Registration Dossier
Registration Dossier
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EC number: 202-785-7 | CAS number: 99-76-3
Toxicological information
Endpoint summary
Administrative data
Key value for chemical safety assessment
Effects on fertility
Description of key information
Effects on male/female reproductive system
- NOAEL (OECD 422 - male/female rats): 1000 mg/kg bw/d
Effects on male/female reproductive system
- NOAEL (OECD 443, female/female rats): 1000 mg/kg bw/d
Link to relevant study records
- 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
- 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. QA statement)
- 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 or test system 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). - 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
- 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.
- Endpoint:
- extended one-generation reproductive toxicity - with F2 generation and developmental neurotoxicity (Cohorts 1A, 1B with extension, 2A and 2B)
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2019-08-28 to 2021-05-05
- Reliability:
- 1 (reliable without restriction)
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
- Version / remarks:
- OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test) - Deviations:
- yes
- Remarks:
- see below: Any Other Information on Results
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- (Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Schwabach, Germany)
- Limit test:
- no
- Justification for study design:
- The Extended one-generation reproductive toxicity study (with all cohort; cohor 1A, 1B, 2A, 2B and 3) was requested by ECHA as standadr information requeriment accordignto Annex IX (Decision number: CCH-D-2114412038-60-01/F).
An extended one-generation reproductive toxicity study provides information on the effects of repeated exposure to a substance during all phases of the reproductive cycle. In particular, the study provides information on the reproductive system, and on development, growth, survival, and functional endpoints of offspring. The test is designed to evaluate the effects of chemicals on pre and postnatal development; the integrity of the male and female reproductive systems; and systemic toxicity in males, pregnant and lactating females, and young and adult offspring. In this study the detailed examination made on key developmental endpoints, such as offspring viability, neonatal health, developmental status at birth, and physical and functional development until adulthood, is expected to identify specific target organs in the offspring. The study provides detailed information about the effects of a test substance on the integrity and performance of the adult male and female reproductive systems, which includes gonadal function, the oestrus cycle, epididymal sperm maturation, mating behaviour, conception, pregnancy, parturition, and lactation. This also includes the information obtained from the developmental neurotoxicity and developmental immunotoxicity assessments. - Specific details on test material used for the study:
- Name: Methyl 4-hydroxybenzoate
CAS No.: 99-76-3 - Species:
- rat
- Strain:
- Wistar
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- Test System
Species/strain: Wistar rats, Crl: WI(Han) (Full Barrier)
Source: e.g. Charles River, 97633 Sulzfeld, Germany
Sex: male and female; the female animals were non-pregnant and nulliparous
Age at the start of the treatment period: approx. 12-13 weeks old
Body weight at the allocation of the animals to the experimental groups: males: 272 - 322 g (mean: 295.74 g, ± 20% =236.59– 354.88 g), females: 189 - 234 g (mean: 195.33 g, ± 20% = 234.39 – 156.26 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 males and females (both parental and F1) and during post-mating period for males (parental and F1) depending on the mating status. During mating period males and females (parental and F1) were housed together in ratio 1:1 (male to female). After the confirmation of mating, females were kept individually during gestation/lactation period in type III H, polysulphone cages and males (parental and F1) were returned to their original cage. In each cage Altromin saw fibre was used as bedding.
- Makrolon tunnels will be provided for all males and for females until GD 18
- Nesting material will be provided latest on GD 18 for all mated females
- Certificates of food, water and bedding are filed for two years at BSL Munich and afterwards archived at Eurofins BioPharma Product Testing Munich GmbH
- Adequate acclimatisation period (at least 5 days) under laboratory conditions - Route of administration:
- oral: gavage
- Vehicle:
- other: 1 % hydroxyethyl-cellulose (viscosity 80-125 cP, 2 % in water at 20 °C)
- Details on exposure:
- Characterisation of Cyclophosphamide
The identity of Cyclophosphamide was inspected upon delivery at the test facility (e.g. name, batch no.) based on the following specifications provided by the supplier.
Name: Cyclophosphamide
Manufacturer: Sigma-Aldrich
Batch No.: LRAC0295
Physical State: white powder in amber vial
Storage Conditions: 2 8 °C, protect from light
Expiry Date: August 2023
Safety Precautions: The routine hygienic procedures were sufficient to assure personnel health and safety.
Characterisation of Keyhole Limpet Haemocyanin (KLH)
The identity of KLH was inspected upon delivery at the test facility (e.g. name, batch no.) based on the following specifications provided by the supplier.
Name: Haemocyanin Megathura crenulata (keyhole limpet)
Manufacturer: Sigma-Aldrich
Batch No.: 029M4885V
Physical State: lyophilized
Storage Conditions: 2 – 8 °C
Retest Date: February 2021
Safety Precautions: The routine hygienic procedures were sufficient to assure personnel health and safety.
Characterisation of the Vehicle for Test Item Formulations
The vehicle used in this study was 1% hydroxyethyl-cellulose (viscosity 80-125 cP, 2% in water at 20 °C. The aqueous solution was prepared with aqua ad injectionem. The specifications provided by the supplier are listed as follows:
Name: hydroxyethyl-cellulose
Manufacturer: Sigma-Aldrich
Batch No.: S7117968 91
Physical State: powder
Colour: beige
Storage Conditions: at room temperature
Retest Date: 30 September 2020
Safety Precautions: The routine hygienic procedures were sufficient to assure personnel health and safety.
Name: aqua ad injectionem (sterile water)
Manufacturer: Deltamedica
Batch No.: 906243
Physical State: liquid
Storage Conditions: at room temperature
Expiry Date: May 2022
Safety Precautions: The routine hygienic procedures were sufficient to assure personnel health and safety.
Characterisation of the Vehicle for Cyclophosphamide
The vehicle for test item 1 was 0.9% NaCl. The specifications provided by the supplier are listed as follows:
Name: 0.9% NaCl
Manufacturer: B. Braun Melsungen
Batch No.: 18517401
Physical State: liquid
Storage Conditions: at room temperature
Expiry Date: November 2021
Safety Precautions: The routine hygienic procedures were sufficient to assure personnel health and safety.
Characterisation of the Vehicle for KLH
The vehicle used for reconstitution of KLH in this study was aqua ad injectionem (sterile water). The specifications provided by the supplier are listed as follows:
Name: aqua ad injectionem (sterile water)
Manufacturer: Deltamedica
Batch No.: 906243
Physical State: liquid
Storage Conditions: room temperature
Expiry Date: May 2022
Safety Precautions: The routine hygienic procedures were sufficient to assure personnel health and safety.
The vehicle used for KLH dilution in this study was PBS (phosphate-buffered saline). The specifications provided by the supplier are listed as follows:
Name: PBS
Manufacturer: Eurofins BioPharma Product Testing Munich GmbH
Batch No.: 24 October 2019
Physical State: liquid
Storage Conditions: room temperature
Expiry Date: 24 October 2020
Safety Precautions: The routine hygienic procedures were sufficient to assure personnel health and safety.
Preparation of the Test Item Formulations
The vehicle has been selected in consultation with the sponsor based on the test item’s characteristics.
The test item, as delivered, was grinded before formulation preparation. Afterwards, test item was weighed into a tared plastic vial on a suitable precision balance and coated with approx. 1/3 of the target volume with 1% aqueous hydroxyethyl-cellulose, the vehicle used in this study. After producing slurry with the glass rod for 1 min, the rest of the vehicle was added to give the appropriate final concentration. The formulation was stirred until visual homogeneity was achieved (at least 30 min).
Based on the results of stability testing (Eurofins Munich Study No. 187385), the test item formulations were prepared once in 4 days as given by Eurofins Munich Study No. 187385. The prepared formulation was stored at room temperature.
Formulates were kept under magnetic stirring during the daily administration.
The vehicle was also used as control item.
Preparation of Cyclophosphamide Formulation
The cyclophosphamide was diluted freshly with 0.9% saline to a concentration of 1 mg/mL. The formulations were vortexed until visual homogeneity was achieved. The test items formulation was prepared freshly on each administration day before the administration.
Preparation of Keyhole Limpet Haemocyanin (KLH)
Lyophilized Keyhole Limpet Haemocyanin (KLH) was reconstituted freshly with aqua ad injectionem (sterile water) to a concentration of 10 mg/mL.
In a second step the KLH solution was diluted further using phosphate-buffered Saline (PBS) to achieve a concentration of 0.4 mg/mL.
The KLH formulation was prepared in a pre-labelled 15 mL blue cap vial (Art. No. 188271, Greiner Bio-One GmbH, Germany).
Administration of Doses
The test item and vehicle were administered at a single dose to the animals by oral gavage. The application volume for all groups (adult / pups) was 5 mL/kg body weight. Pups were dosed from weaning (day 22). For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured.
Dose levels: 0, 100, 300, 1000 mg/kg bw (concentration in vehicle: 0, 20, 60, 200 mg/ml)
Treatment of Cyclophosphamide and KLH
The positive control group (C2) was administered with Cyclophosphamide 7 days before immunization until the day before the last blood sampling. Cyclophosphamide was diluted freshly with 0.9% sodium chloride to a concentration of 1 mg/mL. Cyclophosphamide was administered by oral gavage at a dose of 10 mg/kg and with an application volume of 10 mL/kg.
Approximately one week after the start of the treatment with Cyclophosphamide or vehicle or test item, each animal of group (C, C2, LD, MD and HD) was injected intravenously into the tail vein with 0.300 µg/kg of KLH as single dose (at 0.75 mL/kg of dose volume). On this particular day, the oral gavage treatment with the control or test item was performed after the i.v. treatment of KLH.
On PND 56 ± 3 days, a T-cell dependent antibody response assay was performed. - Details on mating procedure:
- After 2 weeks premating period, one parental male and one parental female from the same dose group were mated (1:1 pairing). Animals from Cohort 1B were maintained on treatment for more than PND 90 and were bred to obtain a F2 generation. F1 males and females (Cohort 1B) of the same dose group were cohabited (1:1 pairing) for up to two weeks beginning on or after PND 90 but not more than PND 120 avoiding the pairing of siblings.
The parental and F1 female were caged with the same male until pregnancy occurred or two weeks have elapsed. On the following morning after pairing and each morning thereafter, the females were examined for the presence of vaginal sperm or a vaginal copulation plug to confirm the mating. In case the vaginal smear of a particular female was not found to be sperm-positive, the actual stage of the oestrus cycle on that day was documented. Animals were separated as soon as possible after evidence of copulation in terms of sperm positive vaginal smear was observed. The day of the vaginal plug and/or sperm was considered as day 0 of gestation.
The cages were arranged in such a way that possible effects due to cage placement were minimized. In case of mating has not occurred after 2 weeks, the animals were separated without further opportunity for mating.
