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Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Based on OCED-conform Extended One-Generation Reproductive Toxicity Study (according to OECD 443):

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

Link to relevant study records

Referenceopen allclose all

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
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.

 

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
Sep 2017- Feb 2018
Reliability:
1 (reliable without restriction)
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Wistar
Details on species / strain selection:
Species/strain: Wistar rats, Crl: WI(Han)
Source: e.g. Charles River, 97633 Sulzfeld, Germany
Sex:
male/female
Details on test animals and environmental conditions:
Species/strain: Wistar rats, Crl: WI(Han) (Full Barrier)
Source: e.g. Charles River, 97633 Sulzfeld, Germany
Sex: male and female; the female animals will be non-pregnant and nulliparous
Age at the start of the treatment period: approx. 8-9 weeks old
Body weight at the allocation of the animals to the experimental groups: interval within ± 20 % of the mean weight, if technically possible.

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)
For the Tolerability Screening the animals will be kept either intermittently individually or in groups of 3 animals in type III H IVC polysulphone cages on Altromin saw fibre bedding
- For the Dose Range Finding Study the animals will be kept in groups of 5 animals / sex / cage in type IV H, polysulphone cages on Altromin saw fibre bedding (except during the mating period when one female will be paired with one male and during gestation/lactation period when females will be housed individually in in type III H, polysulphone cages polysulphone cages)
- Makrolon tunnels will be provided for all males and for females until GD 18
- Nesting material will be provided latest on GD 18 for all mated females
- 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 five days) under laboratory conditions
Route of administration:
oral: gavage
Details on exposure:
Control group: 0 mg/kg body weight/day (5 male/5 female)
Low Dose: 500 mg/kg body weight/day (5 male/5 female)
High Dose: 1000 mg/kg body weight/day (5 male/5 female)

The animals will be treated with the test item formulations or vehicle on 7 days per week. Males will be dosed until the minimum total dosing of 35 days, i.e. during 21 days of pre-mating and maximum 14 days of mating. All females will be dosed during 21 days of pre-mating and up to 14 days of mating. One dam of each treatment group will be dosed up to GD 20 (day of caesarian section). The other dams will be dosed during gestation and up to PND 21. The pups of 3 litters (one from each group) will be treated from PND 13 to PND 21, in accordance with the treatment group of the dam. The last administration of the test item formulations or vehicle will be 30 minutes (+/- 10 minutes) before final necropsy.
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
The animals will be treated with the test item formulations or vehicle on 7 days per week. Males will be dosed until the minimum total dosing of 35 days, i.e. during 21 days of pre-mating and maximum 14 days of mating. All females will be dosed during 21 days of pre-mating and up to 14 days of mating. One dam of each treatment group will be dosed up to GD 20 (day of caesarian section). The other dams will be dosed during gestation and up to PND 21. The pups of 3 litters (one from each group) will be treated from PND 13 to PND 21, in accordance with the treatment group of the dam. The last administration of the test item formulations or vehicle will be 30 minutes (+/- 10 minutes) before final necropsy.
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5 male and 5 femla per dose
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Some female animals of all treatment groups including control showed alopecia on different locations at different periods of the treatment period. 5/10 male HD animals showed alopecia with different duration but starting from treatment day 6 (PMD 6) onwards. 2 HD males were seen with a scratch or cut. 5/10 male HD animals showed moving the bedding right after test item application.
These findings are not considered to reflect any systemic toxicity and are considered either incidental or related to local discomfort (moving the bedding) following oral dosing with the test item.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One LD male (No. 12) was found dead on PMD 8; it is considered that this animal died because it was jammed between the cage and the cage lid. One HD female (No. 50) was found dead on PND 5 it is considered that this animal died due to a technical gavage error and not due to systemic toxicity due to test item administration.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
In both parent males and females, there was no test item treatment related effect observed on body weight and body weight gain in the dose groups during the study period. There were no relevant differences observed for body weight and body weight gain between the dose groups and the control group.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No test item related effect on food consumption was observed in males and females during the whole study period.
Reproductive performance:
no effects observed
Description (incidence and severity):
There were no test item related effects on the reproductive indices (copulation, viability and delivery indices) in the dose groups when compared to the control group.
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
histopathology: non-neoplastic
reproductive performance
Executive summary:

Propylparaben was administered orally (via gavage) at concentrations of:

 

Group 1: 0 mg/kg body weight/day (control group)

Group 2: 500 mg/kg body weight/day (low dose)

Group 3: 1000 mg/kg body weight/day (high dose)

 No specific test item related effects were observed in males or females at any dose level in the parnets and pups. Based on these resuts the NOAEL were set as 1000 mg/kg body weight/ day.

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.

This study was conducted as a dose range finding study for the OECD 443 and based on the results of this study 1000 mg/kg bw per day can e used as the highest dose for the OECD 443.

Endpoint:
extended one-generation reproductive toxicity - with both developmental neuro- and immunotoxicity (Cohorts 1A, 1B without extension, 2A, 2B, and 3)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Qualifier:
according to
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Version / remarks:
Adopted: 25 June 2018
Deviations:
yes
Remarks:
see below: Any Other Information on Results
GLP compliance:
yes (incl. certificate)
Remarks:
(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany)
Limit test:
no
Justification for study design:
This test is performed on the rat. Although several mammalian species may be used,
the rat is the preferred rodent species.
In the assessment and evaluation of the toxic characteristics of chemicals, the
determination of reproduction toxicity using repeated doses by dietary/ oral route may be
carried out after initial information on toxicity has been obtained by sub-acute or chronic
testing.
An Extended One-Generation Reproductive Toxicity Study provides information on the
effects of repeated exposure to a substance during all phases of the reproductive cycle.
In particular, the study provides information on the reproductive system, and on
development, growth, survival, and functional endpoints of offspring. The test is
designed to evaluate the effects of chemicals on pre and postnatal development; the
integrity of the male and female reproductive systems; and systemic toxicity in males,
pregnant and lactating females, and young and adult offspring.
In this study the detailed examination made on key developmental endpoints, such as
offspring viability, neonatal health, developmental status at birth, and physical and
functional development until adulthood, is expected to identify specific target organs in
the offspring.
The study provides detailed information about the effects of a test substance on the
integrity and performance of the adult male and female reproductive systems, which
includes gonadal function, the oestrous cycle, epididymal sperm maturation, mating
behaviour, conception, pregnancy, parturition, and lactation. This also includes the
information obtained from the developmental neurotoxicity and developmental
immunotoxicity assessments.
In this type of study, the test substance is administered daily in graduated doses to
several groups of sexually-mature males and females. The P animals are exposed with
the test item treated diet/ oral gavage 2 weeks premating (males and females), 2 weeks
mating (males and females), 6 weeks post mating up to termination after weaning- 10
weeks total treatment (males), during pregnancy and lactation up to termination after
weaning- 8-10 weeks total treatment (females). In F1 males and females, the direct
exposure to test item is started at weaning till scheduled termination. If a second
generation is assessed, the F1 offspring are maintained on treatment until weaning of
the F2, or until termination of the study. Litter size may be adjusted at postnatal day
(PND) 4, and again at weaning once F1 animals have been assigned to their respective
cohorts.
The litters are examined as soon as possible after birth. To obtain information about the
plasma level of the test item and possibly of metabolites, blood samples can be taken at
several time points.
During the administration period the animals are observed closely each day for signs of
toxicity. Animals which die or are sacrificed during the test period are necropsied and, at
the conclusion of the test, surviving animals are sacrificed and necropsied.
Clinical observations and pathology examinations are performed on all animals for signs
of toxicity, with special emphasis on the integrity and performance of the male and
female reproductive systems and the health, growth, development and function of the
offspring. At weaning, selected offspring are assigned to specific cohorts for the
investigations comprising sexual maturation, reproductive organ integrity and function,
neurological and behavioural endpoints, and immune functions.
Specific details on test material used for the study:
Name: Propyl 4-hydroxybenzoate
Batch No.: BP16102712 (Material No. 16690327141)
Physical State: white solid powder
Active Components: 99.7 % (impurities were not taken into account for final formulation)
Storage Conditions: room temperature
Expiry Date: 27 October 2019
Safety Precautions: The routine hygienic procedures were sufficient to assure personnel health and safety.
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
Test System
Species/strain: Wistar rats, Crl: WI(Han) (Full Barrier)
Source: e.g. Charles River, 97633 Sulzfeld, Germany
Sex: male and female; the female animals were non-pregnant and nulliparous
Age at the start of the treatment period: approx. 13-14 weeks old
Body weight before initiation of pairing: males: 306 - 358 g (mean: 331.30 g, ± 20 % = 265.04 – 397.56 g); females: 177 - 241 g (mean: 214.58 g, ± 20 % = 171.76 – 257.50 g)
The animals were derived from a controlled full barrier maintained breeding system (SPF). According to the German Act on Animal Welfare [10] the animals are bred for experimental purposes.
This study wasperformed 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 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)
- Animals were housed in groups of 5 animals / sex / cage in type IV polysulphone cages or in double decker IVC cages during the premating period for males and females (both parental and F1)
and during post-mating period for males (parental and F1) depending on the mating status. During mating period males and females (parental and F1) were housed together in ratio 1:1 (male to female).
After the confirmation of mating, females were kept individually during gestation/lactation period in type III H, polysulphone cages and males (parental and F1) were returned to their original cage. In each cage Altromin saw fibre was used as bedding.
- Makrolon tunnels will be provided for all males and for females until GD 18
- Nesting material will be provided latest on GD 18 for all mated females
- 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 Formulations
The vehicle had been selected in consultation with the sponsor based on the test item’s characteristics and testing guideline.
The test item, as delivered, was grinded before formulation preparation. Afterwards, test item was 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 slurry with the glass rod for 1 minute, 1 % aqueous hydroxyethyl-cellulose was added to give the appropriate final concentration. The formulation was hen 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 and at 2-8 °C. Formulates were kept under magnetic stirring during the daily administration. The vehicle was also used as control item.

