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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:
From 12 Jan 2022 to 02 Mar 2022
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2022

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
25 June 2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Reference substance name:
12-hydroxy-N-[2-(12-hydroxyoctadecanamido)ethyl]octadecanamide; N-(2-decanamidoethyl)-12-hydroxyoctadecanamide; N-(2-decanamidoethyl)decanamide
EC Number:
907-495-0
Cas Number:
198028-14-7
IUPAC Name:
12-hydroxy-N-[2-(12-hydroxyoctadecanamido)ethyl]octadecanamide; N-(2-decanamidoethyl)-12-hydroxyoctadecanamide; N-(2-decanamidoethyl)decanamide
Test material form:
solid
Details on test material:
Chemical name : Reaction mass of N,N'-ethane-1,2-diylbis(12-hydroxyoctadecan-1-amide) and Octadecanamide, 12-hydroxy-N-[2-[(1-oxodecyl)amino]ethyl]- and Decanamide, N,N'-1,2-ethanediylbis-
Chemical registery number : EC 907-495-0 / CAS : 198028-14-7

Test animals

Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 4 to 6 months.
- Weight at study initiation: between 3080 and 4335 g.
- Housing: The animals were individually housed in noryl cages. For psychological / environmental enrichment, animals were provided with dumbells, hay and as music.
- Diet: Pelleted complete diet (Diet Reference No. 3409, KLIBA) and compacted hay pellets (SSNIFF) were provided ad libitum. During the acclimation period, when reduced food consumption was noted (=50 g/animal/day), carrots were distributed. Carrots were also given to two animals showing little or no food consumption during the treatment period and already noted before treatment initiation.
- Water: municipal tap water provided in water bottles, ad libitum.
- Analyses confirmed the absence in feed and water of known contaminants that could have interfered with the outcome of the study.
- Acclimation period: at least 5 days.

ENVIRONMENTAL CONDITIONS (TARGET)
- Temperature: 15 to 21°C.
- Humidity: 30 to 70%.
- Air changes: Approximately 5 to 15 filtered, non-recycled air changes per hour.
- Photoperiod (hrs dark / hrs light): 8-hrs/16-hrs

IN-LIFE DATES: From 24 Jan 2022 to 02 Mar 2022.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 1% (w/v) Methyl Cellulose in drinking water treated by reverse osmosis.
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Test item dose formulations were prepared daily, on the day of each administration and used within 4 hours after the end of their preparation. Dosing formulation were prepared based on Test Facility Study No. 48783 VAS.

VEHICLE
- Justification for use and choice of vehicle: the test item showed to be soluble and stable in the selected vehicle, and non-toxic to animals.
- Concentration in vehicle: 83, 167 and 333 mg/mL for the low-, mid- and high-dose groups, respectively.
- Amount of vehicle (if gavage): 3 mL/kg bw/day.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
On GD6 and on GD 28 samples for concentration analysis were collected (from middle part) from all groups, while samples for homogeneity (from bottom, middle and top parts) analysis were collected from low- and high-dose groups only.
Analyses described were performed by HPLC/UV using a validated analytical procedure.
For concentration, mean sample concentration results shall be within or equal to 15% of theoretical concentration to meet acceptance criteria. For homogeneity, the relative standard deviation (RSD) of concentrations shall be of =10% for each group to comply with the acceptance criteria.
Details on mating procedure:
- Impregnation procedure: purchased time-mated.
- Other: Day 0 post-coitum is the day of successful mating. Untreated females were between GD0 and GD1 on arrival at the Test Facility.
Duration of treatment / exposure:
From GD6 to GD28.
Frequency of treatment:
Daily.
Duration of test:
24 days (From GD6, first day of administration, to GD29, day of scheduled necropsy).
Doses / concentrationsopen allclose all
Dose / conc.:
250 mg/kg bw/day
Remarks:
Low-dose group
Dose / conc.:
500 mg/kg bw/day
Remarks:
Mid-dose group
Dose / conc.:
1 000 mg/kg bw/day
Remarks:
High-dose group
No. of animals per sex per dose:
22 females per dose level
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The oral route of exposure was selected because this is one of the routes of administration which is requested by the Regulatory Authorities for this type of test item.
The dose levels were selected, based on the results of the following previous study: Dose-Range finding Study No. 48785 RSL where New Zealand White female rabbits (8 mated females per group) received 600, 800 or 1000 mg/kg bw/day, from GD6 to GD28, inclusive. No deaths, clinical signs or relevant effects on body weight or food consumption attributed to the test item were noted. No test item-related macroscopic post-mortem findings were observed in the dams. There were no effects on mean numbers of pre- and post-implantation losses or on the number of live fetuses per dam in any groups. The mean fetal body weight was unaffected. At external examinations, there were no variations or malformations considered to be related to the test item treatment. Based on the results of this study, 1000 mg/kg bw/day was selected as the high-dose level.
The low-dose and mid dose were selected using a ratio representing approximately a 2-fold interval (i.e. 250 and 500 mg/kg bw/day).

