose levels
1. Observations at removal from cage - Animal reactions for external stimuli, such as easiness of removal and vocalization were assessed by scoring method
2. Handling observations- Muscle tone, subnormal temperature, hair appearance (piloerection, staining hair and unkempt hair), skin and mucous colour (paleness, reddening and cyanosis), eyes (lacrimation, exophthalmos and pupillary size), salvation and secretion were observed.
3. Observation in area - Animals were placed on a standard area and observed for posture, motor activity level, respiration, gait characteristics, lid closure, tremor, twitch, convulsion, stereotypical behaviour and abnormal behaviour for one minute or more. Frequencies of defecation and urination were recorded for one minute

BODY WEIGHT: Yes
- All animals were weighed using an electric balance on the following days:
1. On day of grouping
2. On days 1,3,8,12,17,21,26 and 28 of dosing
3. On days 1,5,10 and 14 of recovery period for recovery groups
4. On the necropsy days (before carrying animals from the animal room)

FOOD CONSUMPTION MEASUREMENTS:
- Food weights were measured using an electric balance on the following days for all animals:
1. Feedings weights on the day of grouping
2. Remainder weights on days 1,3,8,15,22 and 28 of dosing
3. Feeding weights on day 1 of recovery and remainder weights on days 4,8 and 14 of recovery for the recovery groups
4. Feedings weights on days 8, 15 and 22 of dosing and day 8 of recovery were also measured after remainder weights measurement and food replenishment. Remainder weights on days 1 and 3 of dosing and day 4 of recovery were regarded as feeding weights of those days and food was not replenished.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood sampling was performed in the day after the last dosing day for main groups and of the last withdrawal day for the recovery groups.
- Anaesthetic used for blood collection: Yes - Isoflurane
- Animals fasted: Yes - 16 to 20 hours
- How many animals: all animals
- Parameters checked in table No.2 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Sampling was performed on the day after the last dosing day for main groups and of the last withdrawal day for the recovery groups.
- Animals fasted: Yes
- How many animals: All groups
- Parameters checked in table no. 2 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: Urine samples were collected for 15 to 16 hours from the afternoon of the last day of the dosing period for the main groups and of the recovery period for the recovery groups to each next morning.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table no. 3 were examined.

NEUROBEHAVIOURAL EXAMINATION: yes
- Time schedule for examinations: Week 4 - day 22 or 23 of dosing
- Dose groups that were examined: all groups were examined
- Battery of functions tested: sensory activity / grip strength / motor activity / other:
1. Reflex
- Visual response (approach contact/touch response)
- Pinna response
- Pain response
- Pupillary reflex
- Air righting reflex
2. Grip strength
3. Locomotor activity counts

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, preformed on all animals after blood sampling. All animals were euthanised by bleeding from ventral aorta and gross necropsy was performed on the dat after last dosing for the main groups and on the day after last withdrawal day for the recovery groups. External surface of the body, all orifices, subcutis, cranial, thoraic, abdominal and pelvic cavities and their contents were observed. For femals, vaginal smears were collecred before the gross necrospy and the stages of estrous cycle were determined with a light microscope after Giemsa staining.

TISSUE COLLECTING AND ORGAN WEIGHT MEASUREMENTS: Yes, see table 4.

HISTOPATHOLOGY: Yes, see table 5.

Statistics:

The data regarding body weights, food consumption, grip strength and locomotor activity counts during the dosing period and parameters of haematological and blood chemical examinations, urine volume, urine specific gravity, organ weights and body weight on the necropsy day for the main groups were analysed by the Bartlett's test for homogeneity of variances.

When the variances were homogeneous at a significant level of 5% in this analysis, the Dunnet's test was performed. When the variances were not homogeneous, the nonparametric Dunnett's test was performed. The frequencies of defecation and urination during the dosing period were analyser by the nonparametric Dunnett's test.

Data regarding body weights, food consumption and locomotor activity count during the recovery period, and parameters of haematological and blood chemical examinations, urine volume, urine specific gravity, organ weights and body weights on the necropsy day for the recovery groups were analysed by the F-test for variance ratio. When there were no significant differences at a significance level of 5% in this analysis, the Students t-test was performed. When there were significant difference the Aspin-Welch t-test was performed. The frequencies if defecation and urination during the recovery period were analysed by the Mann-Whitney U-test.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

General Clinical Observations:

In the male group, salivation in 8/10 animals, decreased spontaneous locomotion in six animals and decreased respiratory rate in 2 animals were observed in the 450 mg/kg group. Recovery was noted in the 14 day recovery period in the 450mg/kg bw/day group.
In females, salivation was observed in 7/10 animals, decreased spontaneous locomotion in 5/10, decreased respiratory rate in 2/10 animals, hypersensitivity in 2/10 animals, and for 1/10 animals incomplete eyelid opening, tremor, lacrimation or sway were present in the 450mg/kg group. Salivation in the 150mg/kg group was observed from immediately after administration of the test item.
Recovery was noted in the 14 day recovery period in the 450mg/kg bw/day group.

Mortality:

not specified

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

1. During the dosing period:

Males: There were significant decreased or decreasing tendencies observed in the 450 mg/kg group from day 3 to 28. The average value on day 3 was 94.0% compared to control value. After that, lower values compared to control were consecutively noted and the average value on 28 day was 93.0% compared to control value. No significant changes were observed in the 50 or 150 mg/kg group.

Females: Significant decreases were observed in the 450mg/kg group on day 3 and day 8 (94.2% and 92.6% compared to control values). No significant changes were observed in the 50 or 150 mg/kg groups.

2. During the recovery period

Males: Decreasing tendencies were observed in the 450 mg/kg group on day 1 and day 5 (92.6% and 93.7% compared to control values).

Females: No significant changes were observed in the 450 mg/kg group.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

1. During dosing period: In males, significant decreases were observed in the 450mg/kg group on day 3 and day 8 (77.6% and 87.8% compared to control values). No significant changes were observed in the 50 or 150 mg/kg groups. In females, significant decreases were observed in the 450 mg/kg group on day 3 and day 8 (75.4% and 83.3% compared to control values). A decreasing tendency was observed in the 150 mg/kg group on day 3 (89.1 compared to control value). No significant changes were observed in the 50 mg/kg group.

2. During recovery period: In either sex, no significant changes were observed in the 450 mg/kg group.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

1. Main Groups

In males there was a significant increase of reticulocyte count ratio in the 150 mg/kg and a significant shortening of the prothrombin time in the 50 mg/kg group observed, although they were not dose dependant. No significant changes were observed in the 450 mg/kg group.

In females, a significant decrease of reticuloytes count ratio was observed in the 50 mg/kg group although it was not dose dependant.

No significant changes were observed in the 150 or 450 mg/kg groups.

2. Recovery groups: In either sex, no significant changes were observed in the 450 mg/kg group.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

1. Main groups


In males, a significant decrease of creatinine in 150 mg/kg group and higher, and a significant increase of triglyceride in the 450 mg/kg group were observed. There were significant decreases in aspartate aminotransferase (AST) in the 50 and 450 mg/kg groups, although they were within the range of the historical control data and were not dose dependant.

In females, significant decrease of creatinine were observed in 150 mg/kg group and higher. A significant increase in inorganic phosphorus and calcium were observed in the 450 mg/kg group, although they were also within the range of historical control data. A significant decrease of chloride was observed although no other electrolytic parameters were significantly changed. A significant decrease of alanine aminotransferase was observed in the 150 mg/kg group, again this was not dose dependant.

No significant changes were observed in the 50 mg/kg group



2. Recovery groups

Males: a significant decrease of AST was observed in the 450mg/kg group, although it was within the range of the historical control data.

Females: a significant increase of alkaline phosphatase and a significant decrease of total bile acid were observed in the 450mg/kg group - this was within the historical control data.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

1. Main groups:
Males: pH7.5 was noted in 1/5 animals in the 450 mg/kg group. A significant increase of the urine volume and a significant decrease of the urine specific gravity were observed in the 150 mg/kg group, this was not dose dependant. In the control group, occult blood (3+) was observed in 1/5 animals. No abnormalities were observed in the 50 mg/kg group.

Females: Occult blood (2+) was observed in 1/5 animals of the 450 mg/kg group. Significant decreases of the urine specific gravity were observed in the 50mg/kg group and higher - within the range of our historical control data and no significant changes were observed in urine volume.


2. Recovery groups
In either sex, no significant changes in the urine volume or urine specific gravity were observed in the 450 mg/kg group. No abnormalities were observed in the control or 450 mg/kg groups.

Behaviour (functional findings):

not specified

Description (incidence and severity):

1. During dosing Period
Males: no significant changes were observed in the grip strength or locomotive activity in any treatment groups, additionally no abnormalities were observed in the control or any treatment groups in the reflex tests.

Females: A significant decrease was observed in the 450 mg/kg group in the locomotor activity count between measurement start and 10 minutes. A significant increase of forelimb grip strength was observed in the 150 mg/kg group - it was not dose dependant. No abnormalities were observed in the control or any treatment group for reflex tests.

2. During the recovery period:
Males: No examinations were performed since no treatment-related abnormalities were suspected in week 4 of dosing.
Females: No significant changes were observed in the locomotor activity count in the 450mg/kg group.

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Organ Weights:
1. Main groups
a. Males: Significant increases in absolute and relative weights of the liver (130.2% and 141.8% compared to control groups). Significant increase of the relative weight of the kidney (113.9% compared to control). Increasing tendency of the absolute weight and a significant increase of the relative weight of the thyroid (122.4% and 133.5% compared to control) and a significant increase of the relative weight of the adrenal (118.6% compared to control) were observed in the 450 mg/kg group. A significant increase of the relative weights of the epididymis was observed - no significant change was observed in the absolute weights. In the 250 mg/kg group, significant increases of the absolute and relative weights of the liver (123.0% and 116.8% compared to control), relative weight of the kidney (107.5% compared to control) and the absolute and relative weights of the thyroid (129.3% and 123.3% compared to control) were observed. In the 50 mg/kg group a significant increase of the absolute weight if the thymus was observed - this was not dose dependant.

b. Females: significant increases in the absolute and relative weights of the liver (129.5% and 134.7% compared to control) , the absolute and relative weights of the thyroid (129.0% and 134.2% compared to control) and relative weights of the adrenal (122.5% compared to control) were observed in the 450 mg/kg group. No significant changes were observed in the 50 or 150 mg/kg groups.

