Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Effects on male reproductive system
- NOAEL (OECD 422 - male rats): 1000 mg/kg bw/d


Effects on female reproductive system
- NOAEL (OECD 422, female rats): 1000 mg/kg bw/d

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-12-03 to 2019-12-23
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted on 29 July 2016
Deviations:
yes
Remarks:
see Overall Remarks
GLP compliance:
yes (incl. certificate)
Remarks:
(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany)
Limit test:
no
Species:
rat
Strain:
Wistar
Details on species / strain selection:
Crl: WI(Han) (Full Barrier)
Sex:
male/female
Details on test animals and environmental conditions:
Test System
Species/strain: healthy Wistar rats, Crl: WI(Han) (Full Barrier)
Source: Charles River, 97633 Sulzfeld, Germany
Sex: male and female; the female animals were non-pregnant and nulliparous
Age at the start of the treatment period: approx. 14-15 weeks old
Body weight at the allocation of the animals to the experimental groups:
males: 326 – 398 g (mean: 360.0 g, ± 20 % = 288.0 – 432.0 g)
females: 207 – 258 g (mean: 227.2 g, ± 20 % = 181.8 – 272.6 g)
The animals were derived from a controlled full-barrier maintained breeding system (SPF). According to the German Act on Animal Welfare the animals were bred for experimental purposes.
This study was performed in an AAALAC-accredited laboratory. According to German animal protection law, the study type has been reviewed and accepted by local authorities.
Furthermore, the study has been subjected to Ethical Review Process and was authorised by the Bavarian animal welfare administration.

Housing and Feeding Conditions
- Full barrier in an air-conditioned room
- Temperature: 22 +/- 3 °C
- Relative humidity: 55 +/- 10 %
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: 10 x / hour
- Free access to Altromin 1324 maintenance diet for rats and mice
- Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- 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 both 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 were provided for all males and for females until GD 18
- Nesting material were 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) under laboratory conditions

Number and Sex of the Animals
80 animals (40 males and 40 females) were included in the study.

Preparation of the Animals
Prior to the start of the treatment period a detailed clinical observation outside the home cage was made. Only healthy animals were used for the study.
Before dosing all females were screened for two weeks for regular oestrous cyclicity and animals (10 females/ group) with regular oestrous cycle (4-5 day cycle)
were used in the study. Before the first administration all animals to be used for the study were weighed and assigned to the experimental groups with achieving
a most homogenous variation in body weight throughout the groups of males and females, respectively (randomisation was performed with IDBS Workbook 10.1.2 software).
Each animal was marked with its identification number by individual ear tattoo or tail marking.



Route of administration:
oral: gavage
Vehicle:
other: 1 % hydroxyethyl-cellulose / aqua ad injectionem
Remarks:
hydroxyethyl-cellulose: Manufacturer: Sigma-Aldrich, Batch No.: MKCD0421, Expiry Date: 05 November 2019 aqua ad injectionem: Manufacturer: Deltamedica, Batch No.: 806148, Expiry Date: May 2021 (each bottle was used for up to one week)
Details on exposure:
The vehicle has been selected in consultation with the sponsor based on the test item’s characteristics.
Based on the results of stability testing (Eurofins Munich Study No. 187385), the test item formulations were prepared at least every 4 days as given by Eurofins Munich Study No. 187385.
The prepared formulation was stored at room temperature.
The test item, as delivered, was grinded before formulation preparation. Afterwards, the 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 1 % aqueous hydroxyethyl-cellulose, the vehicle used in this study. After producing slurry with the glass rod for 1 minute,
the rest of the vehicle was added to give the appropriate final concentration. The formulation was then stirred until visual homogeneity was achieved (at least 30 min).
Formulates were kept under magnetic stirring during the daily administration. The vehicle was also used as control item.

In consultation with the sponsor the following doses were selected for the 3 dose groups (LD = low dose, MD = medium dose, HD = high dose) and 1 control group (C).
The animals were treated with the test item formulation or vehicle on 7 days per week for a maximum period of 63 days in females, i.e. during 14 days of pre-mating
and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day 12 in females.
Males were dosed after the mating period until the minimum total dosing period of 28 days is completed.

Control: 0 mg/kg/d
Low Dose: 100 mg/kg/d
Medium Dose: 300 mg/kg/d
High Dose: 1000 mg/kg/d


The highest dose level was chosen with the aim of inducing toxic effects, but no death or severe suffering.
Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dosage related response and NOAEL.
The animals in the control group were handled in an identical manner to the test group subjects and received the vehicle using the same volume as used for the high dose group.

The test item and vehicle were administered at a single dose to the animals by oral gavage. The application volume for all groups was 5 mL/kg body weight.
For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured.








Details on mating procedure:
Mating was performed using a ratio of 1:1 (male to female). The vaginal smear of the females was checked every morning after the start of the mating period to confirm the mating.
If the vaginal smear of a particular female was not found to be sperm-positive, the actual stage of the oestrous cycle on that day was documented.
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 minimised.




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. 187385).
Prestart homogeneity investigation was included on the samples collected from various levels (top, middle and bottom) of high dose and low dose groups.
The test item was not shown to be homogenous according to Eurofins Study No. 187385. Therefore, 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 study week 1 (pre-mating period),
3 (first week of mating), 5 (gestation) and in the last week of the study (gestation / lactation) (40 samples).
Each sample taken during the study was retained in duplicate (sample A, sample B, each of at least 3 mL). The A-samples were analysed at Eurofins Munich
(Eurofins Munich Study Phase No. 187386) and until then stored under appropriate conditions based on available stability data.
The B-samples were retained at below 15 °C at BSL Munich (test facility) and discarded after completion of the final study report.



Duration of treatment / exposure:
The animals were treated with the test item formulation or vehicle on 7 days per week for a maximum period of 63 days in females, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males
and females, during the gestation period and up to post-natal day 12 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days is completed.
Frequency of treatment:
daily
Details on study schedule:
Arrival of the Test Item: 01 October 2018
Study Initiation Date: 03 December 2018
Date of Amendment to Study Plan: 08 January 2019
Delivery of Animals: 29 November 2018
Acclimatisation Period: 29 November 2018 until 03 December 2018
Experimental Starting Date: 04 December 2018
Treatment Period: 18 December until 10 February 2018
Necropsies: 15 January 2019 – 16 January 2019, 29 January 2019, 05 February 2019 – 11 February 2019
Experimental Completion Date: 11 February 2019
Completion Date of Delegated Phase (Histopathology): 30 August 2019
Completion Date of Delegated Phase (Formulation Analysis):19 December 2019
Study Completion Date: 23 December 2019







Dose / conc.:
0 mg/kg bw/day
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
300 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
No. of animals per sex per dose:
80 animals (40 males and 40 females) were included in the study. 10 male and 10 female animals per group.
Control animals:
yes, concurrent vehicle
Parental animals: Observations and examinations:
Clinical Observations
General clinical observations were made at least once a day, preferably at the same time each day. The health condition of the animals was recorded. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily.
Once before the first exposure, and at least once a week thereafter, detailed clinical observations were made in all animals outside the home cage in a standard arena. Clinical observations included spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size. Changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypes, difficult or prolonged parturition or bizarre behaviour were recorded.

Body Weight and Food Consumption
The animals were weighed once before the assignment to the experimental groups, on the first day of dosing and weekly thereafter as well as at the end of the study.
During pregnancy, females were weighed on gestation days (GD) 0, 7, 14 and 20 and within 24 hours of parturition (day 0 post-partum), on PND 4 and PND 13 along with pups.
All animals were weighed directly before termination.
Food consumption was measured on the corresponding days of the body weight measurements after the beginning of the dose administration.
Food consumption was not measured during the mating period in males and females and the post-mating period in males.

Haematology
Haematological parameters were examined in 5 randomly selected males and females (only lactating females were evaluated) from each group at the end of the treatment
prior to or as part of the sacrifice of the animals. Blood from the abdominal aorta of the animals was collected in EDTA-coated tubes.
The following haematological parameters were examined: haematocrit value (HCT), haemoglobin content (HGB), red blood cell count (RBC), mean corpuscular volume (MCV),
mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), reticulocytes (Ret), platelet count (PLT), white blood cells (WBC), neutrophils (Neut),
lymphocytes (Lym), monocytes (Mono), eosinophils (Eos), basophils (Baso), large unstained cells (Luc)

Blood Coagulation
Coagulation parameters from 5 randomly selected males and females (only lactating females were evaluated) of each group
were examined at the end of the treatment prior to or as part of the sacrifice of the animals.
Blood from the abdominal aorta of the animals was collected in citrate tubes.
The following coagulation parameters were examined: prothrombin time (PT), activated partial thromboplastin time (aPTT)

Clinical Biochemistry
Parameters of clinical biochemistry from 5 randomly selected males and females (only lactating females were evaluated) of each group were
examined at the end of the treatment prior to or as part of the sacrifice of the animals.
Blood from the abdominal aorta of the animals was collected in serum separator tubes.
The following parameters of clinical biochemistry were examined: alanine aminotransferase (ALAT), aspartate-aminotransferase (ASAT), alkaline phosphatase (AP),
creatinine (Crea), total protein (TP), albumin (Alb), urea, total bile acids (TBA), total cholesterol (Chol), glucose (Gluc), sodium (Na), potassium (K)
From 2 female pups per litter on day 4 after birth, from all dams and 2 pups per litter at termination on day 13 and from all adult males at termination, blood samples were collected from the defined site in serum separator tubes. All blood samples were stored under appropriate conditions. Blood samples from the day 13 pups and the adult males were assessed for serum levels for thyroid hormones (T4).
Further assessment of T4 in blood samples from the dams and day 4 pups or other hormones was not deemed necessary as no test item related changes were observed in T4 levels
of adult males and pups on PND 13. Pup blood was pooled by litter for thyroid hormone analysis. Two pups per litter were sacrificed on day 4 after birth and blood samples were taken for possible
serum hormone assessments. The two pups per litter were female pups to reserve male pups for nipple retention evaluations. No pups were eliminated as litter size was below 8 pups (dam no. 60 of the LD group). As there was only one pup available above a litter size of 8, only one pup was sacrificed from dam numbers 43 and 47 from the control group and dam no. 78 from the HD group.
No pups were available from females number 65 and 67 from the MD group as these females were non-pregnant.



















