Registration Dossier

Diss Factsheets

Toxicological information

Developmental toxicity / teratogenicity

Currently viewing:

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 11 Aug 2021 to 06 Oct 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Some females were acclimated for only 4 days. The OECD TG 414 requires a minimum of 5 days of acclimatization. In addition, in each group of the study, including the control group, the four first litters had a low mean fetal weight per litter when compared to all other study litters or to what can be observed usually on GD 21 in Sprague-Dawley rats. It was suspected that this was due to the fact that these females (which were all from a same batch of animals at receipt) arrived on site on GD 1 instead of GD 2 and were therefore euthanized on GD 20 instead of GD 21. In the absence of confirmation of the mistake from the animal supplier, it was decided to not exclude these litters from the group mean litter data. This event was not considered to have impacted the overall integrity of the study or the interpretation of the study results and conclusions because all groups, including controls, were concerned by the same event and had the same low number of affected females (only 4/24 females per group) and because no toxicologically significant effects were identified in the 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:
yes
Remarks:
some females were acclimated for 4 days instead of minimum 5 days as outlined in the OECD TG 414.
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
[4-(4-phenoxybenzoyl)phenyl](4-phenoxyphenyl)methanone
EC Number:
620-097-9
Cas Number:
54299-17-1
Molecular formula:
C32H22O4
IUPAC Name:
[4-(4-phenoxybenzoyl)phenyl](4-phenoxyphenyl)methanone
Test material form:
solid: particulate/powder

Test animals

Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl:CD(SD)
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 10-11 weeks
- Weight at study initiation: 222-349 g
- Housing: single housed in polycarbonate cages with stainless steel lids containing autoclaved sawdust. Individual housing was chosen as group housing can adversely affect gestation. For psychological / environmental enrichment, animals were provided with nylabone and rat hut.
- Diet: ad libitum, SSNIFF rat/mouse pelleted maintenance diet, batch Nos. 62874737 and 36580116 (SSNIFF Spezialdiäten GmbH, Soest, Germany), sterilized by irradiation. Analyses confirmed that there were no known contaminants in the feed that interfered with the objectives of the study.
- Water: ad libitum, municipal tap water filtered with a 0.22 µm filter and dispensed in water bottles. Analyses confirmed that there were no known contaminants in the water that interfered, with the outcome of the study.
- Acclimation period: 4 to 5 days

ENVIRONMENTAL CONDITIONS (TARGET)
- Temperature: 20-24°C
- Humidity: 30-70%
- Air changes: ca. 8 to 15 filtered, non-recycled air changes per hour
- Photoperiod: 12 h dark/12 h light

IN-LIFE DATES: From: 06 September To: 06 October 2021

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Dosing formulations were prepared based on Test Facility Study No. 40920 AHS (Andre, 2015) describing the preparation procedure for a range of concentrations covering the lowest and the highest used in this study, at appropriate concentrations to meet dose level requirements. Test item formulations in vehicle were stored at room temperature.

VEHICLE
- Justification for use and choice of vehicle: suitable for solubility and stability of test item.
- Concentration in vehicle: 0, 20, 60 and 200 mg/mL for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively
- Amount of vehicle: 5 mL/kg bw/day (based on most recent body weight measurement)
- Lot/batch no.: MKCN9742
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For concentration analyses, 3 samples per group were taken from the middle part during the first and the last week of study treatment. Acceptance criteria for concentration are individual sample concentration of ± 15% from the nominal.

Homogeneity analyses performed previously in conjunction with study No.40920 AHS (Andre, 2015) demonstrated that the test item is soluble in the vehicle when prepared under the same mixing conditions at concentrations bracketing those used in the present study. For homogeneity, the acceptance criteria were Relative Standard Deviations (RSD) of concentrations of = 10% for each group.

Stability analyses performed previously in conjunction with study No. 40920 AHS (Andre, 2015) demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study. The stability was demonstrated to be 10 days at a concentration between 2 and 200 mg/mL.

Analyses were performed by HPLC/UV using a validated analytical procedure (40919 VAA.MET).
Details on mating procedure:
- Impregnation procedure: purchased timed pregnant
Duration of treatment / exposure:
from Gestation Day (GD) 6 to GD 20
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day
Remarks:
low-dose group
Dose / conc.:
300 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:
24 females per group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The oral route of exposure was selected because this is the intended route of human exposure. The dose levels were selected based on results from a previous dose-range finding study performed in pregnant rats (Study No. 49120 RSR (Spézia, Draft)). In this previous study, the test item was administered daily by oral gavage to pregnant female Sprague-Dawley rats from GD 6 to 20. There were no effects on the female rat, pregnancy parameters and external fetal development. Therefore, the maximal tolerated dose level in the pregnant female rat was considered to be higher than 1000 mg/kg bw/day.
- Rationale for animal assignment: computerized randomization procedure based on body weight recorded on GD 2
- Other: 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 Sprague Dawley rat was chosen as the animal model for this study as it is an accepted 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
- Time schedule: At least once daily (beginning upon arrival through termination), before dosing when applicable. Animals were also observed 1 and 3 hours post-dose. Mortality was checked at least twice daily (at the beginning and end of working day) beginning upon arrival through termination.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: each weighing day during dosing phase and on the day of euthanasia.

