Registration Dossier

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:
2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study according to guideline

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted 22 january, 2001
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
Composition:
Propyl 4-hydroxybenzoate: 99,7 %
4-Hydroxy-benzoic acid: 0.1 %
unspecified impurity: 0.2 %
Ethanol: < 200 ppm
Propanol: < 200 ppm

melting point: 98 °C

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST SYSTEM
Species/strain: healthy Wistar rats, Crl: WI(Han) (Full Barrier)
Source: Charles River, 97633 Sulzfeld, Germany
Sex: male and female; the female animals were non-pregnant and nulliparous.
Age of the females at the arrival at BSL Munich: approx. 11-12 weeks old
Age of the males at the start of pairing: between 13 weeks and not older than 21 weeks
Body weight before initiation of pairing: males: 303 - 386 g (mean: 344.04 g, ± 20 % = 275.23 – 412.85 g); females: 197 - 264 g (mean: 222.45 g, ± 20 % = 177.96 – 266.94 g)
The animals were derived from a controlled full-barrier maintained breeding system (SPF). According to the German Act on Animal Welfare the animals were bred for experimental purposes.
This study was performed in an AAALAC-accredited laboratory. According to German animal protection law, the study type has been reviewed and accepted by local authorities. Furthermore, the study has been subjected to Ethical Review Process and was authorised by the Bavarian animal welfare administration.


HOUSING AND FEEDING CONDITIONS
- Full barrier in an air-conditioned room
- Temperature: 22 ± 3 °C
- Relative humidity: 55 ± 10 %
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: 10 x / hour
- Free access to Altromin 1324 maintenance diet for rats and mice
- Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- The animals were kept individually in type III H, polysulphone cages on Altromin saw fibre bedding (except during the pre-mating period when females were kept in groups of two animals and during mating period when two females were paired with one male).
- Certificates of food, water and bedding are filed for two years at BSL Munich and afterwards archived at Eurofins Munich
- Adequate acclimatisation period (at least 5 days)


Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 1 % hydroxyethyl-cellulose (viscosity 80-125 cP, 2 % in water at 20 °C)
Details on exposure:
PREPARATION OF THE TEST ITEM FORMULATION
The test item was ground before formulation preparation and afterwards weighed into a tared plastic vial on a suitable precision balance and coated with approx. 1/3 of the target volume with vehicle. After producing a slurry with the glass rod for 1 minute, 1 % aqueous hydroxyethyl-cellulose was added to give the appropriate final concentration. The formulation was then stirred for approximately 30 minutes until visual homogeneity was achieved.
Based on the results of stability testing (Eurofins Munich Study No. 176888), the test item formulations were prepared at least once every 4 days (within stability time frame as given by Eurofins Munich Study No. 176888). The prepared formulation was stored protected from light at 2-8 °C.
Formulates were kept under magnetic stirring during the daily administration.
The vehicle was also used as control item.

CHARACTERISATION OF THE VEHICLE
Based on the characteristics of the test item and solubility check, the vehicle used in this study was 1 % hydroxyethyl-cellulose (viscosity 80-125 cP, 2 % in water at 20 °C). The aqueous solution was prepared with aqua ad injectionem.
The specifications provided by the supplier are listed as follows:
Name: hydroxyethyl-cellulose mittelviskos
Manufacturer: Sigma-Aldrich
Batch No.: MKCD0421
Physical State: powder
Colour: beige
Storage Conditions: at room temperature
Expiry Date: 18 October 2018
Safety Precautions: The routine hygienic procedures were sufficient to assure personnel health and safety.
Name: aqua ad injectionem
Manufacturer: Deltamedica
Batch No.: 612118
Physical State: liquid
Storage Conditions: at room temperature
Expiry Date: November 2019
Safety Precautions: The routine hygienic procedures were sufficient to assure personnel health and safety.



ADMINISTRATION OF DOSES
The test item formulation or vehicle were administered at a single dose to the animals by oral gavage. The application volume for all groups was 5 mL/kg body weight.
For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured.
Inadvertently, the following animals were dosed with less application volume than 5 mL/kg body weight: animal no. 45 (LD), 70 (MD) for one day and animal no. 77 (HD) for three days.

Dose levels: 0, 100, 300, 1000 mg/kg bw (concentration in vehicle: 0, 20, 60, 200 mg/ml)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Before beginning of the treatment period, formulation samples were prepared and analysed in order to obtain knowledge about stability and homogeneity of the test item in the selected vehicle at Eurofins Munich as part of a separate GLP study (Eurofins Munich Study No. 176888).
Study pre start stability analysis was included on the samples from high dose and low dose group and the investigation was made for 0 h, 6 h (RT), 10 day (RT), 10 day (2-8 °C) and 10 day -15 to -35 °C.
Prestart homogeneity investigation was included on the samples collected from various levels (top, middle and bottom) of high dose and low dose groups.
Based on the results from the setup the test item formulation was a suspension.
According to Eurofins Study No. 176888, samples were taken from the top, middle and bottom of prepared formulations from all dose groups and from the middle of the control group in the first and last week of the study and analysed in triplicates (20 samples). Mean value per dose level were reported.
Each sample taken during the study was retained in duplicate (sample A, sample B, each of at least 5 mL). The A-samples were analysed at Eurofins Munich (Eurofins Munich Study Phase No. 176890) and until then stored under appropriate conditions based on available stability data. The B-samples will be retained at -15 to -35 °C at BSL Munich (test facility) and discarded after completion of the final study report. The phase plan was amended to the study plan. The results were reported in the appendix of the final report.

Details on mating procedure:
PREPARATION OF THE ANIMALS
After the acclimatisation period of at least 5 days, females were paired with males as per the ratio of 1:2 (male to female). Prior to the start of the mating a detailed clinical observation outside the home cage was made. Only healthy animals were used in this study.

MATING
Mating was performed using a ratio of 1:2 (male to female). Females were paired for cohabitation in batches in order to control the number of animals for terminal sacrifice on a particular day. At the subsequent mornings, the vaginal smear of the female was checked to confirm the pregnancy. The day on which sperms were observed in the vaginal smear was considered as gestation day ‘0’. Mated females were assigned in an unbiased manner to the control and treatment groups ensuring that group mean body weights were comparable with each other. Each animal was assigned a unique identification number. After getting 100 sperm positive females, the remaining females and males were discarded without any observations.
Duration of treatment / exposure:
The animals were treated with the test item formulation or vehicle on 7 days per week from gestation day 5 until gestation day 19.
Frequency of treatment:
Once per day. 7 days per week
Duration of test:
On GD 20, i.e. the day prior to the expected day of delivery, the presumed pregnant females were subjected to a caesarean section.
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day
Remarks:
20 mg/mL
Dose / conc.:
300 mg/kg bw/day
Remarks:
60 mg/mL
Dose / conc.:
1 000 mg/kg bw/day
Remarks:
200 mg/mL
No. of animals per sex per dose:
156 animals (52 males and 104 females) were included in the study.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Doses were selected according to the results of the dose range finding study (BSL Munich study no. 176886, non-GxP) and in consultation with the sponsor. The highest dose level was chosen with the aim of inducing toxic effects, but not death or severe suffering. Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dose-related response and a NOAEL.
The animals in the control group were handled in an identical manner to the test group subjects and received the vehicle using the same volume as used for the treatment groups.

- Rationale for animal assignment (if not random): Mated females were assigned in an unbiased manner to the control and treatment groups ensuring that the mean body weights were comparable to each other

Examinations

Maternal examinations:
Clinical Observations
General clinical observations were made at least once a day, preferably at the same time each day. The health condition of the animals was recorded. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily.
Clinical observations included spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size. Changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypes, difficult or prolonged parturition or bizarre behaviour were recorded.

Body Weight and Food Consumption
All animals were weighed once before initiation of pairing to ensure that the body weights are within + 20 % variation.
The sperm positive females were weighed during gestations days 0, 5, 8, 11, 14, 17 and 20. Males were not weighed in this study except once before initiation of pairing.
Food consumption of sperm positive females was measured on gestations days 5, 8, 11, 14, 17 and 20.
Food consumption was not measured for males during the entire study or for both male and females during the mating period.

Post-Mortem Examination
On gestation day 20, sperm positive were subjected to a caesarean section after sacrificing the animals using an overdose of pentobarbital injected intraperitoneally at a dosage of approximately 8 mL/kg bw.
At the time of termination or death during the study, the dam (presumably pregnant female) was examined macroscopically for any structural abnormalities or pathological changes which may have influenced the pregnancy. Any macroscopic findings were preserved in 4 % neutral-buffered formaldehyde.
Immediately after the termination or as soon as possible after death, the uteri were removed and the pregnancy status of the dams was confirmed. Uteri that appear non-gravid were further examined by staining with 10 % ammonium sulphide solution to confirm the non-pregnant status.
Each gravid uterus with the cervix was weighed. 
The number of corpora lutea was counted for pregnant animals. The uterine contents were examined for embryonic or foetal deaths as well as the number of viable foetuses. The degree of resorption (late and early) was confirmed in order to help estimate the relative time of death of the conceptus. The position and number of foetuses in each uterine horn was also recorded.
Males were sacrificed without any observations at any time after the completion of the mating or were used for other studies.

Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight including cervix: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter

FOETAL EVALUATIONS
All foetuses from a particular dam were identified by using numbered plates and were weighed and sexed based on the anogenital distance. Each foetus was examined for external anomalies.
One half of each litter was examined for soft tissue anomalies by a microdissection technique. Inadvertently, visceral examination of the pups of animal no. 76 and 77 was performed one day after necropsy.
The remaining foetuses were processed by Alizarin staining and the first 20 litters per group were examined for skeletal alterations.
Craniofacial examination of the heads of the foetuses used for the soft tissue examination of the first 20 litters per group were performed for internal structure including the eyes, brain, nasal passage and tongue by razor blade serial sectioning technique.

EXTERNAL EXAMINATION
Lip and palate were examined for cleft lip and palate by gently opening the mouth with forceps. The head, eyes, ears, jaw and snout was examined for the shape and size. The trunk was examined for any external abnormalities. Limbs were examined for shape, size, position and digits for number and depth of digital furrows. The tail was examined for presence, size, shape and position.

SKELETAL EXAMINATION
Foetuses scheduled for the skeletal examination were eviscerated and the entire litter was transferred into plastic bottles containing 95 % ethanol. These foetuses were processed using the Alizarin red staining technique. After fixation in 95 % ethanol, the foetuses were macerated with a 1 % aqueous potassium hydroxide solution for 1 day and transferred to an Alizarin red solution (0.0025 % in 1 % aqueous potassium hydroxide) for 1 day. After that the foetuses were again transferred to 1 % KOH. Alizarin stained foetuses were then cleared and dehydrated in a solution containing 2 parts of 70 % ethanol, 2 parts of glycerin and one part of benzyl alcohol for 1 day and finally preserved in a 1:1 solution of 70 % ethanol and glycerin.
The stained foetuses were examined under the stereomicroscope, the skull was examined for size, shape and degree of ossification of nasal, parietal, interparietal, supraoccipital, exoccipital, lacrimal, zygomatic (malar), squamosal (temporal), premaxillary, maxillary, basisphenoid, hyoid and tympanic ring (annulus). Similarly, the vertebral centres, ribs and sterna centres were also examined for size, shape and counted for the number of ossification centers. The cervical, thoracic, lumbar, sacral, caudal vertebrae were observed for the ossification of centers and arches. Pelvic girdles, fore limbs and hind limbs were examined for the development of the bones. Any deviation from the normal development was recorded for each foetus.

