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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 9 JANUARY 2019 to 06 APRIL 2019
Reliability:
1 (reliable without restriction)

Data source

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

Materials and methods

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

Test material

Constituent 1
Chemical structure
Reference substance name:
1,1'-[methylenebis(oxy)]dibutane
EC Number:
219-909-0
EC Name:
1,1'-[methylenebis(oxy)]dibutane
Cas Number:
2568-90-3
Molecular formula:
C9H20O2
IUPAC Name:
1,1'-[methylenebis(oxy)]dibutane
Test material form:
liquid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd
- Age at study initiation: Approximately 71 days old
- Weight at study initiation: 211 to 281 g
- Fasting period before study: No
- Housing: Acclimatization: up to four animals; During pairing: one (stock) male and one female ; Gestation: one female
- Diet (e.g. ad libitum): SDS VRF1 Certified pelleted diet. ad libitum
- Water (e.g. ad libitum): Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals. ad libitum
- Acclimation period: Five days before commencement of pairing

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24ºC
- Humidity (%): 40-70%
- Air changes (per hr): Filtered fresh air which was passed to atmosphere and not recirculated
- Photoperiod (hrs dark / hrs light): Artificial lighting, 12 hours dark : 12 hours light

IN-LIFE DATES: From: 16 January 2019 To: 11 to 14 February 2019

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The required amount of test material was weighed out into a suitable container. Approximately 50% of the final volume of vehicle was added to the test material and mixed with a magnetic stirrer until homogenous. The solution was then made up to the required volume with vehicle. The formulation was transferred to a final container and mixed with a magnetic stirrer until homogenous.
A series of suspensions at the required concentrations were prepared by dilution of individual weighings of the test item.


VEHICLE
- Concentration in vehicle: 0, 20, 66, 200 mg/ml
- Amount of vehicle (if gavage): 5 ml/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical method involved extraction and dilution in acetone followed by gas chromatographic analysis with flame ionization detection. Sample concentrations were determined with reference to external standards prepared in the concentration range 10 μg/mL to 100 μg/mL.
An accurate volume of internal standard solution (100 μL) was added to all standard and sample solutions (1000 μL) and mixed by vortex to provide a final concentration of ca. 50 μg/mL of tetradecane.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1 with identified stock males
- Proof of pregnancy: Ejected copulation plugs in cage tray and vaginal smears were checked for the presence of sperm referred to as day 0 of pregnancy
Duration of treatment / exposure:
Females were treated from Day 6 to Day 19 (inclusive) after mating
Frequency of treatment:
Daily (at approximately the same time each day)
Duration of test:
up to day 20 (necropsy)
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
330 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
20 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels of 0, 100, 330 and 1000 mg/kg/day were selected in conjunction with the Sponsor based on findings from a preliminary embryo-fetal development study conducted at these laboratories.
In the preliminary study oral administration of Butylal at 250, 500 and 1000 mg/kg/day was well tolerated with no effects on maternal clinical condition, macropathology, embryo-fetal survival or development. Effects were limited to low maternal body weight gain at 500 and 1000 mg/kg/day and low maternal food consumption at 1000 mg/kg/day.
Based on these findings there was nothing to preclude the use of 1000 mg/kg/day as the high dose level in this study. Dose levels of 100 and 330 mg/kg/day were then selected as low and intermediate dose levels to provide approximate 3-fold dose intervals and to allow assessment of any dose response.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

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: The weight of each adult was recorded on Days 0, 3, 6-20 after mating.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
The weight of food supplied to each adult, that remaining and an estimate of any spilled was recorded for the periods Days 0-2, 3-5, 6-9, 10-13, 14-17 and 18-19 inclusive after mating

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: 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.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Number of fetuses live and dead
Fetal examinations:
- External examinations: Yes: Examination of all viable fetuses and placentae
- Soft tissue examinations: Yes
- Skeletal examinations: Yes
- Head examinations: Yes
Statistics:
For all adult parameters, the analyses were carried out using the individual animal as the basic experimental unit. For litter/fetal findings the litter was taken as the treated unit and the basis for statistical analysis and biological significance was assessed with relevance to the severity of the anomaly and the incidence of the finding within the background control population.
Historical control data:
Historical control data on major fetal examinations, skeletal fetal examinations and visceral fetal examinations

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Two animals receiving 1000 mg/kg/day showed increased salivation; this is often associated with dosing and is considered to relate to the palatability of the formulation rather than a direct effect of treatment
Tables 2 and 3
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Overall group mean body weight gain during treatment (Gestation Days 6 to 19) for females receiving 1000 mg/kg/day was low when compared with Controls (p<0.01) and mean body weight at the end of dosing (GD 20) was significantly low when compared with Controls (p<0.05). For females receiving 100 or 330 mg/kg/day mean body weight gain during treatment was similar to the Controls.
At 1000 mg/kg/day the gravid uterine weight was low when compared with Controls (p<0.05) and the maternal body weight gain following adjustment for the gravid uterine weight was also low for females at the high dose (p<0.01); the gravid uterine weight and adjusted maternal weight gain at 100 or 330 mg/kg/day were similar to the Controls.
Tables 4 and 5
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
During the treatment period group mean food consumption was low for females receiving 1000 mg/kg/day; with the difference attaining statistical significance GD6-9 (p<0.05) and GD10-17 (p<0.01).
Overall food consumption for females receiving 100 or 330 mg/kg/day was similar to the Controls.
Table 6
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Thyroid weight for females on GD20 were unaffected by treatment with Butylal.
Table 7
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic examination of females on Day 20 after mating did not reveal any findings that could be attributed to treatment.
Table 8
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Microscopic examination of the thyroid glands showed no changes that could be related to treatment with Butylal.
Table 9
Other effects:
no effects observed
Description (incidence and severity):
There was no clear effect on serum T3 concentrations and its prohormone T4 in pregnant rats following administration of Butylal.
There was no dose related effect evident on serum TSH concentrations in pregnant rats after the administration of Butylal via oral gavage.

Maternal developmental toxicity

Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
Table 11
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
Table 1
Early or late resorptions:
no effects observed
Description (incidence and severity):
Table 11
Dead fetuses:
no effects observed
Description (incidence and severity):
Tables 10 and 11
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
Table 11

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
changes in number of pregnant
clinical signs
dead fetuses
early or late resorptions
food consumption and compound intake
gross pathology
histopathology: non-neoplastic
maternal abnormalities
mortality
necropsy findings
organ weights and organ / body weight ratios
pre and post implantation loss
total litter losses by resorption

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
Placental, litter or fetal weights were unaffected by maternal treatment.
Table 12
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
Tables 10 and 12
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Table 11
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
Placental, litter or fetal weights were unaffected by maternal treatment.
Table 12
External malformations:
no effects observed
Description (incidence and severity):
Ano-genital of fetuses on GD20 were unaffected by maternal treatment with Butylal.
Table 13
There was no evidence on an increase in major malformations associated with administration of Butylal treatment
Tables 14, 15,16 and 17
Skeletal malformations:
no effects observed
Description (incidence and severity):
At 330 and 1000 mg/kg/day there was an increased incidence of short supernumerary 14th rib when compared with the concurrent Controls; the fetal and litter incidence at 1000 mg/kg/day was outside the Historical Control data (HCD) range. Studies have shown that this finding does not persist in the rat and therefore in isolation the increased incidence in this variant is considered not to be adverse.
In the intermediate and high dose groups, there appeared to be lower incidences of incompletely ossified cranial centres, hyoid, sternebrae and sacrocaudal vertebrae compared to the Control group and in the low dose group (100 mg/kg/day). However, all incidences were within HCD ranges and therefore it was concluded that these findings at 330 and 1000 mg/kg/day are not adverse.
Tables 18, 19, 20 and 21
Visceral malformations:
no effects observed
Description (incidence and severity):
Tables 22, 23, 24 and 25

