Registration Dossier

Administrative data

Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Version / remarks:
adapted Jan 2001
Deviations:
yes
Remarks:
no oestrus cycle and no sperm parameters were evaluated, no sexual maturation parameters were determined, F1 and F2 offspring necropsy data were not reported
GLP compliance:
not specified
Remarks:
study was performed in China and it is not clear whether it was performed according to GLP
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Zhejiang Center of Laboratory Animals, Hangzhou, Zhejiang, China
- Females nulliparous and non-pregnant: yes
- Age at study initiation: P1 generation: males: 4-5 weeks old; females: 8-9 weeks old
- Weight at study initiation: P1 generation: males: 100 - 128 g; females: 200 - 253 g;
- Fasting period before study: not applicable
- Housing: two per cage (polycarbonated cages, stainless steel racks) not further specified whether these conditions were changed during pregancy/lactation period etc.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20±3
- Humidity (%): 30-70
- Air changes (per hr): 10-12
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): no data
- Method: the weighed sample was mixed uniformly with some blank feed and the mixed feed of the sample was mixed with the blank feed gradually to achieve the designated concentration
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: maximum 3 weeks
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): 2 per cage
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
P generation: males: were dosed for 10 weeks prior to mating and continued to be dosed until successful mating
P generation: females: were dosed for 2 weeks prior to mating and dosing continued until the end of lactation
F1 generation: males and females: were dosed for 10 weeks after weaning (about 21 days old), male rats were dosed until successful mating, and female rats were dosed throughout mating, pregnancy and until the end of lactation
Frequency of treatment:
daily, continuously in the diet
Details on study schedule:
- F1 parental animals mated 10 weeks after weaning.
- Selection of parents from F1 generation when pups were about 21 days of age.
- Age at mating of the mated animals in the study: not specified
Doses / concentrationsopen allclose all
Dose / conc.:
625 ppm
Remarks:
equivalent to 71.76 and 61.10 mg/kg bw/day for females and males, respectively, as average of all generations
Dose / conc.:
2 500 ppm
Remarks:
equivalent to 294.88 and 251.19 mg/kg bw/day for females and males, respectively, as average of all generations
Dose / conc.:
10 000 ppm
Remarks:
equivalent to 1236.29 and 1258.55 mg/kg bw/day for females and males, respectively, as average of all generations
No. of animals per sex per dose:
24
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: Based on acute oral LD50 (> 2000 mg/kg bw in male and female rats) and a preliminary dose-range-finding study, 192 SD rats (96 males and 96 females) were randomly assigned to four groups.
Positive control:
no

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations: behavior change, abnormality of excretion, death and other toxi clinical signs

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
Time schedule for examinations: P generations were weighed weekly, the body weights of the pregnant females were ecorded on gestation Day 0, 7, 14, and 21; the body weight of dams and pups (in litter) were measured on Day 0, 4, 7, 14, and 21 (the day pregnant rats delivered was defined as Day 0 post parturition)

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OTHER: signs of difficult or prolonged parturition and all signs of toxicity was recorded during delivery
Estrous cyclicity (parental animals):
not examined
Sperm parameters (parental animals):
Parameters examined in all male parental generations:
testis weight, epididymis weight
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as closely as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 and F2 offspring:
Each litter was examined as soon as possible after delivery (lactation day 0) to establish the number and sex of pups, stillbirths, live births, and presence of gross anomalies. Physical postnatal mortality or behavioural abnormalities were recorded. Body weights were measured on Day 0, 4, 7, 14, and 21 (day 0 is defined as the day of delivery)

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: no

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: all surviving animals after mating
- Maternal animals: all surviving animals after the last litter of each generation was weaned

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

HISTOPATHOLOGY / ORGAN WEIGHTS
Weight of ovaries, testicles, and epididymis were determined. Uterus, ovaries, cervix, testicle, epididymis, seminal vesicle, prostate, penis, pituitary, brain and target organs were preserved for histopathological examination. Histopathological examination was carried out on animals in the highest dose group and control group and on abnormal organs/tissues found in gross necropsy. If no significant pathology changes were found in the highest dose group, histopathological examination was not carried out on other treated groups. If significant pathology changes were found in the highest dose group, the other treated groups were also examined.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed after weaning at 21 days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: all were examined for external abnormalities, only pups with external abnormalities or clinical signs were examined macroscopically for any structural abnormalities or pathological changes.

