Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

rats: 2 -generation toxicity study (similar to OECD 416, oral): NOAEL = 10000 ppm ( 1258.55 and 1236.29 mg/kg bw/day for males and females, respectively)

Link to relevant study records
Reference
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
Reference:
Composition 0
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 information:
Composition 1
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
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
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
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%
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: estrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
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
Key result
Critical effects observed:
no
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: estrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
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
Key result
Critical effects observed:
no
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
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
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
Key result
Critical effects observed:
no
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
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
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
Key result
Critical effects observed:
no
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

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

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 236.29 mg/kg bw/day
Study duration:
chronic
Species:
rat
Quality of whole database:
The available information comprises an adequate, reliable (Klimisch score 2) and consistent study, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII, 8.6, of Regulation (EC) No 1907/2006.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

A two generation toxicity study was conducted according to a protocol similar to OECD guideline 416 (Feng, 2013). The major deviations of this study were: no estrus cycle and no sperm parameters were evaluated, no sexual maturation parameters were determined and no necropsy was performed with F1- and F2 offspring. In this study, groups of 24 male and 24 female Sprague Dawley rats received PEMB via the diet at dose levels of 625, 2500, and 10000 ppm. In the first generation (P0) dosing started for male rats 10 weeks prior to mating and for female rats 2 weeks prior to mating. For male rats the dosing stopped after mating, and females were dosed continuously during mating, gestation and lactation. For the second generation the selected parental animals (P1) received the test substance from weaning for 10 weeks prior to mating and continued to be exposed during mating. The female rats were exposed from weaning for 10 weeks prior to mating, continuing during mating, gestation and lactation. The animals were observed for clinical signs daily. Body weight as well as food consumption was determined for parental (P) and filial (F) generations. At the time of termination or following unexpected mortality during the study, all parental animals (P0 and P1), and all pups with external abnormalities or showing clinical signs were examined macroscopically for any structural abnormalities or pathological changes.  Organs weights of ovaries, testicles, epididymis from P0- and P1-animals were recorded. The uterus, ovaries, cervix, testicles, epididymis, seminal vesicle, prostate, penis, pituitary, brain and target organs from P0- and P1-animals were preserved for histopathological examination.

During the treatment period, the food consumption and behavior of animals were similar between the control and treatment groups, and no clinical signs of significance were observed. No abnormal appearance and abnormalities were observed in pups of each treated generation. During the exposure period one female rat from the P0 generation in the control group died during the lactation period, which is considered to be incidental. The mortality of pups from both generations was not significantly different compared to the respective control groups.

The following results on body weight were observed for the P0 generation:  Males of the high dose group had significantly lower absolute body weights from week 1 (-18.3%) to week 10 (-12.1%) and significantly lower total body weight gain (-16.3%) than the males of the control group. The absolute body weight of female rats in week 9 (-12.0%) and 10 (-9.5%) and the total body weight gain (-125%) was significantly lower than in the female control group. These effects were considered to be treatment-related and toxicologically relevant because it was observed throughout treatment and the average food consumption was not significantly changed compared with the control group. The absolute body weight of male rats in the mid dose group was significantly lower than in the control group at the end of week 1 (-6.5%). This effect was considered to be not of toxicological relevance since it was only observed in week 1 and the difference was below 10%. The absolute body weight in the high dose group of females during the gestation period was significantly lower than in the control group on gestation day 0 (-10.1%), 7 (-12.4%), 14 (-13.3%), 20 (-16.6%), as was the total body weight gain (-28.5%). This effect is considered to be treatment-related and adverse because it was observed throughout the treatment and the average food consumption was not significantly changed compared with the control group. Maternal rats during the lactation period showed significantly lower 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%) compared with the control group. The total body weight gain (+48.3%) during lactation period was significantly higher than in the control group. The 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 with the control group.

Similar results on body weight were observed for the P1 generation:  The absolute body weight as well as the body weight gain (-23.4%) of male rats in the high dose group were significantly lower than in the control group at the end of weeks 0 (-35.8%) to 10 (-25.5%). This effect is considered to be treatment-related and adverse because it was observed throughout treatment and the average food consumption was not significantly changed compared with the control group. The absolute body weight of female rats in the high dose group was significantly lower than in the control group at the end of weeks 0 (-33.3%) to 10 (-13.6%). The total body weight gain was not significantly different compared with the control group. The reduced absolute body weight is considered to be treatment-related and adverse because it was observed throughout treatment and the average food consumption was not significantly changed compared with the control group. However, the total body weight was not changed between high dose group and control group, showing that the initial absolute body weight was lower but the body weight gain was not influenced by the treatment. The absolute body weight and the total body weight gain (-6.8%) of male rats in the mid dose group were significantly lower than in the control group at the end of weeks 3 (-5.8%), 4 (-5.9%), 7 (-6.0), 10 (-5.9%).  These effects are considered not to be toxicological relevant since the changes are less than 10%. The absolute body weight of pregnant rats in the high dose group 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 with the control group. The absolute body weight 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 with the control group. The 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 with the control group. The pups of the high dose groups from both generations also revealed significantly reduced body weights. This observation is considered treatment-related and associated to the reduced body weights of the maternal animals.