Oestrous cyclicity (parental animals):
Oestrous cycles were monitored before treatment initiation to select for the study females with regular oestrous cyclicity.
Vaginal smears were also examined daily from the beginning of the treatment period until evidence of mating.
Sperm parameters (parental animals):
For the testes, a detailed qualitative examination was made; taking into account the tubular stages of the spermatogenic cycle at evaluation of additional haematoxylin-PAS (Periodic Acid Schiff) stained slides.
Litter observations:
The duration of gestation was recorded and is calculated from day 0 of the pregnancy.
Each litter was examined as soon as possible after the delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities.
Live pups were counted and sexed and litters weighed within 24 hours of littering (PND 0) and on PND 4.
Live pups were identified by tattooing. In addition to the observations of the parent animals, any abnormal behaviour of the offspring was recorded.



Postmortem examinations (parental animals):
Pathology
All males were sacrificed after the completion of the mating period (after a minimum dosing period of 28 days) and females were sacrificed on the respective PND 13 by using anaesthesia (ketamine/xylazin).
Vaginal smears were examined on the day of necropsy to determine the stage of oestrous cycle.
Non-pregnant females were sacrificed on study day 26 using the sperm-positive vaginal smear as an evidence of mating.
All adult animals were subjected to a detailed gross necropsy which included careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents.
Special attention was paid to the organs of the reproductive system. The ovaries, uterus with cervix, vagina, testes, epididymides, accessory sex organs (prostate, seminal vesicles with coagulating glands as a whole), the thyroid/parathyroid glands and all organs showing macroscopic lesions of all adult animals were preserved in 4 % neutral-buffered formaldehyde, except for testes and epididymides which were preserved in modified Davidson’s Solution for 24 hours and then transferred to 70 % ethanol.
The number of implantation sites and corpora lutea was recorded for each parental female at necropsy. The number of corpora lutea and implantation sites was recorded for any females sacrificed 26 days after the end of the mating period with no evidence of mating (female no. 67 from the MD group) and for any females sacrificed on day 26 post-coitum due to non-delivery (female no. 65 from the MD group).
The following tissues (adrenal glands, all gross lesions, aorta, brain (incl. medulla/pons, cerebellar and cerebral cortex), caecum, colon, duodenum, epididymides, eyes, femur with knee joint, Harderian glands,
heart, ileum (including Peyer´s patches), jejunum, kidneys, liver, lungs, lymph nodes (axillary), lymph nodes (mandibular), lymph nodes (mesenteric), mammary gland area (male and female), oesophagus, optic nerves, varies, oviducts, pancreas, pituitary, prostate and seminal vesicles with coagulating glands as a whole, rectum, salivary glands (sublingual, submandibular), sciatic nerve, skeletal muscle, skin,
spinal cord (cervical, thoracic and lumbar segments), spleen, sternum (with bone marrow), stomach, testes, thymus, thyroid/parathyroid glands, tongue, trachea, ureters, urinary bladder,
uterus with cervix and vagina) from the five randomly selected male and female animals were preserved in 4 % neutral-buffered formaldehyde except for eyes, testes and epididymides which were fixed in Modified Davidson’s fixative for approximately 24 hours before they were transferred to 70 % ethanol.
Thyroid/parathyroid glands from non-selected adult animals were preserved for potential histopathological examination, if required.

Organ Weights
The wet weight of the organs (testes (paired weight), uterus with cervix, epididymides (paired weight), ovaries (paired weight), prostate, seminal vesicles and coagulating glands (complete weight),
thymus, thyroid/parathyroid glands (from all adult males and females) - were weighed after fixation (complete weight), liver, kidneys (paired weight); spleen; adrenal glands (paired weight); brain,
pituitary gland, heart) of 5 randomly selected male and female animals (only lactating females were evaluated) from each group was recorded as soon as possible. Paired organs were weighed together.
Reproductive organs (testes, epididymides, prostate with seminal vesicles and coagulating glands, uterus with cervix and ovaries) were weighed from all animals.

Histopathology
A full histopathology was carried out on the preserved organs and tissues (see pathology) of the selected animals of the control and high dose group which were sacrificed at the end of the treatment period. Thyroid/parathyroid glands from pups and from the remaining non-selected adult animals were not examined as no statistically significant or relevant findings were observed in thyroid/parathyroid weight
or serum thyroxine hormone (T4) level of adult animals or pups on post-natal day 13.
Testes, epididymides, ovaries, uterus with cervix, vagina, accessory sex organs (prostate, seminal vesicle with coagulating gland) were trimmed, embedded into paraffin,
cut at an approximated thickness of 2-4 µm and stained with haematoxylin and examined in control and HD animals and in non-pregnant female animals of the LD and MD animals.
Testes, epididymides and accessory sex organs (prostate, seminal vesicle with coagulating gland) were also examined in the mating partners of the non-pregnant female LD and MD animals.
Examinations were not extended to animals of all other dosage groups as no treatment-related changes were observed in the high dose group.
Any gross lesion macroscopically identified was examined microscopically in all animals. Discoloration possibly due to the test item was evaluated in the organs of all dose groups.
The histological processing of tissues to microscope slides was performed at the GLP-certified contract laboratory AnaPath GmbH, AnaPath Services, Hammerstraße 49, 4410 Liestal, Switzerland (test site for tissue processing). The histopathological evaluation was performed at the GLP-certified contract laboratory AnaPath GmbH, AnaPath Services, Hammerstrasse 49, 4410 Liestal, Switzerland (test site for histopathology). The study phases from test site 1 and 2 were performed in compliance with the Swiss Ordinance relating to Good Laboratory Practice adopted 18 May 2005 [SR 813.112.1] (Status as of 01 December 2012). Blocking, embedding, cutting, H&E staining and scientific slide evaluation were performed according to the corresponding SOP’s of the test sites.
The principal investigator for histopathological tissue processing sent all raw data (including blocks, slides, paper raw data, statement of compliance and quality assurance statement) to the study director.
The principal investigator for histopathological evaluation provided the histopathology results to the study director by e-mail and sent a pathology phase report to the study director upon the completion of the study.






Postmortem examinations (offspring):
Pathology
All surviving pups were killed by cervical dislocation on PND 13.. Dead pups and all surviving pups sacrificed on PND 13 were carefully examined externally for gross abnormalities before terminal sacrifice.
Thyroid/parathyroid glands from 1 pup/sex/litter/group (except of litter 72 from the HD group) sacrificed on PND 13 were preserved for potential histopathological examination, if required.

Organ Weights
Thyroid/parathyroid glands from 1 pup/sex/litter/group (sacrificed on PND 13) were preserved.
Weight of thyroid/parathyroid glands was measured after fixation.

Brain and Liver Sample Collection for Additional Evaluation
On PND 4 brain (hippocampus) and liver samples were collected from 3 male and 4 female pups from the control group and from 1 male and 2 female
pups from the HD group. On PND 13 brain (hippocampus) and liver samples from 5 pups/sex of the control and HD group were collected for possible
further evaluation. The samples were flashed frozen in liquid nitrogen and then stored at -80 °C at the test facility until shipment.
The stored samples were shipped to the sponsor selected site for potential further analysis.
The data generated from the collected samples were not part of the study report.


