Registration Dossier

Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Link to relevant study records
Reference
Endpoint:
one-generation reproductive toxicity
Remarks:
based on generations indicated in Effect levels
Type of information:
experimental study
Adequacy of study:
key study
Study period:
december 2010 - april 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study has been performed according to OECD guidelines and according to GLP principles.
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc. (Hino Breeding Center)
- Age at study initiation: 9 weeks
- Weight at receipt (8 weeks old): 251.6 to 280.8 g for males and 164.6 to 193.3 g for females
- Housing: stainless-steel cages, except during gestation and lactation period (polymethylpentene cages). One male and one female were housed in a cage during the mating period. One dam and its litter were housed in a cage during the lactation period. As for the other periods, animals were housed individually. Bedding material: satirized wood chip
- Diet: ad libitum autoclave-sterilized pellet diet (CRF-1, Oriental Yeast Co., Ltd)
- Water: ad libitum well water admixed with NaClO (about 2 ppm)
- Acclimation period:9 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.8° to 23.9°C
- Humidity (%): 52.0 to 79.6%
- Air changes (per hr): 10 to 20 times
- Photoperiod (hrs dark / hrs light): 12h/12h
IN-LIFE DATES: From: January 7,2011 To: April 18, 2011
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Dosing solutions were prepared once every 5 or 7 days and stored in a cool place shielded from light.
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: up to 14 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear
- After successful mating each pregnant female was caged: in polymethylpentene cages during the gestation and lactation period. One dam and its litter were housed in a cage during the lactation period.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of the dosing solution at each concentration were analysed. Analytical method validation included linearity, repeatability, specificity, within-run precision and stability testing. All concentrations were within 100 ± 10% as the ratio to the nominal concentration; substance solutions were conformed to be stable after storage in a cool place and shielded from light for 8 days and following 6 hours at room temperature.
Duration of treatment / exposure:
males: from 14 days before mating until necropsy through the mating period (42 days).
females: from 14 days before mating until day 4 of lactation through the mating and pregnancy periods and delivery.
recovery females (satellite group): for 42 days without mating.

Pups were not treated directly, but were potentially exposed to the test substance in utero and through lactational transfer.
Frequency of treatment:
once daily
Details on study schedule:
All copulated females were allowed natural delivery. The observation of delivery was from days 21 to 24 of gestation. The females that did not deliver by 24 days after copulation were regarded as non-delivered females. The delivered dams were allowed to nurse offsprings until day 4 postpartum (day 4 of lactation) and postpartum behavior such as lactation, nesting and presence or absence of cannibalism was observed every day.
Remarks:
Doses / Concentrations:
50, 250, 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
Each test group consisted of 12 males (including 5 males for recovery test) and 12 females (5 females each were added for recovery test in the control and 1000 mg/kg bw/day groups).
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale: based on results obtained in "Fourteen days repeated oral dose toxicity study of E-C104 in rats (No. P101038; dose levels 0, 100, 300 and 1000 mg/kg bw, with 5 rats/sex/dose). As no toxicologically relevant effects were observed, the highest dose was set at 1000 mg/kg bw/day as stated in the guideline and two lower doses were established.
- Post-exposure recovery period in satellite groups: 14 days
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day during the dosing period; once a day during recovery period.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before dosing; once a week until week 6