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

HISTOPATHOLOGY / ORGAN WEIGTHS
not examined
Statistics:
A parameter or non-parameter test was selected based on the results of normality test and homogeneity of variance test. One-way analysis of variance and Dunnett's t-test were used in parameter test, Kruskal-Wallis rank sum test and Wilcoxon-Wilcox rank sum test were used in non-parameter test, the test level of alpha was 0.05. Chi-square test and Fisher exact prabability test were used in enumeration data, the test level of alpha was 0.05, corrected alpha' was 0.0170.
Reproductive indices:
rate of mating success (%) = (number of successful mating animals / number of female animal be mated) x 100%
prenancy rate (%) = (number of pregnant animals / number of female animals be mated) x 100%
live birth rate (%) = (number of female animals producing live offspring / number of pregnant animals) x 100%
Offspring viability indices:
rate of birth livability (%) = (number of offspring survived on day 4 / number of offspring survived on birth day) x 100%
survival rate after weaning (%) = (number of offspring survived on day 21 after weaning / number of offspring survived on day 4) x 100%

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female rat in the control group died during the lactation period.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
high dose group; males: the absolute body weights from week 1 (-18.3%) to week 10 (-12.1%) and the total body weight gain (-16.3%) were significantly lower than in the control group - this effect is considered to be treatment-related and adverse because it is observed throughout treatment and the average food consumption was not significantly changed compared to the control group
high dose group; females: the absolute body weights of week 9 (-12.0%) and 10 (-9.5%) and the total body weight gain (-125%) were significantly lower than in the control group - this effect is considered to be treatment-related and adverse because it is observed throughout treatment and the average food consumption was not significantly changed compared to the control group
mid dose group; males: the absolute body weights at the end of week 1 (-6.5%) were significantly lower than in the control group - this effect is considered to be not of toxicological relevance since it is only observed in week 1 and below 10% and not throughout the treatment
pregnant rats: the absolute body weight in the high dose group on gestation day 0 (-10.1%), 7 (-12.4%), 14 (-13.3%), 20 (-16.6%) and the total body weight gain (-28.5%) during the gestation period were significantly lower than in the control group - this effect is considered to be treatment-related and adverse because it is observed throughout treatment and the average food consumption was not significantly changed compared to the control group
maternal rats during lactation period: the absolute body weight in the high dose group on day 0 (-18.6%), 4 (-19.9%), 7 (-20.3%), 14 (-17.6%), 21 (-11.4%) were significantly lower compared to the control group. The total body weight gain (+48.3%) during lactation period was significantly higher than in the control group - reduced absolute body weight is considered to be treatment-related and adaptive since the total body weight gain is significantly higher than in the control group and the average food consumption was not significantly changed compared to the control group
(see table 3, 5 and 6 in section "any other information on results incl. tables" for more detail)
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
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):
The weight (-20%) and the relative organ weight (-18.8%) of the ovaries in the high dose group of maternal rats were significantly lower than in the control group. This effect is considered to be non-treatment related as the change was <= 20% and no effects on fertility/reproduction was observed.
The epididymis weight (-8.3%) in males at the mid dose group was significantly decreased compared with the control group. Since no dose-response relationship is observed and the change is <= 20% this effect is considered to be incidental.
For more details see table 12 in section "any other information on results incl. tables".
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Atrophia testiculi and spermatogenesis, ductus epididymis without sperms, epididymal epithelial cells vacuolation and prostatic stromal infiltration of inflammatory cells was found in single animals in the control group as well as in the high dose group. Since these effects were only single observations and found in the control group as well as the high dose group these effects are considered to be incidental and not treament-related. See table 14 under section "any other information on results incl. tables".
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: estrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
2 500 ppm
Based on:
test mat.
Remarks:
corresponding to 294.88 and 251.19 mg/kg bw/day for females and males (average of generations), respectively
Sex:
male/female
Basis for effect level:
body weight and weight gain
Key result
Dose descriptor:
NOAEL
Remarks:
fertility
Effect level:
10 000 ppm
Based on:
test mat.
Remarks:
corresponding to 1236.29 and 1258.55 mg/kg bw/day for females and males (average of generations), respectively
Sex:
male/female
Basis for effect level:
reproductive performance