The date of pairing, date of sperm positive vaginal smears and the date of littering were recorded and the pre-coital interval and the duration of gestation were calculated for parental and F1 females. The parental and F1 females from Cohort 1B were carefully examined at the time of expected littering for any signs of dystocia. Any abnormalities in nesting or nursing performances were recorded. - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Dose Formulation Analysis (Test Item)
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).
Study pre start stability analysis included the samples from high dose and low dose group and the investigation was made for 0 h, 6 h (RT), 4 day (RT), 11 day (RT), 11 days at 2-8 °C and 11 days at -15 to -35 °C. The formulation was found to be stable at 6 h (RT), 4 day (RT), and 11 days at 2-8 °C.
Prestart homogeneity investigation included samples collected from various levels (top, middle and bottom) of high dose and low dose groups.
Based on the results from the setup the test item formulation is a suspension.
According to Eurofins Study No. 187385, samples were taken from the top, middle and bottom of prepared formulations from all dose groups and from the middle of the control group in week 1, initiation of month 2, 3, 4 and last week of the study and analysed in triplicates (50 samples). Mean value per dose level is reported.
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. STUGC19AA1018-5) 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.
The phase plan was amended to the study plan. The results are reported in the appendix of the final report.
Dose Formulation Analysis of Cyclophosphamide and KLH
A dose formulation analysis was not performed for the Cyclophosphamide and KLH formulations. Freshly prepared formulations were used to dose the animals. - Duration of treatment / exposure:
- Parental males were dosed until the minimum total dosing of 10 weeks, i.e. during 14 days of pre-mating and maximum 14 days of mating and until terminal sacrifice. All parental females were dosed during 14 days of pre-mating, maximum 14 days of mating, during gestation and until weaning (PND 21).
The selected F1 offspring received further treatment with the test substance from weaning to terminal sacrifice of respective cohort. - Frequency of treatment:
- The animals were treated with the test item formulations or vehicle on 7 days per week.
- Details on study schedule:
- Arrival of the Test Item: 06 November 2018
Study Initiation Date: 28 August 2019
Amendment to Study Plan: 30 August 2019
2nd Amendment to Study Plan: 24 July 2020
Delivery of Animals: 29 August 2019
Acclimatisation Period: 29 August 2019 to 05 September 2019
Experimental Starting Date: 06 September 2019
Treatment Period: 22 September 2019 to
Necropsies:
parental males: 03 October 2019 (animal no. 104), 06 November 2019 (animal no. 38), 09 December 2019 to 12 December 2019
parental females: 26 September 2019 (animal no. 215), 16 October 2019 (animal no. 205), 01 November 2019 (animal no. 153, 161, 219), 15 November 2019 (animal no. 148, 154), 18 November 2019 to 21 November 2019, 27 November 2019 (animal no. 214), 01 December 2019 (animal no. 175, 218)
Experimental Completion Date: 26 March 2020
Completion Date of Delegated Phase (Histopathology): 06 April 2021
Completion Date of Delegated Phase (Formulation Analysis): 30 June 2020
Study Completion Date: 05 May 2021 - 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:
- 220 parental animals (110 males and 110 females) were included in the study (25 male and 25 female animals per group in LD and MD and 30 male and 30 female animals per group in control and HD). In addition some reserve animals (2 per sex) were ordered for possible exchange before study start. However, these animals were not used for the study and euthanized.
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- In consultation with the sponsor and based on the results of 90 day study (BSL Munich / Eurofins Munich Study No. 187383) the doses were selected for the 3 dose groups (LD = low dose, MD= medium dose, HD = high dose) and 1 control group (C).
The highest dose level is chosen with the aim of inducing toxic effects, but not death or severe suffering. Thereafter, a descending sequence of dose levels is selected with a view to demonstrate any dose-related response.
The animals in the control group were handled in an identical manner to the test group subjects and received the vehicle using the same volume as used for the high dose group. - Parental animals: Observations and examinations:
- Body Weight and Food Consumption
Parental animals were weighed once before the assignment to the experimental groups, on the first day of dosing and weekly during the study period as well as at the terminal sacrifice. In addition, during pregnancy, females were weighed on GD 0, 7, 14 and 20 and within 24 hours of parturition (day 0 post-partum) as well as on day 4, 7, 14 and 21 post-partum along with pups.
After weaning, all F1 animals were weighed weekly thereafter and at termination. In addition, F1 females from Cohort 1B were weighed on GD 0, 7, 14 and 20 and within 24 hours of parturition (day 0 post-partum) as well as on day 4, 7, 14 and 21 post-partum along with F2 pups.
Body weights of all cohorts (except Cohort 2B and Cohort 4) were also taken on the day of attainment of vaginal patency or balano-preputial separation.
Body weight was also measured on PND 4, at weaning (surplus pups after selection of cohorts) and weekly until terminal sacrifice.
Food consumption was measured at intervals corresponding to the body weight measurements after the beginning of the dose administration except during mating period for parental and Cohort 1B F1 animals. Food consumption was also not measured during the post-mating period in parental and Cohort 1B F1 males until all females were mated and all males were not back to their original housing cage of 5 males per cage.
Inadvertently, animal no 193 was dosed on gestation day 23, although it was already in PND0. Body weight and food weight were taken on PND1 instead of PND0.
Body weight and food consumption of animal no. 532 (Cohort 1B, HD) was measured on PND 15.
Clinical Observations
For parental and selected F1 cohorts, general clinical observations of the animals were made at least once a day, preferably at the same time each day and considering the peak period of anticipated effects after dosing. The health condition of the animals was recorded. Twice daily all animals (Parental, F1 and F2 generation) were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily. Pertinent behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity, including mortality were recorded, except on 09 December 2019 and 17 January 2020.
Females showing signs of abortion or premature delivery prior to the scheduled scarification of the animals were sacrificed and subjected to a thorough macroscopic examination.
Once before the first exposure and once a week thereafter, detailed clinical observations were made in all P animals and all cohorts (except Cohort 2B and Cohort 4), when animals were weighed outside the home cage. For male animals of the Parental HD group (animal no. 81–110) detailed clinical observations were not recorded in week 8. 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. - Oestrous cyclicity (parental animals):
- Vaginal smears of parental females were examined 2 weeks before beginning of treatment period, during 2 weeks premating period and until the confirmation of mating and/or the end of the 2 weeks mating period to record the oestrus cyclicity and also to confirm the evidence of mating.
Vaginal smears were examined daily for all F1 females in Cohort 1A after the onset of vaginal patency until the first cornified smear was recorded to determine the time interval between these two events. Vaginal smear in Cohort 1A was examined for 2 weeks starting from PND 75.
The vaginal smear in Cohort 1B was examined during mating period to confirm the evidence of mating. The oestrus cycle stage was recorded in P and F1 females of all cohorts at termination (except Cohort 2B, 3 and 4). - Sperm parameters (parental animals):
- At terminal sacrifice, left epididymis, left testis and left vas deferens was separated and used for evaluation of sperm parameters (motility, cauda epididymis sperm count or testicular sperm head count and morphology) from all parental generation males and all Cohort 1A males of each group were performed by using Hamilton Thorn Sperm Analyser (TOX IVOS Version 13.0C). Sperm morphology was performed manually by manual method.
Sperm morphology slides were prepared from all parental generation and all Cohort 1A males. Initially, evaluation was made in male animals of the groups 1 and 4 sacrificed at the end of the treatment period. Sperm morphology examinations were extended to male animals of all other dosage groups if treatment-related changes were observed in the high dose group. For morphology evaluation, sperm from left vas deferens were transferred to 0.1% bovine serum albumin solution.
For staining two drops of 1% aqueous Eosin-Y solution was mixed with six drops of the sperm-suspension. The stained sperm suspension was used to prepare smears on slides. After complete drying, the slides were dipped into 0.1% acetic acid for approximately 30 seconds to intensify the colouring. - Litter observations:
- Each litter of F1 and F2 was examined after parturition (PND 0) to establish the number and sex of pups, stillbirths, live births and the presence of gross external anomalies externally visible abnormalities, including cleft palate, subcutaneous haemorrhages, abnormal skin colour or texture, presence of umbilical cord, lack of milk in stomach and presence of dried secretions. Litter of animal no. 464 and 471 (Cohort 1B, C), animal no. 513 and 520 (Cohort 1B, MD) and animal no. 539 (Cohort 1B, HD) were not examined after parturition.
In addition, the first clinical examination of the neonates included a qualitative assessment of body temperature, state of activity and reaction to handling. Pups found dead on PND 0 or later time were examined for the possible cause of death. Pups were marked with unique identification number or by tattooing.
Live pups were counted and sexed and litters weighed within 24 hours of parturition (PND 0) or PND 1, on PND 4, 7, 14 and PND 21. In addition to the observations on parent animals, any abnormal behaviour of the offspring was recorded.
The clinical signs of pups were recorded on the corresponding days when offspring were weighed.
The anogenital distance (AGD) of each pup was measured once between PND 0 and PND 4. Pup body weight measured on the day of anogenital distance measurement was converted to cube root and used for the calculation of relative AGD (Relative AGD = AGD / Cube root of pup weight).
Male pups were checked for the presence of nipples/areolae on PND 12 or 13.
Litter Size Adjustment
The size of the litter was adjusted on PND 4, by eliminating extra pups by random selection to yield 5 males / 5 females.
If litter adjustment was made and in case there was sufficient pups but an unequal number of males and females such that selection of 5 males/ 5 females cannot be achieved, then litter was standardized to 10 e.g. 2 males / 8 females or 6 males / 4 females.
In case of insufficient number of pups to allow standardization of the litter to 10 or insufficient number of females with pups, the decision was made based on the number of available litters in a treatment group. - Postmortem examinations (parental animals):
- Pathology
At the time of termination or premature death, all P animals were subjected to gross necropsy and examined macroscopically. P males were subjected to necropsy after 10 weeks of dose application and females post weaning. Special attention was paid to the sexual organs for structural abnormalities.
Vaginal smear from adult P females were examined on the day of necropsy to determine the stage of the oestrus cycle.
The uteri of all P females (and Cohort 1B females) were examined for the presence and number of corpora lutea and implantation sites.
Non-pregnant females were sacrificed on day 26 from the day of mating or from the last day of mating period.
Organ Weights
At necropsy, body weights and wet weights of the following organs from all P animals were measured: ovaries, uterus (with oviduct and cervix), testes, epididymides, prostate (dorsolateral and ventral parts combined), axillary lymph noday (Cohort 1A), liver, kidneys, heart, spleen, thymus, pituitary glands, seminal vesicles with coagulating glands and their fluids, thyroid (post-fixation), brain, adrenal glands. Paired organs were weighed together (except testes and epididymides). Organ weights of animals found dead or euthanised for animal welfare reasons were not taken.