For both parental generations there was no significant difference in the rate of mating success, pregnancy, live birth rate and rate of birth livability at any of the tested dose levels compared with the respective control groups. The results of the gross necropsy showed that, compared with the control group, no abnormalities were observed in parental animals in both generations in any of the dose groups. In the high dose group of female rats of the P0 generation a significantly decreased absolute ovary weight (-20%) and relative ovary weight (-19%) compared with the control group was observed. In the P1 generation the testicle weight (-11%) and epididymis weight (-19%) of paternal rats in the high dose group were significantly lower than in the control group. The relative testicle weight (+20%) and epididymis weight (+11.5%) in the high dose group were significantly higher than in the control group. The histological examinations revealed no abnormalities in parental rats of P0 and P1 generation in any dose group compared with the control group. Since no effects were noted in the reproductive performance and no abnormalities in the reproductive organs/tissues were observed, these effects are considered to be incidental.

Based on the food consumption, concentration of test substance in the feed and body weight, the following average food intake over all generations was calculated for the total treatment period: 61.10, 251.19 and 1258.55 mg/kg bw/day for males for the low, mid and high dose respectively and 71.79, 294.88 and 1236.29 mg/kg bw/day for females for the low, mid and high dose group, respectively.

In conclusion, no adverse effect on fertility was observed in any dose group. This leads to a NOAEL fertility of 10000 ppm, equivalent to 1258.55 and 1236.29 mg/kg bw/day for males and females, respectively. Based on the observed effects on body weight gain, the NOAEL systemic is considered to be 2500 ppm, equivalent to 251.19 mg/kg bw/day for males and 294.88 mg/kg bw/day for females, respectively.

Effects on developmental toxicity

Description of key information

rabbits (similar OECD 414, oral): NOAEL maternal toxicity = 126.4 mg/kg bw/day, NOAEL developmental = 126.4 mg/kg bw/day

rats: 2 -generation toxicity study (similar to OECD 416, oral): NOAEL developmental = 2500 ppm (251.19 and 294.88 mg/kg bw/day for males and females, respectively)

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13. Apr. - 30. Jul. 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Reference:
Composition 0
Qualifier:
according to
Guideline:
other: Guidelines for the Testing of Chemicals Section 4: Health effects (ministry of environmental protection of People's Republic of China
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adapted 2001
Deviations:
yes
Remarks:
dosing started on Day 6 instead Day 0, only 12 instead of 20 animals/dose were used, no detailed clinical signs description, no food consumption evaluated, not all fetuses observed for soft tissue or skeletal malformations, no data on individual animals
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 information:
Composition 1
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Jingling Seed Rabbit Farm
- Age at study initiation: 6 month
- Weight at study initiation: day 6 of gestation: 3.23±0.58 kg
- Fasting period before study: no
- Housing: not specified
- Diet: ad libitum
- Water: tap water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 15 - 20
- Humidity (%): 55 - 70
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): not specified
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was mixed with corn oil. Animals were gavaged based on 1 mL/kg bw/day.
Analytical verification of doses or concentrations:
no
Details on mating procedure:
- Impregnation procedure: artificial insemination; the day when the rabbit was artificially fertilized was set as Day 0
- Proof of pregnancy: rabbits were palpated every day to make sure that they were pregnant
Duration of treatment / exposure:
Day 6 to Day 18 of gestation
Frequency of treatment:
daily
Duration of test:
up to Day 29 of gestation
Dose / conc.:
25.3 mg/kg bw/day (actual dose received)
Dose / conc.:
126.4 mg/kg bw/day (actual dose received)
Dose / conc.:
632 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
According to the results of an acute oral toxicity test of female rabbits (LD50 = 3160 mg/kg bw), three dose groups of 25.3, 126.4 and 632.0 mg/kg bw/day were used along with a vehicle control (0 mg/kg bw/day).
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations: checked for general health condition (no further details given)

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: daily during dosing until Day 18, after that on Day 21, 24, 27 and 29

