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Developmental toxicity / teratogenicity

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Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
July 2003 to Jan 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report date:
2004

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
version 2001
Deviations:
yes
Remarks:
treatment stopped 9d before caesarean
Qualifier:
according to guideline
Guideline:
other: Recommendations for "Detection of Toxicity to Reproduction for Medicinal Products" - Guideline prepared within the International Conference on Harmonization (ICH) process, Washington (DC), June 24, 1993
Version / remarks:
June 24,1993
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Reference substance name:
475207-59-1
Cas Number:
475207-59-1
IUPAC Name:
475207-59-1

Test animals

Species:
rabbit
Strain:
Himalayan
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Kißlegg, Germany
- Strain: CHBB:HM
- Age at study initiation: females: 119-206 days
- Weight at study initiation: females: 2009-3153 g
- Housing: individually in Makrolon® cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 50 +/- 20
- Air changes (per hr): >= 10
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: July 2003 - Jan 2004

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: suspension of 0.5 % methylhydroxyethylcellulose (Tylopur MH 300 G4) in demineralized water
Details on exposure:
The male animals were used for mating only and remained untreated.

After copulation, 80 females were allocated to four main experimental groups according to a computer generated randomization plan (random number generator, HP Vectra PC). Nine further females were allocated to three satellite groups (0.41 mg/kg, 1.37 mg/kg, and 4.11 mg/kg) for toxicokinetic investigations according to a computer generated randomization plan (random number generator, HP Vectra PC).

The females were treated daily between 06.00 and 12.00 CET from days 6 to 20 of gestation. The females received the administration formulations orally by gavage.

The females in all experimental groups received a uniform administration volume of 5 ml/kg body weight/day. The dose volume was adjusted to the current body weight, which was determined before each administration, daily from days 6 to 20 p.c. The females of the control group received vehicle, only.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability and homogeneity were confirmed in the investigations on the stability of the active ingredient after 8 days of storage and on homogeneity in samples with concentrations of 0.014, 0.137, and 2.06 mg/ml before the start of the study.

The results of the homogeneity tests and content checks in samples with concentrations of 0.082, 0.274 (content checks, only), and 0.822 mg/ml during the study showed no meaningful deviation of the active ingredient content from the nominal value, and homogeneous distribution was confirmed.
Details on mating procedure:
The mating was performed between 05.00 and 10.00 CET. One male rabbit was mated with one female rabbit under observation. About one hour after the first mating had occurred the same animals were mated again. It was recorded which female was mated with which male.

The day on which the copulation was observed was considered as day 0 of gestation.
Duration of treatment / exposure:
days 6 to 20 of gestation
Frequency of treatment:
once daily
Duration of test:
cesarean section and sacrifice on day 21 p.c. (satellite groups) or day 29 p.c. (main groups)
Doses / concentrations
Remarks:
0.41, 1.37 and 4.11 mg/kg bw/d
No. of animals per sex per dose:
main groups: 20 females per dose
satellite groups for toxicokinetics: 3 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
DOSE SELECTION RATIONALE:
The dose levels were selected according to the results of a previous pilot developmental toxicity study with BAY 54-9085 in rabbits with dose levels of 0, 0.41, 1.37, 4.11 and 6.85 mg/kg bw/day.

Summarizing all findings, slight maternal findings occurred in the 0.41 mg/kg and 1.37 mg/kg groups (cold ears); distinct maternal findings were shown at the 4.11 mg/kg level (cold ears, light colored feces, markedly decreased feed intake, and slight body weight loss), and severe maternal findings were evident at the 6.85 mg/kg level (cold ears, reddish excretion, light colored feces, severely decreased feed intake, and severe body weight loss). All females of the 6.85 mg/kg group showed a total late resorption. The postimplantation loss in the females with successful pregnancy was slightly
increased in the 4.11 mg/kg group, for which a treatment related effect cannot be excluded. Fetal evaluation revealed malformations of the forelimbs in three fetuses and ventricular septal defect of the heart in two fetuses each of the 4.11 mg/kg group, for which a treatment related effect is assumed. A treatment related effect for one fetus of the 0.41 mg/kg group with a ventricular septal defect of the heart, and one fetus of the 1.37 mg/kg group with a malposition of the forelimbs is not assumed, but final evaluation was not possible due to the low number of females in a pilot study.

Examinations

Maternal examinations:
SURVEY OF INVESTIGATIONS

Evaluation of the general tolerance of the test compound by the females was based on appearance and behavior, feed and water intakes, appearance of excretory products, body weight development, and mortality of the animals as well as on gross pathological findings. The data of the females of the satellite groups are documented, but the results were not evaluated, because a control group for the satellite groups with comparable repeated blood sampling stress was not used.