VISCERAL EXAMINATION
Foetuses scheduled for the visceral examination were pinned to a paraffin covered petri dish with the ventral side up under a stereo microscope. The abdominal and thoracic cavities of all foetuses were dissected and examined for visceral anomalies.
The intestine, stomach, spleen and pancreas were examined for size and position. The liver was examined for size, shape, colour and number of lobes. The kidney and adrenal glands were observed for size, position and colour. The kidneys were further observed for the presence of clear fluid-filled cysts, cortical cysts, pitting or granular appearance and then sectioned with a sharp scalpel blade to examine the pelvis for distention or the presence of calculi or white granular material. The left kidney was sectioned with one longitudinal slice just off center and the right kidney was sectioned with one transverse slice directly through the papilla. The capsule, cortex, medulla, renal papilla, and renal pelvis were checked for the presence and the pelvis for distension with fluid.
The reproductive organs were exposed by raising the intestine and the attached viscera from the dorsal wall and examined for any developmental defect.
The rib cage was cut from the side of the sternebrae and xyphisternum (6th sternebra) to examine the thoracic organs. The lung was observed for size, colour and number of lobes. The thymus gland was checked for size and position. The trachea and oesophagus were exposed by removing the thymus gland and examined for fusion or tracheaoesophageal fistula.
The position, size, colour and shape of the heart was recorded. The pericardial sac was opened and the heart was fully exposed and examined for the presence or absence of major blood vessels like aortic arch, pulmonary artery and ductus arteriosus. For an examination of the internal anatomy of the heart, the heart was then repositioned and two cuts through the right ventricle were made using micro-dissecting scissors. The first cut was taken starting from the right of the ventral midline surface at the apex to the base of the pulmonary artery and the second cut was made through the midline surface at the apex extending to the left ventricle in to the ascending aorta. Incisions were opened with fine forceps for the examination of interventricular and auriculo ventricular septum.
After the completion of the visceral examination by the microdissection technique, foetuses were transferred to plastic bottles containing formalin-aceto-alcohol for later craniofacial examination by the razor-blade-serial-section technique.

CRANIOFACIAL EXAMINATION
Before initiating the serial sectioning with a razor blade, foetuses were transferred to the beaker containing tap water for deformalisation. After deformalisation, a single foetus was decapitated and the head of the fetus was subjected to 5-7 sections in order to observe the internal structures of the head including the symmetry of the external nares, nasal conche, nasal septum, palate, the development of the cerebellum and brain stem. Transverse sections of the cephalic region were observed under the stereomicroscope and any anomalies were recorded.
Statistics:
A statistical assessment of the results of the body weight, food consumption was performed by comparing values of dosed with control animals using a one-way ANOVA and a post-hoc Dunnett Test. Foetal evaluation parameters like external, visceral, craniofacial and skeletal parameters were analysed using Fisher’s exact test. The statistics were performed with GraphPad Prism V.6.01 software (p<0.05 is considered as statistically significant).

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
In terminally sacrificed females, predominant clinical signs observed were moving the bedding (16/25 in HD), increased salivation (5/25 in HD) and piloerection (4/25 in Control and LD, 7/25 in MD and 12/25 in HD) were observed on few days during the treatment period of the study.
There were also low incidences of clinical signs like alopecia on various body parts (1/25 in LD MD and HD), crust left ventral thoracic region (1/25 in MD) and sunken flanks (1/25 in LD)
As moving the bedding and salivation was noted mainly immediately after test item administration and just for a short period, this transient sign was considered to be a sign due to a local reaction to the test item rather than a systemic adverse effect. Piloerection was observed on very few days and in all groups including control. Therefore All clinical signs observed in terminally sacrificed treatment group females were of no toxicological relevance or non adverse in nature.
None of the females showed signs of abortion prior to the scheduled sacrifice.
Mortality:
no mortality observed
Description (incidence):
No mortality was observed during the treatment period and all animals survived until end of the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The mean body weight and body weight gain remained unaffected throughout the study period. No statistical significance was achieved in any treatment groups on any day or interval of body weight measurement and all values in treatment groups were comparable with the controls.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
In correlation to the body weight and body weight gain, food consumption in all treatment groups was comparable to the controls and no statistically significant effect was observed on food consumption in treatment groups on any interval of food consumption measurement when compared with the controls.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No gross pathological changes of toxicological relevance were observed during the macroscopic examination of the females of the LD, MD and HD groups.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Details on results:
Clinical Signs
In terminally sacrificed females, predominant clinical signs observed were moving the bedding (16/25 in HD), increased salivation (5/25 in HD) and piloerection (4/25 in Control and LD, 7/25 in MD and 12/25 in HD) were observed on few days during the treatment period of the study.
There were also low incidences of clinical signs like alopecia on various body parts (1/25 in LD MD and HD), crust left ventral thoracic region (1/25 in MD) and sunken flanks (1/25 in LD)
As moving the bedding and salivation was noted mainly immediately after test item administration and just for a short period, this transient sign was considered to be a sign due to a local reaction to the test item rather than a systemic adverse effect. Piloerection was observed on very few days and in all groups including control. Therefore All clinical signs observed in terminally sacrificed treatment group females were of no toxicological relevance or non adverse in nature.
None of the females showed signs of abortion prior to the scheduled sacrifice.

Mortality
No mortality was observed during the treatment period and all animals survived until end of the study.

Body Weight and Weight Changes
The mean body weight and body weight gain remained unaffected throughout the study period. No statistical significance was achieved in any treatment groups on any day or interval of body weight measurement and all values in treatment groups were comparable with the controls.

Food Consumption
In correlation to the body weight and body weight gain, food consumption in all treatment groups was comparable to the controls and no statistically significant effect was observed on food consumption in treatment groups on any interval of food consumption measurement when compared with the controls.

Pathology
No gross pathological changes of toxicological relevance were observed during the macroscopic examination of the females of the LD, MD and HD groups.

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
None of the females showed signs of abortion prior to the scheduled sacrifice.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
No effect observed.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
No effect observed.
Early or late resorptions:
no effects observed
Description (incidence and severity):
No effect observed.
Dead fetuses:
no effects observed
Description (incidence and severity):
No effect observed.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
No female showed signs of premature delivery prior to the scheduled sacrifice on gestation day 20.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
no effect observed.
Details on maternal toxic effects:
Details on maternal toxic effects:


see also: CLINICAL SIGNS
None of the females showed signs of abortion prior to the scheduled sacrifice.

PRENATAL DATA
No test item-related effects of toxicological relevance were noted for any prenatal parameters like terminal body weight, uterus weight, adjusted maternal weight, number of corpora lutea, implantation sites, early and late resorptions, number of live foetuses, number of male and female foetuses, number of foetuses in each uterine horn, sex ratio, and percent pre- and post-implantation loss in treatment groups when compared to the controls. No dead foetuses were noted in any of the groups.
Successful mating resulted in 23/25 pregnancies in the LD group, 21/25 in the MD group and 24/25 in the HD group compared to 20/26 pregnancies in the control group. The marginally low pregnancy rates (no. of pregnancies / no. of females mated or sperm positive x 100) of 76 % in the control group compared to treatment groups (92 % in LD, 84 % in MD and 96 % in HD) was considered to be a biological variation.

Effect levels (maternal animals)

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

Maternal abnormalities

Key result
Abnormalities:
no effects observed
Localisation:
not specified

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
see Details on embryotoxic / teratogenic effects
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
see Details on embryotoxic / teratogenic effects
Changes in sex ratio:
no effects observed
Description (incidence and severity):
see Details on embryotoxic / teratogenic effects
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
see Details on embryotoxic / teratogenic effects
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
see Details on embryotoxic / teratogenic effects
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
see Details on embryotoxic / teratogenic effects
Visceral malformations:
no effects observed
Description (incidence and severity):
see Details on embryotoxic / teratogenic effects
Details on embryotoxic / teratogenic effects:
LITTER DATA
There were no test item-related effects of toxicological relevance observed for the number of live foetuses, mean foetus weight, total litter weight, male and female litter weight and sex ratio in any of the treatment groups when compared with the controls.

EXTERNAL EXAMINATION
There were no external abnormalities considered to be of toxicological relevance in any of the dose groups. Statistical analysis of data revealed no significant differences compared to the control group.
Low incidences of haematoma on various body parts (1 each in control, LD, MD and HD) and short right forelimb (1 in HD) were noted in isolated females. As these findings were observed mostly in single foetuses, they were considered to be incidental in nature and unrelated to the treatment.

VISCERAL EXAMINATION
Internal observation of the foetal viscera by free hand microdissection technique revealed a range of visceral findings in all groups including control.
Visceral findings observed in the dose groups were at frequencies generally comparable to or in some cases slightly higher or lower in frequency compared to controls. As observed findings were either minor variations, within historical control data range and/or due to a lack of dose dependency and consistency, no toxicological significance can be attributed to these findings and they were considered to be spontaneous in nature. 
All litter incidences were statistically insignificant when compared with the control. There was higher litter incidence of umbilical artery transposed, convoluted ureter and bilateral dilatation of ureter observed in all treatment groups including control group. However, values were well within historical control data range (85.71 % - umbilical artery transposed, 73.91 %- convoluted ureter and 87.50 % - dilated ureter). There was discolouration or dark spots on few organs like spleen, lung, liver, adrenal gland, kidneys and thymus observed without dose dependency. Discolouration of organs was considered likely to reflect the consequence of a functional disorder and thus not strictly as developmental anomalies. Due to lack of dose dependency and consistency, these discolouration findings were not considered as toxicologically relevant.
Dilated/convoluted ureter (bilateral) is the common finding in rodent studies and is classified as a variation as it is transient and likely to be postnatally reversible. Furthermore, values were within the historical control data so that no toxicological relevance was attributed to it.

CRANIOFACIAL EXAMINATION
Craniofacial examination by razor blade serial sectioning technique revealed few predominant findings (subcutaneous haematoma and or oedema, slightly dilated nasal cavity, retinal fold, slightly dilated 3rd ventricle and lateral ventricle) at low frequencies generally comparable to or in some cases slightly higher or lower in frequency in the dose groups compared to the controls. These findings were considered to be spontaneous in nature and not related to the treatment with the test item. Statistical analysis of the data revealed no statistical significance of any of the findings.