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No

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

POST-MORTEM EXAMINATIONS: Yes (macroscopic examination)
- Sacrifice on gestation day # 29
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of resorptions: Yes, but not differentiated between early or late resorptions
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: 1/3 per litter
- Skeletal examinations: Yes: 2/3 per litter
- Head examinations: No data
Statistics:
SPSS10.0 was used to perform statistics. All ratios were analyzed by Chi-square test. Study measurments were analyzed by ANOVA or nonparametric statistics. Fetus body length, tail length and body weight were compared by t-test. The data of fetuses was analyzed in the unit of litter.
Indices:
None
Historical control data:
Not reported
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At Day 3 of dosing one rabbit in the high dose group showed cachexia (body becoming thin) and died on Day 9 of dosing.
No abnormalities were detected in the other dosing groups.
These results were described in the summary only. No individual datawas reported.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
One animal in the high dose group died on the day 9 of dosing.
This result was described in the summary only. No individual data was reported.
Body weight and weight changes:
no effects observed
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
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
The dead rabbit in the high dose group did not show any abnormalities in macroscopic examination.
No abnormalities were detected in the other groups.
These results were described in the summary only. No individual data was reported.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
not examined
Dead fetuses:
effects observed, treatment-related
Description (incidence and severity):
In the high dose group in 6 of 11 rabbits all the fetuses died in utero. For 5 of 11 animals in the high dose group all the fetuses were alive at sacrifice of dams. This corresponds to the observation that live fetuses in the high dose group were significantly lower and dead fetuses were significantly higher compared with the control group.
No significant differences were observed in the mid dose and low dose group compared with the control group.
These results were described in the summary and only as a mean ± SD for the group. No individual data was reported.
Changes in pregnancy duration:
not examined
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined
Changes in number of pregnant:
not examined
Details on maternal toxic effects:
The number of corpus luteum and blastocyst implantation and the uterus and fetus weight in all treatment groups showed no significant difference when compared with the control group.
The number of absorptions and sexual ratio in the all treatment groups showed no significant changes when compared to the control group.
These results were described in the summary and only as a mean ± SD for the group. No individual data was reported.
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
126.4 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
mortality
Abnormalities:
not specified
Fetal body weight changes:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined
Reduction in number of live offspring:
effects observed, treatment-related
Description (incidence and severity):
In the high dose group in 6 of 11 rabbits all the fetuses died in utero. For 5 of 11 animals in the high dose group all the fetuses were alive at sacrifice of dams. This corresponds to the observation that live fetuses in the high dose group were significantly lower and dead fetuses were significantly higher compared with the control group.
No significant differences were observed in the mid dose and low dose group compared with the control group.
These results were described in the summary and only as a mean ± SD for the group. No individual data was reported.
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
not examined
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
In the low dose group 1 of 119 fetuses had cephalocele and 1 of 119 fetuses umbilical hernia. This was considered to be an incidental occurence since in all other treatment groups no abnormalities were observed and no significant difference compared with the control group was reported.
Skeletal malformations:
no effects observed
Visceral malformations:
effects observed, treatment-related
Description (incidence and severity):
In the low dose group 1 of 44 (2.3%) fetuses had a brain cavity. In the high dose group 2 of 14 (14.3%) fetuses had a cleft palate. The external malformations in the low-, mid- and high dose group had no significant difference when compared with the control group. However, in the high dose group a treatment-related effect cannot be ruled out.
Other effects:
not examined
Details on embryotoxic / teratogenic effects:
31
Key result
Dose descriptor:
NOAEL
Effect level:
126.4 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
visceral malformations
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
other: cleft palate
Description (incidence and severity):
2/14 (14.3%): no data if these findings were found in one dam or two dams
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
632 mg/kg bw/day
Treatment related:
yes
Relation to maternal toxicity:
developmental effects in the absence of maternal toxicity effects
Dose response relationship:
not specified
Relevant for humans:
not specified

Table 1: Pregnant rabbits of implantation (mean±standard error)

Dosage

(mg/kg bw/day)

No. of pregnant rabbits

No. of corpus luteum

No. of blastocyst implantation

Uterus & fetus weight (g)

0

12

9.2±3.0

7.9±3.1

512.9±179.4

25.3

12

11.0±1.3

10.5±1.3

596.8±115.6

126.4

12

9.3±2.0

8.3±2.3

500.6±139.5

632

11

9.3±2.5

7.8±2.6

380.6±202.0

Table 2: Fetus (mean±standard error)

Dosage

(mg/kg bw/day)

No. of pregnant rabbits

live fetus

dead fetus

no of absorptions

sexual ratio (male:female)

