Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on generations indicated in Effect levels (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study.
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS

- Animals: Rat, RccHanTM: WIST(SPF)
- Rationale: Recognized by international guidelines as a recommended test system.
- Breeder: Harlan Laboratories, B.V., Kreuzelweg 53, 5961 NM Horst / Netherlands
- Number of Animals: 44 males (11 per group) and 44 females (11 per group)
- Age (at Start of Treatment): 11 weeks
- Body Weight Range (at Start of Treatment): 344 to 381 g (males) and 184 to 209 g (females)
- Identification: Parental animals by cage card and individual animal number (ear tattoo). Pups were individually tattooed with Indian ink on day 1 post partum.
- Randomization: Performed after at least three days of acclimatization using a computer-generated random algorithm. Body weights (recorded on the day of allocation) were taken into consideration in order to ensure similar mean body weights in all groups.
- Acclimatization: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS

- Conditions: Standard laboratory conditions. Air-conditioned with 10 - 15 air changes per hour, continuously monitored environmental conditions (temp. range: 22 ± 3 °C; relative humidity range: 30 - 70%). Values outside of these ranges occasionally occurred, usually following room cleaning, which was considered not to have any influence on the study. These data were not reported but were retained in the raw data. There was 12 hour fluorescent light / 12-hour dark cycle with music during the light period.
- Accommodation: In groups of three to five animals in Makrolon type-4 cages with wire mesh tops up to the day of randomization and afterwards individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding (‘Lignocel’ J. Rettenmaier & Söhne GmbH & CoKG, 73494 Rosenberg / Germany, imported by Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) with paper enrichment (ISO-BLOX from Harlan Laboratories B.V., Netherlands). During the pre-pairing period, cages with males were interspersed amongst those holding females to promote the development of regular estrus cycles.
- Diet: Pelleted standard Harlan Teklad 2914C (batch no. 06/12) rodent maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) was available ad libitum. Analyses for contaminants were performed.
- Water: Community tap-water from Füllinsdorf was available ad libitum in water bottles. Bacteriological assay, chemical and contaminant analyses of representative samples were performed.
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
FEED PREPARATION

Dietary admixtures were prepared every three weeks using the test item as supplied by the Sponsor.

Propylparaben was weighed into a tared glass baker on a suitable precision balance, and mixed with microgranulated feed separately for each dose group. An appropriate amount of water was added to aid pelleting. The pellets were dried with air for approximately 48 hours before storage.
Control feed for the animals of group 1 was prepared similarly, but without test item.

STORAGE OF FEED PREPARATION

Stability of test item in feed was at least three weeks if stored at room temperature based upon the results of stability analyses performed prior to the Harlan Laboratories study D52688 (Propylparaben: 14-Day Dose Range-Finding Study in the Han Wistar Rat, non GLP).

Feed preparations were stored at room temperature (17 - 23 °C) in metal containers until use.

TREATMENT

- Method: Oral, by ingestion (dietary administration)
- Rationale for Method: Oral ingestion is a route of human exposure.
- Target Dose Levels: 0 ppm (control group, Group 1), 1500 ppm (Group 2), 4500 ppm (Group 3) and 15000 ppm (Group 4)
- Rationale for Dose Level Selection: The dose levels were selected based on a previous dose range-finding toxicity study in Han Wistar rats, non GLP Harlan Laboratories study D52688, using dose levels of 0, 1200, 3600 and 12000 ppm.
Details on mating procedure:
During the pairing period, females were housed with sexually mature males (1:1) until evidence of copulation was observed. The females were removed and housed individually if the daily vaginal smear was sperm positive, or a copulation plug was observed.

The day on which a positive mating was determined (copulation plug or sperm) was designated day 0 post coitum.

All dams were allowed to give birth and rear their litters (F1 pups) up to day 4 post partum. Day 0 was designated as the day on which a female had delivered all her pups.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
METHOD

Samples for analyses of test item, its content and homogeneity in the feed were drawn at the start of the pre-pairing period and at the end of gestation/start of lactation period (i.e. two occasions). Additionally, samples for analysis of the test item content were drawn from the last dietary admixture. The analyses were performed at Harlan Laboratories Ltd., Zelgliweg 1, 4452 Itingen / Switzerland using a method supplied by the Sponsor. The test item was used as the analytical standard.

Stability (three weeks) of the test item in the feed was determined at the start of the pre-pairing period (i.e. one occasion).

For assessment of content and homogeneity, a 100 g sample was collected from each the top, middle and bottom of every dietary admixture of the respective diet preparation (from group 1 admixtures only one sample was drawn from the middle of the preparation). Samples for assessment of content and homogeneity were collected on the day of preparation and retained frozen between -20 ± 5 °C prior to analysis. For assessment of stability, a 100 g sample was drawn from the middle of the dietary admixture on the day of preparation, stored at room temperature for 3 weeks and consequently frozen as described above.

RESULTS

The Propylparaben peak was assigned in sample chromatograms by comparison to that of working standards. In blank sample chromatograms no peak appeared at the retention time of Propylparaben and, therefore, the absence of the test item in the control diet was confirmed.

The diet samples investigated during the study were found to comprise Propylparaben in the range of 103.8% to 115.7% and, thus, the required content limit of ±20% with reference to the nominal concentration was met. The homogeneous distribution of Propylparaben in the diet preparations was approved because single results found did not deviate more than 2.3% (acceptance criterion: <15%) from the corresponding mean.

In addition, the test item was found to be stable in diet when kept 21 days at room temperature due to recoveries which met the variation limit of 10% from the time-zero (homogeneity) mean.

