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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
(2001)
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Qualifier:
according to
Guideline:
other: Japanese MAFF guidelines: Guideline on the Compiling of Test Results on Toxicity “Teratology Study”, 12-Nousan No. 8147 of November 24, 2000, amended June 26, 2001
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Molecular weight: approximately 235
- Physical state: clear colorless liquid
- purity: 100 %
- Stability under test conditions: analytically verified

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Hsd Cpb:WU (SPF-bred)
- Source: Harlan-Nederlands, 5960 AD Horst, The Netherlands
- Age at study initiation: 12-17 weeks
- Weight at study initiation: 220 - 265 g (females); 428 - 536 g (males)
- Housing: Starting from gestation day 0 individually in Type IIIh Makrolon cages on low-dust wood shavings.
- Diet and water: ad libitum
- Acclimation period: at least 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): approximately 55 %
- Air changes (per hr): > 10 per hour
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: demineralized water
Details on exposure:
Administration volume: 10 mL/kg bw

PREPARATION OF DOSING SOLUTIONS: The formulations were prepared as needed taking into account the analytically determined stability.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test substance was administered as a solution in the vehicle. The formulations were stored at room temperature.
Before the start of treatment, the suitability of the formulation was confirmed by the analysis of concentration and stability of dosage forms prepared in the same way as it was done in the study (0.5 and 300 mg/mL, covering the concentration range used after 8 days of storage). Analyses were carried out before the start of the study. Content checks of the active ingredient in samples with concentrations of 10, 30, and 100 mg/mL were carried out twice during the study.
Details on mating procedure:
The animals were mated by placing two females overnight into a Type IIIh cage together with one male rat. If sperm was detected in the vaginal smear taken on the morning following mating, this day was regarded as day 0 of gestation.
Duration of treatment / exposure:
Day 6 - 20 p. c.
Frequency of treatment:
once daily (between 06:00 and 12:00 CET)
Duration of test:
from days 0 to 21 p.c.
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300, 1000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
25 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on the results of a previous dose toleration study in rats, which revealed no treatment related findings after 1000 mg/kg.

Examinations

Maternal examinations:
CLINICAL EXAMINATIONS: Yes
- Time schedule: The females were inspected from days 0 to 21 p.c. twice daily (once daily only on weekends, on public holidays, and on day 21 p.c.), and all findings were recorded. Attention was paid to disturbances in the general condition of the rats (appearance, behavior), and any alterations concerning their excretory products.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of the females was determined on day 0 p.c. and daily from day 6 to day 21 p.c. Corrected body weight gain was determined by subtracting the uterus weight on day 21 p.c. from the body weight gain from days 0 to 21 p.c.

FOOD CONSUMPTION: Yes
- The food intake of the animals was determined from the difference in weight between the food offered and the food not consumed for the following days of gestation: Days 0 - 3, 3 - 6, 6 - 9, 9 - 12, 12 - 15, 15 - 18, and 18 -21.

WATER CONSUMPTION: Yes
- Water intake was assessed daily by visual estimation of the quantities left over and reported together with clinical findings.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Cesarean sections were performed on day 21 p.c. without knowledge of treatment groups.
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
- Number of dead and live fetuses: Yes
- Other: individual weight and appearance of the placentas
Fetal examinations:
- Sex of live fetuses
- Individual weights of live fetuses
- External examinations: Yes: [findings in alive and dead fetuses are included]
- Soft tissue and head examinations : Yes: [evaluation of about half of alive fetuses per litter]
- Skeletal and cartilage abnormalities : Yes: [evaluation in about half of alive fetuses per litter]
Statistics:
Differences between the control and test item treated groups were considered to be significant when p < 0.05. Significant differences from the control group are indicated with * for p < 0.05 and ** for p < 0.01. Statistical evaluation was performed on an Alpha 800 5/500 computer using the following methods:
Analysis of Variance (ANOVA); in case of significance Dunnett's test for feed intakes, body weights, body weight gains, and corrected body weight gains, uterine weights, number of corpora lutea per female, number of implantations per female, number of live fetuses per female and as percentage of implantations per female, placental weights per female, fetal weights per female.
2 by N CHI2 test; in case of significant differences Fisher's exact test with Bonferroni correction for fertility rate, gestation rate, number of implantations per group, number of preimplantation losses per group, number of postimplantation losses, early resorptions, late resorptions or dead fetuses per group, number of live fetuses per group as percentage of implantations per group, number of male or female fetuses or fetuses with indeterminable sex per group, number of placentas with findings or litters with placental findings per group, number of fetuses or litters with external, visceral or skeletal findings, with malformations or with external or visceral deviations per group.
Kruskal-Wallis test and in case of significant differences Dunn's test for number of preimplantation losses per female, number of postimplantation losses, early resorptions, late resorptions or dead fetuses per female, number of male or female fetuses or fetuses with indeterminable sex per female, number of placentas with findings per female, number of fetuses with external or visceral findings, with malformations or with external or visceral deviations per female.
CHI2 test (correction according to yates) for number of fetuses or litters with cartilaginous tissue observations.
Indices:
gestation rate