2. Recovery groups
a. Male: Significant increase in the relative weight of the liver (109.9% compared to control) and thyroid (118.2% compared to control) were observed in the 450 mg/kg group. A significant increase in the relative weight of the thymus was also observed in the 450 mg/kg group.

b. Females: no significant changes were observed in the 450 mg/kg group.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

1. Main groups
a. Male: Enlargement of the liver in all the 5 animals in the 450 mg/kg group. No abnormalities were observed in the control, 50 mg/kg or 150 mg/kg groups
b. Females: Enlargement of the liver was observed in all five animals in the 450 mg/kg group. Diverticulum in the jejunum was also observed in one animal of the 450 mg/kg group. No abnormalities were noted in the control, 50 mg/kg or 150 mg/kg groups.

2. Recovery groups

In either sexes no abnormalities were observed in the control or 450 mg/kg groups

Neuropathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

1. Males: decreased spontaneous locomotion (6/10 animals) in 450mg/kg group. No abnormalities observed in the recovery period.
2.. Females: decreased spontaneous locomotion (51/0 animals), incomplete eyelid opening (1/10), tremor (1/10), lacrimation (1/10) and sway (1/10) observed in 450mg/kg group. No abnormalities observed in the recovery period.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

1. Main groups
a. Male: Centrilobular hypertrophy of hepatocytes in the liver (slight to moderate) of all 5 animals and diffuse follicular cell hypertrophy of the thyroid (slight) in one animal were observed in the 450 mg/kg group. In the 150 mg/kg group centrilobular hypertrophy of hepatocytes in the liver (slight) was observed in 2/5 animals. Microgranuloma of the liver in two animals and focal atrophy and lymphocyte infiltration of the ventral prostate in one animal each was observed in the control group. Lymphocyte infiltration of the ventral prostate in two animals, capsulitis of the spleen in one animal and ectopic thymic tissue in the thyroid of one animal were observed in the 450 mg/kg group. No abnormalities were observed in the 50 mg/kg group.
b. Females: Centrilobular hypertrophy of hepatocytes in the liver (slight to moderate) of all 5 animals and diffuse follicular hypertrophy of the thyroid (slight) in two animals were observed in the 450 mg/kg group. Additionally, diverticulum in the jejunum was macroscopically observed. In the control group, capsulitis of the spleen was observed in one animal. No abnormalities were observed in the 50 or 150 mg/kg groups.

2. Recovery groups
a. Males: Centrilobular hypertrophy of hepatocytes in the liver (slight) was observed in 1/5 animals of the 450 mg/kg group. No abnormalities were observed in the control groups.

b. Females: No abnormalities observed.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Estrous cycle stage
1. Main Groups
In the control 1/5 animal was in proestrus, 3 were in estrus and one was in diestrus. in the 50 mg/kg group, 2 animals were in proestrus, 1 was in estrus and 2 were in metestrus. In the 150 mg/kg group, 1 animal was in proestrus, 2 were in estrus and 2 were in metestrus. In the 450 mg/kg group 1 animal was in proestrus, 1 was in estrus and 2 were in metestrus and 1 was in diestrus.

2. Recovery groups
In the control group, 1/5 was in estrus, 4 were in metestrus. In the 450 mg/kg group 4 were in estrus and 1 was in metestrus.

Details on results:

Estrous cycle stage
1. Main group. In the control group, one animal out of five was in proestrus, three were in estrus and one was in diestrus. In the 50 mg/kg group, two animals were in proestrus, one was in estrus and two were in diestrus. In the 50 mg/kg group, two animals were in proestrus, one was in estrus and two were in metestrus. In the 150 mg/kg group, one animal was in proestrus, two were in estrus and two were in metestrus. In the 450 mg/kg group, one animal was in proestrus, one was in estrus, two were in metestrus and one was in diestrus.
2. Recovery group: In the control group, one animal out of five was in estrus and four were in metestrus. In the 450 mg/kg group, four animals were in estrus and one was in metestrus.

Key result

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

150 mg/kg bw/day (nominal)

System:

haematopoietic

Organ:

liver

thyroid gland

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

presumably yes

Table 6: 28 day repeat dose oral toxicity study in rats. Summary of general clinical observations 

Sex	Signs		Administration period						Recovery period
		mg/kg	0	0(R)	50	150	450	450(R)	0	450
Male			5^a)	5	5	5	5	5	5	5
	No abnormalities detected		5	5	5	4		1	5	5
	Salivation					1	4	4
	Decreased spontaneous locomotion						3	3
	Decreased respiratory rate						1	1
Female			5	5	5	5	5	5	5	5
	No abnormalities detected		5	5	5	5	1		5	5
	Salivation						4	3
	Decreased spontaneous locomotion						3	2
	Decreased respiratory rate						2
	Incomplete eyelid opening						1
	Tremor						1
	Lacrimation						1
	Sway						1
	Hypersensitivity						1	1

 

 

Table 7: Summary of body weights (g) -Male and female

 

Male						Female
	Dose	Main + Recovery groups	Main group	Main group	Main + Recovery groups	Main + Recovery groups	Main group	Main group	Main + Recovery groups
	mg/kg/day	0	50	150	450	0	50	150	450
Administration period (day)		140.38 ± 5.80 (10)	140.86 ± 5.42 (5)	140.06 ± 5.99 (5)	142.07 ± 6.44 (5)	126.24 ± 4.17 (5)	130.16 ± 7.08 (5)	127.98 ± 5.29 (5)	126.87 ±5.53 (10)
	3	156.59 ±7.42 (10)	157.16 ±4.87 (5)	153.92 ±8.53 (5)	147. 25 * ±6.70 (5)	138.63 ± 4.61 (5)	143.20 ±7.79 (5)	135.48 ­7.07 (5)	130.64 *±6.28 (10)
	8	201.85 ± 10.60 (10)	201.56 ±6.69 (5)	198.56 ±13.99 (5)	185.41 ** ± 10.08 (5)	162.76 ±9.11 (5)	170.84 ±9.82 (5)	165.86 ±12.19 (5)	150.77* ±6.54 (10)
	12	236.16 ±12.85 (10)	235.96 ±6.42 (5)	235.62 ± 17.39 (5)	220.39 * ±13.71 (5)	177.40 ±11.23 (5)	186.26 ±9.24 (5)	183.48 ± 14.84 (5)	168.61 ±7.11 (10)
	17	276.16 ± 13.03 (10)	277.76 ± 6.71 (5)	278.48 ±20.18 (5)	258.32 *±18.05 (5)	192.38 ±13.87 (5)	203.82 ±13.18 (5)	202.88 ±19.06 (5)	187.82 ±8.67 (10)
	21	306.37 ± 16.48 (10)	307.02 ± 7.55 (5)	310.76 ±22.48 (5)	286.11 ±20.57 (5)	207/15 ± 14.70 (5)	218.70 ±12.16 (5)	216.54 ±22.14 (5)	202.15 ±10.90 (10)
	26	336.92 ± 21.39 (10)	336.26 ± 8.39 (5)	343.56 ±24.75 (5)	312.36 * ±24.68 (5)	222.11 ±14.89 (5)	232.06 ±13.51 (5)	232.28 ±21.68 (5)	216.41 ±10.57 (10)
	28	347.02± 23.87 (10)	348.46 ±7.12 (5)	355.80 ± 28.10 (5)	322.60 ±25.56 (5)	227.67 ±16.78 (5)	236.98 ±13.77 (5)	238.96 ±21.90 (5)	221.72 ±12.09 (10)
Recovery period (day)	1	360/04 ± 21.86 (5)	-	-	333.46 ±24.56 (5)	225.74 ±19.97 (5)	-	-	220.06 ±15.45 (5)
	5	381.10 ± 23.28 (5)	-	-	357.18± 29.41 (5)	237.46 ±22.75 (5)	-	-	227.24 ± 16.21 (5)
	10	403.82 ±27.25 (5)	-	-	384.30 ±31.71 (5)	246.34± 17.71 (5)	-	-	238.08 ± 16.02 (5)
	14	423.00 ±28.38 (5)	-	-	407.78 ± 35.52 (5)	255.24 ± 20.38 (5)	-	-	249.46 ±14.45 (5)

Values are shown as Mean ± S.D.

Figure(s) in parentheses indicate numbers of animals used for the statistical analysis

* Significantly different from vehicle control at P<0.05

** Significantly different from vehicle control at P<0.01

 

Table 8: Summary food consumption (g/rat/day) -Male and female

 

Male						Female
	Dose	Main + Recovery groups	Main group	Main group	Main + Recovery groups	Main + Recovery groups	Main group	Main group	Main + Recovery groups
	mg/kg/day	0	50	150	450	0	50	150	450
Administration period (day)	1	17.84 ± 1.44 (10)	18.36 ± 1.16 (5)	18.50 ± 1.97 (5)	18.48 ± 1.47 (10)	15.55 ±1.23 (10)	16.82 ± 2.39 (5)	15.66 ±1.17 (5)	15.78 ±2.08 (10)
	3	20.16 ± 1.63(10)	20.20 ± 0.75 (5)	19.30 ±2.62 (5)	15.64 ** ±1.48 (10)	17.04 ±1.21 (10)	17.76 ±1.23 (5)	15.18 ±1.89 (5)	12.85 ** ±2.26 (10)
	8	22.89 ± 1.67 (10)	23.18 ± 0.92 (5)	23.34 ±2.49 (5)	20.09 ** ±1.97 (10)	17.79 ±1.29 (10)	18.70 ±1.65 (5)	17.92 ± 1.99 (5)	14.82 ** ±1.80 (10)
	15	24.32 ±1.79 (10)	24.72 ±0.37 (5)	25.56 ± 2.06 (5)	24.14 ±2.28 (10)	17.42 ±1.16 (10)	17.58 ±1.15 (5)	18.10 ± 1.79 (5)	18.09 ± 0.90 (10)
	22	25.56 ±1.84 (10)	25.76 ± 0.83 (5)	26.92 ± 2.10 (5)	25.64 ±2.03 (10)	17.82 ±1.56 (10)	17.60 ± 1.53 (5)	18.66 ±1.79 (5)	18.93 ±1.17 (10)
	28	25.28 ±2.69	25.82 ±1.55	26.78 ± 1.73 (5)	25.71 ± 2.69 (10)	18.72 ± 1.82 (10)	18.00 +1.55 (5)	18.66 ±1.65 (5)	19.41 ±0.91 (10)
Recovery period (day)	4	26.50 ± 2.31 (5)	-	-	26.62 ±2.14 (5)	19.26 ±2.09 (5)	-	-	18.76 ±0.91 (5)
	8	26.10 ±2.21 (5)	-	-	27.28 ±2.56 (5)	19.62 ±2.15 (5)	-	-	20.02 ±0.92 (5)
	14	25.96 ±2.30 (5)	-	-	27.50 ±2.79 (5)	19.10 + 1.59 (5)			19.82± 1.29 (5)

Values are shown as Mean ± S.D.

