Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 2017-12-21 to 2018-08-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2016
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, 2000.
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 10 w (m) 13 w (f)
- Weight at study initiation: 266-301 g (m), 190-228 g (f)
- Fasting period before study: no
- Housing: grouped
- Diet (e.g. ad libitum): ad libitim
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 22 °C
- Humidity (%): 42 - 53 %
- Air changes (per hr): at least ten
- Photoperiod (hrs dark / hrs light): 12/12 h

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The oral route of administration was selected because this is a possible route of human exposure during manufacture, handling or use of the test item.
The dose levels were selected based on the results of a 14-day dose range finder with oral gavage administration of the test material in rats, and in an attempt to produce graded responses to the test item. During the dose range finder dose levels of 0 (vehicle only), 600 and 1000 mg/kg were tested. Calmness was
observed 3 hours after dosing in animals treated at 600 mg/kg and to a lesser extent in animals treated at 1000 mg/kg. At 600 mg/kg, no overt signs of toxicity were noted. At 1000 mg/kg the animals showed lower body weights and body weight gain (about 5% from Day 5 onwards), whereas normal food consumption was noted. No toxicologically relevant findings were noted at macroscopic examination or in organ weights. Based on the results of the dose range finder, 0, 100, 300 and 1000 mg/kg were selected as dose levels.
Vehicle:
methylcellulose
Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS:
The vehicle, 1% Methylcellulose, was prepared approximately once a month, stored in a refrigerator set to maintain 4°C, and dispensed daily. The prepared vehicle was removed from the refrigerator and stirred for at least 30 minutes before dosing.


- VEHICLE
- Justification for use and choice of vehicle (if other than water): Vehicle testing was performed at the Test Facility to select the suitable vehicle and to establish a suitable formulation procedure. Vehicle testing formulations were used for dosing and were discarded completion of the assessment. Vehicle testing has a non-GLP status and was carried out in the quality assured environment of the Test Facility.
- Concentration in vehicle: 1% methylcellulose in water
- Amount of vehicle (if gavage): 5 mL
- Lot/batch no. (if required): -
- Purity: -
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
HPLC-UV Method
The analytical method was validated for the following parameters:
• Specificity
• Calibration curve
• Accuracy and repeatability
• Limit of quantification (LOQ)
• Stability of the analytical system and end solutions
• Stability of stock solutions
Formulation Analysis
Trial formulations were prepared in 1% methyl cellulose at 1 mg/g (1 mg/mL) and 200 mg/g (200 mg/mL). Formulations were suspensions.
The concentrations analyzed in the trial formulations were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%).
The formulations were homogeneous (i.e. coefficient of variation ≤ 10%).
Formulations were stable when stored at room temperature protected from light for at least 24 hours, in a refrigerator (2-8°C) for at least 8 days, and in a freezer (≤ -15°C) for at least 21 days (3 weeks).
Duration of treatment / exposure:
Males were treated for 33 days, i.e. 14 days prior to mating, during mating and up to and including the day before scheduled necropsy.
Females that delivered were treated for 50-56 days (most females) or 65 days (one female of Group 2 and one female of Group 4), i.e. 14 days prior to mating (with the objective to coverat least two complete estrous cycles), the variable time to conception, the duration of pregnancy and 13-15 days after delivery, up to and including the day before scheduled necropsy. Females which failed to deliver were treated for 40-42 days.
Frequency of treatment:
once daily 7 days a week
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
40 f, 40 m
Control animals:
yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:
In-life Procedures, Observations, and Measurements – F0-Generation
------------------------------------------------------------------------------------

Mortality/Moribundity Checks – F0-Generation
Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day (for exception see Appendix 8).
Animals were not removed from their cage during observation, unless necessary for identification or confirmation of possible findings. Animals showing pain, distress or discomfort which was considered not transient in nature or was likely to become more severe, were sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/ 2000/7). The circumstances of any death were recorded in detail.