The tissues (brain (cerebrum, cerebellum and pons), spinal cord, eya and optic nerve, liver, kidneys, adrenal glands, oesophagus, stomach, small and large intestines, thymus, thyroid and parathyroid, spleen, lung, trachea, mammary glands, skin, heart, ovaries, uterus with cervix, vagina, testes, epididymides, vas deferens, prostate and seminal vesicles with coagulating glands as a whole, urinary bladder, lymph nodes (mesentric and axillary), peripheral nerve with skeletal muscle, bone with bone marrow (sternum), pituitary gland, oesophagus, spinal cord, gross lesions) of the same selected animals from each group were preserved in 4% neutral buffered formaldehyde except for testes, epididymides and eyes that were fixed in Modified Davidson’s Fixative for approximately 24 hours before they were transferred to 4% neutral buffered formaldehyde.
Full histopathology of the organs listed above was performed for all high-dose and control P animals. Histopathological examinations were not extended to animals of the other dose groups, as no treatment-related changes were observed in the high dose group. Reproductive organs of all animals that failed to deliver healthy offspring, their paired males and all gross lesions were subjected to histopathological evaluation. - Postmortem examinations (offspring):
- Pathology
At the time of termination or premature death, all F1 animals were subjected to gross necropsy and examined macroscopically. The animals of F1 generation were subjected to necropsy depending on the scheduled ages for each cohort (Cohort 1A: week 13, 1B: week 20-25, 2A: week 11-12, 2B: 3 weeks at weaning, Cohort 3: 8 weeks, Cohort 4: 6–7 weeks). Special attention was paid to the sexual organs for structural abnormalities.
Pups not selected for cohorts (including runts) were sacrificed and subjected to gross necropsy at weaning (PND 22) or later when not required for further in-life investigations.
Dead or moribund pups were recorded and examined for possible defects and/or cause of death and preserved.
Vaginal smear from adult F1 females (except females of Cohort 2B, 3 and 4) were examined on the day of necropsy to determine the stage of the oestrus cycle. The uteri of all Cohort 1B females were examined for the presence and number of corpora lutea and implantation sites.
Non-pregnant females were sacrificed on day 26 from the day of mating or from the last day of mating period.
F2 pups from Cohort 1B were sacrificed and subjected to necropsy at weaning.
F1 animals from the Cohorts 3 and 4 were subjected only to gross necropsy and examined macroscopically for any structural abnormalities or pathological changes. Organs with abnormalities were preserved for possible histological evaluation.
Organ Weights
At necropsy, body weights and wet weights of the following organs from all F1 adults (Cohorts 1A) were measured: ovaries, uterus (with oviduct and cervix), testes, epididymides, prostate (dorsolateral and ventral parts combined), axillary lymph noday (Cohort 1A), liver, kidneys, heart, spleen, thymus, pituitary glands, seminal vesicles with coagulating glands and their fluids, thyroid (post-fixation), brain, adrenal glands. Paired organs were weighed together (except testes and epididymides). Organ weights of animals found dead or euthanised for animal welfare reasons were not taken.
The tissues (brain (cerebrum, cerebellum and pons), spinal cord, eya and optic nerve, liver, kidneys, adrenal glands, oesophagus, stomach, small and large intestines, thymus, thyroid and parathyroid, spleen, lung, trachea, mammary glands, skin, heart, ovaries, uterus with cervix, vagina, testes, epididymides, vas deferens, prostate and seminal vesicles with coagulating glands as a whole, urinary bladder, lymph nodes (mesentric and axillary), peripheral nerve with skeletal muscle, bone with bone marrow (sternum), pituitary gland, oesophagus, spinal cord, gross lesions) of the same selected animals from each group were preserved in 4% neutral buffered formaldehyde except for testes, epididymides and eyes that were fixed in Modified Davidson’s Fixative for approximately 24 hours before they were transferred to 4% neutral buffered formaldehyde.
From 10 male and 10 female Cohort 1A animals of each treatment group (1 male or 1 female per litter; randomly selected) one half of the spleen was preserved for histopathological evaluation, while the other half of the spleen was used for the investigation of pre- and post-natally induced immunotoxic effects at termination.
The organs (vagina, uterus with cervix, testes, thyroid, gross lesions, seminal vesicles and coagulating glands, prostate, pituitary, apididymides and adrenal glands) of Cohort 1B animals were weighed and tissues processed to the block. Since the Cohort 1A are not equivocal or suspected reproductive/ endocrine toxicants, so the examination was not extended to Cohort 1B animals.
Cohort 2A animals were terminated between PND 75 and 80 after behavioural testing. The brain weight was recorded and full neurohistopathology was performed. The perfusion fixation was employed for cohort 2A animals. For Cohort 2A, the eyes (retina and optic nerve) and samples of peripheral nerve, muscle and spinal cord were also preserved.
Cohort 2B animals were terminated on PND 21 or 22. The brain weight was recorded and microscopic examination of the brain was performed. The perfusion fixation was employed for Cohort 2B animals.
Brain, spleen, and thymus collected from 10 pups / sex / group (surplus pups not allocated to cohorts and pups of F2 generation) were weighed and preserved along with mammary gland, gross lesion and target tissues for possible histological examination.
For perfusion fixation, approximately 10 minutes before anaesthesia the animals received an intraperitoneal injection of heparin (1000 IU/kg, 2 mL/kg). Each animal was deeply anaesthetized with ketamine and xylazin and subjected to in situ perfusion with 4% neutral buffered formaldehyde to achieve an adequate fixation of the brain according to the below procedure.
After opening the thoracic cavity, a needle (20G) was inserted in the apex of the left ventricle of the heart toward the top where the ascending aorta connects. The needle was connected to the perfusion pump via tubing and the infusion was started at a flow rate of approximately 30 mL/min. A slit in the right atrium was made in order to allow blood to flow. First a pre-flush was performed during 5 minutes with ice cold saline solution (0.9% NaCl solution). After the solution runs clear the perfusion was switched to 4% neutral buffered formaldehyde during 12 minutes. The perfusion solutions were placed in a water bath at approx. 37 °C so that they were administered at a physiological temperature. When the perfusion time had reached the brain, it was carefully examined, sampled and transferred in 4% neutral buffered formaldehyde for histopathological evaluation.
Histopathology
Cohort 1
Full histopathology of the organs listed above was performed on control and high dose adult Cohort 1A animals. Histopathological examinations were not extended to animals of the other dose groups, as treatment-related changes were not observed in the high dose group. All gross lesions were subjected to histopathological evaluation.
Histopathology of lymph nodes (mesenteric and axillary) and bone marrow was performed on 10 male and 10 female Cohort 1A animals. Histopathology of thymus, spleen, and the adrenal glands was performed in all Cohort 1A animals.
As the results from Cohort 1A were not equivocal or suspected reproductive/ endocrine toxicants, the examination were not extended to Cohort 1B animals.
The histopathological examination of ovaries included quantitative evaluation of primordial and small growing follicles and corpora lutea. Follicular enumeration was conducted on control and high-dose animals. As there were no adverse effects, it was not extended to lower dosed.
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.
Cohort 2
Neurohistopathology was performed for all control and high dose Cohort 2A animals after PND 90.
Brain histopathology was performed for all control and high dose Cohort 2B animals per sex on PND 21 or PND 22. Examinations were not extended to animals of the other dose groups, as treatment-related changes were not observed in the high dose group.
For Cohort 2A and 2B animals, multiple sections were made from the brain to examine olfactory bulbs, cerebral cortex, hippocampus, basal ganglia, thalamus, hypothalamus, mid-brain (thecum, tegmentum, and cerebral peduncles), brain-stem and cerebellum.
For Cohort 2A, the eyes (retina and optic nerve) and samples of peripheral nerve, muscle and spinal cord were examined.
Morphometric evaluations were performed in 10 animals / sex / dose (C and HD groups) on representative areas of the brain. At least two consecutive sections were taken at each landmark (level) in order to select the most homologous and representative section for the specific evaluated brain area.
All gross lesions were subjected to histopathological evaluation. All animals (parental and all cohorts) found dead or intercurrently euthanised for animal welfare reasons were subjected to a gross necropsy and the organs were preserved for a histopathological examination.
Brains were stained to determine neuroinflammation and neurodegeneration. The stainings that were used are: haematoxylin & eosin and Flour Jade stain. All two stains were made in control and HD group on different transversal levels (at least 5 levels, including levels 2-6) according to the STP Position paper. - Clinical signs:
- effects observed, non-treatment-related
- Description (incidence and severity):
- In terminally sacrificed parental male and female animals, predominant clinical signs transiently observed in the majority of HD animals were increased salivation and/or moving the bedding.
Low incidences of slight clinical signs like hairless area, piloerection, alopecia on various body parts, over grown teeth, nasal discharge, wound, crust, Chromodacryorrhea, lacrimation and half eyelid closure were observed in few animals on few days in all groups including controls. None of the parental females showed signs of abortion or premature delivery. The clinical signs salivation and moving the bedding in males and females were observed immediately after the dose administration and therefore were considered to be a sign of a local reaction to the test item rather than a systemic adverse effect and has no toxicological relevance. - Dermal irritation (if dermal study):
- not examined
- Mortality:
- mortality observed, non-treatment-related
- Description (incidence):
- From LD group, 1 male (no. 38, on day 46) and from HD group 1 male (no. 104, on day 12) and 2 females (no. 205 on GD 10 and 215 on premating day 5) was euthanised for animal welfare reasons.
Animal no. 38 was having a wound at neck left side from day 11-45 and necrotized on day 46. Animal no. 104 showed abnormal breathing on day 12. Animal no. 215 showed abnormal breathing on day 5. At histopathological evaluation, no cause of death was established for animal no. 38 and 104. Animal no. 205 had hemorrhagic/necrotizing inflammation of esophagus which could be due to misgavage. - Body weight and weight changes:
- effects observed, non-treatment-related
- Description (incidence and severity):
- In both male and female parental animals, there was no test item treatment related effect observed on group mean body weight and mean body weight gain in the dose groups when compared with the controls. There were no statistically significant differences observed for these parameters between the dose groups and the control group; except the following
Statistically significantly lower body weight gain was found between day 14-21 in MD males and between day 21-28 in HD males when compared with the control. In females, statistically significantly lower mean body weight was observed between day GD7-20 in LD and HD and between lactation day 7-14 in LD group when compared with the control. Statistically significantly lower mean body weight gain was observed during premating in females on day 8-14 in LD and MD and lower in HD group when compared with the control. Statistically significantly lower mean body weight gain was observed at GD0-20 in LD group. Due to the lack of dose dependency and consistency, these differences on parental mean body weight and/or gain were not considered as test item related and the mean body weight were found to be within the historical control range of this strain. - Food consumption and compound intake (if feeding study):
- effects observed, non-treatment-related
- Description (incidence and severity):
- In correlation to the body weight and body weight gain, the food consumption in both males and female of parental generation tended to increase with the progress of the study in all study groups.