Figure(s) in parentheses indicate numbers of animals used for the statistical analysis

* Significantly different from vehicle control at P<0.05

** Significantly different from vehicle control at P<0.01

 

Table 9: Summary urinalyses – Male and Female

Male

Female

Items

Dose

Main Groups

Recovery groups

Main Groups

Recovery Group

mg/kg/day

0

50

150

450

0

450

0

50

150

450

0

450

Urine volume

(mL)

7.4± 4.8 (5)

8.6 ± 2.3 (5)

22.6 ((±3.2 (5)

13.2 ±6.1 (5)

7.0 ±3.4 (5)

17.6 ±14.8 (5)

4.0 ± 2.0 (5)

8.2 ±2.8 (5)

11.4 ±7.8

10.8 ±5.2 (5)

6.0 ±1.7 (5)

6.4 ±1.5 (5)

Sp.Gr

(-)

1.0398 ± 0.0171 (5)

1.0302 ±0.0064 (5)

1.0138 ** ± (5)

1.0264 ±0.0089 (5)

1.0430 ±0.0119 (5)

1.0292 ± 0.0192

1.0556 ±0.0237 (5)

1.0246 * ±0.0042 (5)

1.0262 * ±0.0162

1.0278 * ±0.0167

1.0388 ±0.0072 (5)

1.0338 ±0.0060 (5)

Values are shown as Mean ± S.D.

Figure(s) in parentheses indicate numbers of animals used for the statistical analysis

* Significantly different from vehicle control at P<0.05

** Significantly different from vehicle control at P<0.01

 

Table 10: Summary urinalyses -Male 

Male
Items	Group	Main Groups				Recovery groups
	Dose mg/kg/day	0	50	150	450	0	450
Items	No. of animals	5	5	5	5	5	5
Colour
	SY	2	3	5	2	2	4
	Y	3	3	0	3	3	1
Turbidity
	Clear	3	3	5	5	4	5
	Cloudy	2	2	0	0	1	0
pH
	6.5	1	3	4	1	4	1
	7.0	4	2	1	3
	7.5	0	0	0	1	0	0
Protein
	-	1	1	4	2	0	1
	±	1	0	1	1	2	3
	1+	2	4	0	2	2	1
	2+	1	0	0	0	1	0
Glucose
	-	5	5	5	5	5	5
Occult blood
	-	3	5	4	5	4	3
	±	1	0	0	0	1	1
	1+	0	0	1	0	0	1

Colour: SY: Slightly Yellow, Y: Yellow

 

Table 11 Summary urinalyses –Female

Female
Items	Group	Main Groups				Recovery groups
	Dose mg/kg/day	0	50	150	450	0	450
Items	No. of animals	5	5	5	5	5	5
Colour
	SY	1	1	3	4	1	2
	Y	4	4	2	1	4	3
Turbidity
	Clear	5	5	5	5	5	5
pH
	6.0	1	1	1	1	2	0
	6.5	4	2	3	2	2	3
	7.0	0	2	1	2	1	2
Protein
	-	0	4	4	2	1	2
	±	3	1	0	3	4	3
	1+	1	0	1	0	0	0
	2+	1	0	0	0	0	0
Glucose
	-	5	5	5	5	5	5
Occult blood
	-	5	5	4	3	5	5
	±	0	0	1	0	0	0
	1+	0	0	0	1	0	0
	2+	0	0	0	1	0	0

Colour: SY: Slightly Yellow, Y: Yellow

 

Table 12: Summary of Urinalyses -Male

Male
Items	Group	Main Groups				Recovery Group
	Dose mg/kg/day	0	50	150	450	0	450
Items	No. of animals	5	5	5	5	0	0
Urinary Sediment						-	-
Red Blood Cells ^a)						-	-
	0	5	-	-	5	-	-
White Blood cells ^a)						-	-
	0	5	-	-	5	-	-
Epithelial cells ^a)
	0	5	-	-	4	-	-
	1-5	0	-	-	1	-	-
Casts ^b)
	0	5	-	-	5	-	-
Crystals ^c)
	±	5	-	-	4	-	-
	1+	0	-	-	1	-	-

a) Number of cells/10views (x400)

b) Number of casts/18 x18 mm²

c) Incidence of crystals/18 x18 mm

-: not examined

 

Table 13: Summary of Urinalyses -Female

Female
Items	Group	Main Groups				Recovery Group
	Dose mg/kg/day	0	50	150	450	0	450
Items	No. of animals	5	0	0	5	0	0
Urinary Sediment						-	-
Red Blood Cells ^a)						-	-
	0	5	-	-	5	-	-
White Blood cells ^a)						-	-
	0	5	-	-	5	-	-
	1-5	1	-	-	2	-	-
Epithelial cells ^a)
	0	3	-	-	3	-	-
	1-5	2	-	-	2	-	-
Casts ^b)
	0	5	-	-	5	-	-
Crystals ^c)
	-	3	-	-	1	-	-
	±	2	-	-	4	-	-

a) Number of cells/10views (x400)

b) Number of casts/18 x18 mm²

c) Incidence of crystals/18 x18 mm

-: not examined

 

Table 14: Summary of haematological examinations - –Male

male
Items	Dose mg/kg/day	Main Groups				Recovery Group
		0	50	150	450	0	450
Items	No. of animals	5	0	0	5	0	0
RBC	(X10^4/uL)	758.0 ± 30.2 (5)	765.2± 28.1 (5)	760.6 ± 33.0 (5)	767.4 ± 22.6 (5)	788.0 ± 42.3 (5)	793.4 ±20.7 (5)
Hb	(g/dL)	14.82 ± 0.30 (5)	14.64 ±0.55 (5)	14.78 ± 0.33 (5)	14.72±0.50 (5)	14.98 ±0.22 (5)	14.76 ± 0.51 (5)
HI	(%)	42.64 ± 1.26 (5)	41.84 ±1.41 (5)	42.82 ± 0.89 (5)	42.58 ±1.65 (5)	42.16±0.91 (5)	42.34 ±1.90 (5)
MCV	(fL)	56.32 ± 2.77 (5)	54.68 ±1.08 (5)	56.36 ± 1.86 (5)	55.50±2.04 (5)	53.60±2.54 (5)	53.34 ±1.43 (5)
MCH	(pg)	19.56 ± 0.62 (5)	19.14 ± 0.28 (5)	19.46 ± 0.43 (5)	19.16± 0.38 (5)	19.06 ±0.89 (5)	18.80 ±0.42 (5)
MCHC	(g/dL)	34.78 ± 0.64 (5)	34.98 ±0.29 (5)	34.50 ± 0.37 (5)	34.58 ±0.71 (5)	35.54 ±0.55 (5)	34.88 ±0.48 (5)
Platelet	(x10^4/uL)	127.18 ±15.10 (5)	127.82 ±9.67 (5)	133.38 ± 18.08 (5)	137.34 ±19.41 (5)	112.86 ±12.62 (5)	122.08 ± 9.36 (5)
Reticulo	(%)	4.784 ± 0.541 (5)	5.108 ±0.333 (5)	5.940* ± 0.486 (5)	5.384 ±0.847 (5)	3.938 ±0.411 (5)	4.666 ±0.621 (5)
WBC	(x10^2/uL)	107.16 ± 5.64 (5)	120.00 ±27.45 (5)	116.52 ±27.98 (5)	114.54 ±9.30 (5)	108.82 ±8.48 (5)	126.04 ±41.14 (5)
Differentiation of leukocyte
Neutro	(%)	20.48 ± 4.79 (5)	18.38 ±7.14 (5)	16.78 ±5.10 (5)	20.94 ± 4.85 (5)	13.24 ±4.08 (5)	19.26 ± 7.17 (5)
Lymph	(%)	74.15 ± 4.70 (5)	75.58± 8.27 (5)	77.52 ±4.04 (5)	72.76 ± 4.61(5)	81.10 ±4.46 (5)	73.80 ± 8.39 (5)
Eosino	(%)	0.74 ± 0.17 (5)	1.18 ±0.50 (5)	0.70 ±0.24 (5)	0.82 ± 0.20 (5)	0.86 ±0.15 (5)	1.04 ± 0.44 (5)
Baso	(%)	0.08 ± 0.04 (5)	0.08 ±0.04 (5)	0.08 ±0.04 (5)	0.06 ± 0.05 (5)	0.10 ±0.00 (5)	0.06 ± 0.05 (5)
Mono	(%)	4.54 ± 0.90 (5)	4.78 ±0.95 (5)	4.92 ± 1.48 (5)	5.42 ± 0.84 (5)	4.70 ±1.42 (5)	5.84 ±1.37 (5)
PT	(sec)	19.84± 3.02 (5)	16.32 * ±1.99 (5)	17.62 ±1.22 (5)	18.42 ± 1.39 (5)	17.47±1.27 (3)	18.98 ± 2.43 (4)
APTT	(sec)	19.32± 4.15 (5)	16.46 ± 5.60 (5)	18.06 ±4.45 (5)	20.98 ± 2.72 (5)	16.77 ± 5.35 (3)	20.18 ± 2.54 (4)