Clinical Observations – F0-Generation
Clinical observations were performed once daily, beginning during the first administration of the test item and lasting throughout the dosing periods up to the day prior to necropsy.
These clinical observations were conducted at least 3 hours (± 30 minutes) after dosing on the peak period of anticipated effects after treatment (based on dose range finder).
The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight
(grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored. In the data tables, the scored
grades were reported, as well as the percentage of animals affected in summary tables.

Arena Observations – F0-Generation
Clinical observations were conducted in a standard arena beginning before the first administration of the test item and then once weekly throughout treatment. These observations were conducted after clinical observations.

Body Weights – F0-Generation
Animals were weighed individually on the first day of treatment (prior to dosing), and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and
during lactation on PND 1, 4, 7, and 13. In addition, body weights of F0-males were determined on Day 33 (the day prior to necropsy). Terminal body weights were recorded on the day of necropsy (fasted for males, non-fasted for females).

Food Consumption – F0-Generation
Food consumption was quantitatively measured weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13. In addition, food consumption of F0-males was measured in the period from Day 30 to 33 (the 3-day period prior to necropsy).

Water Consumption – F0-Generation
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was expected or noted at visual inspection of the water bottles.

Functional Tests – F0-Generation
Functional tests were performed on two occasions. On the first occasion all animals (spares included) were tested prior to treatment. The results of the animals which did not continue in the study were kept in the raw data but were not reported. On the second occasion, selected animals were tested during the treatment period. The selected 5 males were tested during Week 4 of treatment and the selected 5 females during the last week of lactation (i.e. PND 6-13). These tests were performed prior to dosing.
The following tests were performed (abbreviations mentioned in the respective tables are indicated between brackets):
 Hearing ability (HEARING) (Score 0 = normal/present, score 1 = abnormal/absent).
 Pupillary reflex (PUPIL L/R) (Score 0 = normal/present, score 1 = abnormal/absent).
 Static righting reflex (STATIC R) (Score 0 = normal/present, score 1 = abnormal/absent).
 Fore- and hind-limb grip strength, recorded as the mean of three measurements per animal (Series M4-10, Mark-10 Corporation, J.J. Bos, Gouda, The Netherlands).
 Locomotor activity (recording period: 1-hour under normal laboratory light conditions, using a computerized monitoring system, Kinder Scientific LLC, Poway, USA). Total movements and ambulations were reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or more fine movements like grooming, weaving or movements of the head.

Estrous cycle determination – F0-Generation
Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage.
Daily vaginal lavage was performed for all females beginning 14 days prior to treatment (pretest period), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period. On the day of necropsy, a vaginal lavage was also taken to determine the stage of estrus. This
was done for all females, except for female no.43, which was sacrificed in extremis and female nos. 71 and 78 which had a total litter loss.

Cohabitation/Mating Procedure – F0-Generation
After 14 days of treatment, animals were cohabitated on a 1:1 basis within the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated. A maximum of 14 days was allowed for mating, after which females which had not shown evidence of mating (nos. 56, 71, 78 and 79) were separated from their males. Since less than 9 females of Group 4 showed evidence of mating after 14 days of cohabitation, all females without evidence of mating were re-mated once with a male of proven fertility of the same group for one day.
Detection of mating was not confirmed in first instance for female nos. 48 and 74. Evidence of mating was obtained by palpation and indirectly by delivery of a litter. Apparently, mating was overlooked in the assessment of the vaginal lavage, which explains the continuation of di-estrus during the mating in these females. The mating date of these animals was estimated at 21 days prior to the actual delivery date. This day was designated Day 0 post-coitum.

General Reproduction Data – F0-Generation
From the mating period onwards, the following parameters were recorded for each female: male number paired with, mating date, confirmation of pregnancy and delivery day.
Females were allowed to litter normally. Postnatal day (PND) 1 is defined as the day when a litter is found completed (i.e. membranes and placentas cleaned up, nest built and/or feeding of pups started). The day prior to PND 1 is considered to be the day when the female started to deliver and is defined as PND 0 and used for recording of delivery. Females that were littering were left undisturbed.
Cage debris of pregnant females was examined for evidence of premature delivery and pregnant females were examined to detect signs of difficult or prolonged parturition or deficiencies in maternal care.


