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Key value for chemical safety assessment

Effects on fertility

Description of key information

OECD 422 DOSE RANGE FINDING STUDY (Provivo, 2020)


The aim of the study was to select the dose levels of the test item for the main reproduction/developmental toxicity study (OECD 422).


The rats were treated with 65, 200 or 600 mg test item/kg b.w. daily from test day 1 to test day 14. On test day 8 the high dose level was increased to 1000 mg test item/kg b.w. daily, as no signs of toxicity were noted.


None of the animals died prematurely.


No test item-related observations were noted in behaviour, the external appearance or the appearance of the faeces.


Body weight and food consumption of the male and female animals revealed no test item-related differences.


During the macroscopic inspection at necropsy mandibular lymph nodes with an increased size were noted for nearly all male and female animals of the intermediate and the high dose group (200 or 600/1000 mg test item/kg b.w./day).


After consideration of these data, the following dose levels are suggested for a reproduction/ developmental toxicity study in CD® rats based on OECD guideline 422:


Group 1:    Control (vehicle)


Group 2:    100 mg test item/ kg b.w./day


Group 3:    300 mg test item/ kg b.w./day


Group 4:    1000 mg test item/ kg b.w./day


 


 


OECD 422 MAIN STUDY (Provivo, 2022)


The aim of the study was to obtain information on possible effects of the test item on general toxicity, reproduction and/or development based on OECD guideline 422. The test item was administered orally to rats at dose levels of 100, 300 or 1000 mg/kg b.w./day.


No pathological lesions in the reproductive organs were related to the test item were noted during the histopathological examination in the dams that delivered live pups, regardless of the dose group (100, 300 or 1000 mg test item/kg b.w./day).
Histological differences were noted of the high dose females which lost all implantations. These included implantation sites appearing unphysiological, resorption in the uterus, poorly developed corpora lutea in the ovary and poorly developed mammary glands in 2 of 5 (2/5) females of Group 4. Poorly developed corpora lutea and mammary glands indicate the insufficient progesterone level necessary to maintain pregnancy. There were no abnormalities in the histology of the reproductive organs from the remaining three females of Group 4, as well as from 5 females/group of Groups 2 and 3, examined in this study.
Histopathological examination revealed pathological changes of the adrenal glands of the female high dose animals (1000 mg test item/kg b.w./day) in form of diffuse adrenocortical hypertrophy or multifocal to diffuse fatty vacuoles. Additionally, an increased group mean severity grade for thymus atrophy was observed for the female high dose animals. These results may indicate that the HD females, even in females successful of pregnancy, were under the stressful condition, although no major differences in the terminal body weights were detected. 


At the high dose level (1000 mg test item/kg b.w./day), a reduced number of implants per dam was noted. Additionally, 6 of 9 pregnant high dose females displayed a resorption of all of their implantations, resulting in an increased post-implantation loss and a decreased birth index.
No influence on the viability index was noted. Following the decreased birth index a reduction of the litter weight was noted for the high dose group (1000 mg test item/kg b.w./day). The implantation loss and the subsequently reduced birth index was also a consequence of the high stress level of the high dosed females.
No test item-related abnormalities (malformations or variations) were noted during the external macroscopic examination of the pups at necropsy.


The No Observed Adverse Effect Level (NOAEL) for systemic toxicity was considered to be 300 mg/kg/day for female rats because dosage of of 1000 mg/kg/day resulted in toxicologically significant histopathological findings in the uterus such as unphysiological, resorptions, poorly developed corpora lutea in the ovary and poorly developed mammary glands. Histopathological changes in the adrenal glands were noted for the female high dose animals  in form of diffuse adrenocortical hypertrophy or multifocal to diffuse fatty vacuoles. In addition, the group mean severity grade for thymus atrophy was increased in the high dose females. These may be stress induced effects.


For male rats no toxicological effects could be observed up to the highest tested dose of 1000 mg/kg bw d.

Link to relevant study records

Referenceopen allclose all

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2020-09-30 (Date of experimental Initiation) to 2021-01-06 (End of in-life period)
Reliability:
1 (reliable without restriction)
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted 29 July 2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Crj: CD(SD)
Remarks:
Rat / CD/ Crl:CD(SD) Charles River Laboratories, Research Models and Services, Germany GmbH Sandhofer Weg 7 97633 Sulzfeld Germany
Details on species / strain selection:
Selection of species: The rat is a commonly used rodent species for such studies.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Number and sex of animals (on TD 15): 80 animals (40 males and 40 females )
A sufficient number in order to grant at least 8 pregnant females per group for evaluation of the F0 generation.

- Age at first administration: Males: 99 days, Females: 99 days
- Weight at study initiation: (P) Males: 423.3 - 558.9 g; Females: 232.4 - 374.1g

- Housing: MAKROLON cages (type III plus) with a basal surface of approx. 39 cm x 23 cm and a height of approx. 18 cm

- Diet: ad libitum): ssniff® R/Z V1324, ssniff Spezialdiäten GmbH, 59494 Soest, Germany
- Water (e.g. ad libitum): Tap water was offered ad libitum.
- Acclimation period: 7 adaptation days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 3°C (maximum range)
- Humidity (%): 55% ± 10% (maximum range)
- Air changes (per hr): 15 - 20
- Photoperiod (hrs dark / hrs light): 12 hours dark/12 hours light cycle
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
Route of administration: Oral, by gavage
Frequency of administration: Once daily
Administration volume: 2 mL/kg b.w./day
Vehicle: Tap water

The administration formulations were freshly prepared every day.
The test item was dissolved in the vehicle to the appropriate concentrations and administered orally at a constant administration volume/kg b.w. once daily. The amount of test item was adjusted the animal's current body weight daily.
The control animals received the vehicle at the same administration volume daily in the same way.
Details on mating procedure:
Sexually mature male and female rats of the same dose group were paired monogamously, i.e. 1 male and 1 female animal were placed in one cage during the dark period. The female was placed with the same male until pregnancy had occurred or two weeks had elapsed. Each morning the females were examined for the presence of sperm or a vaginal plug. The day of conception (day 0 of gestation or GD 0) was considered to be the day on which sperm was found. The female animals nos. 60 and 81 with unsuccessful mating during the 14-day mating period were separated thereafter from their mating partner and were placed back in their home-cage in order to rule out that a positive mating sign was overlooked. On day 26 or 24 or their pseudo-gestation period, the female animals nos. 60 and 81 were sacrificed and non-pregnancy status was confirmed by staining according to SALEWSKI.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For the analysis of the test item-vehicle formulations, two (2) aliquots of approximately 3 mL each were taken at the following times and stored at -20°C ± 10% until analysis at the Test Facility:

At start of the treatment period (first dosing day):
Analysis of stability and concentration
Immediately after preparation of the administration formulation as well as after 8 and 24 hours storage at room temperature
(3 samples / dose level group)
Number of samples: 9 (18 aliquots)

Towards the end of the treatment period (when the majority of animals was dosed)
Analysis of concentration
During treatment always before administration to the last animal/dose level group
(1 sample / dose level group).
Number of samples: 3 (6 aliquots)

Sum of all samples: 12 (24 aliquots)

The samples were labelled with the study number, species, type of sample, concentration, aliquot number, test day, sampling time and date.
Only one aliquot was analysed; the second aliquot serves as back-up.
The analytical method was validated by Provivo. The following parameters were determined:
-Linearity
-Accuracy
-Precision
-Sensitivity
-Specificity
-Stability
Duration of treatment / exposure:
Duration of the study: 7 adaptation days and approximately 77 test days

Males: 35 treatment days (from TD 15 to TD 49; sacrifice on TD 50)

Females with litter and not mated females: 49 to 54 treatment days (from TD 15 or 21 to TD 63 or TD 69; sacrifice between TD 64 and TD 71)

Females with resorptions of all implants: 36 or 37 treatment days (from TD 15 to TD 55 or TD 56; sacrifice on TD 56 or TD 57 )
Frequency of treatment:
daily
Details on study schedule:
The study animals were treated during the following periods:
Males:
2 weeks prior to mating (from test day 15 until test day 29), during the mating period (from test day 29 until test day 42#1 at maximum) and during the post-mating period until test day 49 (one day before sacrifice on test day 50).

Females:
2 weeks prior to mating (from test day 15#³ until test day 29), during the mating period (from test day 29 until test day 42#1 at maximum) and during the lactation period until test day 68 to 83 (corresponding to lactation day 13#² or shortly thereafter). The last dosing was always one day before sacrifice. The female animals that lost all of their implantations were treated from test day 15 until test day 55 or 56 (necropsy 2 to 4 days after calculated day of littering, i.e. gestation day 22).

#1: Test days on which a positive sperm detection was noted (mating day or gestation day 0)
#2: The removal days of the female animals in relation to lactation day 1 are given in Table B 'Overview of Female Test Days - Removal Days Related to Lactation Day 1'.
#³: Treatment of animal no. 81 that replaced the prematurely sacrificed animal no. 80 was from test day 21 until test day 70.


Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control group
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Low dose group
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
intermediate dose group
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
High dose group
No. of animals per sex per dose:
10 males and 10 females per dose group
Control animals:
yes, concurrent vehicle
Details on study design:
Justification for dose selection
The dose levels of this study were selected in agreement with the Monitor based on the results of a 14-day dose-range finding study (LPT Study No. 38158, 2020).
The rats were treated with 65, 200 or 600 mg/kg b.w. daily from test day 1 to test day 14. On test day 8, the high dose level was increased to 1000 mg/kg b.w. daily, as no signs of toxicity were noted.
None of the animals died prematurely. No test item-related observations were noted in behaviour, the external appearance or the appearance of the faeces. Body weight and food consumption of the male and female animals revealed no test item-related differences. During the macroscopic inspection at necropsy, mandibular lymph nodes with an increased size were noted for nearly all male and female animals of the intermediate and high dose groups (200 or 600/1000 mg/kg b.w. daily).
Hence, doses of 100, 300 and 1000 mg/kg b.w. were suggested for this study.
Positive control:
not needed
Parental animals: Observations and examinations:
DAILY OBSERVATIONS
Throughout the test period, each animal (parental animals and pups) was observed for clinical signs at least once daily. The frequency was increased when signs of toxicity were observed. Behavioural changes, signs of difficult or prolonged parturition, and all signs of toxicity were recorded. Mortality was recorded twice daily. Animals that were sacrificed prematurely were necropsied after sacrifice.
Individual animals were observed before and after dosing at each time of dosing for any signs of behavioural changes, reaction to treatment, or illness. Any signs of illness or reaction to treatment were recorded for each individual animal.
Cage-side observations included skin/fur, eyes, mucous membranes, respiratory and circulatory systems, somatomotor activity and behaviour patterns. The onset, intensity and duration of any signs observed were recorded.
In addition, animals were checked regularly throughout the working day from 7:00 a.m. to 3:45 p.m. On Saturdays and Sundays animals were checked regularly from 7:00 a.m. to 11:00 a.m. with a final check performed at approximately 3:30 p.m.
Dated and signed records of appearance, change and disappearance of clinical signs were maintained on clinical history sheets for individual animals.

Daily monitoring of vaginal lavages from female were continued throughout the pre-mating period until evidence of mating. A vaginal lavage was also taken in the morning of the day of scheduled necropsy.
Additionally, once before the first exposure (to allow for within-subject comparisons) and once a week thereafter, detailed clinical observations were made in all animals of the F0 generation animals outside the home cage in a standard arena and at the same time, each time preferably by observers unaware of the treatment. Signs noted include changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, and unusual respiratory pattern). Changes in gait, posture and response to handling as well as presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), difficult or prolonged parturition or bizarre behaviour (e.g. self-mutilation, walking backwards) would have been also be recorded.

NEUROLOGICAL SCREENING
Screening of sensory reactivity to stimuli of different types (e.g. auditory, visual and proprioceptive stimuli) (based on Gad ), as well as the assessment of grip strength (Meyer ) and motor activity assessment was conducted as described on the following pages in five males and five females randomly selected from each group. The screening was conducted two hours after dosing and before any blood sampling for laboratory examinations:
5 male F0 animals per group: Shortly before scheduled sacrifice
5 female F0 animals per group: During lactation, shortly before scheduled sacrifice

Righting reflex
The animal was grasped by its tail and flipped in the air approximately 60 cm above the cart surface so that it turned head over heels. The normal animal should land squarely on its feet; for this outcome (0) points were scored. If it landed on its side, 1 point was scored; if it landed on its back, 2 points were scored. This test was repeated five times and the total scores were recorded.
Body temperature
An electronic probe thermometer with a blunt probe was used to take the rectal temperature, being allowed to equilibrate for 30 seconds before the reading was recorded.

Salivation
Discharge of clear fluid from the mouth is most frequently seen as beads of moisture on lips in rats. The normal state is to see none, in which case a zero (0) was recorded in the blank space of the scoring sheet. If present, a plus sign (+) was recorded in the blank.
Startle response
With the animal on the cart, the metal cage was struck with the blunt probe. The normal animal should exhibit a marked but short-lasting response, whereby a zero (0) was recorded in the blank space of the scoring sheet. If there was no response, a plus sign (+) was recorded.

Respiration
While at rest on the cart, the animal's respiration cycle was observed and evaluated in terms of a scale from 1 (reduced) to 5 (increased), with 3 being normal.
Mouth breathing
Rats are normally obligatory nose-breathers. Each animal was observed whether or not it was breathing through its mouth. If the rat was breathing through its nose, a zero (0) was recorded; mouth breathing was documented by a plus sign (+).

Urination
When an animal was removed from its cage, the pan beneath the animal's cage was examined while returning the animal to its cage. The signs of urination were evaluated on a scale of 0 (lacking) to 5 (polyuria) with 3 being normal.

Convulsions
If clonic or tonic convulsions were observed, their intensity was graded on a scale of 1 (minor) to 5 (marked) and the type was recorded. In the normal animal no convulsions should be observed, in which case a score of zero (0) was recorded.
Piloerection
The fur of the animal's back was observed whether it was raised or elevated. In the normal animal no piloerection should be observed and a score of zero (0) was recorded. If piloerection was present, a plus sign (+) was recorded.

Diarrhoea
In examining the pan beneath an animal's cage, it was noted if there were any signs of loose or liquid stools. The normal state is for there to be none (0); in case of diarrhoea the intensity was recorded on a scale of 1 (slight) to 5 (much increased).

Pupil size
It was determined if the pupils were constricted or dilated and the observations were evaluated in terms of a scale from 1 (constricted) to 5 (dilated), with 3 being normal.
Pupil response
The beam of light from the pen light was played across the eyes of the animal and the changes in pupil size were noted. In the normal animal, the pupil is constricted when the beam is on it and then dilates back to normal when the light is removed, whereby a score of zero (0) was recorded. If there was no pupil response, a minus sign (-) was recorded in the blank space.

Lacrimation
The animal was observed for the secretion and discharge of tears. The tears of rats contain a reddish pigment. No discharge is normal, whereby a score of zero (0) was recorded in the blank space of the scoring sheet. If a discharge was present, a plus sign (+) was recorded.

Impaired gait
The occurrence of abnormal gait was evaluated. The most frequent impairments are waddling (W), hunched gait (H), or ataxia (A, the inability of all the muscles to act in unison). The extent of any impairment was recorded on a scale of 1 (slight) to 5 (marked). A normal gait was documented by a score of zero (0).

Stereotypy
Each animal was evaluated for stereotypic behaviour (isolated motor acts or partial sequences of more complex behavioural patterns occurring out of context and with an abnormally high frequency). These were graded on a scale of 1 (slight) to 5 (marked). Normal behaviour was documented by a score of zero (0).

Toe pinch
The blunt probe was used to bring pressure to bear on one of the digits of the hind limb. This should evoke a response from the normal animal. The response or lack thereof was graded on a scale from 1 (absent) to 5 (exaggerated) with 3 being the normal response.

Tail pinch
The toe pinch procedure was utilized with the animal's tail instead of its hind limb and was graded on the same scale from 1 (absent) to 5 (exaggerated), with 3 being the normal response.

Wire manoeuvre
The animal was placed on the metal rod suspended parallel to the cart approximately 60 cm above the cart's surface. The animal's ability to move along the rod was evaluated. If impaired, a score from 1 (slightly impaired) to 5 (unable to stay on wire) was recorded. Normal movement was documented by a score of zero (0).