Values are shown as Mean ± S.D

Figure(s) in parentheses indicate number of animals used for the statistical analysis

*Significantly different from vehicle control at P<0.05

** Significant different from vehicle control at P<0.01

 

Table 15 Summary of haematological examinations - –Female

Female
Items	Dose mg/kg/day	Main Groups				Recovery Group
		0	50	150	450	0	450
Items	No. of animals	5	0	0	5	0	0
RBC	(x10^4/uL)	753.0± 40.9 (5)	748.2± 49.5 (5)	749.4  ±32.8 (5)	770.0 ± 41.7 (5)	776.5 ± 28.5 (5)	763.2 ±12.4 (5)
Hb	(g/dL)	14.32 ±0.83 (5)	14.54 ±0.80 (5)	14.78 ± 0.65 (5)	14.48± 0.29 (5)	14.68 ±0.50 (5)	14.12 ± 0.38 (5)
HI	(%)	41.02 ±1.96 (5)	41.40 ±1.99 (5)	42.00 ± 1.75 (5)	41.28 ±1.27 (5)	40.96± 1.53 (5)	40.04 ±0.86 (5)
MCV	(fL)	54.54 ± 2.13 (5)	55.40 ±1.07 (5)	56.08 ± 1.52 (5)	53.70 ± 2.14 (5)	52.76 ±1.38 (5)	52.46 ±0.77 (5)
MCH	(pg)	19.00 ±0.65 (5)	19.48 ± 0.28 (5)	19.74 ± 0.43 (5)	18.84± 0.70 (5)	18.94 ±0.62 (5)	18.50 ±0.37 (5)
MCHC	(g/dL)	34.92 ±0.56 (5)	35.12 ±0.42 (5)	35.20 ± 0.41 (5)	35.08 ±0.54 (5)	35.86 ±0.70 (5)	35.28 ±0.27 (5)
Platelet	(x10^4/uL)	137.86 ± 19.51 (5)	118.4 ±7.36 (5)	130.94±13.64  (5)	130.90±13.38 (5)	116.94±11.78 (5)	130.30 ±14.89 (5)
Reticulo	(%)	4.884 ±0.896 (5)	3.664* ±0.564 (5)	4.300 ±0.595 (5)	4.692 ±0.388 (5)	3.742 ±0.775 (5)	4.536 ±0.341 (5)
WBC	(x10^2/uL)	109.44 ±19.93 (5)	126.58 ±23.19 (5)	117.42±10.12 (5)	124.14±16.82 (5)	95.22 ±21.47  (5)	95.10 ±25.16 (5)
Differentiation of leukocyte
Neutro	(%)	28.26 +9.90 (5)	17.86 ±8.67 (5)	21.34 ±4.57 (5)	21.82 ±6.48 (5)	20.22 ±4.00 (5)	21.48 ±6.69 (5)
Lymph	(%)	67.08  ± 10.32 (5)	75.90± 8.91 (5)	74.12 ±4.63 (5)	73.12 ±7.17 (5)	74.80 ±4.48 (5)	73.14 ±7.84 (5)
Eosino	(%)	1.10 ± 0.47  (5)	1.02 ± 0.40 (5)	0.70 ±0.29 (5)	0.82 ± 0.33 (5)	1.34  ±0.71 (5)	1.36 ±0.38  (5)
Baso	(%)	0.08 ± 0.04  (5)	0.08 ± 0.04 (5)	0.04 ±0.05 (5)	0.04 ±0.05 (5)	0.02 ±0.04 (5)	0.04 ± 0.05 (5)
Mono	(%)	3.48 ± 1.07  (5)	4.14 ± 0.75 (5)	3.80 ± 0.70 (5)	4.20 ±0.71 (5)	3.62 ±0.89 (5)	3.98 ± 1.30 (5)
PT	(sec)	13.82 ±0.45 (5)	12.82 ± 1.07 (5)	12.93  ±1.14 (5)	14.00 ±0.64 (5)	13.56 ±0.57 (3)	13.74 ±0.50 (4)
APTT	(sec)	16.64 ± 3.13  (5)	14.02 ± 3.85 (5)	12.48 ±3.74 (5)	16.88 ±0.79 (5)	15.80 ± 0.72 (3)	16.44 ±1.45 (4)

Values are shown as Mean ± S.D

Figure(s) in parentheses indicate number of animals used for the statistical analysis

*Significantly different from vehicle control at P<0.05

** Significant different from vehicle control at P<0.01

 

Table 16 Summary of blood chemical examinations - –Male

male
Items	Dose mg/kg/day	Main Groups				Recovery Group
		0	50	150	450	0	450
Items	No. of animals	5	0	0	5	0	0
AST	(IU/L)	74.2 ± 4.8	64.4*± 6.5 (5)	67.2  ± 5.2 (5)	60.2 **± 2.8 (5)	69.2 ± 4.2   (5)	63.6* ±1.8 (5)
ALT	(IU/L)	19.8 ± 1.6 (5)	19.6 ±  3.3 (5)	21.8 ± 3.3 (5)	21.0± 1.2 (5)	19.8 ± 1.1.  (5)	21.0 ± 3.0 (5)
ALP	(IU/L)	503.2 ± 68.2 (5)	493 ± 94.4 (5)	460.6 ± 99.3 (5)	460.8 ±74.7 (5)	350.2  ± 71.9 (5)	334.0 ±24.1 (5)
y-GTP	(IU/L)	0.64 ±0.09 (5)	0.86 ±0.36 (5)	0.80 ± 0.32 (5)	0.84 ± 0.39 (5)	0.60± 0.23 (5)	0.56 ±0.31 (5)
T-Cho	(mg/dL)	52.0 ± 5.3 (5)	52.0  ± 18.9 (5)	66.0 ±4.2 (5)	66.4 ± 6.5   (5)	56.8 ± 6.1 (5)	61.8 ±9.3 (5)
TG	(mg/dL)	44.8 ±16.1 (5)	73.0 ± 35.3 (5)	79.4 ± 21.1  (5)	126.2*± 73.7 (5)	51.0 ± 7.4 (5)	63.6 ± 14.1 (5)
BUN	(mg/dL)	12.82 ± 1.39 (5)	13.70 ±3.59 (5)	12.12 ±0.66 (5)	13.48 ±0.72 (5)	13.92 ±1.28  (5)	13.86 ±1.54 (5)
Creatinine	(mg/dL)	0.292 ±0.022 (5)	0.268 ±0.025  (5)	0.250* ±0.012 (5)	0.230**±0.024  (5)	0.288 ±0.030 (5)	0.274 ±0.011 (5)
T-Protein	(g/dL)	5.48 ±0.23 (5)	5.60 ± 0.1  (5)	5.46 ± 0.12 (5)	5.48 ± 0.13 (5)	5.62 ±0.19 (5)	5.68 ±0.11 (5)
Albumin	(g/dL)	2.64 ± 0.05 (5)	2.64 ± 0.05 (5)	2.66 ± 0.11 (5)	2.54 ± 0.09 (5)	2.54 ±0.09 (5)	2.60 ±0.07 (5)
A/G ratio	(-)	0.932±0.046 (5)	0.894 ±0.039 (5)	0.950 ±0.019 (5)	0.866 ±0.060 (5)	0.826 ±0.032 (5)	0.846 ±0.044 (5)
Glucose	(mg/dL)	115.4 ±20.2 (5)	110.8 ±13.5 (5)	121.6±23.0 (5)	123.6 ± 19.0 (5)	131.2 ±13.3 (5)	121.4 ±15.7 (5)
T-Bil	(mg/dL)	0.064±0.015 (5)	0.066 ±0.005 (5)	0.058 ±0.013 (5)	0.054 ±0.005 (5)	0.076 ±0.015 (5)	0.064 ±0.011 (5)
TBA	(umol/L)	50.82±48.71 (5)	56.28 ±35.6 (5)	19.76 ±15.08 (5)	16.64 ± 12.80 (5)	47.58 ±51.77 (5)	23.02 ±19.90 (5)
IP	(mg/dL)	8.34 ±0.44 (5)	8.18 ±0.37 (5)	8.32 ± 0.24 (5)	8.80 ± 0.67 (5)	7.48 ±0.39  (5)	7.36 ±0.28 (5)
Ca	(mg/dL)	9.78 ±0.47 (5)	10.12 ±0.18 (5)	10.14 ±0.36 (5)	10.22 ±0.16 (5)	9.64 ± 0.23 (5)	9.90 ±0.16 (5)
Na	(mEq/L)	142.4 ±1.3 (5)	141.8 ±1.1 (5)	142.4 ± 1.1 (5)	141.6 ± 0.5 (5)	141.4 ±1.1  (5)	141.0 ±0.7 (5)
K	(mEq/L)	4.58 ± 0.43 (5)	4.72± 0.18 (5)	4.46 ± 0.05 (5)	4.66 ± 0.09 (5)	4.54 ±0.46  (5)	4.66 ±0.18 (5)
Cl	(mEq/L)	104.52±1.41 (5)	103.12±1.60 (5)	103.96 ±0.76 (5)	103.52 ±1.60 (5)	103.52 ±1.37 (5)	103.98 ±1.17 (5)

Values are shown as Mean ± S.D

Figure(s) in parentheses indicate number of animals used for the statistical analysis

*Significantly different from vehicle control at P<0.05

** Significant different from vehicle control at P<0.01

 

