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Diss Factsheets

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Date of Study Plan: 19 November 2019
Start of pre-estrous: 20 November 2019
Start of pre-mating administration: 04 December 2019
Start of mating: 12 February 2020
End of In-life: 18 June 2020
Date final report 17 February 2022
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
According to the ECHA final decision on a compliance check from 2017-04-05 (Decision number: CCH-D-2114356498-35-01/F and submission number: BQ538638-17) the study design of the EOGRTS according to OECD 443 is as follows:
Based on Article 41 of Regulation (EC) No 1907/2006 (the ‘REACH Regulation’), ECHA requests you to submit information on:
Extended one-generation reproductive toxicity study (Annex X, Section 8.7.3.; test method: EU B.56./OECD TG 443) in rats, oral route with the registered substance specified as follows:
- Ten weeks premating exposure duration for the parental (P0) generation;
- Dose level setting shall aim to induce some toxicity at the highest dose level;
- Cohort 1A (Reproductive toxicity);
- Cohort 1B (Reproductive toxicity) without extension to mate the Cohort 1B animals to produce the F2 generation.

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2021
Reference Type:
publication
Title:
Update of OECD DART guidelines with endocrine disruptor relevant endpoints: Practical considerations
Author:
Manon Beekhuijzen et al.
Year:
2016
Bibliographic source:
Manon Beekhuijzen et al.: Update of OECD DART guidelines with endocrine disruptor relevant endpoints: Practical considerations, Reproductive Toxicology (2016) 64: 64 - 71.
Reference Type:
publication
Title:
Interpreting the toxicologic significance of alterations in anogenital distance: potential for confounding effects of progeny body weights
Author:
Gallavan R. H. et al.
Year:
1999
Bibliographic source:
Gallavan R. H. et al.: Interpreting the toxicologic significance of alterations in anogenital distance: potential for confounding effects of progeny body weights, Reproductive Toxicology (1999) 13: 383 - 390.
Reference Type:
study report
Title:
Unnamed
Year:
2022
Report date:
2022

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Version / remarks:
adopted 25 June 2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Justification for study design:
The study design is based on final decision on a compliance check from ECHA dated 5th of April 2017. An Extended one-generation reproductive toxicity study (Annex X, Section 8.7.3.; test method: EU B.56./OECD TG 443) in rats, oral route with the registered substance specified as follows, was requested:
- Ten weeks premating exposure duration for the parental (P0) generation;
- Dose level setting shall aim to induce some toxicity at the highest dose level;
- Cohort 1A (Reproductive toxicity);
- Cohort 1B (Reproductive toxicity) without extension to mate the Cohort 1B animals to produce the F2 generation;

Test material

Constituent 1
Chemical structure
Reference substance name:
3-aminomethyl-3,5,5-trimethylcyclohexylamine
EC Number:
220-666-8
EC Name:
3-aminomethyl-3,5,5-trimethylcyclohexylamine
Cas Number:
2855-13-2
Molecular formula:
C10H22N2
IUPAC Name:
3-aminomethyl-3,5,5-trimethylcyclohexylamine
Test material form:
liquid
Details on test material:
3-aminomethyl-3,5,5-trimethylcyclohexylamine from Evonik, Batch: 19111847

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Details on species / strain selection:
Rat / CD® / Crl:CD(SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS:
- Species / Strain / Stock: Rat / CD® / Crl:CD(SD)
- Breeder: Charles River Laboratories Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Body weight (at start of dosing): Males: 399.1 g - 463.5 g, Females: 230.0 g - 287.7 g
- Age (at start of dosing): Males and females: 69 days
- Selection of species: The rat is a commonly used rodent species for such studies and required by the guideline.
- Number of parental animals: Pre-exposure period:125 female animals were evaluated pre-exposure for estrous cyclicity to yield 96 females (i.e. 24 per group) with a regular estrous cycle for the study. Main study: 192 (96 male and 96 female) animals in order to grant at least 20 pregnant females per group for evaluation of the F0 Generation.
- Adaptation period: 7 days
- Housing: With exception of the mating period, the male and female animals (F0 Generation) are kept singly in MAKROLON cages (type III plus) with a basal surface of approximately 39 cm x 23 cm and a height of approximately 18 cm. Granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt/ Arkeburg, Germany) was used as bedding material in the cages. The cages were changed and cleaned once a week. Periodic analysis of the bedding material for contaminants based on EPA/USA is conducted by LUFA-ITL.
The animals received one piece of wood (certified for animal use) to gnaw on once weekly at change of the cages. Octagon-shaped red-tinted huts (polycarbonate) were placed in the cages to offer the animals a resting and hiding place.
- Diet (ad libitum): ssniff® R-Z V1324, ssniff Spezialdiäten GmbH, 59494 Soest, Germany, Food residue was removed and weighed.
Periodic analysis of the food for contaminants based on EPA/USA is conducted at least twice a year by LUFA-ITL. Certificates of analysis of the composition and for contaminants are provided by the manufacturer and are included in the raw data. No contaminants above the limitations were noted.
- Water: Tap water was offered ad libitum. Samples of the drinking water are taken periodically by the Wasserwerk Wankendorf and periodic analyses are performed by LUFA-ITL according to the 'Deutsche Trinkwasserverordnung, Bundesgesetzblatt 2001' [German Regulations on drinking water, public notice of the law, 2001 ]. In addition, drinking water samples taken at Provivo are analysed by LUFA-ITL once a year for means of bacteriological investigations according to the 'Deutsche Trinkwasserverordnung 2001. No contaminants above the limitations were noted.

ENVIRONMENTAL CONDITIONS
- Temperature: 22°C ± 2 °C (maximum range)
- Humidity: 55% ± 10% (maximum range)
- Photoperiod: The rooms are alternately lit (about 150 lux at approx. 1.50 m room height) and darkened in a 12 hours dark/12 hours light cycle.
- Air changes per hour: 15 to 20

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
purified water
Details on exposure:
Route of administration: Oral, via gavage
Frequency of administration: Once daily
Vehicle: Aqua ad injectabilia (Batch nos. 192838161, 192738002, 180958001 Braun Melsungen AG; Carl-Braun-Str. 1, 34212 Melsungen, Germany.)
Administration volume: 10 mL/kg b.w.
Dosages: 0, 25, 80, 240 (160) mg/kg bw/d; Dose reduction since 9th of January 2020
Frequency of administration: Once daily (from test day 15 until one day before sacrifice)
Selection of route of administration: According to OECD guideline 443.
The test item formulations were administered at a constant administration volume of 10 mL/kg b.w. once daily. The control animals received the vehicle at the same administration volume in the same way.
Details on mating procedure:
Sexually mature male and female rats of the F0 Generation were randomly paired for mating. Mating was monogamous: 1 male and 1 female animal were placed in one cage during the dark period. The female was placed with the same male until evidence of mating was observed or 2 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 GD0) was considered to be the day on which sperm was found.
Females without a positive mating sign were separated from their male partner after 2 weeks without further opportunity for mating.
If there would have been insufficient males, for example due to male death before pairing, then males which had already mated would have been paired with a second female such that all females would have been paired. However, as 3 males and 3 females prematurely deceased before the start of mating, sufficient male animals were always available in this study.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Preparation of test item administration formulations: The administration formulations were freshly prepared every day. The test item was diluted in the vehicle to the appropriate concentrations. The amount of the test item was adjusted to the animal's current body weight before oral administration at a constant volume of 10 mL/kg b.w. once daily.
Test item formulation analysis: For the analysis of the test item-vehicle formulations, two aliquots of approximately 5 mL were taken at the following times and stored at -20°C ± 10% until analysis.
Test item formulation sampling schedule: F0 Generation – Groups 2 to 4:
- At start of the treatment period (first administration day): Analysis of concentration and homogeneity, At the start, during (middle) and before administration to the last animal of each dose level group. (3 samples / dose level group; groups 2 - 4). Number of samples (aliquots): 3 x 3 = 9 (18)
- At the end of the premating period Analysis of concentration, During treatment always before administration to the last animal/dose level group. (1 sample / dose level group; groups 2 - 4).
Number of samples (aliquots): 3 x 1 = 3 (6)
- At dose reduction of group 4 Analysis of concentration and homogeneity, At the start, during (middle) and before administration to the last animal of dose level group 4. (3 samples / dose level group 4). Number of samples (aliquots): 3 x 1 = 3 (6)
- At termination of the F0 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; groups 2 - 4) Number of samples (aliquots): 3 x 1 = 3 -> (6) Total number of samples (aliquots): 18 (36)

Test item formulation sampling schedule: F1 Generation – Groups 2 to 4:
- At start of the treatment period (first administration day), Analysis of concentration and homogeneity, At the start, during (middle) and before administration to the last animal of each dose level group. (3 samples / dose level group; groups 2 - 4). Number of samples (aliquots): 3 x 3 = 9 (18)
- At termination of the Cohort 1 A 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; groups 2 - 4) Number of samples (aliquots): 3 x 1 = 3 (6)
-> Total number of samples (aliquots): 12 (24)

The samples were labelled with study number, test species, generation, cohort no. type of sample, aliquot number, group, concentration, sampling time and date.
The samples were analysed according the method validated by the test institute.
Three months after the issuance of the Final Report, any samples or aliquots still remaining at the testing facility will be destroyed unless the Sponsor requests otherwise.

Test item-formulation analysis: The measured concentrations of the test substance in the test item-formulations were between 101.6 % and 109.6 % of the nominal concentration, indicating correctly prepared and homogeneous test item-formulations.
Duration of treatment / exposure:
The animals were treated with the test item during the following periods:
F0 GENERATION:
- Males: 10 weeks prior to mating, during the mating period and at least until weaning of the F1 Generation (up to and including the day before sacrifice).
- Females: 10 weeks prior to mating, during the mating and lactation period and until termination of weaning of their litters (up to and including the day before sacrifice).

F1 GENERATION:
- F1 Pups: Until weaning, the pups were indirectly exposed to the test item through the breast milk. During the last week of lactation the pups additionally received the test item directly when they commence eating for themselves.
After weaning, each F1 Pup selected for the F1 Cohorts was dosed via gavage.
- Cohort 1A: The male and female animals were dosed for 10 weeks up to and including the day before sacrifice (males and females: PNDs 90 to 92).
- Cohort 1B: The male and female animals were dosed for 11 weeks up to and including the day before sacrifice (males and females: PNDs 98 to 102).
Frequency of treatment:
daily
Details on study schedule:
The animals were treated with the test item during the following periods:
F0 Generation
Males 10 weeks prior to mating, during the mating period and at least until weaning of the F1 Generation (up to and including the day before sacrifice).
Females 10 weeks prior to mating, during the mating and lactation period and until termination of weaning of their litters (up to and including the day before sacrifice).

F1 Generation
F1 Pups Until weaning, the pups were indirectly exposed to the test item through the breast milk. During the last week of lactation the pups additionally received the test item directly when they commence eating for themselves.
After weaning, each F1 Pup selected for the F1 Cohorts was dosed via gavage.
Cohort 1A The male and female animals were dosed for 10 weeks up to and including the day before sacrifice (males and females: PNDs 90 to 92).
Cohort 1B The male and female animals were dosed for 11 weeks up to and including the day before sacrifice (males and females: PNDs 98 to 102).
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
control group
Dose / conc.:
25 mg/kg bw/day (nominal)
Remarks:
low dose group
Dose / conc.:
80 mg/kg bw/day (nominal)
Remarks:
intermediate dose group
Dose / conc.:
240 mg/kg bw/day (nominal)
Remarks:
high dose group; dose reduced on test day 51 (to 160 mg/kg bw/day)
No. of animals per sex per dose:
20 males and females in P generation and 20 males and females in Cohort 1A and Cohort 1B
Control animals:
yes, concurrent vehicle
Details on study design:
The dose levels for this study were selected by the Sponsor based on the results of a 14 day dose range finding study in rats (LPT study no. 37378, 2020) and an OECD 422 study in rats (Provivo study no. 37482, 2021).
In 14-day dose range finding study, the rats were treated orally with 250 or 500/350 mg IPDA/kg b.w./day. The rats of the OECD 422 study were treated orally with 30, 100 or 300/240 mg IPDA/kg b.w./day.
In the OECD 422 study, two female animals of the high dose group treated with 300/240 mg IPDA /kg b.w./day had to be sacrificed on test days 27 or 29 (one day before or one day after dose reduction on test day 28) due to significant body weight loss. Furthermore, piloerection, reduced motility and breathing sounds were noted for these two animals. Two male animals of the high dose group were found dead on test day 41 or 44 (12 or 15 days after dose reduction). Prior to death, breathing sounds, reduced motility and body weight loss occurred.
On test day 48 (i.e. gestation day 17), one further female animal of the high dose had to be prematurely sacrificed due to severe signs of toxicity, i.e. salivation, reduced motility, laboured breathing and prone position.
During the first lactation week, slight reductions in food consumption were noted for the female animals of the intermediate (100 mg IPDA/kg b.w./day) and high dose group (300/240 mg IPDA/kg b.w./day). Furthermore, the remaining 6 females of the high dose group (with live pups) revealed slightly reduced body weights on lactation day 13. However, both findings were statistically not significant. Breathing sounds occurred in 3 of 6, salivation in 2 of 6 and piloerection in 1 of 6 female animals.
Hence, the dose level of 240 mg/kg/day was considered to be the maximum tolerated dose for the F0 Generation with no systemic effects to be expected in the F1 Generation of the present OECD 443 study.