Hind leg splay
The hind paws were marked with ink using an ink pad. The rat was then held 30 cm above a sheet of blotting paper on the cart. The animal was dropped and the distance between the prints of the two hind paws was measured (in cm).
Positional passivity
The animal was observed after being placed in an awkward position, such as on the edge of the top of the wire-bottomed cage on the cart surface. If the animal immediately moved into a normal position, a score of zero (0) was recorded. If not, a score was recorded on a scale of 1 (slightly impaired) to 5 (cataleptic).

Tremors
Periods of continued fine movements, usually starting in the limbs and perhaps limited to them. The normal case is to have none, in which case a score of zero (0) was recorded. If tremors were present, they were graded on a scale of 1 (slight and infrequent) to 5 (continuous and marked).

Positive geotropism
The animal was placed on the inclined (approximately 30°) top surface of the wire cage with its head facing downward. It should turn 180° and face "uphill", in which case a score of zero (0) was recorded in the blank space of the scoring sheet. If this did not occur, a negative sign was recorded in the blank.

Limb rotation
One of the animal's hind limbs was taken and moved through its normal plane of rotation. In the normal state, it should rotate readily but there should be some resistance. The variations from normal were from no resistance (1) to markedly increased resistance or rigidity (5), with 3 being normal.

Auditory function
Each animal was placed in a container and observed for Preyer's reflex (twitching of the pinna) in response to a high frequency sound stimulus. The stimulus was repeated, if necessary, up to 3 times. A normal response was recorded with a plus sign (+); if there was no response a zero (0) was recorded.

Functional tests
Grip strength
Prior to testing, the gauge (Chatillon, Modell DPP - 1.0 kg) was calibrated with a set of known weights and the apparatus adjusted for the size of the animal (about 1 cm clearance on both sides of the animal). After the strain gauge was zeroed and set in the record mode, the animal was placed into the trough with the forepaws inside the triangular grasping ring. Using one hand, the animal was grasped about 2.5 cm of the way up toward the base of the tail and steadily pulled (approx. 2.5 cm/sec) away from the ring until the grip was broken. It was continued to pull the animal along the trough until the hind limbs grasp the T-bar. The trial was completed when grip of the hind limbs was broken. Three successive readings were taken for each animal with an intertrial interval long enough to record the data and zero both meters for the next trial.

Locomotor activity
The motility was measured using the TSE InfraMot system . The infrared sensor was mounted on top of the home cage and any movements were measured for duration of 12 minutes by sensing the body heat image, i.e. the infrared radiation, and its spatial displacement over time.
Any movements within the cage, even brief movement events of only a few milliseconds duration, were detected and included in the activity data.

MORTALITY
Further checks were made early in the morning and again in the afternoon of each working day to look for dead or moribund animals. This allowed post mortem examination to be carried out during the working period of that day. On Saturdays and Sundays, a similar procedure was followed with a final check at approximately 3.30 p.m..

BODY WEIGHT
The adult animals were weighed on each day of dosing for dose adjustment and at sacrifice; the individual body weights were recorded.
The report includes weekly values for the male animals (starting on test day 15) and the body weight on the day of sacrifice.
For the female animals, the body weights on the following days are given in the report:

Pre-mating period Test days 15, 22, 29
Gestation period Gestation days 0, 7, 14, 20
Lactation period Lactation days 1, 4, 8, 13

FOOD AND DRINKING WATER CONSUMPTION
Food intake per rat (g/rat/week) was calculated using the total amount of food given to and left by each rat in each group upon completion of a treatment week (pre-mating and gestation) or treatment period (lactation).
Food residue (or total food left) was weighed and recorded as follows:

Study period Males Females
Pre-mating period TD 15, TD 22, TD 28 TD 15, TD 22, TD 28
Mating period None None
Gestation period Not applicable GD 7, GD 14, GD 20
Lactation period Not applicable LD 1, LD 7, LD 13
Oestrous cyclicity (parental animals):
During a 14-day pre-exposure period, the oestrous cycle of the female animals were monitored to select 40 animals with regular oestrous cycles. Animals that fail to exhibit typical 4-5 day cycles, were not included in the study.
Sperm parameters (parental animals):
Detailed histopathologic examinations with special emphasis on the qualitative stages of spermatogenesis and histopathology of interstitial testicular structure was performed on one testicle and one epididymis of the selected males of groups 1 and 4 following H E and PAS staining.
Litter observations:
EXAMINATION OF THE PUPS
As soon as possible after delivery, each litter was examined to establish the number and sex of pups, stillbirths, live births, runts (pups were considered as runts if their weight was less than 70% of the mean litter weight) and the presence of gross abnormalities. Any abnormal behaviour of the offspring would have been recorded. However, no abnormal behaviour was noted for the pups.
The following examinations/observations were done for the offspring.

Counting, sexing, weighing
Live pups were counted, sexed and weighed on post-natal days 1, 4 and 13.

Ano-genital distance
On PND 4 before litter adjustment, the ano-genital distance (AGD) of all pups was determined. Furthermore, the ano-genital distance was normalized to the cube root of the body weight of the respective pup.

Nipples/areolae counting
Nipples/areolae were counted in all male pups on PND 13.

Litter adjustment
After counting on PND 4, the litters were adjusted to 10 pups per litter (5 pups/sex/litter) by eliminating surplus pups following a randomization scheme. Selective elimination, e.g. based upon body weight was not performed. Whenever the number of male or female pups prevented 5 pups/sex/litter, partial adjustment (e.g. 6 male and 4 female pups) was carried out.
Postmortem examinations (parental animals):
LABORATORY EXAMINATIONS
Blood samples were taken from the retrobulbar venous plexus under isoflurane anaesthesia from animals fasted overnight and collected into tubes as follows:
EDTA anticoagulant (whole blood).......for haematological examinations
Citrate anticoagulant (plasma).............for coagulation tests
Serum.....................................................for clinical chemistry
The following sampling times and animals were employed:
Sampling time
At study termination (before necropsy): 5 males and 5 females randomly selected from each group

HAEMATOLOGY
The following parameters were determined (Instrument: ADVIATM 120, Siemens Diagnostics GmbH, 35463 Fernwald, Germany):
Parameter (in blood)/ Unit
- Haemoglobin content (HGB)/ mmol/L
- Erythrocytes (RBC)/ 10^6/µL
- Leucocytes (WBC)/ 10^3/µL
- Differential blood count : - relative/ % - absolute/ 10^3/µL
- Reticulocytes (Reti)/ %
- Haematocrit value (HCT)/ %
- Platelets (PLT)/ 10^3/µL
- Mean corpuscular volume (MCV)/ fL
- Mean corpuscular haemoglobin (MCH)/ fmol
- Mean corpuscular haemoglobin concentration (MCHC)/ mmol/L
Following the haematological examinations using the ADVIA system, blood smears were prepared from all samples, dried and stained for possible histopathological examinations in case of pathological findings.

Coagulation
The parameters listed below were determined (Instrument: Amax Destiny Plus™, TCoag Deutschland GmbH, 32657 Lemgo, Germany)
Parameter (in plasma)/ Unit
- Prothrombin time (PT)/ sec
- Activated partial thromboplastin time (aPTT)/ sec

CLINICAL CHEMISTRY
The parameters listed below were determined:
Parameter (in serum)/ Unit
Instrument: KONELAB 30i, Thermo Fisher Scientific, 63303 Dreieich, Germany
- Albumin/ g/L
- Bile acids/ µmol/L
- Bilirubin (total)/ µmol/L
- Cholesterol (total)/ mmol/L
- Creatinine/ µmol/L
- Glucose/ mmol/L
- Protein (total)/ g/L
- Blood urea nitrogen (BUN)/ mmol/L
- Calcium/ mmol/L
- Chloride/ mmol/L
- Potassium/ mmol/L
- Sodium/ mmol/L
- Alanine aminotransferase (ALAT)/ U/L
- Alkaline phosphatase (aP)/ U/L
- Aspartate aminotransferase (ASAT)/ U/L
- Lactate dehydrogenase (LDH)/ U/L
by substraction:
- Globulin/ g/L
by calculation:
- Albumin/globulin ratio/ non-dimensional
- Sodium/Potassium ratio/ non-dimensional
- BUN/creatinine ratio/ non-dimensional

THYROID HORMONE DETERMINATION T4
Blood samples were taken always at the same time of day, as scheduled below:
All evaluated dams At sacrifice 32 x 6 Samples Non-fasted Analysis: No Sample volume: 6 x 100 µL
All adult males At sacrifice 40 x 6 Samples Non-fasted Analysis: Yes Sample volume: 6 x 100 µL
The animals scheduled for blood sampling were anaesthetized and euthanized as follows:
Adults: sampling from the retrobulbar venus plexus under isoflurane anaesthesia; euthanized by carbon dioxide (CO2) inhalation.

LH AND PROGESTERONE DETERMINATION
Aliquots of blood samples of the dams were used for the determination of serum levels of LH and progesterone. Serum levels were determined by ELISA employing commercially available kits (IBL International GmbH, 22335 Hamburg; lot. nos: 16K041-2 (LH) and 23K110 (progesterone)).

GROSS NECROPSY
Vaginal lavages prepared on the day of necropsy were examined to determine the stage of the oestrous cycle and allowed correlation with the histopathology of the female reproductive organs.
The animals were euthanised by carbon dioxide (CO2) inhalation, exsanguinated by cutting the aorta abdominalis, weighed at the following times:
Males: On TD 50
Dams (surviving dams): On PND 13 or shortly thereafter

DISSECTION OF ALL ADULT ANIMALS
At the time of sacrifice or premature death during the study, the adult animals were dissected and examined macroscopically for any abnormalities or pathological changes. Special attention was paid to the organs of the reproductive system.
During necropsy, the number of implantation sites was recorded in the female animals.
Apparently non-pregnant uteri were placed in a 10% aqueous solution of ammonium sulfide for about 10 minutes to stain possible implantation sites in the endometrium according to SALEWSKI .
All superficial tissues were examined visually and by palpation and the cranial roof removed to allow observation of the brain pituitary gland and cranial nerved. After ventral midline incision and skin reflection, all subcutaneous tissues were examined. The condition of the thoracic viscera was noted with due attention to the thymus, lymph nodes and heart.
The abdominal viscera were examined externally and by palpation. The gastro-intestinal tract was examined as a whole and the stomach and caecum were incised and examined. The lungs were removed and all pleural surfaces examined under suitable illumination.
The liver and the kidneys were examined. Any abnormalities in the appearance and size of gonads, adrenals uterus, intra-abdominal lymph nodes and accessory reproductive organs were recorded.
The weights of the organs listed below of all adult animals were determined before fixation, where applicable. Thyroid weight was determined after fixation. Paired organs were weighed individually and identified as left or right.

Weight of organs determined:
- Adrenal gland (2)
- Brain
- Epididymis (2)
- Heart
- Kidney (2)
- Liver
- Ovary (2)
- Spleen
- Testicle (2)
- Thyroid (1) (including parathyroids)
- Thymus
- As a whole: prostate and seminal vesicles with coagulating glands
- Uterus including cervix

The organs or parts thereof listed below of all adult animals were preserved in the appropriate fixative.
Fixative: modified Davidson’s solution
- Epididymis (2)
- Testicle (2)

Fixative 7% neutral buffered formalin
- Gross lesions observed
- Ovary and oviduct (2)
- Prostate
- Seminal vesicles with coagulating glands
- Thyroid 1 (including parathyroids)
- Uterus including cervix
- Vagina

DISSECTION OF SELECTED ADULT ANIMALS
The organs listed below or parts thereof of the animal groups listed on the following page were preserved in a suitable fixative:
-All deceased or prematurely sacrificed animals
-5 male and 5 females randomly selected from each group

Fixative: Davidson's solution
- Eye with optic nerve (2)

Fixative: modified Davidson's solution
- Epididymis (2)
- Testicle (2)

Fixative: 7% neutral buffered formalin
- Adrenal gland (2)
- Bone
- Bone marrow (os femoris)
- Brain (cerebrum, cerebellum, pons)
- Gross lesions observed
- Heart (3 levels: right and left ventricle, septum)
- Intestine, small (duodenum, jejunum, ileum, including Peyer's patches, Swiss roll method)
-Intestine, large (colon, rectum)
- Kidney and ureter (2)
- Liver
- Lungs (with mainstem, bronchi and bronchioles), preserved by inflation with fixative and then immersion
- Lymph node (1, cervical)
- Lymph node (1, mesenteric)
- Mammary gland
- Muscle (skeletal)
- Nerve (sciatic)
- Oesophagus
- Ovary and oviduct (2)
- Pituitary
- Prostate
- Seminal vesicles with coagulating glands
- Spinal cord (3 sections)
- Spleen
- Stomach
- Thyroid (including parathyroids)
- Thymus
- Trachea (including larynx)
- Urinary bladder
- Uterus (including cervix)
- Vagina

Any other organs displaying macroscopic changes, possibly in particular mandibular lymph nodes were also preserved.

HISTOPATHOLOGY
Full histopathology was performed on the preserved organs of
-the selected parental animals of groups 1 and 4 and
-all deceased or prematurely sacrificed animals.
The organs listed above are examined histopathologically after preparation of paraffin sections and haematoxylin-eosin staining. Parathyroids could not always be identified macroscopically. They were examined microscopically if in the plane of section and in all cases where they were noted as grossly enlarged.
In addition, frozen sections of the heart, liver and on kidney were prepared, stained with Oil Red O, and examined.
Detailed histopathologic examinations with special emphasis on the qualitative stages of spermatogenesis and histopathology of interstitial testicular structure was performed on one testicle and one epididymis of the selected males of groups 1 and 4 following H E and PAS staining.
After observation of test item-related changes in group 4, the Sponsor was given sufficient notice and the organs listed below of the female animals of the low and intermediate dose groups (groups 2 and 3) were sectioned and examined additionally.

Organ (HE staining only) Number and sex of animals
Adrenal gland (2) 5 females per group
Ovary and oviduct (2) 5 females per group
Thymus 5 females per group
Uterus (including cervix) 5 females per group
Vagina 5 females per group

The blood smears prepared from all animals during the haematological examination are available for possible examination of pathological changes depending on necropsy findings and upon agreement with the Sponsor.

HISTOPATHOLOGICAL EXAMINATIONS
The histotechnique was performed by Provivo. All slides (labelled with study number, species, animal number, block number) prepared at Provivo were dispatched on 08 April 2021 (organs of groups 1 and 4) and on 01 June 2021 (organs of groups 2 and 3 females) to the Test Site 1 for histopathological evaluation. The transport of slides and tissues to the Test Site 1 was arranged by Provivo, whereas the return transport to the Test Facility was arranged by the Test Site 1.
Histopathological evaluation was performed by the Test Site 1 according to all relevant Test Site SOPs.
All observations upon final assessment were reported per animal and the findings considered relevant for the treatment in an incidence and occurrence table. All microscopic findings were recorded, reported and archived with the PathData system version 6.2e2.
The report of this phase of the study comprised a description of objective, materials and methods, results (including results of macroscopic and microscopic changes) and conclusions. The unaudited Draft Phase Report was presented electronically to the Study Director for comments/review. Comments was addressed and the Draft Final Phase Report of the histopathology phase of the study was presented to the TSQAU for auditing.
The audited Draft Final Phase Report of the histopathology of the study will be sent electronically to the Study Director who will provide documented approval on the study contents of the Phase Report by e-mail to the Principal Investigator. An original of the final, signed Phase Report will be sent (on paper and electronically) to the Study Director; a copy will be archived by the Test Site.
The Test Site Phase Draft Report can be found in Section 7 'Histopathology Report'.
Postmortem examinations (offspring):
THYROID HORMONE DETERMINATION T4
On PND 4 and 13, blood samples for T4 hormone level determination were taken from two selected pups, if possible from one male and one female pup. No pups were eliminated, when litter size would have dropped below the culling target (10 pups/litter). If there was only one pup available above the culling target, only one pup was eliminated and used for blood collection for T4 assessments.
Blood samples were taken always at the same time of day, as scheduled below. The blood was processed for serum.