Results and discussion

Results: maternal animals

Maternal developmental toxicity

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

Details on maternal toxic effects:
BODY WEIGHTS:
Statistically significantly decreased body weight gain occurred from days 6-7 p.c. at the 1000 mg/kg level, for which a treatment related adverse effect is not assumed, as only this single interval revealed a decreased body weight gain, and because absolute body weight gain during treatment and gestation periods as well as corrected body weight gain were unaffected at this dose level. Statistically significantly increased body weight gain at the 100 mg/kg and 1000 mg/kg levels from days 20-21 p.c. is caused by the incidentally lower body weight gain of the current control group within this interval.
Thus, absolute and corrected body weight gains were unaffected by treatment at dose levels up to and including 1000 mg/kg. A treatment related adverse effect is not assumed for transiently decreased body weight gain from days 6-7 p.c. at the 1000 mg/kg level.

NECROPSY:
One female of the 1000 mg group showed a slight dilation of the left renal pelvis, for which a treatment related effect is not assumed due to its single occurrence, and since this finding is known as a spontaneous finding in the rat strain used. Furthermore, one female without implantation sites at the 1000 mg/kg level revealed a uterus tightly filled with clear fluid, for which a treatment related effect is not assumed, as this finding was also evident in one female of the current control group, and because this finding is known as a spontaneous finding in the rat strain used.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Basis for effect level:
other: developmental toxicity

Results (fetuses)

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

Details on embryotoxic / teratogenic effects:
WEIGHT and APPEARANCE of PLACENTAS:
Two double placentas out of one litter occurred at the 1000 mg/kg level, for which a treatment related effect is not assumed, as the incidence of this finding lay within the range of historical control data. Furthermore, one pale placenta was shown at the 300 mg/kg level, for which a treatment related effect is not assumed due to its single occurrence, because of a lacking dose dependency, and since this finding is known as a spontaneous finding in the rat strain used.
FETAL MALFORMATIONS:
The overall incidences of fetuses with malformations showed no dose dependency at dose levels up to and including 1000 mg/kg and revealed the highest values on a fetal and a litter basis in the 100 mg/kg group. The overall incidences of affected fetuses and litters lay well within the range of historical control data of the rat strain used .

number of fetuses per group/number of fetuses with malformations/malformed fetuses per group (%)/number of litters per group/number of litters with malformations/malformed litters per group (%)
Vehicle control: 262/0/0.0/21/0/0.0
100 mg/kg b.w. and day: 261/6/2.3/20/4/20.0
300 mg/kg b.w. and day: 288/4/1.4/21/4/19.0
1000 mg/kg b.w. and day: 262/3/1.1/20/3/15.0

FETAL EXTERNAL and VISCERAL DEVIATIONS

Circumscribed hard whitish areas in different localizations of the head occurred in all groups, without showing a dose dependency. Histopathological evaluation of this finding was performed in a recently conducted prenatal developmental toxicity study with the same rat strain and revealed calcium concrements without connection to the underlying tissue in the affected localizations. Calcium might have been dissolved from the fetal bones by the Wilson fixative and precipitated so that these findings were regarded as artifacts. The finding of a small hollow structure in different localizations of the head also occurred in all groups, without showing a dose dependency, and is also considered as a fixation artifact.
The remaining findings of the dose groups revealed no dose dependency in most of the cases or were isolated findings. The incidences were comparable with the current control group and/or lay within the range of historical control data.

FETAL SKELETAL DEVIATIONS INCLUDING CARTIÖAGINOUS DEVIATIONS
Sporadic statistically significant skeletal findings (increased incidence of an unossified round area right at the exoccipital bone at the 100 mg/kg level, decreased incidence of asymmetrical 5th sternebrae at the 300 mg/kg level) showed no dose dependency so that a treatment related effect was not evident

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: no adverse effects with regard to the parameters examined

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Executive summary:

A developmental toxicity study was performed with the test substance according to OECD TG 414. Twenty-five inseminated female Wistar rats each were treated daily orally by gavage with the test substance in deminaralized water from day 6 to day 20 p.c. in the following doses: 0, 100, 300, and 1000 mg/kg body weight (bw), respectively. The fetuses were delivered by cesarean section on day 21 of gestation. Investigation was performed on general tolerance of the test substance by the females as well as on its effect on intrauterine development.

Appearance, behavior, mortality, absolute and corrected body weight gains, food intake, water intake, and fecal and urinary excretions were unaffected at dose levels up to and including 1000 mg/kg bw.

No treatment related gross pathological findings occurred at dose levels up to and including 1000 mg/kg.

The gestation rate, appearance and weights of placentas, postimplantation loss and correspondingly the number of fetuses, fetal sex distribution, and fetal weights were unaffected by treatment at dose levels up to and including 1000 mg/kg.

A treatment related effect on malformations, external, visceral, and skeletal (including cartilage) deviations was not evident at dose levels up to and including 1000 mg/kg.

Summarizing and evaluating all data investigated the following no-observed-adverse-effect levels (NOAELs) were determined: for maternal toxicity 1000 mg/kg bw, for developmental toxicity 1000 mg/kg bw.