In conclusion, the results indicate the accurate use of the test item Propylparaben and the control diet during this study. Diet samples were found to be homogeneously prepared and sufficient stability under storage conditions was approved.
Duration of treatment / exposure:
- Males: Minimum 4 weeks
- Females: Approximately 7 weeks
Frequency of treatment:
Dietary administration, food available ad libitum.
Details on study schedule:
MALES

- Acclimatization: 7 days
- First Test Item Administration: Day 1 of pre-pairing
- Pre-Pairing: 14 days
- Blood Sampling: Day 14 of pre-pairing
- Pairing: 14 days maximum
- Treatment Ends: On day before sacrifice
- Necropsy: After treatment of at least 28 days, when no longer needed for assessment of reproductive effects

FEMALES

- Acclimatization: 7 days
- First Test Item Administration: Day 1 of pre-pairing
- Pre-Pairing: 14 days
- Blood Sampling: Day 14 of pre-pairing
- Pairing: 14 days maximum
- Gestation: Approximately 21 days
- Treatment Ends: On day 3 post partum
- Necropsy: Females and pups on day 4 post partum
Remarks:
Doses / Concentrations:
0, 1500, 4500 and 15000 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
males: corresponding to 98.0, 305.1 and 980.9 mg/kg bw/d (pre-pairing) and 59.3, 178.3 and 605.0 mg/kg bw/d (after pairing)
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
females: corresponding to 116.0, 341.9 and 1076.4 mg/kg bw/d (pre-pairing), 121.6, 349.2 and 1124.6 mg/kg bw/d (gestation) and 137.3, 431.8 and 1380.0 mg/kg bw/d (lactation)
Basis:
nominal in diet
No. of animals per sex per dose:
11
Control animals:
yes, plain diet
Details on study design:
The purpose of this study was to generate information on the possible health hazards likely to arise from repeated exposure over a relatively limited period of time. In addition it provided information on possible effects on male and female reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus and parturition.

Rationale for Choice of Species, Route of Administration and Dose Levels: The rat is a suitable species for repeated dose and reproduction/developmental toxicity studies required by regulatory authorities. The oral route is one possible route for human exposure. Dose levels were selected in agreement with the Sponsor, based on the results of a dose range-finding study (Harlan Laboratories study D52688, non-GLP).
Positive control:
Not required
Parental animals: Observations and examinations:
VIABILITY / MORTALITY

Twice daily

CLINICAL SIGNS

Daily cage-side clinical observations (once daily, during acclimatization and up to day of necropsy). Additionally females were observed for signs of difficult or prolonged parturition, and behavioral abnormalities in nesting and nursing.

FOOD CONSUMPTION

- Males: Pre-Pairing period days 1 - 4, 4 - 8, 8 - 11 and 11 - 13; after pairing period days 1 - 4, 4 - 7 and 7 - 11.

- Females: Pre-Pairing period days 1 - 4, 4 - 8, 8 - 11 and 11 - 13; gestation days 0 - 7, 7 - 14 and 14 - 21 and days 1 - 4 of the lactation.

No food consumption was recorded during the pairing period.

BODY WEIGHTS

Recorded daily from treatment start to day of necropsy.

DETAILED CLINICAL OBSERVATIONS

Detailed clinical observations were performed outside the home cage in all animals. In males, it was performed once prior to the first administration of the test item and weekly thereafter. In females, it was prepared once prior to the first administration of the test item, weekly during the pre-pairing and pairing periods and on days 0, 6, 13 and 20 of the gestation period.

Animals were observed for the following: changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lacrimation, piloerection, pupil size, and unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies or bizarre behavior were also reported.

FUNCTIONAL OBSERVATIONAL BATTERY

At one time during the study (males shortly before the scheduled sacrifice and females on day 3 post partum) relevant parameters were performed with five P generation males and five P generation females from each group. This FOB assessment was conducted following the daily dose administration. Animals were observed for the following:

- Cage-side observations: faeces-balls, urine and posture as well as resistance to removal.

- Hand-held observations: muscle tone, constituation, skin, pupile size, palpebral closure, lacrimation, salivation, reaction to handling and general abnormalities.

- Open field observations: level of ambulatory activity including rearing (one minute evaluation), unusual body movements (e.g. spasms, convulsions), gait evaluation, behavior, hair coat, respiration, quantity of faeces-balls and urine.

- Reflexes: blinking, palpebral closure, pinna reflex, extensor thrust response, paw pinch, responsiveness to sharp noise, righting reflex and hearing ability (Preyer’s reflex).

- Measurements / Counts: hind limb / fore limb grip strength, rectal temperature.

Any abnormal findings were recorded and, where appropriate, graded in severity. Additionally, locomotor activity was measured quantitatively for the same animals. Activity was measured with an Activity Monitor AMS-0151 (FMI, Germany). Activity of the animals (based on beam count) was recorded for 6-minute intervals over a period of 30 minutes. These data and the total activity over 30 minutes were reported.

CLINICAL LABORATORY INVESTIGATIONS

Blood samples were obtained on day 14 of the pre-pairing period from 5 males and 5 females from each group. Blood samples were drawn sublingually from all animals under light isoflurane anesthesia. The animals were fasted for approximately 18 hours before blood sampling but allowed access to water ad libitum. The samples were collected early in the working day to reduce biological variation caused by circadian rhythms.