Pups (2 surplus pups per litter, if possible) on PND 4 Number of samples: 50 x 1 Analysis: no
Pups (2 surplus pups per litter, if possible) on PND 13 Number of samples:60 x 3 Analysis: yes

The animals scheduled for blood sampling were anaesthetized and euthanized as follows:
PND 4 pups: euthanized by decapitation followed by blood collection
PND 13 pups: injection narcosis (Ketamin/Xylazin, i.p.); after blood sampling, euthanized by oral dosage of pentobarbital

NECROPSY
Dead pups and pups sacrificed on day PND 13 were carefully examined externally for gross abnormalities. The external reproductive genitals were examined for signs of altered development.
On PND 13, the thyroid of one male and one female pup from each litter was preserved in 7% neutral buffered formalin and weighed after fixation, preferably from those animals used for T4 ELISA sampling.
Statistics:
A) The statistical evaluation of the parametrical values was done by Provantis (Provantis® integrated preclinical software, version 10.2.1.0, Instem LSS Ltd) using the following settings:
Homogeneity of variances and normality of distribution were tested using the BARTLETT's and SHAPIRO-WILK's test. In case of heterogeneity and/or non-normality of distribution, stepwise transformation of the values into logarithmic or rank values was performed prior to ANOVA. If the ANOVA yielded a significant effect (p ≤ 0.05), intergroup comparisons with the control group were made by the DUNNETT's test (p ≤ 0.01 and p ≤ 0.05).
B) The following statistical methods were used for data not captured by Provantis software (reproductive data):
Homogeneity of variances was test using the BARTLETT's test. In case of homogeneity, intergroup comparison was performed by the DUNNETT's test (p ≤ 0.05 and p ≤ 0.01). In case of heterogeneity of variances, a stepwise comparison of the test groups with the control group was performed using a STUDENT's t-test (p ≤ 0.05 and p ≤ 0.01).
Statistically significantly different data are indicated in the summary tables and in the tables of the result sections.
The mean values and standard deviations were calculated to the highest possible degree of accuracy and then rounded to the reported number of decimals places. Hence, deviations to the last decimal place of up to ± 1 may occur caused by rounding.
Reproductive indices:
Reproductive performance
The following parameters and indices were evaluated:

Reproductive parameters
Pre-coital time and gestation length
- number of pregnant females
- duration of pre-coital time
- gestation length
(The duration of gestation was calculated from gestation day 0 (day of positive sperm detection) until (but not including) lactation day 1 (lactation day 1: morning after littering when no signs of littering were noted anymore).

Implantation sites
- number per dam
- distribution in the uterine horns
- absolute number per group
- mean per group

Number of pups absolute
- at birth (alive and dead)
- after 4 days of life (pre and post-cull), after 13 days

Number of pups per dam
- at birth (alive and dead)
- after 4 days of life (pre and post-cull), after 13 days

Number of male and female pups
- at birth
- after 4 days of life (pre and post-cull), after 13 days

Number of stillbirths
- absolute
- per dam

Number of pups with malformations
(for definition of malformation see Appendix 4)
- absolute
- per dam

REPRODUCTIVE INDICES

Female Fertility Index [%] = (Number of pregnant rats/Number of rats used) x 100

The female fertility index reflects the total number of dams that had achieved pregnancy, including dams that delivered at term, aborted or had fully resorbed litters.

Gestation index [%] = (Number of dams with live pups/Number of pregnant rats) x 100

Offspring viability indices:
For each litter and group the following indices were determined:
Birth Index [%] = (Total number of pups born(alive and dead)/ Number of implantation sites) x 100

Live Birth Index [%] = (Number of pups alive on LD 0/1 / Total number of pups (alive + dead)) x100

Viability Indexpre-cull [%] = (Number of pups alive on LD 4 (pre cull) / Number of pups alive on LD1/0) x 100

Viability Indexpost-cull [%] = (Number of pups alive on LD 13/ Number of pups alive on LD 4 (post cull)) x 100


Post-implantation loss [%] = (Implantation sites - living fetuses/ implantation sites) x 100

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
MALES
In the control group and in the intermediate dose group (300 mg test item/kg b.w./day), no changes in behaviour, external appearance or the faeces were noted.
In the low dose group (100 mg test item/kg b.w./day), the male animal no. 29 displayed moderate salivation on TD 21. Salivation was observed already before administration and disappeared 1 to 2 hours after administration.
The single and transient observation of salivation for one low dose animal on a single day was considered to be spontaneous.
In the high dose group (1000 mg test item/kg b.w./day), slight to moderate salivation was noted for 7 of 10 males for between 1 and 3 days per animal. For 2 animals, the saliva was red discoloured on one day each (see Text Table 4 2 on the following page). Salivation started immediately to 5 minutes after administration or 5 to 20 minutes after administration and was observed not longer than 60 minutes after administration.
Due to only low numbers of occurrences (one to three times) of salivation per animal and due to the short duration, salivation was considered to be test item-related but not adverse.

FEMALES (Pre-mating period, gestation period, lactation period)
In the control group, females no changes in behaviour, external appearance or the faeces were noted.
In the low dose group (100 mg test item/kg b.w./day), the female animal no. 31 was noted with a slight discharge of red-discoloured saliva on TD 24. The single occurrence of one animal with salivation on day test day was considered to be spontaneous.
In the intermediate dose group (300 mg test item/kg b.w./day), the female animal no. 60 displayed slight salivation on TD 19 and animal no. 59 was noted with moderate salivation on GD 0. Both observations of salivation started immediately to 5 minutes after administration. For animal no. 60, salivation disappeared 20 to 60 minutes after administration. For female animal no. 59 salivation was no longer observed 1 to 2 hours after administration. However, the single occurrences of two animals with salivation on one day each were considered to be test item-related but not adverse.
Additionally in the intermediate dose group, animal no. 54 was noted with piloerection, a hemorrhagic vagina and was noted to be pale on the last day of its gestation period (GD 23) and on the first day of the lactation period (LD 1). Furthermore, also the female animal no. 60 was noted with a hemorrhagic vagina on day 9 of its pseudo-gestation period. However, as no observations of a hemorrhagic vagina were noted for the high dose group, the hemorrhagic vagina noted for two intermediate dose animals was considered to be not test item-related. The piloerection and the paleness of animal no. 54 were considered to be spontaneous.
In the high dose group (1000 mg test item/kg b.w./day), salivation was noted for 5 of 10 animals in the pre-mating/mating period and for 2 of 9 females during the gestation period for up to 2 days. Salivation started immediately to 5 minutes after administration and lasted until up to 60 minutes after administration. As salivation was noted for only up to 3 days per animal (female no. 78 displayed salivation for one day in the pre-mating/mating period and two days in the gestation period), salivation was considered to be test item-related but not adverse.

DETAILED CLINICAL OBSERVATIONS
Males and females
No test item-related observations were noted for the male animals of the dose groups (100, 300 or 1000 mg test item/kg b.w./day) and no changes were noted for the male and female animals of the control group and treated females.
Nevertheless, the male low dose animal no. 28 displayed eschar formation in the neck region in test week 4. In the intermediate dose group, male no. 49 revealed a wound at the right ear in test week 3 and eschar formation at the right ear in test week 4. These single occurrences of one male low and intermediate dose animal with a scratch wound and/or eschar formation were considered to be spontaneous.

Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
MALES
No prematurely deceased male animals were noted in the control group and in the dose groups.

FEMALES
No prematurely deceased female animal was noted for the control group and the low and intermediate dose groups.
The female animal no. 80 of the high dose group (1000 mg test item/kg b.w./day) was prematurely sacrificed on TD 19 after a misgavage. Necropsy revealed test item residuals as deposits in the right shoulder accompanied by edematous tissue at the right shoulder. No changes in behaviour, the external appearance or the faeces were noted in the days prior to the premature sacrifice.
This animal was excluded from the evaluation and replaced by animal no. 81.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
MALES
Body weight:
No test item-related differences for the body weight were noted between the control group and the dose groups (100, 300 or 1000 mg test item/kg b.w./day). The difference of the body weight between the control group and the dose groups ranged between 1.9% and +6.0%.
The animals were transferred to the study groups based on their body weight before start of treatment. Hence, the slightly higher body weight in the high dose group developed after transfer to the group and before start of treatment.
Body weight gain:
Accordingly, no test item-related changes in the body weight gain were noted between male animals of the control group and the males of the dose groups (100, 300 or 1000 mg test item/kg b.w./day)

FEMALES
Body weight:
No test item-related influence on body weight was noted for the dose groups (100, 300 or 1000 mg test item/kg b.w./day) during the pre-mating, the gestation and the lactation period.
During the pre-mating period and the first two weeks of the gestation period, no statistically significant differences were noted between the control group and the dose groups. On GD 20, the body weight of the high dose group was distinctly decreased (15.5% below the value of the control group, statistically significant at p < 0.01). However, on LD 1, the difference to the control group was only 1.5%. Further on, during the whole lactation period, no statistically significant differences were noted for the body weight between the control group and the dose groups. Therefore, the decreased body weight of the high dose group on GD 20 was considered to be the result of resorptions of all their implantations in 6 high dose females. Secondly, the resorption occurrence resulted in reduced pup litter weights due to only three litters that were born.

Body weight gain:
In accordance with the reduced body weight of the high dose group during the last week of gestation, also the body weight gain during the gestation period was decreased compared to the other groups. However, as mentioned above, the reduced body weight gain of the high dose group in the gestation period was considered to be due to the loss of all implantations for 6 females and the reduced weight of the three litters born and not due to a test item-related effect on the body weight of the dams.

BODY WEIGHT AT AUTOPSY
Males and females
No test item-related influence on the body weight at autopsy was noted for the male and female animals of any dose group (100, 300 or 1000 mg test item/kg b.w./day) in comparison to the control group.
The body weight at autopsy for the male dose groups ranged between +1.3% (intermediate dose) and +2.6% (low dose) in comparison to the control group and for the female animals of the dose groups, the body weight at autopsy ranged from -4.0% (high dose) to +5.7% (low dose) compared to the value of the control group.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Males and females
During the pre-mating period (TD 15 to TD 29), no statistically significant differences in food consumption were noted between the male rats of the control group and those of the dose groups (100, 300 or 1000 mg test item/kg b.w./day).
No test item-related differences in food consumption were noted between the control group and the treatment groups (100, 300 or 1000 mg test item/kg b.w./day) during the pre-mating, gestation and lactation period.

Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No test item-related changes in the water consumption was were noted for the male and female rats of any dose group.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
MALES
No test item-related differences between the control group and the dose groups (100, 300 or 1000 mg test item/kg b.w./day) for the haematological parameters of the male animals.
Statistically significantly decreased numbers of red blood cells were noted for the low ( 5.8%, p ≤ 0.05) and intermediate dose groups (6.8%, p ≤ 0.01 for the intermediate dose group). Additionally, a statistically significantly increased number of neutrophils was noted for the low dose group (+36%, p ≤ 0.05). However, as no dose-response relationship was present and the values were within the range of the Provivo background data, they were considered to be not test item-related.
In the high dose group, the mean values of the haematological ratio values haematocrit (HCT) and of the mean corpuscular volume (MCV) were slightly below the range of the Provivo background data. As both of these values are ratio values or rather the MCV is calculated from the HCT, it is no wonder that both values are slightly decreased. However, as no statistically significant difference were present, the values of the HCT and MCV below the background data range were considered to be not test item-related.

FEMALES
No test item-related differences for the haematological parameters were noted between the females of the control group and the low and intermediate dose groups (100 or 300 mg test item/kg b.w./day).
In the high dose group (1000 mg test item/kg b.w./day), a dose-dependent decrease was noted for the haemoglobin level (HGB), the number of red blood cells (RBC) and the haematocrit (HCT). For HGB and RBC, the mean values of all groups including the control group, and for HCT the mean values of the intermediate and high dose group were below the range of the Provivo background data. For the above mentioned parameters, the individual values for all of the three evaluated high dose females were below the Provivo background data range. Therefore, the dose-dependently decreased values for the haemoglobin levels, the number of red blood cells and for the haematocrit were considered to be test item-related. Yet, as only three animals could have been investigated and the decrease is only slightly no further conclusion should be drawn and this parameter should not be consulted for the NOAEL determination.
Additionally, mean values of the dose groups above the background data range were noted for the number of large unstained cells (LUC, groups 2 and 3), the activated partial thromboplastin time (aPTT, groups 2 and 4) and for the mean corpuscular volume (MCV, group 2). However, no dose-response relationship was noted for the mentioned parameters and therefore, the values above the background data range were considered to be spontaneous and not test item-related.

Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
MALES
No test item-related differences between the male animals of the control group and the animals of the dose groups (100, 300 or 1000 mg test item/kg b.w./day) were noted for the biochemical parameters.
Mean values slightly outside the Provivo background data range were noted for the levels of glucose, blood urea, sodium and LDH. However, in all cases, no dose-response relationship was present and also individual values of control males were slightly outside the background data range. Therefore, the mean values outside the background data range were considered to be not related to the test item.

FEMALES
No test item-related differences for the biochemical parameters were noted between the females of the control groups and the dose groups (100, 300 or 1000 mg test item/kg b.w./day).
Mean values of the dose groups slightly outside the Provivo background data were noted for the levels of total bilirubin, blood urea, potassium, alanine amino-transferase (ALAT) and alkaline phosphatase (aP). However, more individual values of control females than of the treated females were slightly outside the background data range. Secondly, no dose-response relationship was present. Therefore, the mean values outside the background data range for biochemical parameters of the female animals were considered to be spontaneous and not test item-related.

LH AND PROGESTERONE LEVELS
The serum levels of LH were below the lower limit of quantification for all examined females. Apparently the time window for the LH surge is to narrow to be depicted with one single hormone measurement. Due to the scheduled blood with drawl at necropsy in the morning, potential LH surges for no. 74 have been missed. As the LH surge normally accures during the late afternoon of the day of proestrus . Therefore, no conclusion could be drawn for the influence of the test item on LH serum levels.
For progesterone, no test item-related differences in the serum levels were noted between the control group and the low and intermediate dose groups (100 or 300 mg test item/kg b.w./day). The individual observed progesterone levels for those females are in line with their particular observed stage of oestrus cycle at necropsy.
Although not statistically significant, lower serum levels of progesterone (-55.5%) were noted in the female animals dosed with 1000 mg test item/kg b.w./day. Comparison of the progesterone levels of the individual animals in the high dose group revealed a slight tendency towards lower progesterone levels for females with a high number of resorptions or complete loss of implantations. In retrospect, the progesterone levels were meassured at wrong time point e.g. in blood at necropsy so naturally, the dams which lost their pups one week before normal littering have also lower progesterone levels than the other dams which littered one week later.
Endocrine findings:
no effects observed
Description (incidence and severity):
T4 LEVEL ANALYSIS
No test item-related differences for the serum T4 levels of the adult male animals were noted between the control group and the dose groups (100, 300 or 1000 mg test item/kg b.w./day).
The mean T4 levels of all four groups were below the range of the Provivo background data. However, as no statistical significance and no dose response-relationship were present, the T4 levels were considered to be not influenced by the test item.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Detailed clinical observations
Males and females
No test item-related observations were noted for the male animals of the dose groups (100, 300 or 1000 mg test item/kg b.w./day) and no changes were noted for the male and female animals of the control group and treated females.
Nevertheless, the male low dose animal no. 28 displayed eschar formation in the neck region in test week 4. In the intermediate dose group, male no. 49 revealed a wound at the right ear in test week 3 and eschar formation at the right ear in test week 4. These single occurrences of one male low and intermediate dose animal with a scratch wound and/or eschar formation were considered to be spontaneous.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
MALES
No pathological lesions that were related to the test item were noted during the histopathological examination of the males animals of the high dose group (1000 mg test item/kg b.w./day).
There was no histological evidence of toxicity in the male reproductive organs including testes, epididymides, prostate gland, seminal vesicles and coagulating glands of the high dose males in this study.
Testes were checked on completeness of cell populations and stages, while taking into account the interstitial cell structure and presence/absence of any degenerative changes. As a result, no treatment-related effects on the testicular histomorphology were observed.
Focal and segmental seminiferous tubular degeneration was found unilaterally in the testis of one animal (no. 61) from group 4. This was within the range of normal background lesions that may be observed in this study type and animals of this strain and age, and therefore, was deemed to be incidental changes not related to treatment with the test item.
In males, there were no differences in the incidence and severity of histologic findings recorded in the adrenal glands and thymus, between groups 1 and 4.
All remainder of findings noted for the male reproductive organs and the other organs were within the range of normal background lesions that may be observed in this study type and animals of this strain and age. Therefore, the observed findings were deemed to be incidental changes and not related to treatment with the test item.