- Justification of test system and number of animals: At this time, studies in laboratory animals provide the best available basis for extrapolation to humans and are required to support regulatory submissions. Acceptable models that do not use live animals currently do not exist.
The rabbit was chosen as the animal model for this study as it is an accepted non-rodent species for preclinical toxicity testing by regulatory agencies.
The total number of animals used in this study was considered to be the minimum required to properly characterize the effects of the test item. This study was designed such that it does not require an unnecessary number of animals to accomplish its objectives.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Mortality: at least twice daily beginning upon arrival through termination (on days of receipt and necropsy at least once daily).
- Clinical Observations: at least once daily; beginning upon arrival through termination, before dosing when applicable. In addition to the daily cageside observation, animals were observed between 1 to 3 hours post-dose.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: A full clinical examination was performed once during acclimation, on each weighing day during dosing phase and on the day of euthanasia. Net body weight (carcass weight) was also determined subtracting the gravid uterine weight to the body weight recorded on GD29. Finally, the net body weight change was determined subtracting the gravid uterine weight and the body weight on GD6 to the body weight recorded on GD29.

BODY WEIGHT: Yes
- Time schedule for examinations: Each female was weighed on GD2, GD5, GD6, GD9, GD12, GD15, GD19, GD21, GD24, GD27 and GD29.

FOOD CONSUMPTION: Yes
- Food consumption for each animal was recorded daily between GD2 and GD 29.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on GD29 by an intravenous injection of sodium pentobarbital.
- Organs examined: all animals (including animals euthanised prematurely) were subjected to a macroscopic examination of the abdominal and thoracic cavities to determine pregnancy status, number of corpora lutea and numbers and types of uterine implantations. All macroscopic abnormalities observed were recorded and preserved in 10% formalin (or in another appropriate fixative).
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes

Examinations included:
- Pregnancy status: Yes
- Gravid uterus weight: Yes, for each pregnant female (with at least one live fetus) at hysterectomy. The uterus of apparently non-gravid females was placed in ammonium sulphide solution in order to stain any previously undetected implantation sites
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
Each fetus was examined for external defects and all live fetuses were euthanized by intraperitoneal injection of sodium pentobarbital.
Dead fetuses were examined externally and preserved in Harrison's fixative but not examined further.
The following examinations were performed in all live fetuses on GD29:
- External examinations: Yes, all per litter
- Soft tissue examinations: Yes, all per litter.
- Skeletal examinations: Yes, all per litter
- Head examinations: Yes, all per litter (The head of one half of the fetuses was fixed in Harrison's fluid for subsequent examination by serial sectioning. In the other half of the fetuses, the brain of each fetus was sampled and fixed in Harrison's fluid)
Statistics:
Body weight, food consumption and reproductive data were compared by one-way analysis of variances and Dunnett test (mean values being considered as normally distributed, variances being considered as homogenous) or by Fisher's exact probability test (proportions).
Indices:
CONSTRUCTED VARIABLES:
- Total number of resorptions: Sum of uterine scars + early resorptions + late resorptions
- % of dead fetuses per litter: (Total No. of dead fetuses in litter / No. of implantation sites) x 100
- % of live fetuses per litter: (Total No. of live fetuses in litter / No. of implantation sites) x 100
- Pre-Implantation Loss (%): [(No. of corpora lutea – No. of implantations) / No. of corpora lutea] x 100
- Post-Implantation Loss (%): [(No. of implantations – No. of live fetuses) / No. of implantations] x 100
Historical control data:
HCD was collected from 2019-2020 from a total of 5 studies and reported in the results section. Please also refer to relevant tables under section "Any other information on results incl. tables".