In-life Procedures, Observations, and Measurements – F1-Generation
------------------------------------------------------------------------------------

Mortality/Moribundity Checks – F1-Generation
Pups were observed daily for general health/mortality. The number of live and dead pups were determined on PND 1 and daily thereafter. Pups were not removed from the cage
during observation, unless necessary for identification or confirmation of possible findings.
Pups showing pain, distress or discomfort which was considered not transient in nature or was likely to become more severe, were sacrificed for humane reasons based on OECD guidance
document on humane endpoints (ENV/JM/MONO/ 2000/7). The circumstances of any death were recorded in detail.

Clinical Observations – F1-Generation
Clinical observations were performed at least once daily for all pups. Only days on which clinical signs were present between the first and last litter check were given.

Body Weights – F1-Generation
Live pups were weighed individually on PND 1, 4, 7 and 13.

Sex – F1-Generation
Sex was externally determined for all pups on PND 1 and 4.

Anogenital Distance – F1-Generation
Anogenital distance (AGD) was measured for all live pups on PND 1. The AGD was normalized to the cube root of body weight.

Areola/Nipple Retention – F1-Generation
All male pups in each litter were examined for the number of areola/nipples on PND 13.

Culling – F1-Generation
To reduce variability among the litters, on PND 4 eight pups from each litter of equal sex distribution (if possible) were selected. Blood samples were collected from two of the surplus pups (if possible from one male and one female pup). Selective elimination of pups, e.g. based upon body weight or AGD, was not done. Whenever the number of male or female pups prevented having four of each sex per litter, partial adjustment (for example, five males and three females) was acceptable.


Laboratory Evaluations
----------------------------
Clinical Pathology
--------------------
At the end of the treatment period, urine was collected from the selected F0-males into a specimen vial. During overnight urine collection, the animals were housed in individual metabolism cages (approximately 15-20 hours, starting on the day prior to scheduled necropsy) without food (water was available). After collection, samples were transferred to the appropriate laboratory for processing.
Blood of F0-animals (except for the control female which was sacrificed in extremis and females with total litter loss) was collected on the day of scheduled necropsy. Samples were collected, between 7.00 and 10.30 a.m., from the retro-orbital sinus under anesthesia using isoflurane in the animal facility. Due to clotting of non-serum samples of individual animals, additional blood samples were obtained in the necropsy room. After collection all samples were transferred to the appropriate laboratory for analysis.
F0-males were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. F0- females were not fasted overnight. Blood of F1-animals was collected on PND 4 and PND 14-16, if possible. This was performed in the necropsy room.
On PND 4 at culling, blood was collected from two surplus pups per litter (if possible) by decapitation, between 7.00 and 10.30 a.m., and samples were pooled to one sample per litter.

If available, blood was collected from one male and one female pup. If only one surplus pup per litter was available at culling, as much blood as possible was collected from this single pup.
On PND 14-16, separate blood samples were collected from two pups per litter (from one male and one female, if possible). Any incomplete blood sample was discarded. Blood was drawn, between 7.00 and 10.30 a.m., by aorta puncture under anaesthesia using isoflurane as part of the necropsy procedure.


Hematology
--------------
Blood samples at a target volume of 0.5 mL were collected into tubes containing K3-EDTA as anticoagulant. Blood samples were analyzed for the parameters specified below:
White blood cells (WBC)
Red Blood Cell Distribution Width (RDW)
Neutrophil (absolute)
Haemoglobin
Lymphocyte (absolute)
Haematocrit
Monocyte (absolute)
Mean corpuscular volume (MCV)
Eosinophil (absolute)
Mean corpuscular haemoglobin (MCH)
Basophil (absolute)
Mean corpuscular haemoglobin concentration (MCHC)
Red blood cells
Platelets
Reticulocytes (absolute)

A blood smear was prepared from each hematology sample. Blood smears were labeled, stained, and stored. In case additional examination of blood smears was deemed necessary,
the smears were subsequently evaluated.