SKELETAL EXAMINATION
Skeletal examination of the Alizarin red stained foetuses revealed a range of findings which occurred at an incidence generally comparable to or slightly lower or higher in the dose groups when compared to the control group.
A statistically significant increase in litter incidence for increased ossification of 6th sternebra (25 % compared to 0 % in controls, p< 0.05) in the MD group, 14th rudimentary rib (B) (95 % compared to 60 % in controls, p< 0.05) in HD group and statistically significant decrease in litter incidence for increased ossification of 2nd sternebra (0 % compared to 25 % in controls, p< 0.05) in the LD group compared to the control group was considered to be incidental as frequencies were either within historical control data or not dose dependent. Therefore, these findings were not considered as treatment-related adverse findings and solely spontaneous in nature.
Slightly higher litter incidences compared to the concurrent control group, but without achieving statistical significance were observed in the HD group for incomplete ossification of frontal (R) (15 % compared to 0 % in controls), parietal (R) (30 % compared to 20 % in controls), squamosal (B) ( 30 % compared to 15 % in controls), supraoccipital (90 % compared to 75 % in controls), zygomatic arch (L) (10 % compared to 0 % in controls), zygomatic arch (R) (10 % compared to 5 % in controls), 5th sternebra (75 % compared to 60 % in controls), increased ossification of orbital socket region (B) of skull (40 % compared to 30 % in controls), increased ossification of orbital socket region (R) of skull (25 % compared to 5 % in controls), increased ossification of hindlimb metatarsal (30 % compared to 5 % in controls), unossified 5th sternebra (10 % compared to 5 % in controls), unossified 6th sternebra (10 % compared to 5 % in controls), ribs fused (10 % compared to 0 % in controls), ribs absent (10 % compared to 0 % in controls), full 14th rib (B) (20 % compared to 15 % in controls), full 14th rib (R) (20 % compared to 5 % in controls), rudimentary 14th rib (B) (95 % compared to 65 % in controls), rudimentary 14th rib (L) (65 % compared to 40 % in controls), fused caudal vertebral arch and centrum (35 % compared to 25 % in controls), moderately
irregularly ossified cervical vertebral arch (30 % compared to 5 % in controls), rudimentary 7th cervical rib (B) (10 % compared to 0 % in controls) and slightly irregularly ossified thoracic vertebral centrum (45 % compared to 35 % in controls).  
The observed reduced ossification or unossification without achieving statistical significance of few bones in the HD group that normally exhibit rapid ossification in the last days of gestation and values were within historical control data. Generally delayed ossification is not regarded to persist postnatally and not associated with long-term consequences on survival, general growth and development and therefore is not considered to be adverse.
Rudimentary/short ribs are considered as transient findings. In contrast, full/long ribs are considered permanent and may cause health effects in humans; however, postnatal consequences in rats are unknown but are assumed to be minimal. Furthermore, litter incidences for rudimentary/short or full ribs and all other findings mentioned above were within historical control data range and therefore these findings were not considered to be treatment-related or adverse but spontaneous in nature.
There was no statistical significance and no indication of a test item-related trend in the type and/or incidences of other skeletal findings and they were therefore considered to be spontaneous in nature.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Under the conditions of the study no toxic effects were observed.
Remarks on result:
other: see Conclusion
Remarks:
No effects of Propyl 4-hydroxybenzoate on females and foetuses were found at dose levels up to 1000 mg/kg body weight/day. The NOAEL for both maternal toxicity and foetal toxicity of Propyl 4-hydroxybenzoate in this study is considered to be 1000 mg/kg body weight/day.

Fetal abnormalities

Key result
Abnormalities:
effects observed, non-treatment-related
Localisation:
skeletal: sternum
skeletal: rib
Description (incidence and severity):
A statistically significant increase in litter incidence for increased ossification of 6th sternebra (25 % compared to 0 % in controls, p< 0.05) in the MD group, 14th rudimentary rib (B) (95 % compared to 60 % in controls, p< 0.05) in HD group and statistically significant decrease in litter incidence for increased ossification of 2nd sternebra (0 % compared to 25 % in controls, p< 0.05) in the LD group compared to the control group was considered to be incidental as frequencies were either within historical control data or not dose dependent. Therefore, these findings were not considered as treatment-related adverse findings and solely spontaneous in nature.

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

Prenatal Data

Group

 

Dead Foetuses

No. of Male Foetuses

No. of Female Foetuses

Sex Ratio

Pre Implantation Loss (%)

Post Implantation Loss (%)

C

Mean

0.00

5.45

5.25

1.34

12.57

6.23

SD

0.00

2.26

2.34

1.12

22.40

7.28

N

20

20

20

19

20

20

LD

Mean

0.00

5.96

4.96

1.59

5.93

8.95

SD

0.00

2.51

2.06

1.35

9.26

10.78

N

23

23

23

23

23

23

MD

Mean

0.00

5.86

5.29

1.46

6.95

5.36

SD

0.00

1.77

2.43

0.93

10.98

7.26

N

21

21

21

21

21

21

HD

Mean

0.00

4.92

6.17

0.87

8.71

8.07

SD

0.00

1.38

1.71

0.39

14.40

6.57

N

24

24

24

24

24

24

Group

 

Terminal Body Weight (g)

Uterus Weight (g)

Adjusted Maternal Weight (g)

Corpora Lutea

Implantation Sites

Live Foetuses

Early Resporptions

Late Resporptions

Total Resorptions

C

Mean

340.45

58.84

281.61

12.90

11.40

10.70

0.75

0.00

0.75

SD

24.42

17.15

12.46

1.83

3.42

3.39

0.79

0.00

0.79

N

20

20

20

20

20

20

20

20

20

LD

Mean

335.57

60.24

275.32

12.70

11.96

10.91

1.00

0.04

1.04

SD

27.74

13.23

19.32

1.79

2.08

2.43

1.28

0.21

1.26

N

23

23

23

23

23

23

23

23

23

MD

Mean

332.71

61.63

271.08

12.62

11.76

11.14

0.62

0.00

0.62

SD

15.26

10.42

12.59

1.20

1.81

2.01

0.80

0.00

0.80

N

21

21

21

21

21

21

21

21

21

HD

Mean

334.17

61.78

272.39

13.21

12.04

11.08

1.00

0.00

1.00

SD

19.62

11.48

12.71

1.18

2.18

2.12

0.78

0.00

0.78

N

24

24

24

24

24

24

24

24

24

Group

 

Mean Foetuses Weight (g)

Number of Males

Number of Females

Total No. of Foetuses

Total Litter Weight (g)

Male Litter Weight (g)

Female Litter Weight (g)

C

Mean

3.64

5.45

5.25

10.70

38.24

19.77

18.47

SD

0.25

2.26

2.34

3.39

11.72

8.01

8.04

N

20

20

20

20

20

20

20

LD

Mean

3.59

5.96

4.96

10.91

39.26

21.97

17.29

SD

0.22

2.51

2.06

2.43

9.15

9.65

7.47

N

23

23

23

23

23

23

23

MD

Mean

3.69

5.86

5.29

11.14

40.94

22.11

18.83

SD

0.24

1.77

2.43

2.01

7.32

6.76

8.76

N

21

21

21

21

21

21

21

HD

Mean

3.67

4.92

6.17

11.08

40.56

18.60

21.96

SD

0.23

1.38

1.71

2.12

7.98

5.21

6.49

N

24

24

24

24

24

24

24

Group

Animal No.

No. of Fetuses in left Horn

No. of Fetuses in right Horn

Total

C

Mean

4.8

5.9

10.7

SD

2.0

2.1

3.4

N

20

20

20

LD

Mean

5.7

5.2

10.9

SD

2.1

1.8

2.4

N

23

23

23

MD

Mean

5.1

6.0

11.1

SD

2.3

2.0

2.0

N

21

21

21

HD

Mean

5.3

5.8

11.1

SD

1.4

1.7

2.1

N

24

24

24

Group

 