BODY WEIGHT: Yes
- Time schedule for examinations: males were weighed on days 1, 8, 15, 22, 29, 36, 42 and 43. The recovery males were also weighed on days 50 and 56. The satellite females were weighed in the same manner as the recovery males. The test females were weighed on days 1, 8 and 15, once every 7 days after initiation of copulation, and days 0, 7, 14 and 20 of gestation and days 0 and 4 of lactation for females after parturition. The final body weight was also measured on the day of necropsy.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Food consumption was measured between days 1 to 8, 8 to 15, 22 to 29, 29 to 36 and 36 to 40 for test and recovery males and between days 43 to 50 and 50 to 54 for recovery males only. For females it was measured between days 1 to 8, 8 to 15, 15 to 22, 22 to 29, 29 to 36, 36 to 42, 43 to 50 and 50 to 56 for the satelite females. Food consumption of the test females was measured at the same time as the body weights. Food consumption was not measured for either sex during the mating period. Gross weight of each feeder was weighed, and the main daily food consumption of each animal was calculated for each period.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at necropsy
- Anaesthetic used for blood collection: Yes, sodium pentobarbital (i.p., 30 mg/kg bw)
- Animals fasted: Yes, at least 18 hours
- How many animals: 5 animals with smaller animal numbers for the test males, all animals for the recovery males and satellite females, and 5 animals from the earlier parturition date with small numbers for the test females.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at necropsy
- Animals fasted: Yes, at least 18 hours
- How many animals: same as for haematology

URINALYSIS: Yes
- Time schedule for collection of urine: day 40
- Metabolism cages used for collection of urine: No, fresh urine samples excreted spontaneously on a washed try under the cage were collected.
- Animals fasted: Yes
- Parameters checked: PH, protein, glucose, ketone body and occult blood were determined by means of reagent strip method.
NEUROBEHAVIOURAL EXAMINATION: Yes, FOB
- Time schedule for examinations: week 6
- Dose groups that were examined: 5/sex/dose group
- Battery of functions tested: sensory activity / grip strength / motor activity
Oestrous cyclicity (parental animals):
Vaginal smears were collected with swabs from all test females in the morning from the initial dosing day to the day of mating or end of mating period to confirm the estrous cycle. The obtained smears were collected on a plate for each animal and thionine-eosin-stained. The estrous cycle was classified into diestrus, proestrus, estrus and metestrus. The mean estrous cycle and the number of the estrous period during the test period were calculated. The data obtained during the mating period were treated as referential data.
Sperm parameters (parental animals):
Parameters examined in male parental rats:
testis weight, epididymis weight
Litter observations:
The number of delivered offspring, sex and the presence or absence of external anomalies was examined on day 0 of lactation. Thereafter, clinical signs and mortality were observed daily. Based on the results, the following parameters were calculated: live birth index (%), stillborn rate, viability index on day 4 (%), sex ratio.
All live offspring were weighed individually on days 0 and 4 postpartum.
Postmortem examinations (parental animals):
GROSS NECROPSY
-according to guideline
HISTOPATHOLOGY / ORGAN WEIGHTS
-according to guideline
Postmortem examinations (offspring):
All live offspring were observed for external anomalies including that in the oral cavity on day 4 of lactation, and subsequently euthanized. Stillborn and dead offspring were fixed and stored. Based on the results, the following parameters were calculated: external anomaly index and external anomaly typing index.
Statistics:
Statistical analysis was performed with computer system, MiTOX-PPL with significant levels of 1% and 5% (two-tailed). The data of offspring were handled on a litter-basis. The body weight and food consumption of the female not pregnant, as well as the body weight after initiation of mating in the females failed mating were excluded from evaluation.
For grip strength, motor activity, body weights, food consumption, urinalysis, hematology, blood chemistry, absolute and relative organ weight, the group mean and standard deviation of the following items were calculated and homogeneity of the variance was tested by the Bartlett's test (significant level 5%). When the variance was homogeneous, the Dunnett's multiple comparison was applied, and when it was not homogeneous, the Steel's multiple comparison was applied for control group and each test substance group.
For Histopathological examination, the grades were converted to numerical values, and the Mann-Whitney U test was applied to compare between the control and each test substance group.
Reproductive indices:
Copulation index, fertility index, gestation index and sex ratio were tested with the chi-square test for comparison between the control and each test substance group.
Implantation index and stillborne rate were tested with the Wilcoxon's rank sum test for comparison between the control and each test substance group.
Offspring viability indices:
External anomaly index, external anomaly typing index, live birth index and viability index on day 4 were tested with the Wilcoxon's rank sum test for comparison between the control and each test substance group.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
substance-mixed feces (dark blue color) were noted in all animals of all dose groups
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
in high dose females body weight was higher on day 0 of lactation compared to the control group. No effects on food consumption were observed.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
in high dose females body weight was higher on day 0 of lactation compared to the control group. No effects on food consumption were observed.
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
appearance of pigment-laden macrophages
Other effects:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
In one high dose animal, diestrus remained for 11 days, suggesting an irregular estrous cycle. However, as the cycle recovered normal and copulation was confirmed, it was judged incidental.
In the parental animals, no changes related to the test substance treatement were noted in the estrous cycle, copulation index, fertility index, gestation length, number of corpora lutea and implantation sites, implantation index, or delivery index, as well as in the paturition or lactation conditions.
In the offsprings, no changes related to the test substance treatment were noted in the sex ratio, birth index, or viability index on day 4, as well as in the external examination or body weight.
Dose descriptor:
NOAEL
Remarks:
reproductive toxicity
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No substance related changed were observed on reproductive parameters in the parental animals.
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: increased number globule leukocyte in glandular stomach (m), changes in platelet count, neutrophil and lymphocyte numbers, total cholesterol, plasma glucose and triglyceride levels.
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
Dose descriptor:
NOAEL
Remarks:
offspring
Generation:
F1
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: No substance related changes were observed in offsprings.
Reproductive effects observed:
not specified
Conclusions:
In an OECD 422 study no changes on reproductive toxicity related to the test substance at 50, 250, 1000 mg/kg bw/day were observed in the parental animals, the NOAEL for reproductive toxicity is considered to be at least 1000 mg/kg bw/day.
In the absence of substance related effects in the offspring, the NOAEL offspring is considered to be at least 1000 mg/kg bw/day.
Executive summary:

E-C104 was repeatedly administered to rats (7/sex/dose) by oral gavage at dose level 0, 50, 250 and 1000 mg/kg bw/day from 14 days before mating for 42 days in males and satellite females, and 14 days before mating through gestation and parturition until day 4 of lactation in females to determine the effects of repeated exposure and reproductive and developmental toxicity. Reversibility was studied in satellite groups for controls and high dose (5/sex/dose). As a result of the test substance administration, substance-mixed feces (dark blue color) was noted in all dose groups. Haematological examination showed increases in platelet count and numbers of neutrophils and lymphocytes. At the end of the recovery period, increase in reticulocyte number was observed in males and females showed increase in neutrophils and decrease in lymphocytes. Clinical biochemistry showed in high dose animals decreased levels of total bile acids, total cholesterol and ALT. High dose males showed decreased plasma glucose levels, whereas high dose females showed a decreased plasma triglyceride level. Gross pathology showed blue coloration in the GI contents, mesenteric, submaxillary, cervical and bronchial lymph nodes, lung and kidneys (with no reversibility in coloration in teh lymph nodes and kidney in the mid- and high dose). Histopathological examination showed pigment-laden macrophages in the GI-tract, mesenteric lymph nodes and lungs in rats dosed at and above 250 mg/kg bw/day, but the toxicological relevance is unclear. No reproduction toxicity and no developmental toxicity was observed.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat

Effects on developmental toxicity

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
december 2010 - april 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study has been performed according to OECD guidelines and according to GLP principles.
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:CD(SD)
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc. (Hino Breeding Center)
- Age at study initiation: 9 weeks
- Weight at receipt (8 weeks old): 251.6 to 280.8 g for males and 164.6 to 193.3 g for females
- Housing: stainless-steel cages, except during gestation and lactation period (polymethylpentene cages). One male and one female were housed in a cage during the mating period. One dam and its litter were housed in a cage during the lactation period. As for the other periods, animals were housed individually. Bedding material: satirized wood chip
- Diet (e.g. ad libitum): ad libitum autoclave-sterilized pellet diet (CRF-1, Oriental Yeast Co., Ltd)
- Water (e.g. ad libitum): ad libitum well water admixed with NaClO (about 2 ppm)
- Acclimation period: 9 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.8° to 23.9°C
- Humidity (%): 52.0 to 79.6%
- Air changes (per hr): 10 to 20 times
- Photoperiod (hrs dark / hrs light): 12h/12h
IN-LIFE DATES: From: January 7,2011 To: April 18, 2011
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Dosing solutions were prepared once every 5 or 7 days and stored in a cool [place shielded from light.
Analytical verification of doses or concentrations:
yes
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: up to 14 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear] referred to as
- After successful mating each pregnant female was caged in polymethylpentene cages during the gestation and lactation period. One dam and its litter were housed in a cage during the lactation period.
Duration of treatment / exposure:
males: from 14 days before mating until necropsy through the mating period (42 days).
females: from 14 days before mating until day 4 of lactation through the mating and pregnancy periods and delivery.
recovery females (satellite group): for 42 days without mating.

Pups were not treated directly, but were potentially exposed to the test substance in utero and through lactational transfer.
Frequency of treatment:
once daily
Duration of test:
42 days, with additionally a 14-day recovery period
Remarks:
Doses / Concentrations:
50, 250, 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
Each test group consisted of 12 males (including 5 males for recovery test) and 12 females (5 females each were added for recovery test in the control and 1000 mg/kg bw/day groups).
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on results obtained in "Fourteen days repeated oral dose toxicity study of E-C104 in rats (No. P101038; dose levels 0, 100, 300 and 1000 mg/kg bw, with 5 rats/sex/dose). As no toxicologically relevant effects were observed, the highest dose was set at 1000 mg/kg bw/day as stated in the guideline and two lower doses were established.
- Post-exposure recovery period in satellite groups: 14 days
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day during the dosing period; once a day during recovery period.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before dosing; once a week until week 6

BODY WEIGHT: Yes
- Time schedule for examinations: males were weighed on days 1, 8, 15, 22, 29, 36, 42 and 43. The recovery males were also weighed on days 50 and 56. The satellite females were weighed in the same manner as the recovery males. The test females were weighed on days 1, 8 and 15, once every 7 days after initiation of copulation, and days 0, 7, 14 and 20 of gestation and days 0 and 4 of lactation for females after parturition. The final body weight was also measured on the day of necropsy.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Food consumption was measured between days 1 to 8, 8 to 15, 22 to 29, 29 to 36 and 36 to 40 for test and recovery males and between days 43 to 50 and 50 to 54 for recovery males only. For females it was measured between days 1 to 8, 8 to 15, 15 to 22, 22 to 29, 29 to 36, 36 to 42, 43 to 50 and 50 to 56 for the satelite females. Food consumption of the test females was measured at the same time as the body weights. Food consumption was not measured for either sex during the mating period. Gross weight of each feeder was weighed, and the main daily food consumption of each animal was calculated for each period.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at necropsy
- Anaesthetic used for blood collection: Yes, sodium pentobarbital (i.p., 30 mg/kg bw)
- Animals fasted: Yes, at least 18 hours
- How many animals: 5 animals with smaller animal numbers for the test males, all animals for the recovery males and satellite females, and 5 animals from the earlier parturition date with small numbers for the test females.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at necropsy
- Animals fasted: Yes, at least 18 hours
- How many animals: same as for haematology

URINALYSIS: Yes
- Time schedule for collection of urine: day 40
- Metabolism cages used for collection of urine: No, fresh urine samples excreted spontaneously on a washed try under the cage were collected.
- Animals fasted: Yes
- Parameters checked: PH, protein, glucose, ketone body and occult blood were determined by means of reagent strip method.