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
All clinical signs recorded in surviving females (all pregnant, except for two high dose females) were considered to be unrelated to the test item treatment as they were recorded both in control and test item-treated animals (abnormal feces and/or absent/decreased/soft) and/or are routinely observed in pregnant females (red discharge from the vagina or in the bedding and skin lesions/scabs).
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One control female was euthanized on GD29 due to premature delivery. Two dead fetuses, one live fetus and three placentas with red discharge were found in the bedding. Abnormal gait was noted from GD26, associated with a body weight loss between GD24 and 29 (-8%) and reduced food consumption from GD26 (on average 42 g/day). Four scars were observed in the uterine horns.
A mid-dose female was euthanized on GD28 due to premature delivery. One fetus and two placentas were found in the bedding. A body weight loss was recorded between GD24 and 27 (-5%). Eleven scars were observed in the uterine horns.
A high-dose female was prematurely euthanized on GD24 due to abortion. Four dead fetuses and two placentas were found in the bedding. A body weight loss was recorded between GD21 and 24 (-8%), associated with little food consumption on GD23 (5 g/day vs 147 g in controls). Five scars were observed in the uterine horns.
No macroscopic post-mortem findings were recorded in any of these females.
These isolated cases of abortion/premature delivery, noted irrespective of the dose level, were considered to be incidental.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related effects were noted on mean body weight or mean body weight gain at any dose level.
The slightly lower mean body weight gain recorded at 250 and 500 mg/kg bw/day between GD19 and 21 (+6 and +5 g, respectively, vs +16 g in controls) were considered to be incidental as this was mainly due to the contribution of two isolated females (one at each dose level) and recorded with no dose-level relationship.
No effects on mean carcass weights or mean net body weight changes were observed at any dose level.
Please also refer to Tables 2 and 4 under section "Any other information on results incl. tables".
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Please also refer to Table 3 under section "Any other information on results incl. tables".
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No effects on mean gravid uterus weights were observed at any dose level.
Please also refer to Table 4 under section "Any other information on results incl. tables".
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
None of the macroscopic findings observed were considered to be related to the test item administration as they were noted with no dose-relationship and are findings routinely observed in female rabbits of this strain and age.
Please also refer to Table 5 under section "Any other information on results incl. tables".

Maternal developmental toxicity

Number of abortions:
effects observed, non-treatment-related
Description (incidence and severity):
Three females of the control, mid-dose and high-dose groups were euthanized due to prematurely deliveries on GD29, GD28 and GD24, respectively. For more details please refer also to section "mortality".
These isolated cases of abortion/premature delivery, noted irrespective of the dose level, were considered to be incidental.
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
The higher mean number of post-implantation loss in the low-dose group (11.3 vs 9.1 in controls), was considered to be unrelated to the test item treatment as the differences were not dose-related and mainly due to the contribution of two low-dose females with a high number of early resorptions.
Please also refer to Table 6 under section "Any other information on results incl. tables".
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
No total litter loss was observed in any of the experimental groups.
Early or late resorptions:
effects observed, non-treatment-related
Description (incidence and severity):
The higher mean number of resorptions recorded at 250 mg/kg/day (1.1 vs 0.5 in controls), particularly early, leading to a higher mean post-implantation loss percentage (11.3% vs 9.1% in controls), was considered to be unrelated to the test item treatment as the differences were not dose-related and mainly due to the contribution of two females.
Please also refer to Table 6 under section "Any other information on results incl. tables".
Dead fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
Incidences of dead fetuses were 3.5%, 0.0%, 0.0% and 0.0% for the control, low-, mid- and high-dose groups, respectively, achieving statistical significance in all treated groups. Considering that dead fetuses were observed only in the control group, the finding is considered of no toxicological relevance and not attributable to the test-item administration.
Please also refer to Table 6 under section "Any other information on results incl. tables".
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
At hysterectomy on GD29, 19/22, 21/22, 20/22 and 19/22 females were pregnant with live fetuses in the groups treated at 0, 250, 500 and 1000 mg/kg bw/day, respectively.
Please also refer to Table 1 under section "Any other information on results incl. tables".