0

12

7.6±2.7

0.3±0.6

0.1±0.3

100:113

25.3

12

9.9±1.4

0.4±0.7

0.2±0.6

100:143

126.4

12

7.9±2.2

0.4±0.9

0.0±0.0

100:94

632

11

3.9±5.0**

3.9±3.8**

0.0±0.0

100:96

** p<0.01 compared to controls

Table 3: Growth and development (mean±standard error)

Dosage

(mg/kg bw/day)

No. of pregnant rabbits

Fetus body length (cm)

Fetus tail length (cm)

Fetus bodyweight (g)

0

12

10.504±0.542

1.535±0.070

47.47±7.65

25.3

12

10.236±0.551

1.476±0.083

42.44±7.36

126.4

12

10.525±0.570

1.526±0.130

44.52±7.10

632

5

10.231±0.597

1.445±0.114

41.54±6.97

Table 4: Skeletal malformations in the fetuses

Type                                                                                                         Dose (mg/kg bw/day)

                                                                                   0                     25.3                     126.4           632

                                                                                 (n=60)              (n=75)             (n=62)             (n=29)

Rib malformation                                  9 (15.0%)       9 (12.0%)       16 (25.8%)       3 (10.3%)

Sternum ossification rudimentary or not ossified       12 (20.0%)       12 (16.0%)       5 (8.1%)       1 (3.4%)

Note: number in front of () was number of malformed fetuses, number insed () was ratio of abnormality.

Table 5:  Splanchnic malformations in the fetuses

Type                                                        Dose (mg/kg bw/day)

                                         0              25.3        126.4               632

                                         (n=31)       (n=44)       (n=33)        (n=14)

Cleft tongue, cleft palate       0              0                   0              2 (14.3%)

Brain cavity                          0              1 (2.3%)       0              0

Note: number in front of () was number of malformed fetuses, number insed () was ratio of abnormality.

Conclusions:
In a tetratogenicity study with rabbits performed using a protocol similar to OECD 414 one of 12 females died in the high dose group. In the high dose group 6/11 rabbits showed dead fetuses whereas 5/11 had alive fetuses. Skeletal malformations had no significant difference in the treated groups compared with the control group. In the low dose group 2.3% fetuses had a brain cavity. In the high dose group 14.3% fetuses had a cleft palate. The external malformations in the low-, mid- and high dose group had no significant difference when compared with the control group. However, in the high dose group a treatment-related effect cannot be ruled out. Thus, a NOAEL of 126.4 mg/kg bw/day was established for maternal toxicity and developmental toxicity.
Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
126.4 mg/kg bw/day
Study duration:
subacute
Species:
rabbit
Quality of whole database:
The available information comprises an adequate, reliable (Klimisch score 2) and consistent study, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII, 8.6, of Regulation (EC) No 1907/2006.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

A developmental toxicity study was conducted following a protocol similar to OECD 414, with the following deviations: dosing started on Day 6 of gestation instead of Day 0, only 12 animals per dose instead of 20 animals per dose were treated, no detailed clinical signs were described, food consumption was not recorded, not all live fetuses were examined for soft tissue and skeletal malformations, no dead fetuses were examined for soft tissue or skeletal malformations, and no data on individual animals was reported. Despite the deficiencies, this study is considered to be acceptable for the evaluation of developmental toxicity.

In this study, groups of 12 female  New Zealand White rabbits received PEMB in corn oil orally via gavage at dose levels of 25.3, 126.4, and 623.0 mg/kg/ bw/day from gestation day 6 through 18 (Qi, 2010). At gestation day 29 the rabbits were sacrificed and the uterus was collected and weighed. The number of implantations, corpus luteum, absorption of fetuses, dead fetuses and live fetuses was examined and recorded. The sex, body weight, body length, tail length and external malformations for live fetus were recorded. One third of the fetuses were examined for splanchnic malformations and 2/3 fetus were examined for skeletal malformations.