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
changes in sex ratio
fetal/pup body weight changes
changes in litter size and weights
changes in postnatal survival
external malformations
skeletal malformations
visceral malformations

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables
























 



Control



Butylal



Dose group



1



2



3



4



Dose (mg/kg/day)



0



100



330



1000



 


Table 1: Summary of maternal disposition


































































 



Group 1



Group 2



Group 3



Group 4



 



Number of animals



Number mated



20



20



20



20



Number not pregnant



0



0



0



0



Number pregnant



20



20



20



20



Number of early deliveries



0



0



0



0



Number of litters with dead fetuses



0



0



0



0



Number of litters with resorptions



8



10



7



9



Number of litters subject to fetal pathology



20



20



20



20



 


 


Table 2: Signs associated with dosing – group distribution of observations during gestation






























 



 



 



Number of animals affected



Category



Observation



Group/Sex:


Initial no:



1F


20



2F


20



3F


20



4F


20



Behavior



Salivation



 



0



0



0



2



 


Table 3: Clinical signs – group distribution of observations during gestation

























































 



 



 



Number of animals affected



Category



Observation



Group/Sex:


Initial no:



1F


20



2F


20



3F


20



4F


20



Behavior



Salivation


Vocalization



 



0


0



0


0



0


0



2


1



Coat



Hair loss, Forelimbs


Hair loss, Hindlimbs


Hair loss, Ventral surface



 



3


1


1



1


0


0



0


0


0



1


0


0



Skin



Encrustation, Ear – right


Encrustation, Hindlimb – left



 



0


1



1


0



0


0



0


0



Staining



Abnormal color, Brown, Ears


Abnormal color, Brown, Forelimbs


Abnormal color, Brown, Head


Abnormal color, Brown, Upper dorsal surface



 



1


1


3


1



0


0


2


3



0


0


1


1



0


0


0


1



 


 


 


Table 4: Body weight and body weight change - group mean values (g) during gestation






















































































































































































































































Group


/Sex



 



Day



0



3



6



7



8



9



10



11



12



13



14



15



16



Statistics test



Av



Av



Av



Wi



Wi



Wi



Wi



Wi



Wi



Wi



Wi



Wi



Wi



1F



Mean



245



261



277



278



282



286



291



297



304



307



312



320



331



 



SD



17.8



18.6



19.6



18.4



17.6



18.7



18.7



18.9



20.1



20.7



20.7



21.4



22.3



 



N



20



20



20



20



20



20



20



20



20



20



20



20



20



2F



Mean



247



265



280



281



285



290



295



302



306



311



317



325



336



 



SD



19.3



21.0



22.0



22.6



22.8



22.1



24.1



24.7



25.2



24.7



25.3



26.1



25.8



 



N



20



20



20



20



20



20



20



20



20



20



20



20



20



3F



Mean



244



262



277



276



280



285



291



297



301



306



311



317



328



 



SD



18.6



19.5



19.7



20.7



20.5



21.5



21.9



22.2



22.4



22.6



22.7



23.7



23.2



 



N



20



20



20



20



20



20



20



20



20



20



20



20



20



4F



Mean



244



260



275



271



274



277



284



290



294



298



305



311



319



 



SD



14.4



14.8



15.3



14.5



13.7



16.9



14.8



15.0



14.9



14.9



14.3



14.8



16.1



 



N



20



20



20



20



20



20



20



20



20



20



20



20



20



 


Table 4 (cont): Body weight and body weight change - group mean values (g) during gestation





































































































































































































































Group


/Sex



 



Day


17



 


18



 


19



 


20



Change


0-3



Change


0-6



Change


6-7



Change


6-8



Change


6-9



Change


6-10



Change


6-11



Change


6-12



Statistics test



Wi



Wi



Wi



Wi



Av



Av



Wi



Wi



Sh



Wi



Wi



Wi



1F



Mean



345



360



373



389



16



32



1



4



9



13



20



26



 



SD



23.6



24.9



27.8



29.9



6.1



6.9



3.6



5.2



4.4



5.0



4.7



5.7



 



N



20



20



20



20



20



20



20



20



20



20



20



20



2F



Mean



350



366



381



396



18



34



0



4



10



14



21



26



 



SD



26.5



27.6



29.3



30.2



5.5



6.3



4.2



2.9



4.8



6.0



5.7



6.5



 



N



20



20



20



20



20



20



20



20



20



20



20



20



3F



Mean



341



356



368



385



18



33



-1



3



8



14



19



24



 



SD



27.1



26.0



27.6



28.1



4.4



5.9



4.4



3.8



4.6



5.0



5.9



6.4



 



N



20



20



20



20



20



20



20



20



20



20



20



20



4F



Mean



329*



343*



354*



368*



15



31



-4**



-1**



2**



9*



15**



18**



 



SD



15.5



17.4



16.8



19.0



3.4



5.8



5.0



5.0



9.1



5.4



5.7



5.6



 



N



20



20



20



20



20



20



20



20



20



20



20



20



 


Table 4 (cont): Body weight and body weight change - group mean values (g) during gestation













































































































































































Group


/Sex



 



Change


6-13



Change


6-14



Change


6-15



Change


6-16



Change


6-17



Change


6-18



Change


6-19



Change


6-20



Statistics test



Wi



Wi



Wi



Wi



Wi



Wi



Wi



Wi



1F



Mean



30



35



43



54



68



83



96



111



 



SD



5.1



4.9



5.4



6.5



7.9



9.9



13.6



15.4



 



N



20



20



20



20



20



20



20



20



2F



Mean



30



36



44



55



69



86



100



116



 



SD



6.0



6.8



8.8



9.4



10.4



11.3



14.0



13.2



 



N



20



20



20



20



20



20



20



20



3F



Mean



29



34



40



51



64



79



91



107



 



SD



6.1



5.6



7.4



7.6



11.3



11.2



13.5



14.9



 



N



20



20



20



20



20



20



20



20



4F



Mean



23**



30**



36**



44**



54**



68**



79**



93**



 



SD



6.8



6.4



7.3



8.0



8.9



9.4



11.4



13.1



 



N



20



20



20



20



20



20



20



20



 


Table 5: Gravid uterine weight, adjusted body weight and adjusted body weight change - group mean values (g) on Day 20 of gestation

















































































































































Group


/Sex



 



Body weight


Day 6



Terminal body weight


Day 20



Body weight


change 6-20



Gravid uterine


weight



Adjusted body weight


Day 20



Adjusted body weight


change 6-20



Statistics test



Av



Wi



Wi



Wi



Wi



Wi



1F



Mean



277



389



111



86.4



302



25



 



SD



19.6



30.2



15.6



15.01



25.1



8.5



 



N



20



20



20



20



20



20



2F



Mean



280



397



117



90.1



307



26



 



SD



22.0



30.1



13.6



8.13



25.4



9.7



 



N



20



20



20



20



20



20



3F



Mean



277



385



108



83.0



302



24



 



SD



19.7



28.2



14.9



11.89



24.6



8.3



 



N



20



20



20



20



20



20



4F



Mean



275



368*



93**



78.3*



290



14**



 



SD



15.3



19.0



13.0



11.54



14.0



7.5



 



N



20



20



20



20



20



20



 


Table 6: Food consumption - group mean values (g/animal/day) during gestation













































































































































































Group


/Sex



 



Day


0-2



 


3-5



 


6-9



 


10-13



 


14-17



 