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Remarks:
maternal toxicity
Effect level:
>= 1 000 mg/kg bw/day
Based on:
test mat.
Remarks on result:
other: Absence of adverse effects up to and including the highest dose level tested.

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
Mean fetal weight was unaffected by the test item treatment at any dose level.
Please also refer to Table 7 under section "Any other information on results incl. tables".
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Sex ratio was unaffected by the test item treatment at any dose level.
Please also refer to Table 7 under section "Any other information on results incl. tables".
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related external malformations were observed at any dose level.
Iniencephaly was the only malformation observed in one control fetus.
Please also refer to Table 8 under section "Any other information on results incl. tables".

No test item-related external variations were observed at any dose level.
The few findings recorded were considered to be incidental and not related to the test item treatment as they were noted both in control and test item-treated animals with the same incidence.
Please also refer to Table 11 under section "Any other information on results incl. tables".
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related skeletal malformations were observed at any dose level.
The higher incidence of fused sternebrae at 1000 mg/kg bw/day (three fetuses from two litters vs one fetus in controls) was not attributed to the test item treatment as the difference was noted with no statistical significance and the incidence remained near the limits of our HCD range.
The other findings recorded were considered to be incidental as they were noted both in control and test item-treated animals and/or were noted in isolated fetuses and/or remained within the range of our HCD.
Please also refer to Table 9 under section "Any other information on results incl. tables".

No test item-related effects on cartilages were observed at any dose level.
The higher incidence of non-ossified caudal vertebrae and metacarpal bone (only the respective cartilages were present) at 500 mg/kg bw/day, when compared to controls, was not attributed to the test item treatment as the incidence was not dose-related and/or was near the limits of our HCD range.
The other findings recorded were considered to be incidental as they were noted both in control and test item-treated animals and/or were noted in isolated fetuses and/or remained within the range of our HCD.
Please also refer to Table 13 under section "Any other information on results incl. tables".

No test item-related skeletal variations were observed at any dose level.
The higher incidence of unossification/incomplete ossification of the caudal vertebra centrum and 1st metacarpal bone at 500 mg/kg bw/day, when compared to controls, was not attributed to the test item treatment as the incidence was not dose-related and/or was within or near the limits of our HCD range.
The other findings recorded were considered to be incidental as they were noted both in control and test item-treated animals and/or were noted in isolated fetuses and/or remained within the range of our HCD.
Please also refer to Table 14 under section "Any other information on results incl. tables".
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related visceral malformations were observed at any dose level.
At 1000 mg/kg bw/day, one litter had one fetus with malpositioned right kidney associated with a short right ureter (and dilated renal pelvis, see variations). As these malformations were isolated and noted in a single fetus, a relationship to the test item treatment cannot be ascertained.
The other findings were considered to be incidental and not related to the test item treatment as they were noted in isolated fetuses with no dose-relationship.
Please also refer to Table 10 under section "Any other information on results incl. tables".

No test item-related visceral variations were observed at any dose level.
The higher incidence of hemorrhagic ovary at 1000 mg/kg bw/day (six fetuses from four litters vs one fetus in controls) was not attributed to the test item treatment as this variation was isolated and the incidence remained within the range of our HCD.
The other findings were considered to be incidental and not related to the test item treatment as they were noted both in control and test item-treated animals and/or with no dose-relationship and/or are common findings in fetuses of this strain.
Please also refer to Table 12 under section "Any other information on results incl. tables".

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Effect level:
>= 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: Absence of adverse effects up to and including the highest dose level tested.

Fetal abnormalities

Key result
Abnormalities:
effects observed, non-treatment-related

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

Chemical Analyses of the Dose Formulations


The dose formulations prepared on GD6 and GD28, were all found to be within the acceptance criteria (measured concentrations at ± 15% of the theoretical concentrations). No test item was detected in the control dose formulation.