Preparation of Cyclophosphamide Formulation
The cyclophosphamide was diluted freshly with 0.9 % saline to a concentration of 1 mg/mL. The formulations were vortexed until visual homogeneity was achieved.
The test items formulation was prepared freshly on each administration day before the administration.

Preparation of Keyhole Limpet Haemocyanin (KLH)
Lyophilized KLH was reconstituted freshly with water for injection (aqua ad injectionem) to a concentration of 10 mg/mL.
In a second step the KLH solution was diluted further down using phosphate-buffered Saline (PBS) to achieve a concentration of 0.4 mg/mL.
The KLH formulation was prepared in a pre-labelled 15 mL blue cap vial (Art. No. 188271, Greiner Bio-One GmbH, Germany).

Characterisation of the Vehicle
The vehicle to be 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
Manufacturer: Sigma-Aldrich
Batch No.: MKCD0421
Physical State: powder
Colour: beige
Storage Conditions: at room temperature
Expiry Date: 05/2019
Safety Precautions: The routine hygienic procedures were sufficient to assure personnel health and safety.

Name: aqua ad injectionem (sterile water)
Manufacturer: Delta Medica
Batch No.: 710693, 710745
Physical State: liquid
Storage Conditions: at room temperature
Expiry Date: 09/2020
Safety Precautions: The routine hygienic procedures were sufficient to assure personnel health and safety.

Characterisation of Vehicle for Cyclophosphamide
The vehicle for test item 1 was 0.9 % NaCl. The specifications provided by the supplier are listed as follows:
Name: 0.9 % NaCl
Manufacturer: B. Braun Melsungen
Batch No.: 18144408
Physical State: liquid
Storage Conditions: at room temperature
Expiry Date: 03/2021
Safety Precautions: The routine hygienic procedures were sufficient to assure personnel health and safety.

Characterisation of Vehicle for Keyhole Limpet Haemocyanin (KLH)
The vehicle used for reconstitution of KLH in this study was water for injection (aqua ad injectionem). The specifications provided by the supplier are listed as follows:
Name: aqua ad injectionem
Manufacturer: Sigma Aldrich
Batch No.: 078M4801V
Physical State: liquid
Storage Conditions: room temperature
Expiry Date: July 2020
Safety Precautions: The routine hygienic procedures were sufficient to assure personnel health and safety.

The vehicle used for KLH dilution in this study was PBS (phosphate-buffered saline). The specifications provided by the supplier are listed as follows:
Name: PBS
Manufacturer: BSL Munich
Batch No.: not applicable
Physical State: liquid
Storage Conditions: room temperature
Expiry Date: 11/2018
Safety Precautions: The routine hygienic procedures were sufficient to assure personnel health and safety.


Administration of Doses
The test item and vehicle were administered once daily at a single dose to the animals by oral gavage. The application volume for all groups (adult / pups) was 5 mL/kg body weight.
Animal no. 177 (female, MD group, P generation) was replaced by a reserve animal due to an accident which caused tetraplegia. The new animal kept the same animal no.,
however the application of the test item started 2 days later.
Animal no. 43 (male, LD group, P generation) was not treated with the test item on gestation day 19 due animal welfare reasons.
Inadvertently, one animal from cohort 1B was treated with a higher application volume than 5 mL/kg for 6 days.
Pups were dosed from weaning (day 22). Transfer of the test item into milk had been demonstrated for the period between PND 0 to PND 21 in the dose range finding study (Study No. 176886)
and thus continuous systemic exposure of pups during this period has been confirmed.
For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured.

Dose levels: 0, 100, 300, 1000 mg/kg bw (concentration in vehicle: 0, 20, 60, 200 mg/ml)

Treatment of Cyclophosphamide and KLH
In the positive control group, the immunosuppressant cyclophosphamide (C2) was administered from 7 days before immunization with KLH, until the day before the last blood sampling.
Control group (C) served as negative control group. Approximately one week after the start of the treatment with Cyclophosphamide or vehicle or test item, each animal of group (C, C2, LD, MD & HD)
were injected intravenously into the tail vein at 0.300 µg/kg of KLH as single dose (at 0.75 mL/kg of dose volume). On this particular day, the oral gavage treatment was performed after the i.v. treatment.
Details on mating procedure:
After 2 weeks premating period, one parental male and one parental female from the same dose group were mated (1:1 pairing). Animals from Cohort 1B were maintained on treatment for more than PND 90 and were bred to obtain a F2 generation.
F1 males and females (1B cohort) of the same dose group were cohabited (1:1 pairing) for up to two weeks beginning on or after PND 90 but not more than PND 120 avoiding the pairing of siblings.
The parental and F1 females were caged with the same male until pregnancy occurred or two weeks have elapsed. On the following morning after pairing and each morning thereafter,
the females were examined for the presence of vaginal sperm or a vaginal copulation plug to confirm the mating.
If the vaginal smear of a particular female was not found to be sperm-positive, the actual stage of the estrus cycle on that day was documented.
Animals were separated as soon as possible after evidence of copulation in terms of sperm positive vaginal smear was observed. The day of the vaginal plug and/or sperm was considered as day 0 of gestation.
The cages were arranged in such a way that possible effects due to cage placement were minimized. Mating of all females was completed in or before 2 weeks mating period.
The date of pairing, date of sperm positive vaginal smears and the date of littering were recorded and the precoital interval and the duration of gestation was calculated for parental and F1 females.
The parental and F1 females from cohort 1B were carefully examined at the time of expected littering for any signs of dystocia. Any abnormalities in nesting or nursing performances were recorded.
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 included the samples from high dose and low dose group and the investigation was made for 0 h, 6 h (RT), 4 day (RT),
4 day (2-8 °C) and 4 day -15 to -35 °C.
Prestart homogeneity investigation included 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. 176899, 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 week 1, initiation of month 2, 3, 4 and last week of the study and analysed in triplicates (50 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. 176899) 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.

A dose formulation analysis was not performed for the cyclophosphamide and KLH formulations.
Freshly prepared formulations were used to dose the animals.
Duration of treatment / exposure:
The animals were treated with the test item formulation or vehicle on 7 days per week. Parental males were dosed until the minimum
total dosing of 10 weeks, i.e. during 14 days of pre-mating and maximum 14 days of mating and until terminal sacrifice.
All parental females were dosed during 14 days of pre-mating, maximum 14 days of mating, during gestation and until weaning (PND 21).
The selected F1 offspring received further treatment with the test substance from weaning to terminal sacrifice of respective cohort

Frequency of treatment:
Once per day. 7 days per week
Details on study schedule:
Arrival of the Test Item: 22 August 2017
Study Initiation Date: 06 July 2018
Amendment to Study Plan: 20 July 2018
2nd Amendment to Study Plan: 28 August 2018
3rd Amendment to Study Plan: 27 September 2018
4th Amendment to Study Plan: 02 October 2018
5th Amendment to Study Plan: 03 December 2018
6th Amendment to Study Plan: 12 March 2019
Delivery of Animals: 03 July 2018
Acclimatisation Period: 03 July 2018 – 05 July 2018
Experimental Starting Date: 06 July 2018
Treatment Period: 22 July 2018 to 27 January 2019
Necropsies: 27 August 2018 to 28 January 2019
Experimental Completion Date: 28 January 2019
Completion Date of Delegated Phase (Histopathology): will be included in the final report
Completion Date of Delegated Phase (Formulation Analysis): will be included in the final report
Completion Date of Delegated Phase (Bioanalytics): 18 December 2019
Study Completion Date: date of the study director’s signature
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
220 parental animals (110 males and 110 females) were included in the study (25 male and 25 female animals per group in LD and MD
and 30 male and 30 female animals per group in control and HD). In addition some reserve animals (2 per sex)
were ordered for possible exchange before study start.
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 is chosen with the aim of inducing toxic effects, but not death or severe suffering. Thereafter, a descending sequence of dose levels is selected with a view
to demonstrate any dose-related response.
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 high dose group.


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
Parental animals: Observations and examinations:
Body Weight and Food Consumption
Parental animals were weighed once before the assignment to the experimental groups, on the first day of dosing and weekly during the study period as well as at the terminal sacrifice.
In addition, during pregnancy, females were weighed on gestation days (GD) 0, 7, 14 and 20 and within 24 hours of parturition (day 0 post-partum) as well as on day 4, 7, 14 and 21 post-partum along with pups.
Body weight was also measured on PND 4, at weaning (surplus pups after selection of cohorts) and weekly till terminal sacrifice.
Food consumption was measured at intervals corresponding to the body weight measurements after the beginning of the dose administration except during mating period for parental animals.
Food consumption was also not measured during the post-mating period in parental males until all females were mated and all males were not back to their original housing cage of 5 males/cage.