The following hematology parameters were determined:

- Erythrocyte count
- Hemoglobin
- Hematocrit
- Mean corpuscular volume
- Red cell volume distribution width
- Mean corpuscular hemoglobin
- Mean corpuscular hemoglobin concentration
- Hemoglobin concentration distribution width
- Leukocyte count, total
- Differential leukocyte count
- Platelet count
- Prothrombin time (= Thromboplastin time)
- Activated partial Thromboplastin time

The following clinical biochemistry parameters were determined:

- Glucose
- Urea
- Creatinine
- Bilirubin, total
- Cholesterol, total
- Triglycerides
- Aspartate aminotransferase
- Alanine aminotransferase
- Alkaline phosphatase
- Gamma-glutamyl-transferase
- Bile acids
- Sodium
- Potassium
- Chloride
- Calcium
- Phosphorus
- Protein, total
- Albumin
- Globulin
- Albumin/Globulin ratio
Oestrous cyclicity (parental animals):
From day 1 of the pre-pairing up to day 0 post coitum, vaginal smears were taken and the stage of the estrus cycle recorded.
Sperm parameters (parental animals):
1. SEMINOLOGY AND SPERMATID COUNT

Sperm analysis was performed on the first 5 males per group as follows:

MOTILITY

At necropsy of males, a sperm sample from the left caudal epididymis was obtained from each male. The sample was diluted with a pre-warmed (about 37 - 40 °C) physiological medium, and shortly after being obtained, one hundred sperm were counted in duplicates (two sperm samples) microscopically for determination of percentage of not motile, stationary motile and progressively motile sperm. If there was a variation of more than 10% of the progressive motile sperms between the two determinations a third sperm sample of 100 sperms were counted and motility determined.

MORPHOLOGY

The sperms in the original physiological medium for motility determination were used for morphological assessment after Eosin staining and fixation. The slides were stored at room temperature until analysis. The remaining fixation was stored at 4 °C for maximal 2 weeks for possible re-evaluation. 500 sperms per sample were evaluated microscopically and classified into the following categories:

Code Description
A Normal, complete sperm
B Normal head, abnormal tail
C Normal head only, tail detached
D Abnormal head only, tail detached
E Abnormal head, normal tail

The percentage of categories A to E was determined. Additional findings were added, when noted.

Morphological sperm evaluation was performed initially only for group 1 and 4 males. In the absence of a treatment-related effect the slides for the group 2 and 3 males were not evaluated.

SPERM COUNT

The left testis and the left cauda epididymis were taken for determination of homogenization-resistant sperms (HRS) in testes and epididymis. The testes and cauda epididymis were frozen at -20 ± 5 °C pending evaluation. For evaluation the testes and cauda epididymis were thawed and processed separately. Testes and cauda epididymis were weighed and placed in Triton-X-100 solution and homogenized with a blender (Ultra Turrax) and an ultrasonic water bath. Sperm heads were counted microscopically using a modified Neubauer chamber. Dilution factors for each sample were documented in the raw data.

Sperm count was estimated initially only for group 1 and 4 males. In the absence of a treatment-related effect the slides for the group 2 and 3 males were not evaluated.
Litter observations:
The litters were examined for litter size, live births, still births and any gross anomalies. The sex ratio of the pups was recorded. Pups were weighed individually (without identification) on days 0 (if possible), 1 and 4 post partum.
Postmortem examinations (parental animals):
TERMINATION AND NECROPSY

Males were sacrificed after treatment for at least 28 days, when no longer needed for the assessment of reproductive effects. Dams were sacrificed on day 4 post partum. If birth did not occur on the expected date (day 21 post coitum), the dam was sacrificed on day 25 post coitum.

At the scheduled sacrifice, all animals were weighed and sacrificed by an injection of sodium pentobarbital. All P generation animals were exsanguinated.

All parent animals were examined macroscopically for any structural changes, either at the scheduled necropsy or during the study if death occurred to establish, if possible, the cause of death. For the parent animals, special attention was directed at the organs of the reproductive system.

Specimens of abnormal tissue were fixed in neutral phosphate buffered 4% formaldehyde solution.

The number of implantation sites and corpora lutea was recorded for all dams with litters. The uteri of non-pregnant females were placed in a solution of ammonium sulfide to visualize possible hemorrhagic areas of implantation sites.

ORGAN WEIGHTS

At the scheduled sacrifice, the testes and epididymides of all parental males were weighed separately.

In addition, from five males and five females killed at the end of the study which were selected from each group, the following organs were trimmed from any adherent tissue, as appropriate, and their wet weight taken.

- Adrenal glands (weighed as pairs)
- Brain
- Heart
- Kidneys (weighed as pairs)
- Liver
- Thymus
- Spleen

TISSUE PRESERVATION

The following tissues from all parental males were preserved in neutral phosphate buffered 4% formaldehyde solution:

- Prostate
- Seminal vesicles with coagulating gland
- Testes (in Bouin’s fixative)*
- Epididymides (in Bouin’s fixative)*

* From the first five males in each group which was used for sperm analyses, only the right testis and right epididymis were preserved for histopathological examination.

The following tissues from all parental females were preserved in neutral phosphate buffered 4% formaldehyde solution:

- Ovaries

In addition, from the five males and five females per group selected for organ weights and from all animals found dead or killed in extremis, the following tissues were preserved in neutral phosphate buffered 4% formaldehyde solution:

- Gross lesions
- Brain
- Spinal chord
- Small and large intestines (incl. Peyer’s patches)
- Stomach
- Liver
- Kidneys
- Adrenals
- Spleen
- Heart
- Thymus
- Thyroids, and parathyroids if possible
- Trachea and lungs (preserved by inflation with fixative and then immersion)
- Uterus (with vagina)
- Urinary bladder
- Lymph nodes (mesenterial, mandibular)
- Peripheral nerve (sciatic)
- Bone marrow

HISTOTECHNIQUE

All organ and tissue samples to be examined by the principal investigator were processed, embedded and cut at an approximate thickness of 2 - 4 micrometers and stained with hematoxylin and eosin. Additionally, the testis was stained by PAS-hematoxylin. Special stains were used at the discretion of the study pathologist.