FEMALES
No pathological lesions in the reproductive organs that were related to the test item were noted during the histopathological examination in the dams that delivered live pups, regardless of their dose group (100, 300 or 1000 mg test item/kg b.w./day).
Histologically, there were differences in the histology of the female reproductive organs between groups 1 and 4, but the differences were limited to the high dose (HD) females with loss of visible implantations in spite of Salewski staining positive in the gross observations. These included implantation sites appearing unphysiological, resorption in the uterus, poorly developed corpora lutea in the ovary and poorly developed mammary glands in 2 of 5 (2/5) females of group 4. Poorly developed corpora lutea and mammary glands indicate the insufficient progesterone level necessary to maintain pregnancy. In these animals, the ovaries showed histology similar to proestrus and metoestrus stage, and the vagina appeared mucosal figures corresponding to diestrus/proestrus or metestrus/diestrus stages.
Whereas females gotten normally pregnant showed marked to extensive glandular proliferation in the mammary glands as a physiological alteration. Furthermore, the vaginal mucosa of the female high dose animals with a total loss of implantation sites was noted to be in the di- or proestrus or in the met- or dioestrus. Whereas the vaginal mucosa of the females which delivered live pups was in the lactational dioestrus. The ovaries of these animals showed histology similar to proestrus or metoestrus stage.
Meanwhile, the implantation sites of the uterus in the other three HD females (3 of 5 females examined, nos. 75, 77 and 78) without macroscopic abnormalities showed normal histologic appearance like the control females. These females had well-developed (enlarged) corpora lutea in the ovary. The vaginal mucosa was in lactational dioestrus and the mammary glands developed well. Thus, there were no qualitative differences in histology of ovaries, vagina and mammary glands between these three females and the control females, and the reproductive organs and mammary glands of the HD females without macroscopic abnormalities appeared histological figures corresponding normal pregnant females. There were no abnormalities in the female reproductive organs of animals examined in groups 2 and 3.
Thus, except for failure of maintenance of pregnancy, the microscopic findings recorded in 2/5 HD females were histomorphology corresponding to the pregnant status. There were no abnormalities in the histology of the reproductive organs from the remaining three females of group 4, as well as from 5 females of groups 2 and 3, examined in this study.
In order to investigate if there was a failure of maintenance of pregnancy that led to the high number resorptions, serum progesterone and LH level were measured. In the high dose group, lower serum levels of progesterone were noted for the females which did not delivedered live pups and showed poorly developed corpus lutea. (-55.5%, not statistically significant). However, the progesterone levels were measured at wrong time point e.g. at necropsy.
In addition, pathological changes were noted in the adrenal glands in form of hypertrophy for 4 of 5 examined high dose females (none in the control animals) in which animals with loss of grossly visible implantations were included. In addition, multifocal to diffuse fatty vacuoles were found in 3/5 HD females including two animals with loss of grossly visible implantations, and the incidence and group mean severity grade of this change slightly increased in females of group 4. In the examined female animals of groups 2 and 3, no hypertrophy and no fatty changes were observed in the adrenal glands.
In the thymus, group mean severity grade of atrophy slightly increased in females of group 4. Atrophy was also noted in the thymus of all examined group 2 and 3 females. However, the severity grade was comparable to that of the control females. These findings in adrenals and thymus indicate that the high dose females, even in females successful of pregnancy, were under a stressful condition, although no major differences in the terminal body weights were detected
Histopathological findings: neoplastic:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
STAGE OF OESTRUS CYCLE AT NECROPSY
No test item-related differences for the stage of oestrous cycle at necropsy was noted between the female animals of the control group and the dose groups (100, 300 or 1000 mg test item/kg b.w./day).
The results of the oestrous cycle at necropsy derived from the examination of the vaginal lavages indicate an increased number of estrus stages in the intermediate and high dose group (2 or 4 estrus stages in the intermediate and high dose group compared to none in the control group and the low dose group). In contrast, a slight decrease was noted for the number of diestrus stages (6 diestrus stages of 10 female intermediate animals or 3 of 9 in the high dose group compared to 7 of 9 or 4 of 5 in the control and low dose group, respectively). However, during the microscopic examination of the vaginas of the female animals it was found that all high dose female animals that delivered live pups were in the lactational diestrus stage. Just like the animals in the other dose groups which delivered live pups. Unfortunately, 6 of the 9 high dose dams showed total resorption of implantations, which could explain the lack of a diestrus stage at necropsy. Furthermore, no influence was noted on the number and the length of the oestrous cycles in the intermediate and high dose group. Therefore, the increased number estrus stages and the decreased number of diestrus stages for the females of the intermediate and high dose group were considered to be spontaneous.

OESTRUS CYCLE MONITORING
No test item-related differences for the oestrous cycle length and for the number of oestrous cycles in the period from start of dosing until a positive mating sign were noted between the females of the control group and the dose groups (100, 300 or 1000 mg test item/kg b.w./day).
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Testes were checked on completeness of cell populations and stages, while taking into account the interstitial cell structure and presence/absence of any degenerative changes. As a result, no treatment-related effects on the testicular histomorphology were observed.
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
FEMALE FERTILITY
No test item-related influence on the fertility index of the female rats was noted for any of the dose groups (100, 300 or 1000 mg test item/kg b.w./day).
One female each of the intermediate dose group (no. 60) and of the high dose group (no. 81) did not mate with their male partner during the 14-day mating period.


GESTATION INDEX
No influence on the gestation index was noted for the female rats dosed with 100 or 300 mg test item/kg b.w./day.
In the high dose group (1000 mg test item/mg b.w./day), 6 females (nos. 71, 72, 73, 74, 76 and 79) were noted with the loss of all of their implantations leading to a gestation index of 33%. Histopathological observations in these dams like poorly developed corpora lutea and mammary glands indicate insufficient progesterone levels. A high progesterone level is necessary to maintain pregnancy. All effects are due to stress of the animals in the high dose group.

PRE-COITAL TIME AND GESTATION LENGTH
No test item-related differences were noted in the length of the pre-coital time between the control group and the dose groups (100, 300 or 1000 mg test item/kg b.w./day).
An increased pre-coital time was noted for the intermediate (76.2%) and high dose groups (57.1%, statistically not significant) in comparison to the control group. However, as this is only driven by one animal in each dose group (nos. 60 and 81) which did not got pregnant during the mating period and no dose-response relationship was present, the increase was considered to be not test item-related.
No test item-related differences were noted for the length of the gestation period between the rats of the control group and the rats of the dose groups (100, 300 or 1000 mg test item/kg b.w./day).
General toxicity
Parental male and female animals
No test item-related prematurely deceased animals were observed. One female animal in the high dose group was killed due to animal welfare reasons and replaced.
No changes in behaviour, external appearance or the faeces considered to be adverse were noted for any dose group.
No test item-related changes were noted for the body weight, the body weight gain and for the food consumption.
Decreases in the number of red blood cells, the level of haemoglobin and the haematocrit were noted for the three investigated female high dose animals (1000 mg test item/kg b.w./day).
No test item-related differences were noted for the examination of the biochemical parameters and the T4 levels.

No test item-related significant differences were noted for the body weight at autopsy and the absolute and relative organ weights and no pathological changes considered to be test item-related were observed during necropsy.
No pathological lesions that were related to the test item were noted during the histopathological examination of the male animals of the high dose group (1000 mg test item/kg b.w./day).
No pathological lesions in the reproductive organs were related to the test item were noted during the histopathological examination in the dams that delivered live pups, regardless of the dose group (100, 300 or 1000 mg test item/kg b.w./day).
Histological differences were noted of the high dose females which lost all implantations. These included implantation sites appearing unphysiological, resorption in the uterus, poorly developed corpora lutea in the ovary and poorly developed mammary glands in 2 of 5 (2/5) females of Group 4. Poorly developed corpora lutea and mammary glands indicate the insufficient progesterone level necessary to maintain pregnancy. There were no abnormalities in the histology of the reproductive organs from the remaining three females of Group 4, as well as from 5 females/group of Groups 2 and 3, examined in this study.
Histopathological examination revealed pathological changes of the adrenal glands of the female high dose animals (1000 mg test item/kg b.w./day) in form of diffuse adrenocortical hypertrophy or multifocal to diffuse fatty vacuoles. Additionally, an increased group mean severity grade for thymus atrophy was observed for the female high dose animals. These findings in adrenals and thymus may indicate that the high dose females, even in females successful of pregnancy, were under a stressful condition, although no major differences in the terminal body weights were detected.

Reproductive toxicity
Parental females
No influences were noted on the fertility index, the pre-coital time and the gestation length.
At 1000 mg test item/kg b.w./day, a reduction was noted on the gestation index. This was due to the loss of all implantations of 7 females in the high dose group. All effects are due to stress of the animals in the high dose group

Key result
Dose descriptor:
NOAEL
Remarks:
general toxicity
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
NOAEL
Remarks:
general toxicity
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Effect level:
> 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
Remarks on result:
not determinable due to absence of adverse toxic effects
Critical effects observed:
no
Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
VIABILITY INDEX
No difference between the control group and the dose groups (100, 300 or 1000 mg test item/kg b.w./day) was noted for the viability index (group level) before culling on LD 4 (pre-cull, LD 0/1 to LD 4) after culling (post-cull, LD 4 to LD 13).
In the control group, all pups of dam no. 20 were found dead on LD 2. Female no. 58 of the intermediate dose group lost all pups between LD 2 and LD 4. Therefore, the pre-cull viability index was 85.1% for the control group and 88.0% for the intermediate dose group, respectively. Whereas the pre-cull viability index for the low and high dose group was 100.0% (for the low dose group statistically significant at p ≤ 0.01). The post-cull viability index ranged between 97.2% (control group) and 100.0% (high dose group).
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
BODY WEIGHT OF PUPS
No test item-related difference was noted between the mean body weight of the pups from the dams of the control group and the mean body weight of the pups from the dams of the dose groups (100, 300 or 1000 mg test item/kg b.w./day) on LD 1, LD 4 and LD 13.
For the high dose group, reduced body weights were noted for the female pups (12.9%, 18.1% and 14.6% below the value of the control groups on LD 1, LD 4 or LD 13, respectively. No statistical evaluation was conducted as only two litters with live female pups were present). No difference was noted for the male pups due to one litter consisting of only two male pups that had an increased body weight (of dam no. 75). The male pup body weights of the remaining two litters (dams nos. 77 and 78) were at the lower limit of the range of the male control litters. However, as the mean pup body weight for both sexes combined was within the range of the Provivo background data, the body weights of the high dose pups were considered to be not influenced by the test item.

Runts
One runt each was noted in the control group and the intermediate dose group. The single occurrences of one runt each in the control group and in the intermediate dose group were considered to be spontaneous.


LITTER WEIGHT
No test item-related differences for the litter weight were noted between the control group and the low and intermediate dose groups (100 or 300 mg test item/kg b.w./day).
In the high dose group (1000 mg test item/kg b.w./day), a reduced litter weight was noted for the male and female litters. On the basis of less pups born alive for the dams that born pups at 1000 mg test item/kg b.w./day compared to the control group and the other dose groups. This resulted in a reduced weight for the male and female litters combined on LD 1, 4 and 13 for the high dose group with a statistically significant difference on LD 1 (42.5% below the value of the control group, p ≤ 0.05).
The constantly and distinctly reduced litter weight of the high dose group was below the Provivo background data range. This was due to the test item-relatedly reduced number of pups in the high dose group.

NUMBER OF LIVE PUPS
No test item-related differences were noted between the control group and the dose groups (100, 300 or 1000 mg test item/kg b.w./day) for the number of live pups per dam on the lactation days 1, 4 and 13.
The number of live pups of the male and female pups combined on LD 4 of the intermediate dose group (300 mg test item/kg b.w./day) was below the range of the Provivo background data. However, also the number of live pups of the control group was below the background data range. This was due to one litter each in the control group (12 pups) and the intermediate dose group (13 pups) that was deceased between LD 2 and LD 4. Additionally, the dams in the control group lost more pups between LD 1 and 4, then any of the dose groups. Nearly one pup per dam. Compared to none in the low and high dose groups and one additional pup in the intermediate dose group.
On LD 13, the number of live pups after culling was 8.4 pups per dam for the control group and for the intermediate dose group (which was still below the range of the Provivo background data). As the number of live pups of the intermediate dose group was within the background data range on LD 1 and no difference to the control group was noted on LD 13, the lower number of live pups on LD 4 that was below the background data range was considered not test item-related.
In the high dose group (1000 mg test item/kg b.w./day), a reduction was noted for the number of live pups on LD 1 (79.8% below the value of the control group for the male and female pups combined, statistically significant at p ≤ 0.01). This is a result of the low birth indices in the high dose group. These reduced numbers were due to 6 of 10 dams with a total loss of implantations and due to one dam with only two born pups and were below the range of the Provivo background data. As the number of live born pups was already low at birth, this carries on during the time of lactation. Consequently, reduced numbers of live pups were observed also on LD 4 (76.7% below the value of the control group, statistically significant at p ≤ 0.01) and on LD 13 (70.9% below the value of the control group, statistically significant at p ≤ 0.05). But as there was no further death observed during the lactation period, these reduced numbers of live pups were considered not test item related and only mathematical.

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:
no effects observed
Description (incidence and severity):
THYROID HORMONE LEVELS OF THE PUPS
No test item-related differences were noted for the T4 levels of the male and female pups on LD 13 between the control group and the dose groups (100, 300 or 1000 mg test item/kg b.w./day).
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
No test item-related influence was noted on the ano-genital distance of the male and female pups of the dose groups (100, 300 or 1000 mg test item/kg b.w./day).
The ano-genital distance of the female pups normalized to the cube root of the body weight revealed for all groups a nearly constant correlation with the body weight as it is described in literature for control animals (Gallavan et al., 1999). This indicates that the ano-genital distance for the female pups was only dependent on the body weight of the pups and was not influenced by the test item. However, no constant correlation was noted for the male pups.
In the intermediate dose group (300 mg test item/kg b.w./day), a slight reduction was noted for the absolute (8.1%) and normalized (10.0%) ano-genital distance of the male pups below the value of the control group (statistically not significant). This was due to 6 of 8 male litters that were below the range of the Provivo background data. However, no dose-response relationship was present and no influences were noted on the other parameters pup of development for the intermediate dose group. If the ano-genital distance of the male pups of the intermediate dose group was normalized to the cube root of the body weight, a higher body weight on LD 4 than the control pups was noted but a lower ano-genital distance. Therefore, the lower ano-genital distance for the male intermediate dose pups was considered to be not test item-related.
In the high dose group (1000 mg test item/kg b.w./day), an increase was noted for the absolute ano-genital distances of the male and female pups (12.5% or 10.0% above the values of the control group, statistically not significant for the male pups and not statistically evaluable for the female pups). Also, the relative ano-genital distance of the male and female high dose pups was increased (11.2% or 17.8% above the value of the control group, statistically not significant for the male pups). However, the absolute and relative ano-genital distances for the male and female pups were within the Provivo background data range. The ano-genital distance normalized to the cube root of the male pups of the high dose group displayed only a slightly increased pup body weight (3.3% above the value of the control group) and a stronger increase in ano-genital distance (12.5% above the value of the control group). As already the pup body weight of two male pups in the high dose group showed an increase in body weight this can be the reason for this discrepancy. Therefore, the increased ano-genital distances for the male and female pups were considered to be not test item-related.
Overall, also the ano-genital distance of the male pups was considered to be dependent only on the body weight.

