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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
Study period:
2016-2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: 0001021592
- Expiration date of the lot/batch: December 11th, 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature, protected from light
- Stability under test conditions: Stable
- Solubility and stability of the test substance in the solvent/vehicle: Stable

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Age at study initiation: 12-13 weeks
- Weight at study initiation:
Body weight before the start of pairing:
males: 349 – 381 g (mean: 366.67 g, ± 20% = 293.34 – 440.01 g)
females: 180 - 236 g (mean: 204.17 g, ± 20% = 163.34 – 245.01 g)
- Housing: - Full barrier in an air-conditioned room
- Diet (e.g. ad libitum): - Free access to Altromin 1324 maintenance diet for rats and mice
- Water (e.g. ad libitum): - Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C):22 ± 3 °C
- Humidity (%): 55 ± 10 %
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To:
Delivery of Animals: 03 March 2016
Acclimatisation Period: 03 - 08 March 2016
Experimental Starting Date: 08 March 2016
Experimental Completion Date: 08 July 2016

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): water used
- Concentration in vehicle: 0; 23.5; 117.6 and 313.7 mg/L
- Amount of vehicle (if gavage): 5 mL/kg body weight

Name: aqua ad iniectabilia (sterile water)
Manufacturer: AlleMan Pharma
Batch No.: 503424
Physical State: liquid
Storage Conditions: at room temperature
Expiry Date: 28/02/2018
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The assessment of homogeneity as well as a determination of the nominal concentration of the test item in the vehicle was performed at various intervals using a validated analytic method.
Samples for analysis of concentration analysis of the active ingredient within the vehicle (nominal concentration) were taken in the first and last week of the study for all doses, including controls (8 samples in total). Samples for homogeneity were taken from the top, middle and bottom of the high dose and low dose preparation. Samples were taken in the first and last week of the study (12 samples in total).
Each sample was retained in duplicates (sample A, sample B, each at least 5 mL). The A- sample were analysed. The procedures followed for the study sample analysis were mentioned in a phase plan that was amended to the study plan. The B-samples were retained at -15 to -35°C until the analysis had been performed, and discarded after completion of the final study report. Analytical results are reported in the appendix of the present report (Appendix 3).
Concentration results were considered acceptable if sample concentration results are within or equal to ± 10% of theoretical concentration. For homogeneity, the criteria for acceptability were a relative standard deviation (RSD) of concentrations of ≤10% for each group.
Details on mating procedure:
Mating was performed using a ratio of 1:2 (male to female). Females were paired for cohabitation in batches in order to control the number of animals for terminal sacrifice on a particular day. At the subsequent morning and the next morning onwards, the vaginal smear of each female was checked to confirm the pregnancy. The day on which sperms were observed in the vaginal smear was considered as gestation day ‘0’. Mated females were assigned in an unbiased manner to the control and treatment groups ensuring that group mean body weights were comparable with each other. Each animal was assigned an unique identification number. After obtaining a sufficient number of sperm positive females, the remaining females and males were discarded without any observations.
Duration of treatment / exposure:
The pregnant females were treated with the test item formulation or vehicle on 7 days per week between gestation day 5 until gestation day 19.
Frequency of treatment:
Daily, between gestation day 5 and gestation day 19.
Duration of test:
Until gestation day 20
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control group, group name C
Dose / conc.:
60 mg/kg bw/day (nominal)
Remarks:
Lowest dosage group, group name LD
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Medium dosage group, group name MD
Dose / conc.:
800 mg/kg bw/day (nominal)
Remarks:
High dosage group, group name HD
No. of animals per sex per dose:
The control group had 23 sperm positive females, the LD group had 29 sperm positive females and the MD and HD groups each had 24 sperm positive females. However, at necropsy, the number of gravid females was 21 of 23 in the control group, 21 of 29 in the LD group, 20 of 24 in the MD group and 20 of 24 in the HD group.
Control animals:
yes, concurrent vehicle

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: General clinical observations were made once a day, approximately at the same time each day at 30 min after dosing. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: General clinical observations were made once a day, approximately at the same time each day at 30 min after dosing. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily.