Clinical Observations
For parental general clinical observations of the animals were made at least once a day, preferably at the same time each day and considering the peak period of anticipated effects after dosing.
The health condition of the animals was recorded. Twice daily all animals (Parental) were observed for morbidity and mortality except on weekends and public holidays when observations are made once daily.
Pertinent behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity, including mortality were recorded.
None of the parental females showed signs of abortion or premature delivery prior to the scheduled sacrifice.
Once before the first exposure, and once a week thereafter, detailed clinical observations was made in all P animals when animals were weighed outside the home cage.
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 was recorded.



Oestrous cyclicity (parental animals):
Vaginal smears of parental females were examined 2 weeks before beginning of treatment period, during 2 weeks premating period and until the
confirmation of mating and/ or the end of the 2 weeks mating period to record the estrous cyclicity and also to confirm the evidence of mating.

Sperm parameters (parental animals):
At terminal sacrifice, left epididymis, left testis and left vas deferens were separated and used for evaluation of sperm parameters (Motility, testicular sperm head count and morphology)
from all parental generation males of each group were performed by using Hamilton Thorn Sperm Analyser (TOX IVOS Version 13.0C). Sperm morphology was performed manually by manual method.
Sperm morphology slides were prepared from all parental generation males. Initially, evaluation was made in male animals of the groups 1 and 4 sacrificed at the end of the treatment period.
Sperm morphology examinations were not extended to male animals of all other dosage groups as no treatment-related changes were observed in the high dose group.
For morphology evaluation, sperm from left vas deferens were transferred to 0.1 % bovine serum albumin solution.
Litter observations:
Litter Observations
Each litter of F1 and F2 was examined after parturition (PND 0) to establish the number and sex of pups, stillbirths, live births and the presence of gross external anomalies externally visible abnormalities,
including cleft palate, subcutaneous haemorrhages, abnormal skin colour or texture, presence of umbilical cord, lack of milk in stomach and presence of dried secretions.
In addition, the first clinical examination of the neonates included a qualitative assessment of body temperature, state of activity and reaction to handling.
Pups found dead on PND 0 or later time were examined for the possible cause of death. Pups were marked with unique identification number or by tattooing.
Live pups were counted and sexed and litters weighed within 24 hours of parturition (PND 0) or PND 1, on PND 4, 7, 14 and PND 21. In addition to the observations on parent animals, any abnormal behavior of the offspring was recorded.
The clinical signs of pups were recorded on the corresponding days when offspring were weighed. The anogenital distance (AGD) of each pup was measured on PND 0.
Pup body weight measured on the day of anogenital distance measurement was converted to cube root and used for the calculation of relative AGD (Relative AGD = AGD / Cube root of pup weight).
Male pups were checked for the presence of nipples/areolae on PND 12.

Litter Size Adjustment
The size of the litter was not adjusted on PND 4, by eliminating extra pups by random selection to yield 5 males/ 5 females.
In order to allocate requisite number of offspring to various cohorts required for standard study design as per the guideline or for some additional parameters like determination of test item plasma level, brain liver collection
for additional evaluation or additional cohort for learning and memory evaluation, culling of the pups on PND 4 was not performed as it is not really adding any value to the study. Those pups intended for culling were
used for specific additional evaluations and also more male pups were used for observations of nipple/areolae retention on PND 12.
Postmortem examinations (parental animals):
At the time of termination or premature death, all P were subjected to gross necropsy and examined macroscopically. P males were subjected to necropsy after 10 weeks of dose application and females post weaning.
Special attention was paid to the sexual organs for structural abnormalities.
Vaginal smear from adult P awas examined on the day of necropsy to determine the stage of the oestrous cycle.
The uteri of all P females were examined for the presence and number of corpora lutea and implantation sites.
Non-pregnant females were sacrificed on day 26 from the day of mating or from the last day of mating period.

At necropsy, body weights and wet weights of the organs listed below (ovaries, seminal vesicles with coagulating glands and their fluids, spleen, uterus (with oviduct and cervix), brain, thymus,
testes, liver, pituitary, epididymides, kidneys, thyroid (post-fixation), prostate (dorsolateral and ventral parts combined), heart, adrenal glands) from all P animals were measured.
Paired organs were weighed combined together (except testes and epididymides). Organ weights of animals found dead or euthanised for animal welfare reasons were not taken.

The following tissues (brain (cerebrum, cerebellum and pons), heart, spinal cord, ovaries (females), eye + optic nerve, uterus with cervix (females), liver, vagina (females), kidneys, testes (males),
adrenal glands, epididymides (males), oesophagus, vas deferens (males), stomach, prostate and seminal vesicles with coagulating glands as a whole (males), small and large intestines (including Peyer´s patches),
urinary bladder, thymus, lymphnodes (mesentric and axillary), thyroid and parathyroid, peripheral nerve (e.g. sciatic nerve) with skeletal muscle, spleen, bone with bone marrow (sternum), lung, pituitary gland,
trachea, oesophagus, mammary glands, spinal cord, skin, gross lesions) of the same selected animals from each group were preserved in 4 % neutral buffered formaldehyde except for testes,
epididymides and eyes that were fixed in Modified Davidson’s Fixative for approximately 24 hours before they were transferred to 4 % neutral buffered formaldehyde.


Full histopathology of the organs listed in (brain (cerebrum, cerebellum and pons), heart, spinal cord, ovaries (females), eye + optic nerve, uterus with cervix (females), liver, vagina (females), kidneys, testes (males),
adrenal glands, epididymides (males), oesophagus, vas deferens (males), stomach, prostate and seminal vesicles with coagulating glands as a whole (males), small and large intestines (including Peyer´s patches),
urinary bladder, thymus, lymphnodes (mesentric and axillary), thyroid and parathyroid, peripheral nerve (e.g. sciatic nerve) with skeletal muscle, spleen, bone with bone marrow (sternum), lung, pituitary gland,
trachea, oesophagus, mammary glands, spinal cord, skin, gross lesions) was performed for all high-dose and control P animals. was performed for all high-dose and control P animals.
Histopathological examinations were not extended to animals of the other dose groups, as no treatment-related changes were observed in the high dose group.
Reproductive organs of all animals that failed to deliver healthy offspring, their paired males and all gross lesions were subjected to histopathological evaluation.
Postmortem examinations (offspring):
At the time of termination or premature death, all F1 animals were subjected to gross necropsy and examined macroscopically.
The animals of F1 generation were subjected to necropsy depending on the scheduled ages for each cohorts (cohort 1A: week 13, 1B: week 20-25, 2A: week 11-12, 2B: 3 weeks
(at weaning, cohort 3: 8-10 weeks, Cohort 4: 6-7 weeks). Special attention was paid to the sexual organs for structural abnormalities.
Pups not selected for cohorts (including runts) were killed and subjected to gross necropsy at weaning (PND 22) or later when not required for further in-life investigations.
Dead or moribund pups were recorded and examined for possible defects and/or cause of death.
Vaginal smear from F1 females (except females of cohort 2B, 3 and 4) was examined on the day of necropsy to determine the stage of the oestrous cycle.
The uteri of all Cohort 1B females were examined for the presence and number of corpora lutea and implantation sites.
Non-pregnant females were sacrificed on day 26 from the day of mating or from the last day of mating period.
F2 Pups from Cohort 1B were sacrificed and subjected to necropsy at weaning. F1 animals from the Cohorts 3 and 4 were subjected only to gross necropsy and examined
macroscopically for any structural abnormalities or pathological changes. Organs with abnormalities were preserved for possible histological evaluation.

At necropsy, body weights and wet weights of the organs listed below (ovaries, seminal vesicles with coagulating glands and their fluids, spleen, uterus (with oviduct and cervix),
brain, thymus, testes, liver, pituitary, epididymides, kidneys, thyroid (post-fixation), prostate (dorsolateral and ventral parts combined), heart, adrenal glands, axillary lymphnodes (Cohort 1A)) from all F1 adults (cohorts 1A) were measured.
Paired organs were weighed combined together (except testes and epididymides).
Organ weights of animals found dead or euthanised for animal welfare reasons were not taken.

The following tissues (brain (cerebrum, cerebellum and pons), heart, spinal cord, ovaries (females), eye + optic nerve, uterus with cervix (females), liver, vagina (females), kidneys,
testes (males), adrenal glands, epididymides (males), oesophagus, vas deferens (males), stomach, prostate and seminal vesicles with coagulating glands as a whole (males),
small and large intestines (includingPeyer´s patches), urinary bladder, thymus, lymphnodes (mesentric and axillary), thyroid and parathyroid, peripheral nerve (e.g. sciatic nerve),
skeletal muscle, spleen, bone with bone marrow (sternum), lung, pituitary gland, trachea, oesophagus, mammary glands, spinal cord, skin, gross lesions) of the same selected animals
from each group were preserved in 4 % neutral buffered formaldehyde except for testes, epididymides and eyes that were fixed in Modified Davidson’s Fixative for approximately
24 hours before they were transferred to 4 % neutral buffered formaldehyde.