HISTOPATHOLOGY

Slides of all organs and tissues listed collected at terminal sacrifice from the animals of the control and high-dose groups were examined by the principal investigator. The same applied to all occurring gross lesions.

Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure.

Histological examination of ovaries was carried out on any females that did not give birth. In addition, microscopic examination of the reproductive organs of all infertile males was made, if necessary.

A histopathology peer review was performed and the results included in the histopathology phase report.
Postmortem examinations (offspring):
Pups were sacrificed on day 4 post partum. All animals were weighed and sacrificed by an injection of sodium pentobarbital. All pups, except those excessively cannibalized, were examined macroscopically for any structural changes, either at the scheduled necropsy or during the study if death occurred to establish, if possible, the cause of death. Specimens of abnormal tissue were fixed in neutral phosphate buffered 4% formaldehyde solution.
Statistics:
The following statistical methods were used to analyze food consumption, body weights and reproduction data:

- Means and standard deviations of various data were calculated.
- The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
- Fisher's exact-test was applied if the variables could be dichotomized without loss of information.
Reproductive indices:
From the on-line recorded reproduction data, the following parameters were calculated: fertility indices mean precoital time, post-implantation losses, mean litter size.

For reproduction data, groups mean values were calculated both on a litter basis and on a percentage per group basis.
Offspring viability indices:
From the on-line recorded reproduction data, the following paramters were calculated: dead/live pups at first litter check, pup sex ratio and viability indices
Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Slightly reduced body weight gain in males at 15000 ppm, occasionally reaching statistical significance. No such effect noted in females.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Slightly reduced body weight gain in males at 15000 ppm, occasionally reaching statistical significance. No such effect noted in females.
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
1. IN-LIFE DATA

VIABILITY / MORTALITY

All animals survived scheduled study period.

DAILY CLINICAL SIGNS OR OBSERVATIONS

No test item-related effects were noted in males or females at any dose level.

At the dose level of 1500 ppm, in one female (no. 56) malpositioned hind leg was noted during lactation period. This finding was incidental.

No further findings were noted in males or females at any dose level.

FINDINGS AT DETAILED WEEKLY CLINICAL OBSERVATION

No test item-related effects were noted in males or females at any dose level.

At the dose level of 1500 ppm, malpositioned hind leg in female no. 56 was noted during gestation period.

No further findings were noted in males or females at any dose level.

FUNCTIONAL OBSERVATIONAL BATTERY

No test item-related effects were noted during functional observational battery in males or females at any dose level.

At the dose level of 15000 ppm in males, statistically significantly lower body temperature was noted. Mean body temperature at this dose level was 38.0 °C compared to 38.5 °C in the control group. Because the difference was only minor and value of mean body temperature at the high dose level was in the range of historical controls (containing values from 37.1 to 39.3 °C), this was considered to be a result of biological variability and not test item related.

No further observations were noted during functional observational battery in males or females at any dose level.

LOCOMOTOR ACTIVITY

No effects on locomotor activity were noted in males or females at any dose level.

Mean beam counts during the 30 minutes of measurement at the dose levels of 0, 1500, 4500 and 15000 ppm were respectively: 1452, 1262, 1347 and 1214 in males and 586, 787, 892 and 519 in females.


FOOD CONSUMPTION OF MALES

No effects on food consumption were noted in males at any dose level. In order of ascending dose levels mean food consumption was 24.8, 24.8, 26.0 and 24.5 g/animal/day during the pre-pairing period and 25.4, 25.2, 25.7 and 25.0 g/animal/day during the after pairing period.


FOOD CONSUMPTION OF FEMALES

No effects on food consumption were noted in females at any dose level.

In order of ascending dose levels mean food consumption was 14.9, 15.5, 15.4 and 14.2 g/animal/day during the pre-pairing period, 19.5, 20.6, 19.7 and 18.4 g/animal/day during the gestation period and 26.5, 22.4, 23.1 and 21.7 g/animal/day during the lactation period.

RELATIVE FOOD CONSUMPTION OF MALES

No effects on relative food consumption were noted in males at any dose level.

In order of ascending dose levels mean relative food consumption was 65.2, 65.3, 67.8 and 65.4 g/kg bw/day during the pre-pairing period and 59.8, 59.2, 59.8 and 60.3 g/kg bw/day during the after pairing period.

RELATIVE FOOD CONSUMPTION OF FEMALES

No effects on relative food consumption were noted in females at any dose level.

In order of ascending dose levels mean relative food consumption was 74.9, 77.4, 76.0 and 71.8 g/kg bw/day during the pre-pairing period, 77.3, 81.1, 77.6 and 75.0 g/kg bw/day during the gestation and 109.3, 91.5, 96.0 and 92.0 g/kg bw/day during the lactation period.

BODY WEIGHTS OF MALES

At the dose level of 15000 ppm, body weight gain was slightly reduced if compared to the control values. This reduction was statistically significant on days 2, 5, 7 and 8 of the pre-pairing period and on days 3, 5, 6, 7 and 8 of the pairing period. No statistically significant changes in absolute body weights were noted in males at the high-dose level. The changes in body weight gain were considered to be test item-related but not adverse.