No test item related or statistically significant effect on food consumption was observed in males and females of parental generation during the whole study period except slight but statistically significantly lower group mean food consumption was observed in parental females between day 21 and 28 in the LD males and statistically significantly lower group mean food consumption observed between GD 0 and 7 in LD and between GD 14 and 20 in LD and MD groups when compared with the control. - 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):
- In 10 selected parental males and females per group sacrificed at the end of the treatment period, no test item related adverse effects were observed in haematological parameters in the dose groups when compared to the control group. In males, marginally but statistically significantly higher mean WBC in MD group, mean MCH in LD and HD groups, mean MCHC in HD group were observed when compared to the control. In females, marginally but statistically significantly higher mean percent EOS in the HD group were observed when compared with the control. All these values were without any dose dependency or consistency; hence they are not considered to be adverse.
All other group mean and most of the individual values for haematological parameters in male and females were comparable with the control and within the normal range of variation and also they are in line with historical control data of reproductive and developmental toxicity studies in this strain.
No test item related effect was observed on coagulation parameters in parental males and females when compared with the controls. - Clinical biochemistry findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- In 10 selected parental males and females sacrificed at the end of the treatment period, in males, marginal but statistically significantly lower creatinine in the MD groups was observed when compared with the control. In females, marginal but statistically significantly higher glucose in LD and HD groups, lower potassium in MD and HD groups were observed when compared with the control. As the differences were marginal and all values were within the range of historical control data or without dose-dependency; this is not assumed to be toxicologically relevant.
All other group mean and most of the individual values for clinical chemistry parameters in male and females were comparable with the controls and within the normal range of variation. - Urinalysis findings:
- no effects observed
- Description (incidence and severity):
- The urinalysis performed in 10 selected males and females per group from parental sacrificed at the end of treatment period revealed no test item treatment related effect in the dose groups when compared to control. All urinary parameters were in the normal range of variation. Slightly high protein levels were found in the urine of few male and females of all groups including the control group. Therefore, this effect on urine parameters was not considered to be test item related.
- Behaviour (functional findings):
- effects observed, non-treatment-related
- Description (incidence and severity):
- During the weekly detailed clinical observation, no toxicologically relevant differences between the groups were observed in parental cohorts during the entire study period. There were statistical significances observed in few parameters in parental cohorts on few occasions. However, observed statistical significant difference in few parameters were either before initiation of treatment, not dose dependent/consistent or biologically relevant and therefore these findings were considered to be incidental and not related to the treatment with the test item.
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Histopathological findings: non-neoplastic:
- no effects observed
- Description (incidence and severity):
- Under the conditions of this study, there were P-generation animals sacrificed during the course of the study. The decedents were randomly distributed and, hence, death was not deemed to be related to treatment with the test item. There were no gross lesions and histological findings that could be attributed to treatment. No specific findings were noted that could be related to infertility, and, again these cases were not related to treatment with Methyl 4-hydroxybenzoate.
- Histopathological findings: neoplastic:
- no effects observed
- Reproductive function: oestrous cycle:
- no effects observed
- Description (incidence and severity):
- Parental Females - Pre-Treatment and Premating
Test item had no biologically or statistically significant effect on the oestrus cycle analysed during 2 weeks pre-treatment and 2 weeks premating period after the first administration in treatment groups when compared to the control. 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):
- Motility
Test item had no effect on epididymal sperm motility analysed from all males. Group mean motility values from parental males of the dose groups were comparable to control.
Sperm Morphology
Evaluation of sperm morphology from control and HD parental males did not reveal any test item related findings and percentage of normal and abnormal sperms in treatment groups were comparable to control.
Testicular Sperm Head Count
Test item had no effect on sperm head count in parental males. Group mean sperm head count from parental males of dose groups were comparable to control. - Reproductive performance:
- no effects observed
- Description (incidence and severity):
- There was no test item related effects observed on the reproductive indices (percent male mating index, female mating index, male fertility index, female fertility index, gestation index and live birth index) in parental dose group animals when compared to the respective control group. The survival index of pups during PND 0 to 4, PND 4 (after interim sacrifice) to PND 13 and PND 14 (after interim sacrifice) to PND 21 in parental females remained unaffected and within the range of biological variation in treatment groups when compared with the respective control group.
- 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
- Critical effects observed:
- no
- Clinical signs:
- effects observed, non-treatment-related
- Description (incidence and severity):
- In terminally sacrificed males and females from various F1 cohorts, similar clinical signs like parental animals were observed in HD group but in few animals compared to parental animals.
As these findings showed no dose-dependency and were noted transiently, they were not considered to be toxicologically relevant.
The clinical signs salivation and moving the bedding in males and females were observed immediately after the dose administration and therefore were considered to be a sign of a local reaction to the test item rather than a systemic adverse effect and have no toxicological relevance.
None of the Cohort 1B females showed signs of abortion or premature delivery. - Dermal irritation (if dermal study):
- not examined
- Mortality:
- mortality observed, non-treatment-related
- Description (incidence):
- In Cohort 1B, one MD female (no. 511, on day 112) and one HD male (no. 445, on day 113) found dead during the study period. Both animals did not show any clinical signs beforehand. No cause of death was established at Histopathological evaluations.
- Body weight and weight changes:
- effects observed, non-treatment-related
- Description (incidence and severity):
- In both male and female Cohort 1B animals, there was no test item treatment related effect observed on group mean body weight and mean body weight gain in the dose groups when compared with the control. Slight but statistically significantly lower group mean body weight gain was observed between day 15 and 22 in LD group males when compared with the controls. In females, no statistically significant differences in mean body weight or mean body weight gain were observed during the study (including gestation and lactation) when compared with the control.
Overall, in all parental and F1 cohorts, the body weight and body weight gain remained unaffected by the treatment with test item and values were in the normal range of variation throughout the treatment period when compared to the control group and also the mean body weight were found to be within the historical control range of this strain. - Food consumption and compound intake (if feeding study):
- effects observed, non-treatment-related
- Description (incidence and severity):
- In correlation to the body weight and body weight gain, the food consumption in both males and female of F1 cohorts (Cohort 1A, 1B, 2A, 3 and 4) tended to increase with the progress of the study in all study groups.
No test item related or statistically significant effect on food consumption was observed in males and females of F1 cohorts during the whole study period except. Slight but statistically significantly higher group mean food consumption was observed between day 85-92 in HD groups males of Cohort 1B when compared with the control. Due to the lack of dose dependency or consistency, this effect on food consumption in Cohort 1B animals was not considered to be adverse. - Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- not examined
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- effects observed, non-treatment-related
- Description (incidence and severity):
- During the weekly detailed clinical observation, no toxicologically relevant differences between the groups were observed in F1 cohorts (1A, 1B, 2A and 3) during the entire study period. There were statistical significances observed in few parameters in F1 cohorts on few occasions. However, observed statistical significant difference in few parameters were either before initiation of treatment, not dose dependent/consistent or biologically relevant and therefore these findings were considered to be incidental and not related to the treatment with the test item.
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Description (incidence and severity):
- Organ weight from male and female Cohort 1B animals remained unaffected with the test item treatment and there were no statistically significant differences in absolute and relative organ weights of the dose groups when compared to the control group.
- Gross pathological findings:
- no effects observed
- Description (incidence and severity):
- Few spontaneous gross pathological changes were recorded for male and female animals from various cohorts and were not considered to be treatment-related.
Based on microscopic examination, these findings were not considered to be related to treatment with the test item but deemed incidental. - Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- not examined
- Histopathological findings: neoplastic:
- no effects observed
- Other effects:
- no effects observed
- Reproductive function: oestrous cycle:
- no effects observed
- Description (incidence and severity):
- Cohort 1A Females
In Cohort 1A females, no biologically significant effect was observed on the duration of vaginal opening until first oestrus cycle in treatment groups when compared to control. Statistically significant changes were noticed in MD group animals when compared to control and this could be due to persistent dioestrus of female animal no. 343 and 349.
In this cohort, no biologically significant effect was observed on the oestrus cycle length or sequence of cycle stages between the treatment groups and the control group analysed from PND 75 for 2 weeks. - Reproductive function: sperm measures:
- no effects observed
- Description (incidence and severity):
- Motility
Test item had no effect on epididymal sperm motility analysed from all males. Group mean motility values from Cohort 1A males of the dose groups were comparable to control., however percent static count in Cohort 1A males were found to be statistically significantly lower in LD, MD and HD groups when compared to control. This could be due to slightly higher individual values of static count in few of the control animals.
Sperm Morphology
Evaluation of sperm morphology from control and HD Cohort 1A males did not reveal any test item related findings and percentage of normal and abnormal sperms in treatment groups were comparable to control.
Testicular Sperm Head Count
Test item had no effect on sperm head count in Cohort 1A males. Group mean sperm head count from Cohort 1A males of dose groups were comparable to control. - Reproductive performance:
- no effects observed
- Description (incidence and severity):
- There was no test item related effects observed on the reproductive indices (percent male mating index, female mating index, male fertility index, female fertility index, gestation index and live birth index) in Cohort 1B dose group animals when compared to the respective control group. The survival index of pups during PND 0 to 4, PND 4 (after interim sacrifice) to PND 14 and PND 14 to PND 21 in Cohort 1B females remained unaffected and within the range of biological variation in treatment groups when compared with the respective control group.
- 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
- Critical effects observed:
- no
- Clinical signs:
- effects observed, non-treatment-related
- Description (incidence and severity):
- In terminally sacrificed males and females from various F1 cohorts, similar clinical signs like parental animals were observed in HD group but in few animals compared to parental animals.
As these findings showed no dose-dependency and were noted transiently, they were not considered to be toxicologically relevant.
The clinical signs salivation and moving the bedding in males and females were observed immediately after the dose administration and therefore were considered to be a sign of a local reaction to the test item rather than a systemic adverse effect and have no toxicological relevance. - Dermal irritation (if dermal study):
- not examined
- Mortality / viability:
- mortality observed, non-treatment-related
- Description (incidence and severity):
- Cohort 1A
In Cohort 1A, one LD male (no. 241, on day 43) and one female (no. 325, on day 66-found dead); one MD male (no. 275, on day 60- found dead) and one MD female (no. 350, on day 5); one HD females (no. 379, on day 54) were euthanised for animal welfare reasons. Animal no. 241 showed exophthalmos left eye on day 30-43. Animal no. 350 showed abnormal breathing and moderate, piloerection on day 5. Animal no. 379 did not show any clinical signs beforehand.
Animal no. 325, 275, 379 had hemorrhagic/necrotizing inflammation of esophagus, pleural inflammation in lungs and pericardial inflammation; lung edema respectively, all these findings could be due to misgavage. No cause of death was established at histopathological evaluation for animal no. 241 and 350.
Cohort 2A
In Cohort 2A, no mortality was observed during the study period., except one control male (animal no. 546) was euthanized after identified as female in male control group on day 10.
Cohort 2B
In Cohort 2B, no mortality was observed during the study period.