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed once before initiation of pairing to ensure that the body weights are within + 20% variation.
The sperm positive females were weighed during gestations days 0, 5, 8, 11, 14, 17 and 20. Males were not weighed in this study except once before initiation of pairing.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
Food consumption of sperm positive females was measured on gestations days 5, 8, 11, 14, 17 and 20.
Food consumption was not measured for males during the entire study or for both males and females during the mating period

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: At the time of termination, the dam (presumed pregnant female) was examined macroscopically for any structural abnormalities or pathological changes, which may have influenced the pregnancy. Immediately after the termination, the uteri were removed and the pregnancy status of the dams was confirmed. Uteri that appear non-gravid were further examined by staining with 10 % ammonium sulphide solution to confirm the non-pregnant status.
Each gravid uterus with the cervix was weighed. The number of corpora lutea was counted for pregnant animals. The uterine contents were examined for embryonic or fetal deaths as well as the number of viable fetuses. The degree of resorption (late and early) was confirmed in order to help estimate the relative time of death of the conceptus. The position and number of fetuses in each uterine horn was also recorded.
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 per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]
Craniofacial examination of the heads of the fetuses used for the soft tissue examination were performed for internal structure including the eyes, brain, nasal passage and tongue by razor blade serial sectioning technique.
Fetuses scheduled for skeletal examination were eviscerated and the entire litter was transferred into separate plastic bottles containing 95% ethanol. These fetuses were processed using the Alizarin red staining technique. After fixation in 95% ethanol, the fetuses were macerated with a 1% aqueous potassium hydroxide solution for 1 day and transferred to an Alizarin red solution (0.0025% in 1% aqueous potassium hydroxide) for 1 day. After that, the fetuses were again transferred to 1 % KOH. Alizarin stained fetuses were then cleared and dehydrated in a solution containing 2 parts of 70% ethanol, 2 parts of glycerin and one part of benzyl alcohol for 1 day and finally preserved in a 1:1 solution of 70 % ethanol and glycerin.
The stained fetuses were examined under the stereomicroscope, the skull was examined for size, shape and degree of ossification of nasal, parietal, interparietal, supraoccipital, exoccipital, lacrimal, zygomatic (malar), squamosal (temporal), premaxillary, maxillary, basisphenoid, hyoid and tympanic ring (annulus). Similarly, the vertebral centres, ribs and sternal centres were also examined for size, shape and counted for the number of ossification centers. The cervical, thoracic, lumbar, sacral, caudal vertebrae were observed for the ossification of centers and arches. Pelvic girdles, fore limbs and hind limbs were examined for the development of the bones. Any deviation from the normal development was recorded for each fetus.
- Head examinations: Yes
Statistics:
A statistical assessment of the results of the body weight, food consumption was performed by comparing values of dosed with control animals using a one-way ANOVA and a post-hoc Dunnett Test. Fetal evaluation parameters like external, visceral, craniofacial and skeletal parameters were analysed using a Fisher’s exact test. The statistics will be performed with GraphPad Prism V.6.01 software (p<0.05 is considered as statistically significant).

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
The observation of moving the bedding started with the onset of treatment in MD and HD group animals. Few animals of LD group also showed moving the bedding from GD 12. This behavior was seen immediately post dose administration, was transient and is considered to be a local and non-specific reaction.
Abnormal breathing was observed in 1 of 24 females of MD group and 4 of 24 females of HD group; alopecia was observed in 3 of 29 females of LD group and 1 of 24 females of HD group; crust formation was observed in 1 of 24 females of HD group; piloerection was observed in 4 of 24 females of HD group; salivation was observed in 2 of 29 females of LD group, 1 of 24 females of MD group and 1 of 24 females of HD group. The clinical signs alopecia and salivation in test item treated groups did not show dose dependency and occurred sporadically in single animals. The abnormal breathing was transient, occurred sporadically and was limited to 4 of 24 animals. Therefore, these clinical findings were not considered to be adverse or treatment-related.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
There were no effects on body weight and body weight gain in test-item treated groups compared to the controls. HD animals showed statistically significantly lower weight gain or body weight loss between GD 5-8 however all animals recovered quickly and between GD 8-11 there was a higher weight gain in the HD group compared to the corresponding control. These changes were transient and are not considered to be an adverse effect of test-item treatment.
There were no statistically significant differences in the mean terminal body weights and mean adjusted maternal body weights in test-item treated groups compared to the corresponding control group.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
There was no adverse effects on food consumption during the treatment period in test-item treated groups compared to the control. There was statistically significantly lower food consumption between GD 5-8 in the HD group compared to the corresponding control however all animals recovered quickly and showed food consumption values similar to controls from GD 8 and onwards. This transient change was not considered to be an adverse effect of test-item treatment.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no adverse macroscopic findings in test-item treated group animals. In the LD group there were observations of a cyst on the ovary (1 of 29 females), extra growth in fat tissue (1 of 29 females), and fluid filled and distended uterus (2 of 29 females). In the MD group there was one observation of a fluid filled ovary (1 of 24 females). These findings occurred in a limited number of females and without dose dependency. Therefore, these findings are not considered to be related to test-item treatment.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not specified