NEUROBEHAVIOURAL EXAMINATION: Yes, FOB
- Time schedule for examinations: week 6
- Dose groups that were examined: 5/sex/dose group
- Battery of functions tested: sensory activity / grip strength / motor activity
Ovaries and uterine content:
Vaginal smears were collected with swabs from all test females in teh morning from teh initial dosing day to the day of mating or end of mating period to confirm the estrous cycle. The obtained smears were colelcted on a plate for each animal and thionine-eosin-stained. The estrous cycle was classified into diestrus, proestrus, estrus and metestrus. The mean estrous cycle and the number of the estrous period during the test period were calculated. The data obtained during the mating period were treated as referential data.
All copulated females were allowed natural delivery. The observation of delivery was from days 21 to 24 of gestation. The females that did not deliver by 24 days after copulation were regarded as non-delivered females. The delivered dams were allowed to nurse offsprings until day 4 postpartum (day 4 of lactation) and postpartum behavior such as lactation, nesting, and presence or absence of cannibalism was observed every day.
Gestation length, delivery index and implantation index were recorded.
Fetal examinations:
Observation for offspring included: live birt index, stillborne rate, viability index on day 4, sex ratio, individual body weight of all live offsprings on days 0 and 4 postpartum, external anomaly index and external anomaly typing index.
Statistics:
Statistical analysis was performed with computer system, MiTOX-PPL with significant levels of 1% and 5% (two-tailed). The data of offspring were handled on a litter-basis. The body weight and food consumption of the female not pregnant, as well as the body weight after initiation of mating in the females failed mating were excluded from evaluation.
For grip strength, motor activity, body weights, food consumption, urinalysis, hematology, blood chemistry, absolute and relative organ weigh, the group mean and standard deviation of the following items were calculated and homogeneity of the variance was tested by the Bartlett's test (significant level 5%). When the variance was homogeneous, the Dunnett's multiple comparison was applied, and when it was not homogeneous, the Steel's multiple comparison was applied for control group and each test substance group.

For Histopathological examination, the grades were converted to numerical values, and the Mann-Whitney U test was applied to compare between the control and each test substance group.

Copulation index, fertility index, destation index and sex ratio were tested with the chi-square test for comparison between the control and each test substance group.
Implantation index and stillborne rate were tested with the Wilcoxon's rank sum test for comparison between the control and each testsubstance group.

External anomaly index, external anomaly typing index, live birth index and viability index on day 4 were tested with the Wilcoxon's rank sum test for comparison between the control and each test substance group.
Details on maternal toxic effects:
Maternal toxic effects:yes
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: other:
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: other:
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects
Remarks on result:
other: no effects
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
In an OECD 422 study in rats orally exposed to 50, 250, 1000 mg/kg bw/day E-C104, no substance related effects in the offspring was observed. As a result, the NOAEL offspring is considered to be at least 1000 mg/kg bw/day.
Executive summary:

E-C104 was repeatedly administered to rats (7/sex/dose) by oral gavage at dose level 0, 50, 250 and 1000 mg/kg bw/day from 14 days before mating for 42 days in males and satellite females, and 14 days before mating through gestation and parturition until day 4 of lactation in females to determine the effects of repeated exposure and reproductive and developmental toxicity. Reversibility was studied in satellite groups for controls and high dose (5/sex/dose). As a result of the test substance administration, substance-mixed feces (dark blue color) was noted in all dose groups. Haematological examination showed increases in platelet count and numbers of neutrophils and lymphocytes. At the end of the recovery period, increase in reticulocyte number was observed in males and females showed increase in neutrophils and decrease in lymphocytes. Clinical biochemistry showed in high dose animals decreased levels of total bile acids, total cholesterol and ALT. High dose males showed decreased plasma glucose levels, whereas high dose females showed a decreased plasma triglyceride level. Gross pathology showed blue coloration in the GI contents, mesenteric, submaxillary, cervical and bronchial lymph nodes, lung and kidneys (with no reversibility in coloration in teh lymph nodes and kidney in the mid- and high dose). Histopathological examination showed pigment-laden macrophages in the GI-tract, mesenteric lymph nodes and lungs in rats dosed at and above 250 mg/kg bw/day, but the toxicological relevance is unclear.

No reproduction toxicity and no developmental toxicity was observed.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat

Justification for classification or non-classification

Additional information