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
high dose group; males: the absolute body weight of male rats at the end of weeks 0 (-35.8%) to 10 (-25.5%) and the total body weight gain (-23.4%) were significantly lower than in the control group - this effect is considered to be treatment-related and adverse because it is observed throughout treatment and the average food consumption was not significantly changed compared to the control group
high dose group: females: the absolute body weight of female rats at the end of weeks 0 (-33.3%) to 10 (-13.6%) were significantly lower than in the control group. The total body weight gain was not significantly different compared to the control group - the reduced absolute body weight is considered treatment-related and adverse because it is observed throughout treatment and the average food consumption was not significantly changed compared to the control group however the total body weight was not changed between high dose group and control group showing that that the initial absolute body weight was lower but the body weight gain was not influenced by the treatment
mid dose group: males: the absolute body weight of male rats at the end of weeks 3 (-5.8%), 4 (-5.9%), 7 (-6.0), 10 (-5.9%) and the total body weight gain (-6.8%) were significantly lower than in the control group - these effects are considered not toxicological relevant since the changes are below 10%
pregnant rats: high dose group: the absolute body weight of pregnant rats on gestation day 0 (-14.3%), 7 (-15.4%), 14 (-16.6%), 20 (-18.5%) and the total body weight gain (-26.9%) during the gestation period were significantly lower than in the control group - this effect is considered to be treatment-related and adverse because it is observed throughout treatment and the average food consumption was not significantly changed compared to the control group
maternal rats in lactation period: high dose group: the absolute body weights of maternal rats on day 0 (-13.8%), 4 (-13.1%), 7 (-10.2%), 14 (-9.4%), 21 (-9.0%) during lactation period were significantly lower than in the control group; the total body weight gain increased (+59.5%) but was not significant compared to the control group - reduced absolute body weight is considered to be treatment-related and adaptive since the total body weight gain is significantly higher than in the control group and the average food consumption was not significantly changed compared to the control group
(see table 4, 5 and 6 under section "any other information on results incl. tables" for more detail)
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
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):
The weight of testicles (-11%) and epididymis (-19%) in male rats in the high dose group is significantly decreased compared to the control group. The relative organ weights for both organs are significantly increased (+20% for testicle and +11.5 for epididymis). These effects are considered to be non-treatment related as the changes were <= 20% and no effects on fertility/reproduction were observed.
For detailed information see table 13 under section "any other information on results incl. tables".
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Compared with the control group, no abnormal tissues/organs were observed in the treatment groups. See table 14 under section "any other information on results incl. tables" for more details.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Reproductive function / performance (P1)

Reproductive function: estrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Effect levels (P1)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
systemic toxcity
Effect level:
2 500 ppm
Based on:
test mat.
Remarks:
corresponding to 294.88 mg/kg bw/day (average of generations)
Sex:
female
Basis for effect level:
body weight and weight gain
Key result
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
2 500 ppm
Based on:
test mat.
Remarks:
corresponding to 251.19 mg/kg bw/day (average of generations)
Sex:
male
Basis for effect level:
body weight and weight gain
Key result
Dose descriptor:
NOAEL
Remarks:
fertility
Effect level:
10 000 ppm
Based on:
test mat.
Remarks:
corresponding to 1236.29 and 1258.55 mg/kg bw/day for females and males (average of generations), respectively
Sex:
male/female
Basis for effect level:
reproductive performance

Target system / organ toxicity (P1)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
Survival rate after weaning was not significantly changed between treated groups and control goups. For more detail see table 11 under section "any other information on results incl. tables".
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The average litter weight on day 0 (-23.2%), 4 (-15.3%), 7 (-26.4%), 14 (-26.8%), 21 (-20.3%), and the average body weights of the pups on day 4 (-14.6%), 7 (-25.1%), 14 (-23.0%), 21 (-15.9%) in the high dose group were significantly lower than the control group. These effects are considered to be treatment-related and secondary due to body weight loss of the maternal rats. For more details see tables 7 and 8 under section "any other information on results incl. tables".
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
Other effects:
not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Remarks:
developmental
Generation:
F1
Effect level:
2 500 ppm
Based on:
test mat.
Remarks:
corresponding to 294.88 and 251.19 mg/kg bw/day for females and males (average of generations), respectively
Sex:
male/female
Basis for effect level:
body weight and weight gain