Cohort 3
In Cohort 3, 1 female of the positive control group C2 (no. 794, on day 42), 1 HD female (no. 787, on day 22) were found dead and 1 LD female (no. 761, on day 6) was sacrificed in moribund condition due to animal welfare reasons. Animal no. 761 showed abnormal breathing and cyanosis on day 6.
Overall, the predominant findings observed at necropsy were fluid filled abnormal content in thoracic cavity and abnormal dark red coloured lung (male no. 275, MD- Cohort 1A); abnormal dark red coloured lung with fluid (female no. 350, MD- Cohort 1A); abnormal dark red coloured lung (female no. 325, LD- Cohort 1A) observed. Abnormal dark red coloured, spongy lung was observed (female no. 511, MD- Cohort 1B). Abnormal dark red coloured lung with fluid (female no. 761, LD- Cohort 3) and abnormal dark red coloured lung (female no. 794, C2- Cohort 3) was observed.
Histopathologically, the cause of death was not evident in most of the animals. However, in animals observed with fluid filled thoracic cavity or fluid filled lung/abnormal dark red at necropsy, the cause of death could be related to the technical gavage error.
Cohort 4
No mortality was observed during the study period. - Body weight and weight changes:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Cohort 1A
In both male and female Cohort 1A animals, there was no test item treatment related effect observed on group mean body weight and mean body weight gain in the dose groups when compared with the control. Slight but statistically significantly lower group mean body weight on day 43 in males HD group was observed when compared with the controls. There was also statistically significantly lower mean body weight gain in LD and HD males between 36 and 43 and higher body weight gain in MD group between day 50 and 57 of the study. However, in females, no statistical significance in mean body weight or body weight gain between the groups during the study period. As the observed differences on mean body weight and body weight gain in males was marginal and without dose dependence, they are not considered to be adverse.
Cohort 2A
In both male and female Cohort 2A animals, there was no test item treatment related effect observed on group mean body weight and mean body weight gain in the dose groups when compared with the control. There was no test item related or statistically significant difference on body weight and body weight gain between the dose groups when compared to control in both male and females.
Cohort 3
In both male and female Cohort 3 animals, there was no test item treatment related effect observed on group mean body weight and mean body weight gain in the dose groups when compared with the control. In males and females, no statistically significant differences were observed on body weight between the dose groups and the respective control group. However, statistically significantly lower group mean body weight gain was observed in males between days 29-36 in females between days 1-43 in C2 group when compared to the control.
Cohort 4
In both male and female Cohort 4 animals, there was no test item treatment related effect on group mean body weight and mean body weight gain in the dose groups when compared with the control. There was no statistically significant difference in mean body weight between the dose groups when compared to control in males. In females, there were statistically significantly lower means of body weight on day 1 (8.13% below control) and 8 (13.12% below control) and lower mean body weight gain between day 1 and 8 (19.4% below control) were observed in HD group when compared to control. In males, there was statistically significantly lower mean body weight gain between day 1 (14.53 below control) and day 8, statistically significantly higher mean body weight gain (19.78% above control) were observed. All these statistical significance was due to higher mean body weight of control group; hence this is not considered to be toxicologically relevant.
Overall in all parental and F1 cohorts, mean body weight and mean body weight gain remained unaffected by the treatment with test item and values were in the normal range of variation throughout the treatment period when compared to the control group and they are in line with historical control data of reproductive and developmental toxicity studies in this strain. - Food consumption and compound intake (if feeding study):
- effects observed, non-treatment-related
- Description (incidence and severity):
- In correlation to the body weight and body weight gain, the food consumption in both males and female of F1 cohorts (Cohort 1A, 1B, 2A, 3 and 4) tended to increase with the progress of the study in all study groups.
No test item related or statistically significant effect on food consumption was observed in males and females of F1 cohorts during the whole study period. Slight but statistically significantly lower group mean food consumption was observed between day 50 and 57 in LD and MD groups males of Cohort 1A when compared with the control. Due to the lack of dose dependency or consistency, this effect on food consumption in Cohort 1A animals was not considered to be adverse. - Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Cohort 1A
In 10 selected Cohort 1A males and females per group sacrificed at the end of the treatment period, in males, marginally but statistically significantly higher mean percent neutrophils and monocytes and lower percent lymphocytes were observed in the HD group when compared with the control. In females, there was no statistical significance changes observed when compared with the control. As the differences were marginal and all values were within the range of historical control data; this is not assumed to be toxicologically relevant. All other group mean and most of the individual values for haematological parameters in male and females were comparable with the controls and within the normal range of variation.
In the absence of test item related histopathological findings and effect on splenic lymphocyte subpopulation in the study, above-mentioned increase or decrease in few haematology parameters was not considered to be adverse.
No test item related effect was observed on coagulation parameters in Cohort 1A males and females when compared with the respective control. - Clinical biochemistry findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Cohort 1A
In 10 selected Cohort 1A males and females per group sacrificed at the end of treatment period, there were no statistically significant changes in the dose groups when compared with the control. In the 10 selected females, marginal but statistically significantly lower ASAT and urea in the HD group, lower ALAT and Urea in MD group were observed when compared to control. In absence of test item related histopathological findings and clinical signs, this effect in Cohort 1A animals was not considered to be toxicologically relevant.
All other group mean and most of the individual values for haematological parameters in male and female Cohort 1A animals were comparable with the controls and within the normal range of variation. - Urinalysis findings:
- no effects observed
- Description (incidence and severity):
- The urinalysis performed in 10 selected males and females per group from Cohort 1A sacrificed at the end of treatment period revealed no test item treatment related effect in the dose groups when compared to control. All urinary parameters were in the normal range of variation. Slightly high protein levels were found in the urine of few male and females of all groups including the control group. Therefore, this effect on urine parameters was not considered to be test item related.
- Sexual maturation:
- effects observed, non-treatment-related
- Description (incidence and severity):
- In male pups from parental females on PND 0, marginal but statistically significantly lower pup weight, cube root of pup weight and relative anogenital distance (AGD) in HD groups were observed when compared to the control. No effect was seen on absolute AGD. In MD group, marginal but statistically significantly higher absolute and relative AGD was observed. In female pups from parental females on PND 0, there was no statistical significant difference when compared with the control group. As the differences were only very slight and without any dose dependency and values were also within the range of historical control data, this is not assumed to be toxicologically relevant.
In male pups from Cohort 1B females on PND 0, marginal but statistically significantly lower pup weight, cube root of pup weight and absolute AGD were observed in the LD and HD group when compared to the control group. Statistically significant lower absolute AGD in MD group and lower relative AGD in HD groups were observed. In female pups from Cohort 1B on PND 0, a marginal but statistically significant effect was observed on pup weight and cube root of pup weight parameter. As values were without dose dependency and within the normal range of historical control data, this is not considered toxicologically relevant.
No effect of toxicological relevance was observed on nipple retention in the pups of any of the groups from parental and Cohort 1B females when compared to control. Group mean number of nipple retention in MD group males from parental females was slight but statistically significantly higher than in control. In males from Cohort 1B, nipple retention was statistically significantly lower in MD and HD groups when compared to control. The slightly higher incidence from parental females was attributed with a higher incidence of nipple retention from few dam of MD groups and considered to be incidental and not related to the treatment with test item. - Anogenital distance (AGD):
- no effects observed
- Description (incidence and severity):
- see comments on sexula maturation
- Nipple retention in male pups:
- no effects observed
- Description (incidence and severity):
- see comments on sexula maturation
- Organ weight findings including organ / body weight ratios:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Cohort 1A
In Cohort 1A males, slight but statistically significantly lower absolute and relative kidneys weight in LD group, absolute heart weight in LD and HD group, absolute and relative epididymides weights in LD and HD groups and absolute and relative liver weight in the LD group were observed when compared with the control and they are in line with historical control data of reproductive and developmental toxicity studies in this strain.
In Cohort 1A females, there were no statistically significant differences in the absolute and relative organ weights of the dose groups when compared to control group.
Cohort 2A and 2B
No effect on brain weights were observed in male and females of Cohort 2A and 2B.
F1 Pups not Selected for Cohorts and F2 Cohort (PND 21)
No effect on brain, spleen and thymus weights were observed in F1 pups not selected for cohorts and F2 pups from Cohort 1B females. Slight but statistically significant higher mean absolute weight of thymus in MD group and mean relative organ weight of thymus in LD group was observed in female pups on PND 21 when compared to control.
Weights of lymph nodes, spleen and thymus of Cohort 1A animals revealed no considerable changes that could indicate a test item related immunotoxic effect. - Gross pathological findings:
- no effects observed
- Description (incidence and severity):
- Few spontaneous gross pathological changes were recorded for male and female animals from parental generation and various cohorts and were not considered to be treatment-related.
Based on microscopic examination, these findings were not considered to be related to treatment with the test item but deemed incidental. - Histopathological findings:
- no effects observed
- Description (incidence and severity):
- In the F1-generation, there were also few animals died during the course of the study. In a few cases, the cause of death may have been related to gavage accidents. No gross lesions nor histological lesions could be attributed to treatment with the Methyl 4-hydroxybenzoate. Neuropathology evaluation and evaluation of reproductive organs did not reveal any induced lesion. In summary, based on the pathology evaluation, the NOEL may be established at 1000 mg/kg bw/day.
- Behaviour (functional findings):
- no effects observed
- Description (incidence and severity):
- During the weekly detailed clinical observation, no toxicologically relevant differences between the groups were observed in F1 cohorts (1A, 1B, 2A and 3) during the entire study period. There were statistical significances observed in few parameters in F1 cohorts on few occasions. However, observed statistical significant difference in few parameters were either before initiation of treatment, not dose dependent/consistent or biologically relevant and therefore these findings were considered to be incidental and not related to the treatment with the test item.
There was no test item related effects on learning and memory, auditory startle response, clinical and functional observations and motor activity. Histopathologically, there were no indications of morphological abnormalities in the brain as demonstrated by Haematoxylin & Eosin staining and Fluoro-Jade staining. No morphometric changes were observed in dose groups compared to control. - Developmental immunotoxicity:
- no effects observed
- Description (incidence and severity):
- Cohort 3: C Group (Control Group)
Male: Male animals of the control group C showed an immune response after injection with KLH. All male animals of this group showed elevated anti-KLH IgM levels on day 6 after KLH injection, when compared to pre-levels (in average 6-fold higher) and all 10/10 male animals showed detectable anti-KLH specific IgG levels on 14 days after KLH injection when compared to pre-levels (all BLQ). This supports the expression of an immune response to KLH immunogen with subsequent class-switch from IgM to IgG. At the respective days after immunization total IgM and IgG levels were also elevated in these animals. Detectable levels of total IgM were found in 10/10 animals (approx. 2.1-fold higher) compared to 2/10 animals before immunization and total IgG level detectable in all animals and it was on average 1.6-fold higher than pre-levels.