Nipple retention in male pups:
no effects observed
Description (incidence and severity):
No test item-related influence was noted on the number of nipples of the male pups in the low and intermediate dose groups (100, 300 or 1000 mg test item/kg b.w./day).
In the low and intermediate dose groups, 3 male pups with one nipple each were observed.
In the high dose group (1000 mg test item/kg b.w./day), 2 of 13 pups were noted with one nipple each in comparison to one nipple-bearing pup out of 46 control pups. Yet as only a low number of pups were born at the high dose groups this is considered to be incidental and not test item-related.
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
EXTERNAL EXAMINATION OF PUPS
No gross abnormalities (e.g. malformations or variations) were noted during the macroscopic external examination of the control pups and the pups from the dams treated with 100 or 1000 mg test item/kg b.w./day after terminal sacrifice on lactation day 13.
In the intermediate dose group (300 mg test item/kg b.w./day, all pups of dam no. 59 were noted with necrotic tails and constrictions on the tail. However, as only one intermediate dose litter was affected, the necrotic tails with constrictions were considered to be not test item-related but spontaneous.
The observation of ringtails on young laboratory rodents is described to be spontaneous and may be caused by environmental factors (Recordati et al., 2014). Therefore, and as only one intermediate dose litter was affected, the observations of ringtail were considered to be not test item-related.
Histopathological findings:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
BIRTH INDICES AND POST-IMPLANTAION LOSS
No test item-related differences were noted for the mean number of implantation sites, the mean number of pups born (alive and dead) and the mean number of live born pups between the control group and the low and intermediate dose groups (100 or 300 mg test item/kg b.w./day).
At the high dose level (1000 mg test item/kg b.w./day), the number of implantation sites per dam (13.9) was slightly below the range of the Provivo background data (14.0 - 17.0 implantation sites per dam).
Supplementary, 6 of 9 pregnant high dose females lost all of their implantations. Secondly, 2 of 3 high dose females with live pups had also a high loss of implantations (6 or 17 resorptions for the female nos. 77 and 75, respectively. In total, a loss of 99 of 125 implantations was noted for the high dose group leading to a mean post-implantation loss of 82.2% per dam and a birth index of 18.5% per dam. The dose-dependently decreased number of implantation sites that was below the background data range, the distinctly decreased birth index and the distinctly increased number of resorptions and post-implantation loss were considered to be test item-related.
According to the noticed histopathological changes in the two investigated dams that lost all their implantations, a reduced progesterone level seems responsible for these observations. In fact, the serum progesterone levels of the high dose group were lower ( 55.5%, not statistically significant) in comparison to the control group. The values were even lower for the dams that showed a loss of all implantation and poorly developed corpus lutea compared to the ones (nos. 75, 77, 78) that did deliver some live pups in the highest dose group.
For the live birth index (the number of alive pups at LD 1 divided by the total number of pups born alive and dead), a group value below the background data range (97.1% - 100.0%) was noted for the high dose group (96.3%). However, as no dose-response relationship was present, the live birth index of the high dose group that was slightly below the background data range was considered to be not test item-related.

MALE TO FEMALE RATIO OF THE PUPS
No test item-related influence on the male to female ratio was noted for all treatment groups (100, 300 or 1000 mg test item/kg b.w./day).
The male to female ratios on LD 1 were 1.38 (control dose group), 0.96 (low dose group), 1.05 (intermediate dose group) and 1.89 (high dose group). Due to one litter each that deceased in the control group and the intermediate a slight shift was noted in the male/female ratios on LD 4 for the control and the intermediate dose groups. The ratios were: 1.30 (control group), 0.96 (low dose group), 1.15 (intermediate dose group) and 1.89 (high dose group). The ratio of 1.89 in the high dose group on LD 1 and 4 was due to the low number of pups (17 male pups and 9 female pups in the high dose group).
As there is no dose response relationship present, the slightly lower level at the low dose group was considered to be spontaneous.
#

Behaviour (functional findings):
no effects observed
Developmental immunotoxicity:
no effects observed
Reproductive toxicity

Pups
Pre-natal development:
Nevertheless, at the high dose level (1000 mg test item/kg b.w./day), a reduced number of implants per dam was noted. Additionally, 6 of 9 pregnant high dose females displayed a resorption of all of their implantations, resulting in an increased post-implantation loss and a decreased birth index. However, all these effects are due to stress of the female dams in the high dose group and therefore not test compound related.

Post-natal development:
No influence on the viability index was noted. Following the decreased birth index a reduction of the litter weight was noted for the high dose group (1000 mg test item/kg b.w./day).
No test item-related abnormalities (malformations or variations) were noted during the external macroscopic examination of the pups at necropsy.
Key result
Dose descriptor:
NOAEL
Remarks:
pups
Generation:
F1
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
Critical effects observed:
no
Reproductive effects observed:
no

Text Table 4‑1:      Reproductive outcome of the female animals per group








































































































Test item



Group 1


Control



Group 2


100 mg/kg



Group 3


300 mg/kg



Group 4


1000 mg/kg



Prematurely sacrificed animals



0



0



0



1 #1



Females paired with their male partners



10



10



10



10



Females with evidence of copulation (positive mating sign by sperm detection)



10



10



9



9



Females without evidence of copulation (no positive mating sign)



0



0



1 #²



1 #²



Females not pregnant



0



0



0



0



Females pregnant #³



10



10



9



9



Fertility index



100%



100%



90%



90%



Prematurely deceased females



0



0



0



0



Females with total resorptions



0



0



0



6



Females with litters #4



10



10



9



3



Females that delivered only stillbirth



0



0



0



0



Females with live born pups



10



10



9



3



Females surviving the lactation period until scheduled sacrifice



10



10



9



3



#1:      Female no. 80 was prematurely sacrificed on TD 19 after a misgavage (see Section 4.2 'Mortality') and was excluded from the study and replaced by female no. 81 to obtain a sufficient number of pregnant animals.


#²:      Females nos. 60 and 81 were noted without positive mating sign after a mating period of 14 days. Laparotomy after a pseudo gestation period revealed their non-pregnancy status.


#3:      Number of evaluated animals at start of the gestation period.


#4:      Number of evaluated animals at start of the lactation period.


 


Text Table 4‑5:      Body weight gain of the female animals during the pre-mating, gestation and lactation period









































Body weight gain - Females



Group 1


Control



Group 2


100 mg/kg



Group 3


300 mg/kg



Group 4


1000 mg/kg



 



pre-mating period


(TD 15 to TD 29)



-1.7%



-1.7%



-1.9%



-2.4%



 



gestation period


(GD 0 to GD 20)



+50.7%



+57.5%



+54.1%



+29.7%



 



lactation period


(LD 1 to LD 13)



+13.9%



+13.5%



+11.6%



+8.4%



 



Values taken from Table 9-2 'Body Weight Gain - Summary - Females - Pre-mating Period', Table 9-3 'Body Weight Gain - Summary - Females - Gestation Period' and Table 9‑4 'Body Weight Gain - Summary - Females - Lactation Period'.



 


Text Table 4‑6:      Provivo background data on haematological parameters of the males



















































































Adult Males



Values from Study No. 38159


Mean values per group ± SD


Range of the individual values (n = 5)


(number of individual values outside the background data range in brackets; position: below lower limit = left,


above upper limit = right)



Provivo Background Data #1


obtained from the control groups of 20 OECD 422 studies performed at Provivo between


2016 and 2021



RBC


[x106/µL]



Group 1



9.238 ± 0.248


8.94 - 9.60 (n = 1)



5 to 95% Percentile


7.84 - 9.49



Group 2



8.702 ± 0.201 *


8.48 - 8.95



Group 3



8.608 ± 0.136 **


8.42 - 8.76



Group 4



8.632 ± 0.580


7.95 - 9.19



Neutrophils


[x10³/µL]



Group 1



1.324 ± 0.269


1.07 - 1.63



5 to 95% Percentile


0.76 - 2.89



Group 2



1.800 ± 0.296 *


1.33 - 2.14



Group 3



0.980 ± 0.160


(n = 1) 0.75 - 1.18



Group 4



1.266 ± 0.186


0.98 - 1.46



HCT


[%]



Group 1



47.12 ± 1.61


44.8 - 49.3



5 to 95% Percentile


44.6 - 51.5



Group 2



44.94 ± 1.16


(n = 2) 43.2 - 46.1



Group 3



45.04 ± 2.63


(n = 2) 42.0 - 48.6



Group 4



43.6 ± 2.14


(n = 3) 41.3 - 45.9



MCV


[fL]



Group 1



51.00 ± 0.82


(n = 3) 50.1 - 52.2



5 to 95% Percentile


51.4 - 60.2



Group 2



51.62 ± 1.52


(n = 4) 50.8 - 54.3



Group 3



52.32 ± 2.60


(n = 2) 49.8 - 56.4



Group 4



50.56 ± 1.65


(n = 4) 47.8 - 52.2



#1:       Not audited by the QAU.


Data taken from Table 11-3 'Haematological Parameters - Individual Data - Males'.


Text Table 4‑7:      Provivo background data on haematological parameters of the females





























Adult Females



Values from Study No. 38159


Mean values per group ± SD


Range of the individual values (n = 3 - 5)


(number of individual values outside the background data range in brackets; position: below lower limit = left,


above upper limit = right)



Provivo Background Data #1


obtained from the control groups of 20 OECD 422 studies performed at Provivo between


2016 and 2021



HGB


[mmol/L]



Group 1



8.48 ± 0.37


(n = 3) 8.1 - 9.0



5 to 95% Percentile


8.5 - 10.1



Group 2



8.40 ± 0.14


(n = 4) 8.2 - 8.6



Group 3



8.26 ± 0.59


(n = 3) 7.6 - 9.1



Group 4



7.97 ± 0.21


(n = 3) 7.8 - 8.2



- continued on the following page -


 


 


 


 


 


 


 


 


- continued -
































































































RBC


[x106/µL]



Group 1



7.084 ± 0.261


(n = 4) 6.73 - 7.37



5 to 95% Percentile


7.32 - 9.01



Group 2



7.004 ± 0.317


(n = 5) 6.53 - 7.26



Group 3



6.986 ± 0.404


(n = 3) 6.57 - 7.44



Group 4



6.620 ± 0.286


(n = 3) 6.29 - 6.79



HCT


[%]



Group 1



42.62 ± 2.22


(n = 2) 40.1 - 45.0



5 to 95% Percentile


41.4 - 49.6



Group 2



41.64 ± 0.91


(n = 2) 40.5 - 42.9



Group 3



41.14 ± 3.87


(n = 3) 37.3 - 46.4



Group 4



39.27 ± 1.52


(n = 3) 37.9 - 40.9



LUC


[%]



Group 1



1.10 ± 0.62


0.7 - 2.2 (n = 1)



5 to 95% Percentile


0.3 - 1.1



Group 2



1.20 ± 0.53


0.6 - 1.7 (n = 3)



Group 3



1.46 ± 0.66


0.8 - 2.4 (n = 3)



Group 4



0.67 ± 0.29


0.5 - 1.0



aPTT


[s]



Group 1



15.02 ± 0.53


14.6 - 15.6



5 to 95% Percentile


13.9 - 17.0



Group 2



17.20 ± 4.08


14.1 - 24.1 (n = 2)



Group 3



15.6 ± 0.72


14.7 - 16.4



Group 4



17.57 ± 2.11


16.3 - 20.0 (n = 1)



MCV


[fL]



Group 1



60.14 ± 2.0


57.0 - 62.3 (n = 4)



5 to 95% Percentile


51.9 - 59.3



Group 2



59.54 ± 2.28


57.1 - 62.1 (n = 2)



Group 3



58.78 ± 2.68


55.7 - 62.3 (n = 3)



Group 4



59.27 ± 1.53


57.5 - 60.2 (n = 2)



#1:       Not audited by the QAU.


Data taken from Table 11-4 'Haematological Parameters - Individual Data - Females'.


Text Table 4‑8:      Provivo background data on biochemical parameters of the males

































































Adult Males



Values from Study No. 38159


Mean values per group ± SD


Range of the individual values (n = 5)


(number of individual values outside the background data range in brackets; position: below lower limit = left,


above upper limit = right)



Provivo Background Data #1


obtained from the control groups of 20 OECD 422 studies performed at Provivo between


2016 and 2021



Glucose


[mmol/L]



Group 1



9.648 ± 1.124


8.37 - 11.08 (n = 3)



5 to 95% Percentile


5.95 - 9.22



Group 2



9.036 ± 1.399


7.26 - 10.75 (n = 3)



Group 3



9.006 ± 1.576


7.22 - 10.74 (n = 2)



Group 4



9.860 ± 1.659


8.02 - 11.98 (n = 2)



Blood urea


[mmol/L]



Group 1



6.796 ± 0.349


6.39 - 7.19 (n = 5)



5 to 95% Percentile


3.60 - 6.16



Group 2



8.044 ± 0.958


6.39 - 8.77 (n = 5)



Group 3



7.046 ± 2.175


5.12 - 10.49 (n = 3)



Group 4



6.624 ± 1.122


5.62 - 7.91 (n = 2)



Sodium


[mmol/L]



Group 1



137.8 ± 0.4


(n = 1) 137 - 138



5 to 95% Percentile


138 - 145



Group 2



138.2 ± 0.4


138 - 139



Group 3



138.6 ± 1.1


(n = 1) 137 - 140



Group 4



137.8 ± 0.8


(n = 2) 137 - 139



- continued on the following page -


 


 


 


 


- continued -
























LDH


[U/L]



Group 1



48.2 ± 46.6


(n = 3) 18 - 130



5 to 95% Percentile


35 - 180



Group 2



27.8 ± 7.8


(n = 4) 19 - 40



Group 3



22.2 ± 11.7


(n = 4) 11 - 39



Group 4



31.6 ± 18.4


(n = 3) 16 - 59



#1:       Not audited by the QAU.


Data taken from Table 12-3 'Biochemical Parameters - Individual Data - Males'.


related.


Text Table 4‑9:      Provivo background data on biochemical parameters of the females





























Adult Females



Values from Study No. 38159


Mean values per group ± SD


Range of the individual values (n = 3 - 5)


(number of individual values outside the background data range in brackets; position: below lower limit = left,


above upper limit = right)



Provivo Background Data #1


obtained from the control groups of 20 OECD 422 studies performed at Provivo between


2016 and 2021



Bilirubin (total)


[µmol/L]



Group 1



2.00 ± 0.25


(n = 3) 1.8 - 2.4



5 to 95% Percentile


2.1 - 4.5



Group 2



2.22 ± 0.41


(n = 3) 1.8 - 2.8



Group 3



1.94 ± 0.18


(n = 4) 1.7 - 2.2



Group 4



1.90 ± 0.26


(n = 2) 1.6 - 2.1



 














































































Blood urea


[mmol/L]



Group 1



10.980 ± 1.975


8.88 - 13.50 (n = 3)



5 to 95% Percentile


3.71 - 10.08



Group 2



12.108 ± 0.630


11.20 - 12.92 (n = 5)



Group 3



11.098 ± 2.768


6.79 - 14.25 (n = 4)



Group 4



11.137 ± 1.082


9.93 - 12.02 (n = 2)



Potassium


[mmol/L]



Group 1



4.872 ± 0.420


4.53 - 5.35 (n = 5)



5 to 95% Percentile


3.08 - 4.32



Group 2



4.21 ± 5.66


4.21 - 5.66 (n = 4)



Group 3



4.782 ± 0.294


4.41 - 5.14 (n = 5)



Group 4



4.557 ± 0.689


3.96 - 5.31 (n = 2)



ALAT


[U/L]



Group 1



148.0 ± 28.4


101 - 175 (n = 4)



5 to 95% Percentile


26 - 133



Group 2



140.0 ± 10.9


127 - 150 (n = 3)



Group 3



115 ± 175


115 - 175 (n = 3)



Group 4



118.7 ± 46.7


66 - 155 (n = 2)



aP


[U/L]



Group 1



248.4 ± 121.9


137 - 425 (n = 5)



5 to 95% Percentile


52 - 121



Group 2



235.6 ± 59.2


188 - 339 (n = 5)



Group 3



164.6 ± 112.7


92 - 357 (n = 2)



Group 4



212.0 ± 89.6


112 - 285 (n = 2)



#1:       Not audited by the QAU.


Data taken from Table 12-4 'Biochemical Parameters - Individual Data - Females'.


 


Text Table 4‑10:    Provivo background data on serum T4 levels of adult male animals





























Adult Males



Values from Study No. 38159


Mean values per group ± SD


Range of the individual values


(n = 10)


(number of individual values outside the background data range in brackets; position: below lower limit = left,


above upper limit = right)



Provivo Background Data #1


obtained from the control groups of 20 OECD 422 studies performed at Provivo between


2016 and 2021



Serum T4 levels


[nmol/L] #2



Group 1



41.246 ± 6.586


(n = 3) 28.283 - 47.388



5 to 95% Percentile


42.611 - 90.506



Group 2



37.569 ± 4.830


(n = 7) 31.375 - 44.313



Group 3



36.401 ± 4.996


(n = 8) 31.001 - 46.418



Group 4



38.464 ± 9.866


(n = 7) 27.382 - 57.104



#1:       Not audited by the QAU.


#2:       Data taken from Table 18-2 'T4 Level Analysis - Individual Data - Males'.


 


Text Table 4‑11:    Overview on number of implantation sites, resorptions and stillbirths, number of live pups and progesterone levels of the high dose females






















































































Group 4 (1000 mg test item/kg b.w./day)



Female



No. of


implantation sites



No. of resorptions and stillbirths



Number of live pups



Progesterone level [ng/mL]



71 (NVP)



19



19



0



23.32



72 (NVP)



9



9



0



-



73 (NVP)



14



14



0



-



74 (NVP)



15



15



0



9.18



75



19



17



2



29.89



76 (NVP)



10



10



0



-



77



16



6



10



37.67



78



16



2



14



47.14



79 (NVP)



7



7



0



30.60



81 (NM)



n.a.



n.a.



n.a.



