Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

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.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
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

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

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.

Administration / exposure

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
Doses / concentrationsopen allclose all
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

Examinations

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

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Slightly reduced body weight gain in males at 15000 ppm, occasionally reaching statistical significance. No such effect noted in females.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Slightly reduced body weight gain in males at 15000 ppm, occasionally reaching statistical significance. No such effect noted in females.
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

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.

Effect levels (P0)

open allclose all
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).

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

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.

Effect levels (F1)

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).

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

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

 

Applicant's summary and conclusion

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.