Table 16 Summary of blood chemical examinations - –Female

Female
Items	Dose mg/kg/day	Main Groups				Recovery Group
		0	50	150	450	0	450
Items	No. of animals	5	0	0	5	0	0
AST	(IU/L)	72.0 ± 8.3 (5)	64.40± 5.7 (5)	72.8  ±14.0(5)	62.6 ± 8.4(5)	65.4 ± 9.3(5)	62.0  ±6.4(5)
ALT	(IU/L)	17.2 ± 1.1  (5)	14.6 ±2.1(5)	14.0 * ± 1.65)	18.8± 2.5(5)	16.4 ±1.7(5)	15.6 ± 2.1(5)
ALP	(IU/L)	345.8 ± 58.5 (5)	298.4 ± 32.7 (5)	309.0 ± 53.7 (5)	336.8 ±62.2 (5)	219.4  ± 29.0 (5)	288.2 ** ±26.7 (5)
y-GTP	(IU/L)	1.18 ±0.27(5)	1.62 ±0.45(5)	1.46 ± 0.23(5)	1.48 ±0.27(5)	1.12± 0.36 (5)	1.22 ±0.40 (5)
T-Cho	(mg/dL)	68.8 ±11.9(5)	74.4  ± 9.6(5)	74.3 ±13.1(5)	87.8± 13.2(5)	66.0 ±12.0(5)	69.4 ±1.9(5)
TG	(mg/dL)	21.0 ±8.0(5)	22.6 ± 7.2(5)	28.8 ± 13.6(5)	39.0 ±19.4	22.8 ±5.7 (5)	18.0 ± 5.7 (5)
BUN	(mg/dL)	16.02 ± 2.50(5)	15.18 ±2.88(5)	13.36 ±3.06(5)	14.88 ± 2.52 (5)	17.10 ±2.61(5))	15/62 ±1.86(5)
Creatinine	(mg/dL)	0.302 ±0.033 (5)	0.286 ±0.027(5)	0.252 * ±0.022 (5)	0.248 * ±0.027(5)	0.312 ±0.029(5)	0.302 ±0.038(5)
T-Protein	(g/dL)	5.70 ±0.31(5)	5.46 ±0.31(5)	5.40 ±0.21(5)	5.40 ±0.12(5)	6.10 ±0.30(5)	5.98 ±0.27(5)
Albumin	(g/dL)	2.62 ± 0.11 (5)	2.68 ± 0.19(5)	2.66 ±0.15(5)	2.64 ±0.05(5)	2.94 ±0.28(5)	2.78 ±0.13(5)
A/G ratio	(-)	0.856 ± 0.064(5)	0.968 ±0.088(5)	0.976 * ±0.087(5)	0.958 ±0.044(5)	0.930±0.086(5)	0.870±0.050(5)
Glucose	(mg/dL)	116.0±15.1(5)	107.8 ±5.2(5)	120.2±12.9(5)	117.0 ± 13.1(5)	125.8 ±16.0(5)	129.2±20.8(5)
T-Bil	(mg/dL)	0.050 ± 0.007 (5)	0.058 ±0.008(5)	0.056 ±0.011(5)	0.054 ±0.011 (5)	0.072 ±0.020 (5)	0.060 ±0.007(5)
TBA	(umol/L)	13.82 +8.84(5)	20.18 ±9.72(5)	18.18 ±3.09(5)	15.18 ± 5.42(5)	20.70 ±11.78(5)	13.44 *  ±4.77(5)
IP	(mg/dL)	7.16 ±0.34(5)	7.58 ±0.35(5)	7.84 ±0.74 (5)	8.06 * ±0.15(5)	6.08 ±0.69(5)	6.36 ±0.66(5)
Ca	(mg/dL)	9.78 ±0.225)	9.98 ±0.16(5)	9.82 ±0.31 (5)	10.28 * ±0.26(5)	9.50 ±0.32(5)	9.60 ±0.27(5)
Na	(mEq/L)	141.0 ±0.7(5)	140.6 ±0.9 (5)	140.6 ±1.3(5)	139.4 ±0.9(5)	139.0 ±1.2(5)	139.6 ±0.9(5)
K	(mEq/L)	4.22 ± 0.15(5)	4.52±0.34(5)	4.46 ±0.26(5)	4.52±0.13(5)	4.18 ±0.13(5)	4.12 ±0.19(5)
Cl	(mEq/L)	105.88 ±1.07 (5)	104.96 ±1.96(5)	104.60 ±0.47(5)	103.22 ** ±0.69 (5)	103.82 ±0.86(5)	105.64 ±1.60 (5)

Values are shown as Mean ± S.D

Figure(s) in parentheses indicate number of animals used for the statistical analysis

*Significantly different from vehicle control at P<0.05

** Significant different from vehicle control at P<0.01

 

Table 17 Summary of absolute organ weights - –Male

Items	Dose mg/kg/day	Main Groups				Recovery Group
		0	50	150	450	0	450
Liver	(g)	8.84 ± 0.998(5)	9.848 ±0.301(5)	10.838 *±0.994(5)	11.476** 0.994(5)	10.680 ±0.933(5)	11.284± 1.651(5)
Heaty	(g)	1.122 ±0.093(5)	1.168 ±0.095 (5)	1.158 ±0.040(5)	0.998 ±0.098(5)	1.280 ±0.129(5)	1.286 ±0.124(5)
Kidneys	(g)	2.262 ±0.166(5)	2.458 ±0.058(5)	2.552 ±0.196(5)	2.362 ±0.304(5)	2.748 ±0.239(5)	2.590 ±0.249(5)
Testes	(g)	2.822 ±0.254(5)	2.932 ±0.287(5)	3.086 ±0.190(5)	2.742 ±0.373(5)	3.094 ±0.214(5)	3.048 ± 0.366(5)
Epididymides	(g)	0.634 ±0.092(5)	0.684 ±0.047(5)	0.702 ±0.042(5)	0.716±0.077(5)	0.974 ±0.070(5)	0.870 ±0.144(5)
Prostate	(g)	0.710 ±0.032(5)	0.794 ±0.091(5)	0.744 ±0.096(5)	0.658 ±0.155(5)	1.010 ±0.133(5)	0.832 ±0.156(5)
Seminal vesicles	(g)	1.040 ±10.103(5)	1.004±0.227(5)	1.002 ±0.138(5)	1.080 ±0.302(5)	1.282 ±0.221(5)	1.084 ±0.187(5)
Brain	(g)	1.980 ± 0.090(5)	1.976 ±0.039(5)	2.044±0.046(5)	2.1018 ±0.130(5)	2.032±0.145(5)	2.040 ±0.075(5)
Spleen	(g)	0.626 ± 0.092(5)	0.736 ±0.118(5)	0.658 ±0.072(5)	0.580 ±0.151(5)	0.696 ±0.090(5)	0.716 ±0.135(5)
Thymus	(mg)	496.98 ±59.23(5)	646.46 * ±62.30(5)	577.40 ±91.49(5)	533.40 ±99.36(5)	456.00 ±104.11(5)	582.22 ±80.43(5)
Thyroid	(mg	16.62 ±3.17(5)	19.10 ±2.69(5)	21.52 * ±2.31(5)	20.36 ±1.93(5)	20.08 ±1.02(5)	22.92 ±2.96(5)
Adrenals	(mg)	47.44 ±4.70(5)	51.02 ±3.23(5)	46.46 ±5.56(5)	51.68 ±7.28(5)	49.82 ±10.17(5)	49.96 ±3.28(5)
Final body weight	(g)	312.78 ±23.03(5)	327.02 ±7.59(5)	329.34 ±24.88(5)	287.14 ±28.08(5)	398.34 ±26.29(5)	382.50 ±32.60 (5)

Values are shown as Mean ± S.D

Figure(s) in parentheses indicate number of animals used for the statistical analysis

*Significantly different from vehicle control at P<0.05

** Significant different from vehicle control at P<0.01

 

Table 18 Summary of absolute organ weights - –Female

Items	Dose mg/kg/day	Main Groups				Recovery Group
		0	50	150	450	0	450
Liver	(g)	6.592 ± 0.619(5)	6.844 ±0.499(5)	7.260 *±0.583(5)	8.534** 0.799(5)	6.732 ±0.808(5)	6.982± 0.584(5)
Heatr	(g)	0.850± 0.088	0.844 ±0.097 (5)	1.874 ±0.081(5)	1.796 ±0.082 (5)	1.724 ±0.096(5)	1.770 ±0.187(5)
Kidneys	(g)	1.732 ±0.207 (5)	1.800 ±0.160(5)	1.874 ±0.168(5)	1.796 ±0.082(5)	1.724 ±0.096(5)	1.770 ±0.187(5)
Ovaries	(mg)	100.58 ±20.34 (5)	88.62 ±5.30(5)	89.12 ±12.86(5)	90.02 ±6.46(5)	82.20 ±10.17(5)	88.52 ± 13.94(5)
Uterus	(g)	0.486±0.053 (5)	0.570 ±0.201 (5)	0.504±0.084(5)	0.588±0.285(5)	0.472 ±0.080(5)	0.536±0.094(5)
Brain	(g)	1.892 ± 0.048 (5)	1.878 ±0.029(5)	1.904±0.109(5)	1.942 ±0.052(5)	1.926±0.049 (5)	1.956 ±0.043(5)
Spleen	(g)	0.516 ± 0.087 (5)	0.586±0.086 (5)	0.576 ±0.106(5)	0.496 ±0.031(5)	0.518±0.043(5)	0.546±0.155(5)
Thymus	(mg)	520.38 ±72.07 (5)	508.78  ±55.52(5)	553.64 ±170.16(5)	507.00 ±92.24(5)	467.40 ±162.81(5)	445.78 ±62.4(5)
Thyroid	(mg	15.02 ±2.62 (5)	15.22 ±1.00(5)	18.08 ±3.12(5)	19.38 * ±1.86(5)	18.32 ±2.25(5)	17.64 ±3.57(5)
Adrenals	(mg)	65.92 ±7.40 (5)	65.46 ±9.98(5)	75.52 ±6.98(5)	77.76 ±3.63(5)	61.36 ±7.71(5)	65.68 ±7.03(5)
Final body weight	(g)	217.22 ±15.34 (5)	224.22 ±11.91(5)	221.30 ±21.85(5)	208.92 ±1.77(5)	242.60 ±21.64(5)	227.80±15.00 (5)

Values are shown as Mean ± S.D

Figure(s) in parentheses indicate number of animals used for the statistical analysis