 


Table 1. Pregnancy Status
















































Dose level (mg/kg bw/day)



0



250



500



1000



Number of females



22



22



22



22



Pregnant females



20



21



21



20



Females with live fetuses at termination



19



21



20



19



Female prematurely euthanized (pregnant)



1



0



1



1



Non-pregnant females



2



1



1



2



 


Table 2. Mean body weights/mean body weight changes (g)































































































































































Dose level (mg/kg bw/day)



0



250



500



1000



Body weight (g)



GD 6



3639



3686



3685



3642



GD 9



3694



3740



3749



3698



GD 12



3762



3800



3793



3741



GD 15



3842



3913



3873



3838



GD 19



3883



3940



3905



3874



GD 21



3899



3946



3910



3915



GD 24



3956



3985



3956



3938



GD 27



4007



4045



4015



3982



GD 29



4057



4081



4060



4018



percentage difference vs. controls (%)



 



+1



0



-1



Body weight change (g)



GD 6-9



+55



+55



+64



+56



GD 9-12



+68



+59



+44



+43



GD 12-15



+80



+113



+80



+98



GD 15-19



+41



+27



+32



+36



GD 19-21



+16



+6



+5



+42



GD 21-24



+57



+39



+46



+23



GD 24-27



+50



+60



+59



+52



GD 27-29



+50



+36



+45



+36



GD 6-29



+417



+395



+386



+407



percentage difference vs. controls (%)



 



-5



-7



-2



No statistically significant differences versus controls.


 


Table 3. Mean food consumption (g/animal/day)














































































































































































Dose level (mg/kg bw/day)



0



250



500



1000



GD 6-7



171



183



173



180



GD 7-8



181



185



187



186



GD 8-9



175



179



176



180



GD 9-10



182



191



180



163



GD 10-11



179



186



173



154



GD 11-12



175



178



168



156



GD 12-13



166



169



154



144



GD 13-14



155



166



141



138



GD 14-15



145



167



148



143



GD 15-16



149



185



160



161



GD 16-17



159



174



171



170



GD 17-18



159



166



172



188



GD 18-19



171



178



169



185



GD 19-20



161



157



150



170



GD 20-21



165



157



152



178



GD 21-22



164



155



154



166



GD 22-23



155



150



150



170



GD 23-24



147



137



137



137



GD 24-25



151



127



140



134



GD 25-26



147



125



135



136



GD 26-27



132



136



139



136



GD 27-28



138



126



135



144



GD 28-29



145



135



152



146



No statistically significant differences versus controls.


 


Table 4. Gravid Uterus and Carcass Weights (g)









































Dose level (mg/kg bw/day)



0



250



500



1000



Gravid uterus weight



576.3



541.6



548.8



542.8



percentage difference vs. controls (%)



 



-6



-5



-6



Carcass weight



3517.7



3539.3



3510.8



3475.3



Net weight change from GD6.



-128.4



-146.4



-163.2



-135.4



No statistically significant differences versus controls.


 


Table 5. Macroscopic Post-Mortem Examination























































Dose level (mg/kg bw/day)



0



250



500



1000



Thymus


Reddish color



 



1



 



 



Spleen


Irregular surface



1



 



1



1



Heart


Pericardium, translucent content



1



 



 



 



Liver


Granular consistency



 



2



 



 



Stomach


Depressed area



2



2



 



2



Female gonads


Serous cyst(s)


(connective tissue, periovarian region)



 



2



 



 



 


Table 6. Hysterectomy data





























































































Dose level (mg/kg bw/day)



0



250



500



1000



HCD
min-max



Number of females with live fetuses at termination



19



21



20



19



100



Mean number of corpora lutea per animal



11.2



11.2



12.2



11.5



11.7-13.0



Mean number of implantation sites per animal



9.8



9.6



10.1



9.8



10.1-10.6



Mean percentage of pre-implantation loss (%)



12.1



14.1



16.9



13.9



9.4-21.7



Mean number of live fetuses per animal



8.8



8.5



9.6



9.1



8.9-9.9



Dead fetuses (%)



3.5



0.0**



0.0**



0.0**



0.00-0.39



Mean number of resorptions per female



0.5



1.1*



0.5



0.7



/



Mean number of early resorptions



0.2



0.6*



0.3



0.4



0.55-1.09



Mean number of late resorptions



0.4



0.5



0.3



0.3



Mean percentage of post-implantation loss (%)



9.1



11.3



4.8



7.8



7.3-13.2



Statistically significant vs. controls: *: p<0.05. **: p<0.01.


