Ethyl 4-hydroxybenzoate

Toxicity to reproduction

Administrative data

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)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

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. QA statement)

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.

Test material

Test material information

Constituent 1

Reference substance name:

Propyl 4-hydroxybenzoate

Cas Number:

94-13-3

IUPAC Name:

Propyl 4-hydroxybenzoate

Test material form:

solid

Details on test material:

Composition:
Propyl 4-hydroxybenzoate: 99,7 %
4-Hydroxy-benzoic acid: 0.1 %
unspecified impurity: 0.2 %
Ethanol: < 200 ppm
Propanol: < 200 ppm

melting point: 98 °C

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.

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system 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)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 1 % hydroxyethyl-cellulose (viscosity 80-125 cP, 2 % in water at 20 °C)

Details on exposure:

Preparation of the Test Item 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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 4

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

Examinations

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.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

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.

Details on results (P0)

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 %.

Effect levels (P0)

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: ..

Results: F1 generation

General toxicity (F1)

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.

Developmental neurotoxicity (F1)

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 (F1)

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.

Details on results (F1)

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.

Effect levels (F1)

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: ..

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Applicant's summary and conclusion

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.