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
effects observed, non-treatment-related
Description (incidence and severity):
There were no effects of test-item treatment on prenatal parameters including the number of corpora lutea, number of implantation sites, number of live fetuses and number of resorptions (early and late), number of male and female fetuses, sex ratio and percent pre- and post-implantation losses. Statistically significantly higher mean resorptions (early and total) occurred in the MD group (1.45) compared to the control group (0.48). This finding was not dose dependent and therefore, is not assumed to be related to test-item treatment
Early or late resorptions:
effects observed, non-treatment-related
Description (incidence and severity):
There were no effects of test-item treatment on prenatal parameters including the number of corpora lutea, number of implantation sites, number of live fetuses and number of resorptions (early and late), number of male and female fetuses, sex ratio and percent pre- and post-implantation losses. Statistically significantly higher mean resorptions (early and total) occurred in the MD group (1.45) compared to the control group (0.48). This finding was not dose dependent and therefore, is not assumed to be related to test-item treatment
Dead fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
The finding of 8 dead fetuses in female no. 79 of the HD group was an isolated incidence and is not considered to be a treatment-related effect. The adjusted body weight of this animals was significantly lower compared to the other animals in the group, and the food consumption was reduced from gd 17-20 during which period the animal lost weight
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
not specified

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
800 mg/kg bw/day (nominal)
Based on:
act. ingr.
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
behaviour (functional findings)
gross pathology
number of abortions
pre and post implantation loss
total litter losses by resorption
effects on pregnancy duration
early or late resorptions
dead fetuses
changes in pregnancy duration
changes in number of pregnant

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
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:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were slightly higher incidences of incomplete ossification of supraoccipital bones in MD (30%) and HD (35%) compared to control (23.81%); and slightly higher incidences of full 14th rib (right sided) in MD (10%) and HD (10%) group compared to control (4.76%). In addition, in the HD group there was a higher incidence of misshapen 6th sternebrum (10% in HD and 0% in control); a higher incidence of extra ossification sites at vertebral lumbar arch(es) (10% in HD group and 0% in control); a higher incidence of unossified forelimb metacarpals (63.16% in HD group (and 28.57% in control); a higher incidence of pelvic caudal bone (Bilateral) shift (25% in HD and 19.05% in control) a higher incidence of cervical 7th rudimentary rib (bilateral) (15% in HD (and 4.76% in control); and a higher incidence of cervical 7th rudimentary rib (left) (15% in HD group and 9.52% in control).
Higher incidences of cervical 7th rudimentary rib (right) were noted in LD (14.29%) and HD (10%) groups compared to control (4.76%).
The incidences of all of these observations in the test-item treated groups were without statistical significance, without dose dependency and/or were within the historical control data range, except for the misshapen 6th sternebrum in HD group, for which the incidence was outside the historical control data range (0% to 4.55%). The misshapened sternebrae is a malformation, but considering that only 3 of 112 fetuses were affected, and in the complete absence of any other indication of treatment-related fetal effects within the group, the finding was not considered to be related to the test item, but rather a coincidental finding.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were a range of visceral observations in control and test item treated groups. Most of the observations were noted in single animals and none occurred at statistically significantly higher incidences in test- item treated groups when compared to the corresponding control group. There were higher litter incidences of dilated ureter (bilateral) in MD (30%) and HD (30%) groups compared to control (23.81%) without statistical significance and these were within the historical control data range (see Appendix 4). Therefore, this finding was not an adverse effect of test-item treatment
Other effects:
not specified

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
800 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
not specified
Basis for effect level:
reduction in number of live offspring
changes in sex ratio
fetal/pup body weight changes
changes in litter size and weights
changes in postnatal survival
external malformations
skeletal malformations
visceral malformations

