Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-06-12 and 2016-04-05
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP conform study according to guideline

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)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: solid

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 (8 weeks of age at start of the mating period), males (31-33 weeks of age at start of the mating period)
- Weight at study initiation: The group averages of the body weight of the females were as similar as possible on the first day of gestation
- Housing: before mating: 1-3 females per cage, 1-2 males per cage; mating: 1 male and 1-3 females/cage; during gestation: 2-3 sperm positive females per cage, if not possible 1 sperm positive female per cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 12 days for females, 159 days for males

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-22
- Humidity (%): 43 - 62
- Air changes (per hr): 10 -15
- Photoperiod (hrs dark / hrs light): 12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.5 % aqueous methylcellulose
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test item was formulated in 0.5 % methycellulose in a frequency based on the stability features of the test item in the vehicle (not more than for 3 days at 5 +/- 3°C)


VEHICLE
- Justification for use and choice of vehicle: The test item was not soluble in water therefore 0.5 % aqueous methylcellulose was used for preparing formulations appropriate for oral administration. 0.5 % aqueous methylcellulose is a suitable vehicle to facilitate formulation analysis for the test item.
- Concentration in vehicle: 20, 60 and 200 mg/mL
- Amount of vehicle: 5 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical control of formulations (concentration and homogeneity) in the vehicle was performed in the Analytical Laboratory of Test Facility two times during the study. Five samples were taken from different places from each concentration (Groups 2, 3 and 4) for analysis of concentration and homogeneity on 2 occasions. Similarly, five samples were taken from different places from the control substance (Group 1) at each occasion and measured. The samples were stored at 5 ± 3°C until the analysis. Formulation samples were diluted with tetrahydrofuran and acetonitrile and then analysed by a HPLC method.
Measured concentrations varied between 91 and 112 % of the nominal concentrations and all formulations were considered to be homogeneous.

HPLC conditions:
Detector: 222 nm
Column: Phenomenex, Luna 3μ C18 (2) 100A,
150 x 4.6 mm, 3 μm, No.: 637921-4
Mobil Phase: Acetonitrile : Water = 95 : 5 (v/v)
Flow Rate: 1.2 mL/min
Injection volume: 10 μL
Temperature: 25 °C
Retention time: 9.5 min ±5 %
Run time: 12 minutes
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1:3
- Length of cohabitation: The females were paired to males in the mornings for two to four hours until the number of sperm positive females per group achieves at least twenty two.
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
The sperm positive females were treated from gestational day 5 to 19.
Frequency of treatment:
7 days per week every day at similar time.
Duration of test:
Treatment period: from gestational day 5 to 19
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 300 and 1000 mg/kg bw/day
Basis:
nominal conc.
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 was based on findings obtained in a previous repeated dose toxicity studies with CUROX®CC-P3 in the Rat (Reproduction/Developmental Toxicity Screening Test with CCPIB in the Rat; OECD 421; GLP).

Examinations

Maternal examinations:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily (observations for signs of morbidity and mortality were made twice daily, at the beginning and end of the working period)
- 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 AND COMPOUND INTAKE : 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

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 the 13th gestational day. 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 homogeneity of variance test. Where no significant heterogeneity is detected a one-way analysis of variance (ANOVA) is carried out. If the obtained result is significant Duncan’s Multiple Range test was used to access the significance of inter-group differences. If significance is 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's historical control data.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
None of the female died before scheduled necropsy and there were no clinical signs recorded. No treatment related necropsy findings were observed. No treatment related reduction on food consumption or body weight was observed.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: other:

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Number of implantations, intrauterine mortality and sex distribution of the fetuses were not influenced by the treatment. There were no test item related differences in the fetal- and placental weight, body weight retardation and other external, visceral and skeletal variations. There was one fetus with malformations during external examination at 1000 mg/kg bw/day and five fetuses with malformations during skeletal examinations (one in the control and low dose group, three in the high dose group) .