Gestation

Day 0

Day 5

Day 8

Day 11

Day 14

Day 17

Day 20

C

Mean

235.20

251.55

258.20

268.85

277.35

301.65

340.45

SD

7.41

8.42

8.19

10.56

12.70

14.57

24.42

N

20

20

20

20

20

20

20

LD

Mean

232.87

246.04

251.65

264.74

274.78

299.91

335.57

SD

7.26

15.16

17.76

14.91

15.95

17.10

27.74

N

23

23

23

23

23

22

23

% of control

99

98

97

98

99

99

99

MD

Mean

231.52

246.35

250.95

262.35

271.00

296.95

332.71

SD

7.94

10.92

10.66

12.00

11.87

12.83

15.26

N

21

20

20

20

20

20

21

% of control

98

98

97

98

98

98

98

HD

Mean

231.79

247.35

252.70

263.35

273.35

297.22

334.17

SD

8.51

9.78

11.08

11.92

13.05

14.73

19.62

N

24

23

23

23

23

23

24

% of control

99

98

98

98

99

99

98

Foetal External findings- Summary

External Findings

Group

C

LD

MD

HD

Tail haematoma

No of Incidences

1

0

0

0

Total No of Observed Foetuses

214

251

234

266

% Fetal Incidence

0.47

0.00

0.00

0.00

No of Litters with at least 1 Incidence

1

0

0

0

Total No of Litters Observed

20

23

21

24

% Litter Incidence

5.00

0.00

0.00

0.00

Flank (R) haematoma

No of Incidences

0

1

0

0

Total No of Observed Foetuses

214

251

234

266

% Fetal Incidence

0.00

0.40

0.00

0.00

No of Litters with at least 1 Incidence

0

1

0

0

Total No of Litters Observed

20

23

21

24

% Litter Incidence

0.00

4.35

0.00

0.00

Tip of the nose: cutis lesion

No of Incidences

0

0

1

0

Total No of Observed Foetuses

214

251

234

266

% Fetal Incidence

0.00

0.00

0.43

0.00

No of Litters with at least 1 Incidence

0

0

1

0

Total No of Litters Observed

20

23

21

24

% Litter Incidence

0.00

0.00

4.76

0.00

Head lateral (L) haematoma

No of Incidences

0

0

1

0

Total No of Observed Foetuses

214

251

234

266

% Fetal Incidence

0.00

0.00

0.43

0.00

No of Litters with at least 1 Incidence

0

0

1

0

Total No of Litters Observed

20

23

21

24

% Litter Incidence

0.00

0.00

4.76

0.00

Ventral thoracic region (R) haematoma

No of Incidences

0

0

0

1

Total No of Observed Foetuses

214

251

234

266

% Fetal Incidence

0.00

0.00

0.00

0.38

No of Litters with at least 1 Incidence

0

0

0

1

Total No of Litters Observed

20

23

21

24

% Litter Incidence

0.00

0.00

0.00

4.17

Forelimb (R) short

No of Incidences

0

0

0

1

Total No of Observed Foetuses

214

251

234

266

% Fetal Incidence

0.00

0.00

0.00

0.38

No of Litters with at least 1 Incidence

0

0

0

1

Total No of Litters Observed

20

23

21

24

% Litter Incidence

0.00

0.00

0.00

4.17

Skeletal Finding

Group

C

LD

MD

HD

Skull

Skull auditory ossicles increased ossification

No of Incidences

3

5

7

4

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

2.65

4.20

6.09

3.42

No of Litters with at least 1 Incidence

3

1

3

4

Total No of Observed Litters

20

20

20

20

% Litter Incidence

15.00

5.00

15.00

20.00

Skull basioccipital
incomplete ossification

No of Incidences

3

3

6

1

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

2.65

2.52

5.22

0.85

No of Litters with at least 1 Incidence

3

3

6

1

Total No of Observed Litters

20

20

20

20

% Litter Incidence

15.00

15.00

30.00

5.00

Skull basisphenoid
incomplete ossification

No of Incidences

3

3

1

0

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

2.65

2.52

0.87

0.00

No of Litters with at least 1 Incidence

2

3

1

0

Total No of Observed Litters

20

20

20

20

% Litter Incidence

10.00

15.00

5.00

0.00

Skull bony labyrinth (B) increased ossification

No of Incidences

0

1

0

0

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

0.00

0.84

0.00

0.00

No of Litters with at least 1 Incidence

0

1

0

0

Total No of Observed Litters

20

20

20

20

% Litter Incidence

0.00

5.00

0.00

0.00

Skull exoccipital (L) incomplete ossification

No of Incidences

0

1

0

0

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

0.00

0.84

0.00

0.00

No of Litters with at least 1 Incidence

0

1

0

0

Total No of Observed Litters

20

20

20

20

% Litter Incidence

0.00

5.00

0.00

0.00

Skull frontal (B)
incomplete ossification

No of Incidences

10

7

5

2

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

8.85

5.88

4.35

1.71

No of Litters with at least 1 Incidence

3

4

2

2

Total No of Observed Litters

20

20

20

20

% Litter Incidence

15.00

20.00

10.00

10.00

Skeletal Finding

Group

C

LD

MD

HD

Skull

Skull frontal (L)
incomplete ossification

No of Incidences

1

1

0

2

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

0.88

0.84

0.00

1.71

No of Litters with at least 1 Incidence

1

1

0

2

Total No of Observed Litters

20

20

20

20

% Litter Incidence

5.00

5.00

0.00

10.00

Skull frontal (R)
incomplete ossification

No of Incidences

0

1

2

3

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

0.00

0.84

1.74

2.56

No of Litters with at least 1 Incidence

0

1

1

3

Total No of Observed Litters

20

20

20

20

% Litter Incidence

0.00

5.00

5.00

15.00

Skull hyoid body
incomplete ossification

No of Incidences

1

0

1

1

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

0.88

0.00

0.87

0.85

No of Litters with at least 1 Incidence

1

0

1

1

Total No of Observed Litters

20

20

20

20

% Litter Incidence

5.00

0.00

5.00

5.00

Skull interparietal
incomplete ossification

No of Incidences

42

56

29

42

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

37.17

47.06

25.22

35.90

No of Litters with at least 1 Incidence

16

17

13

16

Total No of Observed Litters

20

20

20

20

% Litter Incidence

80.00

85.00

65.00

80.00

Skull nasal (B)
incomplete ossification

No of Incidences

1

1

1

1

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

0.88

0.84

0.87

0.85

No of Litters with at least 1 Incidence

1

1

1

1

Total No of Observed Litters

20

20

20

20

% Litter Incidence

5.00

5.00

5.00

5.00

Skull nasal (L)
incomplete ossification

No of Incidences

1

0

0

0

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

0.88

0.00

0.00

0.00

No of Litters with at least 1 Incidence

1

0

0

0

Total No of Observed Litters

20

20

20

20

% Litter Incidence

5.00

0.00

0.00

0.00

Skeletal Finding

Group

C

LD

MD

HD

Skull

Skull orbital socket region (B) increased ossification

No of Incidences

15

16

18

13

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

13.27

13.45

15.65

11.11

No of Litters with at least 1 Incidence

6

7

11

8

Total No of Observed Litters

20

20

20

20

% Litter Incidence

30.00

35.00

55.00

40.00

Skull orbital socket region (L) increased ossification

No of Incidences

7

4

3

9

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

6.19

3.36

2.61

7.69

No of Litters with at least 1 Incidence

5

4

3

5

Total No of Observed Litters

20

20

20

20

% Litter Incidence

25.00

20.00

15.00

25.00

Skull orbital socket region (R) increased ossification

No of Incidences

1

1

3

7

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

0.88

0.84

2.61

5.98

No of Litters with at least 1 Incidence

1

1

2

5

Total No of Observed Litters

20

20

20

20

% Litter Incidence

5.00

5.00

10.00

25.00

Skull parietal (B)
incomplete ossification

No of Incidences

22

26

17

16

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

19.47

21.85

14.78

13.68

No of Litters with at least 1 Incidence

9

14

8

9

Total No of Observed Litters

20

20

20

20

% Litter Incidence

45.00

70.00

40.00

45.00

Skull parietal (L)
incomplete ossification

No of Incidences

2

0

0

0

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

1.77

0.00

0.00

0.00

No of Litters with at least 1 Incidence

2

0

0

0

Total No of Observed Litters

20

20

20

20

% Litter Incidence

10.00

0.00

0.00

0.00

Skull parietal (R)
incomplete ossification

No of Incidences

7

12

3

7

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

6.19

10.08

2.61

5.98

No of Litters with at least 1 Incidence

4

10

3

6

Total No of Observed Litters

20

20

20

20

% Litter Incidence

20.00

50.00

15.00

30.00

Skeletal Finding

Group

C

LD

MD

HD

Skull

Skull premaxilla (B)
incomplete ossification

No of Incidences

0

0

0

2

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

0.00

0.00

0.00

1.71

No of Litters with at least 1 Incidence

0

0

0

1

Total No of Observed Litters

20

20

20

20

% Litter Incidence

0.00

0.00

0.00

5.00

Skull squamosal (B)
incomplete ossification

No of Incidences

6

10

5

6

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

5.31

8.40

4.35

5.13

No of Litters with at least 1 Incidence

3

5

3

6

Total No of Observed Litters

20

20

20

20

% Litter Incidence

15.00

25.00

15.00

30.00

Skull squamosal (L)
incomplete ossification

No of Incidences

2

0

1

0

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

1.77

0.00

0.87

0.00

No of Litters with at least 1 Incidence

2

0

1

0

Total No of Observed Litters

20

20

20

20

% Litter Incidence

10.00

0.00

5.00

0.00

Skull squamosal (R)
incomplete ossification

No of Incidences

1

2

1

1

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

0.88

1.68

0.87

0.85

No of Litters with at least 1 Incidence

1

2

1

1

Total No of Observed Litters

20

20

20

20

% Litter Incidence

5.00

10.00

5.00

5.00

Skull supraoccipital
incomplete ossification

No of Incidences

53

60

41

45

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

46.90

50.42

35.65

38.46

No of Litters with at least 1 Incidence

15

17

15

18

Total No of Observed Litters

20

20

20

20

% Litter Incidence

75.00

85.00

75.00

90.00

Skull zygomatic arch (B) incomplete ossification

No of Incidences

7

7

5

2

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

6.19

5.88

4.35

1.71

No of Litters with at least 1 Incidence

4

3

2

2

Total No of Observed Litters

20

20

20

20

% Litter Incidence

20.00

15.00

10.00

10.00

Skeletal Finding

Group

C

LD

MD

HD

Skull

Skull zygomatic arch (L) incomplete ossification

No of Incidences

0

0

0

2

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

0.00

0.00

0.00

1.71

No of Litters with at least 1 Incidence

0

0

0

2

Total No of Observed Litters

20

20

20

20

% Litter Incidence

0.00

0.00

0.00

10.00

Skull zygomatic arch (R) incomplete ossification

No of Incidences

1

3

1

2

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

0.88

2.52

0.87

1.71

No of Litters with at least 1 Incidence

1

2

1

2

Total No of Observed Litters

20

20

20

20

% Litter Incidence

5.00

10.00

5.00

10.00

Skull zygomatic arch (L) slightly fused
(zygomatic bone and zygomatic process (maxilla))

No of Incidences

0

1

0

0

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

0.00

0.84

0.00

0.00

No of Litters with at least 1 Incidence

0

1

0

0

Total No of Observed Litters

20

20

20

20

% Litter Incidence

0.00

5.00

0.00

0.00

Skull zygomatic arch (R)
moderately fused
(zygomatic bone and zygomatic process (maxilla))

No of Incidences

1

0

1

2

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

0.88

0.00

0.87

1.71

No of Litters with at least 1 Incidence

1

0

1

2

Total No of Observed Litters

20

20

20

20

% Litter Incidence

5.00

0.00

5.00

10.00

Skull zygomatic arch (R)
slightly fused
(zygomatic bone and zygomatic process (maxilla))