Fetal abnormalities

Key result
Abnormalities:
no effects observed
Localisation:
external: cranium
external: ear
external: eye
external: face
external: limb
external: paw
external: tail
external: trunk
external: anogenital distance
external: anus
external: genital tubercle
external: large intestine
external: thorax
external: umbilicus
external: pelvic region
skeletal: skull
skeletal: skull, fontanelles
skeletal: skull sutures
skeletal: clavicle
skeletal: scapule
skeletal: forelimb
skeletal: sternum
skeletal: rib
skeletal: supernumerary rib
skeletal: vertebra
skeletal: pelvic girdle
skeletal: hindlimb
visceral/soft tissue: integumentary
visceral/soft tissue: gastrointestinal tract
visceral/soft tissue: hepatobiliary
visceral/soft tissue: urinary
visceral/soft tissue: cardiovascular
visceral/soft tissue: heamatopoietic
visceral/soft tissue: immune system
visceral/soft tissue: musculoskeletal system
visceral/soft tissue: nervous system
visceral/soft tissue: central nervous system
visceral/soft tissue: peripheral nervous system
visceral/soft tissue: somatic nervous system
visceral/soft tissue: autonomic nervous system
visceral/soft tissue: endocrine system
visceral/soft tissue: respiratory system
visceral/soft tissue: male reproductive system
visceral/soft tissue: female reproductive system
visceral/soft tissue: eye
visceral/soft tissue: ear
other:

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
On the basis of this prenatal developmental toxicity study in Wistar pregnant female rats with IBP1-Na at dose levels of 60, 300, and 800 mg/ kg body weight/ day administered on gestation days 5 to 19, the following conclusions can be made:
There were no observed effects on maternal animals of test-item treated groups compared to the corresponding control group. There were no treatment-related adverse effects on embryo-fetal development. Thus, the NOAEL for the maternal toxicity and embryo-fetal developmental toxicity is 800 mg/ kg body weight/ day.

Remark: The test substance as produced and marketet contains NaOH due to the production method.
Executive summary:

The substance was investigated to assess possible effects on pregnant female Wistar rats and embryo-fetal development consequent to repeated exposure of the test item according to OECD Guideline 414 (adopted 22 Janaury 2001). Doses of 0 (control), 60, 300 and 800 mg/kg bw/day were administered orally by gavage to pregnant female rats daily on gestation days 5 to 19 (23 sperm positive females in control group, 29 sperm positive females in low dose group, 24 sperm positive female rats in each of medium dose and high dose groups).


 


There were no mortalities in the control and test-item treated groups. No adverse clinical symptoms related to treatment with the test item were noted during the study. Body weight, body weight gain and food consumption were not affected in test-item treated groups compared to the control. No adverse macroscopic findings were noted during necrospopy in the animals from the test-item treated groups.


 


There were no effects of test-item treatment on prenatal parameters including number of corpora lutea, number of implantation sites, number of live fetuses and number of resorptions (early and late), number of male and female fetuses, sex ratio and percent pre- and post-implantation losses. There were no effects on uterus weight. Fetal deaths (8 fetuses) occurred in a single female of the high dosis group; as an isolated incidence this was considered incidental. In two litters from other high dose females, a total of three fetuses were found with a misshapen 6th sternebrum. In the absence of any other indications of test item related fetal effects, this finding was considered incidental.


 


There were no effects of test-item treatment on litter weight parameters including mean fetus weight, total litter weight, male litter weight or female litter weight. There were no statistically significantly different litter weight values in test-item treated groups compared to corresponding control groups. There were comparable numbers of fetuses in both (left and right) uterine horns of control and test-item treated groups. There was a range of fetal anomalies observed at external, visceral, craniofacial and skeletal examination in the fetuses of the test- item treated groups and control group. None of these anomalies occurred at statistically significantly higher incidence in test item treated groups compared to controls and therefore, were not considered treatment-related.


On the basis of this prenatal developmental toxicity study, the following conclusions can be made:
There were no observed effects on maternal animals of test-item treated groups compared to the corresponding control group. There were no treatment-related adverse effects on embryo-fetal development. Thus, the NOAEL for the maternal toxicity and embryo-fetal developmental toxicity is 800 mg/ kg body weight/ day.