HCD: Historical Control Data (2019-2020), n= 5 studies; /: no data.


 


Table 7. Mean fetal body weight (g) and sex ratio






























Dose level (mg/kg bw/day)



0



250



500



1000



HCD
min-max



Mean fetal body weight (g)



42.5



42.5



39.2



40.9



37.8-42.3



Mean percentage of male fetuses (%)



52.5



55.5



52.0



52.9



42.3-55.3



No statistically significant differences versus controls.


HCD: Historical Control Data (2019-2020), n= 5 studies.


 


Table 8. Mean fetal (F) and litter (L) incidences of external malformations (%)























































Dose level (mg/kg bw/day)



0



250



500



1000



Number of litters



19



21



20



19



Number of fetuses



176



178



193



173



Trunk



 



 



 



 



. Iniencephaly, F (L)



0.6 (5.3)



0.0 (0.0)



0.0 (0.0)



0.0 (0.0)



Litters affected, n (%)



1 (0.6)



0 (0.0)



0 (0.0)



0 (0.0)



Fetus affected, n (%)



1 (5.3)



0 (0.0)



0 (0.0)



0 (0.0)



n: number. No statistically significant differences vs. controls.


 


Table 9. Mean fetal (F) and Litter (L) incidences of skeletal malformations






























































Dose level (mg/kg bw/day)



0



250



500



1000



HCD



Number of litters



19



21



20



19



100



Number of fetuses



168



178



193



173



949



Sternebrae



 



 



 



 



 



. fused sternebrae , F (L)



0.0 (0.0)



0.6 (4.8)



0.0 (0.0)



1.7 (15.8)



0.5-1.4
(4.5-14.3)



Litters affected, n (%)



4 (21.1)



3 (14.3)



5 (25.0)



3 (15.8)



 



Fetus affected, n (%)



4 (2.4)



3 (1.7)



5 (2.6)



3 (1.7)



 



n: number.


No statistically significant differences vs. controls.


HCD: Historical Control Data (2019-2020), n= 5 studies


 


Table 10. Mean fetal (F) and litter (L) incidences of soft tissue malformations (%)






































































































Dose level (mg/kg bw/day)



0



250



500



1000



HCD



Number of litters



19



21



20



19



100



Number of fetuses



168



178



193



173



949



Kidneys



 



 



 



 



 



. marked dilated renal pelvis, F (L)



0.0 (0.0)



0.6 (4.8)



0.0 (0.0)



0.0 (0.0)



0.0-2.9
(0.0-22.2)



. malpositioned kidney, F (L)



0.0 (0.0)



0.0 (0.0)



0.0 (0.0)



0.6 (5.3)



/



Vessels



 



 



 



 



 



. dilated aortic arch, F (L)



0.0 (0.0)



0.0 (0.0)



0.5 (5.0)



0.0 (0.0)



0.0-0.6
(0.0-5.6)



Ureter



 



 



 



 



 



. short ureter, F (L)



0.0 (0.0)



0.0 (0.0)



0.0 (0.0)



0.6 (5.3)



/



Litters affected, n (%)



0 (0.0)



1 (4.8)



1 (5.0)



1 (5.3)



 



Fetus affected, n (%)



0 (0.0)



1 (0.6)



1 (0.5)



1 (0.6)



 



n: number. No statistically significant differences vs. controls.


HCD: Historical Control Data (2019-2020), n= 5 studies; /: no data.


 


Table 11. Mean fetal (F) and litter (L) incidences of external variations (%)






























































































Dose level (mg/kg bw/day)



0



250



500



1000



HCD


min-max



Number of litters



19



21



20



19



100



Number of fetuses



176



178



193



173



949



General



 



 



 



 



 



. abnormal color of amniotic fluid, F (L)



1.1 (10.5)



0.0 (0.0)



0.0 (0.0)



1.7 (10.5)



0.0-1.6
(0.0-5.0)



Paw and digit



 



 



 



 



 



. paw hyperflexion, F (L)



0.6 (5.3)



0.0 (0.0)



0.5 (5.0)



0.6 (5.3)



/



Trunk



 



 



 



 



 



. abdomen, blackish color, F (L)



0.6 (5.3)



0.0 (0.0)



0.0 (0.0)



0.0 (0.0)



0.0-0.5
(0.0-5.0)



Litters affected, n (%)



4 (21.1)



0 (0.0)



1 (5.0)



2 (10.5)



 



Fetus affected, n (%)



4 (2.3)



0 (0.0)



1 (0.5)



4 (2.3)



 



n: number. No statistically significant differences vs. controls.