18-19



Total


6-19



As %


Control



Statistics test



Av



Av



Wi



Wi



Wi



Wi



 



 



1F



Mean



20



22



19



21



24



21



298



-



 



SD



1.9



1.7



1.9



1.9



1.6



3.0



 



 



 



N



20



20



20



20



20



20



 



 



2F



Mean



22



24



20



22



24



22



309



103



 



SD



2.9



2.6



2.6



2.5



2.7



3.0



 



 



 



N



20



20



20



20



20



20



 



 



3F



Mean



21



23



20



21



23



21



298



100



 



SD



2.1



1.9



2.1



2.1



2.4



2.8



 



 



 



N



20



20



20



20



20



20



 



 



4F



Mean



21



23



17*



19**



20**



19



262



88



 



SD



1.6



2.0



1.9



1.8



1.7



2.5



 



 



 



N



20



20



20



20



20



20



 



 



 


 


 


 


Table 7: Organ weights - group mean absolute and adjusted values (g) on Day 20 of gestation

























































































Group


/Sex



 



Terminal


Body weight



Thyroids and


Parathyroids



Statistics test



Wi



 



1F



Mean



389



0.015



 



SD



30



0.002



 



N



20



20



2F



Mean



397



0.014



 



SD



30



0.003



 



N



20



20



3F



Mean



385



0.015



 



SD



28



0.003



 



N



20



20



4F



Mean



368*



0.013



 



SD



19



0.003



 



N



20



20



 


































 



Thyroids and


Parathyroids



Statistics test



Wi



1F



Adjusted Mean



0.015



2F



Adjusted Mean



0.013



3F



Adjusted Mean



0.015



4F



Adjusted Mean



0.013



 


Table 8: Macropathology - group distribution of findings on Day 20 of gestation



























 



 



Number of animals affected



Tissue/Organ and Findings



Group/Sex


No. of animals



1F
20



2F


20



3F
20



4F


20



Vagina


Abnormal contents



 



 


0



 


0



 


1



 


0



 


Table 9: Histopathology - group distribution of findings on Day 20 of gestation



























 



 



Number of animals affected



Tissue/Organ and Findings



Group/Sex


No. of animals



1F
20



2F


20



3F
20



4F


20



Thyroids


Cyst(s), Follicular


 



No. examined


Minimal


Total



20


0


0



20


0


0



20


1


1



20


0


0



 


Table 10: Litter data - summary


































Live offspring compared to



0 mg/kg/day



100 mg/kg/day



300 mg/kg/day



1000 mg/kg/day



Dead fetuses (%)



100



100



100



100



Concurrent controls (%)



100



107



97



95



Number of implantations (%)



100



95



96



94



 


 


Table 11: Litter data - group mean values on Day 20 of gestation

























































































































































































































































Group


/Sex



 



Corpora


lutea



Implantations



Resorption



Implantation loss (%)



Implantation loss#



Live young



Sex ratio


(%M)



Early



Late



Total



Pre-



Post-



Pre-



Post-



Male



Female



Total



Statistics test



Wi



Sh



 



 



 



Wi



Wi



 



 



 



 



Wi



Wi



1F



Mean



16.6



15.2



0.6



0.0



0.6



10.0



3.9



1.4



0.6



7.6



7.0



14.6



50.8



 



SD



2.65



3.17



0.88



0.00



0.88



14.60



5.52



 



 



2.78



1.97



3.03



13.48



 



N



20



20



20



20



20



20



20



20



20



20



20



20



20



2F



Mean



17.1



16.3



0.8



0.0



0.8



5.3



4.6



0.9



0.8



7.7



7.8



15.5



49.5



 



SD



1.37



1.29



0.91



0.00



0.91



6.93



5.67



 



 



1.98



1.77



1.54



10.86



 



N



20



20



20



20



20



20



20



20



20



20



20



20



20



3F



Mean



17.2



14.7



0.6



0.0



0.6



14.0



3.5



2.5



0.6



7.2



6.9



14.1



50.7



 



SD



2.92



2.48



0.94



0.00



0.94



11.55



5.54



 



 



2.24



1.80



2.25



11.92



 



N



20



20



20



20



20



20



20



20



20



20



20



20



20



4F



Mean



15.9



14.8



0.9



0.0



0.9



7.7



6.1



1.2



0.9



7.1



6.8



13.9



50.5



 



SD



2.02



2.31



1.21



0.00



1.21



10.31



8.63



 



 



2.59



2.17



2.37



15.74



 



N



20



20



20



20



20



20



20



20



20



20



20



20



20



# Calculated using the sum of individual values within each group/number of litters per group


 


Table 12: Placental, litter and fetal weights - group mean values (g) on Day 20 of gestation



































































































































Group


/Sex



 



Placental


weight



Total litter


weight



Male fetal


weight



Female fetal


weight



Overall fetal


weight



Statistics test



Wi



Wi



Wi



Wi



Wi



1F



Mean



0.50



54.29



3.90



3.61



3.75



 



SD



0.060



10.113



0.240



0.231



0.224



 



N



20



20



20



20



20



2F



Mean



0.51



56.75



3.80



3.56



3.68



 



SD



0.048



5.861



0.267



0.223



0.231



 



N



20



20



20



20



20



3F



Mean



0.52



52.48



3.78



3.67



3.73



 



SD



0.057



8.020



0.256



0.255



0.247



 



N



20



20



20



20



20



4F



Mean



0.50



50.69



3.76



3.57



3.68



 



SD



0.060



8.117



0.225



0.261



0.247



 



N



20



20



20



20



20



 


Table 13: Ano-genital distance - group mean absolute and adjusted values for fetuses

























































































Group


/Sex



 



Fetal weight (g)



Ano-genital distance (mm)



Statistics test



Wi



 



1M



Mean



3.9



4.1



 



SD



0.24



0.22



 



N



20



20



2M



Mean



3.8



4.1



 



SD



0.27



0.15



 



N



20



20



3M



Mean



3.7



4.1



 



SD



0.28



0.27



 



N



20



20



4M



Mean



3.8



4.1



 



SD



0.23



0.17



 



N



20



20



 


































 



Ano-genital distance



Statistics test



Wi



1M



Adjusted Mean



4.1



2M



Adjusted Mean



4.1



3M



Adjusted Mean



4.1



4M



Adjusted Mean



4.1



 


Table 13 (cont): Ano-genital distance - group mean absolute and adjusted values for fetuses





























































































































Group


/Sex



 



Fetal weight (g)



Ano-genital distance (mm)



Statistics test



Wi



 



1F



Mean



3.6



2.5



 



SD



0.23



0.19



 



N



20



20



2F



Mean



3.6



2.5



 



SD



0.22



0.21



 



N



20



20



3F



Mean



3.7



2.5



 



SD



0.26



0.18



 



N



20



20



4F



Mean



3.6



2.5



 



SD



0.26



0.16



 



N



20



20



 



Ano-genital distance



Statistics test



Wi



1F



Adjusted Mean



2.5



2F



Adjusted Mean



2.5



3F



Adjusted Mean



2.5



4F



Adjusted Mean



2.5


      

 


Table 14: Fetal examinations - major abnormality findings - group incidences




















































































 



 



Fetuses



Litters



Group



 



1



2



3



4



1



2



3



4



Number Examined



 



291*



310**



282***



277



20



20



20



20



Total Number Affected



 



1



3



1



1



1



2



1



1



Head


Skeletal


External



 


Brachygnathia


Cleft palate


Protruding tongue



 


0


0


0



 


0


0


0



 


0


0


0



 


1


1


1



 


0


0


0



 


0


0


0



 


0


0


0



 


1


1


1



Cervical/Thoracic


Skeletal


Visceral



 


Partially split sternum


Retroesophageal aortic arch


Dorsally displaced pulmonary trunk


Muscular ventricular septal defect


Malrotated heart


Thoracic situs inversus



 


0


1


0


0


1


0



 


1


0


1


1


1


0



 


0


0


0


1


0


1



 


0


0


0


0


0


0



 


0


1


0


0


1


0



 


1


0


1


1


1


0



 


0


0


0


1


0


1



 


0


0


0


0


0


0



Lumbar (and abdominal)/Sacral/Caudal


Skeletal


External


 


 


Visceral



 


Termination vertebral column sacral region


Gastroschisis


Threadlike tail


Imperforate anus


Abdominal situs inversus



 


0


0


0


0


0



 


1


1


1


1


1



 


0


0


0


0


0



 


0


0


0


0


0



 


0


0


0


0


0



 


1


1


1


1


1



 


0


0


0


0


0



 


0


0


0


0


0



Note: Individual fetuses/litters may occur in more than one category.