Statistics:
A statistical assessment of the results of body weight, food consumption and litter data was performed for each gender by comparing values
of dosed with control animals using a one-way ANOVA and a post-hoc Dunnett Test. Results of absolute and relative organ weights, parameters
of haematology, blood coagulation and clinical biochemistry were statistically analysed by comparing values of dosed with control animals using
either a parametric one-way ANOVA and a post-hoc Dunnett Test or a non-parametric Kruskal-Wallis Test and a post-hoc Dunn’s Test, based
on the results of homogeneity and normality tests. These statistics were performed with Ascentos 1.3.4 software or
GraphPad Prism V.6.01 software (p<0.05 is considered as statistically significant).
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Moving the bedding was observed in 1/10 females of the control group, 2/10 females of the LD group, 1/10 females of the MD group and 10/10 females and 9/10 males of the HD group mostly in the second half of the treatment period. Increased salivation was noted transiently in 1/10 females of the MD group and in 5/10 males and females of the HD group. Both signs were observed in short timely relation to dose administration or in anticipation thereof and thus were considered to be a sign of discomfort or a local reaction to the test item. These slight signs were not considered as adverse systemic effects.
The clinical sign of piloerection was observed in 4/10 females of the control group, 4/10 females of the LD group, 8/10 females of the MD group, and 7/10 females of the HD group. Due to the absence of dose dependency and the occurrence of piloerection in control animals it was not considered toxicologically relevant. Low incidences of clinical signs without dose dependency (see Details on results P0 ) and thus are not considered test item-related.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
see Details on results P0
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The test item had no statistically significant effect on body weight or body weight change in male animals and body weights of female animals.
Mean body weight gain was slightly but statistically significantly lower in the female LD and female HD group compared to the controls during the first week of the gestation phase. However, without consistency between the groups, without dose dependency and occurrence during only one week of treatment this was not considered adverse. MD females showed no statistically significant differences in body weight change compared to control females. Further slight differences between the groups were within the normal range of variation throughout the treatment period of this study.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
The test item had no toxicologically relevant effect on food consumption in male and female animals of this study.
Slightly but statistically significantly lower food consumption of females of the LD group compared to control females during the second week of gestation was not considered test item related as no differences in food consumption were observed between higher dose groups and the control group. Slightly lower food intake was seen in female animals of the HD group during the premating period. Without achieving statistical significance this is not considered to be an adverse effect.
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):
There was no statistically significant or biologically relevant effect of the test item on haematological parameters of male animals determined at the end of the treatment period of this study. Differences in haematological parameters between test item-treated groups and the corresponding controls followed no dose dependency and within historical control data and thus were not considered test item-related.
There were no statistically significant effects of the test item on coagulation parameters (PT, aPTT) of female animals and on the parameter aPTT of male animals at the end of the treatment period. PT was statistically significantly but slightly higher in males of the HD group compared to control males (13 % above control). However, without the incidence of other clinical findings or macroscopic findings at the time point of necropsy slightly higher PT value of the HD group was not assumed adverse.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
In male animals, mean TBA showed a dose-dependent tendency towards lower TBA levels in test item-treated animals compared to the controls. However, without achieving statistical significance and without a corresponding effect in female animals this difference was not considered adverse. Other parameters of clinical biochemistry showed no statistically significant or biologically relevant differences between males treated with the test item and males of the control group. In female animals there were no statistically significant or biologically relevant differences in any of the parameters of clinical biochemistry when comparing test item treated animals with animals of the control group. The deviation of mean TBA level of HD females from control females (261% deviation from control) was not statistically significant and was mainly based on high TBA levels from two females of the HD group (female numbers 71 and 80) and was not considered adverse.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
see Details on results P0
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):
There was no histological evidence of toxicity in the reproductive organs and tissues including testes, epididymides, prostate gland, seminal vesicles, coagulating glands, ovaries, uterus, cervix, and vagina. No treatment-related effects on the testicular histomorphology and interstitial cell structure were noticed. The treatment with Methyl 4 hydroxybenzoate did not induce histomorphological effects in the reproductive organs of the non-pregnant females (animal nos. 65 and 67) and their pairing partners (animal nos. 25 and 27). Therefore, the histopathological NOEL (no observed effect level) may be established at 1000 mg/kg bw/day.
Histopathological findings: neoplastic:
not examined
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Treatment with the test item had no biologically significant effect on the oestrous cycle analysed during the 2 weeks premating period when comparing test item treated groups 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:
no effects observed
Description (incidence and severity):
Sperm staging - the testes were checked on completeness of cell populations, completeness of
stages and degenerative changes. No treatment-related effects on the testicular histomorphology
were observed. Further, no treatment-related effect on interstitial cell structure was noticed.
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related effects on the reproductive indices including copulation index, fertility index, delivery index, and viability index.
Slight differences in the reproductive indices followed no dose-dependency and were within the range of biological variation
(range of historical control data (±2 fold SD): copulation index: 88.277-108-086, fertility index: 66.806-118.800, delivery index: 90.130-107.029, viability index: 88.977-110.130).

Clinical Observations
Moving the bedding was observed in 1/10 females of the control group, 2/10 females of the LD group, 1/10 females of the MD group and 10/10 females and 9/10 males of the HD group mostly
in the second half of the treatment period. Increased salivation was noted transiently in 1/10 females of the MD group and in 5/10 males and females of the HD group. Both signs were observed
in short timely relation to dose administration or in anticipation thereof and thus were considered to be a sign of discomfort or a local reaction to the test item.
These slight signs were not considered as adverse systemic effects.
The clinical sign of piloerection was observed in 4/10 females of the control group, 4/10 females of the LD group, 8/10 females of the MD group, and 7/10 females of the HD group.
Due to the absence of dose dependency and the occurrence of piloerection in control animals it was not considered toxicologically relevant.
Low incidences of clinical signs like a crust (1/10 males of the HD group and 1/10 females of the MD group), hairless areas (2/10 females of the control group, 3/10 females of the LD group,
6/10 females of the MD group and 1/10 females of the HD group), a scratch/cut (1/10 males of the HD group and 1/10 females of the MD group), an oedema (1/10 males of the control group
and 1/10 females of the MD group), a kinked tail (1/10 females of the LD group), an injured palate (1/10 females of the LD group), hypotonia (1/10 females of the control group),
slow movements (1/10 females of the control group), diarrhoea (1/10 males of the LD group) and aggressiveness (2/10 males of the HD group) were observed in single animals or
without dose dependency and thus are not considered test item-related.

Mortality
No mortality occurred during the treatment period with Methyl 4-hydroxybenzoate in any of the test item-treated groups and the control group. All animals survived the scheduled study period.

Body Weight Development
The test item had no statistically significant effect on body weight or body weight change in male animals and body weights of female animals.
Mean body weight gain was slightly but statistically significantly lower in the female LD and female HD group compared to the controls during the first week of the gestation phase.
However, without consistency between the groups, without dose dependency and occurrence during only one week of treatment this was not considered adverse.
MD females showed no statistically significant differences in body weight change compared to control females.
Further slight differences between the groups were within the normal range of variation throughout the treatment period of this study.

Food Consumption
The test item had no toxicologically relevant effect on food consumption in male and female animals of this study.
Slightly but statistically significantly lower food consumption of females of the LD group compared to control females during the second week of gestation was not considered test item
related as no differences in food consumption were observed between higher dose groups and the control group.
Slightly lower food intake was seen in female animals of the HD group during the premating period. Without achieving statistical significance this is not considered to be an adverse effect.


Haematology and Coagulation
There was no statistically significant or biologically relevant effect of the test item on haematological parameters of male animals determined at the end of the treatment period of this study.
Differences in haematological parameters between test item-treated groups and the corresponding controls followed no dose dependency and thus were not considered test item-related.
There were no statistically significant effects of the test item on coagulation parameters (PT, aPTT) of female animals and on the parameter aPTT of male animals at the end of the treatment period.
PT was statistically significantly but slightly higher in males of the HD group compared to control males (13 % above control). However, without the incidence of other clinical findings
or macroscopic findings at the time point of necropsy slightly higher PT value of the HD group was not assumed adverse.


Clinical Biochemistry
In male animals, mean TBA showed a dose-dependent tendency towards lower TBA levels in test item-treated animals compared to the controls.
However, without achieving statistical significance and without a corresponding effect in female animals this difference was not considered adverse.
Other parameters of clinical biochemistry showed no statistically significant or biologically relevant differences between males treated with the test item and males of the control group.
In female animals there were no statistically significant or biologically relevant differences in any of the parameters of clinical biochemistry when comparing test item treated animals with animals of the control group.
The deviation of mean TBA level of HD females from control females (261% deviation from control) was not statistically significant and was mainly based on high TBA levels from two females of the HD group
(female numbers 71 and 80) and was not considered adverse.

Organ Weights
Slight differences in the mean organ weights between test item-treated groups and the control group are not considered to be caused by treatment with
Methyl 4 hydroxybenzoate as no test item related histopathological findings were observed in any of the organs.
Mean relative pituitary gland weight was slightly higher in the male HD group when compared to the control group (deviation from control: 14 %).
However, due to the lack of dose dependency this was not considered to be adverse and toxicologically relevant.
Absolute (deviation from control: 21 %) and relative (deviation from control: 18 %) mean adrenal gland weight was statistically significantly lower in females of the MD group.
However, this followed no dose dependency and is thereby not considered toxicologically relevant.

Pathology
Only single or occasional macroscopic findings were noted in the groups during necropsy of the animals. These are assumed to be incidental finings without relation to the test item.
Findings were enlarged kidneys (bilateral) of LD male no. 13, small testes (bilateral) of MD male no. 27 and small seminal vesicles (left side) of control male no. 7.
Without corresponding evidence in histopathological examination these macroscopic findings were not considered of toxicological relevance.

Histopathology
There was no histological evidence of toxicity in the reproductive organs and tissues including testes, epididymides, prostate gland, seminal vesicles, coagulating glands, ovaries,
uterus, cervix, and vagina. No treatment-related effects on the testicular histomorphology and interstitial cell structure were noticed. The treatment with Methyl 4 hydroxybenzoate
did not induce histomorphological effects in the reproductive organs of the non-pregnant females (animal nos. 65 and 67) and their pairing partners (animal nos. 25 and 27).
Therefore, the histopathological NOEL (no observed effect level) may be established at 1000 mg/kg bw/day.


Thyroid Hormone (T4) Analysis
Slightly but statistically lower mean thyroxine hormone (T4) level was not considered adverse without corresponding histopathological findings in the thyroid/parathyroid of male animals.

Oestrous Cycles
Treatment with the test item had no biologically significant effect on the oestrous cycle analysed during the 2 weeks premating period when comparing test item treated groups to the controls.
There were no considerable differences in the length or sequence of cycle stages between the treatment groups and the control group.

Precoital Interval and Duration of Gestation
The pre-coital interval and the duration of gestation were not affected by Methyl 4 hydroxybenzoate.
No statistically significant differences were observed when comparing the test item-treated groups with the control group.

Pre- and Postnatal Data
No considerable test item related effects were noted for mean values of number of corpora lutea, implantations sites, live pups on PND 0, 4 and 13 as well as
pre implantation loss and post implantation loss in all dose groups. Slight differences were within the normal range of variation and were not considered toxicologically relevant.