*Significantly different from vehicle control at P<0.05

** Significant different from vehicle control at P<0.01

 

Table 19 Summary of relative organ weights - –male

Items	Dose mg/kg/day	Main Groups				Recovery Group
		0	50	150	450	0	450
Liver	(g/100g)	2.814 ± 0.171(5)	3.012 ±0.073(5)	3.288 ** ±0.0.61(5)	3.990** 0.224(5)	2.676 ±0.103(5)	2.940 *± 0.208(5)
Heart	(g/100g)	0.360± 0.010 (5)	0.356 ±0.023 (5)	0.352 ±0.022(5)	0.350 ±0.019 (5)	0.322 ±0.016(5)	0.338 ±0.033(5)
Kidneys	(g/100g)	0.722±0.013(5)	0.754 ±0.027(5)	0.776 * ±0.0.21(5)	0.822 ±0.044(5)	0.690 ±0.031(5)	0.678 ±0.026(5)
Testes	(g/100g)	0.904 ±0.078(5)	0.896 ±0.069(5)	0.938 ±0.079(5)	0.956 ±0.102(5)	0.776 ±0.029(5)	0.798 ± 0.076 (5)
Epididymides	(g/100)	0.204±0.018(5)	0.208 ±0.016 (5)	0.216±0.021(5)	0.250 *±0.037(5)	0.244 ±0.005(5)	0.228±0.027(5)
Prostate	(g/100g)	0.230±0.012(5)	0.244 ±0.030(5)	0.228 ±0.038(5)	0.228 ±0.036(5)	0.254 ± 0.043(5)	0.218 ±0.036(5)
Seminal vesicle	(g/100g)	0.322±0.023 (5)	0.308 ± 0.076(5)	0.306 ± 0.053(5)	0.374 ± 0.079(5)	0.326 ± 0.081(5)	0.284 ± 0.058(5)
Brain	(g/100g)	0.636 ± 0.046(5)	0.604 ±0.021(5)	0.622±0.56(5)	0.706 ±0.043(5)	0.510±0.020 (5)	0.532 ±0.033(5)
Spleen	(g/100g)	0.200 ± 0.025(5)	0.226±0.033 (5)	0.200 ±0.033(5)	0.200 ±0.0365)	0.176±0.015(5)	0.186±0.029(5)
Thymus	(mg/100g)	159.04 ±16.81(5)	198.04  ±23/02(5)	175.72 ±29.15(5)	186.32 ±31.21(5)	114.08 ±22.57(5)	153.36 *±26.88(5)
Thyroid	(mg/100g)	5.32 ±1.03(5)	5.86 ±0.74(5)	6.56 * ±0.32(5)	7.10 ** ±0.60(5)	5.06 ±0.50(5)	5.98* ±0.49(5)
Adrenals	(mg/100g)	15.18 ±1.05(5)	15.58 ±1.10(5)	14.14 ±1.68(5)	18.00 * ±1.94(5)	12.42 ±2.09(5)	13.20  ±2.00(5)
Final body weight	(g)	312.78 ±23.03(5)	327.02 ±7.59(5)	329.34 ±24.88(5)	287.14 ±28.08(5)	398.34 ±26.29(5)	382.50 ±32.60 (5)

Values are shown as Mean ± S.D

Figure(s) in parentheses indicate number of animals used for the statistical analysis

*Significantly different from vehicle control at P<0.05

** Significant different from vehicle control at P<0.01

Table 20 Summary of relative organ weights - –Female

Items	Dose mg/kg/day	Main Groups				Recovery Group
		0	50	150	450	0	450
Liver	(g/100g)	3.034 ± 0.126(5)	3.054 ±0.163(5)	3.286 ±0.140(5)	4.086** 0.396(5)	2.770 ±0.171(5)	3.072 *± 0.262(5)
Heart	(g/100g)	0.392± 0.024(5)	0.376 ±0.023 (5)	0.376 ±0.023(5)	0.376 ±0.026 (5)	0.364 ±0.026(5)	0.354 ±0.026(5)
Kidneys	(g/100g)	00.798 ±0.063(5)	0.804 ± 0.061(5)	0.850 ±0.056(5)	0.862 ±0.040(5)	0.714 ±0.068(5)	0.776 ±0.072(5)
Ovaries	(mg/100g)	46.02 ±6.74(5)	39.54 ±1.01(5)	40.62 ±6.93(5)	43.10 ±3.085)	32.24 ±6.52(5)	36.86 ± 5.39 (5)
Uterus	(g/100)	0.226±0.033(5)	0.252 ±0.085 (5)	0.228±0.026(5)	0.282 *±0.136(5)	0.196 ±0.042(5)	0.238±0.045(5)
Brain	(g/100g)	0.874 ± 0.055(5)	0.840 ±0.047(5)	0.864±0.54(5)	0.930±0.027(5)	0.7980±0.075 (5)	0.862 ±0.050(5)
Spleen	(g/100g)	0.238 ± 0.04(5)	0.260±0.029 (5)	0.258 ±0.024(5)	0.238 ±0.019(5)	0.216±0.023(5)	0.238±0.055(5)
Thymus	(mg/100g)	214.56 ±46.74(5)	227.60  ±28.92(5)	249.46 ±68.54(5)	242.56 ±43.42	189.68 ±48.91(5))	195.48.36 ±21.40(5)
Thyroid	(mg/100g)	6.90 ±1.08(5)	6.78 ±0.48(5)	8.22 ±1.50(5)	9.26 ** ±0.84(5)	7.64 ±1.34(5)	7.74 ±1.41(5)
Adrenals	(mg/100g)	30.38 ±2.81(5)	29.08 ±3.05(5)	34.52 ±5.78(5)	37.22 * ±1.91(5)	25.46 ±3.97(5)	28.92  ±3.46(5)
Final body weight	(g)	217.22 ±15.34(5)	224.22 ±11.91(5)	221.30 ±21.85(5)	208.92 ±1.77(5)	242.60 ±21.64(5)	227.80±15.00 (5)

Values are shown as Mean ± S.D

Figure(s) in parentheses indicate number of animals used for the statistical analysis

*Significantly different from vehicle control at P<0.05

** Significant different from vehicle control at P<0.01

 

Table 21 Summary of macroscopic examinations – Male and Female

Findings		Male						Female
	Mg/kg/day	0	0 (Recovery)	50	150	450	450(Recovery)	0	0 (Recovery)	50	150	450	450 (Recovery)
		ss	ss	ss	ss	ss	ss	ss	ss	ss	ss	ss	ss
		5 ^a)	5	5	5	5	5	5 ^a)	5	5	5	5	5
No abnormalities detected		5	5	5	5	0	5	5	5	5	5	0	5
Liver enlargement		0	0	0	0	5	0	0	0	0	0	5	0
Jejunum Diverticulum								0	0	0	0	1	0

Ss: Scheduled sacrifice animal

a) Number of animals examined

Table 21 Summary of histopathological examinations – Male

Findings	Grade	Male
		0	0 (Recovery)	50	150	450		450(Recovery)
		ss	ss	ss	ss	ss		ss
		5 ^a)	5	5	5	5		5
Trachea No abnormalities detected		5/5 ^b)	-	-	-	5/5		-
Lung No abnormalities detected		5/5	-	-	-	5/5		0
Submandibular gland No abnormalities detected		5/5	-	-	-	5/5		-
Forestomach No abnormalities detected		5/5	-	-	-	5/5		-
Glandular stomach No abnormalities detected		5/5	-	-	-	5/5		-
Duodenum No abnormalities detected		5/5	-	-	-	5/5		-
Jejunum No abnormalities detected		5/5	-	-	-	5/5		-
Ileum No abnormalities detected		5/5	-	-	-	5/5		-
Cecum No abnormalities detected		5/5	-	-	-	5/5		-
Colon No abnormalities detected		5/5	-	-	-	5/5		-
Rectum No abnormalities detected		5/5	-	-	-	5/5		-
Pancreas No abnormalities detected		5/5	-	-	-	5/5		-
Liver
No abnormalities detected		3/5	5/5	5/5	3/5		0/5	4/5
Hypertrophy of Hepatocytes, centrilobular	+	0/5	0/5	0/5	2/5		2/5	1/5
	++	0/5	0/5	0/5	0/5		3/5	0/5
Microgranuloma	+	2/5	0/5	0/5	0/5		0/5	0/5
Heart No abnormalities detected		5/5	-	-	-	5/5		-
Kidney No abnormalities detected		5/5	-	-	-	5/5		-
Urinary bladder No abnormalities detected		5/5	-	-	-	5/5		-
Testis No abnormalities detected		5/5 ^b)	-	-	-	5/5		-
Epididymis No abnormalities detected		5/5	-	-	-	5/5		-
Ventral prostate								-
No abnormalities detected		3/5	-	-	-	3/5		-
Atrophy focal	++	1/5	-	-	-	0/5		-
Lymphocyte infiltration	+	0/5	-	-	-	1/5		-
	++	1/5	-	-	-	1/5
Dorsolateral prostrate No abnormalities detected		5/5	-	-	-	5/5		-
Coagulating gland No abnormalities detected		5/5	-	-	-	5/5		-
Seminal vesicle No abnormalities detected		5/5	-	-	-	5/5		-
Cerebrum No abnormalities detected		5/5	-	-	-	5/5		-
Cerebellum No abnormalities detected		5/5	-	-	-	5/5		-
Pons No abnormalities detected		5/5	-	-	-	5/5		-
Spinal Cord No abnormalities detected-		5/5	-	-	-	5/5		-
Sciatic cord No abnormalities detected		5/5	-	-	-	5/5		-
Sciatic nerve No abnormalities detected		5/5	-	-	-	5/5		-
Bone Marrow No abnormalities detected		5/5	-	-	-	5/5		-
Axillar lymph node No abnormalities detected		5/5	-	-	-	5/5		-
Mesenteric lymph node No abnormalities detected		5/5	-	-	-	5/5		-
Spleen			-	-	-
No abnormalities detected		5/5	-	-	-		4/5
Capsulitis	+	0/5	-	-	-		1/5
Thymus No abnormalities detected		5/5	5/5	-	-	5/5		5/5
Pituitary gland No abnormalities detected		5/5 ^b)	-	-	-	5/5		-
Thyroid
No abnormalities detected		5/5	5/5	5/5	5/5	3/5		5/5
Ectopic thymic tissue	+	0/5	0/5	0/5	0/5	1/5		0/5
Follicular cell hypertrophy, diffuse	+	0/5	0/5	0/5	0/5	1/5		0/5
Parathyroid No abnormalities detected		5/5	-	-	-	5/5		-
Adrenal No abnormalities detected		5/5	-	-	-	5/5		-
Eyeball No abnormalities detected		5/5	-	-	-	5/5		-
Skeletal muscle No abnormalities detected		5/5	-	-	-	5/5		-
Bone No abnormalities detected		5/5	-	-	-	5/5		-
Mammary Gland No abnormalities detected		5/5	-	-	-	5/5		-