Deviations:
The study was conducted in accordance with the Study Plan and 10 Study Plan Amendments agreed upon.
The following deviations were noted which were not covered by an Amendment:
Test item formulation
Aqua ad iniectabilia was used as vehicle not purified water as stated in the Study Plan.
Clinical observation
Animal no 182 f was excluded from dosing from test day 22 onwards until premature sacrifice on test day 24, due to marked laboured breathing noted during the daily cage-side observation.
Body Weight
Animal no. 454 m (Group 3, Cohort 1B) was overdosed by approximately 17 % on postnatal day 54 (i.e. 01 May 2020) due to a faulty weighing process.
The body weight was not plausible when compared to the body weights noted on the day before or the day after as follows:
PND 53 (i.e. 30 April 2020): 295.8 g
PND 54 (i.e. 01 May 2020): 351.5 g
PND 55 (i.e. 02 May 2020): 310.5 g

Therefore, the body weight on postnatal day 54 of animal no. 454 should have been excluded from reporting. However, this was impossible due to technical reasons.
All deviations were assessed by the Study Director and do not affect the validity and integrity of the scientific results obtained in this study.
Positive control:
no

Examinations

Parental animals: Observations and examinations:
CLINICAL SIGNS
Dated and signed records of appearance, change, and disappearance of clinical signs were maintained on clinical history sheets for each animal. Throughout the test period, each animal (parental animals and pups) was observed for clinical signs at least once daily. Behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity were recorded. In case signs of toxicity occurred the frequency of observations was increased. Each animal was 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. In addition, the animals were checked regularly throughout the working day from 7:00 a.m. to 3:45 p.m. On Saturdays and Sundays, the 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. Cage side observations included skin/fur, eyes, mucous membranes, respiratory and circulatory systems, locomotor activity and behaviour patterns.
A more detailed examination of all F0 animals was conducted on a weekly basis. F0 animals were examined once before the first test item treatment on test day 14 to allow for within-subject comparisons. Thereafter, the examination was performed weekly until termination. Detailed clinical observations were carried out for all animals outside the home cage in a standard arena at approximately the same time of day, each time preferably by observers unaware of the treatment. The observations included in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, pilo-erection, pupil size, and unusual respiratory pattern). Changes in gait, posture and response to handling as well as the 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) were also recorded.

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. On Saturdays and Sundays, a similar procedure was followed with a final check at approximately 3.30 p.m. If necessary, these provisions allowed to record any premortal symptoms in detail and a post mortem examination to be carried out during the working period of a day.

BODY WEIGHT
The body weight the animals was recorded as follows:
Pre-mating period: Daily, starting on the first day of dosing, i.e. test day 15# - report of weekly values
Mating period: Daily - report of weekly values
Post-mating period: Daily - report of weekly values for the males
Gestation period: - report on GD 0, 7, 14, 21 for the females
Lactation period: - report PND 4, 7, 14, 21 for the females
Termination of in-life: Day of sacrifice

FOOD AND DRINKING WATER CONSUMPTION:
Food intake per rat (g) was calculated using the total amount of food given to and left by each rat in each group on those days that are listed below.
Pre-mating period: Weekly
Mating period: None
Post-mating period: Weekly values for the males#
Gestation period: GD 0, 7, 14, 21
Lactation period: PND 1, 7, 14, 21
#: Starting on a suitable day after the mating period to consolidate all male animals (test day 91)

From these data the relative food consumption (in g/kg b.w./day) was determined using the following formula:
Relative food consumption (g/kg b.w./day) = (Total food given (g) - Total food left (g))/ (Number of animal days# x Body weight (kg))
# The term 'animal days' counts one animal day for each animal alive for a whole
day; it is assumed that on the day of death an animal does not eat.

Drinking water consumption was monitored daily by visual appraisal throughout the study.

REPRODUCTIVE PERFORMANCE
The reproductive parameters and reproductive indices listed below were determined to evaluate the reproductive performance.
Reproductive parameters:
- stages of the estrous cycle
- pre-coital time
- number of pregnant females
- gestation length calculated from day 0 of pregnancy
Implantation sites
- number per dam
- distribution in the uterus horns
- absolute number per group
- mean per group
Number of pups per group and per dam
- at birth (live and dead)
- on postnatal days 1, 4 and 13
Number of male and female pups per group and per dam
- at birth (alive and dead)
- on postnatal days 1, 4 and 13
Number of stillbirths
- per group
- per dam
Number of pups with malformations
- per group
- per dam

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:
Time of blood sampling: At necropsy
Animals: 10 males and 10 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)/ ‰ of the erythrocytes
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 PARAMETERS
The following parameters 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

BIOCHEMISTRY
The following parameters were determined (Instrument: KONELAB 30i, Thermo Fisher Scientific , 63303 Dreieich, Germany):
Parameter (in serum)/ Unit
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

Globulin/ g/L -> by substraction
Albumin/globulin ratio/ non-dimensional -> by calculation
Sodium/Potassium ratio/ non-dimensional -> by calculation
BUN/creatinine ratio/ non-dimensional -> by calculation

DETERMINATION OF THYROID HORMONES (T4 and TSH)
Blood samples were taken under isoflurane anaesthesia from animals fasted overnight always at the same time of day (in the morning between 6.30 a.m. and 9.30 a.m. for the adult animals) as scheduled below:
10 males and 10 females per group#1; fasted; Test days 127/129 (at sacrifice)
#1 Animals also selected for laboratory examinations

Blood samples were processed for serum, divided into aliquots and stored -20°C ± 10% until analyses using commercial ELISA kits.

URINALYSIS
The urine was collected for 16 hours in URIMAX funnel cages. The collection of urine was terminated immediately prior to start of blood withdrawals for the haematological and clinical chemistry examinations. The following sampling times and animals were employed:
At the end of dosing period (prior to blood withdrawal), 10 males and 10 females randomly selected from each group
The following parameters were determined by using the instruments given below:
Parameter/ Unit/ Instrument
Volume/ mL/ Graduated vessel
pH/ non-dimensional/ Digital pH meter, type WTW InoLab pH 720
Specific gravity/ g/mL/ Kern Refractometer, type ORA 2PA Sample compared with water (nominal value of 1.000)
The following examinations were also performed using Combur 9 Test (semi-quantitative/qualitative indicators) for determination of analyte concentration in the urine:
Proteins
Glucose
Bilirubin
Urobilinogen
Ketones
Haemoglobin (Hb) (approximate values)
Nitrite

Microscopic examination of urine samples was carried out by centrifuging samples and spreading the resulting deposit on a microscope slide. The deposit was examined for the presence of the following parameters:
E Epithelial cells
L Leucocytes
R Erythrocytes
B Organisms
C Further constituents (i.e. sperm, casts)
A Crystalluria
The frequency of the above parameters were recorded as follows:
0 None found in any field examined
+ Few in some fields examined
++ Few in all fields examined
+++ Many in all fields examined
The colour and the turbidity of the urine were examined visually.
Oestrous cyclicity (parental animals):
Vaginal smears were taken and the oestrus cycle stages were determined at the following time points:

F0 animals during the 14-day pre-exposure period to select 96 animals with regular oestrus cycles (4-5 days) and during 10 weeks of premating until evidence of mating.

F1 animals, Cohort 1A starting after onset of vaginal patency until first appearance of cornified cells and two weeks starting around PND 75.
F0 and F1 animals On the day of sacrifice, Shortly before necropsy.
Sperm parameters (parental animals):
Sperm viability and morphology (spermiogram)
• All F0 males
• All F1 Cohort 1A males
One epididymis and one testicle will be used for the sperm count. The sperm viability is determined and the sperm morphology is examined according to the method described by I. Chahoud and R. Franz (1993) as well as by S. Plassmann and H. Urwyler (2001).
Litter observations:
CLINICAL SIGNS
Dated and signed records of appearance, change, and disappearance of clinical signs were maintained on clinical history sheets for each animal. Throughout the test period, each animal (parental animals and pups) was observed for clinical signs at least once daily. Behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity were recorded. In case signs of toxicity occurred the frequency of observations was increased. Each animal was 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. In addition, the animals were checked regularly throughout the working day from 7:00 a.m. to 3:45 p.m. On Saturdays and Sundays, the 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. Cage side observations included skin/fur, eyes, mucous membranes, respiratory and circulatory systems, locomotor activity and behaviour patterns.
A more detailed examination of all F1 Cohort 1B animals was conducted on a weekly basis. The F1 animals of Cohort 1B were examined weekly after weaning until termination. Detailed clinical observations were carried out for all animals outside the home cage in a standard arena at approximately the same time of day, each time preferably by observers unaware of the treatment. The observations included in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, pilo-erection, pupil size, and unusual respiratory pattern). Changes in gait, posture and response to handling as well as the 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) were also recorded.

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. On Saturdays and Sundays, a similar procedure was followed with a final check at approximately 3.30 p.m. If necessary, these provisions allowed to record any premortal symptoms in detail and a post mortem examination to be carried out during the working period of a day. For the prematurely deceased pups a post mortem examination was performed.

BODY WEIGHT
The body weight the animals was recorded as follows:
Lactation period: PND 1, 4, 7, 14, 21
Time after weaning: Daily, starting on PND 22 - report of weekly values
Termination of in-life: Day of sacrifice

FOOD AND DRINKING WATER CONSUMPTION:
Food intake per rat (g) was calculated using the total amount of food given to and left by each rat in each group on those days that are listed below.
End of weaning: Weekly
From these data the relative food consumption (in g/kg b.w./day) was determined using the following formula:
Relative food consumption (g/kg b.w./day) = (Total food given (g) - Total food left (g))/ (Number of animal days# x Body weight (kg))
# The term 'animal days' counts one animal day for each animal alive for a whole
day; it is assumed that on the day of death an animal does not eat.

Drinking water consumption was monitored daily by visual appraisal throughout the study.

EXAMINATION OF THE PUPS (F1 Generation)
As soon as possible after delivery, each litter was examined to establish the number and sex of pups, stillbirths, live births, runts (i.e. body weight less than 70% of mean litter weight) and the presence of gross abnormalities. Abnormal behaviour or changes in the external appearance of the pups noted during the daily cage side inspections were recorded.
The following examinations/observations were done for the offspring:

COUNTING, SEXING AND WEIGHING
Live pups were counted, sexed and weighed on post-natal days (PND) 1, 4, 7, 14 and 21.

ANO-GENITAL DISTANCE
On PND 4 before litter adjustment the ano-genital distance (AGD) of all pups was determined using a scale. The AGD was normalised to the cube root of body weight.

LITTER ADJUSTMENT
After counting on PND 4 (lactation day 4), the litters were adjusted to 10 pups per litter (5 pups/sex/litter) by eliminating (culling) surplus pups using a randomization scheme generated by Provantis® . Selective elimination of pups, e.g. based upon body weight was not appropriate. In case of unequal gender distribution, a partial litter size adjustment was performed (e.g. 6 male and 4 female pups).
COUTING OF MALE NIPPLES/ AREOLAE
Nipples/areolae were counted in all male pups on PND 13.

SEXUAL MATURATION
All F1 Pups (Cohorts 1A and 1B) were evaluated daily for balano-preputial separation or vaginal opening which indicate sexual maturity of the animals. The genitals were examined for any abnormalities. The body weight was recorded at the time point of balano-preputial separation or vaginal opening. 
Balano-preputial separation:
During this examination the time point of the onset of the function of the preputial glands is determined. A soft pressure is exerted against the root of the penis. If this leads to the observation of small drops of secretion from the preputial glands on both sides of the foreskin, the postnatal day of this observation is determined as the time point of the onset of preputial gland function.