Reproductive Indices
There were no test item-related effects on the reproductive indices including copulation index, fertility index, delivery index, and viability index.
Slight differences in the reproductive indices followed no dose-dependency and were within the range of biological variation
(range of historical control data (±2 fold SD): copulation index: 88.277-108-086, fertility index: 66.806-118.800, delivery index: 90.130-107.029, viability index: 88.977-110.130).


Dose Formulation Analysis
In this study phase 40 samples in total were measured for determination of Methyl 4-hydroxybenzoate in formulation samples received from Eurofins Munich / BSL Munich Study No. 187381 (main study).
Concentration analysis and homogeneity of formulation samples was determined at three concentrations, 20 mg/mL, 60 mg/mL and 200 mg/mL in study weeks 1, 3, 5 and in the last week of the study. The mean recoveries observed for the LD dose group was between 91.6% and 102.6% of the nominal value, between 92.2% and 98.9% for the MD dose group and between 98.6% and 105.3% 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 97.9%, 96.9%, and 102.2% of the nominal concentration, respectively.
Nominal concentrations were confirmed for all dose groups, as measured concentrations were within acceptance criterion of 15%.
The coefficients of variation of the different sampling locations (top, middle, bottom) was between 3.0% and 4.3% in LD dose group, between 1.8% and 5.7% in MD dose group and between 1.0% and 3.7% in HD dose group. All samples were homogenous, as COV was below or equal 15%.









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: see comment
Remarks on result:
other:
Remarks:
No mortality and no clinical signs of systemic toxicity were observed in the animals of this study. Daily oral treatment with Methyl 4-hydroxybenzoate had no effect on body weight and food consumption. At the end of the respective treatment periods of male and female animals there were no changes in clinical pathology results up to 1000 mg/kg bw/day and organ weights were inconspicuous. No relevant macroscopic and no histopathological abnormalities were observed in treated animals. Thus, the NOAEL for general toxicity is determined to be 1000 mg/kg bw/day.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
see Details on results F1
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
No test item-related effect on mean mortality of pups was observed between PND 0 and PND 4 and between PND 4 and 13 in treatment groups
when compared to the control group. Mortality of two pups in the control group was considered incidental.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Treatment with the test item had no statistically significant or biologically relevant effects on litter weight data on PND 0, 4 and 13 when comparing test item-treated groups and the controls. Differences between the groups followed no dose dependency and were within the normal range of variation (range of historical control data (±2 fold SD): PND 0: 4.63-8.141, PND 4: 7.913-13.901, PND 13: 22.947-38.098).
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
effects observed, non-treatment-related
Description (incidence and severity):
No differences of biological relevance were observed in anogenital distance of male and female pups on PND 0 and in nipple retention of male pups on PND 12 when comparing test item treated pups to pups of the control group.
However, mean male pup nipple retention was shown to be statistically significantly lower in the HD group compared to the control group.
As this value was within the normal range of variation (range of historical control data (±2 fold SD): -1.14-1.50), lower nipple retention in the HD group was not considered test item-related.
Female pups treated with the test item were observed with statistically significantly higher mean pup weight (control: 5.76, MD: 6.04) and mean cube root of pup weight (control: 1.79, MD: 1.82) in the MD group and statistically significantly lower absolute (control: 1.31, LD: 1.07) and relative (control: 0.73, LD: 0.60) anogenital distance in the LD group. Without occurrence of these differences in all groups and dose-dependency this was not considered adverse.
Anogenital distance (AGD):
no effects observed
Nipple retention in male pups:
effects observed, non-treatment-related
Description (incidence and severity):
No differences of biological relevance were observed in anogenital distance of male and female pups on PND 0 and in nipple retention of male pups on PND 12
when comparing test item treated pups to pups of the control group.
However, mean male pup nipple retention was shown to be statistically significantly lower in the HD group compared to the control group.
As this value was within the normal range of variation (range of historical control data (±2 fold SD): -1.14-1.50), lower nipple retention in the HD group was not considered test item-related.
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Treatment with Methyl 4-hydroxybenzoate caused no gross external pup findings in any of the test item-treated groups or the control group.
The single external finding of a dark tail in one pup of the LD was not considered test item-related.
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
see Details on results F1: Thyroid Hormone (T4) Analysis, Thyroid Weight on PND 13
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Litter Data
There were no test item-related effects on litter data including total number of male and female pups, sex ratio and number of still births and runts.
There were no statistically significant differences noted for these litter parameters and the values were comparable between dose groups and control group.

Litter Weight Data
Treatment with the test item had no statistically significant or biologically relevant effects on litter weight data on PND 0, 4 and 13 when comparing
test item-treated groups and the controls. Differences between the groups followed no dose dependency and were within the normal range of variation
(range of historical control data (±2 fold SD): PND 0: 4.63-8.141, PND 4: 7.913-13.901, PND 13: 22.947-38.098).

Pup Survival Data
No test item-related effect on mean mortality of pups was observed between PND 0 and PND 4 and between PND 4 and 13 in treatment groups
when compared to the control group. Mortality of two pups in the control group was considered incidental.

Anogenital Distance and Nipple Retention
No differences of biological relevance were observed in anogenital distance of male and female pups on PND 0 and in nipple retention of male pups on PND 12
when comparing test item treated pups to pups of the control group.
However, mean male pup nipple retention was shown to be statistically significantly lower in the HD group compared to the control group.
As this value was within the normal range of variation (range of historical control data (±2 fold SD): -1.14-1.50), lower nipple retention in the HD group was not considered test item-related.
Female pups treated with the test item were observed with statistically significantly higher mean pup weight (control: 5.76, MD: 6.04) and mean cube root of
pup weight (control: 1.79, MD: 1.82) in the MD group and statistically significantly lower absolute (control: 1.31, LD: 1.07) and relative (control: 0.73, LD: 0.60)
anogenital distance in the LD group. Without occurrence of these differences in all groups and dose-dependency this was not considered adverse.


Thyroid Hormone (T4) Analysis and Thyroid/Parathyroid Weight
No test item-related effect of statistical significance was observed on pup thyroid weight and T4 level in PND 13 pups (male and female)
of the test item treated groups when compared to the controls.

Pup External Findings
Treatment with Methyl 4-hydroxybenzoate caused no gross external pup findings in any of the test item-treated groups or the control group.
The single external finding of a dark tail in one pup of the LD was not considered test item-related.




Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see comment
Remarks on result:
other:
Remarks:
No test item-related effects on the reproduction and developmental parameters analysed in this study, i.e. oestrous cycle, copulation, fertility and delivery indices, number of corpora lutea, implantation sites and live pups, pre- and post-implantation loss, number of male and female pups, sex ratio, still births, runts, litter weight data, anogenital distance, nipple retention and external abnormalities. Thus, the NOAEL for reproduction/developmental toxicity is determined to be 1000 mg/kg bw/day.
Reproductive effects observed:
not specified
Conclusions:
On the basis of this combined repeated dose oral toxicity and reproduction/ developmental toxicity screening test with Methyl 4-hydroxybenzoate in male and female Wistar rats with dose levels of 100, 300, and 1000 mg/kg body weight day the following conclusions can be made:
No mortality and no clinical signs of systemic toxicity were observed in the animals of this study. Daily oral treatment with Methyl 4-hydroxybenzoate had no effect on body weight and food consumption. At the end of the respective treatment periods of male and female animals there were no changes in clinical pathology results up to 1000 mg/kg bw/day and organ weights were inconspicuous. No relevant macroscopic and no histopathological abnormalities were observed in treated animals. Thus, the NOAEL for general toxicity is determined to be 1000 mg/kg bw/day.
No test item-related effects on the reproduction and developmental parameters analysed in this study, i.e. oestrous cycle, copulation, fertility and delivery indices, number of corpora lutea, implantation sites and live pups, pre- and post-implantation loss, number of male and female pups, sex ratio, still births, runts, litter weight data, anogenital distance, nipple retention and external abnormalities. Thus, the NOAEL for reproduction/developmental toxicity is determined to be 1000 mg/kg bw/day.
Executive summary:

The aim of this study was to assess the possible effects of Methyl 4-hydroxybenzoateonmale and female fertility and embryo-foetal development after repeated dose administration in Wistar rats.

The test item was administered daily in graduated doses to 3 groups of test animals, one dose level per group for a treatment period of 63 days, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day 12 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days were completed. Animals of an additional control group were handled identically as the dose groups but received 1 % hydroxyethyl-cellulose (viscosity 80-125 cP, 2 % in water at 20 °C), the vehicle used in this study. The 4 groups comprised 10 male and 10 female Wistar rats. Before dosing all females were screened for two weeks for regular oestrous cyclicity and animals (10 females/group) with regular oestrous cycle (4-5 day cycle) were used in the study.

The following doses were evaluated:

Control:                    0 mg/kg body weight

Low Dose:            100 mg/kg body weight

Medium Dose:      300 mg/kg body weight

High Dose:         1000 mg/kg body weight

The test item formulation was prepared at least every 4 days. The test item was suspended in 1 % hydroxyethyl-cellulose and administered daily during14 days of pre-mating and 14 days of mating in both male and female animals, during the gestation period and up to post-natal day 12 in females. Males were dosed for 28 days. Dose volumes were adjusted individually based on weekly body weight measurements. Theadministrationvolumewas 5 mL/kg body weight.

During the period of administration, the animals were observed each day for signs of toxicity. At the conclusion of the test, surviving animals were sacrificed and observed macroscopically.

Body weight and food consumption were measured weekly, except for food consumption measurements which were not taken during the mating period in female animals and the mating and post-mating period in male animals.