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Results: F2 generation

General toxicity (F2)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
Survival rate after weaning was not significantly changed between treated groups and control goups. For more detail see table 11 under section "any other information on results incl. tables".
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The average litter weight on Day 0 (-20.2%), 4 (-11.9%), and the average body weights of pups on Day 14 (-6.5%) and 21 (-9.0%) of high dose group were significantly lower than in the control group. This effect is considered to be treatment-related and secondary due to body weight loss of maternal rats . For more details see tables 7 and 8 under section "any other information on results incl. tables".
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
Other effects:
not examined

Developmental neurotoxicity (F2)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:
not examined

Effect levels (F2)

Key result
Dose descriptor:
NOAEL
Remarks:
developmental
Generation:
F2
Effect level:
2 500 ppm
Based on:
test mat.
Remarks:
corresponding to 294.88 and 251.19 mg/kg bw/day for females and males (average of generations), respectively
Sex:
male/female
Basis for effect level:
body weight and weight gain

Target system / organ toxicity (F2)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
10 000 ppm
Treatment related:
yes
Relation to other toxic effects:
reproductive effects as a secondary non-specific consequence of other toxic effects
Dose response relationship:
yes
Relevant for humans:
not specified

Any other information on results incl. tables

Table 1: Mortality P0-Generation (number of dead animals/total animals)

sex

group

pre-mating

mating

pregnancy

lactation

female

control

0/24

0/24

0/23

1/23

low dose

0/24

0/24

0/21

0/21

mid dose

0/24

0/24

0/23

0/23

high dose

0/24

0/24

0/23

0/23

male

control

0/24

0/24

-

-

low dose

0/24

0/24

-

-

mid dose

0/24

0/24

-

-

high dose

0/24

0/24

-

-

Table 2: Mortality P1-Generation (number of dead animals/total animals)

sex

group

pre-mating

mating

pregnancy

lactation

female

control

0/24

0/24

0/23

0/23

low dose

0/24

0/24

0/22

0/22

mid dose

0/24

0/24

0/21

0/21

high dose

0/24

0/24

0/24

0/24

male

control

0/24

0/24

-

-

low dose

0/24

0/24

-

-

mid dose

0/24

0/24

-

-

high dose

0/24

0/24

-

-

Table 3: Body weight changes P0 Generation (g, mean±SD)

sex

group

week 0

week 1

week 2

week 3

week 4

week 5

week 6

week 7

week 8

week 9

week 10

total body weight gain

male

control

112.15±7.48

168.10±8.63

214.16±17.38

268.63±18.56

315.72±19.84

346.54±22.55

373.45±27.10

396.15±29.29

409.31±31.69

424.40±34.15

436.80±32.67

324.64±33.78

low dose

112.00±7.04

163.11±8.55

213.87±12.34

264.33±16.68

309.95±20.35

333.30±26.72

356.75±29.44

380.08±31.18

396.01±36.81

412.55±30.55

419.78±34.78

307.78±35.97

mid dose

112.49±7.91

157.22±12.74*

208.91±11.71

265.43±13.92

311.17±15.84

344.56±22.01

372.08±25.44

398.18±29.4

414.35±30.55

411.79±73.26

435.95±36.91

323.46±37.59

high dose

112.14±8.60

137.38±8.60*

173.62±12.06*

216.82±16.70*

246.07±23.05*

279.04±27.41*

314.68±28.35*

340.00±27.85*

359.30±29.25*

378.39±27.47*

383.87±29.90*

271.73±28.50*

female

control

-

-

-

-

-

-

-

-

231.13±10.77

239.90±9.98

246.71±11.12

15.58±9.99

low dose

-

-

-

-

-

-

-

-

229.92±10.29

236.30±12.79

243.70±12.75

13.78±11.22

mid dose

-

-

-

-

-

-

-

-

228.96±10.54

232.68±12.69

243.03±14.89

14.07±11.50

high dose

-

-

-

-

-

-

-

-

227.43±10.87

211.22±10.88*

223.38±12.27*

-4.05±11.05

* Dunnett's t-test, compared with control group p < 0.05

Table 4: Body weight changes P1 Generation (g, mean±SD)