BODY WEIGHT: Yes
- Time schedule for examinations: Each female was weighed on GD 2, GD 4, GD 6, GD 9, GD 12, GD 15, GD 18 and GD 21. Body weight change for different intervals was determined. Net body weight (carcass weight) was also reported as body weight recorded on GD 21 minus the gravid uterine weight. Finally, the net body weight change was calculated as the body weight on GD 21 minus the body weight on GD 6 and the gravid uterine weight.

FOOD CONSUMPTION: Yes
- Food consumption of each animal was recorded on periods: GD 2-4, GD 4-6, GD 6-9, GD 9-12, GD 12-15, GD 15-18 and GD 18-21.

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21 by carbon dioxide inhalation and cervical dislocation. Some females could have been euthanized on GD 20 due to a mistake from the animal supplier in providing the correct GD to the test facility.
- Organs examined: Animals were submitted to a macroscopic examination of the abdominal and thoracic cavities and any abnormalities. As remarkable macroscopic lesions were observed on placentae in test item-treated group animals, the placentae of five control animals were sampled and preserved. For each dam, thyroids with parathyroids were weighed wet as soon as possible after fixation. The ratio of organ weight to body weight (presented as carcass weight) was calculated.
Macroscopic lesions and thyroids with parathyroids were collected and preserved in 10% neutral buffered formalin. A microscopic examination was performed on thyroids with parathyroids from all animals.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes, for each female.
Examinations included:
- Gravid uterus weight: Yes. 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
- Other: pregnancy status, live and dead fetuses, uterine scars, individual fetal weight of live fetuses, sex of fetuses at the time of hysterectomy by visual assessment of anogenital distance (confirmed by internal examination of sexual organs at fresh visceral examination or at evisceration).
Blood sampling:
- Plasma: Yes for thyroid hormones analysis
- Serum: No
- Volume collected: 0.5 mL of blood processed to plasma.
- Other: collected from all animals on GD 21 between 7:30 and 9:30 AM under isoflurane anesthesia from the orbital sinus in sampling tubes with K3EDTA (anticoagulant). Animals were not fasted. The levels of the thyroid hormones (T3 and T4) were determined by LC-MS/MS method and those of Thyroid Stimulating Hormone (TSH) by Luminex MAP® technology.
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter (abdominal and thoracic cavities and indication of incomplete testicular descent/cryptorchidism in male fetuses)
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter (from fetuses submitted to soft tissue examinations)
- Anogenital distance of all live rodent fetuses: yes: measured for all fetuses and normalized to the cube root of the body weight of each fetus.
- Other: each fetus was euthanized by oral administration of sodium pentobarbital. The sex of each fetus was confirmed by internal examination of sexual organs.
Statistics:
Body weight, food consumption and reproductive data
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).

Hormones and anogenital distance
Data were compared by analysis of variance in case of a negative result a one-way ANOVA was performed and tests for normality (Shapiro-Wilk test) and for homoscedasticity (Levene test) were performed. If data were not normally or homogenously distributed, a logarithmic or a rank transformation was performed. The transformed data were analyzed again by one-way ANOVA, tests for normality (Shapiro-Wilk test) and for homoscedasticity (Levene test) were performed. If data were normally distributed and homogeneous (p > 0.05) an F-test from previous ANOVA is run. In case a significant difference (p = 0.05) was observed, pairwise group comparisons were performed (Dunnett test). If data were not normally distributed or homogeneous, the overall group differences were analyzed using ranked values (Kruskal-Wallis test). In case a significant difference (p = 0.05) was observed, pairwise group comparisons using ranked values were performed (Dunn test).

