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Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 June 2015 and 21 April 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
(2001)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
(2008)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
(2008)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Toxi-Coop Zrt.,1103 Budapest Cserkesz u. 90., Hungary
- Age at study initiation: females (9 weeks of age at start of the mating period), males (33-35 weeks of age at start of the mating period)
- Weight at study initiation: 160-193 g
- Housing: before mating: 1-3 females per cage, 1-2 males per cage; mating: 1 male and 1-3 females/cage; during gestation: 2 sperm positive females per cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 6 days for females, 174 days for males

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 22
- Humidity (%): 48 - 59
- Air changes (per hr): 10 -15
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: sunflower oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test item was formulated in the vehicle (sunflower oil) in concentrations of 15 mg/mL, 5 mg/mL and 1.5 mg/mL. Formulations were prepared in the formulation laboratory of the Test Facility less than three days before use and stored at 5 ± 3°C.

VEHICLE
- Justification for use and choice of vehicle: The test item was not soluble in water. Therefore sunflower oil was used for preparing formulations appropriate for oral administration. Sunflower oil was a suitable vehicle to facilitate formulation analysis for the test item.
- Concentration in vehicle: 1.5, 5 and 15 mg/mL
- Amount of vehicle: 2 mL/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical control of the dosing solutions (control of concentration) was performed in the Analytical Laboratory of the Test Facility two times during the study. Five samples from different places were taken from each concentration for analysis of concentration and homogeneity on two occasions. Similarly, five samples were taken from the vehicle and analyzed. The suitability of the chosen vehicle for the test item was analytically proven. Recovery was between 101 and 98 % of nominal concentrations at 1 and 200 mg/mL in sunflower oil, respectively.

HPLC Conditions:
Detector: 220 nm
Column: Purospher STAR RP-18e 30-2 mm, 3 μm No.: 014664
Mobil Phase: Acetonitrile : water = 60 : 40 (v/v)
Flow Rate: 0.5 mL/min
Injection volume: 5 μL
Temperature: 25 °C
Retention time of CUROX®CC-DC: 4 min
Run time: 6 minutes
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage:1:1 to 1:3
- Length of cohabitation: The females were paired to males in the morning for two to four hours (one male: one to three females) until the number of sperm positive females per group reached at least twenty two.
- Proof of pregnancy: vaginal plug and/or sperm in the vaginal smear
Duration of treatment / exposure:
The sperm positive females were treated from gestational day 5 to 19.
Frequency of treatment:
7 days/week every day at a similar time
Duration of test:
All sperm positive females were sacrificed by decapitation after anesthesia with Isofluranum on gestation day 20.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
vehicle control
Dose / conc.:
3 mg/kg bw/day (actual dose received)
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Dose / conc.:
30 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
22 sperm positive females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose setting with 3, 10 and 30 mg/kg bw/day was based on findings obtained in a previous repeated dose toxicity studies with CUROX®CC-DC in the rat (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test with CCDFB-90 in the Rat; OECD 422; GLP, study no. 552.422.2950).

Examinations

Maternal examinations:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily (observations for signs of morbidity and mortality were made twice daily, at the beginning and end of the working day)
- Examinations: signs of morbidity, mortality, toxicity as well as behaviour and general conditions

BODY WEIGHT: Yes
- Time schedule for examinations: Body weight of sperm positive females was measured on gestation days 0, 3, 5, 8, 11, 14, 17 and 20. The corrected body weight was calculated for the 20th day of pregnancy (body weight on day 20 minus the weight of the gravid uterus).


FOOD CONSUMPTION: Yes
- Time schedule: between gestation days 0 to 3, 3 to 5, 5 to 8, 8 to 11, 11 to 14, 14 to 17 and 17 to 20 by re-weighing the non-consumed diet

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: uterus with cervix and ovary

OTHER:
- Examination of placental signs: All sperm positive animals were examined for vaginal bleeding (placental sign of gestation) on gestational day 13. If negative on day 13, the examination was repeated on day 14 of gestation.
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 early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: all live fetuses per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter
Statistics:
The homogeneity of variance between groups was checked by Bartlett’s test. Where no significant heterogeneity was detected, a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant, Duncan’s Multiple Range test was used to assess the significance of inter-group differences. If significance was the result of the Bartlett’s test, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test.
Historical control data:
The results were compared to the laboratory historical control data.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs observed.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
None of the animals died before scheduled necropsy.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In the 30 mg/kg bw/day group, the body weight was significantly reduced (5-10%) from gestation day 8 until the end of the in-life phase compared to the control group. The body weight gain of these animals was also significantly lower between gestation days 5 and 11, 17 and 20 as well as for gestation days 0 to 20. The corrected body weight and body weight gain was statistically significantly lower in the 30 mg/kg bw/day group.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In the 30 mg/kg bw/day group, the food consumption was significantly reduced (12 to 24%) from gestational days 5 – 11 and 14 – 17. This reduction was attributed to the treatment.
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):
There were no macroscopic alterations recorded for the maternal animals.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed

Maternal developmental toxicity

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:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
not examined
Other effects:
no effects observed
Details on maternal toxic effects:
There was no effect indicated related to the administration of the test item in the mean percentage of pre-implantation loss, early embryonic, late- and fetal death, the mean number of implantations, the sex distribution of the fetuses as well as in the mean number of viable fetuses in the test item groups.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
10 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
The body weight of the fetuses was slightly but statistically significantly lower in the 30 mg/kg bw/day group versus control. Although the values were within the historical control range (3.0-3.5 g for controls and 2.8-3.5 g if combined with inactive treatments) this reduction was considered to be treatment-related and may be due to the moderate maternal toxicity.
The placental weight (654.9-747.5 mg) was statistically significantly lower in all test item groups than in the controls but stayed within the historical control range or slightly below. Moreover, the control values were slightly above the historical control level. The relative placental weight was statistically significantly lower in the 10 and 30 mg/kg bw/day groups than in the control but within the historical control range (186.9-234.4 mg/g). The statistically significantly lower placental weight values were attributed to the slightly higher current control values.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
There were no malformed fetuses found at external examination.
Body weight retardation (below 2.74 g for males and 2.64 g for females) was evaluated as an external variation. The incidence of body weight retarded fetuses increased moderately and statistically significantly in the 30 mg/kg bw/day dose group, which was attributed to the treatment and might be a consequence of the moderate maternal toxicity.
Placental changes such as fibrinoid degenerated and fused placentas were found with a low incidence and unrelated to the treatment.
Skeletal malformations:
no effects observed
Description (incidence and severity):
Skeletal malformations were found in two control fetuses (bent vertebral column and misaligned spinous process in one and bent scapula as well as short and slightly bent humerus).
The following variations were evaluated: incompletely or not ossified skull bones, incompletely or not ossified, misaligned or bipartite sternebra, wavy ribs, dumb-bell shaped, bipartite, asymmetrically ossified vertebral centra or slightly dumb-bell shaped cartilage, lumbar/sacral asymmetric pelvic articulation, incomplete ossification of pubic bones, slightly bent femur and asymmetric or incomplete ossification of metacarpal and metatarsal. There were no significant increases in the incidence of the skeletal variations in the test item treated groups.
Visceral malformations:
no effects observed
Description (incidence and severity):
Malformation in the form of enlarged perimeningeal space and impressed cerebral hemisphere were found in two fetuses at 10 mg/kg bw/day and in one fetus at 30 mg/kg bw/day, respectively. In addition, a dilated 3rd brain ventricle was observed in another fetus in the 30 mg/kg bw/day group. Variations in the form of slightly dilated lateral ventricles were recorded for one fetus each in the control and in the 3 mg/kg bw/day group.
However, according to the laboratory historical control data similar or identical brain alterations in the form of slightly dilated lateral brain vehicles, slightly dilated 3rd ventricle and enlarged perimeningeal space occurred also in control fetuses with low incidence. Therefore and based on the single occurrence the more than slightly dilated 3rd brain ventricle was finally considered as incidental observation. In addition, there was no dose-response relationship in the incidence of fetuses with enlarged perimeningeal spaces and impressed cerebral hemispheres. Hence, all brain findings were finally judged to be not related to the treatment.
Hydroureter (uni- or bilateral) was evaluated as a variation and was found in the experimental groups without any dose-response relationship. A slightly malpositioned kidney was observed in one fetus in the 10 mg/kg bw/day group. This finding occurs sporadically in control fetuses according to the laboratory historical database.
Other effects:
no effects observed

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
10 mg/kg bw/day (actual dose received)
Basis for effect level:
fetal/pup body weight changes

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
The No Observed Adverse Effect Levels (NOAELs) were determined as follows:
NOAEL (maternal toxicity): 10 mg/kg bw/day
NOAEL (developmental toxicity including teratogenicity): 10 mg/kg bw/day
Executive summary:

The test item was examined for its possible prenatal developmental toxicity. Groups of 22 sperm-positive female Hsd. Wistar rats were treated with the test item by oral administration daily at three dose levels of 3, 10 and 30 mg/kg bw/day from day 5 up to and including day 19 post coitum. A control group of 22 sperm positive females was included and the animals were given the vehicle sunflower oil. The treatment volume was 2 mL/kg bw.

