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Diss Factsheets

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 May - 07 Aug 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adpted in 2018
Deviations:
yes
Remarks:
No special attention paid to external foetal sex (gross examination) compared with internal (gonadal) sex; no indication of incomplete testicular descent /cryptochordism noted in males
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted 1998
Qualifier:
according to guideline
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
2016
Qualifier:
according to guideline
Guideline:
other: JMAFF, 12 Nohsan No. 8147
Version / remarks:
adopted 2000
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
(Z)-N-octadecyldocos-13-enamide
EC Number:
233-226-5
EC Name:
(Z)-N-octadecyldocos-13-enamide
Cas Number:
10094-45-8
Molecular formula:
C40H79NO
IUPAC Name:
(Z)-N-octadecyldocos-13-enamide

Test animals

Species:
rat
Strain:
Wistar
Remarks:
RccHan™;WIST
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS Limited
- Age at study initiation (females): 77 to 83 days old
- Weight at study initiation (females): 180 to 218 g
- Fasting period before study: no
- Housing: Prior to mating, the animals were housed in groups of up to 4/sex in polycarbonate cages with a stainless steel mesh lid and with softwood based bark-free fiber bedding. During mating, males and females were housed in a 1 : 1 ratio in grid bottomed cages with absorbent paper. After mating, females were housed individually. Throughout the study, the cages were enriched with an aspen chew block and a plastic shelter (except during pairing).
- Diet: SDS VRF1 certified pelleted diet, ad libitum
- Water: potable water from the public supply via polycarbonate bottles with sipper tubes, ad libitum
- Acclimation period: Five days before commencement of pairing

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 40-70
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 01 June 2020 To: 25 June 2020

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The required amount of test item was ground in a mortar using a pestle and mixed with some vehicle to form a paste. Further amounts of vehicle were gradually added and mixed to produce a smooth, pourable suspension. The suspension was quantitatively transferred and diluted to volume and finally mixed using a high-shear homogenizer.
A series of suspensions at the required concentrations were prepared by dilution of individual weighings of the test item.
Test item dosing formulations were prepared weekly and stored at ambient temperature (15 to 25°C).

VEHICLE
- Justification for use and choice of vehicle (if other than water):
Due to the low water solubility of the test item, propylene glycol was selected as the vehicle as it had proved suitable for dissolving the test substance and because there was an acceptable analytical method available for analysis of the test formulations.
- Concentration in vehicle: 16.66, 66.66 and 166.66 mg/mL
- Amount of vehicle: 6 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of each of the first and last preparation formulations were analysed for achieved concentration of the test item after extraction in and dilution in tetrahydrofuran (THF) followed by gas chromatographic analysis with flame ionisation detection (FID). Sample concentrations were determined with reference to external standards prepared in the concentration range of 20 µg/mL to 200 µg/mL. The mean concentrations were within 10% of the nominal concentration, confirming the accuracy of formulation, with the exception of Week 1 Group 3 samples (400 mg/kg bw(day) which were +10.1% from nominal. This minor excursion from the accepted range of +/-10% was considered not to have affected the study. The difference from mean remained within 4%, confirming precise analysis. Procedural recoveries remained within the range established during the validation, confirming the continued accuracy of the analytical procedure. The procedural recoveries prepared with last week samples were between 98.6% and 101.6%. The procedural recoveries prepared with first week samples were not reported due to an incorrect vehicle reagent number transcribed in the raw data. This deviation from the study protocol is not considered to have an impact on the outcome of the study as results were not corrected for recoveries.
In addition, the homogeneity and stability of formulations during storage were confirmed as part of another study. It was demonstrated that formulations in the concentration range of 2 and 166.66 mg/mL were stable at ambient temperature (15 to 25°C) for 15 days or refrigerated (2 to 8°C) for 15 days.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1 with identified stock males
- Length of cohabitation: up to 7 days
- Proof of pregnancy: Ejected copulation plugs in cage tray or sperm in vaginal smear referred to as Day 0 of gestation.
Duration of treatment / exposure:
From Gestation Day 6 to Gestation Day 19 (after mating)
Frequency of treatment:
daily, 7 days/week
Duration of test:
From Gestation Day 0 (proof of pregnancy) to Gestation Day 20 (necropsy)
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
400 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
20 females/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels selected for investigation in this main embryo-foetal development study (0, 100, 400 and 1000 mg/kg bw/day) were selected in conjunction with the sponsor and were based on the results of a preliminary embryo-foetal development study and a 14-day repeat dose bridging study with the test substance.
In the preliminary embryo-foetal development study, daily oral gavage doses of 100, 400 and 833 mg/kg bw/day (the highest feasible dose level in the arachis oil vehicle), were administered from Day 6 to 19 of pregnancy. There were no premature deaths, no clinical signs and no effects on food intake. Slightly low body weight gain was observed at 833 mg/kg bw/day on Days 19 and 20 of gestation however, at scheduled termination there were no macroscopic abnormalities detected, no toxicologically significant findings in litter or foetal weights and no findings at the external examination of the foetuses.
The 14-day repeat dose bridging study was employed to determine if there was a change in the toxicity profile of the test substance when formulated using propylene glycol as opposed to arachis oil, which was used in previous toxicology studies. There were no premature deaths and no signs observed in relation to the administration of dose. For males that received 1000 mg/kg bw/day, there was a suggestion of slightly reduced body weight gain from Days 11 to 15 when compared to males that received 400 mg/kg bw/day. There was no effect of treatment on food intake and there were no macroscopic abnormalities detected at scheduled termination on Day 15.
Therefore, 1000 mg/kg bw/day was selected as the high dose level for the current study with 100 and 400 mg/kg bw/day selected as the low and intermediate dose levels, respectively, to assess any dose-response relationship.