8.25



n.a.:                 Not applicable                          NVP:    No viable pups                         NM:                  Not mated


 


Text Table 4‑12:    Body weight of autopsy of the male and female animals



























































Body Weight at Autopsy



Group



Parameter



Males



females



Group 1


control



mean



493.0 g



360.1 g



difference to control



n.a.



n.a.



Group 2


100 mg/kg b.w./day



mean



505.7 g



380.7 g



difference to control



+2.6%



+5.7%



Group 3


300 mg/kg b.w./day



mean



499.4 g



361.9 g



difference to control



+1.3%



+0.5%



Group 4


1000 mg/kg b.w./day



mean



505.6 g



345.8 g



difference to control



+2.5%



-4.0%



n.a.:                 Not applicable


 


Text Table 4‑14:    Stage of the oestrous cycles at necropsy

















































































Stage of oestrous cycle


at necropsy


(F0 Generation) #1



Group 1


Control



Group 2


100 mg/kg



Group 3


300 mg/kg



Group 4


1000 mg/kg



N #2



H



N #2



N



N #2



H



Proestrus



1 of 9



1 of 5



1 of 6



-



1 of 9



1 of 5



Estrus



-



-



-



2 of 10



4 of 9



-



Metestrus



1 of 9



-



-



2 of 10



1 of 9



1 of 5



Diestrus



7 of 9



4 of 5 #3



5 of 6



6 of 10



3 of 9



3 of 5 #5



-:



Not detected.



#1:



The values are taken from Table 15 'Stage of Oestrous Cycle at Necropsy - Individual Data'.



N:



Stage of the oestrous cycle was determined from the vaginal lavage at necropsy.



H:



Stage of the oestrous cycle was determined during the microscopic examination of the vagina (see Section 7 'Histopathological Phase Report').



#2:



The vaginal lavages were not taken for females nos. 16 (group 1), 31, 34, 35, 39 (group 2) and 78 (group 4).



#3:



Termed as lactational dioestrus in the histopathology report.



 


Text Table 4‑15:    Statistically significant or dose-dependent differences of organ weights of male or female animals considered to be not test item-related





























































































































Organ Weights of Male Animals



Organ



Parameter



Group 1


Control



Group 2


100 mg/kg



Group 3


300 mg/kg



Group 4


1000 mg/kg



Prostate and sem. vesic. [g/kg b.w.]



mean



7.9654



6.8860 *



7.8654



7.6447



% diff.



n.a.



-13.6



-1.3



-4.0



Liver [g]



mean



17.93



18.52



19.59



19.94



% diff.



n.a.



+3.3



+9.3



+11.2



Thymus [g]



mean



0.288



0.316



0.316



0.322



% diff.



n.a.



+9.7



+9.7



+11.8



Thyroid, l. [g]



mean



0.0152



0.0146



0.0129



0.0117



% diff.



n.a.



-3.9



-15.1



-23.0



Organ Weights of Female Animals



Ovary, l. [g]



mean



0.0586



0.0595



0.0523



0.0427



% diff.



n.a.



+1.5



-10.7



-27.2



Thymus [g]



mean



0.316



0.282



0.280



0.267



% diff.



n.a.



-10.8



-11.4



-15.6



Thyroid, l. [g]



mean



0.0080



0.0086



0.0087



0.0087



% diff.



n.a.



+7.5



+8.3



+8.3



% diff.: % difference to control                                     l.:         Left


n.a.:                 Not applicable


*:                     Statistically significant at p ≤ 0.05 (Dunnett test)


 


Text Table 4‑16:    Oestrous cycle monitoring during the pre-mating period








































Group / Dose level



Mean cycle length [d]



Number of cycles



TD 15 - TD 29



TD 15 - 29



TD 30 - Mating



Group 1


(Control)



3.92



2.5



0.0



Group 2


(100 mg/kg)



3.87



2.7



0.0



Group 3


(300 mg/kg)



3.84



2.8



0.0



Group 4


(1000 mg/kg)



3.93



2.4



0.0



 


Text Table 4‑17:    Fertility indices per group































Group / Dose level



Fertility index [%]



Pregnant rats /


Females paired with their male partners



Group 1


(Control)



100



10 / 10



Group 2


(100 mg/kg)



100



10 / 10



Group 3


(300 mg/kg)



90



9 / 10



Group 4


(1000 mg/kg)



90



9 / 10



 


Text Table 4‑18:    Gestation indices per group































Group / Dose level



Gestation index [%]



Dams with live pups / pregnant rats



Group 1


(Control)



100



10 / 10



Group 2


(100 mg/kg)



100



10 / 10



Group 3


(300 mg/kg)



100



9 / 9



Group 4


(1000 mg/kg)



33



3 / 9



 


Text Table 4‑19:    Pre-coital time and gestation length



































Group / Dose level



Pre-coital interval [d]



Gestation length [d]



TD 29 - GD 0



GD 0 - LD 1



Group 1


(Control)



2.1



22.7



Group 2


(100 mg/kg)



2.1



22.2



Group 3


(300 mg/kg)



3.7



22.7



Group 4


(1000 mg/kg)



3.3



22.7



 


Text Table 4‑20:    Overview on implantation sites, resorptions and post-implantation loss of the pregnant female animals of the high dose group

















































































Group 4: Overview on implantation sites, resorptions and post-implantation loss



Female



No. of


Implantation sites



No. of resorptions and stillbirths



Post-implantation loss (%)



71 (NVP)



19



19



100.0



72 (NVP)



9



9



100.0



73 (NVP)



14



14



100.0



74 (NVP)



15



15



100.0



75



19



17



89.5



76 (NVP)



10



10



100



77



16



6



37.5



78



16



2



12.5



79 (NVP)



7



7



100.0



Sum:



125



99



n.a.



Mean:



13.9



11.0



82.2



n.a.:                 Not applicable                                      NVP:    No viable pups


 


Text Table 4‑21:    Overview of reproductive parameters

































































































 



Reproductive data



Group 1


(control)



Group 2


(100 mg/kg)



Group 3


(300 mg/kg)



Group 4


(1000 mg/kg)



Parametrical values


(see Section 3.2.6.1 'Reproductive parameters')



Implantation sites (mean per dam) #1



15.7



15.4



15.0



13.9



Pups (born alive and dead)


(mean per dam) #1



14.6



14.7



13.3



3.0 **



Pups born alive (mean per dam) #1



14.3



14.7



13.0



2.9 **



Indices [%]


(see Section 3.2.6.2 'Reproductive indices')



Birth Index



mean per dam #1


group             #2



92.45


92.41



95.56


95.45



89.15


88.89



18.53**


21.60



Live birth Index



mean per dam #1


group             #2



97.07


97.95



100.00


100.00



96.92


97.50



97.78


96.30



Post-implantation loss



mean per dam #1


group             #2



10.37


9.49



4.44


4.55



13.41


13.33



82.16**


79.20



Resorptions and stillbirths



Difference between


no. of implantation sites and no. of pups born alive



mean per dam #3


 


 


sum per group #3



1.5


 


 


15



0.7


 


 


7



2.0


 


 


18



11.0


 


 


99



Number of stillbirths #3



3



0



3



1



#1:



Values taken from Table 24-1 'Birth Indices and Post-implantation loss - Values per Dam - Summary'.



#2:



Taken from Table 23 'Number of Pups and Indices - Overview per Group'.



#3:



Values taken from Table 24-2 'Birth Indices and Post-implantation loss - Values per Dam - Individual Data'; columns 'Stillborn' and 'No. Resorptions + Stillborns'.



 


Text Table 4‑22:    Pre- and post-cull viability index per dam































Group / Dose level



Viability index


LD 4 (pre-cull) [%]



Viability index


LD 13 (post-cull) [%]



Group 1


(Control)



85.1



97.2



Group 2


(100 mg/kg)



100.0 **



98.0



Group 3


(300 mg/kg)



88.0



98.8



Group 4


(1000 mg/kg)



100.0



100.0



**:        Statistically significant at p ≤ 0.01 (Dunnett test).


 


Text Table 4‑24:    Provivo background data on pup body weights

































































Male and female pups



Values from Study No. 38159


Mean values per group ± SD


Range of the individual litter values


(n = 3 - 10)


(number of litter values outside the background data range in brackets; position: below lower limit = left,


above upper limit = right)



Provivo Background Data #1


obtained from the control groups of 20 OECD 422 studies performed at Provivo between


2016 and 2021



Pup body weights


male + female


LD 1


[g]



Group 1



7.20 ± 0.65


6.33 - 8.55 (n = 1)



5 to 95% Percentile


5.75 - 7.87



Group 2



6.97 ± 0.60


6.32 - 8.08 (n = 2)



Group 3



6.88 ± 0.99


(n = 1) 5.00 - 8.46 (n = 2)



Group 4



7.56 ± 1.77


6.38 - 9.6 (n = 1)



Pup body weights


male + female


LD 4


[g]



Group 1



9.73 ± 1.61


8.08 - 13.28 (n = 1)



5 to 95% Percentile


7.71 - 11.70



Group 2



9.65 ± 1.22


8.28 - 12.39 (n = 1)



Group 3



9.89 ± 2.87


(n = 1) 7.27 - 14.56 (n = 2)



Group 4



9.79 ± 2.15


7.94 - 12.15 (n = 1)



Pup body weights


male + female


LD 13


[g]



Group 1



31.98 ± 3.89


27.83 - 37.48 (n = 4)



5 to 95% Percentile


24.34 - 32.75



Group 2



30.63 ± 2.64


26.07 - 34.97 (n = 2)



Group 3



31.05 ± 5.32


24.88 - 41.03 (n = 4)



Group 4



30.88 ± 5.28


26.68 - 36.80 (n = 1)



#1:       Not audited by the QAU.


Values taken from Table 27-2 'Mean Body Weight of the Pups per Dam - Individual Data'.


 


Text Table 4‑25:    Litter weight of the male and female pups per dam on LD 1, LD 4 and LD 13




































































Litter weight for the male and female pups combined



Group



Parameter



LD 1



LD 4



LD 13



Group 1


control



mean per dam



101.8 g



131.5 g



295.8



difference to control



n.a.



n.a.



n.a.



Group 2


100 mg/kg b.w./day



mean per dam



102.2 g



141.0 g



299.8 g



difference to control



+0.4%



+7.2%



+1.3%



Group 3


300 mg/kg b.w./day



mean per dam



88.5 g



124.6 g



292.9



difference to control



-13.1%



-5.3%



-1.0%



Group 4


1000 mg/kg b.w./day



mean per dam



58.5 g



77.9 g



210.7 g



difference to control



-42.5% *



-40.8%



-28.8%



n.a.:   Not applicable


*:       Statistically significant at p ≤ 0.05 (Dunnett test)


Data taken from Table 28-1 'Litter Weight per Dam - Summary'.


Text Table 4‑26:    Provivo background data on litter weights

































































Male and female pups



Values from Study No. 38159


Mean values per group ± SD


Range of the individual litter values


(n = 3 - 10)


(number of litter values outside the background data range in brackets; position: below lower limit = left,


above upper limit = right)



Provivo Background Data #1


obtained from the control groups of 20 OECD 422 studies performed at Provivo between


2016 and 2021



Litter weights


male + female


LD 1


[g]



Group 1



101.79 ± 24.95


(n = 2) 66.3 - 149.7 (n = 1)



5 to 95% Percentile


70.3 - 122.6



Group 2



102.17 ± 10.56


86.4 - 118.3



Group 3



88.48 ± 20.16


(n = 3) 56.9 - 109.1



Group 4



58.50 ± 35.81


(n = 2) 19.2 - 89.3



Litter weights


male + female


LD 4


[g]



Group 1



131.53 ± 34.70


(n = 2) 77.5 - 180.3 (n = 2)



5 to 95% Percentile


104.4 - 164.0



Group 2



140.96 ± 13.57


118.1 - 161.1



Group 3



124.58 ± 28.07


(n = 1) 65.4 - 146.7



Group 4



77.87 ± 52.82


(n = 2) 24.3 - 129.9



Litter weights


male + female


LD 13


[g]



Group 1



295.81 ± 43.76


213.1 - 374.8 (n = 2)



5 to 95% Percentile


205.9 - 322.8



Group 2



299.78 ± 24.03


260.7 - 345.5 (n = 1)



Group 3



292.93 ± 39.01


226.3 - 330.8 (n = 3)



Group 4



210.67 ± 119.35


(n = 1) 73.6 - 291.6



#1:       Not audited by the QAU.


Data taken from Table 28-2 'Litter Weight per Dam - Individual Data'.


 


Text Table 4‑27:    Provivo background data on number of live pups

































































Male and female pups



Values from Study No. 38159


Mean values per group ± SD


Range of the individual litter values


(n = 3 - 10)


(number of litter values outside the background data range in brackets; position: below lower limit = left,


above upper limit = right)



Provivo Background Data #1


obtained from the control groups of 20 OECD 422 studies performed at Provivo between


2016 and 2021



Number of live pups


male + female


LD 1



Group 1



14.3 ± 3.7


(n = 3) 8.0 - 20.0 (n = 2)



5 to 95% Percentile


13.0 - 16.0



Group 2



14.7 ± 1.3


13.0 - 17.0 (n = 1)



Group 3



13.0 ± 3.0


(n = 3) 8.0 - 16.0



Group 4



2.9 ± 5.3 **


(n = 8) 0.0 - 14.0



Number of live pups


male + female


LD 4



Group 1



12.4 ± 5.7


(n = 4) 0.0 - 19.0 (n = 2)



5 to 95% Percentile


13.0 - 16.0



Group 2



14.7 ± 1.3


13.0 - 17.0 (n = 1)



Group 3



11.4 ± 5.2


(n = 5) 0.0 - 16.0



Group 4



2.9 ± 5.3 **


(n = 8) 0.0 - 14.0



Number of live pups


male + female


LD 13



Group 1



8.4 ± 3.2


(n = 3) 0.0 - 10.0



5 to 95% Percentile


10.0 - 10.0



Group 2



9.8 ± 0.4


(n = 2) 9.0 - 10.0



Group 3



8.4 ± 0.8


(n = 4) 0.0 - 10.0



Group 4



2.4 ± 4.3 *


(n = 7) 0.0 - 10.0



#1:       Not audited by the QAU.


*:          Statistically significant at p ≤ 0.05 (Dunnett test).


Text Table 4‑28:    Provivo background data on pup ano-genital distance on LD 4



















































































Male and female pups



Values from Study No. 38159


Mean values per group ± SD


Range of the individual litter values


(n = 3 - 10)


(number of litter values outside the background data range in brackets; position: below lower limit = left,


above upper limit = right)



Provivo Background Data #1


obtained from the control groups of 20 OECD 422 studies performed at Provivo between


2016 and 2021



Ano-genital distance of male pups


[ocular units]



Group 1



33.43 ± 3.38


31.6 - 39.7



5 to 95% Percentile


29.74 - 43.43



Group 2



33.78 ± 3.19


(n = 1) 28.8 - 40.4



Group 3



30.74 ± 7.79


(n = 6) 24.4 - 45.0 (n = 1)



Group 4



37.61 ± 5.05


33.0 - 43.0



Ano-genital distance of female pups


[ocular units]



Group 1



16.49 ± 2.09


14.8 - 19.9



5 to 95% Percentile


13.30 - 25.0



Group 2



17.24 ± 1.96


14.3 - 20.6



Group 3



15.44 ± 4.08


(n = 2) 11.8 - 22.7



Group 4



18.15 ± 3.32


15.8 - 20.5



Ano-genital distance of male pups


[ocu. u./(g b.w.)]



Group 1



15.73 ± 1.13


14.1 - 17.9



5 to 95% Percentile


13.94 - 20.27



Group 2



15.80 ± 1.55


(n = 1) 13.5 - 18.6



Group 3



14.6 ± 2.50


(n = 6) 12.1 - 18.4



Group 4



17.49 ± 1.75


15.5 - 18.7



Ano-genital distance of female pups


[ocu. u./(g b.w.)]



Group 1



7.75 ± 1.00


(n = 1) 6.2 - 9.4



5 to 95% Percentile


6.30 - 11.80



Group 2



8.20 ± 0.96


6.8 - 9.9



Group 3



7.23 ± 1.37


(n = 1) 5.7 - 9.4



Group 4



9.13 ± 1.96


7.7 - 10.5



#1:       Not audited by the QAU.