Female: Also, female animals of the control group C showed an immune response after injection with KLH. All 10/10 female animals showed increased anti-KLH IgM titers on day 6 when compared to pre-levels (in average 6.5-fold higher). 8/10 female animals also showed detectable anti-KLH IgG levels on 14 days after injection of KLH, when compared to pre-levels (all BLQ). This supports the expression of an immune response to KLH immunogen with subsequent class-switch from IgM to IgG. At the respective days after immunization total IgM and IgG levels were also elevated in these animals. Mean total IgM level was approx. 2.7-fold higher and mean total IgG level was 1.7-fold higher than pre-levels.
Cohort 3: C2 Group (Positive Control Group)
Male: Decreased anti-KLH IgM titers after immunization were observed in all 10/10 male animals treated with immunosuppressant cyclophosphamide. Levels of 5/10 animals were even below the level of quantification. With the exception of one animal, anti-KLH IgG levels on day 14 after immunization were also below the level of quantification and baseline (BLQ). In contrast to the immunocompetent negative control group no considerable changes were observed in total IgM and IgG levels of these animals. Taken together, this shows that the immune response to KLH in male animals treated with cyclophosphamide was effectively decreased.
Female: In female animals of this group KLH-specific IgM titer was lower in 8/10 animals after treatment with the immunogen when compared to pre-levels. Levels of anti-KLH IgG was below quantification limit before KLH injection. After immunization on day 14 levels were still below quantification limit in 5/9 animals and the remaining 4/9 animals showed lower levels when compared to test item groups. These results clearly show a lack of or reduced immune-stimulation in this group. In line with this, no considerable changes were observed in total IgM and IgG levels of these animals on the respective day 6 and 14 when compared to pre-levels.
Cohort 3: Dose Groups
Male: All male animals treated with Methyl 4-hydroxybenzoate (10/10 for each dose group; with the exception of a single male of the HD group with a nearly unchanged level) showed elevated anti-KLH IgM levels on day 6 after KLH-injection, when compared to pre-treatment levels. This shows a clear immunological response in these animals which is further supported by increases in total IgM and IgG levels between 70% and 110% and between 20 and 100 %, respectively, in LD, MD and HD groups.
Female: Elevated anti-KLH IgM levels were also observed in all female animals treated with Methyl 4-hydroxybenzoate (10/10 for each dose group) on day 6 after KLH-injection, when compared to pre-treatment levels. Again, this shows a clear immunological response to KLH antigen. As in male animals, this coincided in dosed females with increases in total IgM (between 44% and 126%) and IgG levels (between 38% and 50%) on the respective days, when compared to pre-levels. This also indicates an immunological reaction in the animals. - Key result
- Dose descriptor:
- NOAEL
- Generation:
- F1 (cohort 1A)
- 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
- Dose descriptor:
- NOAEL
- Generation:
- F1 (cohort 1B)
- 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
- Dose descriptor:
- NOAEL
- Generation:
- F1 (cohort 2A)
- 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
- Dose descriptor:
- NOAEL
- Generation:
- F1 (cohort 2B)
- 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
- Dose descriptor:
- NOAEL
- Generation:
- F1 (cohort 3)
- 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
- Critical effects observed:
- no
- Key result
- Reproductive effects observed:
- no
- Conclusions:
- On the basis of the present study, the Extended One-Generation Reproductive Toxicity Study after oral administration in male and female Wistar rats with Methyl 4-hydroxybenzoate with dose levels of 100, 300 and 1000 mg/kg body weight day the following conclusions can be made:
General Toxicity
Few mortalities/morbidities were randomly distributed throughout the majority of groups of parental animals and various cohorts. Based on histopathology the cause of death was not evident in few animals whereas in others, the cause of death could be related to the technical gavaging error and may not be related to the test item effects.
There were no clinical signs of toxicological relevance observed in the treatment groups.
Body weight and food consumption were not affected by treatment with test item. At termination no test item related effects on clinical pathology (haematology, blood coagulation, clinical biochemistry and urinalysis) were observed in parental generation and in Cohort 1A. Furthermore, no test item related gross pathological findings, effect on organ weights and histopathological findings were observed in the study.
Developmental and Reproduction Toxicity
There were no considerable differences in the length or sequence of oestrus cycle stages, duration of precoital interval, duration of gestation of the parental generation and Cohort 1A between the treatment groups and the control group. There were no signs of abortion or premature delivery, in litter parameters, i.e. number of still births, runts, total number of pups, sex ratio, number of alive pups, weight of pups, survival index. Corpora lutea, implantation sites, percent preimplantation loss and post implantation loss in parental generation and Cohort 1B were unaffected by the test item. There was no test item related effects on reproductive indices (male mating index, female mating index, male fertility index, female fertility index, gestation index and live birth index) in parental generation and in Cohort 1B. No test item related external findings were observed in the pups of this study. There was no test item related effect on anogenital distance and nipple retention and sexual maturity parameters (i.e. vaginal opening and balano-preputial separation). There was no test item related effects on sperm motility and morphology as well as for sperm head count of parental generation and Cohort 1A males. Histopathologically, no effects on reproductive organs were detected. The test item had no effect on serum T4 and TSH levels in parental generation (males and females) and in Cohort 1A animals on PND4, PND 21 and at adult age. There was no indication of endocrine disruptive properties of the test item in this study.
Neurotoxicity
There was no test item related effects on learning and memory, auditory startle response, clinical and functional observations and motor activity. Histopathologically, there were no indications of morphological abnormalities in the brain as demonstrated by Haematoxylin & Eosin staining and Fluoro-Jade staining. No morphometric changes were observed in dose groups compared to control.
Immunotoxicity
There was no sign of immunotoxicity in this study. The results of the TDAR indicate a functional immune system. KLH-specific IgM levels indicate some variability similar to the negative control and did not show any sign of effect on the specific immune response. An integrated evaluation of all immunologically relevant data of the study comes to the conclusion that this is not considered clinically relevant.
These data comprise clinical observations including phenotyping of splenocytes subpopulations, clinical pathology parameters, weight of immune organs, macroscopic and histopathological evaluation of lymph nodes, peyer’s patches, spleen and thymus of parental and Cohort 1A animals, where no test item related effects were observed.
In the absence of indication of toxicity, the NOAEL for developmental and reproductive toxicity, developmental neurotoxicity and developmental immunotoxicity is determined as 1000 mg/kg body weight/day. - Executive summary:
The aim of this study was to assesspossible adverse effects of the test item Methyl 4-hydroxybenzoatevia oral administration (gavage)after repeated exposure during all phases of the reproductive cycle including pre and postnatal development with sexual maturation, the integrity of the male and female reproductive systems and systemic toxicity in males, pregnant and lactating females and young/adult offspring (development, growth, survival, and functional endpoints). In a single study, it tests parental (P) fertility and reproductive function, and offspring (F1) development through sexual maturity, including assessment of sexual landmarks (Cohort 1), nervous system - neuropathological and behavioural endpoints (Cohort 2), immune function (Cohort 3), learning and memory (Cohort 4) and effect on F2 generation by mating of F1 offspring if there are indications of potential adverse effects on F1 offspring.
The P-generation groups contained thirty sexually matured animals per sex in control and HD group and 25 per sex in LD and MD groups.
The test item was administered daily in graduated doses to 3 groups of test animals, one dose level per group. Animals of an additional control group were handled identically as the dose groups but received1% hydroxyethyl-cellulose, the vehicle used in this study.
The test item formulation was prepared at least once at least every 4 days andthe prepared formulations were stored protected from light at 2-8 °C.The test item was suspended in 1% aqueous hydroxyethyl-cellulose. Dose volumes were adjusted individually based on recent body weight measurements. The test item was administered daily in graduated doses to 3 groups of test animals at dose volume of 5 mL/kg bw. Animals of the control group were handled identically as the dose groups, but received 1% aqueous hydroxyethyl-cellulose (viscosity 80-125 cP, 2% in water at 20 °C), the vehicle used in this 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 P-generation animals were exposed with the test item by oral gavage 2 weeks during premating (males and females), 2 weeks during mating (males andfemales), 6 weeks post mating up to termination after weaning- 10 weeks total treatment (males), during pregnancy and lactation up to termination after weaning- 8-10 week’s total treatment (females).
At weaning, selected F1 offspring were assigned to specific cohorts for the investigations comprising sexual maturation, reproductive organ integrity and function, neurological and behavioural endpoints, and immune functions. In F1 males and females, the direct exposure to test item was started at weaning until the scheduled termination, i.e., until an age of 13 weeks (Cohort 1A, twenty animals per sex and group) or until study termination (weeks 20-25: Cohort 1B, twenty animals per sex and group). Furthermore, Cohort 2A animals were sacrificed at an age of 12 weeks (Cohort 2A, ten animals per sex and group). Cohort 2B animals served for developmental neurotoxicity and were sacrificed at weaning (ten animals per sex and group). Cohort 3 animals underwent evaluation of developmental immunotoxicity and were sacrificed at an age of 8-10 weeks (ten animals per sex and group). Cohort 4 contained ten animals per groups and sex for learning and memory testing that was sacrificed after completion of the test on post-natal day 38-39.
During the period of administration, the animals were observed each day for signs of toxicity. Animals that died were examined macroscopically and at the conclusion of the test, surviving animals were sacrificed and observed macroscopically.
Once before the first exposure, and once a week thereafter, detailed clinical observations were made in all P animals and all cohorts (exceptCohort 2B andCohort 4).
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.
10 male and 10 female Cohort 2Aand 2Banimals from each treatment group were used for neurotoxicity assessments. Cohort 2Afrom each treatment group was subjected to auditory startle, functional observational battery, motor activity, and neuropathology assessments. An auditory startle test was performed on PND 24 (±1 day) using animals in Cohort 2A. The Cohort 2A animals (between PND 63 and PND 75), were subjected to multiple detailed behavioural observations using functional observational battery of tests and test of motor activity.Cohort 4 included10 males and 10 females from control and HD group for learning and memory testing between PND 21-42 by using Y water maze.
After 2 weeks premating period, parental male and female from the same dose group were mated (1:1 pairing). F1 males and females from Cohort 1B were bred (1:1 pairing) after minimum treatment up to PND 90 to obtain a F2 generation.
Each F1 and F2 litter was examined as soon as possible after the delivery of the dam (PND 0) 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 (day 0 post-partum) and on days 4, 7, 14 and 21 post-partum.
The anogenital distance (AGD) of each F1 and F2 (Cohort 1B) pup was measured on PND 0 and all male pups were checked for the presence of nipples/areolae on PND 12.
All selected F1 male and female pups from all cohorts (except Cohort 2B and Cohort 4) were checked daily for balano-preputial separation or vaginal patency, respectively starting from PND 30 in males and PND 25 in females.
Vaginal smears of parental females were examined 2 weeks before beginning of treatment period, during 2 weeks premating period and until the confirmation of mating.Vaginal smears were examined daily for all F1 females in Cohort 1A after the onset of vaginal patency until the first cornified smear was recorded.Vaginal smear in Cohort 1Awas also examined for 2 weeks starting from PND 75. The vaginal smear in Cohort 1Bwas examined during mating period to confirm the evidence of mating.