HCD: Historical Control Data (2019-2020), n= 5 studies; /: no data.


 


Table 12. Mean Fetal (F) and Litter (L) incidences of soft tissues variations (%)














































































































































































Dose level (mg/kg bw/day)



0



250



500



1000



HCD



Number of litters



19



21



20



19



100



Number of fetuses



168



178



193



173



949



Spleen



 



 



 



 



 



. misshapen, F (L)



0.6 (5.3)



0.0 (0.0)



0.0 (0.0)



0.0 (0.0)



/



Gall bladder



 



 



 



 



 



. small, F (L)



0.0 (0.0)



0.0 (0.0)



0.5 (5.0)



0.0 (0.0)



0.0-3.9
(0.0-18.2)



Kidneys



 



 



 



 



 



. dilated renal pelvis, F (L)



4.2 (31.6)



2.8 (19.0)



1.0 (10.0)



0.6 (5.3)



0.0-2.9
(0.0-22.2)



. enlarged kidney, F (L)



1.2 (10.5)



0.0 (0.0)



0.0 (0.0)



0.0 (0.0)



/



Gonads



 



 



 



 



 



. hemorrhagic ovary, F (L)



0.6 (5.3)



0.0 (0.0)



0.5 (5.0)



3.5 (21.1)



0.0-3.5
(0.0-16.7)



Vessels



 



 



 



 



 



. absent brachiocephalic trunk, F (L)



36.3 (89.5)



50.0* (100.0)



34.7 (80.0)



41.6 (94.7)



29.3-36.0
(68.2-94.4)



. short innominate artery, F (L)



1.8 (15.8)



0.6 (4.8)



1.6 (15.0)



1.7 (10.5)



0.0-3.8
(0.0-23.8)



Trunk



 



 



 



 



 



. thin abdominal wall, F (L)



0.0 (0.0)



0.0 (0.0)



0.0 (0.0)



0.6 (5.3)



0.0-0.5
(0.0-5.0)



Thymus



 



 



 



 



 



. enlarged, F (L)



0.6 (5.3)



0.0 (0.0)



0.0 (0.0)



0.0 (0.0)



/



Litters affected, n (%)



18 (94.7)



21 (100)



17 (85.0)



18 (94.7)



 



Fetus affected, n (%)



70 (41.7)



94* (52.8)



71 (36.8)



77 (44.5)



 



n: number. Statistically significant vs. controls: *: p<0.05.


HCD: Historical Control Data (2019-2020), n= 5 studies; /: no data.


 


Table 13. Mean fetal (F) and Litter (L) incidences of cartilages findings














































































Dose level (mg/kg bw/day)



0



250



500



1000



HCD



Number of litters



19



21



20



19



100



Number of fetuses



168



178



193



173



949



Caudal vertebrae



 



 



 



 



 



. cartilage of caudal vertebra(e): present, F (L)



1.8 (15.8)



5.6 (23.8)



7.8* (40.0)



3.5 (26.3)



0.0-3.4
(0.0-27.3)



Metacarpal bone



 



 



 



 



 



. cartilage of metacarpal bone(s): present, F (L)



10.7 (52.6)



11.8 (42.9)



21.8** (55.0)



10.4 (42.1)



0.0-21.2
(0.0-52.4)



Litters affected, n (%)



19 (100)



21 (100)



20 (100)



19 (100)



 



Fetus affected, n (%)



147 (87.5)



159 (89.3)



168 (87.0)



150 (86.7)



 



n: number.


Statistically significant vs. controls: *: p<0.05, **: p<0.01.


HCD: Historical Control Data (2019-2020), n= 5 studies.