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:
Time of blood sampling: At necropsy
Animals: 10 males and 10 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)/ ‰ of the erythrocytes
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 PARAMETERS
The following parameters 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

BIOCHEMISTRY
The following parameters were determined (Instrument: KONELAB 30i, Thermo Fisher Scientific , 63303 Dreieich, Germany):
Parameter (in serum)/ Unit
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

Globulin/ g/L -> by substraction
Albumin/globulin ratio/ non-dimensional -> by calculation
Sodium/Potassium ratio/ non-dimensional -> by calculation
BUN/creatinine ratio/ non-dimensional -> by calculation

DETERMINATION OF THYROID HORMONES (T4 and TSH)
Blood samples were taken under isoflurane anaesthesia from animals fasted overnight always at the same time of day (in the morning between 6.30 a.m. and 9.30 a.m. for the adult animals) as scheduled below:
F1 Generation: 2 surplus pups per litter#2; non-fasted; PND 4; T4 only
F1 Generation: 2 surplus pups per litter#2#3; non-fasted; PND 21/22; T4 + TSH
F1 Generation: 10 males and 10 females per Cohort 1 A group#1; fasted; PND 90 - 92 (at sacrifice); T4 + TSH
#1 Animals also selected for laboratory examinations
#2 If the individual volume obtained from the pups was insufficient for analysis, the samples may be pooled by litter.
#3 Pups were selected from all available litters.

Blood samples were processed for serum, divided into aliquots and stored -20°C ± 10% until analyses using commercial ELISA kits.

URINALYSIS
The urine was collected for 16 hours in URIMAX funnel cages. The collection of urine was terminated immediately prior to start of blood withdrawals for the haematological and clinical chemistry examinations. The following sampling times and animals were employed:
At the end of dosing period (prior to blood withdrawal), F1 Generation Cohort 1A 10 males and 10 females randomly selected from each group
The following parameters were determined by using the instruments given below:
Parameter/ Unit/ Instrument
Volume/ mL/ Graduated vessel
pH/ non-dimensional/ Digital pH meter, type WTW InoLab pH 720
Specific gravity/ g/mL/ Kern Refractometer, type ORA 2PA Sample compared with water (nominal value of 1.000)
The following examinations were also performed using Combur 9 Test (semi-quantitative/qualitative indicators) for determination of analyte concentration in the urine:
Proteins
Glucose
Bilirubin
Urobilinogen
Ketones
Haemoglobin (Hb) (approximate values)
Nitrite

Microscopic examination of urine samples was carried out by centrifuging samples and spreading the resulting deposit on a microscope slide. The deposit was examined for the presence of the following parameters:
E Epithelial cells
L Leucocytes
R Erythrocytes
B Organisms
C Further constituents (i.e. sperm, casts)
A Crystalluria
The frequency of the above parameters were recorded as follows:
0 None found in any field examined
+ Few in some fields examined
++ Few in all fields examined
+++ Many in all fields examined
The colour and the turbidity of the urine were examined visually.
Postmortem examinations (parental animals):
NECROPSY
On the day of necropsy, vaginal lavages of the adult animals were obtained and examined to determine the stage of estrous cycle and allow correlation with the histopathology of the female reproductive organs. The animals were euthanized by carbon dioxide (CO2) inhalation, exsanguinated by cutting the aorta abdominalis.
A gross or full necropsy of the animals was carried out. At gross necropsy the animals were inspected externally and/or internally for gross abnormalities. The full necropsy additionally included sampling and weighting of selected organs.
Blood samples for determination of haematological and biochemical parameter as well as for thyroid hormone determination were taken. The animals were weighed, dissected and inspected macroscopically (gross necropsy) as follows:
Animals/ Examination after sacrifice/ Time of necropsy/ Vaginal lavage
All males#1/ Full necropsy/ TD 127 - 129/ No
All dams#1/ Full necropsy /TD 129 - 134/ Yes
#1 After weaning of F1 Generation

In the case the time of dissection was fallen on a weekend or bank holiday, necropsy would have been postponed to the next working day and dosing would have been continued up to and including the day before sacrifice.
Animals which were prematurely sacrificed or died during the study were necropsied as soon as possible after exitus.

NECROPSY OF ADULT ANIMALS
At the time of sacrifice or premature death during the study, all adult animals were examined macroscopically for any abnormalities or pathological changes. Special attention was paid to the organs of the reproductive system.
All superficial tissues were examined visually and by palpation and the cranial roof 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 condition to the thoracic viscera was 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 the 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, adrenals, uterus, intra-abdominal lymph nodes and accessory reproductive organs were recorded.

WEIGHING/ PRESERVATION OF ORGANS - F0 Generation
During necropsy the number of implantation sites in the uteri was recorded in the female animals and used to evaluate reproductive performance. Apparently non-pregnant uteri of the F0 animals 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 The following organs of the male and female F0 animals were weighed before fixation except for the thyroid. Paired organs were weighed individually and identified as left or right:
Adrenal gland (2)
Ovary (2)
Prostate (dorsolateral and ventral parts combined)
Brain
Oviducts (2)
Seminal vesicles with coagulating glands
Epididymis (2)
Spleen
Pituitary
Heart
Testis (2)
Uterus incl. cervix
Kidney (2)
Thyroid, parathyroids (after fixation) (1, left)
Liver
Thymus
# Weighing before fixation except for thyroids. Paired organs were weighed individually and identified as left or right.

The following organs or parts thereof obtained from the male and female animals of the F0 Generation were preserved in an appropriate fixative:
Fixative: Davidson's solution
Eye with optic nerve (2)
Fixative: modified Davidson's solution
Epididymis (1)#
Testis (1)#
Fixative: 7%buffered formalin
Adrenal gland (2)
Ovary (2)
Bone
Oviducts
Bone marrow (os femoris)
Pituitary
Brain (cerebrum, cerebellum, pons)
Prostate
Gross lesions observed
Seminal vesicles with coagulating glands
Heart (3 levels: right and left ventricle, septum)
Spinal cord (3 sections)
Intestine, small (duodenum, jejunum, ileum, incl. Peyer’s patches, Swiss roll method)
Spleen
Stomach
Intestine, large (colon, rectum)
Thyroid (2, incl. parathyroids)
Kidney and ureter (2)
Thymus
Liver
Trachea (incl. larynx)
Lungs (with mainstem bronchi and bronchioles)
Urinary bladder
Mammary gland
Uterus (incl. cervix)
Muscle (skeletal)
Vagina
Nerve (sciatic)
Vas deferens
Oesophagus
# The second epididymis and testis was not preserved but used for the spermiogram.
Any other organs displaying macroscopic changes were also be preserved. In addition, sperm viability and morphology were evaluated for all male F0 animals and bone marrow smears were prepared for selected F0 animals (same as used for laboratory examinations).

SPEERMIOGRAM
One epididymis and one testis of all F0 males were used for the sperm count. The sperm viability was determined and the sperm morphology was examined according to the method described by I. Chahoud and R. Franz (1993) as well as by S. Plassmann and H. Urwyler (2001).

BONE MARROW EXAMINATION
During dissection fresh bone marrow was obtained from the os femoris (3 air-dried smears/animal) of male and female F0 animals and stained according to PAPPENHEIM. The myeloid : erythroid ratio was determined by cell differentiation (counting of 200 nuclei-containing cells) for the animals of groups 1 and 4.

HISTOPATHOLOGY
The blood smears prepared from all animals during the haematological examination are available for possible examination of pathological changes but examined and evaluated only depending on necropsy findings and upon agreement with the Sponsor.
Full histopathology was performed on the preserved organs of the control and high dosed animals of groups 1 and 4:
- F0 Generation (parents): 20 male and 20 female animals per group
A full histopathology was also performed for all deceased or prematurely sacrificed animals, from the F0 Generation.
The organs "Organs/Tissues to be preserved - F0 Generation"and "Organs/Tissues to be preserved - F1 Generation Cohort 1A" were examined histopathologically after preparation of paraffin sections and haematoxylin-eosin (H&E) staining. Parathyroids could not always identified macroscopically. They were examined microscopically if in the plane of section. In addition, frozen sections of the heart, liver and one kidney were examined after staining with Oil Red O.
Detailed histopathological examination with special emphasis on the qualitative stages of spermatogenesis and histopathology of interstitial testicular structure was performed on one testis and one epididymis of the control and high dosed animals of groups 1 and 4 following H&E and PS staining:
- F0 Generation (parents): 20 male animals per group
In case of test item-related changes in the high dose level group 4, the Sponsor would have given sufficient notice before the corresponding organs of the animals of the F0 Generation of the intermediate and low dose level groups (group 2 and 3) are sectioned and examined histopathologically.

HISTOPATHOLOGICAL EVALUATION
Histotechnique was performed by the test institute. The slides were labelled with study number, test species, animal number and block number and were dispatched on August 07, 2020 to the Test Site for histopathological evaluation.
The transport of slides to the histopathology Test Site (TS) was arranged by the test institute, whereas the return transport to the Test Facility will be arranged by the TS. The study phase was recorded under the TS reference number 12848C. All observations upon final assessment were reported per animal and the findings considered to be relevant for the treatment were recorded in incidence and occurrence tables. All microscopic findings are recorded, reported and archived with the with the PathData system .

The report of this study phase comprises a description of objective, materials, methods and results (macroscopic and microscopic changes) and conclusions. The unaudited Draft Phase Report was presented electronically to the Study Director for comments/review. Comments will be addressed and the Draft Final Phase Report of the histopathology phase of the study will be presented to the TS Quality Assurance Unit (TSQAU) for auditing.
The audited Draft Final Phase Report of the histopathology phase of the study will be sent electronically to the Study Director who will provide documented approval on the 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.

Postmortem examinations (offspring):
NECROPSY
On the day of necropsy, vaginal lavages of the adult animals were obtained and examined to determine the stage of estrous cycle and allow correlation with the histopathology of the female reproductive organs. The animals were euthanized by carbon dioxide (CO2) inhalation, exsanguinated by cutting the aorta abdominalis. The pups were euthanized by decapitation (PND4) or by carbon dioxide (CO2) inhalation (PND 21/22). A gross or full necropsy of the F1 animals was carried out. At gross necropsy the animals were inspected externally and/or internally for gross abnormalities. The full necropsy additionally included sampling and weighting of selected organs.
Blood samples for determination of haematological and biochemical parameter as well as for thyroid hormone determination were taken. The animals were weighed, dissected and inspected macroscopically (gross necropsy) as follows:
Animals/ Examination after sacrifice/ Time of necropsy/ Vaginal lavage
All 'surplus' (= culled) pups/ Gross necropsy/ PND 4/ No
10 ‘surplus’ pups/sex/group/ Gross necropsy, partial organ/tissue preservation/ PND 21 - 22/ No
All remaining 'surplus' pups/ Gross necropsy/ PND 21 - 22/ No
Cohort 1A All animals#2/ Full necropsy/ PND 90-92/ Yes
Cohort 1B All animals#2/ Full necropsy/ PND 98-102/ Yes
#1 After weaning of F1 Generation
#2 At the end of the dosing period

In the case the time of dissection was fallen on a weekend or bank holiday, necropsy would have been postponed to the next working day and dosing would have been continued up to and including the day before sacrifice.
Animals which were prematurely sacrificed or died during the study were necropsied as soon as possible after exitus.

NECROPSY OF DEAD OR "SURPLUS PUPS"
External inspection for gross abnormalities:
Dead pups and culled F1 Pups on PND 4 were carefully examined externally for gross abnormalities. The external reproductive genitals were examined for signs of altered development.
External and internal inspection for gross abnormalities:
Pups not selected for the F1 Cohorts were sacrificed on PND 21/22 and examined macroscopically for any abnormalities or pathological changes.

NECROPSY OF ADULT ANIMALS
At the time of sacrifice or premature death during the study, all adult animals were examined macroscopically for any abnormalities or pathological changes. Special attention was paid to the organs of the reproductive system.
All superficial tissues were examined visually and by palpation and the cranial roof 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 condition to the thoracic viscera was 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 the 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, adrenals, uterus, intra-abdominal lymph nodes and accessory reproductive organs were recorded.

WEIGHING / PRESERVATION OF ORGANS "SURPLUS PUPS"
Ten ‘surplus’ pups per sex and group were used for organ weighing and preservation. The following organs/tissues of the F1 Pups were weighed and/or preserved in 7% formalin:
Brain#
Spleen#
Gross Abnormalities
Stomach
Kidney (left, right) #
Thymus#
Mammary gland
# Organs were weighed before fixation. Paired organs were weighed individually and identified as left or right.

WEIGHING / PRESERVATION OF ORGANS - F1 Generation Cohort 1A
The following organs of all adult male and female F1 Cohort 1A animals were weighed before fixation except for the thyroid. Paired organs were weighed individually and identified as left or right.
Adrenal gland (2)
Lymph node (1, cervical)
Thymus
Brain
Lymph node (1, mesenteric)
Prostate (dorsolateral and ventral parts combined)
Epididymis (2)
Ovary (2)
Seminal vesicles with coagulating glands
Heart
Oviducts (2)
Pituitary
Kidney (2)
Spleen
Uterus incl. cervix
Liver
Testis (2)
Thyroid incl. parathyroids (after fixation) (1, left)
# Weighing before fixation except for thyroids. Paired organs were weighed individually and identified as left or right.