APPEARANCE, BEHAVIOR, FEED AND WATER INTAKES, EXCRETORY PRODUCTS AND MORTALITY

The females of the main groups were inspected from days 0 to 29 p.c. and the females of the satellite groups from day 0 to day 21 p.c. twice daily (once daily only on weekends, on public holidays, and on day 29 p.c./21 p.c.), and all findings were recorded. Attention was paid to disturbances in the rabbits' general condition (appearance, behavior) and alterations concerning their excretory products. The feed intake of the animals was determined from the difference in weight between the feed offered and the feed not consumed for the following days of gestation: days 0-3, 3-6, 6-9, 9-12, 12-15, 15-18, 18-20, 20-21, 21-24, 24-27, and 27-29 p.c. for the main groups, and days 0-3, 3-6, 6-9, 9-12, 12-15, 15-18, 18-20, and 20-21 p.c. for the satellite groups. Water intake was assessed daily by visual estimation of the quantities left over.



BODY WEIGHT DEVELOPMENT

The body weight of the females was determined on day 0 p.c. and daily from day 6 to day 29 p.c. (main groups) or to day 21 p.c. (satellite groups). Corrected body weight gain was detennined by subtracting the uterus weight on day 29 p.c. from the body weight gain from days 0 to 29 p.c. (main groups, only).


TOXICOKINETIC INVESTIGATIONS

Venous blood samples (extremity veins) were taken from all females of the 0.41mg/kg, 1.37 mg/kg, and 4.11 mg/kg satellite groups on days 6 and 20 p.c., 1, 2, 4, 7, and 24 hours after administration. The blood of the females was cooled (Eppendorf-Isotherm-System), and plasma samples were prepared by centrifugation. The plasma samples were subsequently deep frozen, and plasma samples were stored at < -20°C until transfer to the Department of Preclinical Pharmacokinetics of BHC-PH-PD-P, 42096 Wuppertal, Germany.


Analyses of BAY 54-9085 in maternal plasma were performed at the Department of Preclinical Pharmacokinetics of BHC-PH-PD-P, 42096 Wuppertal, Germany.


GROSS PATHOLOGICAL INVESTIGATIONS

The females were subjected to gross pathological evaluation at cesarean section on day 29 p.c. (main groups) or on day 21 p.c. (satellite groups). Necropsy was performed without knowledge of treatment groups except for the females of the satellite groups. Females which aborted were killed and necropsied after abortion was evident.


The females were sacrificed by intravenous injection of 2 ml T61 ® ad us. vet. (Intervet Germany GmbH, 85701 Unterschleißheim).
Ovaries and uterine content:
INVESTIGATIONS AT CESAREAN SECTION
Cesarean sections were performed on gestation day 21 (satellite groups) or 29 (main groups), respectively. The following parameters were determined and assessed at cesarean section on day 29 p.c. in the main groups (except for the females , which showed no implantation sites, an uterine anomaly or which aborted):
- Number of corpora lutea
- Number of implantations (in females without visible implantation sites after staining of the uterus with a solution of 10 % ammoniumsulfide)
- Uterine weights
- Individual weight and appearance of the placentas
- Number of early resorptions (only implantation site visible), late resorptions (fetal or placental remnant visible), and dead fetuses (fetuses without signs of life, but without maceration)

The term of combined finding of the skeleton or viscera was used in case a comprehensive description was suitable or necessary. The term does not include a classification of the finding (malformation or deviation).

During cesarean section on day 21 p.c. the following data were ascertained in the satellite groups, but the results were not evaluated, because a control group for the satellite groups with comparable repeated blood sampling stress was not used:
- Existence of implantations
Fetal examinations:
- Number of live fetuses
- Sex of live fetuses
- Individual weights of live fetuses
- Occurrence of external findings in the fetuses
- Occurrence of findings in abdominal, pelvic, and thoracic organs and in the brain
- Occurrence of findings in the skeletal system
Statistics:
All data were recorded online using the TASC-system (Scientific Computer Consultants, Inc., 547 Stonetown Road, Ringwood, NJ 07456, USA) on an Alpha 8005/500 computer, with the exception of the data of gross necropsy of the females of the satellite groups.

Females without implantation sites, with an uterine anomaly as well as females which aborted were not taken into account for calculation of mean values. Females with total resorption were not taken into account for calculation of group mean values of body weights, body weight gains, feed intakes, and uterine weights.

The mean values in the tables calculated by computer are the rounded results of the calculations with unrounded raw data values.

In case of skeletal localizations with mechanical damage, these were excluded from the calculation of percentages of affected localizations. The tables of individual skeletal findings enumerate those findings for which the affected localizations were excluded from calculation.