- Rationale for animal assignment: Females showing at least two copulation plugs as positive proof of mating were allocated to group and cage position in the sequence of mating. Females mating on any one day were evenly distributed amongst the groups.
- Fasting period before blood sampling for (rat) dam thyroid hormones: no
- Time of day for (rat) dam blood sampling: To minimize any potential confounding effect of the time of day of blood sampling, the time of blood sampling was controlled to allow satisfactory inter-group comparisons.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily
- Cage side observations included: signs associated with dosing, clinical signs of ill-health and mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: A detailed physical examination was performed on each animal on Days 0, 5, 12, 18 and 20 after mating to monitor general health.

BODY WEIGHT: Yes
- Time schedule for examinations: on Days 0, 3 and 6-20 after mating

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
For the periods Days 0-3, 3-6, 6-10, 10-14, 14-18 and 18-20 after mating inclusive

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.
- Organs examined:
All abnormalities and the thyroid gland were fixed in 10% neutral buffered formalin. The thyroid gland was weighed, fixed and subjected to a gross and histopathological examination
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes, including cervix and ovaries
For each ovary/uterine horn:
- Number of corpora lutea: Yes
- Number of implantations sites: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Number of foetuses (live and dead).

For apparently non pregnant animals:
The number of uterine implantation sites were checked after staining with ammonium sulphide [modification of the Salewski staining technique].
Blood sampling:
- Plasma: No
- Serum: No

- Other: Thyroid Hormone Analysis
Blood samples were collected at termination from the sublingual vein under isoflurane anaesthesia. Parameters investigated were:
Triiodothyronine (T3), Thyroxine (T4) and Thyroid stimulating hormone (TSH).
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter] (fixed in Bouin's fluid)
- Skeletal examinations: Yes: [half per litter] (fixed in Industrial Methylated Spirit (IMS) and stained with Alizarin Red)
- Head examinations: No
- Anogenital distance of all live rodent pups: Yes

Detailed foetal examinations:
Findings observed were classified, according to severity and incidence, as:
Major abnormalities: normally rare, definitely detrimental to normal subsequent development, possibly lethal, e.g. ventricular septal defect.
Minor abnormalities: minor differences from normal that are detected relatively frequently considered to have little detrimental effect and may be a transient stage in development e.g. bipartite centrum, dilated ureter.
Variants: alternative structures or stages of development occurring regularly in the control population, e.g. number of ribs, incomplete ossification of 5th and 6th sternebrae.
Observations on repeated structures like ribs, vertebrae and sternebrae are reported as the first and last affected element, in the form ‘5th 13th bilateral ribs’, which should be interpreted as ‘5th to 13th bilateral ribs’.
Statistics:
Please refer to the document "Statistical Analyis_OECD 414" under "Attached background material".
Indices:
- Pre-implantation loss (%) = (Number of corpora lutea - Number of implantations / Number of corpora lutea) x 100;
- Post-implantation loss (%) = (Number of implantations - Number of live foetuses / Number of implantations) x 100