In the high dose group (632 mg/kg bw/day) one rabbit showed at day 3 of dosing cachexia (body becoming thin). On day 9 during dosing the rabbit died. At necropsy no abnormalities were observed in this rabbit. No further clinical signs were reported for the remaining rabbits. In the high dose group in 6 of 11 rabbits all the fetuses died in utero. For 5 of 11 animals in the high dose group all the fetuses were alive until the dams were sacrificed. The in utero mortality leads to a significant decrease in the mean viability of fetuses in the high-dose group, compared with the control group. The large standard deviation in the high-dose group indicates the large inter-individual variability. The number of absorptions, corpus luteum, number of blastocyst implantations, and the combined uterus and fetus weight did not show significant differences compared with the control group. However, the combined uterus and fetus weight in the high dose group showed clearly lower weight (380.6±202.0 g) compared with the control group (512.9±179.4 g). This is probably a result of the fetus mortality in utero in 6 of 11 rabbits. The number of sexual ratio, fetus body length, fetus tail length and fetus bodyweight did not show any significant differences in the treated groups compared with the control group.  For external malformation 91, 119, 95, and 43 fetuses were examined in the control group, low dose (25.3 mg/kg/bw/day), mid dose (126.4 mg/kg bw/day) and high dose (632 mg/kg bw/day) group, respectively. One of 119 (corresponding to 0.8%) fetuses in the low dose group showed cephalocele and one of 119 (corresponding to 0.8%) fetuses showed umbilical hernia. These external malformations are regarded as incidental since it was only observed in the low dose group with a low incidence. For skeletal malformation 60, 75, 62, and 29 fetuses were examined in the control group, low dose (25.3 mg/kg/bw/day), mid dose (126.4 mg/kg bw/day) and high dose (632 mg/kg bw/day) group, respectively. No differences in skeletal malformations were observed between the treated groups and the control group. For splanchnic malformations 31, 44, 33, and 14 fetuses were examined in the control group, low dose (25.3 mg/kg/bw/day), mid dose (126.4 mg/kg bw/day) and high dose (632 mg/kg bw/day) group, respectively. In the high dose group 2 of 14 (corresponding to 14.3%) fetuses showed a cleft palate and 1 of 44 (corresponding to 2.3%) in the low dose group had a brain cavity. These splanchnic malformations were not statistically significant compared to the control group. However, a treatment-related effect in the high dose group cannot be ruled out due to the low number of examined fetuses in the high dose group which was the result of the high mortality of fetuses. This again leads to a high incidence of 14.3% of cleft palate. Furthermore, a treatment-related effect cannot be ruled out since it is not known if the offspring exhibiting a cleft palate were born in one or 2 litters. Altogether, this increases the uncertainty around the relevance of the cleft palate incidence.

In conclusion, a treatment-related effect on the mortality of the one dam cannot be ruled out and therefore the NOAEL for maternal toxicity is set to the mid dose of 126.4 mg/kg bw/day. Since only one dam died and no further systemic toxicity of the dams are described the reduced viability of the fetuses cannot solely be attributed to the maternal toxicity. This leads to a developmental NOAEL of 126.4 mg/kg bw/day and a classification for toxicity to reproduction (Category 2). Due to the high incidence of cleft palate observed in the high dose group and a complete lack of details on individual animals a NOAEL (teratogenicity) of 126.4 mg/kg bw/day is concluded.

A two-generation study was performed in rats similar to OECD 416 (Feng, 2013). The major deviations of this study were: no estrus cycle and no sperm parameters were evaluated, no sexual maturation parameters were determined and no necropsy was performed with F1- and F2 offspring. In this study, groups of 24 male and 24 female Sprague Dawley rats received PEMB via the diet at dose levels of 625, 2500, and 10000 ppm. In the first generation (P0) dosing started for male rats 10 weeks prior to mating and for female rats 2 weeks prior to mating. For male rats the dosing stopped after mating, and females were dosed continuously during mating, gestation and lactation. For the second generation the selected parental animals (P1) received the test substance from weaning for 10 weeks prior to mating and continued to be exposed during mating. The female rats were exposed from weaning for 10 weeks prior to mating, continuing during mating, gestation and lactation. The animals were observed for clinical signs daily. Body weight as well as food consumption was determined for parental (P) and filial (F) generations. At the time of termination or following unexpected mortality during the study, all parental animals (P0 and P1), and all pups with external abnormalities or showing clinical signs were examined macroscopically for any structural abnormalities or pathological changes.

No abnormal appearance and abnormalities were observed in pups of each treated generation. The mortality of pups from both generations was not significantly different compared to the respective control groups. The pups of the high dose groups from both generations revealed significantly reduced body weights. This observation is considered treatment-related and associated to the reduced body weights of the maternal animals.

In summary, reduced body weights of F1 and F2 offspring were observed as secondary effect on maternal toxicity. Therefore, a NOAEL for developmental toxicity in rats is set to 2500 ppm which is equivalent to 251.19 and 294.88 mg/kg bw/day for male and female rats, respectively.

Mode of Action Analysis / Human Relevance Framework

not applicable

Justification for classification or non-classification

The available data on toxicity to reproduction of the test substance meet the criteria for classification according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and are therefore classified as toxic to reproduction Category 2 (H361d) due to the developmental effects observed in the developmental toxicity study with rabbits.