Differences between the control and BAY 54-9085-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 (TASCsystem) using the following methods:

a. Analysis of variance (ANOVA), and in case of significant results Dunnett's test
b. 2 by N CHI2 test; in case of significant differences Fisher's exact test with Bonferroni correction
c. Kruskal-Wallis test, and in case of significant differences Dunn's test

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
One female aborted at the 4.11 mg/kg level after it had shown signs of mild systemic toxicity. Furthermore, an increased incidence of females with cold ears transiently occurred at the 4.11 mg/kg level. A treatment related effect for the increased incidence of females with cold ears at the 0.41 mg/kg and 1.37 mg/kg levels is unlikely. It can, however, not completely be excluded.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Absolute body weight gain during the treatment period was marginally decreased at the 4.11 mg/kg level, for which a treatment related effect cannot totally be excluded.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Transiently marginally decreased mean feed intake correlating with reduced, soft, and light colored feces, and reddish excretion occurred at the 4.11 mg/kg level.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No treatment related gross pathological findings were evident at dose levels up to and including 4.11 mg/kg.

Maternal developmental toxicity

Number of abortions:
effects observed, treatment-related
Description (incidence and severity):
The gestation rate of the 4.11 mg/kg group was decreased by the abortion of one female and by three females, which showed total resorptions.
Pre- and post-implantation loss:
effects observed, treatment-related
Description (incidence and severity):
The postimplantation loss in females with viable fetuses was increased at the 4.11 mg/kg level, mainly caused by late resorptions. Correspondingly, the mean number of fetuses was decreased at the 4.11 mg/kg level.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Increased incidences of placental findings (partly necrotic placentas) occurred at the 4.11 mg/kg level. The placental weights in all dose groups were unaffected by treatment with BAY 54-9085.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Remarks on result:
not determinable
Remarks:
A NOAEL cannot be established conservatively due to cold ears already in lowest dose-group

Maternal abnormalities

Abnormalities:
effects observed, treatment-related
Localisation:
placenta
Description (incidence and severity):
decreased gestation with one abortion and three total resorptions, increased placental findings, increased postimplantation loss with subsequently reduced number of fetuses in the high dose group

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
The fetal weights in all dose groups did not differ to a meaningful extent from the control value.
Reduction in number of live offspring:
effects observed, treatment-related
Description (incidence and severity):
The postimplantation loss in females with viable fetuses was increased at the 4.11 mg/kg level, mainly caused by late resorptions. Correspondingly, the mean number of fetuses was decreased at the 4.11 mg/kg level.
Changes in sex ratio:
effects observed, treatment-related
Description (incidence and severity):
Fetal sex distribution was shifted to 40.4 % males at the 4.11 mg/kg level. Although fetal sex is already determined before start of treatment, and the value lay only marginally below the normal range of scattering, a treatment related effect, based on marginally higher lethality of male fetuses, cannot totally be excluded.
External malformations:
no effects observed
Description (incidence and severity):
A treatment related effect on external deviations (findings other than malformations) was not evident at dose levels up to and including 4.11 mg/kg.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
A treatment related effect is assumed for the increased incidence of fetal malformations (mainly malformations of vertebrae, and ribs) at the 4.11 mg/kg level, known as common malformations in the rabbit strain used.

Skeletal development (retardations/variations) revealed an increased incidence of fused sternebrae and retarded ossification of cervical vertebral bodies and frontal bones at the 4.11 mg/kg level.
Visceral malformations:
effects observed, treatment-related
Description (incidence and severity):
A treatment related effect is assumed for the increased incidence of fetal malformations (kidneys) at the 4.11 mg/kg level, known as common malformations in the rabbit strain used.

A treatment related effect on visceral deviations (findings other than malformations) was not evident at dose levels up to and including 4.11 mg/kg.
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
1.37 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
external malformations
skeletal malformations

Fetal abnormalities

Abnormalities:
not specified
Localisation:
skeletal: rib
skeletal: vertebra
visceral/soft tissue: urinary
Description (incidence and severity):
A treatment related effect is assumed for the increased incidence of fetal malformations (mainly malformations of kidneys, vertebrae, and ribs) at the 4.11 mg/kg level, known as common malformations in the rabbit strain used.

Skeletal development (retardations/variations) revealed an increased incidence of fused sternebrae and retarded ossification of cervical vertebral bodies and frontal bones at the 4.11 mg/kg level.