Sufficient stability and homogeneity in the chosen vehicle were verified over the range of relevant concentrations at the appropriate frequency of preparation. The test item was proved to be stable in sunflower oil formulations at ~1 and ~200 mg/mL concentration levels at least for one day at room temperature and at least for 3 days in the refrigerator (5 ± 3°C). Analytical control of dosing solutions was performed on the first and last week of treatment. Concentrations of the test item in the dosing formulations varied in the acceptable range between 104 and 106 % of nominal concentrations at both analytical occasions confirming proper dosing. During the study, mortality was checked and clinical observations were performed. Body weight and food consumption of the dams were also recorded. The day, when sperm was detected in the vaginal smear, was regarded as day 0 of gestation. Caesarean section and gross pathology were performed on gestational day 20. The number of implantations, early and late resorptions, live and dead fetuses in each uterine horn and the number of corpora lutea were recorded. Each fetus was weighed and examined for sex and gross external abnormalities. The placentas were weighed and examined externally. About half of each litter was preserved for visceral examination and the other half of the litters were preserved for skeletal evaluation. At visceral examination, the bodies were micro dissected with a dissecting microscope. The heads were examined by Wilson's free-hand razor blade method. After cartilage-bone staining the skeletons were examined with a dissecting microscope. All abnormalities found during the fetal examinations were recorded.

On gestation day 20, a total of 20, 20, 21 and 21 litters in the control, 3, 10 and 30 mg/kg bw/day groups, respectively, were evaluated. None of the dams died before scheduled necropsy. No clinical signs of toxicity or treatment related necropsy findings were observed. In the 30 mg/kg bw/day group, the body weight was significantly reduced from gestation day 8 up to the end of the in-life phase, the body weight gain was clearly decreased between gestation days 5 to 11and from days 0 to 20. The corrected body weight/body weight gain were clearly reduced in the high dose group (30 mg/kg bw/day). In the high dose group there was also a clearly statistically significantly lower food consumption of the dams from gestational days 5-11 and 14-20. The observed clear reductions in body weight parameter and food consumption are considered to be a consequence of the treatment. The mean number of implantations, the intrauterine mortality and sex distribution of the fetuses was not influenced by the treatment.

The body weight of the fetuses was slightly but statistically significantly lower in the 30 mg/kg bw/day group versus control. Although the values were within the historical control range, this reduction was considered to be a consequence of the treatment, maybe due to the moderate maternal toxicity. The placental weight was statistically significantly lower in all test item groups than in the control but stayed within the historical control range or slightly below. The relative placental weight was statistically significantly lower in the 10 and 30 mg/kg bw/day groups than in the control, but within the historical control range. The statistically significance indicated in the lower placental weight parameters might be attributed to the relatively higher current control values, which were slightly above the historical control range.

There were no malformed fetuses found at external examination. The body weight retardation (below 2.74 g for males and 2.64 g for females) was evaluated as an external variation. The incidence of body weight retarded fetuses increased moderately and statistically significantly in the 30 mg/kg bw/day dose group which was attributed to the treatment and might be a consequence of the moderate maternal toxicity. The result of the statistical analysis was not significant if the number of affected litters was evaluated. Placental changes were found at a low incidence and unrelated to the treatment.

At visceral examination, malformation in the form of enlarged perimeningeal space and impressed cerebral hemisphere were found in two fetuses at 10 mg/kg bw/day and in one fetus at 30 mg/kg bw/day, respectively. In addition, a dilated 3rd brain ventricle was observed in another fetus in the 30 mg/kg bw/day group. Variations in the form of slightly dilated lateral ventricles were recorded for one fetus each in the control and in the 3 mg/kg bw/day group. However, according to the laboratory historical control data similar or identical brain alterations in the form of slightly dilated lateral brain vehicles, slightly dilated 3rd ventricle and enlarged perimeningeal space occurred also in control fetuses with low incidence. Therefore and based on the single occurrence the more than slightly dilated 3rd brain ventricle was finally considered as incidental observation. In addition, there was no dose-response relationship in the incidence of fetuses with enlarged perimeningeal spaces and impressed cerebral hemispheres. Hence, all brain findings were finally judged to be not related to the treatment. Visceral variations were found in the experimental groups without any dose-response relationship.

Skeletal malformations were only found in two control fetuses. There were no significant increases in the incidence of the skeletal variations in the test item treated groups.

Based on these observations, the No Observed Adverse Effect Levels (NOAELs) were determined as follows:

NOAEL (maternal toxicity): 10 mg/kg bw/day

NOAEL (developmental toxicity including teratogenicity): 10 mg/kg bw/day