*Litter 13, 7 fetuses heads only examined, included in tabulated data.


**Litter 26 head only examined for one fetus, included in tabulated data.


***Litter 48 ID tags detached for 2 fetuses, ID’s randomly allocated.


Table 14 (cont): Fetal examinations - major abnormality findings - group incidences




























































 



 



Fetuses



Litters



Group



 



1



2



3



4



1



2



3



4



Number Examined



 



291*



310**



282***



277



20



20



20



20



Total Number Affected



 



1



3



1



1



1



2



1



1



Appendicular


Skeletal



 


Short/bent/thickened long bones


Clavicle bent/incompletely ossified



 


0


0



 


0


0



 


0


0



 


1


1



 


0


0



 


0


0



 


0


0



 


1


1



Note: Individual fetuses/litters may occur in more than one category.


*Litter 13, 7 fetuses heads only examined, included in tabulated data.


**Litter 26 head only examined for one fetus, included in tabulated data.


***Litter 48 ID tags detached for 2 fetuses, ID’s randomly allocated


 


Table 15: Fetal examinations - major abnormality findings - group percentage incidences
































































































 



 



Fetuses



Litters



Group



 



1



2



3



4



1



2



3



4



Number Examined



 



291*



310**



282***



277



20



20



20



20



Total Number Affected



 



1



3



1



1



1



2



1



1



Head


Skeletal


External



 


Brachygnathia


Cleft palate


Protruding tongue



 


0.0


0.0


0.0



 


0.0


0.0


0.0



 


0.0


0.0


0.0



 


0.7


0.4


0.4



 


0.0


0.0


0.0



 


0.0


0.0


0.0



 


0.0


0.0


0.0



 


5.0


5.0


5.0



Cervical/Thoracic


Skeletal


Visceral



 


Partially split sternum


Retroesophageal aortic arch


Dorsally displaced pulmonary trunk


Muscular ventricular septal defect


Malrotated heart


Thoracic situs inversus



 


0.0


0.7


0.0


0.0


0.7


0.0



 


0.6


0.0


0.6


0.6


0.6


0.0



 


0.0


0.0


0.0


0.7


0.0


0.7



 


0.0


0.0


0.0


0.0


0.0


0.0



 


0.0


5.0


0.0


0.0


5.0


0.0



 


5.0


0.0


5.0


5.0


5.0


0.0



 


0.0


0.0


0.0


5.0


0.0


5.0



 


0.0


0.0


0.0


0.0


0.0


0.0



Lumbar (and abdominal)/Sacral/Caudal


Skeletal


External


 


 


Visceral



 


Termination vertebral column sacral region


Gastroschisis


Threadlike tail


Imperforate anus


Abdominal situs inversus



 


0.0


0.0


0.0


0.0


0.0



 


0.6


0.3


0.3


0.3


0.6



 


0.0


0.0


0.0


0.0


0.0



 


0.0


0.0


0.0


0.0


0.0



 


0.0


0.0


0.0


0.0


0.0



 


5.0


5.0


5.0


5.0


5.0



 


0.0


0.0


0.0


0.0


0.0



 


0.0


0.0


0.0


0.0


0.0



Appendicular


Skeletal



 


Short/bent/thickened long bones


Clavicle bent/incompletely ossified



 


0.0


0.0



 


0.0


0.0



 


0.0


0.0



 


0.7


0.7



 


0.0


0.0



 


0.0


0.0



 


0.0


0.0



 


5.0


5.0



Note: Individual fetuses/litters may occur in more than one category.


*Litter 13, 7 fetuses heads only examined, included in tabulated data.


**Litter 26 head only examined for one fetus, included in tabulated data.


***Litter 48 ID tags detached for 2 fetuses, ID’s randomly allocated


Table 16: Fetal examinations - Major Historical Control Data - incidence






















Species/strain/source



Rat/ Crl:CD(SD) / Charles River UK



Necropsy date range



Aug 2018– Apr 2019



Number of studies



8



Study types



Main



 






































































































































































































































































































 



 



GB01PV



HCD Range



 



 



Fetuses



Litters



Fetuses



Litters



Group



1



2



3



4



1



2



3



4



 



 



Number Examined



291



310



282



277



20



20



20



20



2098



141



Head



 



 



 



 



 



 



 



 



 



 



Skeletal



Brachygnathia



0



0



0



1



0



0



0



1



0-1



0-1



External



Cleft palate



0



0



0



1



0



0



0



1



0-0



0-0



 



Protruding tongue



0



0



0



1



0



0



0



1



0-1



0-1



Cervical/Thoracic



 



 



 



 



 



 



 



 



 



 



Skeletal



Partially split sternum



0



1



0



0



0



1



0



0



0-1



0-1



Visceral



Dorsally displaced pulmonary trunk



0



1



0



0



0



1



0



0



0-0



0-0



 



Muscular ventricular septal defect



0



1



1



0



0



1



1



0



0-0



0-0



 



Thoracic situs inversus



0



0



1



0



0



0



1



0



0-0



0-0



Lumbar (and abdominal)/Sacral/Caudal



 



 



 



 



 



 



 



 



 



 



Skeletal



Termination vertebral column sacral region



0



1



0



0



0



1



0



0



0-0



0-0



External



Gastroschisis



0



1



0



0



0



1



0



0



0-0



0-0



 



Threadlike tail



0



1



0



0



0



1



0



0



0-0



0-0



 



Imperforate anus



0



1



0



0



0



1



0



0



0-0



0-0



Visceral



Abdominal situs inversus



0



1



0



0



0



1



0



0



0-0



0-0



Appendicular



 



 



 



 



 



 



 



 



 



 



Skeletal



Short/bent/thickened long bones



0



0



0



1



0



0



0



1



0-1



0-1



 



Clavicle bent/incompletely ossified



0



0



0



1



0



0



0



1



0-1



0-1



 


Table 17: Fetal examinations - Major Historical Control Data - percentage (%)






















Species/strain/source



Rat/ Crl:CD(SD) / Charles River UK



Necropsy date range



Aug 2018– Apr 2019



Number of studies



8



Study types



Main



 






































































































































































































































































































 



 



GB01PV



HCD Range



 



 



Fetuses



Litters



Fetuses



Litters



Group



1



2



3



4



1



2



3



4



 



 



Number Examined



291



310



282



277



20



20



20



20



2098



141



Head



 



 



 



 



 



 



 



 



 



 



Skeletal



Brachygnathia*



0.0



0.0



0.0



0.7



0.0



0.0



0.0



5.0



0.0-0.7



0.0-5.3



External



Cleft palate



0.0



0.0



0.0



0.4



0.0



0.0



0.0



5.0



0.0-0.0



0.0-0.0



 



Protruding tongue



0.0



0.0



0.0



0.4



0.0



0.0



0.0



5.0



0.0-0.3



0.0-5.3



Cervical/Thoracic



 



 



 



 



 



 



 



 



 



 



Skeletal



Partially split sternum*



0.0



0.6



0.0



0.0



0.0



5.0



0.0



0.0



0.0-1.0



0.0-16.7



Visceral



Dorsally displaced pulmonary trunk**



0.0



0.6



0.0



0.0



0.0



5.0



0.0



0.0



0.0-0.0



0.0-0.0



 



Muscular ventricular septal defect**



0.0



0.6



0.7



0.0



0.0



5.0



5.0



0.0



0.0-0.0



0.0-0.0



 



Thoracic situs inversus!