No of Incidences

0

0

1

0

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

0.00

0.00

0.87

0.00

No of Litters with at least 1 Incidence

0

0

1

0

Total No of Observed Litters

20

20

20

20

% Litter Incidence

0.00

0.00

5.00

0.00

Skeletal Finding

Group

C

LD

MD

HD

Scapula

Scapula (B) markedly bent

No of Incidences

0

1

1

0

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

0.00

0.84

0.87

0.00

No of Litters with at least 1 Incidence

0

1

1

0

Total No of Observed Litters

20

20

20

20

% Litter Incidence

0.00

5.00

5.00

0.00

Scapula (B) incomplete ossification

No of Incidences

0

0

0

1

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

0.00

0.00

0.00

0.85

No of Litters with at least 1 Incidence

0

0

0

1

Total No of Observed Litters

20

20

20

20

% Litter Incidence

0.00

0.00

0.00

5.00

Scapula (B) slightly bent

No of Incidences

1

0

0

0

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

0.88

0.00

0.00

0.00

No of Litters with at least 1 Incidence

1

0

0

0

Total No of Observed Litters

20

20

20

20

% Litter Incidence

5.00

0.00

0.00

0.00

Scapula (L) moderately bent

No of Incidences

1

1

0

0

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

0.88

0.84

0.00

0.00

No of Litters with at least 1 Incidence

1

1

0

0

Total No of Observed Litters

20

20

20

20

% Litter Incidence

5.00

5.00

0.00

0.00

Scapula (L) slightly bent

No of Incidences

0

2

0

0

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

0.00

1.68

0.00

0.00

No of Litters with at least 1 Incidence

0

2

0

0

Total No of Observed Litters

20

20

20

20

% Litter Incidence

0.00

10.00

0.00

0.00

Scapula (R) markedly bent

No of Incidences

4

2

0

0

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

3.54

1.68

0.00

0.00

No of Litters with at least 1 Incidence

4

2

0

0

Total No of Observed Litters

20

20

20

20

% Litter Incidence

20.00

10.00

0.00

0.00

Skeletal Finding

Group

C

LD

MD

HD

Scapula

Scapula (R) moderately bent

No of Incidences

2

1

1

0

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

1.77

0.84

0.87

0.00

No of Litters with at least 1 Incidence

2

1

1

0

Total No of Observed Litters

20

20

20

20

% Litter Incidence

10.00

5.00

5.00

0.00

Scapula (R) slightly bent

No of Incidences

2

2

0

1

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

1.77

1.68

0.00

0.85

No of Litters with at least 1 Incidence

2

2

0

1

Total No of Observed Litters

20

20

20

20

% Litter Incidence

10.00

10.00

0.00

5.00

Scapular spine(s) bent

No of Incidences

5

4

2

0

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

4.42

3.36

1.74

0.00

No of Litters with at least 1 Incidence

4

4

1

0

Total No of Observed Litters

20

20

20

20

% Litter Incidence

20.00

20.00

5.00

0.00

Sternebra

Sternebra (1st)
incomplete ossification

No of Incidences

0

0

0

2

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

0.00

0.00

0.00

1.71

No of Litters with at least 1 Incidence

0

0

0

1

Total No of Observed Litters

20

20

20

20

% Litter Incidence

0.00

0.00

0.00

5.00

Sternebra (2nd)
incomplete ossification

No of Incidences

7

0

2

2

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

6.19

0.00

1.74

1.71

No of Litters with at least 1 Incidence

5

0*

2

2

Total No of Observed Litters

20

20

20

20

% Litter Incidence

25.00

0.00

10.00

10.00

Sternebra (2nd)
unossified

No of Incidences

0

0

0

2

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

0.00

0.00

0.00

1.71

No of Litters with at least 1 Incidence

0

0

0

1

Total No of Observed Litters

20

20

20

20

% Litter Incidence

0.00

0.00

0.00

5.00

Skeletal Finding

Group

C

LD

MD

HD

Sternebra

Sternebra (3rd)
incomplete ossification

No of Incidences

0

0

0

2

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

0.00

0.00

0.00

1.71

No of Litters with at least 1 Incidence

0

0

0

1

Total No of Observed Litters

20

20

20

20

% Litter Incidence

0.00

0.00

0.00

5.00

Sternebra (4th)
incomplete ossification

No of Incidences

0

0

0

1

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

0.00

0.00

0.00

0.85

No of Litters with at least 1 Incidence

0

0

0

1

Total No of Observed Litters

20

20

20

20

% Litter Incidence

0.00

0.00

0.00

5.00

Sternebra (4th)
misaligned ossification

No of Incidences

0

0

1

1

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

0.00

0.00

0.87

0.85

No of Litters with at least 1 Incidence

0

0

1

1

Total No of Observed Litters

20

20

20

20

% Litter Incidence

0.00

0.00

5.00

5.00

Sternebra (5th)
incomplete ossification

No of Incidences

32

28

17

31

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

28.32

23.53

14.78

26.50

No of Litters with at least 1 Incidence

12

14

8

15

Total No of Observed Litters

20

20

20

20

% Litter Incidence

60.00

70.00

40.00

75.00

Sternebra (5th)
misaligned ossification

No of Incidences

1

0

0

0

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

0.88

0.00

0.00

0.00

No of Litters with at least 1 Incidence

1

0

0

0

Total No of Observed Litters

20

20

20

20

% Litter Incidence

5.00

0.00

0.00

0.00

Sternebra (5th) unossified

No of Incidences

2

0

0

3

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

1.77

0.00

0.00

2.56

No of Litters with at least 1 Incidence

1

0

0

2

Total No of Observed Litters

20

20

20

20

% Litter Incidence

5.00

0.00

0.00

10.00

Skeletal Finding

Group

C

LD

MD

HD

Sternebra

Sternebra (6th)
incomplete ossification

No of Incidences

90

84

84

79

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

79.65

70.59

73.04

67.52

No of Litters with at least 1 Incidence

20

17

20

19

Total No of Observed Litters

20

20

20

20

% Litter Incidence

100.00

85.00

100.00

95.00

Sternebra (6th)
increased ossification

No of Incidences

0

3

5

2

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

0.00

2.52

4.35

1.71

No of Litters with at least 1 Incidence

0

3

5*

2

Total No of Observed Litters

20

20

20

20

% Litter Incidence

0.00

15.00

25.00

10.00

Sternebra (6th) unossified

No of Incidences

1

0

0

3

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

0.88

0.00

0.00

2.56

No of Litters with at least 1 Incidence

1

0

0

2

Total No of Observed Litters

20

20

20

20

% Litter Incidence

5.00

0.00

0.00

10.00

Ribs

Ribs fused

No of Incidences

0

0

0

2

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

0.00

0.00

0.00

1.71

No of Litters with at least 1 Incidence

0

0

0

2

Total No of Observed Litters

20

20

20

20

% Litter Incidence

0.00

0.00

0.00

10.00

Rib(s) branched

No of Incidences

0

0

0

1

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

0.00

0.00

0.00

0.85

No of Litters with at least 1 Incidence

0

0

0

1

Total No of Observed Litters

20

20

20

20

% Litter Incidence

0.00

0.00

0.00

5.00

Rib(s) absent

No of Incidences

0

0

0

2

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

0.00

0.00

0.00

1.71

No of Litters with at least 1 Incidence

0

0

0

2

Total No of Observed Litters

20

20

20

20

% Litter Incidence

0.00

0.00

0.00

10.00

A statistically significant increase in litter incidence for increased ossification of 6thsternebra (25 % compared to 0 % in controls, p< 0.05) in the MD group, 14thrudimentary rib (B) (95 % compared to 60 % in controls, p< 0.05)in HD group and statistically significant decrease in litter incidence for increased ossification of 2ndsternebra (0 % compared to 25 % in controls, p< 0.05) in the LD group compared to the control group was considered to be incidental as frequencies were either within historical control data or not dose dependent. Therefore, these findings were not considered as treatment-related adverse findings and solely spontaneous in nature

Skeletal Finding

Group

C

LD

MD

HD

Ribs

Rib (14th) (B) full

No of Incidences

5

2

6

6

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

4.42

1.68

5.22

5.13

No of Litters with at least 1 Incidence

3

2

4

4

Total No of Observed Litters

20

20

20

20

% Litter Incidence

15.00

10.00

20.00

20.00

Rib (14th) (B) rudimentary

No of Incidences

28

28

35

36

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

24.78

23.53

30.43

30.77

No of Litters with at least 1 Incidence

12

14

17

19*

Total No of Observed Litters

20

20

20

20

% Litter Incidence

60.00

70.00

85.00

95.00

Rib (14th) (L) full

No of Incidences

4

5

3

4

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

3.54

4.20

2.61

3.42

No of Litters with at least 1 Incidence

3

4

3

3

Total No of Observed Litters

20

20

20

20

% Litter Incidence

15.00

20.00

15.00

15.00

Rib (14th) (L) rudimentary

No of Incidences

9

9

12

15

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

7.96

7.56

10.43

12.82

No of Litters with at least 1 Incidence

8

8

9

13

Total No of Observed Litters

20

20

20

20

% Litter Incidence

40.00

40.00

45.00

65.00

Rib (14th) (R) full

No of Incidences

1

1

3

4

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

0.88

0.84

2.61

3.42

No of Litters with at least 1 Incidence

1

1

3

4

Total No of Observed Litters

20

20

20

20

% Litter Incidence

5.00

5.00

15.00

20.00

Rib (14th) (R) rudimentary

No of Incidences

15

16

12

9

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

13.27

13.45

10.43

7.69

No of Litters with at least 1 Incidence

11

8

9

7

Total No of Observed Litters

20

20

20

20

% Litter Incidence

55.00

40.00

45.00

35.00

Skeletal Finding

Group

C

LD

MD

HD

Ribs

Rib (15th) (R) rudimentary

No of Incidences

0

0

0

1

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

0.00

0.00

0.00

0.85

No of Litters with at least 1 Incidence

0

0

0

1

Total No of Observed Litters

20

20

20

20

% Litter Incidence

0.00

0.00

0.00

5.00

Rib(s)
incomplete ossification

No of Incidences

0

0

0

1

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

0.00

0.00

0.00

0.85

No of Litters with at least 1 Incidence

0

0

0

1

Total No of Observed Litters

20

20

20

20

% Litter Incidence

0.00

0.00

0.00

5.00

Rib(s) wavy

No of Incidences

43

44

38

37

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

38.05

36.97

33.04

31.62

No of Litters with at least 1 Incidence

17

15

16

15

Total No of Observed Litters

20

20

20

20

% Litter Incidence

85.00

75.00

80.00

75.00

Vertebra

Vertebra caudal arch (es) incomplete ossification

No of Incidences

1

0

0

0

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

0.88

0.00

0.00

0.00

No of Litters with at least 1 Incidence

1

0

0

0

Total No of Observed Litters

20

20

20

20

% Litter Incidence

5.00

0.00

0.00

0.00

Vertebra caudal fused
(arch(es) and centrum(-tra))