Ss: Scheduled sacrifice animal

a) Number of animals autopsied

b) Number of animals affected/Number of animals examined

-: Not examined

+: Slight, ++: moderate

Table 22 Summary of histopathological examinations – Female

Findings	Grade	Female
		0	0 (Recovery)	50	150	450	450(Recovery)
		ss	ss	ss	ss	ss	ss
		5 ^a)	5	5	5	5	5
Trachea No abnormalities detected		5/5 ^b)	-	-	-	5/5	-
Lung No abnormalities detected		5/5	-	-	-	5/5	-
Submandibular gland No abnormalities detected		5/5	-	-	-	5/5	-
Forestomach No abnormalities detected		5/5	-	-	-	5/5	-
Glandular stomach No abnormalities detected		5/5	-	-	-	5/5	-
Duodenum No abnormalities detected		5/5	-	-	-	5/5	-
Jejunum		-	-	-			-
No abnormalities detected		5/5			4/5		-
Diverticulum		0/5			1/5		-
Ileum No abnormalities detected		5/5	-	-	-	5/5	-
Cecum No abnormalities detected		5/5	-	-	-	5/5	-
Colon No abnormalities detected		5/5	-	-	-	5/5	-
Rectum No abnormalities detected		5/5	-	-	-	5/5	-
Pancreas No abnormalities detected		5/5	-	-	-	5/5	-
Liver
No abnormalities detected		5/5	5/5	5/5	5/5	0/5	5/5
Hypertrophy of Hepatocytes, centrilobular	+	0/5	0/5	0/5	0/5	4/5	0/5
	++	0/5	0/5	0/5	0/5	1/5	0/5
Heart No abnormalities detected		5/5	-	-	-	5/5	-
Kidney No abnormalities detected		5/5	-	-	-	5/5	-
Urinary bladder No abnormalities detected		5/5	-	-	-	5/5	-
Ovary No abnormalities detected		5/5 ^b)	-	-	-	5/5	-
Uterus (horn) No abnormalities detected		5/5	-	-	-	5/5	-
Uterus (Cervix) No abnormalities detected		5/5	-	-	-	5/5	-
Vagina No abnormalities detected		5/5	-	-	-	5/5	-
Cerebrum No abnormalities detected		5/5	-	-	-	5/5	-
Cerebellum No abnormalities detected		5/5^b)	-	-	-	5/5	-
Pons No abnormalities detected		5/5	-	-	-	5/5	-
Spinal Cord No abnormalities detected-		5/5	-	-	-	5/5	-
Sciatic cord No abnormalities detected		5/5	-	-	-	5/5	-
Sciatic nerve No abnormalities detected		5/5	-	-	-	5/5	-
Bone Marrow No abnormalities detected		5/5	-	-	-	5/5	-
Axillar lymph node No abnormalities detected		5/5	-	-	-	5/5	-
Mesenteric lymph node No abnormalities detected		5/5	-	-	-	5/5	-
Spleen			-	-	-
No abnormalities detected		4/5	-	-	-	5/5
Capsulitis	+	1/5	-	-	-	0/5
Thymus No abnormalities detected		5/5	5/5	-	-	5/5	5/5
Pituitary gland No abnormalities detected		5/5 ^b)	-	-	-	5/5	-
Thyroid
No abnormalities detected		5/5	5/5	5/5	5/5	3/5	5/5
Follicular cell hypertrophy, diffuse	+	0/5	0/5	0/5	0/5	2/5	0/5
Parathyroid No abnormalities detected		5/5	-	-	-	5/5	-
Adrenal No abnormalities detected		5/5	-	-	-	5/5	-
Eyeball No abnormalities detected		5/5	-	-	-	5/5	-
Skeletal muscle No abnormalities detected		5/5	-	-	-	5/5	-
Bone No abnormalities detected		5/5	-	-	-	5/5	-
Mammary Gland No abnormalities detected		5/5	-	-	-	5/5	-

a) Number of animals autopsied

b) Number of animals affected/Number of animals examined

-: Not examined

+: Slight, ++: moderate

Table 23: Summary estrous cycle stage

 

Stage	Female
	0	0 (Recovery)	50	150	450	450(Recovery)	mg/kg/day
	ss	ss	ss	ss	ss	ss
	5 ^a)	5	5	5	5	5
Proestrus	1	0	2	1	1	0
Estrus	3	1	1	2	1	4
Metestrus	0	4	2	2	2	1
Diestrus	1	0	0	0	1	0

ss: scheduled sacrifice animal

a) Number of animals examined

Conclusions:

This study was performed in accordance with OECD TG 407 under GLP conditions. General clinical observations, detailed clinical observations, blood chemical examinations, organ weight measurement, necropsy and histopathological examination were used to reach an outcome. It was determined that the test substance causes treatment-related adverse effects in the liver, with secondary effects on the thyroid, and a potential effect on the nervous system. Effects seen in the liver, thyroid and nervous system were all noted as reservable as they were not noted during the recovery period.

Based on the results presented, it was concluded that 28 days of exposure to the test substance exhibits target organ toxicity to the liver and thyroid at doses of 150mg/kg and above in males, and 450mg/kg in females. The NOAEL was therefore considered to be 50 mg/kg/day.

Executive summary:

This study was conducted in accordance with OECD TG 407 under GLP conditions. The systemic toxic potential of the test item was assessed in a 28-day oral gavage study using Crl: CD(SD) rats; recovery from any effects were evaluated during a subsequent 14-day recovery period. Three groups, each comprising of five male and five female rats received the test item at doses of 50, 150 and 450 mg/kg/day with an additional recovery group receiving 450 mg/kg/day. Two control groups (one used in the recovery analysis) of five male and five females were dosed with the vehicle alone (purified water). The recovery groups were dosed as per study groups and then maintained without treatment for a further 14 days. 

Clinical signs (general and detailed), sensorimotor function, body weight change and food and water consumption were monitored during the study. Haematology, blood chemistry and urinalysis were evaluated for all non-recovery groups at the end of the treatment and for all animals in the recovery group animals at the end of the examination period. Histopathology examinations were performed with no unscheduled mortalities. 

Liver enlargement was observed in males and females of the 450 mg/kg groups with increases in absolute and relative weights in males of the 150 mg/kg group and higher and females of the 450 mg/kg group at the end of the dosing period. Centrilobular hypertrophy of hepatocytes was observed in males in the 150mg/kg group and higher and in the females of the 450 mg/kg group. Additionally, a decrease in creatinine was observed in males and females of the 150 mg/kg group and higher with an increase in triglycerides in males of the 450 mg/kg group following blood chemical analysis. In this study, the concomitant changes in liver size, weight and pathology along with the presence of altered blood chemistry (lipid metabolism changes), and associated systemic alterations in homeostasis leading to additional thyroid effects (outlined below) were considered to be due to hepatotoxicity.

Decreased spontaneous locomotion, decreased respiratory rate and suppression of body weight gain in males and females of the 450 mg/kg groups including decreased food consumption in males of the 450 mg/kg group and females of the 150 mg/kg group and higher were considered to be a reflection of the liver effects noted during the dosing period. Lower body weight of males was also observed in the recovery period with this recovering gradually. Transient salivation which was observed in one male of the 150 mg/kg group and females of the 150mg/kg group and higher was considered to be a result of the taste of the test material and not an adverse effect on the nervous system due to the short sporadic observation time period. 

The effect on the nervous system included incomplete eyelid opening, tremor, lacrimation, sway and hypersensitivity which was observed in one or two females of the 450 mg/kg group during the dosing period. However, no abnormalities were observed in the histopathological examination with the effects on the nervous system noted being reversible due to the changes not being noted during observation in the recovery period.

Increases in the absolute and relative organ weights of the thyroid in males in the 150 mg/kg group and higher and females of the 450 mg/kg group were observed at the end of the dosing period. Diffuse follicular cell hypertrophy of the thyroid was observed in histopathological examination in males and females of the 450 mg/kg groups. The effects on the thyroid were considered to be reversible, since no changes in the thyroid were observed at the end of the recovery period. These effects were determined to be related to the test substance and therefore toxicologically relevant.

At the end of the dosing period an increase in the relative adrenal weights were observed in males and females in the 450 mg/kg group, although histopathological examinations did not reveal any abnormalities. The changes were therefore noted to be a result of stress related to the other adverse effects outlined, and not considered to be of toxicological importance. 

Increases in relative renal weight was observed in males in the 450 mg/kg group. These were considered to be an incidental change, as no abnormalities were detected in the blood chemical or histopathological examinations. 

No treatment-related effects were observed in the sensorimotor function examinations, urinalysis, haematological examinations or estrous cycle states.

Additional changes noted during the dosing period were determined not to be of toxicological concern but rather adaptive changes following administration of the test substance. These changes were also noted to be within the historical control data and were not determined to be dose dependant. 

Adverse effects on the liver were concluded to be the main adverse effect of the test substance, with secondary effects on the thyroid. Under the conditions of the study, No-Observed-Adverse-Effect-Level (NOAEL) was regarded to be 50 mg/kg/day for males/females. 