sex

group

week 0

week 1

week 2

week 3

week 4

week 5

week 6

week 7

week 8

week 9

week 10

total body weight gain

male

control

81.32±10.22

131.81±14.39

173.16±24.51

243.99±21.74

294.58±23.58

337.72±24.37

371.80±33.17

403.90±27.67

427.83±29.36

453.53±34.70

467.33±37.56

386.01±34.61

low dose

81.75±8.02

129.66±12.51

174.45±14.16

238.73±17.48

286.89±17.61

331.82±19.92

367.80±22.76

395.48±26.57

420.86±28.76

435.39±35.36

450.57±32.37

368.81±28.66

mid dose

79.70±12.40

123.90±17.35

169.84±21.99

229.89±25.23*

277.15±30.41*

324.35±31.31

358.65±36.39

379.79±50.50*

407.18±44.18

431.90±46.39

439.65±51.51*

359.95±44.83*

high dose

52.21±8.57*

77.63±10.05*

107.03±13.24*

151.77±17.54*

187.23±19.89*

220.75±22.44*

261.80±24.42*

285.65±28.06*

300.17±28.10*

327.91±32.09*

348.04±35.73*

295.83±30.82*

female

control

74.80±8.91

115.86±12.09

148.50±13.31

177.35±14.42

190.72±14.49

214.78±14.94

230.55±15.56

238.94±16.76

248.94±16.24

259.16±19.89

264.15±21.66

189.35±17.41

low dose

77.10±9.42

118.14±12.97

151.65±12.58

179.60±13.07

191.28±14.42

213.55±16.67

231.36±18.05

236.19±17.20

250.86±16.48

256.81±18.28

262.86±18.45

185.75±16.02

mid dose

70.56±11.08

110.25±11.88

143.45±11.23

176.83±11.32

188.94±12.26

211.34±13.08

229.30±15.22

237.65±13.40

247.83±11.72

257.36±12.99

259.81±14.06

189.25±15.86

high dose

49.90±8.65*

72.43±9.41*

98.28±12.08*

131.78±13.04*

149.80±14.29*

173.10±14.83*

192.56±15.38*

200.63±15.87*

217.08±23.57*

222.70±18.62*

228.20±19.33*

178.30±14.78

* Dunnett's t-test, compared with control group p < 0.05

Table 5: Body weight changes of pregnant rats (g, mean±SD)

maternal rats

group

day 0

day 7

day 14

day 20

total body weight gain

P0

control

253.39±10.71

290.56±14.40

325.08±17.59

393.50±31.57

140.11±33.00

low dose

251.76±12.43

283.54±14.28

313.21±18.64

375.79±35.50

124.02±33.55

mid dose

252.56±15.38

281.00±18.90

316.15±21.54

380.26±29.44

127.70±21.94

high dose

227.90±14.46*

254.65±19.96*

281.71±21.10*

328.05±23.99*

100.15±19.63*

P1

control

268.66±20.93

293.85±22.73

324.56±26.91

402.37±33.52

133.71±17.80

low dose

267.99±20.20

295.00±22.66

327.13±24.15

405.77±30.33

137.78±14.49

mid dose

263.82±11.38

282.79±14.28

313.40±16.16

388.47±20.92

124.65±12.39

high dose

230.35±18.86*

248.60±19.42*

270.68±21.51*

328.05±27.39*

97.70±15.36

* Dunnett's t-test, compared with control group p < 0.05

Table 6: Body weight changes of maternal rats during lactation (g, mean±SD)