No of Incidences

10

12

8

15

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

8.85

10.08

6.96

12.82

No of Litters with at least 1 Incidence

5

7

6

7

Total No of Observed Litters

20

20

20

20

% Litter Incidence

25.00

35.00

30.00

35.00

Vertebra cervical arch(es) incomplete ossification

No of Incidences

4

1

2

2

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

3.54

0.84

1.74

1.71

No of Litters with at least 1 Incidence

3

1

1

2

Total No of Observed Litters

20

20

20

20

% Litter Incidence

15.00

5.00

5.00

10.00

Skeletal Finding

Group

C

LD

MD

HD

Vertebra

Vertebra cervical arch(es) slightly
irregular ossified

No of Incidences

3

1

2

3

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

2.65

0.84

1.74

2.56

No of Litters with at least 1 Incidence

1

1

2

3

Total No of Observed Litters

20

20

20

20

% Litter Incidence

5.00

5.00

10.00

15.00

Vertebra cervical arch(es) moderately
irregular ossified

No of Incidences

1

3

5

6

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

0.88

2.52

4.35

5.13

No of Litters with at least 1 Incidence

1

3

4

6

Total No of Observed Litters

20

20

20

20

% Litter Incidence

5.00

15.00

20.00

30.00

Vertebra cervical arch(es) misshapen

No of Incidences

0

0

0

1

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

0.00

0.00

0.00

0.85

No of Litters with at least 1 Incidence

0

0

0

1

Total No of Observed Litters

20

20

20

20

% Litter Incidence

0.00

0.00

0.00

5.00

Vertebra cervical centrum(-tra) unossified

No of Incidences

87

96

89

86

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

76.99

80.67

77.39

73.50

No of Litters with at least 1 Incidence

19

20

20

19

Total No of Observed Litters

20

20

20

20

% Litter Incidence

95.00

100.00

100.00

95.00

Vertebra cervical rib (7th) (L) full

No of Incidences

0

1

1

0

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

0.00

0.84

0.87

0.00

No of Litters with at least 1 Incidence

0

1

1

0

Total No of Observed Litters

20

20

20

20

% Litter Incidence

0.00

5.00

5.00

0.00

Vertebra cervical rib (7th) (L) rudimentary

No of Incidences

2

2

3

0

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

1.77

1.68

2.61

0.00

No of Litters with at least 1 Incidence

2

2

2

0

Total No of Observed Litters

20

20

20

20

% Litter Incidence

10.00

10.00

10.00

0.00

Skeletal Finding

Group

C

LD

MD

HD

Vertebra

Vertebra cervical rib (7th) (R) full

No of Incidences

0

1

0

0

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

0.00

0.84

0.00

0.00

No of Litters with at least 1 Incidence

0

1

0

0

Total No of Observed Litters

20

20

20

20

% Litter Incidence

0.00

5.00

0.00

0.00

Vertebra cervical rib (7th) (R) rudimentary

No of Incidences

1

2

2

2

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

0.88

1.68

1.74

1.71

No of Litters with at least 1 Incidence

1

2

2

2

Total No of Observed Litters

20

20

20

20

% Litter Incidence

5.00

10.00

10.00

10.00

Vertebra cervical rib (7th) (B) full

No of Incidences

0

0

0

1

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

0.00

0.00

0.00

0.85

No of Litters with at least 1 Incidence

0

0

0

1

Total No of Observed Litters

20

20

20

20

% Litter Incidence

0.00

0.00

0.00

5.00

Vertebra cervical rib (7th) (B) rudimentary

No of Incidences

0

1

0

2

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

0.00

0.84

0.00

1.71

No of Litters with at least 1 Incidence

0

1

0

2

Total No of Observed Litters

20

20

20

20

% Litter Incidence

0.00

5.00

0.00

10.00

Vertebra lumbar centrum(-tra) hemicentric ossification

No of Incidences

0

0

0

1

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

0.00

0.00

0.00

0.85

No of Litters with at least 1 Incidence

0

0

0

1

Total No of Observed Litters

20

20

20

20

% Litter Incidence

0.00

0.00

0.00

5.00

Vertebra lumbar arch(es) increased ossification

No of Incidences

4

7

2

3

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

3.54

5.88

1.74

2.56

No of Litters with at least 1 Incidence

3

5

2

3

Total No of Observed Litters

20

20

20

20

% Litter Incidence

15.00

25.00

10.00

15.00

Skeletal Finding

Group

C

LD

MD

HD

Vertebra

Vertebra lumbar arch(es) unossified

No of Incidences

0

0

0

1

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

0.00

0.00

0.00

0.85

No of Litters with at least 1 Incidence

0

0

0

1

Total No of Observed Litters

20

20

20

20

% Litter Incidence

0.00

0.00

0.00

5.00

Vertebra lumbar centrum(-tra)
 split

No of Incidences

0

0

0

1

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

0.00

0.00

0.00

0.85

No of Litters with at least 1 Incidence

0

0

0

1

Total No of Observed Litters

20

20

20

20

% Litter Incidence

0.00

0.00

0.00

5.00

Vertebra lumbar centrum(-tra) asymmetric ossification

No of Incidences

0

1

0

0

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

0.00

0.84

0.00

0.00

No of Litters with at least 1 Incidence

0

1

0

0

Total No of Observed Litters

20

20

20

20

% Litter Incidence

0.00

5.00

0.00

0.00

Vertebra lumbar centrum(-tra) dumbbell ossification

No of Incidences

0

0

0

1

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

0.00

0.00

0.00

0.85

No of Litters with at least 1 Incidence

0

0

0

1

Total No of Observed Litters

20

20

20

20

% Litter Incidence

0.00

0.00

0.00

5.00

Vertebra sacral arch(es) incomplete ossification

No of Incidences

1

0

0

0

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

0.88

0.00

0.00

0.00

No of Litters with at least 1 Incidence

1

0

0

0

Total No of Observed Litters

20

20

20

20

% Litter Incidence

5.00

0.00

0.00

0.00

Vertebra sacral centrum(-tra) incomplete ossification

No of Incidences

0

0

0

1

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

0.00

0.00

0.00

0.85

No of Litters with at least 1 Incidence

0

0

0

1

Total No of Observed Litters

20

20

20

20

% Litter Incidence

0.00

0.00

0.00

5.00

Skeletal Finding

Group

C

LD

MD

HD

Vertebra

Vertebra sacral centrum(-tra) slightly irregular ossification

No of Incidences

0

0

0

1

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

0.00

0.00

0.00

0.85

No of Litters with at least 1 Incidence

0

0

0

1

Total No of Observed Litters

20

20

20

20

% Litter Incidence

0.00

0.00

0.00

5.00

Vertebra sacral fused
(centrum(-tra) and arch(es))