Data taken from Table 29-2 'Ano-genital Distance of the Pups - Mean Values per Dam - Individual Data'.


Text Table 4‑29:    Overview on nipple-bearing pups












































































Pups with nipples #1



Group 1


(Control)



Group 2


100 mg/kg



Group 3


300 mg/kg



Group 4


1000 mg/kg



Dam no.



Pup no.


(number of nipples) #2



dam no.



Pup no.


(number of nipples)



dam no.



Pup no.


(number of nipples)



dam no.



Pup no.


(number of nipples)



14



14-04 (1)



33



33-01 (1)



51



51-08 (1)



77



77-01 (1)



 



 



37



37-04 (1)



52



52-02 (1)



78



78-01 (1)



 



 



39



39-03 (1)



55



55-01 (1)



 



 



Summary



1



1 Pup



3



3 Pups



3



3 Pups



2



2 Pups



#1



See table A ‘Individual Pup Data’ from the listed dams.



#2



The number of nipples that were noted for the listed pup are given in brackets.



 


Text Table 4‑30:    Results of the test item formulation analysis.





















Parameter



Sampling / dealing



Range of


% nominal concentration



Stability and Concentration



immediately, 8 h and 24 h after preparation of the test item formulations



104.5% - 107.5%



Concentration



before administration to the last animal of the group at the end of the study



101.8% - 104.7%



 

Conclusions:
General toxicity
Parental male and female animals
No test item-related prematurely deceased animals were observed. One female animal in the high dose group was killed due to animal welfare reasons and replaced.
No changes in behaviour, external appearance or the faeces considered to be adverse were noted for any dose group.
No test item-related changes were noted for the body weight, the body weight gain and for the food consumption.
Decreases in the number of red blood cells, the level of haemoglobin and the haematocrit were noted for the three investigated female high dose animals (1000 mg test item/kg b.w./day).
No test item-related differences were noted for the examination of the biochemical parameters and the T4 levels.
Although not statistically significant, lower serum levels of progesterone were noted for the female animals dosed with 1000 mg test item/kg b.w./day compared to the control and other two dose groups. Nevertheless, even lower values were observed for the dams which did not delivered live pups. This was in line with their histopathology observed poorly developed corpus lutea..
No test item-related significant differences were noted for the body weight at autopsy and the absolute and relative organ weights and no pathological changes considered to be test item-related were observed during necropsy.
No pathological lesions that were related to the test item were noted during the histopathological examination of the male animals of the high dose group (1000 mg test item/kg b.w./day).
No pathological lesions in the reproductive organs were related to the test item were noted during the histopathological examination in the dams that delivered live pups, regardless of the dose group (100, 300 or 1000 mg test item/kg b.w./day).
Histological differences were noted of the high dose females which lost all implantations. These included implantation sites appearing unphysiological, resorption in the uterus, poorly developed corpora lutea in the ovary and poorly developed mammary glands in 2 of 5 (2/5) females of Group 4. Poorly developed corpora lutea and mammary glands indicate the insufficient progesterone level necessary to maintain pregnancy. The implantation loss of 6 dams in the high dose group was considered to be due to stress of the animals during the study (see amendment to final report). There were no abnormalities in the histology of the reproductive organs from the remaining three females of Group 4, as well as from 5 females/group of Groups 2 and 3, examined in this study.
Histopathological examination revealed pathological changes of the adrenal glands of the female high dose animals (1000 mg test item/kg b.w./day) in form of diffuse adrenocortical hypertrophy or multifocal to diffuse fatty vacuoles. Additionally, an increased group mean severity grade for thymus atrophy was observed for the female high dose animals. These findings in adrenals and thymus may indicate that the high dose females, even in females successful of pregnancy, were under a stressful condition, although no major differences in the terminal body weights were detected

Reproductive toxicity
Parental females
No influences were noted on the fertility index, the pre-coital time and the gestation length.
At 1000 mg test item/kg b.w./day, a reduction was noted on the gestation index.

Pups
Pre-natal development:
Nevertheless, at the high dose level (1000 mg test item/kg b.w./day), a reduced number of implants per dam was noted. Additionally, 6 of 9 pregnant high dose females displayed a resorption of all of their implantations, resulting in an increased post-implantation loss and a decreased birth index. These effects were considered to occur due to stress of the dams in the high dose group.
Post-natal development:
No influence on the viability index was noted. Following the decreased birth index a reduction of the litter weight was noted for the high dose group (1000 mg test item/kg b.w./day).
No test item-related abnormalities (malformations or variations) were noted during the external macroscopic examination of the pups at necropsy.


General toxicity (parental animals)

NOAEL (female) 300 mg test item/kg b.w./day, p.o.
Based on histopathological changes for female animals treated with 1000 mg test item/kg b.w./day.


NOAEL (male) above 1000 mg test item/kg b.w./day, p.o.


Reproductive toxicity

a) adverse effects on the reproductive parameters of the parental females
NOAEL above 1000 mg test item/kg b.w./day, p.o.


b) adverse effects on pre-natal development
NOAEL above 1000 mg test item/kg b.w./day, p.o.


c) adverse effects on post-natal development
NOAEL above 1000 mg test item/kg b.w./day, p.o.
Executive summary:

The aim of the study was to obtain information on possible effects of the test item on general toxicity, reproduction and/or development based on OECD guideline 422. The test item was administered orally to rats at dose levels of 100, 300 or 1000 mg/kg b.w./day.


 


























































































































































































































































































General toxicity - Parental animals



Mortality (test item-related)



Males and females


No test item-related premature deaths were noted for the male and female animals of any dose group (100, 300 or 1000 mg test item/kg b.w./day).


  

Clinical signs



Males and females


Non-adverse salivation was noted for 2 females of the intermediate dose group (300 mg test item/ kg b.w./day) and for 7 males and 7 females of the high dose group (1000 mg test item/kg b.w./day).


Additionally, in the intermediate dose group, animal no. 54 was noted with piloerection, a hemorrhagic vagina and the animal was noted to be pale on the last day of its gestation period (GD 23) and on the first day of the lactation period (LD 1). Furthermore, also the female animal no. 60 was noted with a hemorrhagic vagina on one day of its pseudo-gestation period.


  

Observational Screening


(TD 48/49 for the males,


TD 63 (LD 8-12) for the females)



Males and females


No test item-related influence was noted.


  

Grip strength


(TD 48/49 for the males,


TD 63 (LD 8-12) for the females)



Males and females


No test item-related influence was noted.


  

Spontaneous motility


(TD 48/49 for the males,


TD 63 (LD 8-12) for the females)



Males and females


No test item-related influence was noted.


  

Body weight and


body weight gain



Males and females


No test item-related influence on the body weight and body weight gain was noted.


  

Food consumption



Males and females


No test-item related differences were noted.


  

Drinking water consumption



Males and females


No test-item related changes were noted by visual appraisal.


  

Haematology



Males


No test item-related differences were noted.


Nevertheless, statistically significantly decreased numbers of red blood cells were noted for the low (‑5.8%; p ≤ 0.05) and intermediate dose groups (‑6.8%; p ≤ 0.01). In addition, a statistically significantly increased number of neutrophils was noted for the low dose.


Females


In the high dose group (1000 mg test item/kg b.w./day), consisting of three female animals, decreases were noted for the levels of red blood cells and haemoglobin and for the haematocrit (6.5%, 6.1% and 7.9% below the value of the control group, not statistically significant)


 

Clinical biochemistry



Males and females


No test item-related differences were noted.


 

T4 level analysis



Males and females


No test item-related differences were noted.


However, the mean T4 levels of all four groups were below the range of the Provivo background data. Yet, there was not statistically significant change or dose dependency noticed.


 
  
 


Examination at termination



Body weight at autopsy



Males and females


No influence on body weight at autopsy was noted.


  

Macroscopic post mortem examination



Males and females


No test item-related pathological changes were noted.


  

Oestrous stage at necropsy



No test item-related differences were noted.


  

Organ weights



Males and females


No test item-related differences were noted.


  

Histopathological examinations



Males


No test item-related pathological changes were noted.


 


Females


No pathological lesions in the reproductive organs that were related to the test item were noted during the histopathological examination in the dams that delivered live pups. The two investigated high dose females with a total loss of implantations showed some changes such as implantation sites appearing unphysiological, resorption in the uterus, poorly developed corpora lutea in the ovary and poorly developed mammary glands in 2 of 5 (2/5) females of Group 4. Poorly developed corpora lutea and mammary glands indicate an insufficient progesterone level, which is necessary to maintain pregnancy. The implantation loss was due to stress of the high dose females (see Amendment to final report).


There were no abnormalities in the histology of the reproductive organs from the remaining three females of Group 4, as well as from 5 females/group of Groups 2 and 3, examined in this study.


Histopathological changes in the adrenal glands were noted for the female high dose animals (1000 mg test item/kg b.w./day) in form of diffuse adrenocortical hypertrophy or multifocal to diffuse fatty vacuoles. In addition, the group mean severity grade for thymus atrophy was increased in the high dose females. These findings in adrenals and thymus may indicate that the high dose females, even in females successful of pregnancy, were under a stressful condition, although no major differences in the terminal body weights were detected.


  


Reproductive toxicity


Reproductive Parameters of the Parental Females



Oestrous cycle examination



No test item-related differences for the oestrous cycle length and for the number of oestrous cycles in the period were noted from start of dosing until a positive mating sign was noted.


  

Fertility index



No test item-related influence was noted.


  

Gestation index



For the females of the high dose group (1000 mg test item/kg b.w./day), a decrease was noted for the gestation index (33% compared to 100% in the control group) due to 6 high dose females with a total loss of implantations.


  

Pre-coital time



No test item-related influence was noted.


Nevertheless, an increased pre-coital time was noted for the intermediate (76.2%) and high dose groups (57.1%, statistically not significant) driven by the one animal in each (nos. 60 and 81) which did not get pregnant during mating period.


  

Gestation length



No test item-related influence was noted.


  

Pups - Pre- and Postnatal Development



Prenatal development (from conceptus to birth)



Reproductive parameters



For the dams of the high dose group (1000 mg test item/kg b.w./day), a slight reduction was noted for the number of implantation sites per dam (13.9 implantation sites per dam in comparison to 15.7 in the control).


Also, an increased number of resorptions per dam (11.0 in comparison to 1.5 in the control group), leading to a statistically significantly increased post-implantation loss (82.2% compared to 10.4% in the control group). The birth index was statistically significantly decreased to 18.5% (control group: 92.5%).


No significant influence was noted at the live birth index. For all test groups, three stillbirths each were noted in the control and low dose group and one stillbirth in the high dose group.


 

Postnatal development (pup), LD 1 - LD 4



Mortality (Viability index)



No test item-related influence was noted.


Nevertheless, in the control (dam 20) and the intermediate dose group (dam 58) lost all pups between LD 2 and 4.


  

Male to female ratio



No test item-related influence was noted.


  

Pup body weight



No test item-related influence was noted.


  

Litter weight



Due to the low number of live born pups in the high dose group (1000 mg test item/kg b.w./day), reduced litter weights were noted for the male and the female litters on LD 1 LD 4 and LD 13 (42.5%, 40.8% and 28.8% below the value of the control group, statistically significant for the male and female litters combined on LD 1 at p ≤ 0.05).


This reduction was considered to be caused by the reduced number of implants.


  

Number of live pups



Due to the low number of live born pups at 1000 mg test item/kg b.w./day, this consequently carried on as a reduced number of live pups on LDs 1, 4 and 13 (79.8%, 76.7% or 70.9% below the value of the control group, statistically significant at p ≤0.05 or 0.01).


  

Ano-genital distance



No test item-related influence was noted.


  

Nipple retention



No test item-related influence was noted.


  

External examination



The external macroscopic examination of the pups after sacrifice revealed no gross abnormalities besides for one dam in the intermediate dose group. The pups of dam no. 59 showed ringtails, which are the results of environmental factors and not test item-related.


  

Thyroid hormone levels of the pups



 


No test item-related influence was noted.


  

Analysis of test item formulation



Stability and concentration


 



The analysis of the test item formulation revealed concentrations that ranged between 101.8% and 107.5% of the nominal concentration. This confirms correctly prepared test item formulations.


Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
23.06.2020 to 07.07.2020
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
Short term study with reduced scope of examination
Qualifier:
equivalent or similar to guideline
Guideline:
other: dose range finding study for OECD 422 Selection of route of administration: According to OECD guidelines 421 and 422.
Version / remarks:
2016
Principles of method if other than guideline:
- Principle of test:
14-day dose-range-finding study to select the dose levels for a reproduction/developmental toxicity study of the test item administered by oral administration to rats daily over 14 days.

- Short description of test conditions:
24 animals (12 males and 12 females)
Oral administration by gavage
2 mL/kg b.w./day , vehicle: water
single housing in MAKROLON cages
temperature 22°C ± 3°C and the relative humidity 55% ± 10%

Duration of the study:
6 adaptation days
14 days of treatment
Necropsy on test day 15

- Parameters analysed / observed:
Clinical signs
Mortality
Body weight
Food and drinking water consumption
Necropsy
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
other:
Details on species / strain selection:
CD/ Crl:CD(SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Strain: Rat / CD / Crl:CD(SD)
- Sex: male and female
- Age: 62 days
- Body weight (at 1st administration): Males: 340,6 g - 369,9 g, Females: 216,3 g - 249,0 g
- Housing: singly in MAKROLON cages (type III plus) with a basal surface of approx. 39 cm × 23 cm and a height of approx. 18 cm, Granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt, Germany) was used as bedding material for the cages. The cages were changed and cleaned once a week
- Diet (ad libitum): ssniff® R/Z V1324, ssniff Spezialdiäten GmbH, 59494 Soest, Germany
- Water (ad libitum): tap water
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS:
- Temperature: 22°C ± 3°C
- Humidity: 55% ± 10%
- Photoperiod: 12 hours dark/12 hours light
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
Frequency of administration: Once daily for 14 days
Administration volume: 2 mL/kg b.w./day
Vehicle: Tap water
Dose levels : Group 1: Control (vehicle), Group 2: 65 mg/kg b.w./day, Group 3: 200 mg/kg b.w./day, Group 4: 600/1000 mg/kg b.w./day
The test item formulations were administered orally at a constant volume once daily. The control animals received the vehicle at the same administration volume daily in the same way.
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
For each test item mixed with the vehicle, tests by appropriate analytical methods will be carried out for the main study to determine the concentration and, if needed, stability of the test item in the formulations.
Duration of treatment / exposure:
14 days
Frequency of treatment:
once daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
vehicle control
Dose / conc.:
65 mg/kg bw/day (nominal)
Remarks:
low dose level
Dose / conc.:
200 mg/kg bw/day (nominal)
Remarks:
intermediate dose level
Dose / conc.:
600 mg/kg bw/day (nominal)
Remarks:
high dose level until test day 8
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
high dose level after test day 8
No. of animals per sex per dose:
3
Control animals:
yes, concurrent vehicle
Details on study design:
The rat was selected because of its proven suitability in toxicology and reproduction studies and its compliance with regulatory requirements for testing in a rodent animal species.

The dose levels were selected by the Monitor based on available data. The initial high dose level of 600 mg/kg b.w./day was selected due to an oral LD50 value of 918 mg/kg b.w./day. The initial high dose level of 600 mg/kg b.w./day was increased to 1000 mg/kg b.w./day as of test day 8 onwards as none of the animals of the high dose group revealed any signs of toxicity during the first test week employing 600 mg/kg b.w./day.
Positive control:
no
Parental animals: Observations and examinations:
CLINICAL SIGNS
Each animal was observed before and after dosing at each time of dosing for any signs of behavioural changes, reaction to treatment or illness. In addition, the animals were checked regularly throughout the working day from 7.00 a.m. to 3.45 p.m. by cageside observations. On Saturdays and Sundays, the animals were checked regularly from 7.00 a.m. to 11.00 noon with a final check performed at approximately 3.30 p.m. Cageside observations included skin/fur, eyes, mucous membranes, respiratory and circulatory systems, somatomotor activity and behaviour patterns. The onset, intensity and duration of any signs observed were recorded. Dated and signed records of appearance, change and disappearance of clinical signs of each animal were maintained on clinical history sheets.