One fetus was found with agnathy in the 1000 mg/kg bw/day dose group at external examination which finding occurs sporadically also in control fetuses according to the Historical control data of Toxi-Coop Zrt. No clear pattern was observed between the observed malformations. Therefore these observations were judged as not adverse. No clear pattern was observed between the observed malformations. Therefore these observations were judged as not adverse.

Skeletal malformations were found in one control fetus (bipartite cartilage of a thoracic centrum), in one fetus in the 100 mg/kg bw/day group (a displaced thoracic centrum and a supernumerary hemicentric vertebral centrum) and in three fetuses in the 1000 mg/kg bw/day group (one pair of bifurcate ribs, a rudimentary 13th rib and a misshapen thoracic arch in one fetus, split and fused sternebra and bent humerus in a second fetus as well as markedly hypoplastic, medially fused mandible in a third fetus (which was judged to have agnathy at external examination). According to the Historical control data of Toxi-Coop Zrt. and the experience of this laboratory, similar mandibular, sternal, vertebral, rib- and humerus malformations occur sporadically in control fetuses, hence the malformations found in the 100 and 1000 mg/kg bw/day groups were judged to be unrelated from the treatment with the test item. No clear pattern was observed between the observed malformations. Therefore these observations were judged as not adverse. This is also in line with historical control data of another strain of rats, i.e. CrL:CD(R) BR rats (Lang, P.L. (1993) Historical control data for developmental and reproductive toxicity studies using the Crl:CD®BR rat, compiled by MARTA (Middle Atlantic Reproduction and Teratology Association), Charles River Laboratories, September 1993).

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Based on these observations the No Observed Adverse Effect Level (NOAEL) was determined as follows:
NOAEL (maternal toxicity): 1000 mg/kg bw/day
NOAEL (developmental toxicity including teratogenicity): 1000 mg/kg bw/day
Executive summary:

The test item was examined for its possible prenatal developmental toxicity. Groups of 22 sperm-positive female Hsd. Han: Wistar rats were treated with the test item by oral administration daily at three dose levels of 100, 300 and 1000 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 0.5 % methylcellulose. The treatment volume was 5 mL/kg bw.

A 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 in 0.5 % methylcellulose was stable at room for 3 days in a refrigerator (5 +/- 3 oC) at the concentrations of 1 and 200 mg/mL. 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 91 and 112 % 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 by means of a dissecting microscope. The heads were examined by Wilson's free-hand razor blade method.

After cartilage-bone staining the skeletons were examined by means of a dissecting microscope. All abnormalities found during the fetal examinations were recorded.

In total, on gestation day 20 there were 21 evaluated litters each in the control and 300 mg/kg bw/day group, and 19 and 20 in the 100 and 1000 mg/kg bw/day group.

None of the female died before scheduled necropsy and there were no clinical signs recorded. No treatment related necropsy findings were observed.

There was no treatment related reduction on food consumption and body weight indicated.

The test item did not influence the number of implantations, intrauterine mortality and sex distribution of the fetuses.

There were no test item related differences in the fetal- and placental weight, body weight retardation and other external, visceral and skeletal variations. Agnathy observed at external and proved at skeletal examination (as a markedly hypoplastic and fused mandible) and other two different skeletal malformations (one pair of bifurcate ribs, a rudimentary 13thrib and a misshapen thoracic arch in one fetus, split and fused sternebra and bent humerus in another fetus) in the 1000 mg/kg bw/day dose group as well as a displaced- and a supernumerary hemicentric thoracic centrum in one fetus in the 100 mg/kg bw/day group were judged to be unrelated from the treatment with the test item considering the historical control database and the experience with this strain at the lab. This is also in line with historical control data of another strain of rats, i.e. CrL:CD(R) BR rats ( Lang, P.L. (1993) Historical control data for developmental and reproductive toxicity studies using the Crl:CD®BR rat, compiled by MARTA (Middle Atlantic Reproduction and Teratology Association), Charles River Laboratories, September 1993).

 

Based on these observations the No Observed Adverse Effect Level (NOAEL) was determined as follows:

NOAEL (maternal toxicity): 1000 mg/kg bw/day

NOAEL (developmental toxicity including teratogenicity): 1000 mg/kg bw/day