maternal rats

group

day 0

day 4

day 7

day 14

day 21

total body weight gain

P0

control

307.07±21.21

321.84±21.49

333.67±16.38

338.23±21.81

341.51±20.23

35.45±20.84

low dose

301.11±22.93

319.34±21.20

325.50±21.91

328.90±26.32

336.21±21.21

35.10±17.16

mid dose

294.80±23.78

313.67±23.21

319.74±24.83*

327.29±18.03

331.83±22.37

37.03±13.69

high dose

249.84±18.40*

257.90±18.72*

265.91±17.51*

278.87±27.06*

302.43±32.25

52.59±32.19*

P1

control

316.86±26.24

341.02±29.0

346.23±29.23

351.52±23.69

339.16±25.96

22.30±18.25

low dose

315.21±26.71

338.86±27.4

345.82±23.08

355.38±24.47

339.14±20.82

23.93±12.59

mid dose

303.06±22.91

332.25±20.06

340.80±20.29

342.13±18.05

336.94±13.13

30.88±14.11

high dose

272.98±33.23*

296.38±33.93*

311.02±30.13*

318.45±22.60*

308.55±25.41*

35.57±26.07

* Dunnett's t-test, compared with control group p < 0.05

Table 7: Litter weight changes of pups (g, mean±SD)

Filial generation

group

day 0

day 4

day 4 after adjusting

day 7

day 14

day 21

F1

control

84.12±18.73

133.30±27.86

86.57±13.36

132.07±23.96

258.97±38.97

425.77±61.61

low dose

85.51±12.35

133.46±16.94

88.67±9.54

128.81±22.12

236.62±57.74

415.74±71.37

mid dose

84.40±15.27

127.43±25.49

86.49±10.21

127.66±26.41

227.07±66.23

382.08±121.39

high dose

64.62±11.42*

90.96±15.67*

73.35±11.91*

97.19±20.39*

189.65±42.80*

339.47±65.81*

F2

control

89.54±10.69

133.05±32.13

85.05±13.41

137.75±19.85

273.84±27.49

464.59±52.67

low dose

91.99±12.14

147.19±19.72

85.10±7.59

138.76±12.29

274.83±41.10

454.40±70.83

mid dose

87.64±8.44

140.10±20.95

85.3±9.54

134.85±13.86

269.01±33.22

446.64±57.33

high dose

71.43±11.00*

117.17±13.33*

83.66±5.05

130.80±9.03

260.55±20.01

429.67±33.32

* Dunnett's t-test, compared with control group p < 0.05

Table 8: Body weight changes of pups (g, mean±SD)

Filial generation

group

day 0

day 4

day 4 after adjusting

day 7

day 14

day 21

F1

control

6.93±0.49

11.07±1.19

11.21±1.29

17.19±2.33

33.77±3.22

55.92±4.85

low dose

6.87±0.57

11.04±1.16

11.08±1.19

16.90±1.89

32.37±3.26

56.65±4.50

mid dose

7.03±0.57

10.91±1.79

11.05±1.66

16.65±3.09

32.89±4.69

54.89±9.68

high dose

6.67±0.69

9.56±1.50*

9.57±1.50*

12.87±2.35*

26.00±5.09*

47.05±8.63*

F2

control

6.57±0.52

10.12±2.38

10.45±1.76

17.37±2.19

34.99±2.69

59.35±5.39

low dose

6.43±0.51

10.87±1.59

10.64±0.95

17.34±1.54

35.93±2.84

59.29±4.51

mid dose

6.48±0.33

10.66±1.18

10.66±1.19

17.07±1.71

36.15±2.63

59.94±3.79

high dose

6.35±0.23

10.37±0.63

10.46±0.63

16.35±1.13

32.73±2.13*

53.98±3.63*

* Dunnett's t-test, compared with control group p < 0.05

Table 9: Average food consumption of P0 generation (g/kg bw/day)

sex

group

pre-mating

mating period

pregnancy period

lactation

average

male

control

94.51

73.82

-

-

92.63

low dose

94.63

74.11

-

-

92.77

mid dose

98.54

75.56

-

-

96.45

high dose

127.52

84.79

-

-

123.64

female

control

92.49

73.82

125.69

182.73

113.28

low dose

94.47

74.11

99.55

185.24

110.1

mid dose

94.95

75.56

100.68

191.08

111.69

high dose

96.04

84.79

111.86

207.53

117.79

Table 10: Average food consumption of P1 generation (g/kg bw/day)

sex

group

pre-mating

mating period

pregnancy period

lactation

average

male

control

106.41

62.54

-

-

103.48

low dose

105.31

67.19

-

-

102.77

mid dose

107.1

68.12

-

-

104.5

high dose

131.76

76.47

-

-

128.07

female

control

111.37

62.54

85.31

184.24

117.41

low dose

115.89

67.19

86.48

179.38

119.64

mid dose

121.49

69.12

88.72

183.86

124.22

high dose

127.38

76.47

94.81

185.32

129.46

Table 11: Reproduction index of P generation

parental generation

group

succsessful mating (%)

pregnancy (%)

live birth rate (%)

rate of birth livability (%)

survival rate after weaning (%)