MORTALITY
Further checks were made early in the morning and again in the afternoon of each working day to look for dead or moribund animals. This would have allowed post mortem examination to be carried out during the working period of that day. On Saturdays and Sundays, a similar procedure was followed with a final check at approximately 3.30 p.m.

BODY WEIGHT
The body weight of each rat was recorded at the time of group allocation and daily thereafter during the treatment period for dose adjustment. Weekly values are stated in the report.

FOOD AND DRINKING WATER CONSUMPTION
The quantity of food left by individual animals was recorded on a weekly basis throughout the experimental period. The food intake per animal (g/animal/week) was calculated using the total amount of food given to and left by each animal in each group on completion of a treatment week.
The relative food consumption (in g/kg b.w./day) was determined using the following formula: (total food given [g] - total food left [g]) / number of animal days x body weight [kg])

The drinking water consumption was monitored daily by visual appraisal throughout the study.
Postmortem examinations (parental animals):
On test day 15 (approximately 24 hours after the last administration) all animals were euthanized by carbon dioxide (CO2) inhalation, exsanguinated by cutting the aorta abdominalis, weighed, dissected and inspected macroscopically under the direction of a pathologist.
All superficial tissues were examined visually and by palpation. The cranial roof was removed to allow observation of the brain, pituitary gland, and cranial nerves. After ventral midline incision and skin reflection, all subcutaneous tissues were examined. The conditions of the thoracic viscera were noted with due attention to the thymus, lymph nodes, and heart.
The abdominal viscera were examined before and after removal. The urinary bladder was examined externally and by palpation. The gastro-intestinal tract was examined as a whole, and the stomach and caecum were incised and examined. The lungs were removed and all pleural surfaces were examined under suitable illumination. The liver and the kidneys were examined. Any abnormalities in the appearance and size of the gonads, adrenal glands, uterus, intra-abdominal lymph nodes, and accessory reproductive organs were recorded.
The weights of the following organs of all animals were determined before fixation:
Adrenal gland (2)
Brain
Epididymis (2)
Heart
Kidney (2)
Liver
Ovary (2)
Spleen
Prostate and seminal vesicle with coagulating glands (as a whole)
Testicle (2)
Thymus
Uterus including cervix
Paired organs were weighed individually and identified as left or right. Special attention was paid to the organs of the reproductive system.
The following organs or parts of organs of all animals were fixed in 7% buffered formalin except for the testes and epididymides, which were preserved in modified Davidson's solution:
Epididymis (2)
Organs showing macroscopic lesions (if any)
Ovary (2)
Prostate and seminal vesicle with coagulating glands (as a whole)
Testicle (2)
Uterus including cervix
Vagina
Statistics:
Text table 3-1: Group size / Dose levels

Group Propan-1-ol, 2-amino-2-
methyl-, 4-methylbenzenesulphonate
dose
[mg/kg b.w./day, p.o.] No. and sex of animals Animal number
1 0 (control) 3 m/ 3 f 1-3/ 4-6
2 65 (low dose) 3 m/ 3 f 7-9/ 10-12
3 200 (intermediate dose) 3 m/ 3 f 13-15/ 16-18
4 600 / 1000# (high dose) 3 m/ 3 f  19-21/ 22-24
m: male
f: female
#: The initial high dose level of 600 mg/kg b.w./day was increased to 1000 mg/kg
b.w./day as of test day 8 onwards as none of the animals of the high dose group
revealed any signs of toxicity during the first test week employing 600 mg/kg
b.w./day.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related changes in behaviour, the external appearance were noted during the daily cage side observations of the male and female animals that were treated with 65, 200 or 600/1000 mg test item/kg b.w./day.
However, slight salivation was observed for the high dosed male no. 21 on test day 11 in the morning at work start before dosing, lasting up to 60 min after dosing. Further, a decreased water consumption was noted for 1 of 3 females of the low dose group, and for 2 of 3 females each of the intermediate and the high dose group on test day 6. Both observations (salivation and decreased water consumption) were regarded to be incidental and of no toxicological relevance. Since these changes were noted only on a single test day during the 14-day treatment period. The faeces of all animals showed a normal consistency during the course of the study.
Mortality:
no mortality observed
Description (incidence):
No premature deaths were noted for the male and female animals treated with 65, 200 or 600/1000 mg test item/kg b.w. daily for 14 days.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No test item-related difference in body weight and body weight gain were noted between the control group and the treatment groups (65, 200 or 600/1000 mg test item/kg b.w./day). No test item-related difference in the body weight at autopsy.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No test item-related difference in food consumption was noted between the control group and the treatment groups (65, 200 or 600/1000 mg test item/kg b.w./day).
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, non-treatment-related
Description (incidence and severity):
Male: The visual appraisal of the drinking water consumption did not reveal any test item related influence on the water intake of the male animals at any tested dose level.
Female: A decreased water consumption was noted for 1 of 3 females of the low dose group and for 2 of 3 females each of the intermediate and the high dose group on one test day. The visual appraisal of the drinking water consumption on the other test days did not reveal any test item-related influence on the water intake of the animals at any tested dose level.
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):
Slight salivation was observed for the high dosed male no. 21 on test day 11 in the morning at work start before dosing, lasting up to 60 min after dosing. Further, a decreased water consumption was noted for 1 of 3 females of the low dose group, and for 2 of 3 females each of the intermediate and the high dose group on test day 6.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
not examined
Dose descriptor:
other: no descriptor determined; dose range finding study for OECD 422
Sex:
male/female
Critical effects observed:
yes
Lowest effective dose / conc.:
200 mg/kg bw/day (nominal)
Organ:
lymph node
Treatment related:
yes
Dose response relationship:
not specified
Relevant for humans:
not specified
Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality / viability:
not examined
Body weight and weight changes:
not examined
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:
not examined
Anogenital distance (AGD):
not examined
Nipple retention in male pups:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined

Text table 3‑1:      Group size / Dose levels




































Group



dose


[mg/kg b.w./day, p.o.]



No. and sex


of animals



Animal


number



1



0


(control)



3 m


3 f



1 - 3


4 - 6



2



65


(low dose)



3 m


3 f



7 - 9


10 - 12



3



200


(intermediate dose)



3 m


3 f



13 - 15


16 - 18



4



600 / 1000#


(high dose)



3 m


3 f



19 - 21


22 - 24



m:   male


f:     female


#:    The initial high dose level of 600 mg/kg b.w./day was increased to 1000 mg/kg b.w./day as of test day 8 onwards as none of the animals of the high dose group revealed any signs of toxicity during the first test week employing 600 mg/kg b.w./day.


 


Text table 3‑2:      Weighed organs














Weighed organs



Adrenal gland (2)


Brain


Epididymis (2)


Heart


Kidney (2)


Liver



Ovary (2)


Spleen


Prostate and seminal vesicle


  with coagulating glands


  (as a whole)


Testicle (2)



Thymus


Uterus including cervix


 


 


 


 



 


Text table 3‑3:      Preserved organs













Preserved organs



Epididymis (2)


Organs showing macroscopic lesions (if any)


Ovary (2)


Prostate and seminal vesicle


  with coagulating glands (as a whole)



Testicle (2)


Uterus including cervix


Vagina


 


 



 


Text table 3‑4:      Statistics














Test



Parameter



Multiple t-test based on


DUNNETT, C. W.


New tables for multiple


comparisons with a control.


Biometrics, 482-491


(September 1964)



Body weight / Food consumption /


Relative and absolute organ weights


(p ≤ 0.05 and p ≤ 0.01)



 


Text table 4‑1:      Body weight gain of male animals during the treatment period
























Males



Group 1


Control



Group 2


65 mg/kg



Group 3


200 mg/kg



Group 4


600/


1000 mg/kg



Body weight gain #


(test day 1 to test day 14)



+13.7 %



+14.9 %



+16.9 %



+14.4 %



#:



Values taken from table 3-1 'Body Weight Gain - Summary - Males'



 


Text table 4‑2:      Body weight gain of female animals during the treatment period
























Females



Group 1


Control



Group 2


65 mg/kg



Group 3


200 mg/kg



Group 4


600/


1000 mg/kg



Body weight gain #


(test day 1 to test day 14)



+7.1 %



+11.3 %



+10.7 %



+8.6 %



#:



Values taken from table 3-2 'Body Weight Gain - Summary - Females'



 


Text table 4‑3:      Macroscopic post mortem findings

































Macroscopic organ changes


(number of animals affected / examined)



Organ / Finding



Group 1


Control



Group 3


200 mg/kg



Group 4


600/1000 mg/kg



 



m



f



m



f



m



f



Mandibular lymph nodes


  enlarged



 


0 / 3



 


0 / 3



 


3 / 3



 


3 / 3



 


2 / 3



 


3 / 3



 


Text table 4‑4:      Organs with noticeable changes in comparison to control; statistically significant from control or not (not considered to be test item-related)












































































































































































 



Changes in comparison to the control group [%]



Group 2



Group 3



Group 4



Males



 



Adrenal gland (left)



(g) #1



- 4.3



- 8.6



- 19.0



(g/kg) #2



- 1.6



- 4.7



- 16.0



 



Adrenal gland (right)



(g) #1



- 16.9



- 11.9



- 28.8



(g/kg) #2



- 14.5



- 7.8



-26.1



 



Thymus



(g) #1



-15.3



-16.0



-22.2



(g/kg) #2



- 13.3



- 12.7



- 19.1



 



Spleen



(g) #1



+13.6



+11.4



+4.5



(g/kg) #2



+16.5**



+15.9*



+8.3



 



Females



 



Adrenal gland (left)



(g) #1



+19.3



+27.2



+12.3



(g/kg) #2



+16.0



+27.5



+10.3



Testis left



Adrenal gland (right)



(g) #1



+20.6



+23.4



+8.4



(g/kg) #2



+17.6



+24.3



+6.3



 



Thymus



(g) #1



+65.0



+28.0



+51.0



(g/kg) #2



+66.6



+33.4



+53.1



 



Spleen



(g) #1



+0.6



- 8.4



- 2.4



(g/kg) #2



- 0.8



- 7.8



- 3.3



*/**:



Statistically significant from control at p ≤ 0.05 / 0.01 (Anova / Dunnett test).



#1:



Values taken from table 8-1 'Absolute Organ Weights - Sum. - Males' and


table 8-2 'Absolute Organ Weights - Sum. - Females'



#2:



Values taken from table 7-1 'Relative Organ Weights - Sum. - Males' and


table 7-2 'Relative Organ Weights - Sum. - Females'


      

 

Conclusions:
The aim of the study was to select the dose levels of the test item for a reproduction/developmental toxicity study (OECD 422). The rats were treated with 65, 200 or 600 mg test item/kg b.w. daily from test day 1 to test day 14. On test day 8 the high dose level was increased to 1000 mg /kg b.w. daily, as no signs of toxicity were noted.
Executive summary:

The aim of the study was to select the dose levels of the test item for a reproduction/developmental toxicity study (OECD 422).


The rats were treated with 65, 200 or 600 mg test item/kg b.w. daily from test day 1 to test day 14. On test day 8 the high dose level was increased to 1000 mg test item/kg b.w. daily, as no signs of toxicity were noted.


None of the animals died prematurely.


No test item-related observations were noted in behaviour, the external appearance or the appearance of the faeces.


Body weight and food consumption of the male and female animals revealed no test item-related differences.


During the macroscopic inspection at necropsy mandibular lymph nodes with an increased size were noted for nearly all male and female animals of the intermediate and the high dose group (200 or 600/1000 mg test item/kg b.w./day).


After consideration of these data, the following dose levels are suggested for a reproduction/ developmental toxicity study in CD® rats based on OECD guideline 422:


Group 1:    Control (vehicle)


Group 2:    100 mg test item/ kg b.w./day


Group 3:    300 mg test item/ kg b.w./day


Group 4:    1000 mg test item/ kg b.w./day

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Klimisch 1
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

OECD 422 Main study


 


General toxicity
Parental male and female animals
No test item-related prematurely deceased animals were observed. One female animal in the high dose group was killed due to animal welfare reasons and replaced.
No changes in behaviour, external appearance or the faeces considered to be adverse were noted for any dose group.
No test item-related changes were noted for the body weight, the body weight gain and for the food consumption.
Decreases in the number of red blood cells, the level of haemoglobin and the haematocrit were noted for the three investigated female high dose animals (1000 mg test item/kg b.w./day).
No test item-related differences were noted for the examination of the biochemical parameters and the T4 levels.

No test item-related significant differences were noted for the body weight at autopsy and the absolute and relative organ weights and no pathological changes considered to be test item-related were observed during necropsy.
No pathological lesions that were related to the test item were noted during the histopathological examination of the male animals of the high dose group (1000 mg test item/kg b.w./day).
No pathological lesions in the reproductive organs were related to the test item were noted during the histopathological examination in the dams that delivered live pups, regardless of the dose group (100, 300 or 1000 mg test item/kg b.w./day).
Histological differences were noted of the high dose females which lost all implantations. These included implantation sites appearing unphysiological, resorption in the uterus, poorly developed corpora lutea in the ovary and poorly developed mammary glands in 2 of 5 (2/5) females of Group 4. Poorly developed corpora lutea and mammary glands indicate the insufficient progesterone level necessary to maintain pregnancy.  There were no abnormalities in the histology of the reproductive organs from the remaining three females of Group 4, as well as from 5 females/group of Groups 2 and 3, examined in this study.
Histopathological examination revealed pathological changes of the adrenal glands of the female high dose animals (1000 mg test item/kg b.w./day) in form of diffuse adrenocortical hypertrophy or multifocal to diffuse fatty vacuoles. Additionally, an increased group mean severity grade for thymus atrophy was observed for the female high dose animals.These results may indicate that the HD females, even in females successful of pregnancy, were under the stressful condition, although no major differences in the terminal body weights were detected. 


Reproductive toxicity
Parental females
No influences were noted on the fertility index, the pre-coital time and the gestation length.
At 1000 mg test item/kg b.w./day, a reduction was noted on the gestation index. This was due to loss of all implantations of 6 high dose females and considered to be due to stress of the high dosed females and therefore not test item related.


Pups
Pre-natal development:
Nevertheless, at the high dose level (1000 mg test item/kg b.w./day), a reduced number of implants per dam was noted. Additionally, 6 of 9 pregnant high dose females displayed a resorption of all of their implantations, resulting in an increased post-implantation loss and a decreased birth index. This was due to stressed dams in the high dose group which lost all implantations. This effect is regarded as not test item related.
Post-natal development:
No influence on the viability index was noted. Following the decreased birth index a reduction of the litter weight was noted for the high dose group (1000 mg test item/kg b.w./day).
No test item-related abnormalities (malformations or variations) were noted during the external macroscopic examination of the pups at necropsy.


 

Effects on developmental toxicity

Description of key information

No studies have been performed to explicitly address the question of developmental toxicity in animals caused by the test item.


However, daily administration of test item by oral gavage to Sprague-Dawley Crl.:CD (SD) strain rats at dose levels of 100, 300 and 1000 mg/kg bw/day (Low, Mid and High dose groups, respectively) during the conditions of the Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test according to OECD 422 (Provivo 2022) did not result in any test item-related effects on the development of the F1 offspring.


At the high dose level (1000 mg test item/kg b.w./day), a reduced number of implants per dam was noted. Additionally, 6 of 9 pregnant high dose females displayed a resorption of all of their implantations, resulting in an increased post-implantation loss and a decreased birth index. These effects occured due to stress of the high dose females and are therefore not test item related.
No influence on the viability index was noted. Following the decreased birth index a reduction of the litter weight was noted for the high dose group (1000 mg test item/kg b.w./day).
No test item-related abnormalities (malformations or variations) were noted during the external macroscopic examination of the pups at necropsy.


The No Observed Adverse Effect Level (NOAEL) for systemic toxicity was considered to be 300 mg/kg/day for female rats because dosage of of 1000 mg/kg/day resulted in toxicologically significant histopathological findings in the uterus such as unphysiological, resorptions, poorly developed corpora lutea in the ovary and poorly developed mammary glands.  Histopathological changes in the adrenal glands were noted for the female high dose animals  in form of diffuse adrenocortical hypertrophy or multifocal to diffuse fatty vacuoles. In addition, the group mean severity grade for thymus atrophy was increased in the high dose females. These may be stress induced effects.


For male rats no toxicological effects could be observed up to the highest tested dose of 1000 mg/kg bw d.


 


 

Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the results of the performed studies OECD 422 DRF study (Provivo, 2020) and the main OEC D422 study (Provivo 2022), the test material has not be classied according to the criteria of EC Regulation 1272/2008.

Additional information