Organ weight
Data were analyzed for normality (Kolmogorov test). If all groups showed a normal distribution, the homogeneity of variance was tested (Bartlett test / F-test). If variance was homogenous, group differences were analyzed by one-way analysis of variance and in case of a significant result (p < 0.05 or 0.01) a Dunnett test (if > 2 groups) or t-test (if 2 groups) were performed. In case of non-normal distribution in at least one group or a non-homogeneous variance, group differences were analyzed by Kruskal-Wallis test. If a significant result (p < 0.05 or 0.01) was achieved, a Dunn test (if > 2 groups) or a Wilcoxon test (if 2 groups) were performed.
Indices:
For each litter:
- total number of resorptions: Sum of uterine scars + Early resorptions + Late resorptions
- total number of dead fetuses: sum of dead fetuses
- % of dead fetuses per litter: (Total number of dead fetuses / Number of implantation sites) x 100
- total number of live fetuses: (Sum of live male + Live female fetuses)
- % of live fetuses per litter: (Total number of live fetuses / Number of implantation sites) x 100
- % of pre-implantation loss: [(Number of corpora lutea – Number of implantation sites) / Number of corpora lutea] x 100
- % of post-implantation loss: [(Number of implantation sites – Number of live fetuses) / Number of implantation sites] x 100
- average fetal body weight: Sum of individual fetal weights / Number of live fetuses
- AGD normalized to the cube root of fetal body weight: AGD / ³vBody weight
Historical control data:
Fetal weight
Mean fetal weight per litter on GD 21 in CRL:CD(SD) rats at Charles River Laboratories Montreal (2007-2018): mean of 5.77 g.

External examinations
Malformations: anal atresia and thread-like tail in rat Crl:CD(SD) at Charles River Laboratories Montreal (2007-2018); Thread-like tail in rat RjHan at Charles River Laboratories Evreux (2016-2018).

Soft tissue examinations
Variations: dilated ureter in rat Crl:CD(SD) at Charles River Laboratories Montreal (2007-2018); Dilated cerebral ventricle, dilated renal pelvis, absent innominate artery and dilated ureter in rat RjHan at Charles River Laboratories Evreux (2016-2018).

Skeletal examinations
Abnormalities: Absent cartilage of lumbar vertebra(e) in rat Crl:CD(SD) at Charles River Laboratories Montreal (2007-2018); Absent cartilage of caudal vertebra(e) and fused cartilage of ribs in rat RjHan at Charles River Laboratories Evreux (2016-2018).

Variations: incomplete ossification (frontal, interparietal) of head, unossified and incomplete ossification of hyoid, incomplete ossification of 1th to 4th sternebra(e) and missahapen sternebra(e) in rat Crl:CD(SD) at Charles River Laboratories Montreal (2007-2018); incomplete ossification (frontal, interparietal) of head, incomplete ossification of hyoid, incomplete ossification of centrum of cervical vertebra(e), unossified centrum of caudal vertebra(e), incomplete ossification of 1th to 4th sternebra(e) and missahapen sternebra(e) in rat RjHan at Charles River Laboratories Evreux (2016-2018).

Malformations: absent sacral vertebrae, absent caudal vertebrae, fused sternebrae in rat Crl:CD(SD) at Charles River Laboratories Montreal (2007-2018); absent lumbar vertebrae, absent caudal vertebrae, fused sternebrae and fused cartilage of ribs in rat RjHan at Charles River Laboratories Evreux (2016-2018); skullcap hole in Rat RjHan and Crl:CD(SD) at Charles River Laboratories Evreux (2006-2009).

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related clinical signs (the clinical sings were not dose-related and were reported in single animals).
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Refer also to Table 1 and Table 2 under section "Any other information on results incl. tables". There were no test item-related effects on mean body weights or mean body weight changes at any dose level.
There were no test item-related effects on mean mean carcass weight or mean net body weight change at any dose level.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no test item-related effects on mean food consumption at any dose level.
Endocrine findings:
no effects observed
Description (incidence and severity):
Refer also to Table 3 under section "Any other information on results incl. tables". The mean thyroid hormone levels (T3, T4 and TSH) were considered to be unaffected by the test item as changes are not dose-related, were without statistical significance and were of low magnitude.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Refer also to Table 2 under section "Any other information on results incl. tables". There were no test item-related effects on mean gravid uterus weight at any dose level.
The gravid uterus weights of the four first females per group were often among the lowest and there were no relevant differences between groups for these females. This was probably due to the fact that these females (which were all from a same batch of animals at receipt) arrived on site on GD 1 instead of GD 2 and were therefore euthanized on GD 20 instead of GD 21.

There were no test item-related thyroid gland weight changes.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
None of the macroscopic observations were considered as test item-related as they were distributed randomly among groups and are commonly recorded in the rat.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no test item-related microscopic observations in the thyroid or parathyroid glands.