0.0



0.0



0.7



0.0



0.0



0.0



5.0



0.0



0.0-0.0



0.0-0.0



Lumbar (and abdominal)/Sacral/Caudal



 



 



 



 



 



 



 



 



 



 



Skeletal



Termination vertebral column sacral region*



0.0



0.6



0.0



0.0



0.0



5.0



0.0



0.0



0.0-0.0



0.0-0.0



External



Gastroschisis



0.0



0.3



0.0



0.0



0.0



5.0



0.0



0.0



0.0-0.0



0.0-0.0



 



Threadlike tail



0.0



0.3



0.0



0.0



0.0



5.0



0.0



0.0



0.0-0.0



0.0-0.0



 



Imperforate anus



0.0



0.3



0.0



0.0



0.0



5.0



0.0



0.0



0.0-0.0



0.0-0.0



Visceral



Abdominal situs inversus



0.0



0.6



0.0



0.0



0.0



5.0



0.0



0.0



0.0-0.0



0.0-0.0



Appendicular



 



 



 



 



 



 



 



 



 



 



Skeletal



Short/bent/thickened long bones*



0.0



0.0



0.0



0.7



0.0



0.0



0.0



5.0



0.0-0.7



0.0-5.3



 



Clavicle bent/incompletely ossified*



0.0



0.0



0.0



0.7



0.0



0.0



0.0



5.0



0.0-0.7



0.0-5.3




*
Findings noted at skeletal examination, therefore % was calculated from number of skeletons examined 


** Findings noted at visceral examination, therefore % was calculated from number of fixed fetuses.


 


 


Table 18: Fetal examinations - minor skeletal abnormality and variants findings - group incidences




























































 



 



Fetuses



Litters



Group



 



1



2



3*



4



1



2



3



4



Number Examined



 



144



154



141



138



20



20



20



20



Minor skeletal abnormalities


Cranial


Vertebral element abnormality


Ribs


Sternebrae


 


Costal cartilage


 


 


Total affected by one or more of the above



 


Sutural bone(s)


Thoracic


Medially thickened/kinked


Misaligned ossification sites


Misshapen


Partially fused


Misaligned


7th not connected to sternum



 


0


0


0


1


0


1


1


0


2



 


0


1


1


0


1


0


0


0


3



 


0


0


0


0


0


0


0


0


0



 


1


1


2


1


0


0


0


1


5



 


0


0


0


1


0


1


1


0


2



 


0


1


1


0


1


0


0


0


3



 


0


0


0


0


0


0


0


0


0



 


1


1


2


1


0


0


0


1


5



Rib and vertebral configuration


Cervical rib


1st rib


13th rib


Number of 14th ribs


 


Thoracolumbar vertebrae


Pelvic girdle



 


Short supernumerary


Short with costal cartilage


Short with/without costal cartilage


Short supernumerary


Total


20


Unilateral cranial shift



 


0


1


5


3


3


1


0



 


0


0


1


6


6


0


1



 


0


0


4


14


14


0


0



 


1


0


0


23


23


1


0



 


0


1


4


3


3


1


0



 


0


0


1


6


6


0


1



 


0


0


3


8


8


0


0



 


1


0


0


12


12


1


0



Note: Individual fetuses/litters may occur in more than one category.


*Litter 48 ID tags detached for 2 fetuses, ID’s randomly allocated.


Table 18 (cont): Fetal examinations - minor skeletal abnormality and variants findings - group incidences
















































 



 



Fetuses



Litters



Group



 



1



2



3*



4



1



2



3



4



Number Examined



 



144



154



141



138



20



20



20



20



Delayed/Incomplete ossification/unossified


Cranial


 


 


Sternebrae


 


 


Vertebrae


 


 


 


 


Ribs


Appendicular


 


 


 


Increased ossification


Cervical vertebral centra



 


Large nasofrontal suture


Cranial centres


Hyoid


5th and/or 6th


Other


Total


Cervical


Thoracic


Lumbar


Sacrocaudal


Caudal


Any


Scapula(e)


Pelvic bones


Metacarpals


Metatarsals


 


All ossified



 


0


15


17


92


14


93


4


10


1


13


1


0


0


8


3


2


 


0



 


1


10


17


113


22


115


2


5


1


14


2


0


0


10


1


1


 


2



 


0


4


8


71


7


73


0


5


0


4


3


0


0


5


0


0


 


0



 


0


1


0


50


8


52


1


17


1


1


1


1


1


1


0


0


 


0



 


0


8


7


20


9


20


4


9


1


9


1


0


0


5


2


2


 


0



 


1


5


11


20


11


20


2


5


1


7


2


0


0


5


1


1


 


2



 


0


2


6


18


3


18


0


5


0


2


1


0


0


3


0


0


 


0



 


0


1


0


18


8


18


1


11


1


1


1


1


1


1


0


0


 


0



Note: Individual fetuses/litters may occur in more than one category.


*Litter 48 ID tags detached for 2 fetuses, ID’s randomly allocated.


 


Table 19: Fetal examinations - minor skeletal abnormality and variants findings - group percentage incidences




























































 



 



Fetuses



Litters



Group



 



1



2



3*



4



1



2



3



4



Number Examined



 



144



154



141



138



20



20



20



20



Minor skeletal abnormalities


Cranial


Vertebral element abnormality


Ribs


Sternebrae


 


Costal cartilage


 


 


Total affected by one or more of the above



 


Sutural bone(s)


Thoracic


Medially thickened/kinked


Misaligned ossification sites


Misshapen


Partially fused


Misaligned


7th not connected to sternum



 


0.0


0.0


0.0


0.7


0.0


0.7


0.7


0.0


1.4



 


0.0


0.6


0.6


0.0


0.6


0.0


0.0


0.0


1.9



 


0.0


0.0


0.0


0.0


0.0


0.0


0.0


0.0


0.0



 


0.7


0.7


1.4


0.7


0.0


0.0


0.0


0.7


3.6



 


0.0


0.0


0.0


5.0


0.0


5.0


5.0


0.0


10.0



 


0.0


5.0


5.0


0.0


5.0


0.0


0.0


0.0


15.0



 


0.0


0.0


0.0


0.0


0.0


0.0


0.0


0.0


0.0



 


5.0


5.0


10.0


5.0


0.0


0.0


0.0


5.0


25.0



Rib and vertebral configuration


Cervical rib


1st rib


13th rib


Number of 14th ribs


 


Thoracolumbar vertebrae


Pelvic girdle



 


Short supernumerary


Short with costal cartilage


Short with/without costal cartilage


Short supernumerary


Total


20


Unilateral cranial shift



 


0.0


0.7


3.5


2.1


2.1


0.7


0.0



 


0.0


0.0


0.6


3.9


3.9


0.0


0.6



 


0.0


0.0


2.8


9.9


9.9


0.0


0.0



 


0.7


0.0


0.0


16.7


16.7


0.7


0.0



 


0.0


5.0


20.0


15.0


15.0


5.0


0.0



 


0.0


0.0


5.0


30.0


30.0


0.0


5.0



 


0.0


0.0


15.0


40.0


40.0


0.0


0.0



 


5.0


0.0


0.0


60.0


60.0


5.0


0.0



Note: Individual fetuses/litters may occur in more than one category.