Haematological and clinical biochemistry evaluations were performedon blood samples collected at terminal sacrifice fromfive randomly selected males and females from each group.

After 14 days of treatment to both male and female, animals were mated (1:1) for a maximum period of 14 days. The subsequent morning onwards the vaginal smears of females were checked to confirm the evidence of mating. After the confirmation of the mating, females were separated and housed individually. Each litter was examined as soon as possible after delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities. Live pups were counted, sexed and litters weighed within 24 hours of parturition, on day 4 and day 13 post-partum. The anogenital distance (AGD) of each pup was measured on PND 0. The number of nipples/areolae in male pups was counted on PND 12. Blood samples from the day 13 pups and the adult males were assessed for serum levels for thyroid hormones (T4).

The males were sacrificed after completion of the mating period on treatment day 29 and the females along with their pups were sacrificed on post-natal day 13. Non-pregnant females were sacrificed on day 26.

The number of implantation sites and corpora lutea was recorded for each parental female at necropsy.

Pups sacrificed on post-natalday 4 or 13 and those found dead, were carefully examined for gross external abnormalities.

A full histopathological evaluation of the preserved tissues was performed on five selected high dose and control animals, in non-pregnant female animals and male mating partners of the LD and MD animals. These examinations were not extended to animals of all other dosage groups as treatment-related changes were not observed in the high dose group. For the testes, a detailed qualitative examination was made; taking into account the tubular stages of the spermatogenic cycle at evaluation ofadditional haematoxylin-PAS (Periodic Acid Schiff)stained slides.All gross lesions macroscopically identified were examined microscopically in all animals.

Summary Results

No test item related mortality or clinical signs of systemic toxicity were observed during daily observations. The clinical signs of moving the bedding and increased salivation observed were observed in short timely relation to dose administration andwere considered to be a sign of discomfort or a local reaction to the test item. Other clinical signs like hairless areas, crusts, scratches/cuts, oedema, kinked tail, hypotonia, slow movements, diarrhoea and aggressiveness were observed without dose dependency in single animals and were not considered adverse.

The test item had no statistically significant or toxicologically relevant effect on body weight or body weight gain and on food consumption in both sexes. However, mean body weight gain was observed to be slightly but statistically significantly lower in the female LD and female HD group compared to the controls during the first week of the gestation phase. However, without consistency between the groups, without dose dependency and occurrence during only one week of treatment this was not considered adverse. Slightly but statistically significantly lower food consumption of females of the LD group during the second week of gestation was not considered test item related as no differences in food consumption were observed between higher dose groups and the control group.

There were no considerable differences in the length or sequence of oestrous cycle stages between the dose groups and the control group. No toxicologically relevant changes in pre-coital interval and duration of gestation were observed in the dose groups when compared to the controls.

No toxicologically relevant effects were noted for corpora lutea, implantations sites, live pups, pre implantation loss and post implantation loss in all dose groups. All values were within the normal range of variation.

There were no test item-related effects on total number of male and female pups, sex ratio and number of still births and runts. Values of these litter parameters were comparable between dose groups and control group.

Litter weight data showed no test item related changes. Slight differences between the groups were within the normal range of variation.

Pre implantation loss and post implantation loss were within the normal range of variation and not relevantly different between dose groups and control group. There was no test item-related effect on mean mortality of pups between PND 0 and PND 4 and during PND 4-13.

The test item had no relevant effect onreproductive indices (copulation, fertility, viability, and delivery index).

Statistically significantly lower mean male pup nipple retention was observed in the HD group. However, as this value was within the normal range of variation it was not considered test item-related. Female pups in the MD group were observed with statistically significantly higher mean pup weight and mean cube root of pup weight and statistically significantly lower anogenital distance in the LD group. Without occurrence of these differences in all groups and dose-dependency this was not considered adverse.

No test item-related effect was observed on pup thyroid weight and thyroxine hormone (T4) level in males and PND 13 pups of the dose groups when compared to the controls.

Two pups were found dead in the control group and no mortality occurred in any of the test item-treated groups. Mortality in the control group was considered to be within the normal range of background findings.A single finding of an external abnormality (dark tail) in one pup of the control group was considered incidental.

There were no statistically significant or toxicologically relevant effects of the test item on haematological, clinical biochemistry and coagulation parameters determined at the end of the treatment period of this study.

No test item related macroscopic findings were noted in the groups during necropsy of the animals. Single observed macroscopic findings and slight differences in organ weights between test item treated groups and the controls were considered incidental without the evidence of corresponding histopathological findings.

No test item related changes were observed during the histopathological evaluation. All findings recorded were deemed to be incidental or were within the range of background alterations that may be recorded in Wistar rats.There was no histological evidence of toxicity in the reproductive organs and tissues including testes, epididymides, prostate gland, seminal vesicles, coagulating glands, ovaries, uterus, cervix, and vagina. No treatment-related effects on the testicular histomorphology were observed. Further, no treatment-related effect on interstitial cell structure was noticed.

Conclusion

On the basis ofthiscombined repeated dose oral toxicity and reproduction/ developmental toxicity screening test withMethyl 4-hydroxybenzoatein male and femaleWistarrats with dose levels of 100, 300, and 1000 mg/kg body weight day the following conclusions can be made:

No mortality and no clinical signs of systemic toxicity were observed in the animals of this study. Daily oral treatment withMethyl 4-hydroxybenzoate had no effect on body weight and food consumption. At the end of the respective treatment periods of male and female animals there were no changes in clinical pathology results up to 1000 mg/kg bw/day and organ weights were inconspicuous. No relevant macroscopic and no histopathological abnormalities were observed in treated animals. Thus, the NOAEL for general toxicity is determined to be 1000 mg/kg bw/day.

No test item-related effects on the reproduction and developmental parameters analysed in this study, i.e. oestrous cycle, copulation, fertility and delivery indices, number of corpora lutea, implantation sites and live pups, pre- and post-implantation loss, number of male and female pups, sex ratio, still births, runts, litter weight data, anogenital distance, nipple retention and external abnormalities. Thus, the NOAEL forreproduction/developmental toxicity isdetermined to be1000 mg/kg bw/day.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
1 (reliable without restriction)
Additional information

NOEL (uterotrophic assay, female mice): > 100 mg/kg bw/d (highest dose tested)

Effects on developmental toxicity

Description of key information

NOAEL (OECD 422): 1000 mg/kg bw /d
NOEL (developmental/maternal, oral, rat): 550 mg/kg bw/d (highest dose tested)
NOEL (developmental/maternal, oral, mouse): 550 mg/kg bw/d (highest dose tested)

Based on a combined repeated dose oral toxicity and reproduction/ developmental toxicity screening test with Methyl 4-hydroxybenzoate in male and female Wistar rats (according to OECD 442) with dose levels of 100, 300, and 1000 mg/kg body weight day the following conclusions can be made:

No mortality and no clinical signs of systemic toxicity were observed in the animals of this study. Daily oral treatment with Methyl 4-hydroxybenzoate had no effect on body weight and food consumption. At the end of the respective treatment periods of male and female animals there were no changes in clinical pathology results up to 1000 mg/kg bw/day and organ weights were inconspicuous. No relevant macroscopic and no histopathological abnormalities were observed in treated animals. Thus, the NOAEL for general toxicity is determined to be 1000 mg/kg bw/day.

No test item-related effects on the reproduction and developmental parameters analysed in this study, i.e. oestrous cycle,  copulation, fertility and delivery indices, number of corpora lutea, implantation sites and live pups, pre- and post-implantation loss, number of male and female pups, sex ratio, still births, runts, litter weight data, anogenital distance, nipple retention and external abnormalities. Thus, the NOAEL for reproduction/developmental toxicity is determined to be 1000 mg/kg bw/day.


Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-12-03 to 2019-12-23
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: OECD 422
Version / remarks:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
Test System
Species/strain: healthy Wistar rats, Crl: WI(Han) (Full Barrier)
Source: Charles River, 97633 Sulzfeld, Germany
Sex: male and female; the female animals were non-pregnant and nulliparous
Age at the start of the treatment period: approx. 14-15 weeks old
Body weight at the allocation of the animals to the experimental groups:
males: 326 – 398 g (mean: 360.0 g, ± 20 % = 288.0 – 432.0 g)
females: 207 – 258 g (mean: 227.2 g, ± 20 % = 181.8 – 272.6 g)
The animals were derived from a controlled full-barrier maintained breeding system (SPF). According to the German Act on Animal Welfare the animals were bred for experimental purposes.
This study was performed in an AAALAC-accredited laboratory. According to German animal protection law, the study type has been reviewed and accepted by local authorities.
Furthermore, the study has been subjected to Ethical Review Process and was authorised by the Bavarian animal welfare administration.

Housing and Feeding Conditions
- Full barrier in an air-conditioned room
- Temperature: 22 +/- 3 °C
- Relative humidity: 55 +/- 10 %
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: 10 x / hour
- Free access to Altromin 1324 maintenance diet for rats and mice
- Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- 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 both 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 were provided for all males and for females until GD 18
- Nesting material were 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) under laboratory conditions

Number and Sex of the Animals
80 animals (40 males and 40 females) were included in the study.

Preparation of the Animals
Prior to the start of the treatment period a detailed clinical observation outside the home cage was made. Only healthy animals were used for the study.
Before dosing all females were screened for two weeks for regular oestrous cyclicity and animals (10 females/ group) with regular oestrous cycle (4-5 day cycle)
were used in the study. Before the first administration all animals to be used for the study were weighed and assigned to the experimental groups with achieving
a most homogenous variation in body weight throughout the groups of males and females, respectively (randomisation was performed with IDBS Workbook 10.1.2 software).
Each animal was marked with its identification number by individual ear tattoo or tail marking.