From 10 male and 10 female cohort 1A animals of each treatment group (1 male or 1 female per litter; randomly selected) one half of the spleen were preserved for
histopathological evaluation, the other half of the spleen was used for the investigation of pre- and postnatally induced immunotoxic effects at termination.
The following organs (vagina (not weighed), seminal vesicles and coagulating glands, uterus with cervix, prostate, ovaries, pituitary, testes, epididymides, thyroid (post fixation),
adrenal gland, gross lesion) of Cohort 1B animals were weighed and tissues processed to the block.
As the results from cohort 1A were not equivocal or suspected reproductive/ endocrine toxicants, the examination was not extended to Cohort 1B animals.


Cohort 2A animals were terminated between PND 75 and 80 after behavioral testing. The brain weight was recorded and full neurohistopathology was performed.
The perfusion fixation was employed for cohort 2A animals.
For cohort 2A, the eyes (retina and optic nerve) and samples of peripheral nerve, muscle and spinal cord were also preserved.
Cohort 2B animals were terminated on PND 21 or 22. The brain weight was recorded and microscopic examination of the brain was performed.
The perfusion fixation was employed for cohort 2B animals.
Brain, spleen, and thymus was collected from 10 pups/ sex/ group (surplus pups not allocated to cohorts and pups of F2 generation)
were weighed and preserved along with mammary gland, gross lesion and target tissues for possible histological examination.
For perfusion fixation, approximately 10 minutes before anesthesia the animals received an intraperitoneal injection of heparin (1000 IU/kg, 2 mL/kg).
Each animal was deeply anaesthetized with ketamine and xylazine and subjected to in situ perfusion with 4 % neutral buffered formaldehyde to achieve an adequate
fixation of the brain according to the below procedure.
After opening the thoracic cavity a needle (20G) was inserted in the apex of the left ventricle of the heart toward the top where the ascending aorta connects.
The needle was connected to the perfusion pump via tubing and the infusion was started at a flow rate of approximately 30 mL/min.
A slit in the right atrium was made in order to allow blood to flow. First a pre-flush was performed during 5 minutes with ice cold saline solution (0.9 % NaCl solution).
After the solution runs clear the perfusion was switched to 4 % neutral buffered formaldehyde during 12 minutes. The perfusion solutions were placed in a water bath at approx. 37 °C
so that they were administered at a physiological temperature. When the perfusion time had reached the brain, it was carefully examined, sampled and transferred in 4 % neutral buffered
formaldehyde for histopathological evaluation.

Cohort 1: Full histopathology of the organs listed in Table 9 was performed on control and high dose adult cohort 1A animals. Histopathological examinations were not extended to animals of the other dose groups, as treatment-related changes were not observed in the high dose group. All gross lesions were subjected to histopathological evaluation.
Histopathology of lymph nodes (mesenteric and axillary) and bone marrow were performed on 10 male and 10 female cohort 1A animals. Histopathology of thymus, spleen, and the adrenal glands were performed in all cohort 1A animals.
As the results from cohort 1A were not equivocal or suspected reproductive/ endocrine toxicants, the examination were not extended to Cohort 1B animals.
The histopathological examination of ovaries included quantitative evaluation of primordial and small growing follicles and corpora lutea. Follicular enumeration was conducted on control and high-dose animals, and extended to lower dosed in the event of an adverse effect.
For the testes, a detailed qualitative examination were made; taking into account the tubular stages of the spermatogenic cycle at evaluation of additional hematoxylin-PAS (Periodic Acid Schiff) stained slides.
Cohort 2: Neurohistopathology were performed for all control and high dose cohort 2A animals after PND 90.
Brain histopathology was performed for all control and high dose cohort 2B animals per sex on PND 21 or PND 22. Examinations were not extended to animals of the other dose groups, as treatment-related changes were not observed in the high dose group.
For cohort 2A and 2B animals, multiple sections were made from the brain to examine olfactory bulbs, cerebral cortex, hippocampus, basal ganglia, thalamus, hypothalamus, mid-brain (thecum, tegmentum, and cerebral peduncles), brain-stem and cerebellum.
For cohort 2A, the eyes (retina and optic nerve) and samples of peripheral nerve, muscle and spinal cord were examined.
Morphometric evaluations were performed in 10/sex/dose (C and HD) on representative areas of the brain. At least two consecutive sections were taken at each landmark (level) in order to select the most homologous and representative section for the specific brain area to be evaluated.
All gross lesions were subjected to histopathological evaluation.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
In terminally sacrificed parental male and female animals, predominant clinical signs transiently observed in the majority of HD animals and few MD parental females were increased salivation and moving the bedding.
Low incidences of slight clinical signs like hairless area, piloerection, alopecia on various body parts, wound, crust, regurgitation of the test item and half eyelid closure were observed in few animals on few days in all groups including controls.