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
No abortion was observed.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
Refer also to Table 4 under section "Any other information on results incl. tables". There were no test item-related effects on pre- and post-implantation loss, and number of females with post-implantation loss.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
Refer also to Table 4 under section "Any other information on results incl. tables". There were no female with total resorption.
Early or late resorptions:
no effects observed
Description (incidence and severity):
Refer also to Table 4 under section "Any other information on results incl. tables". There were no test item-related effects on early or late resorptions.
Dead fetuses:
no effects observed
Description (incidence and severity):
Refer also to Table 4 under section "Any other information on results incl. tables". There were no test item-related effects on the number of dead fetuses.
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
Refer also to Table 4 under section "Any other information on results incl. tables". All females except one of the low- and one of the mid-dose groups were not pregnant. The effect was not dose-dependent and considered not related to the treatment.
Other effects:
no effects observed
Description (incidence and severity):
Refer also to Table 5 under section "Any other information on results incl. tables". The number of corpora lutea and implantation sites were unaffected by treatment. No implantation scars were observed in all females. The mean number of live fetuses was similar among all groups.

Effect levels (maternal animals)

Key result
Dose descriptor:
LOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Remarks on result:
other: absence of adverse toxic effects.

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Refer also to Table 6 under section "Any other information on results incl. tables". The four first litters had a low mean fetal weight per litter (between 3.73 and 4.50 g) when compared to all other study litters (between 5.15 and 6.90 g) or to what can be observed usually on GD 21 in Sprague-Dawley rats (HCD in RJHan:SD rats at Charles River Laboratories Evreux (2016-2018): mean of 5.8 g; Historical Control Data (HCD) in CRL:CD(SD) rats at Charles River Laboratories Montreal (2007-2018): mean of 5.77 g). They were probably at GD 20 instead of GD 21 at the time of hysterectomy. There were no major differences between groups for mean fetal weight in these litters.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Refer also to Table 7 under section "Any other information on results incl. tables". There were no test item-related effects on mean sex ratio at any dose level.
Anogenital distance of all rodent fetuses:
no effects observed
Description (incidence and severity):
Refer also to Table 8 under section "Any other information on results incl. tables". There were no test item-related effects on mean anogenital distance at any dose level.
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Refer also to Table 9 under section "Any other information on results incl. tables". There were no test item-related fetal external variations. The only external variations observed were limb hyperflexion in a single fetus at 100 mg/kg bw/day and local edema in a single fetus at 300 mg/kg bw/day; these findings were considered to be incidental.
There were no test item-related fetal external malformations. The only malformed fetus noted in the study was considered as incidental even though it was in the 1000 mg/kg bw/day group, as it was isolated and its malformation can happen spontaneously as also revealed by the incidence in HCD.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Refer also to Table 10, Table 11 and Table 12 under section "Any other information on results incl. tables". None of these skeletal malformations were considered to be test item-treatment-related.
When compared to controls, there were several sternebrae malformations (sternoschisis, fused sternebrae, split cartilage): in a total of 4 fetuses from one litter at 300 mg/kg bw/day, and three fetuses from one litter at 1000 mg/kg bw/day. In view of the single litter incidence in each group, a test item relationship was considered to be unlikely. Otherwise, the other skeletal malformations in the 1000 mg/kg bw/day group were only observed in a single fetus (with anal atresia and thread-like tail at external examination). They were therefore considered to be incidental.
All the skeletal malformations at 300 mg/kg bw/day concerned only 1/23 litters and were therefore considered to be linked to the litter and not the test item.
The skeletal malformations at 100 mg/kg bw/day were noted in isolated fetuses only, they were not recorded in the other groups and/or they were within HCD.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Refer also to Table 13 under section "Any other information on results incl. tables". None of the observed fetal visceral variations were considered to be test item-related, as they were within the HCD, lower than control incidences, of low incidences and/or were not dose-related. There were no fetal soft tissue malformations in any group.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: absence of adverse toxic effects.

Fetal abnormalities

Key result
Abnormalities:
no effects observed
Localisation:
other: refer to "Description (incidence and severity)".
Description (incidence and severity):
Refer also to Table 14 under section "Any other information on results incl. tables". There was no dose-related increase in the frequency of litters with malformed fetuses. Overall at 100, 300 and 1000 mg/kg bw/day, the incidences of malformed litters observed at external, visceral and skeletal examinations were close to the control data (up to 2 litters with malformation(s) vs. none in controls). This would therefore indicate an absence of a test item relationship.

Overall developmental toxicity

Developmental effects observed:
no
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
Treatment related:
no

Any other information on results incl. tables

DOSE FORMULATION ANALYSES


The test item concentrations in the administered dose formulations analyzed in the first and last weeks of the dosing period were within an acceptable range of variations (-12.2% to +11.5%) when compared to the nominal values (± 15% required). The acceptance criteria were therefore met.