Route of administration:
oral: gavage
Vehicle:
other: 1 % hydroxyethyl-cellulose / aqua ad injectionem
Remarks:
hydroxyethyl-cellulose: Manufacturer: Sigma-Aldrich, Batch No.: MKCD0421, Expiry Date: 05 November 2019 aqua ad injectionem: Manufacturer: Deltamedica, Batch No.: 806148, Expiry Date: May 2021 (each bottle was used for up to one week)
Details on exposure:
The vehicle has been selected in consultation with the sponsor based on the test item’s characteristics.
Based on the results of stability testing (Eurofins Munich Study No. 187385), the test item formulations were prepared at least every 4 days as given by Eurofins Munich Study No. 187385.
The prepared formulation was stored at room temperature.
The test item, as delivered, was grinded before formulation preparation. Afterwards, the 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 1 % aqueous hydroxyethyl-cellulose, the vehicle used in this study. After producing slurry with the glass rod for 1 minute,
the rest of the vehicle was added to give the appropriate final concentration. The formulation was then stirred until visual homogeneity was achieved (at least 30 min).
Formulates were kept under magnetic stirring during the daily administration. The vehicle was also used as control item.

In consultation with the sponsor the following doses were selected for the 3 dose groups (LD = low dose, MD = medium dose, HD = high dose) and 1 control group (C).
The animals were treated with the test item formulation or vehicle on 7 days per week for a maximum period of 63 days in females, i.e. during 14 days of pre-mating
and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day 12 in females.
Males were dosed after the mating period until the minimum total dosing period of 28 days is completed.

Control: 0 mg/kg/d
Low Dose: 100 mg/kg/d
Medium Dose: 300 mg/kg/d
High Dose: 1000 mg/kg/d


The highest dose level was chosen with the aim of inducing toxic effects, but no death or severe suffering.
Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dosage related response and NOAEL.
The animals in the control group were handled in an identical manner to the test group subjects and received the vehicle using the same volume as used for the high dose group.

The test item and vehicle were administered at a single dose to the animals by oral gavage. The application volume for all groups was 5 mL/kg body weight.
For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured.








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. 187385).
Prestart homogeneity investigation was included on the samples collected from various levels (top, middle and bottom) of high dose and low dose groups.
The test item was not shown to be homogenous according to Eurofins Study No. 187385. Therefore, 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 study week 1 (pre-mating period),
3 (first week of mating), 5 (gestation) and in the last week of the study (gestation / lactation) (40 samples).
Each sample taken during the study was retained in duplicate (sample A, sample B, each of at least 3 mL). The A-samples were analysed at Eurofins Munich
(Eurofins Munich Study Phase No. 187386) and until then stored under appropriate conditions based on available stability data.
The B-samples were retained at below 15 °C at BSL Munich (test facility) and discarded after completion of the final study report.
Details on mating procedure:
Mating was performed using a ratio of 1:1 (male to female). The vaginal smear of the females was checked every morning after the start of the mating period to confirm the mating.
If the vaginal smear of a particular female was not found to be sperm-positive, the actual stage of the oestrous cycle on that day was documented.
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 minimised.
Duration of treatment / exposure:
The animals were treated with the test item formulation or vehicle on 7 days per week for a maximum period of 63 days in females, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males
and females, during the gestation period and up to post-natal day 12 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days is completed.
Frequency of treatment:
daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
80 animals (40 males and 40 females) were included in the study. 10 male and 10 female animals per group.
Control animals:
yes, concurrent vehicle
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Moving the bedding was observed in 1/10 females of the control group, 2/10 females of the LD group, 1/10 females of the MD group and 10/10 females and 9/10 males of the HD group mostly in the second half of the treatment period. Increased salivation was noted transiently in 1/10 females of the MD group and in 5/10 males and females of the HD group. Both signs were observed in short timely relation to dose administration or in anticipation thereof and thus were considered to be a sign of discomfort or a local reaction to the test item. These slight signs were not considered as adverse systemic effects.
The clinical sign of piloerection was observed in 4/10 females of the control group, 4/10 females of the LD group, 8/10 females of the MD group, and 7/10 females of the HD group. Due to the absence of dose dependency and the occurrence of piloerection in control animals it was not considered toxicologically relevant. Low incidences of clinical signs without dose dependency (see Details on results P0 ) and thus are not considered test item-related.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The test item had no statistically significant effect on body weight or body weight change in male animals and body weights of female animals.
Mean body weight gain was slightly but statistically significantly lower in the female LD and female HD group compared to the controls during the first week of the gestation phase. However, without consistency between the groups, without dose dependency and occurrence during only one week of treatment this was not considered adverse. MD females showed no statistically significant differences in body weight change compared to control females. Further slight differences between the groups were within the normal range of variation throughout the treatment period of this study.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
The test item had no toxicologically relevant effect on food consumption in male and female animals of this study.
Slightly but statistically significantly lower food consumption of females of the LD group compared to control females during the second week of gestation was not considered test item related as no differences in food consumption were observed between higher dose groups and the control group. Slightly lower food intake was seen in female animals of the HD group during the premating period. Without achieving statistical significance this is not considered to be an adverse effect.
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):
There was no statistically significant or biologically relevant effect of the test item on haematological parameters of male animals determined at the end of the treatment period of this study. Differences in haematological parameters between test item-treated groups and the corresponding controls followed no dose dependency and within historical control data and thus were not considered test item-related.
There were no statistically significant effects of the test item on coagulation parameters (PT, aPTT) of female animals and on the parameter aPTT of male animals at the end of the treatment period. PT was statistically significantly but slightly higher in males of the HD group compared to control males (13 % above control). However, without the incidence of other clinical findings or macroscopic findings at the time point of necropsy slightly higher PT value of the HD group was not assumed adverse.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
In male animals, mean TBA showed a dose-dependent tendency towards lower TBA levels in test item-treated animals compared to the controls. However, without achieving statistical significance and without a corresponding effect in female animals this difference was not considered adverse. Other parameters of clinical biochemistry showed no statistically significant or biologically relevant differences between males treated with the test item and males of the control group. In female animals there were no statistically significant or biologically relevant differences in any of the parameters of clinical biochemistry when comparing test item treated animals with animals of the control group. The deviation of mean TBA level of HD females from control females (261% deviation from control) was not statistically significant and was mainly based on high TBA levels from two females of the HD group (female numbers 71 and 80) and was not considered adverse.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
see Details on results P0
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Slight differences in the mean organ weights between test item-treated groups and the control group are not considered to be caused by treatment with Methyl 4 hydroxybenzoate as no test item related histopathological findings were observed in any of the organs.
Mean relative pituitary gland weight was slightly higher in the male HD group when compared to the control group (deviation from control: 14 %). However, due to the lack of dose dependency this was not considered to be adverse and toxicologically relevant.
Absolute (deviation from control: 21 %) and relative (deviation from control: 18 %) mean adrenal gland weight was statistically significantly lower in females of the MD group. However, this followed no dose dependency and is thereby not considered toxicologically relevant.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Only single or occasional macroscopic findings were noted in the groups during necropsy of the animals. These are assumed to be incidental finings without relation to the test item. Findings were enlarged kidneys (bilateral) of LD male no. 13, small testes (bilateral) of MD male no. 27 and small seminal vesicles (left side) of control male no. 7. Without corresponding evidence in histopathological examination these macroscopic findings were not considered of toxicological relevance.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
There was no histological evidence of toxicity in the reproductive organs and tissues including testes, epididymides, prostate gland, seminal vesicles, coagulating glands, ovaries, uterus, cervix, and vagina. No treatment-related effects on the testicular histomorphology and interstitial cell structure were noticed. The treatment with Methyl 4 hydroxybenzoate did not induce histomorphological effects in the reproductive organs of the non-pregnant females (animal nos. 65 and 67) and their pairing partners (animal nos. 25 and 27). Therefore, the histopathological NOEL (no observed effect level) may be established at 1000 mg/kg bw/day.
Histopathological findings: neoplastic:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Abnormalities:
no effects observed
Details on embryotoxic / teratogenic effects:
Litter Data
There were no test item-related effects on litter data including total number of male and female pups, sex ratio and number of still births and runts.
There were no statistically significant differences noted for these litter parameters and the values were comparable between dose groups and control group.

Litter Weight Data
Treatment with the test item had no statistically significant or biologically relevant effects on litter weight data on PND 0, 4 and 13 when comparing
test item-treated groups and the controls. Differences between the groups followed no dose dependency and were within the normal range of variation
(range of historical control data (±2 fold SD): PND 0: 4.63-8.141, PND 4: 7.913-13.901, PND 13: 22.947-38.098).

Pup Survival Data
No test item-related effect on mean mortality of pups was observed between PND 0 and PND 4 and between PND 4 and 13 in treatment groups
when compared to the control group. Mortality of two pups in the control group was considered incidental.

Anogenital Distance and Nipple Retention
No differences of biological relevance were observed in anogenital distance of male and female pups on PND 0 and in nipple retention of male pups on PND 12
when comparing test item treated pups to pups of the control group.
However, mean male pup nipple retention was shown to be statistically significantly lower in the HD group compared to the control group.
As this value was within the normal range of variation (range of historical control data (±2 fold SD): -1.14-1.50), lower nipple retention in the HD group was not considered test item-related.
Female pups treated with the test item were observed with statistically significantly higher mean pup weight (control: 5.76, MD: 6.04) and mean cube root of
pup weight (control: 1.79, MD: 1.82) in the MD group and statistically significantly lower absolute (control: 1.31, LD: 1.07) and relative (control: 0.73, LD: 0.60)
anogenital distance in the LD group. Without occurrence of these differences in all groups and dose-dependency this was not considered adverse.