Dermal irritation (if dermal study):
not examined
Description (incidence):
From LD group 3 females (no. 143 on gestation day 21, 151 on premating day 6 and 158 on post-natal day 4) and from MD group 1 female (no. 189 on post-natal day 18) was euthanised for animal welfare reasons.From LD group 3 females (no. 143 on gestation day 21, 151 on premating day 6 and 158 on post-natal day 4) and from MD group 1 female (no. 189 on post-natal day 18) was euthanised for animal welfare reasons.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
In both male and female parental animals, there was no test item treatment related effect observed on group mean body weight and body weight gain in the dose groups when compared with the controls. There were no statistically significant differences observed for body weight and body weight gain between the dose groups and the control group except statistically significantly lower body weight gain during day 42-49 in LD and during day 63-70 in LD and MD males when compared with the controls. Due to the lack of dose dependency and consistency, this effect on parental male body weight gain was not considered as test item related.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
IIn correlation to the body weight and body weight gain, the food consumption in both males and female of parental generation tended to increase with the progress of the study in all study groups.
No test item related or statistically significant effect on food consumption was observed in males and females of parental generation during the whole study period.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
IIn 10 selected parental males per group sacrificed at the end of the treatment period, no test item related adverse effects were observed for haematological parameters in dose groups when compared to the control group. Marginally but statistically significantly higher LUC (large unstained cells) rate in HD were observed when compared to the controls. This is not considered to be adverse.
In 10 selected parental females per group sacrificed at the end of treatment period, marginally but statistically significantly lower group mean HCT and WBC in the HD group, higher MCHC in the MD and HD groups and higher neutrophils in the LD and MD groups were observed when compared with the controls but not considered to be adverse as this difference was attributed to very high values from just one control and very low values from one HD female.
No test item related effect was observed on coagulation parameters in parental females when compared with the controls except slight but statistically significantly higher prothrombin time (PT) in MD group when compared with the control which was not considered to be of toxicological relevance.
All other group mean and most of the individual values for haematological parameters in male and females were comparable with the controls and within the normal range of variation.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
In 10 selected parental males sacrificed at the end of the treatment period, statistically significantly lower alkaline phosphatase (AP) in the MD and HD groups, total protein (TP) and urea in the HD group and total bile acids (TBA) in the LD group were observed when compared with the control group, which were either not dose dependent or clinically irrelevant.
In 10 selected parental females sacrificed at the end of the treatment period, statistically significantly higher ALAT and lower creatinine in HD group were observed when compared with the control. As an increase in ALAT was minimal (not 2-3 fold increase to show some adversity) and no histopathological findings were observed in the liver, this effect is assumed to be incidental.
All other group mean and most of the individual values for clinical chemistry parameters in male and females were comparable with the controls and within the normal range of variation.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
The urinalysis performed in 10 selected male and female animals per group from parental and cohort 1A sacrificed at the end of treatment period revealed no test item treatment related effect in treatment groups when compared with the controls and all urinary parameters were in the normal range of variation. Slightly high protein levels were found in the urine of few male and females of all groups including the control group. Therefore, this effect on urine parameters was not considered to be test item related.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
During the weekly detailed clinical observation, no relevant biological or toxicologically relevant differences between the groups were observed in parental during the entire study period.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Under the conditions of this study, there were P-generation animals sacrificed during the course of the study. The decedents were randomly distributed and, hence, death was not deemed to be related to treatment with the test item. There were no gross lesions and histological findings that could be attributed to treatment. Non-pregnant females were randomly distributed throughout the groups including controls. No specific findings were noted that could be related to infertility, and, again these cases were not related to treatment with Propyl 4-hydroxybenzoate.
Histopathological findings: neoplastic:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Test item had no biologically significant effect on the estrous cycle analysed during 2 weeks pre-treatment and 2 weeks premating period after the first administration in treatment groups
when compared to the controls. There were no considerable differences in the length or sequence of cycle stages between the treatment groups and the control group.
Reproductive function: sperm measures:
effects observed, non-treatment-related
Description (incidence and severity):
Test item had no effect on epididymal sperm motility count analysed from all males at terminal sacrifice by using Hamilton Thorn Sperm Analyser (TOX IVOS Version 13.0C). Although group mean motility values from parental males were marginally lower in HD group, this effect was attributed to very low values from one each parental male and sperm motility values from all other males were comparable with control and therefore this marginal decrease in group mean motility in the HD group was not considered as adverse.
Evaluation of sperm morphology from control and HD parental males did not reveal any indicator for toxicity induced by the test item, and percentage of normal and abnormal sperms in treatment groups were comparable with the controls.
Test item had no effect on sperm head count in the dose groups of this study. No considerable and statistically significant differences were observed between dose and control groups of parental animals.
Reproductive performance:
no effects observed
Description (incidence and severity):
There were no test item related effects observed on the reproductive indices (percent male mating index, female mating index, male fertility index, female fertility index, gestation index and live birth index) in parental dose group animals when compared to the respective control group. The survival index of pups during PND 0 to 4, PND 4 after interim sacrifice to PND 13 and PND 13 after interim sacrifice to PND 21 in parental females and during PND 0 to 4, PND 4 after interim sacrifice to PND 14 remained unaffected and within the range of biological variation in treatment groups when compared with the respective controls.
Dose Formulation Analysis
Concentration Analysis
Concentration analysis of formulation samples was determined at three concentrations, 20 mg/mL, 60 mg/mL and 200 mg/mL in study weeks 1, initiation of month 2 (week 5), 3 (week 9), 4 (week 13) and in the last week of the study. However measurement of initiation of month 3 (week 13) had to be repeated due to an invalid run. Repetition of measurement was done in week 16. The mean recoveries observed for the LD dose group was between 94.6 % and 105.8% of the nominal value, between 92.4 % and 106.8 % for the MD dose group and between 91.9 % and 105.2 % of the nominal value for HD dose group. The mean recoveries observed in the low dose (LD), medium dose (MD) and high dose (HD) groups were 100.0 %, 101.4 %, and 96.6 % of the nominal concentration, respectively.
Nominal concentrations were confirmed for all dose groups, as measured concentrations were within acceptance criterion of 15 %.
Homogeneity
Homogeneity of formulation samples was determined at three concentrations, 20 mg/mL, 60 mg/mL and 200 mg/mL, in study weeks 1, initiation of month 2, 3, 4 and in the last week.
The coefficients of variation of the different sampling locations (top, middle, bottom) was between 1.0 % and 3.7 % in LD dose group, between 1.0 % and 3.3 % in MD dose group and between 0.5 % and 5.6 % in HD dose group. All samples were homogenous, as COV was below or equal 10 %.
Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: ..
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
In terminally sacrificed males and females from various F1 cohorts, similar clinical signs like parental animals were observed in HD group but in few animals compared to parental animals.
As these findings showed no dose-dependency and were noted transiently, they were not considered to be toxicologically relevant.
The clinical signs salivation and moving the bedding in males and females were observed immediately after the dose administration and therefore were considered to be a sign of a local reaction to the test item rather than a systemic adverse effect and has no toxicological relevance.
None of the parental or cohort 1B females showed signs of abortion or premature delivery.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
Cohort 1A:
In cohort 1A, 2 HD males (no. 282 on day 32 and 294 on day 62), 1 MD male (no. 266 on day 41) and 2 HD females (no. 373 on day 12 and 380 on day 29) were found dead.
Cohort 1B:
In cohort 1B, 1 control female (no. 480 on day 2) was sacrificed in moribund condition due to accidental femur fracture. There was also one control male (no. 392 on day 24) and one MD male (no.424 on day 51) found dead during the study period.
Cohort 2A:
In cohort 2A, one MD female (no. 608 on day 28) was sacrificed in moribund condition due to animal welfare reasons.
Cohort 3:
In cohort 3, 1 LD male (no. 715 on day 24). 1 HD male (no. 741 on day 26), 1 LD female (no. 777 on day 19) and one HD female (no. 793 on day 18) were found dead during the study period.
No specific clinical signs were observed in these animals before death or moribund sacrifice except abnormal breathing on the day of mortality or moribund sacrifice in 3 females (no. 777 from LD and no. 793 from HD, cohort 3 and 608 from MD cohort 2A). Predominant findings observed at necropsy were fluid filled lung (female no. 777 from LD cohort 3 and female no. 380 from HD cohort 1A), fluid filled thoracic cavity (male no. 424 from MD cohort 1B) and abnormal white or dark discoloured lung (male no. 424 from MD cohort 1B and male no. 294 from HD cohort 1A).
Histopathologically, the cause of death was not evident in most animals. However, in animals observed with fluid filled thoracic cavity or fluid filled lung at necropsy, the cause of death could be related to the technical gavage error and not due to systemic toxicity due to test item administration.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
In cohort 1A animals, marginally but statistically significantly lower group mean body weight on day 1 (post weaning), day 36 to 64 in males and on day 1 in females was observed in HD group when compared with the controls. There was also statistically significantly higher body weight gain during day 50-57 in LD males and lower body weight gain during day 29-36 and overall period of day 1-64 was observed in HD males. However, in females, no such trend of statistically significantly higher body weight gain during day 22-29 and overall period of day 1-64 in LD and HD group was observed when compared with the controls. As this observed effect on male body weight and body weight gain in HD group was marginal (< 10%) and could be attributed to already low initial body weights on day 1 before initiation of direct dose administration as compared to the controls and therefore this effect was not considered to be adverse.
In cohort 1B animals, statistically significantly higher group mean body weight gain was observed during day 92-99 in MD and HD group males and lower body weight gain during day 99-106 in HD males when compared with the controls. In females, statistically significantly higher group mean body weight gain was observed during day 15-22, 29-36, 1-71 in HD group, during day 57-64 in LD and MD group and during day 1-71 in MD group when compared with the controls which was not considered to be adverse. No test item related effect observed on body weight and body weight gain in cohort 1B females during gestation and lactation period.
In cohort 2A males and females, no statistically significant differences were observed on body weight and body weight gain between the dose groups and the control group except statistically significantly lower body weight gain during day 1-8 in LD and during day 36-43 in HD males when compared with the controls. Due to the lack of dose dependency and consistency this effect on cohort 2A male body weight gain was not considered to be adverse.

In cohort 3 males and females, no statistically significant differences were observed on body weight between the dose groups and the respective control group. However, statistically significantly higher group mean body weight gain was observed during day 1-8 and 29-43 in LD, day 15-22 in C2 and statistically significantly lower body weight gain during day 36-43 in C2 males when compared to the controls. There was also statistically significantly lower group mean body weight gain observed during day 29-43 in C2 group females which was not considered to be test item related.
In cohort 4 males and females, there was no test item related or statistically significant effect observed on mean body weight and body weight gain between the control and HD group.
Overall in all parental and F1 cohorts animals, body weight and body weight gain remained unaffected by the treatment with test item and values were in the normal range of variation throughout the treatment period when compared to the control group.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
In correlation to the body weight and body weight gain, the food consumption in both males and female of F1 cohorts (Cohort 1A, 1B, 2A, 3 and 4) tended to increase with the progress of the study in all study groups.
No test item related or statistically significant effect on food consumption was observed in males and females of F1 cohorts during the whole study period except statistically significantly lower group mean food consumption was observed during day 36-43 in the MD and HD males of cohort 1A and statistically significantly higher group mean food consumption during day 29-36 in LD group females of cohort 1A when compared with the controls. Due to the lack of dose dependency or consistency, this effect on food consumption in cohort 1A animals was not considered to be adverse.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
F1-1A cohort:
In 10 selected cohort 1A males per group sacrificed at the end of the treatment period, marginally but statistically significantly higher HGB in the HD group, WBC and basophils in MD and HD groups and lower platelets and neutrophils in the HD group was observed when compared with the controls. As the differences were very slight and values were within the range of historical control data or without dose-dependency this is not assumed to be toxicologically relevant.
In 10 selected cohort 1A females per group sacrificed at the end of the treatment period, marginally but statistically significantly higher HCT and HGB in the MD group, RBC in MD, WBC, monocytes and basophils in the LD, MD and HD groups were observed when compared with the controls. As the differences were very slight and values were within the range of historical control data or without dose-dependency this is not assumed to be toxicologically relevant.
In the absence of test item related histopathological findings and effect on splenic lymphocyte subpopulation in the study, above-mentioned increase or decrease in few haematology parameters was not considered to be adverse.
No test item related effect was observed on coagulation parameters in cohort 1A males and females when compared with the respective controls. Marginal but statistically significantly higher group mean prothrombin time (PT) in HD females was observed when compared with the controls, but is not considered toxicologically relevant.
All other group mean and most of the individual values for haematological parameters in male and females were comparable with the controls and within the normal range of variation.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
F1-1A cohort:
In 10 selected cohort 1A males sacrificed at the end of treatment period, statistically significantly lower creatinine in HD and urea in the LD, MD and HD groups were observed when compared with the control.
In 10 selected cohort 1A females sacrificed at the end of treatment period, statistically significantly lower AP and urea in the MD and HD groups and higher potassium in the HD group were observed when compared with the control. In absence of test item related histopathological findings and clinical signs, this effect on few clinical chemistry parameters in cohort 1A animals was not considered to be toxicologically relevant.
All other group mean and most of the individual values for haematological parameters in male and female cohort 1A animals were comparable with the controls and within the normal range of variation.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
The urinalysis performed in 10 selected male and female animals per group from cohort 1A sacrificed at the end of treatment period revealed no test item treatment related effect in treatment groups when compared with the controls and all urinary parameters were in the normal range of variation. Slightly high protein levels were found in the urine of few male and females of all groups including the control group. Therefore, this effect on urine parameters was not considered to be test item related.
Sexual maturation:
effects observed, non-treatment-related
Description (incidence and severity):
F1-1A cohort:
No significant difference in day of onset of vaginal opening in females and balano-preputial separation in males was observed. However marginally but statistically significantly lower male group mean body weight on the day of attainment of balano-preputial separation was observed in the MD and HD group when compared with the controls.
F1-1B cohort:
No significant difference in group mean body weight and day of onset of the vaginal opening in females and balano-preputial separation in males was observed in treatment groups when compared with respective controls.
F1-2A cohort:
No statistically significant difference in group mean body weight and day of onset of the vaginal opening in females was observed in treatment groups when compared with controls. However, in males, moderately higher group mean body weight and day of attainment of balano-preputial separation was observed in the HD group when compared with the controls which is not considered to be adverse.
F3-3 cohort:
No statistically significant difference in group mean body weight and day of onset of the vaginal opening in females and balano-preputial separation in males was observed in treatment groups when compared with respective controls.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
F1-1A cohort:
In cohort 1A males, slight but statistically significantly lower absolute and relative liver weights in the MD group, absolute liver, heart and adrenal gland weights in the HD group were observed when compared with the controls.
In Cohort 1A females, there were no statistically significant differences in the absolute and relative organ weights of the dose groups when compared to the control group.
Weights of lymph nodes, spleen and thymus of cohort 1A animals revealed no considerable changes that could indicate a test item related immunotoxic effect.
F1-1B cohort:
Organ weight from male and female cohort 1B animals remained unaffected due to treatment with the test item.
F1-2A and F1-2B
No effect on brain weights were observed in male and females of cohort 2A and 2B.
F1 pups not selected for cohorts and F2 cohort
No effect on brain, spleen and thymus weights were observed in F1 pups not selected for cohorts and F2 pups from cohort 1B females at weaning.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Few spontaneous gross pathological changes were recorded for male and female animals from parental generation and various cohorts and were not considered to be treatment-related.
Based on microscopic examination, these findings were not considered to be of test item treatment relevance but deemed incidental. (histo not available yet and will be adjusted accordingly)
Histopathological findings:
no effects observed
Description (incidence and severity):
In the F1-generation, there were also few animals that died during the course of the study. In a few cases, the cause of death may have been related to gavage accidents. Neither gross lesions nor histological lesions could be attributed to treatment with the Propyl 4-hydroxybenzoate. Neuropathology evaluation and evaluation of reproductive organs did not reveal any induced lesion.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
During the weekly detailed clinical observation, no relevant biological or toxicologically relevant differences between the groups were observed in F1
cohorts (1A, 1B, 2A and 3) during the entire study period. There were statistical significances observed in few parameters in F1 cohorts on few occasions. However, observed statistical significance in few parameters were either before initiation of treatment, not dose dependent/consistent or biologically relevantand therefore these findings were considered to be incidental and not related to the treatment with the test item.