*Litter 48 ID tags detached for 2 fetuses, ID’s randomly allocated.


Table 19 (cont): Fetal examinations - minor skeletal abnormality and variants findings - group percentage incidences
















































 



 



Fetuses



Litters



Group



 



1



2



3*



4



1



2



3



4



Number Examined



 



144



154



141



138



20



20



20



20



Delayed/Incomplete ossification/unossified


Cranial


 


 


Sternebrae


 


 


Vertebrae


 


 


 


 


Ribs


Appendicular


 


 


 


Increased ossification


Cervical vertebral centra



 


Large nasofrontal suture


Cranial centres


Hyoid


5th and/or 6th


Other


Total


Cervical


Thoracic


Lumbar


Sacrocaudal


Caudal


Any


Scapula(e)


Pelvic bones


Metacarpals


Metatarsals


 


All ossified



 


0.0


10.4


11.8


63.9


9.7


64.6


2.8


6.9


0.7


9.0


0.7


0.0


0.0


5.6


2.1


1.4


 


0.0



 


0.6


6.5


11.0


73.4


14.3


74.7


1.3


3.2


0.6


9.1


1.3


0.0


0.0


6.5


0.6


0.6


 


1.3



 


0.0


2.8


5.7


50.4


5.0


51.8


0.0


3.5


0.0


2.8


2.1


0.0


0.0


3.5


0.0


0.0


 


0.0



 


0.0


0.7


0.0


36.2


5.8


37.7


0.7


12.3


0.7


0.7


0.7


0.7


0.7


0.7


0.0


0.0


 


0.0



 


0.0


40.0


35.0


100.0


45.0


100.0


20.0


45.0


5.0


45.0


5.0


0.0


0.0


25.0


10.0


10.0


 


0.0



 


5.0


25.0


55.0


100.0


55.0


100.0


10.0


25.0


5.0


35.0


10.0


0.0


0.0


25.0


5.0


5.0


 


10.0



 


0.0


10.0


30.0


90.0


15.0


90.0


0.0


25.0


0.0


10.0


5.0


0.0


0.0


15.0


0.0


0.0


 


0.0



 


0.0


5.0


0.0


90.0


40.0


90.0


5.0


55.0


5.0


5.0


5.0


5.0


5.0


5.0


0.0


0.0


 


0.0



 


Table 20: Fetal examinations - Skeletal Historical Control Data - incidence






















Species/strain/source



Rat/ Crl:CD(SD) / Charles River UK



Necropsy date range



Aug 2018– Apr 2019



Number of studies



8



Study types



Main



 






























































































































































































































































































































































 



 



GP01PV



HCD Range



 



 



Fetuses



Litters



Fetuses



Litters



Group



1



2



3



4



1



2



3



4



 



 



Number Examined



144



154



141



138



20



20



20



20



1105



141



Minor skeletal abnormalities



 



 



 



 



 



 



 



 



 



 



Cranial



sutural bone(s)



0



0



0



1



0



0



0



1



0-0



0-0



Vertebral element abnormality



thoracic



0



1



0



1



0



1



0



1



0-1



0-1



Ribs



medially thickened/kinked



0



1



0



2



0



1



0



2



0-1



0-1



Sternebrae



misshapen



0



1



0



0



0



1



0



0



0-0



0-0



Costal cartilage



7th not connected to sternum



0



0



0



1



0



0



0



1



0-1



0-1



Rib and vertebral configuration



 



 



 



 



 



 



 



 



 



 



Cervical rib



short supernumerary



0



0



0



1



0



0



0



1



0-2



0-1



Number of 14th ribs



short supernumerary



3



6



14



23



3



6



8



12



1-14



1-8



Pelvic girdle



unilateral cranial shift



0



1



0



0



0



1



0



0



0-1



0-1



Delayed/Incomplete ossification/unossified



 



 



 



 



 



 



 



 



 



 



Cranial



large nasofrontal suture



0



1



0



0



0



1



0



0



0-3



0-2



 



cranial centres



15



10



4



1



8



5



2



1



1-20



1-11



 



hyoid



17



17



8



0



7



11



6



0



10-29



4-13



Sternebrae



5th and/or 6th



92



113



71



50



20



20



18



18



42-99



6-20



 



other



14



22



7



8



9



11



3



8



4-11



1-9



 



total



93



115



73



52



20



20



18



18



45-99



6-20



Vertebrae



sacrocaudal



13



14



4



1



9



7



2



1



0-18



0-8



Ribs



any



0



0



0



1



0



0



0



1



0-1



0-1



Appendicular



Scapula



0



0



0



1



0



0



0



1



0-0



0-0



Increased ossification



 



 



 



 



 



 



 



 



 



 



Cervical vertebral centra



all ossified



0



2



0



0



0



2



0



0



0-0



0-0



 


 


Table 21: Fetal examinations - Skeletal Historical Control Data - percentage (%)


























 



Species/strain/source



Rat/ Crl:CD(SD) / Charles River UK



 



Necropsy date range



Aug 2018– Apr 2019



 



Number of studies



8



 



Study types



Main



 






























































































































































































































































































































































 



 



GP01PV



HCD Range



 



 



Fetuses



Litters



Fetuses



Litters



Group



1



2



3



4



1



2



3



4



 



 



Number Examined



144



154



141



138



20



20



20



20



1105



141



Minor skeletal abnormalities



 



 



 



 



 



 



 



 



 



 



Cranial



sutural bone(s)



0.0



0.0



0.0



0.7



0.0



0.0



0.0



5.0



0.0-0.0



0.0-0.0



Vertebral element abnormality



thoracic



0.0



0.6



0.0



0.7



0.0



5.0



0.0



5.0



0.0-0.7



0.0-5.3



Ribs



medially thickened/kinked



0.0



0.6



0.0



1.4



0.0



5.0



0.0



10.0



0.0-0.7



0.0-5.3



Sternebrae



misshapen



0.0



0.6



0.0



0.0



0.0



5.0



0.0



0.0



0.0-0.0



0.0-0.0



Costal cartilage



7th not connected to sternum



0.0



0.0



0.0



0.7



0.0



0.0



0.0



5.0



0.0-0.7



0.0-5.3



Rib and vertebral configuration



 



 



 



 



 



 



 



 



 



 



Cervical rib



short supernumerary



0.0



0.0



0.0



0.7



0.0



0.0



0.0



5.0



0.0-1.3



0.0-5.6



Number of 14th ribs



short supernumerary



2.1



3.9



9.9



16.7



15.0



30.0



40.0



60.0



0.7-10.9



5.3-50.0



Pelvic girdle



unilateral cranial shift



0.0



0.6



0.0



0.0



0.0



5.0



0.0



0.0



0.0-0.7



0.0-5.3



Delayed/Incomplete ossification/unossified



 



 



 



 



 



 



 



 



 



 