Thyroid Hormone (T4) Analysis and Thyroid/Parathyroid Weight
No test item-related effect of statistical significance was observed on pup thyroid weight and T4 level in PND 13 pups (male and female)
of the test item treated groups when compared to the controls.

Pup External Findings
Treatment with Methyl 4-hydroxybenzoate caused no gross external pup findings in any of the test item-treated groups or the control group.
The single external finding of a dark tail in one pup of the LD was not considered test item-related.




Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
Treatment related:
no
Conclusions:
On the basis of this combined repeated dose oral toxicity and reproduction/ developmental toxicity screening test with Methyl 4-hydroxybenzoate in male and female Wistar rats with dose levels of 100, 300, and 1000 mg/kg body weight day the following conclusions can be made:
No mortality and no clinical signs of systemic toxicity were observed in the animals of this study. Daily oral treatment with Methyl 4-hydroxybenzoate had no effect on body weight and food consumption. At the end of the respective treatment periods of male and female animals there were no changes in clinical pathology results up to 1000 mg/kg bw/day and organ weights were inconspicuous. No relevant macroscopic and no histopathological abnormalities were observed in treated animals. Thus, the NOAEL for general toxicity is determined to be 1000 mg/kg bw/day.
No test item-related effects on the reproduction and developmental parameters analysed in this study, i.e. oestrous cycle, copulation, fertility and delivery indices, number of corpora lutea, implantation sites and live pups, pre- and post-implantation loss, number of male and female pups, sex ratio, still births, runts, litter weight data, anogenital distance, nipple retention and external abnormalities. Thus, the NOAEL for reproduction/developmental toxicity is determined to be 1000 mg/kg bw/day.
Executive summary:

The aim of this study was to assess the possible effects of Methyl 4-hydroxybenzoate on male and female fertility and embryo-foetal development after repeated dose administration in Wistar rats.

The test item was administered daily in graduated doses to 3 groups of test animals, one dose level per group for a treatment period of 63 days, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day 12 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days were completed. Animals of an additional control group were handled identically as the dose groups but received 1 % hydroxyethyl-cellulose (viscosity 80-125 cP, 2 % in water at 20 °C), the vehicle used in this study. The 4 groups comprised 10 male and 10 female Wistar rats. Before dosing all females were screened for two weeks for regular oestrous cyclicity and animals (10 females/group) with regular oestrous cycle (4-5 day cycle) were used in the study.

The following doses were evaluated:

Control:                    0 mg/kg body weight

Low Dose:            100 mg/kg body weight

Medium Dose:      300 mg/kg body weight

High Dose:         1000 mg/kg body weight

The test item formulation was prepared at least every 4 days. The test item was suspended in 1 % hydroxyethyl-cellulose and administered daily during14 days of pre-mating and 14 days of mating in both male and female animals, during the gestation period and up to post-natal day 12 in females. Males were dosed for28 days. Dose volumes were adjusted individually based on weekly body weight measurements. Theadministrationvolumewas 5 mL/kg body weight.

During the period of administration, the animals were observed each day for signs of toxicity. At the conclusion of the test, surviving animals were sacrificed and observed macroscopically.

Body weight and food consumption were measured weekly, except for food consumption measurements which were not taken during the mating period in female animals and the mating and post-mating period in male animals.

Haematological and clinical biochemistry evaluations were performedon blood samples collected at terminal sacrifice fromfive randomly selected males and females from each group.

After 14 days of treatment to both male and female, animals were mated (1:1) for a maximum period of 14 days. The subsequent morning onwards the vaginal smears of females were checked to confirm the evidence of mating. After the confirmation of the mating, females were separated and housed individually. Each litter was examined as soon as possible after delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities. Live pups were counted, sexed and litters weighed within 24 hours of parturition, on day 4 and day 13 post-partum. The anogenital distance (AGD) of each pup was measured on PND 0. The number of nipples/areolae in male pups was counted on PND 12. Blood samples from the day 13 pups and the adult males were assessed for serum levels for thyroid hormones (T4).

The males were sacrificed after completion of the mating period on treatment day 29 and the females along with their pups were sacrificed on post-natal day 13. Non-pregnant females were sacrificed on day 26.

The number of implantation sites and corpora lutea was recorded for each parental female at necropsy.

Pups sacrificed on post-natalday 4 or 13 and those found dead, were carefully examined for gross external abnormalities.

A full histopathological evaluation of the preserved tissues was performed on five selected high dose and control animals, in non-pregnant female animals and male mating partners of the LD and MD animals. These examinations were not extended to animals of all other dosage groups as treatment-related changes were not observed in the high dose group. For the testes, a detailed qualitative examination was made; taking into account the tubular stages of the spermatogenic cycle at evaluation ofadditional haematoxylin-PAS (Periodic Acid Schiff)stained slides.All gross lesions macroscopically identified were examined microscopically in all animals.

Summary Results

No test item related mortality or clinical signs of systemic toxicity were observed during daily observations. The clinical signs of moving the bedding and increased salivation observed were observed in short timely relation to dose administration andwere considered to be a sign of discomfort or a local reaction to the test item. Other clinical signs like hairless areas, crusts, scratches/cuts, oedema, kinked tail, hypotonia, slow movements, diarrhoea and aggressiveness were observed without dose dependency in single animals and were not considered adverse.

The test item had no statistically significant or toxicologically relevant effect on body weight or body weight gain and on food consumption in both sexes. However, mean body weight gain was observed to be slightly but statistically significantly lower in the female LD and female HD group compared to the controls during the first week of the gestation phase. However, without consistency between the groups, without dose dependency and occurrence during only one week of treatment this was not considered adverse. Slightly but statistically significantly lower food consumption of females of the LD group during the second week of gestation was not considered test item related as no differences in food consumption were observed between higher dose groups and the control group.

There were no considerable differences in the length or sequence of oestrous cycle stages between the dose groups and the control group. No toxicologically relevant changes in pre-coital interval and duration of gestation were observed in the dose groups when compared to the controls.

No toxicologically relevant effects were noted for corpora lutea, implantations sites, live pups, pre implantation loss and post implantation loss in all dose groups. All values were within the normal range of variation.

There were no test item-related effects on total number of male and female pups, sex ratio and number of still births and runts. Values of these litter parameters were comparable between dose groups and control group.

Litter weight data showed no test item related changes. Slight differences between the groups were within the normal range of variation.

Pre implantation loss and post implantation loss were within the normal range of variation and not relevantly different between dose groups and control group. There was no test item-related effect on mean mortality of pups between PND 0 and PND 4 and during PND 4-13.

The test item had no relevant effect onreproductive indices (copulation, fertility, viability, and delivery index).

Statistically significantly lower mean male pup nipple retention was observed in the HD group. However, as this value was within the normal range of variation it was not considered test item-related. Female pups in the MD group were observed with statistically significantly higher mean pup weight and mean cube root of pup weight and statistically significantly lower anogenital distance in the LD group. Without occurrence of these differences in all groups and dose-dependency this was not considered adverse.

No test item-related effect was observed on pup thyroid weight and thyroxine hormone (T4) level in males and PND 13 pups of the dose groups when compared to the controls.

Two pups were found dead in the control group and no mortality occurred in any of the test item-treated groups. Mortality in the control group was considered to be within the normal range of background findings.A single finding of an external abnormality (dark tail) in one pup of the control group was considered incidental.

There were no statistically significant or toxicologically relevant effects of the test item on haematological, clinical biochemistry and coagulation parameters determined at the end of the treatment period of this study.

No test item related macroscopic findings were noted in the groups during necropsy of the animals. Single observed macroscopic findings and slight differences in organ weights between test item treated groups and the controls were considered incidental without the evidence of corresponding histopathological findings.

No test item related changes were observed during the histopathological evaluation. All findings recorded were deemed to be incidental or were within the range of background alterations that may be recorded in Wistar rats.There was no histological evidence of toxicity in the reproductive organs and tissues including testes, epididymides, prostate gland, seminal vesicles, coagulating glands, ovaries, uterus, cervix, and vagina. No treatment-related effects on the testicular histomorphology were observed. Further, no treatment-related effect on interstitial cell structure was noticed.

Conclusion

On the basis ofthiscombined repeated dose oral toxicity and reproduction/ developmental toxicity screening test withMethyl 4-hydroxybenzoatein male and femaleWistarrats with dose levels of 100, 300, and 1000 mg/kg body weight day the following conclusions can be made:

No mortality and no clinical signs of systemic toxicity were observed in the animals of this study. Daily oral treatment withMethyl 4-hydroxybenzoate had no effect on body weight and food consumption. At the end of the respective treatment periods of male and female animals there were no changes in clinical pathology results up to 1000 mg/kg bw/day and organ weights were inconspicuous. No relevant macroscopic and no histopathological abnormalities were observed in treated animals. Thus, the NOAEL for general toxicity is determined to be 1000 mg/kg bw/day.

No test item-related effects on the reproduction and developmental parameters analysed in this study, i.e. oestrous cycle, copulation, fertility and delivery indices, number of corpora lutea, implantation sites and live pups, pre- and post-implantation loss, number of male and female pups, sex ratio, still births, runts, litter weight data, anogenital distance, nipple retention and external abnormalities. Thus, the NOAEL forreproduction/developmental toxicity isdetermined to be1000 mg/kg bw/day.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May 1973
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well performed and reported study. Relevant aspects (treatment, examinations etc.) are in line with the current guideline.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
only 12 animals in high dose group, body weight every 6 days recorded
GLP compliance:
no
Remarks:
performed before GLP guidelines
Limit test:
no
Species:
rabbit
Strain:
other: Dutch-belted
Details on test animals and environmental conditions:
Husbandry:
Virgin, adult female rabbits were individually housed in mesh bottom cages in temperature and humidity-controlled quarters with free access to food and fresh tap water.