Coagulation
--------------
Blood samples at a target volume of 0.45 mL were collected into tubes containing Citrate as anticoagulant. Blood samples were processed for plasma, and plasma was analyzed for the
parameters below:
- Prothrombin Time (PT)
- Activated Partial Thromboplastin Time (APTT)


Clinical Chemistry
-------------------
Blood samples at a target volume of 0.5 mL (plasma) were collected into tubes containing Li-Heparin as anticoagulant. Serum samples at a target volume of 0.25 mL were collected in tubes without anticoagulant. Blood samples were processed for plasma or serum (bile acids), which was analyzed for the parameters specified below:

Alanine aminotransferase (ALAT)
Creatinine
Aspartate aminotransferase (ASAT)
Glucose
Alkaline Phosphatase (ALP)
Cholesterol
Total protein
Sodium
Albumin
Potassium
Total Bilirubin
Chloride
Bile Acids
Calcium
Urea Inorganic
Phosphate (Inorg. Phos)

Urinalysis
-------------
The following parameters were recorded for analyis:
Volume
Bilirubin
Specific gravity
Urobilinogen
Clarity
Protein
Colour
Ketones
pH
Glucose
Blood
Nitrite
White blood cells (WBC)

Sediment:
White blood cells (WBC-sed.)
Red blood cells (RBC-sed.)
Casts
Epithelial cells
Crystals
Bacteria
Other

Thyroid hormone
---------------------
Blood samples were processed for serum, and serum was analyzed for the parameters listed below:
Thyroxine (T4)
Thyroid Stimulating Hormone (TSH)

Measurement of total T4 was conducted for F0-males and PND 14-16 pups. On account of the treatment-related reduction in total T4 in F0-males assessment of thyroid hormone in parental animals was extended to T4 in females and TSH in both sexes. Assessment of T4 for PND 4 pups and TSH for PND 14-16 pups was considered not relevant because no treatment-related changes in T4 were noted in pups at PND 14-16.
Serum samples retained for possible future analysis were maintained by the Test Facility in the freezer (≤-75°C). Under these storage conditions, samples are stable for 6 months. Any remaining samples were discarded.
Sacrifice and pathology:
Scheduled Euthanasia
Animals surviving until scheduled euthanasia were weighed, and deeply anaesthetized using isoflurane and subsequently exsanguinated and subjected to a full post mortem examination.
Scheduled necropsies were conducted on the following days:
Males (which sired and failed to sire): Following completion of the mating period (a minimum of 28 days of administration).
Females which delivered: PND 14-16.
Females which failed to deliver with evidence of mating: Post-coitum Day 25-26.
All males were fasted overnight with a maximum of 24 hours before necropsy. Water was available. F0-females were not fasted.

Necropsy – F0-Generation
All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs. Necropsy procedures were performed by qualified personnel with appropriate training and
experience in animal anatomy and gross pathology. A veterinary pathologist, or other suitably qualified person, was available. The numbers of former implantation sites and corpora lutea were recorded for all females.
In case no macroscopically visible implantation sites were present, nongravid uteri were stained using the Salewski technique in order to detect any former implantation sites and the number of corpora lutea was recorded in addition.

Organ Weights – F0-Generation
The organs identified as shown below were weighed at necropsy for all scheduled euthanasia animals. In addition, for two female the thyroid weight was determined at necropsy. Organ weights were not recorded for the control female that was euthanized in extremis. Paired organs were weighed together. Organ to body weight ratios (using the terminal body weight) were calculated.

Brain
Cervix
Epididymis
Gland, adrenal
Gland, coagulation
Gland, parathyroid
Gland, prostate
Gland, seminal vesicle
Gland, thyroid
Heart
Kidney
Liver
Ovaries
Spleen
Testes
Thymus
Uterus

Tissue Collection and Preservation – F0-Generation
Representative samples of the tissues identified in the table below were collected from all animals and preserved in 10% neutral buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands), unless otherwise indicated.