No test item-related effects on absolute body weights or body weight gain were noted in males at the dose levels of 1500 and 4500 ppm.

At the low- and mid-dose levels, statistically significantly lower body weight gains were noted on day 7 of the pairing period. If compared to the control group, the differences were only minor and not clearly dose dependent. Mean body weight gain was 3% in both dose groups and 4% in the control group. For these reasons changes in body weight gain at low- and mid-dose levels were considered not to be related to the treatment.

In order of ascending dose levels, mean body weight gain was 11%, 12%, 12% and 10% from day 1 to 13 of the pre-pairing period, 4%, 3%, 3% and 3% during the pairing period and it was 7% in all dose groups during the after pairing period.

BODY WEIGHTS OF FEMALES

No test item-related effects on absolute body weights or body weight gain were noted in females at any dose level.

At the dose levels of 1500 and 4500 ppm, statistically significantly higher body weight gain was noted on several days during the pre-pairing period. In the absence of any dose dependency, these changes were considered not to be related to the treatment with the test item.

In order of ascending dose levels, mean body weight gain was 5%, 5%, 7% and 4% from day 1 to 13 of the pre-pairing period, 54%, 51%, 53% and 49% during the gestation period and 6%, 5%, 4% and 2% during the lactation period.

2. CLINICAL LABORATORY INVESTIGATIONS

HEMATOLOGY

- Males: No test item-related effects on hematology parameters were noted in males at any dose level. At the dose level of 15000 ppm, red cell volume distribution width (RDW) was statistically significantly lower than the control value. Mean relative value of RDW was 0.115 at the high-dose level and 0.126 in the control group. Value at the high-dose level was within 95% of tolerance limit (containing values from 0.115 to 0.234) and therefore this difference was considered to be a result of biological variability and not related to the treatment with the test item.
At the dose level of 4500 ppm, there was a statistically significantly higher amount of eosinophils (EOS). Mean number of EOS was 0.11 x 109/l in the mid-dose group compared to 0.07 x 109/l in the control group. In the absence of statistically significant changes at the high-dose level, this effect was considered not to be related to the treatment with the test item.
No further statistically significant changes in hematology parameters were noted in males at any dose level.

- Females: No test item-related effects on hematology parameters were noted in females at any dose level. At the dose level of 1500 ppm, statistically significantly higher amount of lymphocytes (LYMPH) was noted. Mean relative value of LYMPH was 0.848 at the low-dose level and 0.806 in the control group. In the absence of any dose dependency, this difference was considered not to be related to the treatment with the test item.
No further statistically significant changes in hematology parameters were noted in females at any dose level.

BIOCHEMISTRY

- Males: At the dose level of 15000 ppm, statistically significant increase in triglicerydes concentration was noted. Mean triglicerydes concentration was 1.03 mmol/l at the high-dose level and 0.71 mmol/l in the control group. In the absence of any histopathological changes the reason for this observation was not evident. However, value of the triglicerydes concentration at the high dose level was above the range of the historical control values (0.75 ± 0.22 mmol/l) and therefore this finding was considered to be related to the treatment.
Remaining statistically significant differences were higher concentration of creatinine (CREAT) noted at the low- and mid-dose levels and lower concentration of alanine aminotransferase (ALAT) activity at the low-dose level. Mean concentration of CREAT was 22.2 and 24.2 µmol/l at the low- and mid-dose levels, respectively, and 19.2 µmol/l in the control group. Mean ALAT activity was 15.8 U/l at the low-dose level and 25.1 U/l in the control group. In the absence of any dose dependency, these effects were considered not to be related to the treatment with the test item.

- Females: No effects on biochemistry parameters were note in females at any dose level.

3. TERMINAL FINDINGS

SEMINOLOGY AND SPERMATID COUNT

No changes were noted during sperm analyses. Sperm motility was similar in all dose groups. All morphological categories of sperms were represented with similar frequency as well as sperm count was similar in the control group and at the high-dose level.

ORGAN WEIGHTS

No test item-related changes in organ weights were noted in males or females at any dose level.

Following statistically significant changes were noted in males: lower absolute weights of kidneys at the dose levels of 1500 and 15000 ppm, lower kidney weight to body weight ratio at the dose levels of 1500 and 4500 ppm and lower kidney weight to brain weight ratio at the dose level of 1500 ppm. The differences to the respective controls were only minor and the changes did not follow a dose dependency. For these reasons they were considered not to be test item-related.

Further statistically significant changes in males were higher absolute weights of right epididymides at the dose levels of 4500 and 15000 ppm, higher right epididymides weight to body weight ratio at the dose level of 15000 ppm and higher right epididymides weight to brain weight ratios at the dose levels of 4500 and 15000 ppm. Also these differences were not dose dependent and in addition they were noted only in the right organ. For these reasons they were considered not to be test item-related.

In females statistically significantly lower adrenal weight to brain weight ratio was noted. Because this effect occurred in the absence of statistically significant changes in absolute adrenal weights or adrenal weight to body weight ratio, it was considered not to be test item-related.

MACROSCOPICAL FINDINGS

- Males: No test item-related effects were noted during macroscopical examination of males at any dose level. Enlarged liver was noted in one male at the dose level of 1500 ppm, 2 males at the dose level of 4500 and 4 males at the dose level of 15000 ppm whereas in the control group this finding was noted in one male. Because no effects on liver weights and no histopathological changes in the liver were noted, this finding was considered not to be related to the treatment.
Remaining findings were noted in individual males and therefore gave no indication of a test item-related effect: pelvic dilation found in one male at the dose level of 4500 ppm and foci on thymus in one male at the dose level of 15000 ppm.