Historical control data:
Historical Control data supplied for selected observations where this information is considered to assist interpretation of study data. In this instance minor skeletal historical control data is included

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
On Day 16 and 17 of gestation, piloerection was recorded in one female that received 400 mg/kg bw/day however an association of this finding with treatment was considered unlikely in the absence of similar signs in other females at this or higher dose levels. There were no clinical signs recorded throughout the duration of study that were attributable to treatment with the test material.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
There were no premature decedents.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Minor variations in body weight gain were observed in some instances attaining statistical significance, however these marginal variations were considered incidental and of no biological or toxicological significance. Thus, there was no effect of treatment on group mean body weight gain from Day 6 to 20 of gestation.
For details please refer to Table no. 1 under “Any other information on results incl. tables”.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
There were minor variations observed in food consumption attaining in one instance statistical significance, however these marginal variations were considered incidental and of no biological or toxicological significance.
Thus, there was no effect of treatment on group mean food intake for females that received the test substance at doses up to and including 1000 mg/kg bw/day.
For details please refer to Table no. 2 under “Any other information on results incl. tables”.
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:
no effects observed
Description (incidence and severity):
There was no effect of treatment on thyroid gland weights at any dose level investigated.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
All macroscopic findings were considered spontaneous and/or incidental because they occurred at a low incidence, were randomly distributed across groups (including concurrent controls), and/or were as expected for Han Wistar rats of this age; therefore, they were considered not treatment-related.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
At terminal sacrifice, an increase in the incidence of thyroid follicular cell hypertrophy was seen in females administered 1000 mg/kg bw/day.
However, the finding was minimal and not affecting the functionality of the follicular cells and consequently considered non-adverse.
For details please refer to Table no. 3 under “Any other information on results incl. tables”.
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Thyroid Hormone Analysis:
Administration of the test material at 400 or 1000 mg/kg bw/day was associated with a dose dependent statistically significant increase in serum TSH concentrations when compared to controls. However, the extent of the increase was not considered adverse, and in the absence of a concomitant decrease in serum T3/T4 concentrations or any associated effects on maternal reproductive performance or the survival and development of the foetuses, these differences in serum TSH concentration were considered to be not toxicologically relevant.
There were no changes in serum T3 or T4 concentrations when compared to the controls in the 100, 400 or 1000 mg/kg bw/day groups.
For details please refer to Table no. 4 under “Any other information on results incl. tables”.

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
There was no effect of treatment on pre- and post-implantation loss.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Description (incidence and severity):
There was no effect of treatment on resorptions (early or late).
Dead fetuses:
no effects observed
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
Two control females (No. 19 and 81) were found to be not pregnant at scheduled termination, all other females assigned to study were confirmed as being pregnant. The following assessment of litter data is therefore based on a total of 18, 20, 20 and 20 females with live young at termination on Day 20 of gestation in the Control group and at 100, 400 and 1000 mg/kg bw/day respectively.
Other effects:
no effects observed
Description (incidence and severity):
Placental weight:
There was no effect of maternal treatment on mean placental weight at any dose level investigated.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Remarks:
maternal toxicity
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: no adverse effects observed

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
There was no effect of maternal treatment on mean foetal weights at any dose level investigated. It was noted that mean foetal weights at 1000 mg/kg bw/day were marginally higher than control, with differences in female foetal weights attaining statistical significance, however this marginal increase was considered incidental and of no biological or toxicological significance.
For details please refer to Table no. 5 under “Any other information on results incl. tables”.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
There was no effect of maternal treatment on the number of live young.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
There was no effect of maternal treatment on the sex ratio.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
There was no effect of maternal treatment on litter size and weights.
Anogenital distance of all rodent fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
The adjusted mean anogential distance of male foetuses at 1000 mg/kg bw/day was marginally shorter than control, with the difference attaining statistical significance, however in the absence of a similar difference in female foetuses, this marginal decrease was considered incidental and of no biological or toxicological significance.
Thus, there was no effect of maternal treatment on foetal anogenital distance at all dose levels investigated.
For details please refer to Table no. 6 under “Any other information on results incl. tables”.
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
Detailed external foetal examination did not reveal any abnormalities which were considered to be related to maternal treatment with the test substance.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/day there was an increased foetal incidence of 14th short supernumerary ribs and ossified cervical vertebral centra, both of which were within the Historical Control Data range.
As short supernumerary 14th ribs are variants and ossified cervical centra is a marker of foetal maturity, both findings were considered not to be adverse.
For details please refer to Table no. 7 and Table no. 9 under “Any other information on results incl. tables”.
Visceral malformations:
effects observed, treatment-related
Description (incidence and severity):
Detailed visceral foetal examinations revealed an increased incidence of effects on umbilical artery (left) in treatment groups compared with controls. As no clear dose-response-relatioship was observed with regard to this finding (incidence: 16, 20, 18 and 21 at 0, 100, 400 and 1000 mg/kg bw/day), this finding was considered to be of no biological or toxicological relevance.
For details please refer to Table no. 8 under “Any other information on results incl. tables”.
Other effects:
no effects observed
Details on embryotoxic / teratogenic effects:
no effects observed