No of Incidences

18

19

10

13

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

15.93

15.97

8.70

11.11

No of Litters with at least 1 Incidence

8

9

8

8

Total No of Observed Litters

20

20

20

20

% Litter Incidence

40.00

45.00

40.00

40.00

Vertebra thoracic arch(es) malpositioned

No of Incidences

0

0

0

1

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

0.00

0.00

0.00

0.85

No of Litters with at least 1 Incidence

0

0

0

1

Total No of Observed Litters

20

20

20

20

% Litter Incidence

0.00

0.00

0.00

5.00

Vertebra thoracic arch(es) incomplete ossification

No of Incidences

1

0

0

0

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

0.88

0.00

0.00

0.00

No of Litters with at least 1 Incidence

1

0

0

0

Total No of Observed Litters

20

20

20

20

% Litter Incidence

5.00

0.00

0.00

0.00

Vertebra thoracic arch(es) small

No of Incidences

0

0

0

1

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

0.00

0.00

0.00

0.85

No of Litters with at least 1 Incidence

0

0

0

1

Total No of Observed Litters

20

20

20

20

% Litter Incidence

0.00

0.00

0.00

5.00

Vertebra thoracic arch(es) unossified

No of Incidences

0

0

0

1

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

0.00

0.00

0.00

0.85

No of Litters with at least 1 Incidence

0

0

0

1

Total No of Observed Litters

20

20

20

20

% Litter Incidence

0.00

0.00

0.00

5.00

Skeletal Finding

Group

C

LD

MD

HD

Vertebra

Vertebra thoracic centrum(-tra)
moderately irregular ossified

No of Incidences

3

2

4

4

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

2.65

1.68

3.48

3.42

No of Litters with at least 1 Incidence

3

2

4

4

Total No of Observed Litters

20

20

20

20

% Litter Incidence

15.00

10.00

20.00

20.00

Vertebra thoracic centrum(-tra)
slightly irregular ossified

No of Incidences

17

12

12

15

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

15.04

10.08

10.43

12.82

No of Litters with at least 1 Incidence

7

8

11

9

Total No of Observed Litters

20

20

20

20

% Litter Incidence

35.00

40.00

55.00

45.00

Vertebra thoracic centrum(-tra) asymmetric ossification

No of Incidences

1

1

1

2

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

0.88

0.84

0.87

1.71

No of Litters with at least 1 Incidence

1

1

1

2

Total No of Observed Litters

20

20

20

20

% Litter Incidence

5.00

5.00

5.00

10.00

Vertebra thoracic centrum(-tra) bipartite ossification

No of Incidences

1

0

0

1

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

0.88

0.00

0.00

0.85

No of Litters with at least 1 Incidence

1

0

0

1

Total No of Observed Litters

20

20

20

20

% Litter Incidence

5.00

0.00

0.00

5.00

Vertebra thoracic centrum(-tra) dumbbell ossification

No of Incidences

0

2

1

0

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

0.00

1.68

0.87

0.00

No of Litters with at least 1 Incidence

0

1

1

0

Total No of Observed Litters

20

20

20

20

% Litter Incidence

0.00

5.00

5.00

0.00

Vertebra thoracic centrum(-tra) hemicentric ossification

No of Incidences

0

0

0

1

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

0.00

0.00

0.00

0.85

No of Litters with at least 1 Incidence

0

0

0

1

Total No of Observed Litters

20

20

20

20

% Litter Incidence

0.00

0.00

0.00

5.00

Skeletal Finding

Group

C

LD

MD

HD

Vertebra

Vertebra thoracic centrum(-tra) large

No of Incidences

0

0

0

1

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

0.00

0.00

0.00

0.85

No of Litters with at least 1 Incidence

0

0

0

1

Total No of Observed Litters

20

20

20

20

% Litter Incidence

0.00

0.00

0.00

5.00

Vertebra thoracic centrum(-tra)
unossified

No of Incidences

0

0

0

1

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

0.00

0.00

0.00

0.85

No of Litters with at least 1 Incidence

0

0

0

1

Total No of Observed Litters

20

20

20

20

% Litter Incidence

0.00

0.00

0.00

5.00

Vertebra thoracic centrum(-tra)
malpositioned

No of Incidences

0

0

0

1

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

0.00

0.00

0.00

0.85

No of Litters with at least 1 Incidence

0

0

0

1

Total No of Observed Litters

20

20

20

20

% Litter Incidence

0.00

0.00

0.00

5.00

Vertebra thoracic arches
fused

No of Incidences

0

0

0

1

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

0.00

0.00

0.00

0.85

No of Litters with at least 1 Incidence

0

0

0

1

Total No of Observed Litters

20

20

20

20

% Litter Incidence

0.00

0.00

0.00

5.00

Forelimb

Forelimb humerus(-i)
markedly misshapen

No of Incidences

0

0

0

1

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

0.00

0.00

0.00

0.85

No of Litters with at least 1 Incidence

0

0

0

1

Total No of Observed Litters

20

20

20

20

% Litter Incidence

0.00

0.00

0.00

5.00

Forelimb humerus(-i)
moderately misshapen

No of Incidences

4

4

1

0

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

3.54

3.36

0.87

0.00

No of Litters with at least 1 Incidence

2

3

1

0

Total No of Observed Litters

20

20

20

20

% Litter Incidence

10.00

15.00

5.00

0.00

Skeletal Finding

Group

C

LD

MD

HD

Forelimb

Forelimb humerus(-i)
slightly misshapen

No of Incidences

3

0

1

1

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

2.65

0.00

0.87

0.85

No of Litters with at least 1 Incidence

3

0

1

1

Total No of Observed Litters

20

20

20

20

% Litter Incidence

15.00

0.00

5.00

5.00

Forelimb metacarpal(s) unossified

No of Incidences

11

9

3

12

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

9.73

7.56

2.61

10.26

No of Litters with at least 1 Incidence

8

5

2

8

Total No of Observed Litters

20

20

20

20

% Litter Incidence

40.00

25.00

10.00

40.00

Forelimb phalanges
unossified

No of Incidences

112

119

114

118

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

99.12

100.00

99.13

100.85

No of Litters with at least 1 Incidence

20

20

20

20

Total No of Observed Litters

20

20

20

20

% Litter Incidence

100.00

100.00

100.00

100.00

Forelimb radius(-ii)
 bent

No of Incidences

1

0

0

0

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

0.88

0.00

0.00

0.00

No of Litters with at least 1 Incidence

1

0

0

0

Total No of Observed Litters

20

20

20

20

% Litter Incidence

5.00

0.00

0.00

0.00

Hindlimb

Hindlimb metatarsal(s) increased ossification

No of Incidences

1

11

7

8

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

0.88

9.24

6.09

6.84

No of Litters with at least 1 Incidence

1

4

4

6

Total No of Observed Litters

20

20

20

20

% Litter Incidence

5.00

20.00

20.00

30.00

Hindlimb metatarsal(s) unossified

No of Incidences

0

0

0

1

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

0.00

0.00

0.00

0.85

No of Litters with at least 1 Incidence

0

0

0

1

Total No of Observed Litters

20

20

20

20

% Litter Incidence

0.00

0.00

0.00

5.00

Skeletal Finding

Group

C

LD

MD

HD

Hindlimb

Hindlimb calcaneus(-i)
increased ossification

No of Incidences

2

2

0

0

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

1.77

1.68

0.00

0.00

No of Litters with at least 1 Incidence

2

2

0

0

Total No of Observed Litters

20

20

20

20

% Litter Incidence

10.00

10.00

0.00

0.00

Hindlimb femur(-ora)
 slightly bent

No of Incidences

0

1

0

0

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

0.00

0.84

0.00

0.00

No of Litters with at least 1 Incidence

0

1

0

0

Total No of Observed Litters

20

20

20

20

% Litter Incidence

0.00

5.00

0.00

0.00

Hindlimb femur(-ora)
incomplete ossification

No of Incidences

0

0

0

2

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

0.00

0.00

0.00

1.71

No of Litters with at least 1 Incidence

0

0

0

1

Total No of Observed Litters

20

20

20

20

% Litter Incidence

0.00

0.00

0.00

5.00

Hindlimb fibula(-ae) incomplete ossification

No of Incidences

0

0

0

2

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

0.00

0.00

0.00

1.71

No of Litters with at least 1 Incidence

0

0

0

1

Total No of Observed Litters

20

20

20

20

% Litter Incidence

0.00

0.00

0.00

5.00

Hindlimb phalanges increased ossification

No of Incidences

0

1

0

0

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

0.00

0.84

0.00

0.00

No of Litters with at least 1 Incidence

0

1

0

0

Total No of Observed Litters

20

20

20

20

% Litter Incidence

0.00

5.00

0.00

0.00

Hindlimb tibia(-ae) incomplete ossification

No of Incidences

0

0

0

2

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

0.00

0.00

0.00

1.71

No of Litters with at least 1 Incidence

0

0

0

1

Total No of Observed Litters

20

20

20

20

% Litter Incidence

0.00

0.00

0.00

5.00

Skeletal Finding

Group

C

LD

MD

HD

Pelvic girdle

Pelvic girdle (B) caudal shift

No of Incidences

6

1

5

4

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

5.31

0.84

4.35

3.42

No of Litters with at least 1 Incidence

3

1

4

3

Total No of Observed Litters

20

20

20

20

% Litter Incidence

15.00

5.00

20.00

15.00

Pelvic girdle (L) caudal shift

No of Incidences

1

4

3

3

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

0.88

3.36

2.61

2.56

No of Litters with at least 1 Incidence

1

3

2

2

Total No of Observed Litters

20

20

20

20

% Litter Incidence

5.00

15.00

10.00

10.00

Pelvic girdle (R) caudal shift

No of Incidences

1

0

2

0

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

0.88

0.00

1.74

0.00

No of Litters with at least 1 Incidence

1

0

2

0

Total No of Observed Litters

20

20

20

20

% Litter Incidence

5.00

0.00

10.00

0.00

Pelvic girdle ilium (B) incomplete ossification

No of Incidences

0

0

0

2

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

0.00

0.00

0.00

1.71

No of Litters with at least 1 Incidence

0

0

0

1

Total No of Observed Litters

20

20

20

20

% Litter Incidence

0.00

0.00

0.00

5.00

Pelvic girdle pubis (B) incomplete ossification

No of Incidences

5

0

0

2

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

4.42

0.00

0.00

1.71

No of Litters with at least 1 Incidence

3

0

0

1

Total No of Observed Litters

20

20

20

20

% Litter Incidence

15.00

0.00

0.00

5.00

Pelvic girdle ischium (B) incomplete ossification

No of Incidences

0

0

0

2

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

0.00

0.00

0.00

1.71

No of Litters with at least 1 Incidence

0

0

0

1

Total No of Observed Litters

20

20

20

20

% Litter Incidence

0.00

0.00

0.00

5.00

Skeletal Finding

Group

C

LD

MD

HD

Clavicula

Clavicula (R) slightly misshapen

No of Incidences

1

0

0

0

Total No of Observed Foetuses

113

119

115

117

% Foetal Incidence

0.88

0.00

0.00

0.00

No of Litters with at least 1 Incidence

1

0

0

0

Total No of Observed Litters

20

20

20

20

% Litter Incidence

5.00

0.00

0.00

0.00

Visceral Findings

Group

C

LD

MD

HD

Ureter (L) convoluted

No of Incidences

7

15

11

12

Total No of Observed Foetuses

101

120

113

126

% Fetal incidence

6.93

12.50

9.73

9.52

No of Litters with at least 1 incidence

3

10

8

7

Total No of Observed Litters

19

23

21

24

% Litter incidence

15.79

43.48

38.10

29.17

Ureter (R) convoluted

No of Incidences

0

1

0

1

Total No of Observed Foetuses

101

120

113

126

% Fetal Incidence

0.00

0.83

0.00

0.79

No of Litters with at least 1 Incidence

0

1

0

1

Total No of Observed Litters

19

23

21

24

% Litter Incidence

0.00

4.35

0.00

4.17

Ureter (B) convoluted

No of Incidences

2

4

1

3

Total No of Observed Foetuses

101

120

113

126

% Fetal Incidence

1.98

3.33

0.88

2.38

No of Litters with at least 1 Incidence

2

4

1

2

Total No of Observed Litters

19

23

21

24

% Litter Incidence

10.53

17.39

4.76

8.33

Ureter (L) dilated

No of Incidences

5

9

5

7

Total No of Observed Foetuses

101

120

113

126

% Fetal Incidence

4.95

7.50

4.42

5.56

No of Litters with at least 1 Incidence

5

6

5

6

Total No of Observed Litters

19

23

21

24

% Litter Incidence

26.32

26.09

23.81

25.00

Ureter (R) dilated

No of Incidences

6

4

2

6

Total No of Observed Foetuses

101

120

113

126

% Fetal Incidence

5.94

3.33

1.77

4.76

No of Litters with at least 1 Incidence

4

3

1

5

Total No of Observed Litters

19

23

21

24

% Litter Incidence

21.05

13.04

4.76

20.83

Ureter (B) dilated

No of Incidences

17

23

19

23

Total No of Observed Foetuses

101

120

113

126

% Fetal Incidence

16.83

19.17

16.81

18.25

No of Litters with at least 1 Incidence

8

11

11

14

Total No of Observed Litters

19

23

21

24

% Litter Incidence

42.11

47.83

52.38

58.33

Visceral Findings

Group

C

LD

MD

HD

Umbilical artery transposed

No of Incidences

13

14

15

18

Total No of Observed Foetuses

101

120

113

126

% Fetal Incidence

12.87

11.67

13.27

14.29

No of Litters with at least 1 Incidence

9

11

11

13

Total No of Observed Litters

19

23

21

24

% Litter Incidence

47.37

47.83

52.38

54.17

Urinary bladder spotted red

No of Incidences

1

3

4

2

Total No of Observed Foetuses

101

120

113

126

% Fetal Incidence

0.99

2.50

3.54

1.59

No of Litters with at least 1 Incidence

1

3

3

2

Total No of Observed Litters

19

23

21

24

% Litter Incidence

5.26

13.04

14.29

8.33

Spleen discoloured white

No of Incidences

0

0

0

1

Total No of Observed Foetuses

101

120

113

126

% Fetal Incidence

0.00

0.00

0.00

0.79

No of Litters with at least 1 Incidence

0

0

0

1

Total No of Observed Litters

19

23

21

24

% Litter Incidence

0.00

0.00

0.00

4.17

Lung discolored red (R)

No of Incidences

0

1

0

0

Total No of Observed Foetuses

101

120

113

126

% Fetal Incidence

0.00

0.83

0.00

0.00

No of Litters with at least 1 Incidence

0

1

0

0

Total No of Observed Litters

19

23

21

24

% Litter Incidence

0.00

4.35

0.00

0.00

Liver discoloured dark

No of Incidences

3

2

0

3

Total No of Observed Foetuses

101

120

113

126

% Fetal Incidence

2.97

1.67

0.00

2.38

No of Litters with at least 1 Incidence

3

1

0

3

Total No of Observed Litters

19

23

21

24

% Litter Incidence

15.79

4.35

0.00

12.50

Liver spotted white

No of Incidences

1

0

0

0

Total No of Observed Foetuses

101

120

113

126

% Fetal Incidence

0.99

0.00

0.00

0.00

No of Litters with at least 1 Incidence

1

0

0

0

Total No of Observed Litters

19

23

21

24

% Litter Incidence

5.26

0.00

0.00

0.00

Visceral Findings

Group

C

LD

MD

HD

Renal pelvis dilated (R)

No of Incidences

1

1

1

1

Total No of Observed Foetuses

101

120

113

126

% Fetal Incidence

0.99

0.83

0.88

0.79

No of Litters with at least 1 Incidence

1

1

1

1

Total No of Observed Litters

19

23

21

24

% Litter Incidence

5.26

4.35

4.76

4.17

Renal pelvis dilated (L)

No of Incidences

0

1

0

0

Total No of Observed Foetuses

101

120

113

126

% Fetal Incidence

0.00

0.83

0.00

0.00

No of Litters with at least 1 Incidence

0

1

0

0

Total No of Observed Litters

19

23

21

24

% Litter Incidence

0.00

4.35

0.00

0.00

Renal pelvis dilated (B)