P0

control

100.0 (24/24)

95.8 (23/24)

100.0 (23/23)

99.6 (281/282)

94.4 (168/178)

low dose

87.5 (21/24)

87.5 (21/24)

100.0 (21/21)

97.7 (258/265)

92.3 (155/168)

mid dose

95.5 (23/24)

95.8 (23/24)

100.0 (23/23)

97.8 (272/278)

86.2 (156/181)

high dose

100.0 (24/24)

95.8 (23/24)

100.0 (23/23)

98.7 (224/227)

94.9 (168/177)

P1

control

95.8 (23/24)

95.8 (23/24)

100.0 (23/23)

92.1 (290/315)

95.6 (172/180)

low dose

91.7 (22/24)

91.7 (22/24)

100.0 (22/22)

95.9 (302/315)

96.0 (169/176)

mid dose

87.5 (21/24)

87.5 (21/24

100.0 (21/21)

96.8 (276/285)

93.5 (157/168)

high dose

100.0 (24/24)

100.0 (24/24)

100.0 (24/24)

95.3 (261/274)

99.5 (183/184)

Table 12: Organ weight (g) and Organ coefficient (%) of P0 generation (mean±SD)

organ weights (g)

organ coefficient (%)

sex

group

body weight (g)

testicle/ovary

epididymis

testicle/ovary

epididymis

male

control

440.55±32.21

3.49±0.54

1.21±0.12

0.79±0.12

0.28±0.03

low dose

425.98±35.97

3.45±0.74

1.17±0.17

0.81±0.16

0.27±0.03

mid dose

445.10±35.09

3.65±0.56

1.31±0.10*

0.82±0.14

0.30±0.03

high dose

390.03±30.95*

3.47±0.54

1.13±0.14

0.90±0.16

0.29±0.04

female

control

322.48±34.49

0.15±0.03

-

0.048±0.012

-

low dose

332.74±21.58

0.16±0.03

-

0.047±0.009

-

mid dose

331.96±21.50

0.16±0.02

-

0.046±0.008

-

high dose

301.70±31.74*

0.12±0.02*

-

0.039±0.008*

-

* Dunnett's t-test, compared with control group p < 0.05

Table 13: Organ weight (g) and Organ coefficient (%) of P1 generation (mean±SD)

organ weights (g)

relative organ weights (%)

sex

group

body weight (g)

testicle/ovary

epididymis

testicle/ovary

epididymis

male

control

480.33±36.39

3.54±0.23

1.26±0.10

0.74±0.06

0.26±0.02

low dose

467.01±36.22

3.63±0.24

1.30±0.12

0.78±0.07

0.28±0.03

mid dose

458.83±51.92

3.49±0.26

1.25±0.12

0.77±0.08

0.28±0.04

high dose

355.31±34.81*

3.15±0.27*

1.02±0.10*

0.89±0.09*

0.29±0.03*

female

control

340.52±26.64

0.16±0.03

-

0.047±0.009

-

low dose

338.67±20.60

0.17±0.02

-

0.051±0.007

-

mid dose

341.23±24.02

0.17±0.03

-

0.049±0.009

-

high dose

308.56.25.40*

0.15±0.03

-

0.049±0.009

-

Table 14: Histopathology of male rats

parental generation

group

total animal number

animal number effected

A

B

C

D

P0

control

24

1

1

0

2

low dose

24

-

-

-

-

mid dose

24

-

-

-

-

high dose

24

1

1

1

1

P1

control

24

0

0

0

2

low dose

24

-

-

-

-

mid dose

24

-

-

-

-

high dose

24

0

0

0

3

A = Atrophia testiculi and spermatogenesis

B = ductus epididymis without sperms

C = epididymal epithelial cells vacuolation

D = prostatic stromal infiltration of inflammatory cells

Applicant's summary and conclusion