 


Table 14. Mean Fetal (F) and Litter (L) incidences of skeletal variations (%)






























































































Dose level (mg/kgbw/day)



0



250



500



1000



HCD



Number of litters



19



21



20



19



100



Number of fetuses



168



178



193



173



949



Caudal vertebrae



 



 



 



 



 



. incomplete ossification of centrum, F (L)



1.2 (10.5)    



5.1 (23.8)                               



7.8** (35.0)                               



3.5 (26.3)             



1.0-4.7
(4.8-30.0)



. unossified centrum, F (L)



0.6 (5.3)



0.6 (4.8)



5.2* (30.0)



1.2 (10.5)



0.0-3.4
(0.0-27.3)



Metacarpal bone



 



 



 



 



 



. unossified 1st metacarpal, F (L)



4.2 (21.1)



1.7 (14.3)



9.8* (30.0)



1.7 (15.8)



2.9-8.4
(18.2-38.1)



. incomplete ossification of 1st metacarpal, F (L)



6.5 (42.1)



10.1 (42.9)



14.5* (55.0)



8.7 (36.8)



8.3-20.7
(27.3-42.9)



Litters affected, n (%)



19 (100)



21 (100)



20 (100)



19 (100)



 



Fetus affected, n (%)



162 (96.4)



168 (94.4)



189 (97.9)



161 (93.1)



 



n: number.


Statistically significant vs. controls: *: p<0.05, **: p<0.01.


HCD: Historical Control Data (2019-2020), n= 5 studies.

Applicant's summary and conclusion

Conclusions:
Based on the results obtained in this study, the No Observed Adverse Effect Level (NOAEL) for maternal parameters and for embryo-fetal development was considered to be 1000 mg/kg bw/day.
Executive summary:

The objective of this study was to provide general information concerning the effects of prenatal exposure on the pregnant test animal and on the developing organism of 99422018 when administered by the oral (gavage) route from implantation to the day prior to the scheduled hysterectomy [from Gestation Day (GD) 6 to GD28].


Three groups of 22 time-mated female New-Zealand White rabbits received the test item, once daily by the oral route (gavage) at dose levels of 250, 500 or 1000 mg/kg/day from GD6 to GD28 inclusive. A control group of 22 time-mated females received the vehicle only (1% methylcellulose in drinking water) under the same experimental conditions. A constant dosage volume of 3 mL/kg/day was used.
The actual test item concentrations were determined in the dose formulations on two occasions, using a validated HPLC/UV analytical method.
The animals were checked daily for mortality and clinical signs. Body weight was recorded at designated intervals. Food consumption was recorded daily.
On GD29, the females were euthanized and a macroscopic post mortem examination was performed. Hysterectomies were performed and the numbers of corpora lutea, implantation sites, early and late resorptions, and live and dead fetuses were recorded. Placentas were observed. The fetuses were weighed and sexed by internal examination of the gonads. They were subjected to detailed external, soft tissue and skeletal (bones + cartilages) examinations.


Actual concentrations of the test item in the dose formulations analyzed during the study were within the accepted range of ± 15% of the theoretical concentrations.
At hysterectomy on GD29, 19/22, 21/22, 20/22 and 19/22 females were pregnant with live fetuses in the groups treated at 0, 250, 500 and 1000 mg/kg/day, respectively.
No test item-related deaths or clinical signs were recorded.
Body weight, body weight change and food consumption were unaffected by the test item treatment.
At necropsy of the dams, no test item-related macroscopic findings were observed.
Gravid uterus weight, carcass weight and net body weight change were not impacted by the test item treatment. No test item-related effects on the numbers of corpora lutea, implantations or resorptions were noted at any dose level.
The fetal body weight and sex ratio were unaffected at all dose levels.
At external, soft tissue or skeletal examinations of the fetuses, no variations or malformations attributable to the test item treatment were noted.


The test item, 99422018, was administered daily to time-mated female NZW rabbits by the oral route (gavage) at the dose level of 250, 500 or 1000 mg/kg/day from GD6 to GD28, inclusive.
Based on the results obtained in this study, the No Observed Effect Level (NOEL) for maternal parameters and embryo-fetal development was considered to be 1000 mg/kg/day.