No of Incidences

0

1

0

1

Total No of Observed Foetuses

101

120

113

126

% Fetal Incidence

0.00

0.83

0.00

0.79

No of Litters with at least 1 Incidence

0

1

0

1

Total No of Observed Litters

19

23

21

24

% Litter Incidence

0.00

4.35

0.00

4.17

Thymus cranial (R) long

No of Incidences

1

1

3

0

Total No of Observed Foetuses

101

120

113

126

% Fetal Incidence

0.99

0.83

2.65

0.00

No of Litters with at least 1 Incidence

1

1

3

0

Total No of Observed Litters

19

23

21

24

% Litter Incidence

5.26

4.35

14.29

0.00

Adrenal glands (B) spotted red

No of Incidences

7

6

4

7

Total No of Observed Foetuses

101

120

113

126

% Fetal incidence

6.93

5.00

3.54

5.56

No of Litters with at least 1 incidence

3

5

4

4

Total No of Observed Litters

19

23

21

24

% Litter incidence

15.79

21.74

19.05

16.67

Adrenal glands (L) spotted red

No of Incidences

4

1

2

1

Total No of Observed Foetuses

101

120

113

126

% Fetal incidence

3.96

0.83

1.77

0.79

No of Litters with at least 1 incidence

3

1

2

1

Total No of Observed Litters

19

23

21

24

% Litter incidence

15.79

4.35

9.52

4.17

Visceral Findings

Group

C

LD

MD

HD

Adrenal glands (R) spotted red

No of Incidences

0

0

1

0

Total No of Observed Foetuses

101

120

113

126

% Fetal Incidence

0.00

0.00

0.88

0.00

No of Litters with at least 1 Incidence

0

0

1

0

Total No of Observed Litters

19

23

21

24

% Litter Incidence

0.00

0.00

4.76

0.00

Adrenal glands (B) spotted dark

No of Incidences

0

0

1

0

Total No of Observed Foetuses

101

120

113

126

% Fetal Incidence

0.00

0.00

0.88

0.00

No of Litters with at least 1 Incidence

0

0

1

0

Total No of Observed Litters

19

23

21

24

% Litter Incidence

0.00

0.00

4.76

0.00

Kidney (R) discoloured red

No of Incidences

0

1

0

0

Total No of Observed Foetuses

101

120

113

126

% Fetal Incidence

0.00

0.83

0.00

0.00

No of Litters with at least 1 Incidence

0

1

0

0

Total No of Observed Litters

19

23

21

24

% Litter Incidence

0.00

4.35

0.00

0.00

Innominate artery short

No of Incidences

1

0

1

0

Total No of Observed Foetuses

101

120

113

126

% Fetal Incidence

0.99

0.00

0.88

0.00

No of Litters with at least 1 Incidence

1

0

1

0

Total No of Observed Litters

19

23

21

24

% Litter Incidence

5.26

0.00

4.76

0.00

Innominate artery absent

No of Incidences

1

0

0

0

Total No of Observed Foetuses

101

120

113

126

% Fetal Incidence

0.99

0.00

0.00

0.00

No of Litters with at least 1 Incidence

1

0

0

0

Total No of Observed Litters

19

23

21

24

% Litter Incidence

5.26

0.00

0.00

0.00

Thymus spotted red

No of Incidences

0

0

2

0

Total No of Observed Foetuses

101

120

113

126

% Fetal Incidence

0.00

0.00

1.77

0.00

No of Litters with at least 1 Incidence

0

0

2

0

Total No of Observed Litters

19

23

21

24

% Litter Incidence

0.00

0.00

9.52

0.00

Visceral Findings

Group

C

LD

MD

HD

Thymus cranial (L) long

No of Incidences

0

1

3

4

Total No of Observed Foetuses

101

120

113

126

% Fetal Incidence

0.00

0.83

2.65

3.17

No of Litters with at least 1 Incidence

0

1

3

4

Total No of Observed Litters

19

23

21

24

% Litter Incidence

0.00

4.35

14.29

16.67

Thymus cranial (B) long

No of Incidences

0

2

2

0

Total No of Observed Foetuses

101

120

113

126

% Fetal Incidence

0.00

1.67

1.77

0.00

No of Litters with at least 1 Incidence

0

2

2

0

Total No of Observed Litters

19

23

21

24

% Litter Incidence

0.00

8.70

9.52

0.00

Craniofacial Findings

Group

C

LD

MD

HD

Ventricle (3rd) slightly dilated

No of Incidences

0

0

1

0

Total No of Observed Foetuses

101

103

108

105

% Fetal Incidence

0.00

0.00

0.93

0.00

No of Litters with at least 1 Incidence

0

0

1

0

Total No of Observed Litters

19

20

20

20

% Litter Incidence

0.00

0.00

5.00

0.00

Ventricle (3rd) dilated

No of Incidences

0

1

1

2

Total No of Observed Foetuses

101

103

108

105

% Fetal Incidence

0.00

0.97

0.93

1.90

No of Litters with at least 1 Incidence

0

1

1

1

Total No of Observed Litters

19

20

20

20

% Litter Incidence

0.00

5.00

5.00

5.00

Ventricle (lateral) (R) dilated

No of Incidences

0

1

0

0

Total No of Observed Foetuses

101

103

108

105

% Fetal Incidence

0.00

0.97

0.00

0.00

No of Litters with at least 1 Incidence

0

1

0

0

Total No of Observed Litters

19

20

20

20

% Litter Incidence

0.00

5.00

0.00

0.00

Ventricle (lateral) (B) dilated

No of Incidences

0

1

1

0

Total No of Observed Foetuses

101

103

108

105

% Fetal Incidence

0.00

0.97

0.93

0.00

No of Litters with at least 1 Incidence

0

1

1

0

Total No of Observed Litters

19

20

20

20

% Litter Incidence

0.00

5.00

5.00

0.00

Ventricle (lateral) (B) slightly dilated

No of Incidences

8

10

8

14

Total No of Observed Foetuses

101

103

108

105

% Fetal Incidence

7.92

9.71

7.41

13.33

No of Litters with at least 1 Incidence

5

5

3

4

Total No of Observed Litters

19

20

20

20

% Litter Incidence

26.32

25.00

15.00

20.00

Ventricle (lateral) (L) slightly dilated

No of Incidences

2

4

6

4

Total No of Observed Foetuses

101

103

108

105

% Fetal Incidence

1.98

3.88

5.56

3.81

No of Litters with at least 1 Incidence

2

3

5

4

Total No of Observed Litters

19

20

20

20

% Litter Incidence

10.53

15.00

25.00

20.00

Craniofacial Findings

Group

C

LD

MD

HD

Ventricle (lateral) (R) slightly dilated

No of Incidences

0

0

1

3

Total No of Observed Foetuses

101

103

108

105

% Fetal Incidence

0.00

0.00

0.93

2.86

No of Litters with at least 1 Incidence

0

0

1

1

Total No of Observed Litters

19

20

20

20

% Litter Incidence

0.00

0.00

5.00

5.00

Retinal (L) fold

No of Incidences

2

2

3

3

Total No of Observed Foetuses

101

103

108

105

% Fetal Incidence

1.98

1.94

2.78

2.86

No of Litters with at least 1 Incidence

2

2

3

2

Total No of Observed Litters

19

20

20

20

% Litter Incidence

10.53

10.00

15.00

10.00

Retinal (R) fold

No of Incidences

3

2

2

1

Total No of Observed Foetuses

101

103

108

105

% Fetal Incidence

2.97

1.94

1.85

0.95

No of Litters with at least 1 Incidence

2

2

2

1

Total No of Observed Litters

19

20

20

20

% Litter Incidence

10.53

10.00

10.00

5.00

Retinal (B) fold

No of Incidences

2

2

2

0

Total No of Observed Foetuses

101

103

108

105

% Fetal Incidence

1.98

1.94

1.85

0.00

No of Litters with at least 1 Incidence

2

2

1

0

Total No of Observed Litters

19

20

20

20

% Litter Incidence

10.53

10.00

5.00

0.00

Head - subcutaneous haematoma

No of Incidences

3

1

1

0

Total No of Observed Foetuses

101

103

108

105

% Fetal Incidence

2.97

0.97

0.93

0.00

No of Litters with at least 1 Incidence

3

1

1

0

Total No of Observed Litters

19

20

20

20

% Litter Incidence

15.79

5.00

5.00

0.00

Head - subcutaneous oedema

No of Incidences

0

0

0

1

Total No of Observed Foetuses

101

103

108

105

% Fetal Incidence

0.00

0.00

0.00

0.95

No of Litters with at least 1 Incidence

0

0

0

1

Total No of Observed Litters

19

20

20

20

% Litter Incidence

0.00

0.00

0.00

5.00

Craniofacial Findings

Group

C

LD

MD

HD

Nasal cavity (B) slightly dilated

No of Incidences

2

0

5

6

Total No of Observed Foetuses

101

103

108

105

% Fetal Incidence

1.98

0.00

4.63

5.71

No of Litters with at least 1 Incidence

2

0

2

3

Total No of Observed Litters

19

20

20

20

% Litter Incidence

10.53

0.00

10.00

15.00

Subdural haematoma

No of Incidences

1

0

1

0

Total No of Observed Foetuses

101

103

108

105

% Fetal Incidence

0.99

0.00

0.93

0.00

No of Litters with at least 1 Incidence

1

0

1

0

Total No of Observed Litters

19

20

20

20

% Litter Incidence

5.26

0.00

5.00

0.00

Macroscopic Findings- females - Summary

Organ

Finding

C

LD

MD

HD

Total number of animals examined

26

25

25

25

-

-

-

-

-

-

Historical data are attached as PDF

Applicant's summary and conclusion

Conclusions:
On the basis of this prenatal developmental toxicity study in pregnant Wistar female rats with Propyl 4-hydroxybenzoate at dose levels of 100, 300, and 1000 mg/kg body weight/ day administered on gestation days 5 to 19, the following conclusions can be made:
No mortality was observed in the study and there were no clinical signs of toxicological relevance observed in the treatment groups.
The body weight, food consumption, prenatal, litter data and gross pathology of terminally sacrificed females remained unaffected in the treatment groups when compared to the controls.
Furthermore, no treatment-related and toxicologically relevant external, visceral or craniofacial findings were observed in the HD group and other treated groups. However, reduced ossification of some bones and few other skeletal finings within historical control data range were observed. Generally delayed ossification is not regarded to persist postnatally and not associated with long-term consequences on survival, general growth and development and therefore is not considered to be adverse.
No effects of Propyl 4-hydroxybenzoate on females and foetuses were found at dose levels up to 1000 mg/kg body weight/day. The NOAEL for both maternal toxicity and foetal toxicity of Propyl 4-hydroxybenzoate in this study is considered to be 1000 mg/kg body weight/day.
Executive summary:

The aim of this study was to assess possible adverse effects on pregnant females and embryo-foetal development which could arise from repeated exposure of Propyl 4-hydroxybenzoate via oral administration (gavage) to female rats during gestation days 5 to 19. Nulliparous and non-pregnant females were mated with males (2:1 ratio) and divided into four groups based on their body weights on the day of sperm positive vaginal smears (GD 0).The 4 groups comprised 25 female Wistar rats.

The following doses were evaluated:

Control:                         0         mg/kg body weight/day

Low Dose:                    100     mg/kg body weight/day

Medium Dose:             300     mg/kg body weight/day

High Dose:                   1000   mg/kg body weight/day

Summary Results

Maternal Findings

No mortality ans toxicological relevant clinical signs were observed during the treatment period and all animals survived until end of the study. None of the females showed signs of abortion prior to the scheduled sacrifice. No test item-related effects of toxicological relevance were noted for any prenatal parameters like terminal body weight, uterus weight, adjusted maternal weight, number of corpora lutea, implantation sites, early and late resorptions, number of live foetuses, number of male and female foetuses,number of foetuses in each uterine horn,sex ratio, and percent pre- and post-implantation loss in treatment groups when compared to the controls. No dead foetuses were noted in any of the groups.The marginally low pregnancy rates (no. of pregnancies / no. of females mated or sperm positive x 100) of 76 % in the control group compared to treatment groups (92 % in LD, 84 % in MD and 96 % in HD) was considered to be a biological variation. No gross pathological changes of toxicological relevance were observed during the macroscopic examination of the females of the LD, MD and HD groups.

Foetal Findings

There were no test item-related effects of toxicological relevance observed for the mean foetus weight, total litter weight, male and female litter weight in any of the treatment groups when compared with the controls. There were no external abnormalities considered to be of toxicological relevance in any of the dose groups. Internal observation of the foetal viscera by free hand microdissection technique revealed a range of visceral findings in all groups including control.Visceral findings observed in the dose groups were at frequencies generally comparable to or in some cases slightly higher or lower in frequency compared to controls. As observed findings were either minor variations, within historical control data range and/or due to a lack of dose dependency and consistency, no toxicological significance can be attributed to these findings and they were considered to be spontaneous in nature. All litter incidences were statistically insignificant when compared with the control. Spontaneous findings regarding craniofacial examination by razor blade serial sectioning technique were observed at low frequencies generally . These findings were considered to be s not related to the treatment with the test item. Statistical analysis of the data revealed no statistical significance of any of the findings. Skeletal examination of the Alizarin red stained foetuses revealed a range of findings which occurred at an incidence generally comparable to or slightly lower or higher in the dose groups when compared to the control group. Slightly higher litter incidences compared to the concurrent control group, but without achieving statistical significance were observed in the HD group for incomplete ossification, increased ossification in few bones, unossified few bones and few rib, vertebra related findings but all the differences in ossification or unossification were within historical control data. Therefore, these finding were not to be considered as treatment-related and solely spontaneous in nature.There was no statistical significance and no indication of a test item-related trend in the type and/or incidences of other skeletal findings and they were therefore considered to be spontaneous in nature.

Conclusion

On the basis of this prenatal developmentaltoxicity study in pregnant Wistar female rats with Propyl 4-hydroxybenzoateat dose levels of 100, 300, and 1000 mg/kg body weight/ day administered on gestation days 5 to 19, the following conclusions can be made:

No mortality was observed in the study and there were no clinical signs of toxicological relevance observed in the treatment groups.

The body weight, food consumption, prenatal, litter data and gross pathology of terminally sacrificed females remained unaffected in the treatment groups when compared to the controls.

Furthermore, no treatment-related and toxicologically relevant external, visceral or craniofacial findings were observed in the HD group and other treated groups. Findings of reduced ossification of some bones and few other skeletal findings were well within the historical control data range for this strain of rats and not considered to be a substance related effect. Generally delayed ossification is not regarded to persist postnatally and not associated with long-term consequences on survival, general growth and development and therefore is not considered to be adverse.

No effects of Propyl 4-hydroxybenzoateon females and foetuses were found at dose levels up to 1000 mg/kg body weight/day. The NOAEL for both maternal toxicity and foetal toxicity of Propyl 4-hydroxybenzoatein this study is considered to be 1000 mg/kg body weight/day.