No test item was detected in the control dose formulations.


 


Table 1 - Mean Body Weight and Body Weight Changes (g)






























































Dose level (mg/kg bw/day)



0



100



300



1000



Body weight (g)



 



 



 



 



GD 6.



283



282



284



281



 



-



(0)



(0)



(-1)



GD 21



439



447



439



444



 



-



(+2)



(0)



(+1)



Body weight change (g)



 



 



 



 



GD 6 - 21



+155



+165



+155



+162



-: not applicable; ( ): in brackets, percentage difference vs. controls; No statistical significance.


 


 


Table 2 - Mean Carcass Weights, Net Body Weight Changes and Gravid Uterus Weights (g)























































Dose level (mg/kg bw/day)



0



100



300



1000



Gravid uterus weight



109.6



114.7



109.0



109.4



 



-



(+5)



(-1)



(0)



Carcass weight



329.2



332.2



329.9



334.4



 



-



(+1)



(0)



(+2)



Net body weight change from GD 6



+45.8



+50.0



+45.8



+52.9



 



-



(+9)



(0)



(+16)



-: not applicable; ( ): in brackets, percentage difference vs. controls; No statistical significance.


 


 


Table 3 - Mean Thyroid Hormone Levels (T3, T4 and TSH) in Females












































































Dose level (mg/kg bw/day)



0



100



300



1000



T3 (ng/mL)



0.28



0.27



0.33



0.32



SD



0.083



0.147



0.101



0.095



 



-



(-5)



(+18)



(+12)



T4 (ng/mL)



12.62



13.43



14.04



14.73



SD



3.465



7.638



5.823



3.481



 



-



(+6)



(+11)



(+17)



TSH (pg/mL)



621



638



518



652



SD



307.2



253.2



196.8



244.6



 



-



(+3)



(-17)



(+5)



-: not applicable; ( ): in brackets, percentage difference vs. controls; No statistical significance.


 


 


Table 4  - Pregnancy Status









































Dose level (mg/kg bw/day)



0



100



300



1000



Number of time-mated females



24



24



24



24



Pregnant females



24



23



23



24



Females with live fetuses



24



23



23



24



Non-pregnant females



0



1



1



0



 


 


Table 5 - Hysterectomy Data








































































































Dose level (mg/kg bw/day)



0



100



300



1000



Number of pregnant females at hysterectomy



24



23



23



24



Number of females with live fetuses at termination



24



23



23



24



Number of females with total resorption



0



0



0



0



Mean number of corpora lutea



15.4



16.4



15.4



15.1



Mean number of implantation sites



14.5



15.0



14.8



14.3



Mean pre-implantation loss (%)



5.4



7.9



3.8



5.3



Mean number of live fetuses



14.0



14.7



14.2



14.0



Dead fetuses (%)



0.0



0.0



0.0



0.0



Mean number of implantation scars



0.0



0.0



0.0



0.0



Mean number of early resorptions



0.3



0.3



0.7



0.4



Mean number of late resorptions



0.2



0.0



0.0



0.0



Mean post-implantation loss (%)



3.4



2.6



4.3



2.8



Number of females with post-implantation loss



8



6



11



9



 


 


Table 6 - Mean Fetal Body Weights



























Dose level (mg/kg bw/day)



0



100



300



1000



Mean fetal body weight (g)



5.60



5.58



5.44



5.66



 



-



(0)



(-3)



(+1)



-: not applicable; ( ): in brackets, percentage difference vs. controls; No statistical significance.


 


 


Table 7 - Mean Percentages of Male Fetuses



























Dose level (mg/kg bw/day)



0



100



300



1000



Mean percentage of male fetuses (%)



53.2



54.8



47.9



50.5



 



-



(+3)



(-10)



(-5)



-: not applicable; ( ): in brackets, percentage difference vs. controls; No statistical significance.


 


 


Table 8 - Mean Anogenital Distance (AGD mm) and Mean Anogenital Distance Corrected for Body Weight Cubed Root






























































































Sex



Male



 



 



 



Female



 



 



 



Dose level (mg/kg bw/day)



0



100



300



1000



0



100



300



1000



Mean AGD



3.74



3.75



3.67



3.74



1.72



1.72



1.70



1.75



SD



0.315



0.345



0.222



0.322



0.157



0.160



0.110



0.155



 



-



(0)



(-2)



(0)



-



(0)



(-1)



(+2)



AGD/BW1/3



2.09



2.10



2.07



2.08



0.98



0.98



0.98



0.99



SD



0.160



0.181



0.116



0.161



0.092



0.095



0.072



0.090



 



-



(+1)



(-1)



(0)



-



(0)



(0)



(+1)



-: not applicable; ( ): in brackets, percentage difference vs. controls; No statistical significance.