Cranial



large nasofrontal suture



0.0



0.6



0.0



0.0



0.0



5.0



0.0



0.0



0.0-2.2



0.0-11.1



 



cranial centres



10.4



6.5



2.8



0.7



40.0



25.0



10.0



5.0



1.0-13.7



15.8-55.0



 



hyoid



11.8



11.0



5.7



0.0



35.0



55.0



30.0



0.0



7.5-20.4



31.6-66.7



Sternebrae



5th and/or 6th



63.9



73.4



50.4



36.2



100.0



100.0



90.0



90.0



41.6-67.8



88.9-100.0



 



other



9.7



14.3



5.0



5.8



45.0



55.0



15.0



40.0



2.7-7.7



16.7-45.0



 



total



64.6



74.7



51.8



37.7



100.0



100.0



90.0



90.0



44.6-67.8



88.9-100.0



Vertebrae



sacrocaudal



9.0



9.1



2.8



0.7



45.0



35.0



10.0



5.0



0.0-13.4



0.0-44.4



Ribs



any



0.0



0.0



0.0



0.7



0.0



0.0



0.0



5.0



0.0-0.7



0.0-5.6



Appendicular



scapula



0.0



0.0



0.0



0.7



0.0



0.0



0.0



5.0



0.0-0.0



0.0-0.0



Increased ossification



 



 



 



 



 



 



 



 



 



 



Cervical vertebral centra



all ossified



0.0



1.3



0.0



0.0



0.0



10.0



0.0



0.0



0.0-0.0



0.0-0.0



 


 


 


 


 


 


 


 


 


 


 


 


 


 


 


Table 22: Fetal examinations - minor visceral abnormality and necropsy findings - group incidences




























































 



 



Fetuses



Litters



Group



 



1



2



3



4



1



2



3



4



Number Examined



 



147*



156**



141***



139



20



20



20



20



Total Number Affected



 



31



20



24



26



16



13



14



14



Visceral abnormalities


Brain


Eyes


 


 


Thymus


 


Lungs


Caudal vena cava


Diaphragm


Liver


Adrenal(s)


Ureter(s)


Testis(es)


 


Umbilical artery



 


Dilated interventricular foramen


Folded retina


Dilated orbital sinus


Variation in lens shape


Partially undescended lobe


Thymic remnant


Absent accessory lobe


Anomalous confluence/left hepatic vein


Thinning with liver protrusion


Fissured posterior caudate lobe


Dark


Dilated


Undescended


Malpositioned


Left



 


0


1


1


1


5


0


0


2


2


1


0


1


0


2


2



 


0


0


0


0


5


0


0


0


0


0


0


0


0


1


3



 


0


2


1


0


1


1


1


0


0


0


1


0


1


3


1



 


1


0


0


0


5


0


0


0


0


0


0


0


1


1


2



 


0


1


1


1


4


0


0


1


2


1


0


1


0


2


2



 


0


0


0


0


4


0


0


0


0


0


0


0


0


1


3



 


0


2


1


0


1


1


1


0


0


0


1


0


1


3


1



 


1


0


0


0


3


0


0


0


0


0


0


0


1


1


2



Note: Individual fetuses/litters may occur in more than one category.


*Litter 13, 7 fetuses heads only examined, included in tabulated data.


**Litter 26 head only examined for one fetus, included in tabulated data.


***Litter 48 ID tags detached for 2 fetuses, ID’s randomly allocated


Table 22 (cont): Fetal examinations - minor visceral abnormality and necropsy findings - group incidences








































































 



 



Fetuses



Litters



Group



 



1



2



3



4



1



2



3



4



Number Examined



 



147*



156**



141***



139



20



20



20



20



Total Number Affected



 



31



20



24



26



16



13



14



14



Haemorrhages


Head


 


 


Neck/Thorax


 


Abdomen


 


General



 


Brain


Vitreous humour eye


Eye lid(s)


Dorsal fat pad


Thoracic cavity


Abdomen cavity


Liver lobes


Subcutaneous



 


3


1


1


1


0


12


1


1



 


5


0


0


0


0


6


0


0



 


3


0


0


1


0


5


0


2



 


6


0


0


1


1


6


0


1



 


3


1


1


1


0


8


1


1



 


5


0


0


0


0


4


0


0



 


2


0


0


1


0


3


0


2



 


6


0


0


1


1


5


0


1



Necropsy observations (external)


Skin


 


Head


Tail



 


Shiny


Subcutaneous edema


Short snout


Curled



 


1


0


0


0



 


0


0


0


0



 


3


1


0


0



 


0


0


1


1



 


1


0


0


0



 


0


0


0


0



 


2


1


0


0



 


0


0


1


1



Note: Individual fetuses/litters may occur in more than one category.


*Litter 13, 7 fetuses heads only examined, included in tabulated data.


**Litter 26 head only examined for one fetus, included in tabulated data.


***Litter 48 ID tags detached for 2 fetuses, ID’s randomly allocated


 


 


Table 23: Fetal examinations - minor visceral abnormality and necropsy findings – group percentage incidences




























































 



 



Fetuses



Litters



Group



 



1



2



3



4



1



2



3



4



Number Examined



 



147*



156**



141***



139



20



20



20



20



Total Number Affected



 



31



20



24



26



16



13



14



14



Visceral abnormalities


Brain


Eyes


 


 


Thymus


 


Lungs


Caudal vena cava


Diaphragm


Liver


Adrenal(s)


Ureter(s)


Testis(es)


 


Umbilical artery



 


Dilated interventricular foramen


Folded retina


Dilated orbital sinus


Variation in lens shape


Partially undescended lobe


Thymic remnant


Absent accessory lobe


Anomalous confluence/left hepatic vein


Thinning with liver protrusion


Fissured posterior caudate lobe


Dark


Dilated


Undescended


Malpositioned


Left



 


0.0


0.7


0.7


0.7


3.4


0.0


0.0


1.4


1.4


0.7


0.0


0.7


0.0


1.4


1.4



 


0.0


0.0


0.0


0.0


3.2


0.0


0.0


0.0


0.0


0.0


0.0


0.0


0.0


0.6


1.9



 


0.0


1.4


0.7


0.0


0.7


0.7


0.7


0.0


0.0


0.0


0.7


0.0


0.7


2.1


0.7



 


0.7


0.0


0.0


0.0


3.6


0.0


0.0


0.0


0.0


0.0


0.0


0.0


0.7


0.7


1.4



 


0.0


5.0


5.0


5.0


20.0


0.0


0.0


5.0


10.0


5.0


0.0


5.0


0.0


10.0


10.0



 


0.0


0.0


0.0


0.0


20.0


0.0


0.0


5.0


10.0


5.0


0.0


5.0


0.0


10.0


10.0



 


0.0


10.0


5.0


0.0


5.0


5.0


5.0


0.0


0.0


0.0


5.0


0.0


5.0


15.0


5.0



 


5.0


0.0


0.0


0.0


15.0


0.0


0.0


0.0


0.0


0.0


0.0


0.0


5.0


5.0


10.0



Note: Individual fetuses/litters may occur in more than one category.


*Litter 13, 7 fetuses heads only examined, included in tabulated data.