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Remarks:
embryo-foetal survival and development
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

Table 1: Body weight (g) and body weight gain (g) group mean values ± SD during gestation

















































































































Sex



Dose group (mg/kg bw/day)



Days



Change



 



 



 



0



6



10



16



19



20



0-3



3-6



7-8



9-10



15-16



19-20



6-20



Female



0



199 ± 8.1



218 ± 8.9



231 ± 10.4



265 ± 11.4



294 ± 14.3



306 ± 15.9



10 ± 4.0



9 ± 3.7



4 ± 2.8



3 ± 3.3



9 ± 3.7



12 ± 3.7



88 ± 10.8



100



196 ± 7.5



214 ± 7.4



226 ± 7.7



255 ± 8.3



282 ± 9.3



294 ± 9.7



10 ± 3.1



9 ± 3.3



3 ± 2.8



5 ± 2.3*



8 ± 2.4



12 ± 3.2



80 ± 6.3



400



199 ± 7.2



217 ± 9.0



228 ± 9.8



258 ± 14.3



287 ± 18.0



299 ± 21.1



9 ± 2.8



9 ± 3.1



3 ± 2.0



5 ± 3.1*



8 ± 2.6



12 ± 4.6



83 ± 14.7



1000



200 ± 7.4



219 ± 10.0



230 ± 11.9



262 ± 14.8



291 ± 17.5



303 ± 19.8



10 ± 3.3



9 ± 3.6



3 ± 3.0*



4 ± 2.3*



8 ± 3.8



11 ± 4.5



83 ± 12.4


                

* and **: statistical significance at p < 0.05 and p < 0.01


 


Table 2: Food consumption (g/animal/day) group mean values ± SD during gestation


























































Sex



Dose group (mg/kg bw/day)



Change



 



 



0-3



3-6



6-10



10-14



14-18



18-20



Female



0



16 ± 2.1



18 ± 1.7



15 ± 1.7



18 ± 1.7



19 ± 1.5



19 ± 1.8



100



15 ± 1.7



17 ± 1.5



15 ± 1.5



17 ± 2.1



18 ± 1.5*



18 ± 1.9



400



15 ± 2.7



18 ± 2.0



15 ± 1.8



17 ± 2.2



19 ± 2.0



18 ± 2.7



1000



16 ± 2.1



18 ± 1.4



16 ± 2.2



18 ± 2.4



20 ± 1.9



19 ± 3.2



* and **: statistical significance at p < 0.05 and p < 0.01


 


Table 3: Microscopic findings in females on Day 20 of gestation














































Parameter



Females



Dose group (mg/kg bw/day)



0



100



400



1000



Thyroids



 



Number examined



20



20



20



20



Follicular cell hypertrophy



 



Minimal



1



1



2



8



Total



1



1



2



8



 


 


Table 4: Serum TSH, T3 and T4 concentration data, mean ± SD






































Parameter



Females



Dose group (mg/kg bw/day)



0



100



400



1000



TSH (pg/mL)



1150 ± 531



1130 ± 798



1700 ± 806*



2000 ± 867**



T3 (pg/mL)



449 ± 105



435 ± 104



416 ± 109



445 ± 86.1



T4 (pg/mL)



21800 ± 4680



21400 ± 3630



21300 ± 6450



20100 ± 3960



* and **: statistical significance at p < 0.05 and p < 0.01


Williams test (two-tailed) ANOVA model


 


Table 5: Litter and foetal weight (g) on Day 20 of gestation, group mean values ± SD











































Sex



Dose group (mg/kg bw/day)