 


 


Table 9 - Fetal (Litter) incidences (%) of External Malformations














































































Dose level (mg/kg bw/day)



0



100



300



1000



HCD



Dams with live fetuses, n



24



23



23



24



 



Live fetuses, n



337



337



326



335



 



Trunk



 



 



 



 



 



. anal atresia, F (L)



 



 



 



0.3 (4.2)



0.37 (5.00)a



Tail



 



 



 



 



 



. thread-like tail, F (L)



 



 



 



0.3 (4.2)



0.31 (4.0)a


0.3 (4.2)b



Litters with external malformations, n (%)c



0 (0.0)



0 (0.0)



0 (0.0)



1 (4.2)



 



Fetuses with external malformations, n (%)c



0 (0.0)



0 (0.0)



0 (0.0)



1 (0.3)



 



n: number.


HCD: Historical Control Data, Sprague-Dawlay rat, a: Rat Crl:CD(SD) at Charles River Laboratories Montreal (2007-2018); b: Rat RjHan at Charles River Laboratories Evreux (2016-2018); upper fetal (litter) incidence per study; c: all malformations combined; No statistical significance.


 


 


Table 10 - Fetal (Litter) incidences (%) of Skeletal Cartilages






















































































































Dose level (mg/kg bw/day)



0



100



300



1000



HCD



Dams with live fetuses, n



24



23



23



24



 



Live fetuses, n



175



175



168



172



 



Cartilage of sternebra(e)



 



 



 



 



 



. split, F (L)



 



 



0.6 (4.3)



 



/



Cartilage of sacral vertebra(e)



 



 



 



 



 



. absent, F (L)



 



 



 



0.6 (4.2)d



/



Cartilage of caudal vertebra(e)



 



 



 



 



 



. absent, F (L)



 



 



 



0.6 (4.2)d



0.7 (4.2)b



. fused, F (L)



 



0.6 (4.3)e



 



 



/



Cartilage of lumbar vertebra(e)



 



 



 



 



 



. absent, F (L)



 



0.6 (4.3)e



 



0.6 (4.2)d



0.6 (4.3)a



Cartilage of ribs



 



 



 



 



 



. fused, F (L)



 



0.6 (4.3)



 



 



0.9 (5.0)b



n: number; HCD: Historical Control Data Sprague-Dawlay rat, a: Rat Crl:CD(SD) at Charles River Laboratories Montreal (2007-2018); b: Rat RjHan at Charles River Laboratories Evreux (2016-2018); upper fetal (litter) incidence per study; d: same fetus, e: same fetus. No statistical significance; /: not available in HCD.


 


 


Table 11 - Fetal (Litter) incidences (%) of most relevant Skeletal Variations






















































































































































Dose level (mg/kg/day)



0



100



300



1000



HCD



Dams with live fetuses, n



24



23



23



24



 



Live fetuses, n



175  



175



168



172



 



Head



 



 



 



 



 



. frontal: incomplete ossification, F (L)



 



 



 



1.2 (4.2)



2.68 (16.67)a


0.9 (5.0)b



. interparietal: incomplete ossification, F (L)


 

 



1.8 (8.7)



2.9* (12.5)



44.29 (80.95)a


4.5 (21.1)b



. skullcap: extra ossification bone, F (L)


 

 



 



0.6 (4.2)



/



. hyoid: unossified, F (L)



0.6 (4.2)



1.1 (8.7)



 



2.3 (4.2)



5.26 (22.73)a


 



. hyoid: incomplete ossification, F (L)



0.6 (4.2)



1.7 (13.0)



 



3.5 (20.8)



32.14 (86.36)a


6.5 (30.0)b



Cervical vertebra(e)


 

 



 



 



 



. incomplete ossification of centrum, F (L)



12.0 (41.7)



10.3 (39.1)



10.1 (43.5)



15.1 (62.5)



 


14.5 (45.0)b



Caudal vertebra(e)


 

 



 



 



 



.unossified centrum, F (L)



2.9 (4.2)



1.7 (8.7)



0.6 (4.3)



1.2 (8.3)



 


0.9 (5.6)b



Sternebra(e)



 



 



 



 



 



. incomplete ossification of 1th to 4th sternebra(e), F (L)



0.6 (4.2)



 



 



1.2 (8.3)



12.21 (42.86)a


3.4 (15.0 )b



. misshapen sternebra(e), F (L)


 

 



0.6 (4.3)



1.2 (4.2)