Haematological, coagulation, thyroid hormone analysis (T4 and TSH) andclinical biochemistry parameters were determined with blood samples obtained from10 randomly selected parental males and females and 10 randomly selected F1 male and female animals of Cohort 1Aat their terminal sacrifice.A urinalysis was also performed on samples collected from these animals prior to or as part of their terminal sacrifice. Thyroid hormone analysis (T4 and TSH) was also performed on 10 pups/sex/group at PND 4 and after weaning (pups not allocated to cohorts)on PND 22.
To evaluate possible toxic effects on male fertility, sperm motility and testicular sperm head count was performed at the end of the treatment period from all parental generation males and all Cohort 1Amales of each group by using Hamilton Thorn sperm analyser. Sperm morphology was evaluated at the end of the treatment period from all parental generation males and all Cohort 1Amales from control and high dose groups.
Cohort 3 animals of 10 male and 10 female (on PND 56±3 days), from each treatment group were used for a T-cell dependent antibody response assay to measure KLH-specific IgM and IgG antibodies. Pre- and post-natally induced immunotoxic effects at termination from 10 male and 10 female Cohort 1A animals from each treatment group was evaluated by analysis of splenic lymphocyte subpopulation (CD4+ and CD8+ T lymphocytes, B lymphocytes, and natural killer cells) using one half of the spleen.
At the conclusion of the treatment period of parental animals and various cohorts, all animals were sacrificed and subjected to necropsy. The wet weight of a subset of tissues was taken and a set of organs/tissues was preserved. Animals that died or were sacrificed in a moribund condition were examined macroscopically and histopathologically.
A full histopathological evaluation of the collected tissues was performed on high dose and control parental and Cohort 1A animals.The histopathological examination of Cohort 1A female ovaries included quantitative evaluation of primordial and small growing follicles and corpora lutea. From Cohort 1A males, forthe 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.These examinations were not extended to animals of the other dosage groups as no treatment-related changes were observed in the HD group.
Any gross lesion macroscopically identified was examined microscopically in all animals including found dead or moribund sacrificed animals. Neurohistopathology was performed for all control and high dose Cohort 2A animals sacrificed after PND 90. Brain histopathology was performed for all control and high dose Cohort 2B animals sacrificed on PND 21.
Referenceopen allclose all
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%.
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.
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.
Oestrous Cyclicity
Cohort 1A Females
In Cohort 1A females, no biologically significant effect was observed on the duration of vaginal opening until first oestrus cycle in treatment groups when compared to control. Statistically significant changes were noticed in MD group animals when compared to control and this could be due to persistent dioestrus of female animal no. 343 and 349.
In this cohort, no biologically significant effect was observed on the oestrus cycle length or sequence of cycle stages between the treatment groups and the control group analysed from PND 75 for 2 weeks.
Litter Data
Cohort 1B Females
Litter parameters of 1B females, like group mean total number of pups born, number of male pups, number of female pups, sex ratio, number of live pups, stillbirth, runt on PND 0 as well as number of live pups, male pups, number of female pups and sex ratio on PND 4 7, 14 and PND 21 remained unaffected when compared with the controls. Statistical analysis of litter data revealed no significant effects in treatment groups when compared with the controls.
Litter Weight Data
Parental Generation and Cohort 1B Females
There was no test item related effect on pup mean weight, total litter weight, male and female litter weight on PND 0, PND 4, 7, 14 and PND 21 observed in parental and Cohort 1B treatment groups when compared with the controls. There was no statistically significant change in dose groups compared to control, except for slight but statistically significantly lower pup mean weight from females of Cohort 1B on PND 0 and mean litter weight of male on day 7 in HD group in parental females when compared to control.
Precoital Interval and Duration of Gestation
Parental Generation and Cohort 1B Females
There was no test item related effect observed on the duration of pre-coital interval and the duration of gestation in the parental and Cohort 1B female dose groups when compared to control. A single, but slightly lower (21.94 days) duration of gestation was observed in LD- cohort 1B group females when compared to control (22.33 days). Due to the single incidence of the effect and the absence of any dose-response relationship this effect is not considered to be toxicologically relevant.
Pre- and Post-Natal Data
Parental Generation and Cohort 1B Females
There was no test item treatment related effects observed on the number of corpora lutea, implantation sites, live pups on PND 0, percent preimplantation loss and post implantation loss in parental and Cohort 1B treatment group females when compared with the corresponding control group, except slight but statistically significant lower mean corpora lutea in LD and MD groups and statistically significantly higher percent post implantation loss in LD group of parental females when compared to control. This is not considered toxicologically relevant due to missing does-response-relationship and because all data are also in line with historical control data of reproductive and developmental toxicity studies in this strain.
Reproductive Indices
Parental Generation and Cohort 1B Females
There was no test item related effects observed on the reproductive indices (percent male mating index, female mating index, male fertility index, female fertility index, gestation index and live birth index) in parental and Cohort 1B dose group animals when compared to the respective control group. The survival index of pups during PND 0 to 4, PND 4 (after interim sacrifice) to PND 13 and PND 14 (after interim sacrifice) to PND 21 in parental females and during PND 0 to 4, PND 4 (after interim sacrifice) to PND 14 and PND 14 to PND 21 in Cohort 1B females remained unaffected and within the range of biological variation in treatment groups when compared with the respective control group.
Pup Survival Data
Parental Generation and Cohort 1B Females
No test item related effect on mean mortality of pups from PND 0 to 4, PND 7 (after interim sacrifice) to PND 14 and PND 14 (after interim sacrifice) to PND 21 in parental females and from PND 0 to 4, PND 4 to 21 were observed in Cohort 1B female treatment groups when compared to the control group.
Mean mortality of pups of the dose groups was comparable with the respective control and slight differences are considered as incidental and not related to the treatment with the test item and they are in line with historical control data of reproductive and developmental toxicity studies in this strain.
Anogenital Distance and Nipple Retention
Parental Generation and Cohort 1B Females
In male pups from parental females on PND 0, marginal but statistically significantly lower pup weight, cube root of pup weight and relative anogenital distance (AGD) in HD groups were observed when compared to the control. No effect was seen on absolute AGD. In MD group, marginal but statistically significantly higher absolute and relative AGD was observed. In female pups from parental females on PND 0, there was no statistical significant difference when compared with the control group. As the differences were only very slight and values were also within the range of historical control data, this is not assumed to be toxicologically relevant.
In male pups from Cohort 1B females on PND 0, marginal but statistically significantly lower pup weight, cube root of pup weight and absolute AGD were observed in the LD and HD group when compared to the control group. Statistically significant lower absolute AGD in MD group and lower relative AGD in HD groups were observed. In female pups from Cohort 1B on PND 0, a marginal but statistically significant effect was observed on pup weight and cube root of pup weight parameter. As values were within the normal range of historical control data, this is not considered toxicologically relevant.
No effect of toxicological relevance was observed on nipple retention in the pups of any of the groups from parental and Cohort 1B females when compared to control. Group mean number of nipple retention in MD group males from parental females was slight but statistically significantly higher than in control. In males from Cohort 1B, nipple retention was statistically significantly lower in MD and HD groups when compared to control. The slightly higher incidence from parental females was attributed with a higher incidence of nipple retention from few dam of MD groups and considered to be incidental and not related to the treatment with test item.
Pup External Findings
Parental Generation and Cohort 1B Females
No test item related gross external abnormalities of toxicological relevance on PND 0-20 were observed in the pups of any of the groups from parental and Cohort 1B females.
Few specific findings like right eye scratched, murky/dull/encrusted eye, pale skin, haematoma, swollen hind limb, haematoma on head/snout, dark abdomen were observed in few pups from parental and Cohort 1B females and considered to be spontaneous in nature and not related to test item treatment.
Thyroid Hormone (T4 and TSH) Analysis
Parental Generation
In parental males and females (10/sex/group), group mean T4 and TSH levels in dose groups were comparable with the control, except statistically significantly higher group mean TSH values in LD group parental females. As this was not found at higher doses and not associated with the microscopic finding, this is not assumed to be toxicologically relevant, but more likely an incidental finding and also they are in line with historical control data of reproductive and developmental toxicity studies in this strain.
F1 pups on PND 4 and PND 21
In pups sacrificed on PND 4 (10/sex/group - pooled samples), T4 and TSH levels in treatment groups were comparable with the controls. In pups sacrificed on PND 21 (10/sex/group), T4 levels in treatment groups were comparable with the controls.
Cohort 1A
In males and females of Cohort 1A (10/sex/group), group mean T4 and TSH levels in treatment groups were comparable with the controls. Moreover, no test item related effect of toxicological relevance was observed on thyroid weight and thyroid histopathology.
Sexual Maturity
All selected F1 male and female pups from all cohorts (except Cohort 2B and Cohort 4) were checked daily for balano-preputial separation or vaginal patency, respectively starting from PND 30 in males and PND 25 in females.
No abnormalities of genital organs like persistent vaginal thread, hypospadia or cleft penis were observed in any of the pup.
Cohort 1A
No significant difference in group mean body weight and day of onset of vaginal opening in females and balano-preputial separation in males was observed in treatment groups when compared to control.
Cohort 1B
No significant difference in group mean body weight and day of onset of the vaginal opening in females and balano-preputial separation in males was observed in treatment groups when compared to control; however marginal but statistically significant higher mean balano-preputial separation in males were observed in LD (PND 32.90) and HD (PND 33.80) groups when compared to control (PND 31.15). It is not considered to be adverse and these values are within the normal biological variation in this strain.
Cohort 2A
No statistically significant difference in group mean body weight and day of onset of the vaginal opening in females and balano-preputial separation in males was observed in treatment groups when compared to control.
Cohort 3
No statistically significant differences in group mean body weight and day of onset of the vaginal opening in females and balano-preputial separation in males were observed in treatment groups when compared to control, however marginal but statistically significant lower mean body weight in all treated groups and balano-preputial separation in males were observed in LD (PND 32.00) and MD (PND 32.60) groups when compared to control (PND 35.20). It is not considered to be adverse and these values are within the normal biological variation in this strain.
Learning and Memory
Cohort 4
Analysis of learning and memory data from Cohort 4 males and females revealed that there were no statistically significant differences in escape latency time observed during the learning phase (PND 28/29) between HD group and control group in both genders by considering 6 rounds of mean escape latency. Similarly, HD group animals subjected to the test during the memory phase on PND35/36 showed no statistically significant difference in escape latency time when compared to the control.
Auditory Startle-Response
Cohort 2A
Analysis of data from auditory startle test (Kinder Scientific Startle Reflex Measuring System) performed on PND 24 using male and female animals in Cohort 2A revealed no toxicologically significant changes observed in mean startle response in animals treated with test item groups as compared to the control group. The mean response amplitude on each block of 10 trials (5 blocks of 10 trials) was determined, with test conditions optimized to produce intra-session habituation.