Total litter weight



Male foetal weight



Female foetal weight



Overall foetal weight



Female



0



40.98 ± 6.833



3.54 ± 0.186



3.31.± 0.212



3.42 ± 0.199



100



35.43 ± 5.465



3.46 ± 0.338



3.26 ± 0.297



3.35 ± 0.287



400



38.33 ± 9.299



3.58 ± 0.163



3.36 ± 0.180



3.47 ± 0.142



1000



40.11 ± 7.979



3.62 ± 0.266



3.47 ± 0.232*



3.55 ± 0.239



* and **: statistical significance at p < 0.05 and p < 0.01


 


Table 6: Anogenital distance - Absolute and adjusted values (mm) on Day 20 of gestation




























































Sex



Dose group (mg/kg bw/day)



Absolute (mean ± SD)



Adjusted (mean)



Male



0



3.9 ± 0.55



3.9



 



100



3.7 ± 0.54



3.7



 



400



3.6 ± 0.55



3.6



 



1000



3.5 ± 0.53



3.5*



Female



0



2.6 ± 0.36



2.6



 



100



2.4 ± 0.33



2.5



 



400



2.4 ± 0.36



2.4



 



1000



2.4 ± 0.35



2.4



* and **: statistical significance at p < 0.05 and p < 0.01


 


Table 7: Foetal examinations – minor skeletal abnormalities and variant findings (group incidences)
































































































Parameter



Foetuses



Litters



Number examined



110



105



111



114



18



20



20



20



Dose group (mg/kg bw/day)



0



100



400



1000



0



100



400



1000



Minor skeletal abnormalities



 



Number of 14th ribs:



 



 



 



 



 



 



 



 



Short supernumerary



18



23



28



32



10



12



13



14



Full supernumerary



0



2



0



4



0



2



0



3



Total



18



23



28



33



10



12



13



14



Increased ossification



 



Cervical vertebral centra (all ossified)



1



2



7



13



1



1



5



4



 


Table 8: Foetal examinations – minor visceral abnormalities and necropsy findings (group incidences)
















































Parameter



Foetuses



Litters



Number examined



106



107



110



114



18



20



20



20



Dose group (mg/kg bw/day)



0



100



400



1000



0



100



400



1000



Visceral abnormalities



 



Umbilical artery (left)



16



20



18



21



11



14



12



14



 


Table 9: Fetal examinations – minor skeletal historical control data


 




















































































































































































































































































































































 



 



8437167



HCD Range



 



 



Fetuses



Litters



Fetuses



Litters



Group



 



1



2



3



4



1



2



3



4



 



 



Number Examined



 



110



105



111



114



18



20



20



20



1016



187



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



Minor skeletal abnormalities



 



 



 



 



 



 



 



 



 



 



Cranial



sutural bone(s)



0



1



1



0



0



1



1



0



0-1



0-1



Ribs



branched with costal cartilage



0



1



0



0



0



1



0



0



0-1



0-1



Sternebrae



hemisternebra



0



0



1



0



0



0



1



0



0-1



0-1



 



 



 



 



 



 



 



 



 



 



 



 



Rib and vertebral configuration



 



 



 



 



 



 



 



 



 



 



 



 



short supernumerary



18



23



28



32



10



12



13



14



15-36



5-17



Number of 14th ribs



full supernumerary



0



2



0



4



0



2



0



3



0-2



0-2



 



total



18



23



28



33



10



12



13



14



15-37



5-17



Thoracolumbar vertebrae



20



0



1



1



1



0



1



1



1



0-3



0-2



 



 



 



 



 



 



 



 



 



 



 



 



Delayed/Incomplete ossification/unossified



 



 



 



 



 



 



 



 



 



 



Vertebrae



cervical



0



0



0



2



0



0



0



2



0-1



0-1



Appendicular



metatarsals



0



1



0



0



0



1



0



0



0-1



0-1



 



 



 



 



 



 



 



 



 



 



 



 



Increased ossification



 



 



 



 



 



 



 



 



 



 



 



Cervical vertebral centra



all ossified



1



2



7



13



1



1



5



4



4-29



3-10



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 


 

Applicant's summary and conclusion

Conclusions:
Based on the results of this study, the No-Observed-Adverse-Effect-Level (NOAEL) for maternal toxicity and embryo-foetal survival and development was concluded to be >= 1000 mg/kg bw/day.