.
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
Beginning on Day 6 and continuing daily through Day 18 the females were dosed with 3, 14, 65 or 300 mg Methylparaben/kg bw or 2.5 mg 6-Aminonicotinamide/kg bw (positive control on Day 9). The negative controls were treated with the vehicle (water) at a level equivalent to the group receiving the highest dose level.
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
No data
Details on mating procedure:
On Day 0, each doe was given an injection of 0.4 mL human chorionic gonadotropin (400 IU) via the marginal ear vein. 3 hours later, each doe was inseminated artificially with 0.3 mL of diluted semen from a proven donor buck using approximately 20 x 10(exp 6) motile sperm.
Duration of treatment / exposure:
Methylparaben:
Day 6 to Day 18 of gestation

6-Aminonicotinamide:
on Day 9 of gestation
Frequency of treatment:
Daily
Duration of test:
29 days
No. of animals per sex per dose:
Control: 14 mated animals (11 pregnant animals)
6-Aminonicotinamide: 17 mated animals (10 pregnant animals)
3.0 mg/kg bw/d Methylparaben: 20 mated animals (9 pregnant animals)
14.0 mg/kg bw/d Methylparaben: 20 mated animals (9 pregnant animals)
65.0 mg/kg bw/d Methylparaben: 14 mated animals (10 pregnant animals)
300.0 mg/kg bw/d Methylparaben: 12 mated animals (9 pregnant animals)
Control animals:
yes, concurrent vehicle
Details on study design:
None
Maternal examinations:
Body weight: on Days 0, 6, 12, 18 and 29 of gestation
Clinical signs/mortality: daily
Food consumption
Ovaries and uterine content:
On Day 29 of gestation all does were subjected to Caesarean section under surgical anethesia and the numbers of Corpora lutea, implantation sites, resorption sites and dead fetuses were recorded. The urogenital tract of each animal was examined in detail for normality.
Fetal examinations:
Body weights of the live pups were recorded. All fetuses underwent a detailed gross examination for the presence of external congenital abnormalities. The live fetuses of each litter were then placed in an incubator for 24 hours for the evaluation of the neonatal survival. All surviving pups were sacrificed and examined for visceral abnormalities (by dissection). All fetuses were cleared in potassium hydroxide, stained with Alizarin S dye and examined for skeletal defects.
Statistics:
None
Indices:
No data
Historical control data:
No data
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
- All pregnant animals (except of 1 female in the control group) survived until scheduled necropsy and no clinical signs were noted
- Body weights were not affected by treatment
- The relevant reproduction parameters (no. of Corpora lutea, implantation sites/dam, resorptions etc.) were not affected by treatment with the test item
Key result
Dose descriptor:
NOEL
Effect level:
300 mg/kg bw/day (actual dose received)
Basis for effect level:
other: maternal toxicity
Key result
Dose descriptor:
NOEL
Effect level:
300 mg/kg bw/day (actual dose received)
Basis for effect level:
other: developmental toxicity
Key result
Abnormalities:
no effects observed
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
- Sex ratio and fetal body weight were not affected by treatment
- No dose related skeletal findings or soft tissue abnormalities
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no changes observed
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
Conclusions:
The administration of Methylparaben up to 300 mg/kg bw/d to pregnant rabbits (on days 6 to 18 of gestation) did not have any effect on maternal or developmental parameters. Based on the result of this study the NOEL for maternal and developmental effects can be set at 300 mg/kg bw/d.
Executive summary:

Methylparaben was administered to female pregnant rabbits from day 6 to day 18 of gestation. The test item was administered orally at dose levels of 3, 14, 65 and 300 mg/kg bw/d.

The administration up to 300 mg Methylparaben/kg bw/d had no clearly discernable effect on nidation or maternal or fetal survival. The number of abnormalities seen in either soft or skeletal tissues of the test groups did not differ from the number occuring spontaneously in the vehicle treated controls.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
October 1972
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well performed and reported study. Relevant aspects (treatment, exminations etc.) are in line with the current guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
body weights every 5 days recorded
GLP compliance:
no
Remarks:
performed before GLP guidelines
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
Husbandry:
Virgin adult female rats were individually housed in mesh bottom cages in temperature and humidity-controlled quarters with free access to food and fresh tap water.
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
Beginning on Day 6 and continuing daily through Day 15 of gestation, the females were dosed with different dosages of Methylparaben dissolved in water by oral intubation. Controls were treated with corn oil.
Dosage volume: 1 mL/kg body weight
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
No data
Details on mating procedure:
Virgin adult female rats were mated with young adult males, and observation of the vaginal sperm plug was considered Day 0 of gestation.
Duration of treatment / exposure:
Day 6 to Day 15 of gestation
Frequency of treatment:
Daily
Duration of test:
20 days
No. of animals per sex per dose:
Control: 24 mated animals (23 pregnant animals)
Aspirin: 24 mated animals (22 pregnant animals)
5.5 mg/kg bw/d Methylparaben: 24 mated animals (23 pregnant animals)
25.5 mg/kg bw/d Methylparaben: 24 mated animals (23 pregnant animals)
118.0 mg/kg bw/d Methylparaben: 24 mated animals (24 pregnant animals)
550.0 mg/kg bw/d Methylparaben: 24 mated animals (23 pregnant animals)
Control animals:
yes, sham-exposed
Details on study design:
Sham group was dosed with corn oil.
Maternal examinations:
Body weight: on Days 0, 6, 11, 15 and 20 of gestation
Clinical signs/Mortality: daily
Food consumption
Ovaries and uterine content:
On Day 20 all dams were subjected to Caesarean section under surgical anesthesia, and the numbers of Corpora lutea, implantation sites, resorption sites and live and dead fetuses were recorded. The urogenital tract of each dam was examined in detail for anatomical normality.
Fetal examinations:
Body weights of the live pups were recorded. All fetuses were examined grossly for the presence of external congenital abnormalities. One-third of the fetuses of each litter underwent detailed visceral examinations employing 10x magnification. The remaining two-thirds were cleared in potassium hydroxide, stained with alizarin red S dye and examined for skeletal defects.
Statistics:
None
Indices:
No data
Historical control data:
No data
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
- All pregnant animals survived until scheduled necropsy and no clinical signs were noted
- Body weights were not affected by treatment
- The relevant reproduction parameters (no. of Corpora lutea, implantation sites/dam, resorptions etc.) were not affected by treatment with the test item
Key result
Dose descriptor:
NOEL
Effect level:
550 mg/kg bw/day (actual dose received)
Basis for effect level:
other: maternal toxicity
Key result
Dose descriptor:
NOAEL
Effect level:
550 mg/kg bw/day (actual dose received)
Basis for effect level:
other: developmental toxicity
Key result
Abnormalities:
no effects observed
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
- Sex ratio and fetal body weight were not affected by treatment
- No dose related significant skeletal findings or soft tissue abnormalities
Key result
Dose descriptor:
NOAEL
Effect level:
550 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no changes observed
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
Conclusions:
The administration of Methylparaben up to 550 mg/kg bw/d to pregnant rats (on days 6 to 15 of gestation) did not have any significant effect on maternal or developmental parameters. Based on the result of this study the NO(A)EL for maternal and developmental effects can be set at 550 mg/kg bw/d.
Executive summary:

Methylparaben was administered to female pregnant rats from day 6 to day 15 of gestation. The test item was administered orally at dose levels of 5.5, 25.5, 118.0 and 550.0 mg/kg bw/d.

The administration up to 550 mg Methylparaben/kg bw/d had no clearly discernable effect on nidation or maternal or fetal survival. The number of abnormalities seen in either soft or skeletal tissues of the test groups did not differ from the number occuring spontaneously in the corn oil treated controls.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
1 (reliable without restriction)
Additional information

The developmental toxicity of Methylparaben was evaluated in four studies in rabbits, rats, mice and hamsters similar to OECD guideline 414 study and in according to OECD 422 in rats. No maternal and developmental effects were observed up to the highest tested dosages (1000 mg/kg bw/d - rats, 300 mg/kg bw/d – rabbit; 550 mg/kg bw/d – rat; 550 mg/kg bw/d – mouse; 300 mg/kg bw/d – hamster).
Therefore, it is concluded that Methylparaben is not subject to classification and labelling according to Directive 67/548/EEC and Regulation 1272/2008/EC regarding reproductive toxicity.

Toxicity to reproduction: other studies

Additional information

Methylparaben was tested for its estrogenic activity and properties to affect the fertility in several in vivo studies.

Methylparaben did not induce effects on reproductive organs and had no influence on sperm parameters and testosterone, LH/FSH blood levels when applied up to 1000 mg/kg bw/d to male rats in a 56 day dietary study and did not show any significant effect in the uterotrophic assay up to 800 mg/kg bw/d (rat)

Justification for classification or non-classification

There is no evidence to suggest that a classification for reproductive and developmental toxicity is appropriate.

With reference to the developmental studies performed with four different species (rabbit, rat, mouse and hamster) and the lack of maternal/developmental effects, it is concluded that Methylparaben is not subject to classification and labelling according to Directive 67/548/EEC and Regulation 1272/2008/EC regarding reproductive toxicity/teratogenicity. Furthermore an OECD 443 study was requested by ECHA based on compliance check (Decision number: CCH-D-2114412038-60-01/F). Experimental study is started.