Motor Activity
Cohort 2A
Based on data from males and females from Cohort 2A, no relevant effects were observed on motor activity (slow and fast animal movements, number of slow and fast stereotypies and number of slow and fast rearings) in treatment groups during evaluation between PND 63 and 75 when compared with the controls.
Analysis of Splenic Lymphocyte Subpopulation
Cohort 1A
Cohort 1A
Analysis of splenic lymphocyte subpopulation (CD4+ and CD8+ T lymphocytes, B lymphocytes, and natural killer cells) from 10 male and 10 female animals per group from Cohort 1A revealed that there was a slight increase in mean lymphocytes and T cell populations in both male and females when compared to controls. There was no test item related change in the number of splenic B cells which was found to be comparable between all dose groups and control group in both male and female. Thus, there was no indication of immunosuppressive effect of the test item on lymphocytes sub-populations.
Cohort 1A Females
In Cohort 1A females, no biologically significant effect was observed on the duration of vaginal opening until first oestrus cycle in treatment groups when compared to control. Statistically significant changes were noticed in MD group animals when compared to control and this could be due to persistent dioestrus of female animal no. 343 and 349.
In this cohort, no biologically significant effect was observed on the oestrus cycle length or sequence of cycle stages between the treatment groups and the control group analysed from PND 75 for 2 weeks.
Litter Data
Cohort 1B Females
Litter parameters of 1B females, like group mean total number of pups born, number of male pups, number of female pups, sex ratio, number of live pups, stillbirth, runt on PND 0 as well as number of live pups, male pups, number of female pups and sex ratio on PND 4 7, 14 and PND 21 remained unaffected when compared with the controls. Statistical analysis of litter data revealed no significant effects in treatment groups when compared with the controls.
Litter Weight Data
Parental Generation and Cohort 1B Females
There was no test item related effect on pup mean weight, total litter weight, male and female litter weight on PND 0, PND 4, 7, 14 and PND 21 observed in parental and Cohort 1B treatment groups when compared with the controls. There was no statistically significant change in dose groups compared to control, except for slight but statistically significantly lower pup mean weight from females of Cohort 1B on PND 0 and mean litter weight of male on day 7 in HD group in parental females when compared to control.
Precoital Interval and Duration of Gestation
Parental Generation and Cohort 1B Females
There was no test item related effect observed on the duration of pre-coital interval and the duration of gestation in the parental and Cohort 1B female dose groups when compared to control. A single, but slightly lower (21.94 days) duration of gestation was observed in LD- cohort 1B group females when compared to control (22.33 days). Due to the single incidence of the effect and the absence of any dose-response relationship this effect is not considered to be toxicologically relevant.
Pre- and Post-Natal Data
Parental Generation and Cohort 1B Females
There was no test item treatment related effects observed on the number of corpora lutea, implantation sites, live pups on PND 0, percent preimplantation loss and post implantation loss in parental and Cohort 1B treatment group females when compared with the corresponding control group, except slight but statistically significant lower mean corpora lutea in LD and MD groups and statistically significantly higher percent post implantation loss in LD group of parental females when compared to control. This is not considered toxicologically relevant due to missing does-response-relationship and because all data are also in line with historical control data of reproductive and developmental toxicity studies in this strain.
Reproductive Indices
Parental Generation and Cohort 1B Females
There was no test item related effects observed on the reproductive indices (percent male mating index, female mating index, male fertility index, female fertility index, gestation index and live birth index) in parental and Cohort 1B dose group animals when compared to the respective control group. The survival index of pups during PND 0 to 4, PND 4 (after interim sacrifice) to PND 13 and PND 14 (after interim sacrifice) to PND 21 in parental females and during PND 0 to 4, PND 4 (after interim sacrifice) to PND 14 and PND 14 to PND 21 in Cohort 1B females remained unaffected and within the range of biological variation in treatment groups when compared with the respective control group.
Pup Survival Data
Parental Generation and Cohort 1B Females
No test item related effect on mean mortality of pups from PND 0 to 4, PND 7 (after interim sacrifice) to PND 14 and PND 14 (after interim sacrifice) to PND 21 in parental females and from PND 0 to 4, PND 4 to 21 were observed in Cohort 1B female treatment groups when compared to the control group.
Mean mortality of pups of the dose groups was comparable with the respective control and slight differences are considered as incidental and not related to the treatment with the test item and they are in line with historical control data of reproductive and developmental toxicity studies in this strain.
Anogenital Distance and Nipple Retention
Parental Generation and Cohort 1B Females
In male pups from parental females on PND 0, marginal but statistically significantly lower pup weight, cube root of pup weight and relative anogenital distance (AGD) in HD groups were observed when compared to the control. No effect was seen on absolute AGD. In MD group, marginal but statistically significantly higher absolute and relative AGD was observed. In female pups from parental females on PND 0, there was no statistical significant difference when compared with the control group. As the differences were only very slight and values were also within the range of historical control data, this is not assumed to be toxicologically relevant.
In male pups from Cohort 1B females on PND 0, marginal but statistically significantly lower pup weight, cube root of pup weight and absolute AGD were observed in the LD and HD group when compared to the control group. Statistically significant lower absolute AGD in MD group and lower relative AGD in HD groups were observed. In female pups from Cohort 1B on PND 0, a marginal but statistically significant effect was observed on pup weight and cube root of pup weight parameter. As values were within the normal range of historical control data, this is not considered toxicologically relevant.
No effect of toxicological relevance was observed on nipple retention in the pups of any of the groups from parental and Cohort 1B females when compared to control. Group mean number of nipple retention in MD group males from parental females was slight but statistically significantly higher than in control. In males from Cohort 1B, nipple retention was statistically significantly lower in MD and HD groups when compared to control. The slightly higher incidence from parental females was attributed with a higher incidence of nipple retention from few dam of MD groups and considered to be incidental and not related to the treatment with test item.
Pup External Findings
Parental Generation and Cohort 1B Females
No test item related gross external abnormalities of toxicological relevance on PND 0-20 were observed in the pups of any of the groups from parental and Cohort 1B females.
Few specific findings like right eye scratched, murky/dull/encrusted eye, pale skin, haematoma, swollen hind limb, haematoma on head/snout, dark abdomen were observed in few pups from parental and Cohort 1B females and considered to be spontaneous in nature and not related to test item treatment.
Thyroid Hormone (T4 and TSH) Analysis
Parental Generation
In parental males and females (10/sex/group), group mean T4 and TSH levels in dose groups were comparable with the control, except statistically significantly higher group mean TSH values in LD group parental females. As this was not found at higher doses and not associated with the microscopic finding, this is not assumed to be toxicologically relevant, but more likely an incidental finding and also they are in line with historical control data of reproductive and developmental toxicity studies in this strain.
F1 pups on PND 4 and PND 21
In pups sacrificed on PND 4 (10/sex/group - pooled samples), T4 and TSH levels in treatment groups were comparable with the controls. In pups sacrificed on PND 21 (10/sex/group), T4 levels in treatment groups were comparable with the controls.
Cohort 1A
In males and females of Cohort 1A (10/sex/group), group mean T4 and TSH levels in treatment groups were comparable with the controls. Moreover, no test item related effect of toxicological relevance was observed on thyroid weight and thyroid histopathology.
Sexual Maturity
All selected F1 male and female pups from all cohorts (except Cohort 2B and Cohort 4) were checked daily for balano-preputial separation or vaginal patency, respectively starting from PND 30 in males and PND 25 in females.
No abnormalities of genital organs like persistent vaginal thread, hypospadia or cleft penis were observed in any of the pup.
Cohort 1A
No significant difference in group mean body weight and day of onset of vaginal opening in females and balano-preputial separation in males was observed in treatment groups when compared to control.
Cohort 1B
No significant difference in group mean body weight and day of onset of the vaginal opening in females and balano-preputial separation in males was observed in treatment groups when compared to control; however marginal but statistically significant higher mean balano-preputial separation in males were observed in LD (PND 32.90) and HD (PND 33.80) groups when compared to control (PND 31.15). It is not considered to be adverse and these values are within the normal biological variation in this strain.
Cohort 2A
No statistically significant difference in group mean body weight and day of onset of the vaginal opening in females and balano-preputial separation in males was observed in treatment groups when compared to control.
Cohort 3
No statistically significant differences in group mean body weight and day of onset of the vaginal opening in females and balano-preputial separation in males were observed in treatment groups when compared to control, however marginal but statistically significant lower mean body weight in all treated groups and balano-preputial separation in males were observed in LD (PND 32.00) and MD (PND 32.60) groups when compared to control (PND 35.20). It is not considered to be adverse and these values are within the normal biological variation in this strain.
Learning and Memory
Cohort 4
Analysis of learning and memory data from Cohort 4 males and females revealed that there were no statistically significant differences in escape latency time observed during the learning phase (PND 28/29) between HD group and control group in both genders by considering 6 rounds of mean escape latency. Similarly, HD group animals subjected to the test during the memory phase on PND35/36 showed no statistically significant difference in escape latency time when compared to the control.
Auditory Startle-Response
Cohort 2A
Analysis of data from auditory startle test (Kinder Scientific Startle Reflex Measuring System) performed on PND 24 using male and female animals in Cohort 2A revealed no toxicologically significant changes observed in mean startle response in animals treated with test item groups as compared to the control group. The mean response amplitude on each block of 10 trials (5 blocks of 10 trials) was determined, with test conditions optimized to produce intra-session habituation.
Motor Activity
Cohort 2A
Based on data from males and females from Cohort 2A, no relevant effects were observed on motor activity (slow and fast animal movements, number of slow and fast stereotypies and number of slow and fast rearings) in treatment groups during evaluation between PND 63 and 75 when compared with the controls.
Analysis of Splenic Lymphocyte Subpopulation
Cohort 1A
Cohort 1A
Analysis of splenic lymphocyte subpopulation (CD4+ and CD8+ T lymphocytes, B lymphocytes, and natural killer cells) from 10 male and 10 female animals per group from Cohort 1A revealed that there was a slight increase in mean lymphocytes and T cell populations in both male and females when compared to controls. There was no test item related change in the number of splenic B cells which was found to be comparable between all dose groups and control group in both male and female. Thus, there was no indication of immunosuppressive effect of the test item on lymphocytes sub-populations.
Effect on fertility: via oral route
- Endpoint conclusion:
- no adverse effect observed
- Dose descriptor:
- NOAEL
- 1 000 mg/kg bw/day
- Study duration:
- subchronic
- 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
- 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
- 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. QA statement)
- Limit test:
- no
- Species:
- rat
- Strain:
- Wistar
- Details on test animals or test system 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
- 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 or test system 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
- 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 or test system 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 - 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.
Referenceopen allclose all
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.
Additional information
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Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