Organs/Tissues of 'surplus' F1 Pups (PND 4) to be preserved and/or weighed
Weighing and preservation of the following organs/tissues (fixative 7% formalin)
Brain#
Intestine, large (colon, rectum)
Mammary gland
Gross Abnormalities
Kidney (left, right) #
Spleen#
Heart#
Liver#
Stomach
Intestine, small (duodenum, jejunum, ileum)##
Lungs#
Thymus#
# Organs were weighed before fixation. Paired organs were weighed individually and identified as left or right.
## Swiss roll method

The following organs or parts thereof obtained from the male and female animals of F1 Generation Cohort 1 A were preserved in an appropriate fixative:
Fixative: Davidson’s solution
Eye with optic nerve (2)
Fixative: modified Davidson’s solution
Epididymis (1)#1
Testis (1)#1
Fixative: 7%buffered formalin
Adrenal gland (2)
Oesophagus
Bone
Ovary (2)#4
Bone marrow (os femoris)
Oviducts
Brain (cerebrum, cerebellum, pons)
Pituitary
Gross lesions observed
Prostate
Heart (3 levels: right and left ventricle, septum)
Seminal vesicles with coagulating glands
Intestine, small (duodenum, jejunum, ileum, incl. Peyer’s patches, Swiss roll method) Spinal cord (3 sections)
Spleen#3
Intestine, large (colon, rectum)
Stomach
Kidney and ureter (2)
Thyroid (2, incl. parathyroids)
Liver
Thymus
Lungs (with mainstem bronchi and bronchioles)
Trachea (incl. larynx)
Lymph node (1, cervical)#2
Urinary bladder
Lymph node (1, mesenteric)#2
Uterus (incl. cervix)
Mammary gland
Vagina
Muscle (skeletal)
Vas deferens
Nerve (sciatic)
#1 The second epididymis and testis was not preserved but used for the spermiogram
#2 Lymph nodes were preserved for all Cohort 1A animals, however, histopathology was only carried out for 10 animals/sex/group (1 animal per litter, all litters represented by at least 1 pup; randomly selected).
#3 For 10 animals/sex/group of Cohort 1A, randomly selected (same as selected for laboratory examination): One half of the spleen was preserved for histopathoogical evaluation, the second half was used for splenic lymphocyte subpopulation analysis
#4: One ovary of the F1 Cohort 1A females of groups 1 and 4 ( 20 females per group) was processed for detailed histopathological examination as follows:
- Ten (10) 2-4-μm step sections with 50 μm steps in between;
- Each slide was labelled with the slide number to follow the sequence.
Any other organs displaying macroscopic changes were also be preserved. In addition, sperm viability and morphology were evaluated for all male animals of F1 Cohort 1A and bone marrow smears were prepared for selected animals of F1 Cohort 1A animals.

WEIGHING/ PRESERVATION OF ORGANS - F1 Generation Cohort 1B
The following organs of male and female F1 Cohort 1B animals were weighed before fixation except for the vagina. Paired organs were weighed individually and identified as left or right.
Organ/ Weigh/ Fixative/ Block
Endocrine system:
Adrenal gland (2)/ Yes/ 7% formalin/ No
Pituitary/ Yes/ 7% formalin/ Yes
Thyroid (2) (including parathyroids)/1, post-fixation/ 7% formalin/ No
Reproductive system:
Epididymis (2)/ Yes/ Modified Davidson’s/ Yes
Ovary (2)/ Yes/ 7% formalin/ Yes
Prostate/ Yes/ 7% formalin/ Yes
Seminal vesicles with coagulating glands/ Yes/ 7% formalin/ Yes
Testicle (2)/ Yes/ Modified Davidson’s/ Yes
Uterus (including oviducts and cervix)#/ Yes/ 7% formalin/ Yes
Vagina/ No/ 7% formalin/ Yes
Vas deferens/ No/ 7% formalin/ No
# The oviducts of Cohort 1B were weighed separately as in Cohort 1A.

The adrenals, thyroids and vas deferens are not processed to block stage as this is not required by the current OECD TG 443 (paragraph 67) adopted 25 June 2018.
In the case of test item-related changes in the corresponding organs of the F0 and F1 Cohort 1A animals, the Study Monitor will be given sufficient notice before the respective organs of F1 Cohort 1B animals are sectioned and examined histopathologically, if possible.

SPERMIOGRAM - F1 Generation Cohort 1A
One epididymis and one testis of all F0 and F1 Cohort 1A males were used for the sperm count. The sperm viability was determined and the sperm morphology was examined according to the method described by I. Chahoud and R. Franz (1993) as well as by S. Plassmann and H. Urwyler (2001).

BONE MARROW EXAMINATION - F1 Generation Cohort 1A
During dissection fresh bone marrow was obtained from the os femoris (3 air-dried smears/animal) of male and female F0 and F1 Generation Cohort 1 A animals and stained according to PAPPENHEIM. The myeloid : erythroid ratio was determined by cell differentiation (counting of 200 nuclei-containing cells) for the animals of groups 1 and 4.

PHENOTHYPIC ANALYSIS OF SPLEEN CELLS - F1 Generation Cohort 1A
The spleens of the male and female F1 Cohort 1A animals were split in two halves. The portion of the spleen not preserved for histopathology was minced using a mechanic dissociator to prepare single cell suspensions. The following parameters were determined in the samples( Instrument: MACSQuant® Analyzer 10/16, Miltenyi Biotec GmbH, Friedrich-Ebert-Str. 68, 51429 Bergisch Gladbach, Germany):
CD4+ T-Lymphocytes
CD8+ T-Lymphocytes
Pan-T-lymphocytes (CD3+)
B-lymphocytes (CD45RA+)
Natural killer cells (CD161+)
Evaluation was performed by the test institute.

HISTOPATHOLOGY
The blood smears prepared from all animals during the haematological examination are available for possible examination of pathological changes but examined and evaluated only depending on necropsy findings and upon agreement with the Sponsor.
Full histopathology was performed on the preserved organs of the control and high dosed animals of groups 1 and 4:
- F1 Generation (Cohort 1A): 20 male and 20 female animals per group
A full histopathology was also performed for all deceased or prematurely sacrificed animals, from Cohort 1A.
The organs listed in"Organs/Tissues to be preserved - F1 Generation Cohort 1A" were examined histopathologically after preparation of paraffin sections and haematoxylin-eosin (H&E) staining. Parathyroids could not always identified macroscopically. They were examined microscopically if in the plane of section.
In addition, frozen sections of the heart, liver and one kidney were examined after staining with Oil Red O.
Detailed histopathological examination with special emphasis on the qualitative stages of spermatogenesis and histopathology of interstitial testicular structure was performed on one testis and one epididymis of the control and high dosed animals of groups 1 and 4 following H&E and PS staining:
- F1 Generation (Cohort 1A): 20 male animals per group
Detailed histopathological examination with quantitative evaluation of primordial and small growing follicles as well as corpora lutea was performed on one ovary of the following control and high dosed animals of groups 1 and 4:
- F1 Generation (Cohort 1A): 20 female animals
In case of test item-related changes in the high dose level group 4, the Sponsor would have given sufficient notice before the corresponding organs of the animals of the F1 Generation Cohort 1A of the intermediate and low dose level groups (group 2 and 3) are sectioned and examined histopathologically.

HISTOPATHOLOGICAL EVALUATION
Histotechnique was performed by the test institute.
The slides were labelled with study number, test species, animal number and block number and were dispatched on August 07, 2020 to the Test Site for histopathological evaluation.
The transport of slides to the histopathology Test Site (TS) was arranged by Provivo, whereas the return transport to the Test Facility will be arranged by the TS. The study phase was recorded under the TS reference number 12848C. All observations upon final assessment were reported per animal and the findings considered to be relevant for the treatment were recorded in incidence and occurrence tables. All microscopic findings are recorded, reported and archived with the with the PathData system .

The report of this study phase comprises a description of objective, materials, methods and results (macroscopic and microscopic changes) and conclusions. The unaudited Draft Phase Report was presented electronically to the Study Director for comments/review. Comments will be addressed and the Draft Final Phase Report of the histopathology phase of the study will be presented to the TS Quality Assurance Unit (TSQAU) for auditing.
The audited Draft Final Phase Report of the histopathology phase of the study will be sent electronically to the Study Director who will provide documented approval on the 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.
Statistics:
DATA ACQUISITION: The following data were captured or calculated by the departmental computerized system Provantis: Clinical signs, body weight, body weight gain, food consumption, haematological and biochemical parameters. Data maintained on paper (e.g. pup data, ELISA experiments) were entered in Provantis in a retrospective manner using the laboratory records according to the appropriate SOPs.

STATISTICS: The statistical evaluation of the parametrical values was done by Provantis® 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).
For the statistical evaluation of the histopathological data FISHER's exact test was used.
The mean values and standard deviations were calculated to the highest possible degree of accuracy and then rounded to the reported number of decimal places. Hence, deviations to the last decimal place of up to +/- 1 may occur caused by rounding.
Reproductive indices:
The following indices were calculated for each group:
Female Fertility Index [%] = (Number of pregnant rats/ Number of rats paired with a male) x 100
The female fertility index reflects the total number of dams that had achieved pregnancy, including dams which delivered at term, aborted or had fully resorbed litters.
Gestation Index [%] = (Number of dams with live pups/ Number of pregnant rats) x 100
For each litter and group the following indices were determined:
Birth Index [%] #1 = (Total number of pups born (alive + dead)/Number of implantation sites) x 100
Live Birth Index [%] = (Number of pups alive on day 0/1 of lactation/ Total number of pups (alive + dead)) x 100
Viability Index [%] pre-cull = (Number of pups alive on day 4 (pre cull)/ Number of pups alive on day 0/1) x 100
Viability Index [%] post cull = (Number of pups alive on day 13/ Number of pups alive on day 4 (post cull)) x 100
Post-implantation loss [%]#1 = ((Implantations - number of pups born alive)/ Implantation sites) x 100
#1: Twins (shared placentae) are considered as additional implantation sites in the calculation of the birth index and the post-implantation loss, to avoid a birth index above 100% or a negative post-implantation loss.
Offspring viability indices:
Birth and live birth index, post-impantation loss and viability indices of the pups.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Male animals
No test item-related changes in behaviour and the external appearance were noted for the male animals of the low dose group (25 mg IPDA/kg b.w./day). Nevertheless, in this dose group a haemorrhagic nose/snout (no. 71) or pultaceous faeces (no. 64) were noted for one animal each on one test day each. Furthermore, an increased water consumption (no. 52) was noted for one animal on seven test days. Due to the low incidence of only one affected animal for the observed symptoms they were considered to be spontaneous.

At the intermediate dose level (80 mg IPDA/kg b.w./day) a post-dosing salivation was noted for 17 of 24 male animals. The observation of a post dosing salivation was considered to be test item-related but not adverse (or of toxicological relevance), as in nearly all cases it did not last longer than 60 min (see section 7.3.1. ‘Start and duration of observations’). Additionally, an increased water consumption was noted for one animal (no. 120) on 4 test days.

At the high dose level (240/160 mg IPDA/kg b.w./day) a post-dosing salivation was noted for all surviving male animals. Furthermore, breathing sounds were noted for 10 of 22 animals, piloerection for 5 of 22 animals, an increased water consumption for 2 animals (nos. 146 and 158) and haemorrhagic urine (no. 159) and pultaceous faeces (no. 145) for one animal each.
The observations of breathing sounds and piloerection that were noted at the high dose level were considered to be test item related, as they were noted for several animals. Furthermore, breathing sounds were in most cases noted at the start of the working day before dosing and lasted consistently for the whole day.

Female animals
No signs of toxicological relevance were noted at the low and the intermediate dose level (25 or 80 mg IPDA/kg b.w./day) during the pre-mating, the gestation and the lactation period. The observation of post dosing salivation for one animal of the low dose group and several animals of the intermediate dose group was considered to be of no toxicological relevance.