1.96 (14.29)a


1.6 (10.3 )b



Litters with skeletal variations, n (%)c



20 (83.3)



21 (91.3)



20 (87.0)



19 (79.2)



 



Fetuses with skeletal variations, n (%)c



110 (62.9)



113 (64.6)



90 (53.6)



91 (52.9)



 



n: number; Statistical significance vs. controls *: p<0.05 for the number of fetus;


HCD: Historical Control Data, Sprague-Dawlay rat, a: Rat Crl:CD(SD) at Charles River Laboratories Montreal (2007-2018); b: Rat RjHan at Charles River Laboratories Evreux (2016-2018); upper fetal (litter) incidence per study; c: all variations combined; /: none in HCD.


 


 


Table 12 - Fetal (Litter) incidences (%) of Skeletal Malformations






























































































































































































































Dose level (mg/kg bw/day)



0



100



300



1000



HCD



Dams with live fetuses, n



24



23



23



24



 



Live fetuses, n



175  



175



168



172



 



Head skull



 



 



 



 



 



. skullcap: hole, F (L)



 



 



 



0.6 (4.2)f



0.8 (5.0)c



Thoracic vertebrae



 



 



 



 



 



. open arch, F (L)



 



 



1.2 (4.3)



 



/



Lumbar vertebrae



 



 



 



 



 



. absent, F (L)



 



0.6 (4.3)



 



0.6 (4.2)f



2.4 (5.3)b



. open arch, F (L)



 



 



1.2 (4.3)



 



/



Sacral vertebrae



 



 



 



 



 



. absent, F (L)



 



 



 



0.6 (4.2)f



0.60 (4.17)a



. open arch, F (L)



 



 



0.6 (4.3)



 



/



Caudal vertebrae



 



 



 



 



 



. absent, F (L)



 



 



 



0.6 (4.2)f



0.60 (4.17)a


0.7 (4.2)b



. open arch, F (L)



 



 



0.6 (4.3)



 



/



Sternebrae



 



 



 



 



 



. sternoschisis, F (L)



 



 



 



0.6 (4.2)



/



. fused sternebrae, F (L)



 



 



1.8 (4.3)



1.2 (4.2)



0.78 (5.0)a


0.9 (5.3)b



Cartilage of sternebra(e)



 



 



 



 



 



. split, F (L)



 



 



0.6 (4.3)



 



/



Cartilage of caudal vertebra(e)



 



 



 



 



 



. fused, F (L)



 



0.6 (4.3)



 



 



/



Cartilage of ribs



 



 



 



 



 



. fused, F (L)



 



0.6 (4.3)



 



 



0.9 (5.0)b



Fetuses affected, n (%)d,e



0 (0.0)



2 (1.1)



4 (2.4)



4 (2.3)



 



Litters affected, n (%)d,e



0 (0.0)



2 (8.7)



1 (4.3)



2 (8.3)



 



n: number.


HCD: Historical Control Data, Sprague-Dawlay rat, a: Rat Crl:CD(SD) at Charles River Laboratories Montreal (2007-2018); b: Rat RjHan at Charles River Laboratories Evreux (2016-2018); upper fetal (litter) incidence per study; c: Rat RjHan and Crl:CD(SD) at Charles River Laboratories Evreux (2006-2009); /: none in HCD. d: all malformations combined; e: including the fetuses with malformed cartilages mentioned in the table 10; f: same fetus.


 


 


Table 13 - Fetal (Litter) Incidences (%) of Soft Tissue Variations






























































































































Dose level (mg/kg bw/day)



0



100



300



1000



HCD



Dams with live fetuses, n



24



23



23



24



 



Live fetuses, n



162



162



158



163



 



Brain



 



 



 



 



 



. dilated cerebral ventricle, F (L)



 



 



 



0.6 (4.2)



0.9 (5.0)b



Kidneys



 



 



 



 



 



. dilated renal pelvis, F (L)



0.6 (4.2)



 



1.3 (8.7)



 



9.1 (30.8)b



Adrenal glands



 



 



 



 



 



. colored, F (L)



0.6 (4.2)



1.2 (8.7)



1.3 (4.3)



0.6 (4.2)



/



Vessels



 



 



 



 



 



. Absent innominate artery, F (L)



4.9 (20.8)



2.5 (13.0)



1.9 (13.0)



0.6 (4.2)



3.6 (20.8)b



Ureter



 



 



 



 



 



. dilated, F (L)



6.2 (33.3)



8.6 (30.4)



13.3* (47.8)



10.4 (41.7)



4.24 (23.53)a


15.1 (48.7)b



Litters with soft tissue variations, n (%)c



11 (45.8)



10 (43.5)



13 (56.5)



11 (45.8)



 



Fetuses with soft tissue variations, n (%)c



17 (10.5)



19 (11.7)



26 (16.5)



20 (12.3)



 



n: number; Statistical significance vs. controls *: p<0.05 for the number of fetus.