Animal identification
Artery, aorta
Body cavity, nasopharynx
Bone marrow
Bone, femur
Bone, sternum
Brain (seven levels)
Cervix
Epididymisa
Esophagus
Eyea
Gland, adrenal
Gland, coagulation
Gland, harderiana, b
Gland, lacrimal
Gland, mammary
Gland, parathyroidc
Gland, pituitary
Gland, prostate
Gland, salivary
Gland, seminal vesicle
Gland, thyroid
Gross lesions/masses
Gut-associated lymphoid tissue
Heart
Kidney
Large intestine, cecum
Large intestine, colon
Large intestine, rectum
Larynx
Liver
Lung
Lymph node (mandibular and mesenteric site)
Muscle, skeletal
Nerve, optica, b
Nerve, sciatic
Ovaries
Pancreas
Skin
Small intestine, duodenum
Small intestine, ileum
Small intestine, jejunum
Spinal cord
Spleen
Stomach
Testesa
Thymus
Tongue
Trachea
Urinary bladder
Uterus
Vagina


Histology – F0-Generation
The following tissues were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin:
Selected animals and unscheduled death: Tissues identified in Text
Males that failed to sire (except for males which were selected) and females that failed to deliver pups: Cervix, epididymis, coagulation gland, prostate gland, seminal vesicles, ovaries, testes, uterus and vagina.
Remaining animals: Gross lesions/masses.

Histopathology – F0-Generation
All tissues as defined under Histology – F0-Generation were examined by a board-certified toxicological pathologist with training and experience in laboratory animal pathology. Target tissues identified by the study pathologist during microscopic evaluation were communicated to the Study Director; tissues were evaluated and reported.
For the testes of all males of Groups 1 and 4, and all males that failed to sire, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle.
A peer review on the histopathology data was performed by a second pathologist.

Method of Euthanasia - F1-Generation
Pups, younger than 7 days were euthanized by decapitation. All remaining pups (PND 14-16), except for the two pups per litter selected for blood collection were euthanized by an intraperitoneal injection of sodium pentobarbital (Euthasol® 20%).
The pups selected for blood collection on PND 14-16 were anesthetized using isoflurane followed by exsanguination

Unscheduled Deaths - F1-Generation
Pups and recognizable fetuses of one female that was euthanized in extremis were examined externally, sexed (both externally and internally), and euthanized by decapitation.
Pups that died or were euthanized before scheduled termination were examined externally and sexed (both externally and internally), and their stomachs were examined for the presence of
milk. Some pups were found dead during the weekend and were fixed in identified containers containing 70% ethanol as they were not necropsied on the same day.


Scheduled Euthanasia - F1-Generation
On PND 4, the surplus pups (> 8 pups per litter) were euthanized by decapitation. From two surplus pups per litter, blood was collected, if possible. All remaining pups were euthanized on PND 14-16. Sex was determined both externally and internally. Descriptions of all external abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development. External
abnormalities were collected and fixed in 10% buffered formalin at discretion of the Study Director.
In addition, blood was collected from two pups per litter, and the thyroid from two pups per litter (if possible one male and one female pup) was preserved in 10% buffered formalin. The pups selected for blood sampling were the same pups as selected for thyroid preservation.

Histopathology thyroid – F1-Generation
For two PND 14-16 pups (one/sex, if possible) of each litter of all groups, routinely prepared hematoxylin-eosin stained paraffin sections of the thyroid were examined microscopically at request of the Sponsor.