At the high dose level (240/160 mg IPDA/kg b.w./day) 4 female animals had to be prematurely sacrificed due to poor health condition. In detail 3 of the 4 prematurely sacrificed animals were sacrificed during the pre-mating period on test days 49, 59, 24 (nos. 174, 178 and 182, respectively) and no. 187 was prematurely sacrificed at the end of the gestation period (gestation day 20).
At the high dose group post dosing salivation was noted for all animals, piloerection and / or breathing sounds were observed for 5 and 10 animals, respectively. Furthermore, an increased respiratory rate (no. 181) and an increased water consumption (no. 188) were noted during the pre-mating / mating period for altogether 2 females. Female no. 187, which was prematurely sacrificed on gestation day 20 revealed gasping, a haemorrhagic nose/snout, laboured breathing and a thickened abdomen during the pre-mating / mating period.
The 4 prematurely sacrificed high dose females (nos. 174, 178, 182 and 187) all showed breathing sounds before death. A haemorrhagic nose/snout was additionally noted for nos. 182 and 187. No. 182 furthermore revealed laboured breathing and no. 187 piloerection on the day and two days before the sacrifice, respectively (see Text Tables 7-3 and 7-4B in section 7.2 ‘Mortality’).
As a result of the increased number of recorded observations the 4 females were prematurely sacrificed. The later histopathology assessment revealed an accident during the process of dosing (influx of test item formulation into the respiratory tract by accidental influx or incidental regurgitation) for female no.187 (prematurely sacrificed on gestation day 20). This can also be the cause of gasping, laboured breathing and the thickened abdomen that were noted for female no. 187 during the pre-mating / mating period.
The observation of breathing sounds are in nearly all cases long lasting observations which lasted for several hours or for the whole day.
The observations at the high dose level that were noted for the female animals were considered to be test item-related and, with the exception of post-dosing salivation, of toxicological relevance.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
Males and females
No test item-related premature death was noted in the control group, low dose group (25 mg IPDA/kg b.w./day) and intermediate dose group (80 mg IPDA/kg b.w./day) for the male and female animals.
Three test item-related deaths were noted at the high dose level (240/160 mg IPDA/kg b.w./day).
At the high dose level (240/160 mg IPDA/kg b.w./day) one male (no. 153) and 2 female animals (nos. 178 and 182) had to be prematurely sacrificed due to poor health condition on test day 73, 59 and 24, respectively (see Text Table 7-2). The noted observations during the daily cage side observations before deaths are listed in Text Table 7-3.
All recorded microscopic findings were stress-related changes or non-specific changes that are frequently observed in animals with deteriorated general conditions, and the findings that could be specific to the test item treatment were not identified. No signs of misgavage, an accidental influx or incidental regurgitation of the test item-formulation into the respiratory tract were observed for these 3 animals. Thus, histomorphologically, a direct cause of animals morbidity could not be established (see section 3.4.1. ‘Microscopic Findings in Decedents’ of the Histopathology Phase Report).
The macroscopic findings that were noted for the prematurely deceased animals (no gross lesions were considered to be specific to the test item) are listed in Text Table 5 in section 3.1 ‘Mortality’ of the Histopathology Phase Report.

Not test item-related premature deaths
The premature death of six animals (two male (nos. 151, 164) and 4 female (nos. 40, 126, 174, 187)) was caused by an accident during the process of dosing (see text table 7-4A).
Among other findings, reddened lungs were noted for 3 (nos. 126, 151, 174) of these six animals at necropsy.
The microscopic lesions that were noted for these animals were considered to be produced by accidental influx or incidental regurgitation of the test item-formulation into the respiratory tract and not to be adverse, but deemed to be the direct cause(s) of animals death or morbidity (see section 3.4.1. ‘Microscopic Findings in Decedents’ of the Histopathology Phase Report).
The male animal no. 164 of the high dose group (240/160 mg IPDA/kg b.w./day) was found dead on TD 16 after a misgavage. No changes in behaviour, the external appearance of the faeces were noted in the days prior to the premature sacrifice.
The animal was excluded from the evaluation and replaced by animal no. 193.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Males: Pre-mating-, mating- and post-mating period
No test item-related changes in body weight and body weight gain were noted for the male rats between the control group and the treatment groups (25, 80 or 240/160 mg IPDA/kg b.w./day).

Females: Pre-mating-, gestation- and lactation period
No test item-related changes in body weight and body weight gain were noted for the female rats between the control group and the treatment groups (25, 80 or 240/160 mg IPDA/kg b.w./day).

Males and females:
No test item-related differences between the control group and the treatment groups (25, 80 or 240/160 mg IPDA/kg b.w./day) were noted for the body weight at autopsy for the male and female animals.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Males & Females
No test item-related difference in food consumption was noted between the control group and in the treatment groups (25, 80 or 240/160 mg IPDA/kg b.w./day).
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Water consumption was performed by visual appraisal during the daily cage side observations. For nearly all animals, no decreased or increased water consumption was noted.
However, for 4 males and 1 female an increased water consumption was noted during the daily cage side observations on a few test days. Due to the low incidence this was considered spontaneous.

Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
Males
No test item-related differences for the examined haematological parameters were noted between the control group and the treatment groups (25, 80 or 240/160 mg IPDA/kg b.w./day).

Females
No test item-related differences for the examined haematological parameters were noted between the control group and the treatment groups (25, 80 or 240/160 mg IPDA/kg b.w./day).
Decreased values in comparison to the control group were noted for the number of white blood cells for the female animals of the low ( 12.2 %, statistically not significant) and the intermediate dose group (27.2 % below the control; p ≤ 0.05).

However, these changes were considered spontaneous, as no dose response-relationship was noted considering all treatment groups and no changes were noted for the male animals. Additionally, the opposite picture was noted for the number of white blood cell in female animals of the F1 Generation (Cohort 1A), which were exposed to the test item during their whole life time. Finally, with one exception, all individual values of the low dose group (5.80 to 10.43 x10E3 cells/µL) and the intermediate dose group (4.70 to 9.91 x10E3 cells/µL) were in the range of the Provivo background data (4.65 to 11.38 x10E3 cells/µL).

In the number of eosinophilic granulocytes, statistically significant reductions were noted for the female animals of the low, the intermediate and the high dose group (52.3 %, 46.1 % and 51.5 %, respectively, below the control, p ≤ 0.05 or 0.01). No dose response-relationship was noted and the values in all three treatment groups were similar to each other. The noticed reductions in the number of eosinophilic granulocytes in the treated groups can be considered to be due to an increased value in the control group.
Furthermore, only two animals (one control and one intermediate dosed female) exhibit eosinophilic granulocytes values outside of the Provivo background range (0.05 to 0.42 x10E3 cells/µL). Hence, the decreased values in the treatment groups are considered as not relevant.

A statistically significantly reduced number of basophilic granulocytes was noted at the intermediate dose level (50.0 % below the control, p ≤ 0.05).
However, firstly all individual values of the control group and the treatment groups were within the Provivo background range. Secondly, no dose response-relationship was noted and thirdly no changes were noted for the male animals. Therefore, the statistically significantly decreased value that was noted at the intermediate dose level was considered to be spontaneous.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Males
No test item-related differences for the examined biochemical parameters were noted between the control group and the treatment groups (25, 80 or 240/160 mg IPDA/kg b.w./day) for the male animals.

Females
No test item-related differences for the examined biochemical parameters were noted between the control group and the treatment groups (25, 80 or 240/160 mg IPDA/kg b.w./day) for the female animals.
However, a statistically significantly increased bilirubin concentration was noted at the low dose level (15.1 % above the control, p ≤ 0.05). However, as no dose response-relationship was noted and all individual values of the low dose group (2.5 to 4.4 µmol/L) were within the Provivo backround range (2.4 to 4.5 µmol/L). The increased bilirubin concentration that was noted at the low dose level was considered to be spontaneous.
A statistically significantly decreased glucose concentration was noted at the high dose level (18.7 % below the control, p ≤ 0.05). The glucose concentrations of 5 individual females of the high dose level (5.43 to 5.72 mmol/L) were marginally below the Provivo background range (5.88 to 9.72 mmol/L). However, no changes were noted for the high dosed male animals of the F0 Generation (2.2 % below the control). Also in Cohort 1A, no changes were noted for the high dosed male and female animals (6.8 % above or 5.0 % below the control, statistically not significant), which were exposed to the test item during their whole life time. Hence, the decreased glucose concentration that was noted for the female animals of the high dose group was considered to be spontaneous.
Endocrine findings:
no effects observed
Description (incidence and severity):
Males and females
No test item-related differences for the examined thyroid hormone levels (T4 and TSH) were noted between the control group and the treatment groups (25, 80 or 240/160 mg IPDA/kg b.w./day). Nearly all T4 concentrations of the individual animals were within the Provivo background range. Four animals one control male and female, one female of the low and the intermediate dose each showed a value slightly above the Provivo background range.
Differences between the control group and the treatment groups were noted for the TSH concentration of the female animals. The male animals treated with 25, 80 or 240/160 mg IPDA/kg b.w./day showed reductions in TSH concentration of about 51.5 %, 38.7 % and 14.2 %, respectively. The changes in the female animals were for the low dose ( 7.0 %) below and for the intermediate (+39.4 %) and high dose (+30.4 %) above the control. A comparison with the Provivo background data revealed that for the male animals a few individual values from the treatment groups were below the Provivo background range (1.506 to 6.964 ng/mL, 25 % to 95 %). In this case 25 % percentile was used as the lower value of the background range, as the 5 % percentile was at the low limit of detection (LOD = 0.081 ng/mL). Nearly all individual female animals of the treatment groups were within the Provivo background range (0.238 to 4.123 ng/mL, 25 % to 95 % percentile) (see Text Table 7 16A). As this was the case and the observed TSH differences were not statistically significant due to a high variability of the individual values, no dose response relationship was noted, and the changes of the male and female animals showed opposite directions, the observations were not considered to be test item-related.
Furthermore, no influence was noted on the thyroid weight in the male animals. For the female animals a statistically significantly increased relative organ weight was noted at the low dose level. However, this difference was considered to be spontaneous (see section ‘Organ weights (relative and absolute).
Finally, no changes were noted during the histopathological examination of the thyroid glands from the high dosed males and females. It is generally known that histopathological examination of the thyroid is usually more sensitive than thyroid weight and hormone levels ( Beekhuijzen et. al., 2016). The validation report of OECD 407 states that “thyroid histopathology was consistently the most reliable and most sensitive endpoint for the detection of thyroid modulation. Thyroid weigth was reliable, but was somewhat less sensitive when compared to thyroid histopathology. Circulating thyroid hormone levels (T3, T4, and TSH) were not always reliable and sensitive, but the standard operating procedures for blood sampling and for thyroid hormone analyses were not standardized to reduce stress induced variability and to reduce analytical variability, respectively. Circulating T4 levels were the most promising of the three thyroid hormonal values. As observed in this study the T4 values are nearly all in the range of the Provivo background range for the males and female animals.
Urinalysis findings:
no effects observed
Description (incidence and severity):
Males and females
No test item-related differences for the examined urinalysis parameters were noted between the control group and the treatment groups (25, 80 or 240/160 mg IPDA/kg b.w./day).
Behaviour (functional findings):
not examined
Immunological findings:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Males and females
No test item-related changes were observed for the examined male and female animals of the control and high dose group (240/160 mg IPDA/kg b.w./day) during the histopathological examination.
Among the dead animals, the lesions considered to be produced by accidental influx or incidental regurgitation of the test item formulation into the respiratory tract were observed in a female of control (no. 40), a female of intermediate dose group (no. 126), and two males (nos. 151, 164) and two females of high dose group (nos. 174, 187). The respiratory lesions are not considered to be adverse, but were deemed to be the direct cause(s) of animals’ death or morbidity.
The remainders of microscopic findings were stress-related changes or non-specific changes that are frequently observed in animals with deteriorated general conditions, and the findings that could be specific to the test item treatment were not identified.
Thus, histomorphologically, the direct cause(s) of animals’ morbidity was not established in male no. 153 and female nos. 178 and 182 of high dose group, and the effects to animal’s general conditions by the exposure with higher dose level of the test item were not excluded.
In conclusion, since the animals’ deaths occurred in this study were due to accidental/ incidental events, it was considered that there was no lethal effect in the dose levels of the test item selected for this study. However, the high dose level (240/160 mg/kg b.w./day) may affect the animals' general conditions, because of presence of stress-related microscopic findings in decedents.
In survivors, toxicologically relevant treatment-related changes were not observed in any organs and tissues in the control and high dose animals of the F0 generation.

In the previous study (OECD 422 study) in which the treatment in the high-dose group was started at a dose of 300 mg/kg b.w./day (thereafter it was reduced to 240 mg/kg during the treatment period), the treatment-related microscopic changes were observed in the stomach and kidneys. However, the histopathological examination of the present study revealed that the test item did not produce treatment-related microscopic changes not only in the stomach and kidneys but also in any other organs including male and female reproductive system of the high dose animals that were treated at 240/160 mg/kg b.w./day in F0 generation
.
Male reproductive organs:
No test item-related changes were noted. No histomorphological changes to be toxicologically concerned in the view of reproductive performance were found in the male reproductive organs including testes, epididymides, prostate glands, seminal vesicles and coagulating glands in the control and high dose group.
All testes showed completeness of stages and cell population. All the other findings recorded in the male reproductive organs were within the range of normal background changes that may be observed in this type of study or animals of this strain and age, and hence, deemed not to be treatment-related.

Female Reproductive Organs:
No test item-related histomorphological changes were noted in the female reproductive organs including ovaries, oviducts, uterus, uterine cervix and vagina. In summary no histopathological changes was observed which would have lead to an test-item induced change in the female reproductive performance.
All findings recorded were within the range of normal background lesions or physiological alterations that may be observed in this type of study or animals of this strain and age.



Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
bone marrow
Males and females
No test item-related differences for the myeloid/erythroid ratio of the bone marrow were noted between the control group and the high dose group (240/160 mg IPDA/kg b.w./day).

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
ADULT P0
Monitoring of estrous cycles - Exposure period
After the allocation of the animals to the test groups and the start of treatment on test day 15, the estrous cycles were further monitored during the pre-mating and mating period until one day before a positive mating sign (verification of copulation) was noted.
No test item-related differences were noted for the mean length and the mean number of estrous cycles per dam during the pre-mating period between the female animals of the control group and the treatment groups (25, 80 or 240/160 mg IPDA/kg b.w./day).
A slightly but statistically significantly increased mean cycle length of 5.05 days in comparison to 4.50 days in the control group was noted at the high dose level.
However, this difference was only small and only one female of the high dose group (the high dosed female no. 187 with a mean cycle length of 7.7 test days) was outside the range of the mean cycle length from the females of the control group (4.0 to 6.8 days).
A comparison with the Provivo background range revealed that the mean cycle length from one female of the control group (6.8 days) and one female of the low dose group (6.6 days) were slightly above the Provivo background range (3.8 to 6.4 days). In the high dose group 2 females were noted with mean cycle lengths that were above the Provivo background range (no. 190 with 6.7 days and the already mentioned no. 187 with 7.7 days).
Hence, the isolated observation of the elevated mean cycle length of female no 187 can be considered spontaneous. Secondly, no increase of the mean cycle length was observed in the F1 Co1A and lastly no differences in the mating (pre-coital time) were observed in the F0 generation.



Stage of estrous cycle at necropsy
No test item-related differences were noted between the control group and the treatment groups (25, 80 or 240/160 mg IPDA/kg b.w./day) in the distribution of the stages of the estrous cycle. A diestrus stage was noted for more than half of the animals of each group after examination of the vaginal lavage taken at necropsy form the females of the control and the treatment groups. This was in compliance with the results of the histopathological examination of the vagina.

ADULT F1 Cohort 1A
The stages of the estrous cycle of the cohort 1A animals were monitored on 14 test days between test days 49 and 62 of the F1 Generation Study.
No test item-related differences were noted for the mean length and the mean number of estrous cycles per female animal between the females of the control group and the treatment groups (25, 80 or 160 mg IPDA/kg b.w./day) (see Text Table 8-15 on the following page).
A slight, but statistically significantly elongated mean cycle length was noted at the high dose level (4.5 days compared to 4.12 days in the control group, p ≤ 0.05). However, the difference was only small and the mean cycle length of the individual females of the high dose group were in the range of the mean cycle length of the individual females of the control group. Secondly, the physiological cycle length of a rat lies between 4 and 5 days. Hence, the observed increase in the mean cycle length is still in physiological range. Therefore, this observation was considered to be spontaneous.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Sperm number
No test item-related difference was noted between the rats of the control group and the rats treated with 25, 80 or 240/160 mg IPDA/kg b.w./day for the number of ultrasound-resistant sperm heads (sperm count) per gram testicular tissue.

Sperm motility
No test item-related differences were noted between the rats of the control group and the treatment groups (25, 80 or 260/140 mg IPDA/kg b.w./day) for the percentage of motile spermatozoa in the epididymal cauda on the total number of motile and non-motile spermatozoa.
A slightly increased percentage of motile spermatozoa in comparison to the control group was noted at the intermediate dose level (75.8 % motile spermatozoa in comparison to 71.3 % in the control group). As the difference was only small and no dose-response relationship was noted, the difference was not considered to be test item-related. Furthermore, an increased number of motile spermatozoa is not an adverse effect.
Finally, the percentages of sperm motility of all individual males of the control group, the low dose group and the high dose group were within the Provivo background range. With a motility range between 51 to 86 %. In the intermediate dose group one male animal (no. 105) showed 87 % motile spermatozoa, which was marginally above the Provivo background range.

Sperm morphology
The examination of spermatozoa from the epididymal cauda revealed no increased numbers of spermatozoa with a malformation in the treatment groups (25, 80 or 240/160 mg IPDA/kg b.w./day) in comparison to the control group.
In detail, the only observed malformation that was noted was banana-like sperm heads (7 were noted in the control group, 11 in the low dose group, 3 in the intermediate dose group and 14 in the high dose group).

Males that did not inseminate their female partner
No verified copulation (no sperm detection in the vaginal smear) was noted for 3 pairs. However, the investigated sperm parameter for the 3 male animals did not show any changes that could explain the missing sperm detection during the mating period and the resulting not pregnant females.

Reproductive performance:
no effects observed
Description (incidence and severity):
Female fertility
No test item-related influence on the fertility index was noted for the female rats of the treatment groups (25, 80 or 240/160 mg IPDA/kg b.w./day).
One non-pregnant female was noted in the control group (no. 28; with a verified copulation) and the low dose group (no. 95, without a verified copulation). Two non-pregnant females each in the intermediate (nos. 137, 140, both with a verified copulation) and the high dose group (nos. 177, 189, both without a verified copulation) were additionally observed.

Gestation index
No test item-related influence on the gestation index was noted for the female rats of the treatment groups (25, 80 or 240/160 mg IPDA/kg b.w./day).
One pregnant female each in the control (no. 40), intermediate (no. 126) and high dose (no. 187) deceased during the gestation period were excluded in the calculation of the gestation index. All other pregnant females delivered live pups leading to a gestation index of 100% in all groups.

Pre-coital time
No test item-related differences were noted in the length of the pre-coital time between the control group and the treatment groups (25, 80 or 240/160 mg IPDA/kg b.w./day).

Gestation length
No test item-related differences were noted for the length of the gestation period between the rats of the control group and the treatment groups (25, 80 or 240/160 mg IPDA/kg b.w./day).


Details on results (P0)

General toxicity
Parental male and female animals (F0 Generation)
Three test item-related deaths were noted at the high dose level (240/160 mg IPDA/kg b.w./day). In the histopathological examination no direct cause of morbidity of these animals could be established.
Adverse test item-related changes in behaviour and the external appearance were noted for the male and female animals at 240/160 mg IPDA/kg b.w./day in the form of breathing sound and piloerection.
No test item-related influence was noted for the male and female animals on body weight, food consumption, drinking water consumption, the haematological and biochemical parameters, urinalysis, the levels of the thyroid hormones and the sperm parameters.
No test item-related changes were noted during the macroscopic examination, the examination of bone marrow and for the relative and absolute organ weights.
No test item-related observations were noted during the histopathological examination of the male and female animals of the high dose group.

Reproductive toxicity
Parental females
No test item-related influence was noted on the length and numbers of the estrous cycles during the pre-mating period, the fertility index, the gestation index, the duration of the pre-coital time interval and the gestation period.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
80 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Cohort 1A: Males
No changes in behaviour, the external appearance and the consistency of the faces were noted for the male animals of the control group and the low dose group (25 mg IPDA/kg b.w./day).
At the intermediate dose level (80 mg IPDA/kg b.w./day) a post dosing salivation was noted for 3 of 20 animals on 1 or 3 test days.
At the high dose level (160 mg IPDA/kg b.w./day) post dosing salivation was noted for 11 of 19 animals and breathing sounds for 2 of 19 animals. Animal no. 326 revealed breathing sounds, laboured breathing and hunched position for several days.
The observation of a post dosing salivation was considered to be not of toxicological relevance, as in nearly all cases it did not last longer than 60 min.
Females
No changes in behaviour, the external appearance and the consistency of the faces were noted for the female animals of the control group and the low dose and the intermediate dose group (25 or 80 mg IPDA/kg b.w./day).
At the high dose level (160 mg IPDA/kg b.w./day) post dosing salivation was noted for 15 of 20 animals and breathing sounds for 2 of 20 animals. The observation of a post dosing salivation was considered to be not of toxicological relevance, as in nearly all cases it did not last longer than 60 min.

Males and females: Start and duration of observations
Salivation started immediately to 5 min after the administration and disappeared between 5 to 60 min after administration.
Breathing sounds are in nearly all cases long lasting observations which lasted for several hours or for the whole day.


Cohort 1B: Males
No changes in behaviour, the external appearance and the consistency of the faces were noted for the male animals of the control group.
In the low dose group (25 mg IPDA/kg b.w./day) one (no. 406) of 20 males showed a post dosing salivation on one test day.
At the intermediate dose level (80 mg IPDA/kg b.w./day) one animal each showed post dosing salivation (no. 452), piloerection (no. 456) or a haemorrhagic urine (no. 460) on one test day.
At the high dose level (160 mg IPDA/kg b.w./day) post dosing salivation was noted for 16 of 20 animals. Breathing sounds were noted for 2 (nos. 492, 493) animals and a haemorrhagic urine for one (no. 488) animal on one test day each.
The observation of a post dosing salivation was considered to be not of toxicological relevance, as in nearly all cases it did not last longer than 60 min.

Females
No changes in behaviour, the external appearance and the consistency of the faces were noted for the female animals of the control group and the low dose group (25 mg IPDA/kg b.w./day).
At the intermediate dose level (80 mg IPDA/kg b.w./day) a post dosing salivation was noted for 2 of 20 animals on one test day each.
At the high dose level (160 mg IPDA/kg b.w./day) a post dosing salivation was noted for all animals. One animal (no. 502) showed breathing sounds on 16 consecutive test days and animal no. 506 showed piloerection on one test day.
The observation of a post dosing salivation was considered to be not of toxicological relevance, as in nearly all cases it did not last longer than 60 min.



Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
PUP
Viability index - F1 Pups
Pre- and post-cull period
No test item-related differences between the control group and the treatment groups (25, 80 or 240/160 mg IPDA/kg b.w./day) were noted for the viability indices of the pre- and the post-cull period. In detail, the viability index for the intermediate dose group was the lowest with 97.44% compared to the control group 99.36%.



ADULT
Cohort 1A: Males and females
One prematurely sacrificed male animal was noted at the high dose level (160 mg IPDA/kg b.w./day) (see Text Table 8-1 below). There were no signs of an accident during the dosing process. A direct cause of animals morbidity could not be established by histopathology as it was also stated for 3 prematurely sacrificed high dosed animals of the F0 Generation. The microscopic findings were stress-related changes or non-specific changes that are frequently observed in animals with deteriorated general conditions, and histopathological findings that could be specific to the test item treatment were not identified.

Cohort 1B: Males and females
No test item-related premature death was noted in any dose group (25, 80 or 160 mg IPDA/kg b.w./day) for the male and female animals.
However, male no. 443 of the intermediate dose group was found dead on its postnatal day 84. Piloerection and a pale body were noted on several test days before death. Furthermore, body weight loss was noted. Necropsy revealed an increased spleen, beige liquid in the abdomen, reddish liquid in the thorax, reddened lungs and a haemorrhagic nose/snout.
An accidental death (caused by the accidental influx or incidental regurgitation) was considered as the cause of death due to the observation of reddened lungs at necropsy, though no histopathological examination was performed (only requested for prematurely deceased animals of the F0 Generation and Cohort 1A in the Study Plan) to verify that observation.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
PUP
No test item-related influence on the body weight of pups was noted for all treatment groups (25, 80 or 240/160 mg IPD/kg b.w./day).
Slightly reduced pup body weights were noted for all treatment groups in comparison to the control group on lactation day 14 (between 3.7 % and 4.5 % below the control for the male and the female animals combined; statistically not significant) (see Text Table 7-30 on the following page).
However, as the differences that were noted on lactation day 14 were only small and the differences between the control group and the treatment groups were smaller on lactation day 21 as on lactation day 14, the differences that were noted on lactation day 14 were considered to be spontaneous.
Statistically significant differences in pup body weight were noted for the female pups on lactation day 14 for the low dose with 4.8 % and for the high dose at 5.1 % below the control (p ≤ 0.05). As the differences were only small and no dose response-relationship was noted (on lactation day 21 the pup body weight of the female animals was 3.9 % at the low dose and 3.0 % at the high dose below the control, statistically not significant), they were considered to be spontaneous.

No test item-related influence on the litter weight was noted for all treatment groups (25, 80 or 240/160 mg IPDA/kg b.w./day).
Slight, but statistically significantly reduced litter weights were noted for the female pups of the low (12.2 % below the control, p ≤ 0.05) and intermediate dose group (13.3 % below the control, p ≤ 0.05) on lactation day 14. This was due to the slightly reduced body weights on lactation day 14 that were considered to be spontaneous and due to a slightly reduced number of female pups per dam. Hence, the reduced litter weights of the female pups on lactation day 14 were considered to be spontaneous. On LD 21, no statistically significant difference was noted anymore between the treatment groups and the control group.