- Females: Type and distribution of findings noted in females during necropsy did not indicate any test item-related effect. Following findings were noted: pelvic dilation in one female at the dose level of 1500 ppm, discoloration of ovaries in one female at the dose level 4500 ppm and foci on thymus in two females at the dose level of 15000 ppm.

HISTOPATHOLOGY FINDNGS

All microscopic findings recorded were within the range of normal background lesions which may be recorded in animals of this strain and age.

No differences on the completeness of stages or cell populations of the testes were recorded between the control animals and the high dose animals.

No indication of a test item-related effect was found during examination of reproductive organ in males which mating did not result in pregnancy of a female.

4. REPRODUCTION AND BREEDING

ESTROUS CYCLES

No effects on estrus cycles were noted at any dose level.

In all groups mean number of days between estrus was 4, no irregular cycles were noted in any group.

MATING PERFORMANCE AND FERTILITY

Mating performance and fertility were not affected by the treatment at any dose level.

Percentage of mating was 100% in all groups. Mating of all females was recorded during first nine days of the pairing period.

Mean (median) precoital times were 2.6 (3), 3.3 (3), 4.0 (3) and 2.9 (3) days at the dose levels of 0, 1500, 4500 and 15000 ppm, respectively.

CORPORA LUTEA COUNT

No test item-related effects on corpora lutea count were observed at any dose level.

Mean number of corpora lutea per dam was 12.3, 13.2, 13.3 and 12.0 in order of ascending dose levels.

DURATION OF GESTATION

No effects on duration of gestation were observed at any dose level.

Mean duration of gestation was 21.2, 21.6, 21.2 and 21.1 days, in order of ascending dose level.

IMPLANTATION RATE AND POST IMPLANTATION LOSS

No effects on implantation rate or post-implantation loss were observed at any dose level.

In order of ascending dose levels, mean number of implantations was 11.9, 10.5, 12.3 and 11.3 per dam whereas mean incidence of post-implantation loss was 0.7, 0.7, 0.6 and 1.4 per dam.

Mean post-implantation loss at the high dose level was higher than in the control group. The difference was not statistically significant and the high-dose level value was within the range of historical controls (containing values from 0.5 to 1.7). For these reasons the difference in post-implantation loss was considered not to be related to the treatment with the test item.

LITTER SIZE AT FIRST LITTER CHECK

No effects on litter size were noted at any dose level.

Mean number of living pups per dam at first litter check was 11.2, 9.8, 11.6 and 9.9 in order of ascending dose levels.

Birth index (number of pups born alive as a percentage of implantations) was 94.1%, 93.3%, 94.8% and 87.6% at the dose level of 0, 1500, 4500 and 15000 ppm, respectively.

POSTNATAL LOSS DAYS 0 – 4 POST PARTUM

Postnatal loss noted in this study was considered not to give any indication of a test item-related effect.

At the dose level of 1500 ppm two pups from litter no. 59 died spontaneously on days 2 and 4 of the lactation period. At the dose level of 4500 ppm, one further pup from litter no. 67 was missing on day 3 of the lactation period. No further post-natal loss was noted at any dose level.
Dose descriptor:
NOAEL
Remarks:
for general toxicity
Effect level:
980.9 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Corresponding to 15000 ppm (highest dose tested).
Dose descriptor:
NOAEL
Remarks:
for general toxicity
Effect level:
1 076.4 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Corresponding to 15000 ppm (highest dose tested).
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
EXTERNAL EXAMINATION AT FIRST LITTER CHECK AND DURING LACTATION

No test item-related findings were noted during external examination at first litter check or during lactation at any dose level.

At the dose level of 1500 ppm, in all pups of one litter no milk in the stomach was noted at first litter check. At the dose level of 15000 ppm, hematoma and wound were noted in one pup.

No further findings were noted in pups at any dose level.

SEX RATIOS

Pups sex ratio was not affected by exposure to the test item at any dose level.

At first litter check, percentages of male pups were 45%, 50%, 44% and 51% at the dose levels of 0, 1500, 4500 and 15000 ppm, respectively.

BODY WEIGHTS TO DAY 4 POST PARTUM

Body weights of pups were not affected by the treatment with the test item at any dose level.

Mean body weights of pups at the dose levels of 0, 1500, 4500 and 15000 ppm were: 6.3 g, 6.3 g, 6.0 g and 6.1 g on day 1 post partum and 8.7 g, 8.9 g, 8.1 g and 8.5 g on day 4 post partum.

MACROSCOPICAL FINDINGS

No findings were noted during macroscopical examination of pups at any dose level.
Dose descriptor:
NOAEL
Remarks:
reproduction/developmental toxicity
Generation:
F1
Effect level:
1 124.6 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Corresponding to 15000 ppm (highest dose tested).
Reproductive effects observed:
not specified

TEST ITEM INTAKE OF MALES

 

During Pre-Pairing Period

 

Generation

Concentration
(ppm)

Mean achieved dose level
(mg/kg bw/day)

P

0

0

 

1500

98.0

 

4500

305.1

 

15000

980.9

 

During After Pairing Period

 

Generation

Concentration
(ppm)

Mean achieved dose level
(mg/kg bw/day)

P

0

0

 

1500

59.3

 

4500

178.3

 

15000

605.0

 

TEST ITEM INTAKE OF FEMALES

 