Learning and Memory

Cohort 4:
Analysis of learning and memory data from cohort 4 males and females revealed that there were no statistically significant differences in escape latency time observed during the learning phase (PND 28/29) between HD group and control group in both genders by considering 6 rounds of mean escape latency. Similarly, HD group animals subjected to the test during the memory phase on PND35/36 showed no statistically significant difference in escape latency time when compared to the controls. In female HD rats, the overall mean escape latency of 6 rounds was statistically significantly lower when compared control female. This indicates rather an improved than impaired learning and memory as demonstrated by the Y maze-learning and memory test.

Auditory Startle response
Cohort 2A:
Analysis of data from auditory startle test (Kinder Scientific Startle Reflex Measuring System) performed on PND 24 using male and female animals in
cohort 2A revealed no toxicologically significant changes observed in mean startle response in animals treated with test item groups as compared to the
control group. The mean response amplitude on each block of 10 trials (5 blocks of 10 trials) was determined, with test conditions optimized to
produce intra-session habituation


Functional Observations
Cohort 2A:
In males and females from cohorts 2A, no relevant or statistically significant effects were observed in any of the parameters of the functional observation
battery during evaluation between PND 63 and 75 when compared with the controls. There were no biologically relevant differences observed in body
temperature between the groups.

Motor Activity
Cohort 2A:
Based on data from males and females from cohort 2A, no relevant effects were observed on motor activity (slow and fast animal movements,
number of slow and fast stereotypies and number of slow and fast rearings) in treatment groups during evaluation between PND 63 and 75 when
compared with the controls.
Developmental immunotoxicity:
no effects observed
Description (incidence and severity):
Results reported here were obtained using a KLH concentration of approx. 0.06 mg/kg. Thus, the dose that was applied to the animals intravenously was approx. only 20 % of the intended and validated dose of 0.3 mg/kg bw (see attached expert statement). Therefore, although visible in the data, the immunological stimulus used in this study was weaker than intended which has to be taken into consideration when interpreting the test results. An expected immunosuppressive effect after immunization with KLH was shown after administration of positive control substance cyclophosphamide in female animals (based on IgM and IgG values). In male animals of this group KLH-specific IgM levels were lower than in negative controls, however, they were mostly slightly increased when compared to pre-immunization levels. Nevertheless, even under consideration of lower KLH-specific IgG antibody levels an immunosuppressive effect could also be demonstrated in males of this positive control group. In almost all male animals of the test item dose groups KLH-specific IgM (day 6) and IgG (day 14) level responses were higher than before immunization and also higher than in the positive control group but lower than in the negative control group. Although the interpretation of the data with regard to a potential immunosuppressive effect is hampered this demonstrates a functioning immune system. Due to the administered lower KLH concentration and the variability of data, interpretation of the data is only possible to a limited degree but does not point to a biologically meaningful immunsuppressive effect of the test item in male animals. In females of the test item dose groups, specific IgM levels following KLH administration compared to before immunization were increased in less animals and not quite as clear as in the negative control group. However, KLH-specific IgG levels in these animals were more similar to negative control animals which indicates a functioning immune system. Increased total IgM and IgG serum levels in these animals indicate a normal immune response to administration with the immunogen. Overall, the results obtained allow to conclude the correct functioning of the immune system. This conclusion is supported by the absence of any other effect on the immune system in this study (clinical observations including body temperature measurements, phenotyping of splenocyte subpopulations, clinical pathology parameters, macroscopic and histopathological evaluation of lymph nodes, peyer patches, spleen and thymus) which provides strong evidence with regard to the absence of an immunotoxic effect. The observed findings did not show any clinical relevance or signs of an impaired functioning of the immune system and are thus not considered to be toxicologically relevant.
Estrous Cyclicity
F1-1A cohort females:
In cohort 1A females, no statistically or biologically significant effect observed on the duration of vaginal opening till first estrous cycle in treatment groups when compared with the controls.
In these animals no biologically significant effect was observed on the estrous cycle length or sequence of cycle stages between the treatment groups and the control group analysed from PND 75 for 2 weeks.

Litter Data
Parental females:
In parental females, there were no test item treatment related or statistically significant effects of of Propyl 4-hydroxybenzoate on litter data parameters like group mean total number of pups born, number of male pups, number of female pups, sex ratio, number of live pups, stillbirth, runt on PND 0 as well as number of live pups, male pups, number of female pups and sex ratio on PND 4, 7, 13 and PND 21 when compared with the controls.
F1-1B cohort females:
From 1B females, litter parameters like group mean total number of pups born, number of male pups, number of female pups, sex ratio, number of live pups, stillbirth, runt on PND 0 as well as number of live pups, male pups, number of female pups and sex ratio on PND 4 7, 14 and PND 21 remained unaffected when compared with the controls. Statistical analysis of litter data revealed no statistically significant effects observed in treatment groups when compared with the controls.


Litter Weight Data
Parental generation and F1-1B cohort females:
There was no test item related effect on pup mean weight, total litter weight, male and female litter weight on PND 0, PND 4, 7, 14 and PND 21 observed in parental and cohort 1B treatment groups when compared with the controls. There was no statistically significant change in dose groups compared to corresponding controls except for statistically significantly lower pup mean weight from parental females on PND 14 in the HD group.


Parental generation and F1-1B cohort females:
There was no test item related or statistically significant effect observed on the duration of precoital interval and the duration of gestation in the parental and cohort 1B female dose groups when compared to the respective control group.


Pre- and Post-Natal Data
Parental generation and F1-1B cohort females:
There were no test item treatment related effects observed on the number of corpora lutea, implantation sites, live pups on PND 0, percent preimplantation loss and post implantation loss in parental and cohort 1B treatment group females when compared with the corresponding control group.


Reproductive Indices
Parental generation and F1-1B cohort:
There were no test item related effects observed on the reproductive indices (percent male mating index, female mating index, male fertility index, female fertility index, gestation index and live birth index) in parental and cohort 1B dose group animals when compared to the respective control group. The survival index of pups during PND 0 to 4, PND 4 after interim sacrifice to PND 13 and PND 13 after interim sacrifice to PND 21 in parental females and during PND 0 to 4, PND 4 after interim sacrifice to PND 14 and PND 14 to PND 21 in cohort 1B females remained unaffected and within the range of biological variation in treatment groups when compared with the respective controls.


Pup Survival Data
Parental generation and F1-1B cohort females:
No test item related effect on mean mortality of pups from PND 0 to 4, PND 4 after interim sacrifice to PND 13 and PND 13 after interim sacrifice to PND 21 in parental females and from PND 0 to4, PND 4 after interim sacrifice to PND 14 and PND 14 to PND 21 in cohort 1B female treatment groups when compared to the control group.
A mean mortality of pups from parental and cohort 1B females was comparable with the respective controls and slight differences are considered as incidental and not related to the treatment with the test item.