ADULT
Cohort 1A:
Males and females
No test item-related differences in body weight and body weight gain were noted for the male and female animals between the control group and the treatment groups (25, 80 or 160 mg IPDA/kg b.w./day).
A marginally reduced body weight was noted for the female animals on postnatal day 26, which remained during the whole study period of Cohort 1A until postnatal day 89. However, these marginal differences that failed statistical significance were considered to be spontaneous.


Cohort 1B: Males
No test item-related differences in body weight and body weight gain were noted for the male animals between the control group and the treatment groups (25, 80 or 160 mg IPDA/kg b.w./day). Only marginal and statistically not significant differences in body weight were noted between the control group and the treatment groups at start of Cohort 1B on PND 26 and near the end of Cohort 1B on PND 98.
No test item-related differences in body weight gain were noted between the control group and the treatment groups for the male animals.

Females
No test item-related differences in body weight and body weight gain were noted for the female animals between the control group and the treatment groups (25, 80 or 160 mg IPDA/kg b.w./day).
However, marginal reductions in body weight were noted for the female animals of the three treatment groups compared to the control group animals from postnatal day 26 on until near the end of the study on postnatal day 98 (see Text Table 9-8A below). These marginal differences were considered to be non-advers, as there was no correspondence in the body weight gain noticed.
Furthermore, the differences temporarily decreased for the female animals during the course of the study between postnatal days 40 and 96. Statistically significant differences were noted in this period in all dose groups (at maximum 8.4 % below the control on postnatal day 47 in group 4; p ≤ 0.01). As these slight but statistically significant differences were without any influence on body weight gain they were considered to be spontaneous. Moreover, no statistically significant differences in body weight were noted for the female animals of Cohort 1A.

Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Cohort 1A: Males
No test item-related difference in food consumption was noted in the control group and in the treatment groups (25, 80 or 160 mg IPDA/kg b.w./day).
A slight but statistically significant increased food consumption was noted for the high dose males between the test days 43 to 50 and 50 to 57 (6.5 % and 6.4 % below the control, respectively, p ≤ 0.05). These slight and transient differences were considered to be spontaneous.

Females
No test item-related difference in food consumption was noted in the control group and in the treatment groups (25, 80 or 160 mg IPDA/kg b.w./day).


Cohort 1B:
Males
No test item-related difference in food consumption was noted between the control group and the treatment groups (25, 80 or 160 mg IPDA/kg b.w./day) for the male animals.
However, a statistically significant differences in food consumption was noted for the males of the high dose group (5.0 % below the control, p ≤ 0.05) between test days 57 and 64. This was considered not to be test item-related but spontaneous.

Females
No test item-related difference in food consumption was noted between the control group and the treatment groups (25, 80 or 160 mg IPDA/kg b.w./day) for the female animals.



Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Cohort 1A & 1B:
No decreased or increased water consumption was noted by visual appraisal during the daily cage side observations.
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
Cohort 1A: Males and females
No test item-related differences for the examined haematological parameters were noted between the control group and the treatment groups (25, 80 or 160 mg IPDA/kg b.w./day) for the male and female animals.
However, a statistically significantly increased concentration was noted for the male animals of the high dose group for the number of platelets (28.3 % above the control, p ≤ 0.01). These is only due to the increased number of platelets of 2 individual high dosed males (no. 329 with 1386 and no. 330 with 1727 platelets x10E3/µL). The values they both exhibit were above the Provivo background range of 562 to 1268 platelets x10E3/µL.
As only 2 individual values of the high dose group were slightly above the Provivo background data and no statistically significant changes were noted for the female animals, the observed increased platelet concentration that was noted for the male animals of the high dose group was considered to be spontaneous. Finally, no changes were noted for the other haematological parameters for the male animals.
For the high dosed female animals a marginally but statistically significantly decreased MCV value was noted (2.6 % below the control, p ≤ 0.05). Nevertheless, only the MCV value from one high dosed female (49.9 fL) was marginally below the Provivo background range (50.7 to 55.8 fL). As furthermore no statistically significantly decreased MCV value was noted for the male animals, the decreased MCV value that was noted for the female animals of the high dose group was considered to be spontaneous.

Lymphocyte typing in spleen: Males and females
No test item-related influence was noted in the proportion of the examined lymphocyte subtypes to each other between the male and female animals of the control group and the treatment groups (25, 80 or 160 mg IPDA/kg b.w./day).

Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
PUP
Thyroid hormone determination - F1 Pups
T4 level (PND 4 and PND 21/22)
No test item-related differences between the control group and the treatment groups (25, 80 or 240/160 mg IPDA/kg b.w./day) were noted for the T4 level on lactation days 4 and 21/22 for the male and female pups.
Post-natal day 4:
On lactation day 4 increased T4 levels were noted at the low dose level and the high dose level. This was the case for the male and the female pups alone (statistically significant and not, respectively) and for the male and female pups combined (18.6 % or 26.0 %, p ≤ 0.05 / 0.01). In detail, the mean T4 concentrations of the pups (both sexes) from 6 individual dams of the low dose group, 4 individual dams of the intermediate dose group and 8 individual dams of the high dose group were above the Provivo background range.
However, no dose response relationship was noted.
Furthermore, no increased T4 levels were noted for the pups on lactation days 21/22 (see text below)
Hence, the increased T4 levels that were noted for the pups on lactation day 4 were considered to be spontaneous.
Post-natal days 21/22:
On lactation day 22 statistically significantly decreased T4 levels were noted at the intermediate dose level for the male and female pups (15.4 % and 16.7 % below the control, respectively, p ≤ 0.01). This was also noted for the low dose female pups alone (11.5 % below the control, p ≤ 0.05). For the male and female pups combined, a statistically significantly decreased T4 level was noted at the intermediate and the high dose level ( 15.7 % and 9.6 %, respectively, p ≤ 0.05 / 0.01) (see Text Table 7-38A below).
However, the mean T4 concentrations of the pups from the individual dams from all treatment groups were within the Provivo background range (see Text Table 7-38B below).
Hence, as no dose response relationship was noted, and all individual values of the treatment groups were within the background range, the decreased T4 concentrations that were noted on lactation days 21/22 are not considered to be test item-related, but spontaneous.


ADULT
Cohort 1A:
Clinical biochemistry: Males and females
No test item-related differences for the examined biochemical parameters were noted between the control group and the treatment groups (25, 80 or 160 mg IPDA/kg b.w./day).
A statistically significantly decreased sodium concentration was noted for the female animals of the intermediate dose group (1.1 % below the control, p ≤ 0.5). In detail, the sodium concentrations from 4 individual females of the intermediate dose group (134 to 135 mmol/L) were slightly below the Provivo background range (136 to 141 mmol/L). Additionally, one female each in the control and low dose and two females in the high dose groups also show similar sodium values, which were also slightly below the Provivo background range. Therefore, as this was observed in all female groups, no statistically significant changes were noted for the male animals and no dose-response relationship was noted, this observation was considered to be spontaneous.

Thyroid hormone levels: Males
No test item-related differences for the examined thyroid hormone levels (T4 and TSH) were noted between the control group and the treatment groups (25, 80 or 160 mg IPDA/kg b.w./day).
All individual T4 concentrations of the control group and all individual T4 concentrations of the intermediate and the high dose level, with one exception each, were within the Provivo background range. Only in case of the low dose group 4 individual values (79.006 to 93.925 nmol/L) were above the Provivo background range (42.528 to 76.219 nmol/L), which was considered to be spontaneous as no statistical significance and no dose response-relationship was noted.
An increased TSH concentration was observed for the male animals of the intermediate and the high dose group (50.0 % and 80.8 % above the control, respectively, statistically not significant). The rise appeared to be dose dependent but in the treatment groups the values are highly variable. In detail, 2 or 3 of the individual male TSH values (group 2: 1.732 and 2.589 ng/mL, group 3: 1.790 and 5.126 ng/mL, group 4: 2.416, 3.219 and 4.043 ng/mL) were above the range of the control group (0.081 to 1.530 ng/mL). Additionally, none of individual value was above the upper range of the Provivo background data (upper range = 5.699 ng/mL). Finally, an opposite trend was noted for the female animals with decreased TSH values in all treatment groups in comparison to the control. Hence, the observed increased values that were noted for the TSH values of the male animals at the intermediate and the high dose level were considered to be spontaneous.

Females
No test item-related differences for the T4 and TSH thyroid hormone levels were noted between the control group and the treatment groups (25, 80 or 160 mg IPDA/kg b.w./day).
Nearly all T4 concentrations of the individual animals were within the Provivo background range.
A dose dependent decrease of the TSH concentration was noted for all treatment groups (group 2: 31.5 %, group 3: 66.6 % and group 4: 79.5 % (p ≤ 0.05) below the control). At the intermediate and the high dose level the TSH concentrations from 7 of 10 females each were below the lower limit of detection (LOD = 0.081 ng/mL). Corresponding to the hormonal decrease, a weight increase of the pituitary gland (absolute and relative), the gland which produces TSH, was noted at the low and the high dose level. But no weight increase of the pituitary gland was noted at the intermediate dose level. Nevertheless, the histopathology observations did not report any histopathological changes in the pituitary gland compared to the control.
The female animals of the F0 generation showed a direct opposite trend. The females of the F0 generation show increased values in comparison to the control group. There the males on the other hand exhibited decreased values in comparison to the control. Due to this opposite trend, the high variability between the individual values, no histopathological changes noted for the pituitary gland and the males of the Co1A also showed a direct opposite development a decreased TSH, the differences that were noted between the control group and the treatment groups were considered to be spontaneous and toxicological not relevant.
Urinalysis findings:
no effects observed
Description (incidence and severity):
Cohort 1A: Males and females
No test item-related differences for the examined urinalysis parameters were noted between the control group and the treatment groups (25, 80 or 160 mg IPDA/day).
Sexual maturation:
no effects observed
Description (incidence and severity):
Males
Cohort 1A
No test item-related differences between the control group and the test item-treated groups (25, 80 or 160 mg IPDA/kg b.w./day) were noted for the time point of balano-prepuital separation and the body weight at the time point of balano-prepuital separation.
However, a marginally and statistically not significantly earlier time point of balano-preputial separation was noted at the intermediate and the high dose level. The intermediate dosed males showed a balano-preputial separation on PND 22.2 ± 0.4, the high dosed males at PND 22.3 ± 0.7 in comparison, the control group exhibit it at PND 22.6 ± 0.7.
As these differences were only small and as slightly earlier sexual maturation is not an adverse effect, these observations were considered to be spontaneous and not toxicological relevant.

Cohort 1A and 1B combined
A combined analysis of the time-points of balano-preputial separation from animals Cohort 1A and 1B together results in similar values as the analysis for Cohort 1A alone. However, due to the increased number of analyzed animals, the earlier time points of balano-preputial separation that were noted at the intermediate and the high dose level were now statistically significant. In detail, for Cohorts 1A and 1B combined, the time point of balano-preputial separation was 22.2 ± 0.5 for the intermediate and the high dose level, p ≤ 0.05 and p ≤ 0.01, respectively. In the control group the time-point of balano-preputial separation was on post-natal day 22.6 ± 0.7.
However, as stated for Cohort 1A alone, these differences were considered to be spontaneous, as the difference was only marginal and a slightly earlier sexual maturation is not an adverse effect, these observations were considered to be spontaneous and not toxicological relevant.


Females: Females
No test item-related differences between the control group and the test item-treated groups (25, 80 or 160 mg IPDA/kg b.w./day) were noted for the time point of vaginal opening and the body weight at the time point of vaginal opening.

Time point (postnatal day) of vaginal opening:
Cohort 1A
A slight delay for the time point of vaginal opening was noted at the low dose level (post-natal day 34.5 ± 3.4, statistically not significant) and the high dose level (post-natal day 34.9 ± 2.7, p ≤ 0.05) in comparison to the control group (postnatal day 33.1 ± 3.0) for the females of Cohort 1A. However, no dose response relationship was noted, as the time point of vaginal opening. Then the time point for the vaginal opening at the intermediate dose group (postnatal day 33.1 ± 1.7) was again in the range of the control group.

Cohort 1B:
Males
No test item-related differences between the control group and the test item-treated groups (25, 80 or 160 mg IPDA/kg b.w./day) were noted for the time point of balano-preputial separation and no differences were noted for the body weight at the time point of balano-preputial saparation.
However, for the male animals of Cohort 1B the time point of balano-preputial separation was slightly, but statistically significantly earlier for the high dose (postnatal day 22.1 ± 0.2) than for the control group (postnatal day 22.6 ± 0.7). As this difference was only small and is non-adverse, the slightly earlier time point of balano-preputial separation that was noted for the males of cohort 1B was considered to be spontaneous.

Females
No test item-related differences between the control group and the test item-treated groups (25, 80 or 160 mg IPDA/kg b.w./day) were noted for the time point of vaginal opening and no differences were noted for the body weight at the time point of vaginal opening.