HCD: Historical Control Data, Sprague-Dawlay rat, a: Rat Crl:CD(SD) at Charles River Laboratories Montreal (2007-2018); b: Rat RjHan at Charles River Laboratories Evreux (2016-2018); upper fetal (litter) incidence per study; c: all variations combined; /: not available in HCD.


 


 


Table 14 - Malformed fetuses























































Dose level (mg/kg bw/day)



0



100



300



1000



 



 



Fetus having absent lumbar vertebra(e), fused cartilage of caudal vertebrae



Fetus having fused sternebrae



Fetus having fused sternebrae



 



 



Fetus having fused cartilage of ribs (distal)



Fetus having fused sternebrae, thoracic and lumbar vertebrae open arch



Fetus having fused sternebrae



 



 



 



Fetus having fused sternebrae, sacral, caudal, thoracic and lumbar vertebrae open arch



Fetus having sternoschisis



 



 



 



Fetus having split cartilage of sternebrae



Fetus having anal atresia, thread-like tail, skullcap hole, absent lumbar, sacral and caudal vertebrae



Total litters affected (%)



0%



8.7%



4.3%



8.3%



Total fetuses affected (%)



0%



1.1%



2.4%



2.0%


Applicant's summary and conclusion

Conclusions:
No adverse findings were observed up to 1000 mg/kg bw/day, the highest dose tested, in dams and fetuses. The NOAELs for maternal toxicity and for developmental toxicity were set at 1000 mg/kg bw/day.
Executive summary:

The effects of the test item on pregnant females and on embryo-fetal development after prenatal exposure, when administered to the dams by the oral (gavage) route from implantation until the day before scheduled hysterectomy [from Gestation Day (GD) 6 to GD 20], were investigated in a GLP-compliant study conducted according to the OECD TG 414 (25th of June 2018). During the course of the study it was noted that some females were likely to be euthanized on GD 20 instead of GD 21 since they were arrived at test Facility on GD 1 instead of GD 2. This event was not considered to have impacted the overall integrity of the study or the interpretation of the study results and conclusions because all groups, including controls, were concerned by the same event and had the same low number of affected females (only 4/24 females per group) and because no toxicologically significant effects were identified in the study. 


The test item concentrations in the dose formulations were determined on two occasions using a validated HPLC/UV analytical method. The females were checked daily for mortality and clinical signs. Body weight and food consumption were recorded at designated intervals. At termination, thyroid hormones (TSH, T3 and T4) were determined in all females. On GD 21, the females were euthanized and a macroscopic post-mortem examination was performed. Hysterectomy was performed and the numbers of corpora lutea, implantation sites, uterine scars, early/late resorptions and live/dead fetuses were recorded. Placentas were observed. The fetuses were weighed and sexed by internal examination of the gonads. The anogenital distance was measured. Detailed external, soft tissue and skeletal examinations (including cartilage) were performed in the fetuses. Macroscopic lesions and thyroids with parathyroids were collected from the dams, preserved in 10% buffered formalin and microscopically examined. Thyroids with parathyroids were weighed. 


The concentrations of the test item in the dose formulations, determined in the first and last weeks of the treatment period, were within an acceptable range of variations (± 15% of the nominal concentrations). No test item was detected in control dose formulations.


On GD 21, there were 24, 23, 23 and 24 pregnant females at 0, 100, 300 and 1000 mg/kg bw/day, respectively. There were no unscheduled deaths, no test item-related clinical signs, no macroscopic or microscopic findings and no test item-related effects on mean body weights, mean food consumption, mean thyroid hormone levels, mean thyroid weights, mean carcass weights or mean net body weight changes from GD 6 at any dose level. There were no test item-related effects on mean gravid uterus weights or mean hysterectomy data.


There were no test item-related effects on mean fetal body weights, mean sex ratios or mean anogenital distances, and no test item-related fetal external variations/malformations, visceral variations/malformations or skeletal malformations and there were no toxicologically significant fetal skeletal variations.


In conclusion, on the basis of the experimental conditions and results obtained in this study:



  • the No Observed Effect Level (NOEL) for maternal parameters was considered to be 1000 mg/kg bw/day based on the absence of findings up to this dose level,

  • the No Observed Adverse Effect Level (NOAEL) for embryo-fetal development was considered to be 1000 mg/kg bw/day based on the absence of adverse findings up to this dose level.