Anogenital Distance and Nipple Retention
Parental generation and F1-1B cohort females:
In male pups from parental females on PND 0, marginal but statistically significantly lower pup weight and absolute anogenital distance (AGD) in HD groups were observed when compared to the controls. No effect was seen on relative AGD. In female pups from parental females on PND 0, minimal but statistically significantly lower absolute and relative anogenital distance in LD, MD and HD groups was observed when compared with the controls. As the differences were only very slight, the animals developed normally and values were within the range of historical control data this is not assumed to be toxicologically relevant.
In male pups from cohort 1B females on PND 0, statistically significantly shorter absolute and relative AGD were observed in the HD group when compared to the controls. As values were within the normal range of historical control data this is not considered toxicologically relevant. In female pups from cohort 1B females on PND 0, no statistically significant effect was observed on any AGD or pup weight parameter.

Pup External Findings
Parental generation and F1-1B cohort females:
No test item related gross external abnormalities of toxicological relevance on PND 0-20 were observed in the pups of any of the groups from parental and Cohort 1B females.
Few specific findings like dark snout, dark spot on various body locations were observed in few pups from parental and Cohort 1B females and considered to be spontaneous in nature and not related to test item treatment.


Thyroid Hormone (T4 and TSH) Analysis

PParental generation:
In parental males and females (10/sex/group), group mean T4 and TSH levels were comparable with the controls except statistically significantly higher group mean TSH values in HD group parental females. As this was not associated with the microscopic finding of hypertrophy or an increased weight of the pituitary gland, this is not assumed to be toxicologically relevant.
F1 pups on PND 4 and PND 21:
In pups sacrificed on PND 4 (10/sex/group - pooled samples), T4 and TSH levels in treatment groups were comparable with the controls. In pups sacrificed on PND 21 (10/sex/group), T4 levels in treatment groups were comparable with the controls.
F1-1A cohort:
In males and females of cohort 1A (10/sex/group), group mean T4 values were comparable with the controls. Statistically significantly higher group mean T4 values in MD and HD females are not considered toxicologically relevant as individual values were within the range of historical control data. Moreover, no test item related effect of toxicological relevance was observed on thyroid weight and thyroid histopathology. There was also an increase in group mean TSH values in HD group males when compared with the controls. As this was not associated with the microscopic finding of hypertrophy or an increased weight of the pituitary gland, this is not assumed to be toxicologically relevant.
The measured hormone concentrations in all males and females were observed with high individual variation.


Sexual Maturity
All selected F1 male and female pups from all cohorts (except cohort 2B, cohort 4 and surplus not selected for cohorts) were checked daily for balano-preputial separation or vaginal patency, respectively starting from PND 30 in males and PND 25 in females.
No abnormalities of genital organs like persistent vaginal thread, hypospadia or cleft penis were observed in any of the pup.
F1-1A cohort:
No significant difference in day of onset of vaginal opening in females and balano-preputial separation in males was observed. However marginally but statistically significantly lower male group mean body weight on the day of attainment of balano-preputial separation was observed in the MD and HD group when compared with the controls.
F1-1B cohort:
No significant difference in group mean body weight and day of onset of the vaginal opening in females and balano-preputial separation in males was observed in treatment groups when compared with respective controls.
F1-2A cohort:
No statistically significant difference in group mean body weight and day of onset of the vaginal opening in females was observed in treatment groups when compared with controls. However, in males, moderately higher group mean body weight and day of attainment of balano-preputial separation was observed in the HD group when compared with the controls which is not considered to be adverse.
F3-3 cohort:
No statistically significant difference in group mean body weight and day of onset of the vaginal opening in females and balano-preputial separation in males was observed in treatment groups when compared with respective controls.


Analysis of splenic lymphocyte subpopulation
F1-1A cohort:
Analysis of splenic lymphocyte subpopulation (CD4+ and CD8+ T lymphocytes, B lymphocytes, and natural killer cells) from 10 male and 10 female animals per group from cohort 1A revealed that there was a slight increase in mean lymphocytes and T cell populations in both male and females when compared to controls. There was no test item related change in the number of splenic B cells which was found to be comparable between dose groups and control group in both male and female. Thus, there was no indication of immunosuppressive effect of the test item on lymphocytes sub-populations


Determination of Test Item Plasma Level
The systemic exposure to Propyl 4-hydroxybenzoate (test item) and 4-hydroxybenzoic acid (metabolite) assessed from parental and cohort 1A male and female rats (30 ± 10 minutes post dose) were demonstrated at LD (100 mg/kg/day), MD (350 mg/kg/day) and HD (1000 mg/kg/day) groups. Whereas in parental animals the test item was detected at low concentrations or below the level of quantification, the metabolite was found in almost all dosed animals at high concentrations. In addition, exposure to Propyl 4-hydroxybenzoate (test item) and 4-hydroxybenzoic acid (metabolite) was demonstrated in F1 pups (PND4, 13, 21), Cohort 1B-F2 pups (PND4, 21) and Cohort 4 pups (PND38/39). Exposure at PND4 demonstrated transfer of test item via milk.
Parental generation:
In total, 40 males and 40 females (10 animal/sex/group) of Parent (P) were subjected to blood sampling 1 week before final necropsy (30 ± 10 minutes post dose) and collected plasma samples were analysed for Propyl 4-hydroxybenzoate (test item) and 4-Hydroxybenzoic acid (metabolite). The results revealed that male (test item - C-1/10, LD-2/10, MD-3/10 & HD-3/10; metabolite -C-1/10, LD-10/10, MD-10/10 & HD-10/10) and female (test item- C-1/10, LD-0/10, MD-5/10 & HD-8/10; metabolite- C-0/10, LD-9/10, MD-10/10 & HD-10/10) were exposed to the test item. There was a higher plasma concentration of metabolite in both genders; however females were more exposure to metabolite in HD than in males. Detailed investigation revealed that contamination of test item in one male and one female control group may be due to environmental exposure (ex-vivo) or during sample handling.
F1-1A cohort:
In total, 40 males and 40 females (10 animal/sex/group) of F1 pups were subjected to blood sampling on PND4 and 21 and also on PND13 with 10 males and 10 females. The plasma samples were analysed for Propyl 4-hydroxybenzoate and 4-Hydroxybenzoic acid. Results revealed that most of the pups from treated groups (PND4, 13 & 21) were exposed to test item and metabolite in both genders. In addition, there were control sample contaminations which are almost similar to test item group plasma concentration. The detailed investigation revealed that contamination of test item in male and female of control pups (PND4-12/20, PND13-10/10 & 19/20) was due the xylazin/ketamine (i.p) which was used to anaesthetize pups for blood sampling. The compositional analysis of anaesthetic drug revealed that it has both propyl and methyl paraben as preservative.
Thus, the plasma contamination in control pups is considered to be a contamination at terminal sacrifice using anaesthesia xylazin/ketamine. Finally, it was hypothetized that it is incorrect to interpret these results as confirmation of systemic exposure to the test item.
In order to confirm this, plasma from Cohort 4 rats was sampled at PND38/39 after anaesthetizing with xylazin/ketamine (5/sex/group) and after cervical dislocation, without anaesthesia (5/sex/group). Results of plasma analysis revealed that those control rats which received xylazin/ketamine showed similar plasma concentration like F1-1A pups and those rats sacrificed by cervical dislocation did not show plasma concentration of test item or metabolite. The HD group showed sufficient exposure of test item and metabolite in plasma. This showed that contamination of F1-1A control pup plasma was due to xylazin/ketamine anaesthesia at terminal sampling.
In total, 40 males and 40 females (10 animal/sex/group) of Cohort 1A were subjected to blood sampling 1 week before final necropsy (30 ± 10 minutes post dose) and collected plasma sample analysed for test item and metabolite. The results revealed that male (test item - C-3/10, LD-3/10, MD-2/10 & HD-6/10; metabolite -C-0/10, LD-9/10, MD-10/10 & HD-10/10) and female (test item- C-2/10, LD-9/10, MD-10/10 & HD-8/10; metabolite- C-1/10, LD-10/10, MD-10/10 & HD-10/10) were exposed to the test item. In addition a detailed investigation revealed that contamination of test item in three male and two female control groups may be due to environmental exposure (ex-vivo) or during sample handling.
F2 cohort:
In addition, Cohort 1B-F2 pups were subjected to blood sampling on PND4 and 21 with 20 males and 20 females on respective PND’s. The plasma samples were analysed for Propyl 4-hydroxybenzoate and 4-Hydroxybenzoic acid. Results revealed that only few pups showed plasma concentration of test item and metabolite on PND4 which includes 4 control pups. Considerably more animals were observed in the HD group (test item- C-4/20, LD-4/20, MD-3/20 & HD-9/20; metabolite- C-0/20, LD-1/20, MD-3/20 & HD-17/20). Control sample contamination in 4 pups may be due to environmental exposure (ex-vivo) or during sample handling. On PND21, no animal of the control and LD groups, one pup from the MD group showed metabolite exposure and 6/20 pups of the HD group showed test item and metabolite exposure. These data indicate measurable test item concentration levels at the HD level.