During Pre-Pairing Period

 

Generation

Concentration
(ppm)

Mean achieved dose level
(mg/kg bw/day)

P

0

0

 

1500

116.0

 

4500

341.9

 

15000

1076.4

 

During Gestation Period

 

Generation

Concentration
(ppm)

Mean achieved dose level
(mg/kg bw/day)

P

0

0

 

1500

121.6

 

4500

349.2

 

15000

1124.6

 

During Lactation Period

 

Generation

Concentration
(ppm)

Mean achieved dose level
(mg/kg bw/day)

P

0

0

 

1500

137.3

 

4500

431.8

 

15000

1380.0

 

MATING PERFORMANCE

 

P Animals Breeding for F1 Litters

 

Group
(ppm)

1
(0)

2
(1500)

3
(4500)

4
(15000)

Female numbers

45 - 55

56 - 66

67 - 77

78 - 88

Number of females paired

11

11

11

11

Number of females mated

11

11

11

11

Number of females not pregnant (A)

1

1

0

1

Number of females which reared their pups until day 4 post partum

10

10

11

10

(A)       Female Nos. 53, 61 and 81

 

Conclusions:
This study is a valid investigation of the toxicological effects resulting from repeated oral-gavage administration of the test item Propylparaben to rats. Propylparaben was administered in dietary mixtures daily at concentrations of: 0, 1500, 4500 and 15000 ppm. Test item was administered to male rats for 28 days and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum.

No signs of adverse toxicity were observed in males or females at any dose level.

At the dose level of 15000 ppm in males, reduced body weight gain was noted in the absence of statistically significant changes in absolute body weights.

At the dose level of 15000 ppm, increase in triglicerydes concentration was noted in the absence of any histopathological or other changes related to the finding.

No further test item-related effects were noted in males or females at any dose level.

Sperm motility, morphology and sperm count, estrus cycle, mating performance, fertility, duration of gestation, corpora lutea count, implantation rate, post implantation and postnatal loss and litter size were similar in the control and all dose groups.

There were no test item-related findings in pups noted during the first litter check, the first 4 days post partum or during the necropsy.


Based on these data, the NOAEL (No Observed Adverse Effect Level) for general toxicity and the NOEL (No Observed Effect Level) for reproduction/developmental toxicity were established at the dose level of 15000 ppm, the highest dose level used in the study.
Executive summary:

Propylparaben was administered orally in the feed at concentrations of:

 

Group 1: 0 ppm (control group)

Group 2: 1500 ppm

Group 3: 4500 ppm

Group 4: 15000 ppm

 

The following results were obtained:

 

MORTALITY AND GENERAL TOLERABILITY IN PARENTAL ANIMALS

 

All animals survived scheduled study period.

 

No test item-related effects were noted in males or females at any dose level.

 

FUNCTIONAL OBSERVATIONAL BATTERY IN PARENTAL ANIMALS

 

No test item-related effects were noted during functional observational battery or locomotor activity measurement in males or females at any dose level.

 

FOOD CONSUMPTION AND RELATIVE FOOD CONSUMPTION OF PARENTAL ANIMALS

 

No effects on food consumption or relative food consumption were noted in males or females at any dose level.

 

BODY WEIGHTS OF PARENTAL ANIMALS

 

In males at the dose level of 15000 ppm, body weight gain was slightly reduced if compared to the control values. This reduction was statistically significant on several days without statistically significant effect on absolute body weights. Changes in body weight gain in males at the high dose level were considered to be test item-related but not adverse.

 

No test item-related effects on absolute body weights or body weight gain were noted in males at the dose levels of 1500 and 4500 ppm or in females at any dose level.

 

CLINICAL LABORATORY INVESTIGATIONS IN PARENTAL ANIMALS

 

At the dose level of 15000 ppm, increase in triglicerydes concentration was noted. This finding was considered to be related to the treatment with the test item.

 

No further test item-related effects on hematology or biochemistry parameters were noted in males or females at any dose level.

 

REPRODUCTION AND BREEDING DATA

 

Estrus cycles, mating performance, fertility, corpora lutea count, duration of gestation, implantation rate and post-implantation loss, litter size or postnatal loss were not affected by the treatment with the test item at any dose level.

 

SEMINOLOGY AND SPERMATID COUNT

 

No changes were noted during sperm analyses.

 

ORGAN WEIGHTS OF PARENTAL ANIMALS

 

No test item-related changes in organ weights were noted in males or females at any dose level.

 

MACROSCOPICAL FINDINGS AND HISTOPATHOLOGICAL EXAMINATIONS OF PARENTAL ANIMALS

 

No test item-related effects were noted during macroscopical examination of males or females at any dose level.

 

All microscopic findings were within the range of normal background lesions which may be recorded in animals of this strain and age.

 

FINDINGS IN PUPS AT FIRST LITTER CHECK AND DURING LACTATION

 

No test item-related findings were noted during external examination at first litter check or during lactation at any dose level.

 

PUP WEIGHTS TO DAY 4 POST PARTUM

 

Body weights of pups were not affected by the treatment with the test item at any dose level.

 

MACROSCOPICAL FINDINGS OF PUPS

 

No findings were noted during macroscopical examination of pups at any dose level.

 

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Species:
rat
Quality of whole database:
The available information comprises adequate, reliable (Klimisch score 1) and consistent studies, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.7, of Regulation (EC) No 1907/2006.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

In a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test according to OECD 422 and GLP, propylparaben was administered to 11 rats per sex and group in doses of up to and including 980 and 1076 mg/kg bw/d in the diet to males and females (Harlan, 2012). The duration of the treatment period was 4 weeks for males and approximately 7 weeks for females.