Statistics:
Standard statistical methods have been applied for data processing.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Piloerection was noted in 8/10 females at 1000 mg/kg, starting after five weeks of treatment (i.e. towards the end of the gestation period for most animals) and generally lasting 2-9 days. In most females the piloerection was transient and not accompanied by other signs of toxicity. As such, this clinical finding was regarded as non-adverse. One 1000 mg/kg female (no. 79) showed piloerection on 11 consecutive days during lactation and a hunched posture on the last ten days of the lactation period. The piloerection noted in one female at 100 mg/kg on a single day was regarded as unrelated to treatment. No other clinical signs were noted in treated females. Male rats treated up to 1000 mg/kg showed no clinical signs. No clinical signs were noted during the weekly arena observations.
Mortality:
no mortality observed
Description (incidence):
Two females of the 1000 mg/kg group were sacrificed prematurely (together with their healthy offspring) on PND 1 due to abnormal maternal behavior. One control female was euthanized in extremis on post-coitum Day 23 due to delivery difficulties. On the day prior to sacrifice, she showed a pale appearance, hunched posture and piloerection. She showed no abnormalities in clinical appearance, growth or food
consumption prior to this day. Prior to necropsy, the female had delivered two pups, one alive male and one dead female pup. Necropsy findings consisted of a pale appearance and
the presence of four dead male fetuses (all without external abnormalities) and dark red contents in the uterus. Microscopic examination of a limited number of organs (reproductive organs only) showed no related abnormalities. As this concerned a control females, it was not test-item related.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males at 300 and 1000 mg/kg gained statistically significantly less weight than controls throughout the study. Males at 100 mg/kg showed a similar trend but statistical significance was not achieved. The magnitude of the differences in body weight gain tended to increase with dose. The resulting differences in mean body weights at the end of the treatment period (study Day 33) were 4, 5 and 6% at 100, 300 and 1000 mg/kg, respectively (statistically significant at 300 and 1000 mg/kg). Females at 300 and 1000 mg/kg had gained slightly, but statistically significantly less weight than controls on the first day of the mating period. The reductions in weight gain showed no dose-related trend and did not result in significantly lower mean body weights at the end of the pre-mating period.
Body weight gain of 1000 mg/kg females remained slightly lower than that of controls throughout gestation (statistically significant on post-coitum Day 17), resulting in a 6% difference in mean body weights on post-coitum Day 20 (not statistically significant). During lactation, body weight gain was not affected but mean body weights at 1000 mg/kg were lower throughout the lactation period (up to 10% difference from control values, statistically significant from Day 4 of lactation onwards).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In 1000 mg/kg males, food consumption (before and after correction for body weight) was reduced in the first week of the treatment period (25% relative difference for absolute food consumption).
In 1000 mg/kg females, absolute food consumption was reduced between Days 0-11 of the post-coitum period (by about 11-14%, statistically significant up to Day 7) and throughout the lactation period (by 23% between lactation Days 1-4 and about 15% thereafter, statistically significant up to Day 7). Food consumption relative to body weight was reduced between post-coitum Days 0-11 (to about the same extent as absolute food consumption) and between lactation Days 1-4 (by 13%, not statistically significant). Food consumption of males and females at 100 and 300 mg/kg was considered unaffected by treatment.
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):
No treatment-related changes were noted in hematology parameters (red and white blood cell parameters, number of platelets). An isolated, statistically significant intergroup difference observed in males was regarded as unrelated to treatment due to the lack of a dose-related response (higher mean corpuscular haemoglobin at 100 mg/kg).

Coagulation parameters (prothrombin time (PT) and activated partial thromboplastin time (APTT)) were considered not to be affected by treatment. The statistically significantly lower mean APTT value noted in males at 1000 mg/kg was considered to be the result of a slightly high concurrent control value rather than effect of treatment with the test item (mean APTT in 1000 mg/kg males was close to the historical control mean whereas 3/5 controls had values approaching or above the P95-limit of the historical range)
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The following changes, all at 1000 mg/kg, distinguished treated animals from control animals. The differences were statistically significant unless indicated otherwise. Relative changes in mean values (or fold change) compared to the control group are indicated between parentheses.
 Higher concentration of cholesterol in males (40%) and females (41%). Values at 1000 mg/kg generally remained within the historical control ranges.
 Higher activity of alanine aminotransferase (ALAT) in females (48%). Mean ALAT activity at 1000 mg/kg remained within the historical control range, values in 2/5 animals (nos. 73 and 76) were just below or slightly above the P95-limit of the historical range.
 Higher concentrations of urea (19%) and creatinine (16%) in males. Values at 1000 mg/kg generally remained within the historical control ranges.
 Higher concentration of total bilirubin in females (35%, not statistically significant). Mean bilirubin concentration at 1000 mg/kg exceeded the P95-limit of the historical range. It was noted that the concurrent control group mean was at the higher end of the historical range.
 Higher concentration of bile acids in females (2-fold increase in mean value, not statistically significant). Mean bile acids concentration at 1000 mg/kg remained within the historical control range, values in 2/5 animals exceeded the P95-limit of the historical control range