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Toxicokinetics:


Toxicokinetic investigations showed, that for BAY 43-9006 AUC(0-24) and Cmax on day 20 p.c. increased about dose-proportionally after administration of 0.41 and 1.37 mg/kg bw. The increase in AUC(0-24) and Cmax was slightly more than dose-proportional, when the dose was increased from 1.37 to 4.11 mg/kg bw. In case of M-4 metabolite a dose linear increase in AUC(0-24) and Cmax was observed for the low and medium dose administered. Further increase in dose to the high dose of 4.11 mglkg (BAY 54-9085) was accompanied by a moderately more than dose-linear increase in AUC(0-24) and Cmax. Peak plasma concentrations for BAY 43-9006 and M-4 on day 20 p.c. were reached in a time interval between 2 and 7 h for BAY 43-9006 and between 2 and 24 h for M -4, respectively, after oral administration for all 3 applied doses. A flat concentration time profile for BAY 43-9006 and M-4 was observed in this time interval. The residual concentrations found for both analytes were high in all dose groups. The observed concentrations amounted to 50 % of Cmax in case of BAY 43-9006 and up to 100 % of Cmax in case of M-4. After repeated dosing of BAY 54-9085 a slight increase in AUC(0-24) and Cmax of BAY 43-9006 by a factor of approximately 1.3 was observed for all dose groups comparing day 20 p.c. to day 6 p.c. For BAY 43-9007 (M-4) a marked increase in exposure after repeated dosing was observed regarding both Cmax and AUC(0-24). Accumulation factors in terms of AUC(0-24) were 2.87 to 3.45-fold for all doses administered. The increase in Cmax was as well distinct. Cmax values increased by a factor of about 2.47 to 3.24 when day 20 p.c. to day 6 p.c. are compared.

Applicant's summary and conclusion

Conclusions:
Based on a possibly increased incidence of females with cold ears at 0.41 and 1.37 mg/kg, for which a treatment related effect is unlikely, but cannot be excluded, a conservative systemic maternal no-observed-adverse-effect-level (NOAEL) was not established in this study.Therefore, under the conditions described the NOAEL for systemic maternal toxicity (cold ears) in rabbits was < 0.41 mg/kg/day and the NOAEL for intrauterine development in rabbits was 1.37 mg/kg/day.
Executive summary:

BAY 54-9085 is the tosylate salt of BAY 43-9006 (sorafenib base), which is used as an anti-cancer drug.


 


Twenty inseminated female Himalayan rabbits each were treated orally by gavage with BAY 54-9085 with daily doses of 0, 0.41, 1.37, or 4.11 mg/kg (corresponding to 0, 0.3, 1.0 or 3.0 mg/kg/day BAY 43-9006) from day 6 to day 20 post conceptionem. On day 29 of gestation the fetuses were delivered by cesarean section. Three additional females per group were treated with BAY 54-9085 doses of 0.41, 1.37, and 4.11 mg/kg/day from day 6 to day 20 post conceptionem for determination of BAY 43-9006 and metabolites in the plasma of the females.


 


One female at 4.11 mg/kg aborted after signs of mild systemic toxicity. Furthermore, an increased incidence of females with cold ears transiently occurred at 4.11 mg/kg. Transiently marginally decreased mean feed intake correlating with reduced, soft, and light colored feces, and reddish excretion occurred at 4.11 mg/kg. Absolute body weight gain during the treatment period was marginally decreased at 4.11 mg/kg. No treatment-related gross pathological findings were evident at dose levels up to and including 4.11 mg/kg.


 


The gestation rate of the 4.11 mg/kg group was decreased by the abortion of one female, and by three females, which showed total resorptions. Increased incidences of placental findings (partly necrotic placentas) occurred at 4.11 mg/kg. The placental weights in all dose-groups were unaffected by treatment.


 


The postimplantation loss in females with viable fetuses was increased at 4.11 mg/kg, mainly caused by late resorptions. Correspondingly, the mean number of fetuses was decreased at 4.11 mg/kg. Fetal sex distribution was shifted to 40 % males at 4.11 mg/kg. Although fetal sex is already determined before start of treatment, and the value are only marginally below the normal range of historical controls, a treatment related effect, based on marginally higher lethality of male fetuses, cannot be excluded. The fetal weights in all dose groups did not differ to a meaningful extent from the control value.


 


A treatment-related effect is assumed at 4.11 mg/kg for the increased incidence of fetal malformations (mainly malformations of kidneys, vertebrae, and ribs), known as common malformations in the rabbit strain used. A treatment-related effect on external and visceral deviations (findings other than malformations) was not evident at doses up to and including 4.11 mg/kg. Skeletal development (retardations/variations) revealed an increased incidence of fused sternebrae and retarded ossification of cervical vertebral bodies and frontal bones at 4.11 mg/kg.


 


Based on a possibly increased incidence of females with cold ears at 0.41 and 1.37 mg/kg, for which a treatment related effect is unlikely, but cannot be excluded, a conservative systemic maternal no-observed-adverse-effect-level (NOAEL) was not established in this study.Therefore, under the conditions described the NOAEL for systemic maternal toxicity (cold ears) in rabbits was < 0.41 mg/kg/day and the NOAEL for intrauterine development in rabbits was 1.37 mg/kg/day.