**Litter 26 head only examined for one fetus, included in tabulated data.


***Litter 48 ID tags detached for 2 fetuses, ID’s randomly allocated


Table 23 (cont): Fetal examinations - minor visceral abnormality and necropsy findings – group percentage incidences








































































 



 



Fetuses



Litters



Group



 



1



2



3



4



1



2



3



4



Number Examined



 



147*



156**



141***



139



20



20



20



20



Total Number Affected



 



31



20



24



26



16



13



14



14



Haemorrhages


Head


 


 


Neck/Thorax


 


Abdomen


 


General



 


Brain


Vitreous humour eye


Eye lid(s)


Dorsal fat pad


Thoracic cavity


Abdomen cavity


Liver lobes


Subcutaneous



 


2.0


0.7


0.7


0.7


0.0


8.2


0.7


0.7



 


3.2


0.0


0.0


0.0


0.0


3.8


0.0


0.0



 


2.1


0.0


0.0


0.7


0.0


3.5


0.0


1.4



 


4.3


0.0


0.0


0.7


0.7


4.3


0.0


0.7



 


15.0


5.0


5.0


5.0


0.0


40.0


5.0


5.0



 


25.0


0.0


0.0


0.0


0.0


20.0


0.0


0.0



 


10.0


0.0


0.0


5.0


0.0


15.0


0.0


10.0



 


30.0


0.0


0.0


5.0


5.0


25.0


0.0


5.0



Necropsy observations (external)


Skin


 


Head


Tail



 


Shiny


Subcutaneous edema


Short snout


Curled



 


0.3


0.0


0.0


0.0



 


0.0


0.0


0.0


0.0



 


1.1


0.4


0.0


0.0



 


0.0


0.0


0.4


0.4



 


5.0


0.0


0.0


0.0



 


0.0


0.0


0.0


0.0



 


10.0


5.0


0.0


0.0



 


0.0


0.0


5.0


5.0



Note: Individual fetuses/litters may occur in more than one category.


*Litter 13, 7 fetuses heads only examined, included in tabulated data.


**Litter 26 head only examined for one fetus, included in tabulated data.


***Litter 48 ID tags detached for 2 fetuses, ID’s randomly allocated


Table 24: Fetal examinations - Visceral Historical Control Data - incidence






















Species/strain/source



Rat/ Crl:CD(SD) / Charles River UK



Necropsy date range



Aug 2018– Apr 2019



Number of studies



8



Study types



Main



 



















































































































































































































 



 



GB01PV



HCD Range



 



 



Fetuses



Litters



Fetuses



Litters



Group



1



2



3



4



1



2



3



4



 



 



Number Examined



147



156



141



139



20



20



20



20



1094



141



Visceral abnormalities



 



 



 



 



 



 



 



 



 



 



Brain



dilated interventricular foramen



0



0



0



1



0



0



0



1



0-3



0-3



Thymus



thymic remnant



0



0



1



0



0



0



1



0



0-1



0-1



Lungs



absent accessory lobe



0



0



1



0



0



0



1



0



0-0



0-0



Adrenal(s)



dark



0



0



1



0



0



0



1



0



0-0



0-0



Testis(es)



undescended



0



0



1



1



0



0



1



1



0-1



0-1



Haemorrhages



 



 



 



 



 



 



 



 



 



 



Neck/Thorax



thoracic cavity



0



0



0



1



0



0



0



1



0-0



0-0



Necropsy observations (external)



 



 



 



 



 



 



 



 



 



 



Skin



subcutaneous edema



0



0



1



0



0



0



1



0



0-0



0-0



Head



short snout



0



0



0



1



0



0



0



1



0-1



0-1



Tail



curled



0



0



0



1



0



0



0



1



0-0



0-0



 


Table 25: Fetal examinations - Visceral Historical Control Data - percentage (%)


 






















Species/strain/source



Rat/ Crl:CD(SD) / Charles River UK



Necropsy date range



Aug 2018– Apr 2019



Number of studies



8



Study types



Main



 


 



















































































































































































































 



 



GB01PV



HCD Range



 



 



Fetuses



Litters



Fetuses



Litters



Group



1



2



3



4



1



2



3



4



 



 



Number Examined



147



156



141



139



20



20



20



20



1094



141



Visceral abnormalities



 



 



 



 



 



 



 



 



 



 



Brain



dilated interventricular foramen



0.0



0.0



0.0



0.7



0.0



0.0



0.0



5.0



0.0-2.2



0.0-15.8



Thymus



thymic remnant



0.0



0.0



0.7



0.0



0.0



0.0



5.0



0.0



0.0-0.6



0.0-5.0



Lungs



absent accessory lobe



0.0



0.0



0.7



0.0



0.0



0.0



5.0



0.0



0.0-0.0



0.0-0.0



Adrenal(s)



dark



0.0



0.0



0.7



0.0



0.0



0.0



5.0



0.0



0.0-0.0



0.0-0.0



Testis(es)



undescended



0.0



0.0



0.7



0.7



0.0



0.0



5.0



5.0



0.0-0.7



0.0-5.3



Haemorrhages



 



 



 



 



 



 



 



 



 



 



Neck/Thorax



thoracic cavity



0.0



0.0



0.0



0.7



0.0



0.0



0.0



5.0



0.0-0.0



0.0-0.0



Necropsy observations (external)



 



 



 



 



 



 



 



 



 



 



Skin



subcutaneous edema



0.0



0.0



0.4



0.0



0.0



0.0



5.0



0.0



0.0-0.0



0.0-0.0



Head



short snout



0.0



0.0



0.0



0.4



0.0



0.0



0.0



5.0



0.0-0.3



0.0-5.3



Tail



curled



0.0



0.0



0.0



0.4



0.0



0.0



0.0



5.0



0.0-0.0



0.0-0.0



 

Applicant's summary and conclusion

Conclusions:
Oral administration of Butylal to pregnant Sprague-Dawley rats during organogenesis and the fetal growth phase at 100, 330 and 1000 mg/kg/day was well tolerated with no effects on maternal clinical condition, thyroid weight and associated hormones or macropathology. Embryo-fetal survival, fetal weight and ano-genital distance were unaffected by maternal treatment.
Maternal effects were limited to low body weight gain, adjusted maternal weight gain and food consumption at 1000 mg/kg/day. In addition the gravid uterine weight for females at 1000 mg/kg/day was low but in the absence of any effect on litter size or fetal weight this was not considered to be toxicologically significant.
Fetal effects were limited to a non-adverse increase in the fetal/litter incidence of the skeletal variant of short supernumerary 14th rib at 1000 mg/kg/day.
Based on the findings in this study the no observed adverse effect level for both maternal animals and embryo-fetal development was considered to be 1000 mg/kg/day.
Executive summary:

The purpose of this study was to assess of the influence of Butylal (an industrial chemical) on embryo-fetal survival and development when administered during the organogenesis and fetal growth phases of pregnancy in the Sprague-Dawley rat.


Three groups of 20 females received Butylal at doses of 100, 330 or 1000 mg/kg/day by oral gavage administration, from Day 6 to 19 after mating.  A similarly constituted Control group received the vehicle, corn oil at the same volume dose as treated groups.  Animals were killed on Day 20 after mating for reproductive assessment and fetal examination.


Clinical observations, body weight and food consumption were recorded.  On Day 20 after mating adult females were subject to macroscopic and microscopic pathology investigation and the gravid uterine weight and thyroid weight recorded.  Ano-genital distance were measured for all fetuses and were examined macroscopically at necropsy and subsequently by detailed internal visceral examination or skeletal examination.


Results and conclusion


Oral administration of Butylal to pregnant Sprague-Dawley rats during organogenesis and the fetal growth phase at 100, 330 and 1000 mg/kg/day was well tolerated with no effects on maternal clinical condition, thyroid weight and associated hormones or macropathology. Embryo-fetal survival, fetal weight and ano-genital distance were unaffected by maternal treatment.


Maternal effects were limited to low body weight gain, adjusted maternal weight gain and food consumption at 1000 mg/kg/day.  In addition the gravid uterine weight for females at 1000 mg/kg/day was low but in the absence of any effect on litter size or fetal weight this was not considered to be toxicologically significant.


Fetal effects were limited to a non-adverse increase in the fetal/litter incidence of the skeletal variant of short supernumerary 14th rib at 1000 mg/kg/day.


Based on the findings in this study the no observed adverse effect level for both maternal animals and embryo-fetal development was considered to be 1000 mg/kg/day.