Any other statistically significant variations noted in clinical chemistry values were considered to be unrelated to treatment due to the lack of a dose-related response (alkaline phosphatase and total protein in low or mid-dose males, urea in low- and mid-dose females).

Thyroid hormone analyses:
Mean serum T4 levels were reduced (statistically significantly) at 1000 mg/kg in both sexes (differences from controls: 41% and 33%. respectively). Mean T4 values at 1000 mg/kg were below the historical control ranges. Serum TSH levels showed wide variation, both in control and treated rats. Mean TSH of 1000 mg/kg males was 33% lower compared to controls. As statistical significance was not achieved and mean TSH of 1000 mg/kg males remained within the historical control range, this difference was considered to reflect normal background variation rather than an effect of the test item. For the same reasons, the higher mean TSH noted in females at 300 mg/kg (52% difference from controls) was regarded as unrelated to treatment. Moreover, TSH values in females showed no dose-related trend.

Urinalysis findings:
no effects observed
Description (incidence and severity):
No treatment-related changes were noted in urinalysis parameters.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Functional observation parameters were considered not to be affected by treatment.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The following statistically significant changes, all at 1000 mg/kg, distinguished treated animals from control animals. Relative changes in mean values compared to the control group are indicated between parentheses.
 Higher adrenal weight in males (absolute weight 22%, relative to body weight 29%). Mean relative weight at 1000 mg/kg was at the P95-limit of the historical control range (4/5 individual values were at or above the P-95 limit) while the absolute weights were within the historical range. Although absolute and relative adrenal weights of one control male were slightly below the P5-limit of the historical range, mean adrenal weights of 1000 mg/kg males remained higher than the control group mean when this control male was excluded (16 and 20% for absolute and relative adrenal weight, respectively).
 Higher liver weight in females (relative to body weight only, 13%). Mean relative weight at 1000 mg/kg was at the upper end of the historical control range (3/5 individual values were at or above the P-95 limit).
 Lower absolute weights of the heart (12%), epididymides (7%) and seminal vesicles (18%) in males. Similar decreases in absolute weights of these organs were noted in males at 300 mg/kg (not statistically significant for absolute heart weight). As the relative weights of these organs did not differ statistically significantly from control values, the lower absolute weights were considered to be secondary to the test item-related reduction in (terminal) body weights of 1000 mg/kg males.
The statistically significantly lower absolute heart weight noted in low-dose (100 mg/kg) females was regarded as unrelated to treatment due to the lack of a dose-related response.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Test item-related macroscopic findings were present in the mesenteric lymph node in the form of reddish discoloration in 3/10 females at 1000 mg/kg. The microscopic correlate was intrasinusoidal erythrocytes.
The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain. These findings were therefore considered to be unrelated to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related microscopic observations in male rats. Microscopic examination showed a test item-related increase in the incidence and severity of sinus erythrocytes (macroscopically correlating with reddish discoloration) and histiocytosis in the mesenteric lymph node of females treated at 1000 mg/kg. This morphological change, which was also present in a few control animals, was regarded as non-adverse based on its mild degrees (up to slight) and the absence of correlating test item-related changes in other parameters (e.g. test item-related hemorrhages in the gastro-intestinal tract).
Histopathological findings: neoplastic:
not examined

Effect levels

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:
clinical signs

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
In conclusion, based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the following No Observed Adverse Effect levels (NOAELs) were established for the test material: Parental NOAEL: 300 mg/kg bw/day.