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

50 mg/kg bw/day

Study duration:

subacute

Experimental exposure time per week (hours/week):

0

Species:

rat

Quality of whole database:

This endpoint was composed of one quality study with a Klimish score of 1 performed in accordance with OECD TG 407 and GLP.

System:

haematopoietic

Organ:

liver

thyroid gland

Justification for classification or non-classification

ENDPOINT SUMMARY: REPEATED DOSE TOXICITY

 

Key information:

• OECD TG407; 28 day repeated dose toxicity; Hoshuyama al. 2018

 

Hoshuyama et al (2018) was undertaken in accordance with OECD TG 407 under GLP conditions. General clinical observations, detailed clinical observations, blood chemical examinations, organ weight measurement, necropsy and histopathological examination were used to reach an outcome. The specific liver, thyroid and related blood chemistry effects observed are summarised below.

 

Liver effects:

• Enlargement in liver size in males and females


• Dose dependent increases in absolute and relative weight in males dosed at 150mg/kg and higher, and females dosed at 450mg/kg (41% increase in relative weight at a dose of 450mg/kg for male; 34% increase at a dose of 450mg/kg for female).


• Centrilobular (regional) hypertrophy of hepatocytes (Males - observed in all 5 animals (‘slight’=2, ‘moderate’=3) at the 450mg/kg dose. Females - observed in all animals (‘slight’=1, ‘moderate’=4) at the 450mg/kg dose.


• There were no notable histopathological changes associated with degeneration or irreversible damage observed.

 

Thyroid effects:

• increases in the absolute and relative weights of the thyroid in males dosed at 150mg/kg and over, and females dosed at 450mg/kg, and


• diffuse follicular cell hypertrophy of the thyroid in males and females dosed at 450mg/kg.

 

Blood chemistry:

• A significant decrease in creatinine was observed in male and female animals dosed with 150mg/kg and higher.


• In males, a significant increase in triglyceride was also present in the 450mg/kg group. No significant change was observed in female animals.

 

The effects seen in the liver, thyroid and nervous system were all noted as reversable as they were not noted during the recovery period. Based on the results presented, Hoshuyama et al. concluded that 28 days of exposure to the test substance results in target organ toxicity to the liver and thyroid at doses of 150mg/kg and above in males, and 450mg/kg in females. The NOAEL was therefore considered to be 50 mg/kg/day.

 

The CLP guidance value range for STOT RE classification into category 1 is ≤ 30mg/kg and category 2 is ≤ 300mg/kg bw/day. Adverse effects considered to be related to treatment were reported at doses of 150mg/kg bw/day and above, therefore the effects are within the value range for classification of the test substance as STOT RE Category 2 (liver, thyroid).

 

In forming a justification for classification, it is pertinent to consider both the current knowledge base on liver toxicity in rodent models alongside the available guidance on determining classification, as part of a weight-of-evidence approach.

 

Rats are widely considered to be more sensitive to xenobiotic-induced perturbation of liver and thyroid homeostasis than humans (Lewandowski et al., 2004; Ennulat et al., 2010; Curran & Degroot., 1991; Colnot & Dekant, 2017). This can manifest as varied adaptive changes for which defining relevance to humans is not always straightforward (Hall et al., 2012).

 

As an example, one recognised Adverse Outcome Pathway (AOP) known to hold little relevance to humans is that linking enhanced hepatic clearance of thyroid hormones to thyroid follicular cell adenomas and carcinomas in the rat and mouse (Society for Advancement of AOPs, 2020). This AOP is initiated by the interaction of xenobiotics with members of the Nuclear Receptor superfamily constitutive androstane receptor (CAR) and the pregnane X receptor (PXR) in the liver, and typically presents as increased liver weight coupled with cellular hypertrophy and increased liver enzyme levels and activities (alkaline phosphatase, gamma glutamyl transferase and T4- glucuronytransferase activity), accompanied by thyroid effects (increased weight with histopathological changes, increased thyroid-stimulating hormone (TSH) levels and decreased thyroid hormone levels).

 

Despite similarity between the effects reported by Hoshuyama et al (2018) and the species-specific adaptive changes outlined within the AOP above, the authors reached a conclusion that these were treatment related rather than adaptive response. In support of this decision, the study authors quote the guidance of Yoshida (2014), which states that effects can be considered to be the result of hepatotoxicity when liver hypertrophy is accompanied by changes in the lipid metabolism system, which in this case is represented by the triglyceride changes observed in male animals.

 

In order to establish whether classification as STOT-RE Cat. 2 is justified, the evidence presented within the key study was compared to both the criteria for hepatotoxicity provided by Yoshida (2014) and guidance utilised within Europe, via publications relevant to REACH from the European Society on Toxicological Pathology (ESTP) (Hall et al, 2012) and cross-country and cross-regulatory specialist working groups (BPR Human Health WG, 2018; WHO, 2015) to form a weight-of evidence approach. It is clear that there is much discussion around the toxicological relevance to humans of hepatocellular hypertrophy and/or increased liver weight in rats following exposure to xenobiotics, and that a clear-cut guide for decisions around whether liver effects are considered adverse or adaptive is hard to identify.

 

In relation to liver enlargement, ESTP suggests increases in relative liver weight up to 50% may be considered adaptive if there are no other indicators of toxicity, where others are far more conservative at 10% or 20%. Although there is a wide range in recommended cut off for adaptive vs adverse response, it is most appropriate here to consider the increase of up to 41% in relative liver weight observed within this study adverse as it is accompanied by other indicators of toxicity.

 

For hepatocellular hypertrophy of the liver to be considered an adverse effect, the available guidance states that it should be accompanied by other hepatic responses identifiable by histology. As no notable histopathological changes associated with degeneration or irreversible damage were observed alongside hepatocellular hypertrophy, this observation supports adaptive response more than adverse.

 

In relation to biochemistry, the changes related to clinical pathology (creatinine and triglyceride) contribute to a weight of evidence approach to classify the substance as a Cat. 2 STOT RE. As mentioned above, Yoshida (2014) consider that changes in lipid metabolism alongside hypertrophy may be considered adverse, therefore triglyceride level changes contribute toward the case for STOT RE Cat.2. The only biomarker that changes consistently in a dose-response manner is creatinine. This could be due to decreases in liver function, as creatinine levels are known to drop in chronic liver disease (a consideration which is also noted by Hoshuyama et al. in the study report). Alternatively, it could be attributable to the thyroid effects noted, as a link between hyperthyroidism in humans and decreased creatinine levels has been previously highlighted (Basu et al, 2012; Rhee, 2016). Without further investigation, it is not possible to conclude on the organ driving this change.

 

In relation to the observed thyroid effects, without additional information relating to circulating thyroid hormone levels, it is not possible to elucidate whether these are due to stand alone effects on the thyroid, or a result of metabolic enzyme activation in the liver. However, WHO (2015) note that if effects are seen which may be secondary to those observed in the liver, evaluation of the mode of action for these effects should be considered to determine the relevance of these effects to humans and the establishment of health-based guidance values. It may be relevant to consider this in any future testing on this substance.

 

Notably, WHO (2015) state that “If there is insufficient information to determine whether observed liver hypertrophy is an adaptive or an adverse response, then the default is to assume that the effect is adverse.” Therefore, although the evidence presented within the key study means that classification for target organ toxicity is not clear cut, a weight of evidence approach combined with the precautionary principle lead to the conclusion that classification of STOT RE Cat.2 (liver, thyroid) is the most suitable outcome.

References:

Basu, G., & Mohapatra, A. (2012). Interactions between thyroid disorders and kidney disease; Indian journal of endocrinology and metabolism, 16(2), 204–213. doi: 10.4103/2230-8210.93737

 

Colnot, T. and Dekant, W. (2017). Approaches for grouping of pesticides into cumulative assessment groups for risk assessment of pesticide residues in food. Regulatory Toxicology and Pharmacology. 83: 89-99.

 

Curran, P.G. and Degroot, L.J. (1991). The effect of hepatic enzyme- inducing drugs on thyroid hormones and the thyroid gland. Endocrine Reviews. 12(2): 135–50.

 

Ennulat, D., Walker, D., Clemo, F., Magid-Slav, M., Ledieu, D., Graham, M., Botts, S. and Boone, L. (2010). Effects of hepatic drug-metabolizing enzyme induction on clinical pathology parameters in animals and man. Toxicologic Pathology 38(5): 810–28.

 

Hall, A. P. et al. (2012) Liver Hypertrophy: A Review of Adaptive (Adverse and Non-adverse) Changes—Conclusions from the 3rd International ESTP Expert Workshop; Toxicologic Pathology, 40(7), pp. 971–994. doi: 10.1177/0192623312448935.

 

 

Lewandowski, T.A., Seeley, M.R. and Beck, B.D. (2004). Interspecies differences in susceptibility to perturbation of thyroid homeostasis: a case study with perchlorate. Regulatory Toxicology and Pharmacology 39(3): 348-362.

 

Rhee C. M. (2016). The interaction between thyroid and kidney disease: an overview of the evidence; Current opinion in endocrinology, diabetes, and obesity, 23(5), 407–415. doi: 10.1097/MED.0000000000000275

 

Society for Advancement of AOPs (2020), AOP 162: Enhanced hepatic clearance of thyroid hormones leading to thyroid follicular cell adenomas and carcinomas in the rat and mouse, AOP-Wiki, Available from http://aopwiki.org (accessed 21/10/20).

 

UK contributors to the BPR Human Health WG (WG-IV-2018) (2018); Annex to the document “Interpretation of liver effects”; accessible at https://webgate.ec.europa.eu/s-circabc/faces/jsp/extension/wai/navigation/container.jsp (accessed 26/11/20)

 

WHO Core Assessment Group on Pesticide Residues (2015); Pesticide residues in food - Guidance document for WHO monographers and reviewers; WHO/HSE/GOS/2015.1, 1-106pp

 

Yoshida, M. et al. (2015); [Basic principles of interpretation of hepatocellular hypertrophy in risk assessment in Japan] Shokuhin eiseigaku zasshi; Journal of the Food Hygienic Society of Japan, 56(2), 42–48. doi: 10.3358/shokueishi.56.42