Key result
Dose descriptor:
NOAEL
Generation:
other: Cohort 1A/ 1B/2B and 3
Effect level:
> 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: ..
Key result
Reproductive effects observed:
no
Conclusions:
On the basis of the present study, the Extended One-Generation Reproductive Toxicity Study after oral administration in male and female Wistar Rats with Propyl 4-hydroxybenzoate with dose levels of 100, 300, and 1000 mg/kg body weight day the following conclusions can be made:
General toxicity:
Few mortalities/morbidities were randomly distributed throughout the majority of groups of parental animals and various cohorts. Based on histopathology the cause of death was not evident in most animals whereas in few, the cause of death could be related to the technical gavaging error and not due to systemic toxicity caused by the test item.
There were no clinical signs of toxicological relevance observed in the treatment groups.
Body weight and food consumption were not affected by treatment with Propyl 4-hydroxybenzoate. At termination no effects on clinical pathology (hematology, blood coagulation, clinical biochemistry and urinalysis) were observed in parental generation and in cohort 1A. Furthermore, no test item related gross pathological findings, effect on organ weight and histopathological findings were observed in the study.
Developmental and Reproduction toxicity:
There were no considerable differences in the length or sequence of oestrous cycle stages, duration of precoital interval and the duration of gestation of the parental generation and cohort 1A between the treatment groups and the control group. There were no signs of abortion or premature delivery, in litter parameters, i.e. number of still births, runts, total number of pups, sex ratio, number of live pups, weight of pups, survival index. Corpora lutea, implantation sites, percent preimplantation loss and post implantation loss in parental generation and cohort 1B were unaffected by Propyl 4-hydroxybenzoate. There was no toxicological effect of the test item on reproductive indices (male mating index, female mating index, male fertility index, female fertility index, gestation index and live birth index) in parental generation and in cohort 1B. No test item related external findings were observed in the pups of this study. There was no toxicological effect on anogenital distance and nipple retention and sexual maturity parameters (i.e. vaginal opening and balano-preputial separation).
There were no effects on sperm motility and morphology as well as for sperm head count of parental generation and cohort 1A males.
The test item had no effect on serum T4 and TSH levels in parental generation (males and females) and in cohort 1A animals on PND4, PND 21 and at adult age.
There was no indication of endocrine disruptive properties of the test item in this study.
Neurotoxicity:
There was no test item related effects on learning and memory, auditory startle response, clinical and functional observations and motor activity. Histopathologically, there were no indications of morphological abnormalities in the brain as demonstrated by Haematoxylin & Eosin staining and Fluoro-Jade staining. No morphometric changes were observed in dose groups compared to controls.
Immunotoxicity:
There was no sign of immunotoxicity in this study. The results of the TDAR indicate a functional immune system. Although, KLH-specific IgM levels indicate that a marginal immunosuppressive effect of the test item in males and females cannot be fully excluded, the subsequent IgG response – similar to the negative control - does not show any sign of effect on the specific immune response. An integrated evaluation of all immunologically relevant data of the study comes to the conclusion that this is not considered clinically relevant. These data comprise clinical observations including body temperature measurements, phenotyping of splenocyte subpopulations, clinical pathology parameters, weight of immune organs, macroscopic and histopathological evaluation of lymph nodes, Peyer’s patches, spleen and thymus of parental and cohort 1A animals, where no test item related effects were observed.
In the absence of indication of toxicity, the NOAEL for developmental and reproductive toxicity, neurotoxicity and immunotoxicity is determined as 1000 mg/kg body weight/day.
Executive summary:

On the basis of the present study, the Extended One-Generation Reproductive Toxicity Study after oral administration in male and female Wistar Rats with Propyl 4 - hydroxybenzoate with dose levels of 100, 300, and 1000 mg/kg body weight day the following conclusions can be made: General toxicity: Few mortalities/morbidities were randomly distributed throughout the majority of groups of parental animals and various cohorts. Based on histopathology the cause of death

was not evident in most animals whereas in few, the cause of death could be related to the technical gavaging error and not due to systemic toxicity caused by the test item. There were no clinical signs of toxicological relevance observed in the treatment groups. Body weight and food consumption were not affected by treatment with Propyl 4-

hydroxybenzoate. At termination no effects on clinical pathology (hematology, blood coagulation, clinical biochemistry and urinalysis) were observed in parental generation and in cohort 1A. Furthermore, no test item related gross pathological findings, effect on organ weight and histopathological findings were observed in the study. Developmental and Reproduction toxicity:

There were no considerable differences in the length or sequence of oestrous cycle stages, duration of precoital interval and the duration of gestation of the parental generation and cohort 1A between the treatment groups and the control group. There were no signs of abortion or premature delivery, in litter parameters, i.e. number of still births, runts, total number of pups, sex ratio, number of live pups, weight of pups, survival index. Corpora lutea, implantation sites, percent preimplantation loss and post implantation loss in parental generation and cohort 1B were unaffected by Propyl 4 - hydroxybenzoate. There was no toxicological effect of the test item on reproductive indices (male mating index, female mating index, male fertility index, female fertility index, gestation index and live birth index) in parental generation and in cohort 1B. No test item related external findings were observed in the pups of this study. There was no

toxicological effect on anogenital distance and nipple retention and sexual maturity parameters (i.e. vaginal opening and balano-preputial separation). There were no effects on sperm motility and morphology as well as for sperm head count of parental generation and cohort 1A males. The test item had no effect on serum T4 and TSH levels in parental generation (males and females) and in cohort 1A animals on PND4, PND 21 and at adult age. There was no indication of endocrine disruptive properties of the test item in this study. Neurotoxicity: There was no test item related effects on learning and memory, auditory startle response, clinical and functional observations and motor activity. Histopathologically,

there were no indications of morphological abnormalities in the brain as demonstrated by Haematoxylin & Eosin staining and Fluoro-Jade staining. No morphometric changes were observed in dose groups compared to controls.

Immunotoxicity: There was no sign of immunotoxicity in this study. The results of the TDAR indicate a functional immune system. KLH-specific IgM levels indicate some variability – similar to the negative control – but do not show any sign of effect on the specific immune response. An integrated evaluation of all immunologically relevant data of the study

comes to the conclusion that this is not considered clinically relevant. These data comprise clinical observations including body temperature measurements, phenotyping of splenocyte subpopulations, clinical pathology parameters, weight of immune organs, macroscopic and histopathological evaluation of lymph nodes, Peyer’s patches, spleen

and thymus of parental and cohort 1A animals, where no test item related effects were observed.

In the absence of indication of toxicity, the NOAEL for developmental and reproductive toxicity, neurotoxicity and immunotoxicity is determined as 1000 mg/kg body weight/day.

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

On the basis of the Extended One-Generation Reproductive Toxicity Study (according to OECD 443) after oral administration in male and female Wistar Rats with Propyl 4-hydroxybenzoate with dose levels of 100, 300, and 1000 mg/kg body weight day the following conclusions can be made:

General toxicity:

Few mortalities/morbidities were randomly distributed throughout the majority of groups of parental animals and various cohorts. Based on histopathology the cause of death was not evident in most animals whereas in few, the cause of death could be related to the technical gavaging error and not due to systemic toxicity caused by the test item. There were no clinical signs of toxicological relevance observed in the treatment groups. Body weight and food consumption were not affected by treatment with Propyl 4-hydroxybenzoate. At termination no effects on clinical pathology (hematology, blood coagulation, clinical biochemistry and urinalysis) were observed in parental generation and in cohort 1A. Furthermore, no test item related gross pathological findings, effect on organ weight and histopathological findings were observed in the study.

Developmental and Reproduction toxicity:

There were no considerable differences in the length or sequence of oestrous cycle stages, duration of precoital interval and the duration of gestation of the parental generation and cohort 1A between the treatment groups and the control group. There were no signs of abortion or premature delivery, in litter parameters, i.e. number of still births, runts, total number of pups, sex ratio, number of live pups, weight of pups, survival index. Corpora lutea, implantation sites, percent preimplantation loss and post implantation loss in parental generation and cohort 1B were unaffected by Propyl 4-hydroxybenzoate. There was no toxicological effect of the test item on reproductive indices (male mating index, female mating index, male fertility index, female fertility index, gestation index and live birth index) in parental generation and in cohort 1B. No test item related external findings were observed in the pups of this study. There was no toxicological effect on anogenital distance and nipple retention and sexual maturity parameters (i.e. vaginal opening and balano-preputial separation).

There were no effects on sperm motility and morphology as well as for sperm head count of parental generation and cohort 1A males.

The test item had no effect on serum T4 and TSH levels in parental generation (males and females) and in cohort 1A animals on PND4, PND 21 and at adult age. There was no indication of endocrine disruptive properties of the test item in this study.

Neurotoxicity:

There was no test item related effects on learning and memory, auditory startle response, clinical and functional observations and motor activity. Histopathologically, there were no indications of morphological abnormalities in the brain as demonstrated by Haematoxylin & Eosin staining and Fluoro-Jade staining. No morphometric changes were observed in dose groups compared to controls.

In the absence of indication of toxicity, the NOAEL for developmental and reproductive toxicity, neurotoxicity and immunotoxicity is determined as 1000 mg/kg body weight/day.

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 based on actual analytical determined dose.

 

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 studies, performed according to OECD 443 OECD 422, paid special attention to sperm parameters (OECD 443, 2018; 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 1000 mg/kg bw/d was taken forward for hazard assessment.




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.