No mortality occurred during the study period and no clinical signs of toxicity were observed. No effects were found on food consumption, body weight and body weight gain. No test substance-related findings were noted at necropsy with special attention to prostate, testes, seminal vesicles with coagulating glands, epididymis and ovaries. No test substance-related effects were observed on organ weights and during histopathology. No changes were noted during sperm analyses. Sperm motility was similar in all dose groups. All morphological categories of sperms were represented with similar frequency. Sperm count was similar in the control group and at the high dose group. No effects on estrus cycles were noted at any dose level. Mating performance and fertility were not affected by the treatment at any dose level. No test substance-related effects on corpora lutea count, duration of gestation, implantation rate or post-implantation loss were observed at any dose level. No effects on litter size were noted at any dose level and postnatal loss noted in this study was considered not to give any indication of a test substance-related effect. The sex ratio of the pups was not affected by test substance treatment. External examination of the pups did not reveal any findings and pup body weights were comparable to the control.

Based on the results of this study, the NOAEL for toxicity to reproduction (fertility) of propylparaben was considered to be 980 mg/kg bw/d for males and 1076 mg/kg bw/d for females.

 

In a published fertility study, propylparaben was administered in the diet at doses of approximately 12.4, 125 and 1290 mg/kg bw/d to male rats for 4 weeks (Oishi, 2002). No statistically significant differences and biological relevant effects in the weights of the testes, epididymis, ventral prostates, seminal vesicles and preputial glands (either absolute or relative to body weight) were observed between the control group and the test substance groups. In the highest dose group, the body weight was slightly but statistically significantly lower compared to the control group. No differences in food uptake were observed. The cauda epididymal sperm reserves (no. of sperms/cauda epididymis) and sperm concentrations (no. of sperms/g cauda epididymis) decreased in a dose-dependent manner and the difference was statistically significant in the mid and high dose groups. The daily sperm production and its efficiency in the testis also decreased dose-dependently and the decrease was statistically significant in all treated groups. The serum concentration of testosterone decreased in a dose-dependent manner and was statistically significant in the high dose group. Based on the results of this study, the LOAEL for male fertility of propylparaben was considered to be 12.4 mg/kg bw/d.

 

In a further fertility study, male rats were orally treated with test substance doses of 3, 10, 100 and 1000 mg/kg bw/d for 8 weeks (Gazin et al., 2012). Thereafter, one half of each group was sacrificed and investigated. The other half was left untreated for a 26 week recovery period and was then sacrificed. No mortality occurred until the end of the study period. Hypersalivation was noted in the high dose group through the end of the study period. No other treatment-related clinical signs were observed. Treatment with the test substance did not influence body weight, body weight gain, time of sexual maturation and LH, FSH and testosterone serum levels in any dose group. No treatment-related effect was observed on testicular and epididymal weights or on sperm count and motility data in any of the treated groups. A decrease tendancy in the highest dose group was observed without any statistical significance. At this dose level, no trend was observed on motility parameters. No test substance-related histopathological changes were observed at the end of the study period in any dose group. Based on the results of this study, the NOAEL for male fertility of propylparaben was considered to be 1000 mg/kg bw/d.

 

There are three studies available investigating the effects of propylparaben on reproduction performance. In the publication by Oishi (2002), a LOAEL of 12.4 mg/kg bw/d was set based on decrease in sperm reserves, sperm concentrations, daily sperm production and efficiency as well as serum testosterone concentration. However, these findings are not supported by two reliable studies. The most recent study, performed according to OECD 422, paid special attention to sperm parameters (Harlan, 2012). No changes were noted during sperm analyses. Sperm count, motility and morphology were not affected by test substance treatment. In a further fertility study in male rats with subsequent recovery period, also no effects were noted on sperm count and motility and on serum testosterone level (Gazin et al., 2012).

As a consequence, a NOAEL of 980 mg/kg bw/d was taken forward for hazard assessment.


Short description of key information:
NOAEL (male fertility, rat, OECD 422): 980 mg/kg bw/d
NOAEL (female fertility, rat, OECD 422): 1076 mg/kg bw/d

Justification for selection of Effect on fertility via oral route:
The selected study is the most adequate and reliable study based on overall assessment of quality, duration and dose descriptor level (refer to the endpoint discussion for further details).

Effects on developmental toxicity

Description of key information

Based on OCED-conform prenatal developmental toxicity sudy (OECD 414):

NOAEL (developmental toxicity, rat): 1000 mg/kg bw/d

Link to relevant study records
Reference
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
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
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)


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.
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
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).
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.
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.
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
Key result
Abnormalities:
no effects observed
Localisation:
not specified
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.
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.
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.
Key result
Developmental effects observed:
no

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

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.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Species:
rat
Quality of whole database:
Guideline study according to GLP available. No derivations and/or confounders. Klimisch rating 1 representing reliability without restrictions. Information valid and meets data requirements.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

In an OECD-conform parental developmental toxicity sudy (OECD 414) pregnant Wistar female rats were dosed with Propyl 4-hydroxybenzoate at dose levels of 100, 300, and 1000 mg/kg body weight/ day 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. 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.

Justification for selection of developmental toxicity endpint:

Guideline study according to GLP with a Klimisch rating 1.

 

Justification for classification or non-classification

The available data on toxicity to reproduction and on developmental toxicity/teratogenicity of the test substance does not meet the criteria for classification according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and is therefore conclusive but not sufficient for classification.