Cohort 1A and 1B combined
Evaluation of the time points of vaginal opening from Cohort 1A and 1B combined resulted in nearly the same time points as for Cohort 1A alone. However, the combination showed an increased statistical significance at the high dose level. In detail, the time points of vaginal opening were on post-natal day 33.1 ± 2.5 for the control, 34.2 ± 2.8 for the low dose group, 33.1 ± 1.9 for the intermediate dose group and 34.6 ± 2.3 for the high dose group. Statistically significant at the high dose level (p ≤ 0.01).
Nevertheless, also in the combination of both cohorts, no dose response relationship was noted. Secondly, nearly all individual values were within the Provivo background range. In detail, only the time points of vaginal opening of one female each of the control group (postnatal day 43), high dose group (postnatal day 43) and 2 females of the low dose group (postnatal days 41 and 44) were above the range of the Provivo background data (postnatal days 30 to 37).
Hence, the observed delays that were noted for the time points of vaginal opening at the low and the intermediate dose groups were considered to be spontaneous.

Body weight at the time point (postnatal day) of vaginal opening:
Cohort 1A and 1B combined
A slightly increased body weight was noted at the time point of vaginal opening for the females of the low and the high dose group (4.3 % or 3.9 % above the control, statistically not significant). These slight differences were considered to be spontaneous.

Appearance of cornified cells in the vaginal lavage
(first time the cycle of the animals reaches the stage of estrus)
In the control group, the low dose group and the intermediate dose group the interval between the time point of vaginal opening and the time point the rat cycle reaches the stage of estrus for the first time (identified by the appearance of cornified cells in the vaginal lavage) was between 1.9 and 1.7 test days. This time interval was reduced to 0.7 test days in the high dose group. In detail, 15 of 20 females of the high dose group were in the stage of estrus at the day of vaginal opening in comparison to 5 of 20 females from the control group.
However, the reduced time interval between the time point of vaginal opening and the time point for the appearance of cornified cells was not considered to be test item-related. The reason is that there is no dose-response relationship for the time point for the appearance of cornified cells. The postnatal day for the appearance of cornified cells at the low dose level was later as at the high dose level (postnatal day 36.3 in comparison to postnatal day 35.6). Hence, the reduced time interval that was noted at the high dose level was only due to a delay for the time point of vaginal opening at the high dose level, which was considered as spontaneous.
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
PUP
No test item-related difference in the absolute and the relative ano-genital distance (value normalized to cube root of pup body weight) of the male and the female pups was noted between the control group and the treatment groups (25, 80 or 240/160 mg IPDA/kg b.w./day).
Slightly reduced absolute and relative values (statistically significant or not) were noted at the intermediate and the high dose level for the male and female animals. However, the similar distance reduction in the ano-genital distance for male and female pups are only showing a developmental delay at LD 4, which normalised until LD21. Hence, these slight differences were considered to be spontaneous and have no toxicological relevance.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
No test item-related difference in the number of nipples was noted between the male pups of the control group and in the male pups of the treatment groups (25, 80 or 240/160 mg IPDA/kg b.w./day).
Number and pups that were noted with nipples are given in Text Table 7-34 on the following page. For most of the affected male pups only 1 nipple was noted per pup. The number of these pups (male pup with one nipple) is nearly identical in each group (8 in the control group and between 7 and 8 in the treatment groups).
Male pups with 2 nipples were noted in all treatment groups (2 pups in each group) but not in the control group.
One male pup with 3 nipples was noted in the control group and 2 pups with 3 nipples each in the high dose group.
The maximum number of nipples that was noted for a male pup were 4 nipples. Pups with 4 nipples were noted in the low dose group (4 pups) and in the high dose group (1 pup), but not in the control group and in the intermediate dose group.
In summary, there is a slightly increased number of more severely affected pups (pups with 3 or 4 nipples) in the low and the high dose group (4 or 3 pups with 3 or 4 nipples) than in the control group and in the intermediate dose group (1 or no pup with 3 or 4 nipples). However, as no dose response relationship was observed, these slight differences were considered to be spontaneous.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
PUP
Pup organ weights (absolute) - F1 Pups
No test item-related differences for the examined absolute organ weights were noted between the control group and the treatment groups (25, 80 or 240/160 mg IPDA/kg b.w./day).


ADULT
Cohort 1A:
Males
No test item-related differences for the examined absolute and relative organ weights were noted between the control group and the treatment groups (25, 80 or 160 mg IPDA/kg b.w./day).

Nevertheless, decreased relative and absolute prostate weights were noted at the intermediate and the high dose level. In detail, at the intermediate dose level, the relative prostate weight was 9.2 % below the control (statistically not significant) and at the high dose level 13.9 % below the control at the high dose level (p ≤ 0.05). The absolute prostate weight at the intermediate dose level was 8.9 % below the and 8.4 % below the control at the high dose level (both not statistically significant).
A comparison with the Provivo background data revealed that the relative prostate weights from 2 individual animals of the control group, 5 individual animals of the low dose group, 6 individual animals of the intermediate dose group and 11 individual animals of the high dose group were below the Provivo background range.
However, only marginally reduced relative and absolute prostate weights were noted for the high dosed males of Cohort 1B (5.5 % and 4.3 % below the control, respectively, statistically not significant).
Furthermore, the absolute prostate weights did not show a dose dependent reduction in neither Cohort and also for the combination of Cohorts 1A and 1B.
Finally, there were no qualitative and semi-quantitative differences in the histomorphology between Groups 1 and 4.
The combination of the organ weights of Cohorts 1A and 1B revealed a statistically significantly decreased relative weight of the left epididymis in the low dose group ( 7.7%, p ≤ 0.05). However, no dose-response relationship and no histopathological changes were noted.
Hence, the differences observed in the prostate and epididymis weight were considered to be spontaneous and not test item-related.


Females
No test item-related differences for the examined absolute and relative organ weights were noted between the control group and the treatment groups (25, 80 or 160 mg IPDA/kg b.w./day).

Statistically significant weight changes were detected for the pituitary, the heart and the mesenteric lymph node.
For the pituitary gland statistically significantly increased relative and absolute weights were noted at the low dose level (17.5 % and 18.9 % above the control, respectively, p ≤ 0.05) and at the high dose level (23.2 % and 20.0 % above the control, respectively, p ≤ 0.05). However, all absolute weights of the pituitary glands from the individual animals of the low dose group were within the Provivo background range. At the high dose level only one animal with a pituitary gland weight of 0.025 g was marginally above the Provivo background range (0.010 to 0.022 g). Furthermore, only slightly increased weights were noted at the intermediate dose level. Hence, there was no dose response-relationship for the changes of the pituitary weights. Also the combination of the relative and absolute pituitary weights of Cohorts 1A and 1B, did not reveal a dose-response relationship.
Statistically significant reductions in the relative and absolute heart weight were noted at the intermediate dose level (9.1 % and 11.1 % below the control, respectively, p ≤ 0.05 / 0.01). The absolute heart weights of 4 individual animals of the intermediate dose group were below the Provivo background range. The reductions in the relative and absolute heart weight were declined at the high dose level but not as much as in the intermediate dose (4.5 % or 7.2 % below the control, statistically not significant). Hence, there was no dose-response-relationship present for the changes in the heart weights.
At the intermediate dose group a statistically significant reduction was also noted for the relative and absolute weight of the mesenteric lymph nodes (37.3 % and 36.8 % below the control, respectively, p ≤ 0.05). The absolute mesenteric lymph node weights from 3 females of the intermediate dose group were below the Provivo background range. The differences in the mesenteric lymph node weights between the control group and the high dose group were like with the heart weights smaller in comparison to the intermediate dose group and were no longer statistically significant. Therefore, also in this case no dose-response relationship was observed.
The combination of the relative and absolute organ weights of Cohort 1A and 1B, led to a statistically significantly decreased absolute weight of the left adrenal gland in the high dose group (-10.7%, p ≤ 0.05) and a statistically significantly increased absolute weight of the left ovary in the intermediate and high dose group (+12.8% and +15.6%, respectively, p ≤ 0.05). However, for the right adrenal gland, no dose-response relationship was present and for the left ovary, the weights were within the range of the Provivo background data.
Finally, the histopathological examination of the animals of group 4, revealed no histomorphological findings that could correlate with the weight changes that were noted for the pituitary gland, the heart, the mesenteric lymph nodes, the adrenal glands and the ovaries.

Therefore, it was considered that there is no toxicological significance in the weight changes detected in the pituitary gland, the heart, the mesenteric lymph nodes, the adrenal glands and the ovaries.

Cohort 1B:
Males
No test item-related differences for the examined absolute and relative organ weights were noted between the control group and the treatment groups (25, 80 or 160 mg IPDA/kg b.w./day).
Increased values were noted for the relative and absolute organ weights of the pituitary gland at the low dose level (5.6 % and 8.0% above the control, respectively, statistically not significant) and at the high dose level (21.9 % and 22.6 % above the control, respectively, statistically significant at p ≤ 0.05 / 0.01). However, as no dose response-relationship was noted and no such observations were recorded for the male animals of Cohort 1A, the changes that were noted for the males of Cohort 1B were considered to be spontaneous.

Females
No test item-related differences for the examined absolute and relative organ weights were noted between the control group and the treatment groups (25, 80 or 160 mg IPDA/kg b.w./day).
Decreased relative and absolute organ weights were noted for the right adrenal gland from the female animals of the low dose group (14.5 % (p ≤ 0.05) and 12.4 % (not statistically significant) below the control, p ≤ 0.05 or not statistically significant) and the high dose group (15.4 % (p ≤ 0.01) and 11.5 % (not statistically significant) below the control, respectively).
However, no significant changes were noted for the left adrenal weight from the females of Cohort 1B and the right and the left adrenal weights from the females of Cohort 1A. Furthermore, no dose-response relationship was noted. Hence, the decreased weights of the right adrenal gland, noted for the females of Cohort 1B were considered to be spontaneous.
Statistically significantly increased values were noted for the relative organ weights of the left ovary at all dose levels (16.6 %, 20.1 % or 20.8 % above the control value at the low, the intermediate or the high dose level, respectively; p ≤ 0.05 / 0.01).
The absolute organ weights of the left ovary were also increased but failed statistical significance (13.8 %, 15.2 % or 15.0 % above the control at the low, the intermediate or the high dose level; respectively).
However, no statistically significant changes were noted for the relative and absolute organ weights of the right ovary of the females from Cohort 1B and the left and the right ovary weights from the females of Cohort 1A. Additionally, no histomorphological changes to be toxicologically concerned in the view of reproductive performance were found in the female reproductive organs including ovaries in the F1 Cohort 1A. All findings recorded were within the range of normal background lesions or physiological alterations that may be observed in this type of study or animals of this strain and age.
Hence, the increased weights of the left ovary that were noted for the females of Cohort 1B were considered to be spontaneous and not of toxicological relevance.

Gross pathological findings:
no effects observed
Description (incidence and severity):
PUP
External and internal examination of the pups – F1 Pups
No gross abnormalities (e.g. malformations or variations) were noted during the macroscopic external examination of the pups that were terminally sacrificed after weaning, the pups that were found dead or for the stillbirths from the control group and the treatment groups (25, 80 or 240/160 mg IPDA/kg b.w./day).
Furthermore, no malformations were noted during the macroscopic examination of the inner organs and tissues of the control pups and the pups of the treatment groups.


ADULT
Cohort 1A:
Males
No observations were noted for the surviving male animals of the control group and the treatment groups (25, 80 or 160 mg IPDA/kg b.w./day).

Females
No observations were noted for the female animals of the treatment groups (25, 80 or 160 mg IPDA/kg b.w./day).
There were no findings were noted in the treatment groups. However, 2 females (nos. 223, 230) of the control showed a dilated uterus (filled with clear liquid) and one other control female (no. 235) exhibited a reddened thymus. Another female of the control group (no. 237) revealed an enlarged mesenteric lymph node, an enlarged spleen and intestines with a thickened mucosa.
All the macroscopic findings recorded at necropsy were lesions within the range of normal background changes or physiological alterations which maybe observed in this type of study or animals of this strain and age, or incidental gross appearances without corresponding histomorphological changes.

Cohort 1B:
Males
No observations were noted for the surviving male animals of the control group and the treatment groups (25, 80 or 160 mg IPDA/kg b.w./day).

Females
No observations were noted for the female animals of the treatment groups (25, 80 or 160 mg IPDA/kg b.w./day).
In the control group one female animal (no. 398) with a dilated uterus was noted. As described for the macroscopic findings of Cohort 1A, a dilated uterus belongs to the normal background changes or physiological alterations which maybe observed in this type of study or animals of this strain and age, or incidental gross appearances without corresponding histomorphological changes.
Histopathological findings:
no effects observed
Description (incidence and severity):
Cohort 1A:
Males
No test item-related changes were observed for the examined male animals of the high dose group (160 mg IPDA/kg b.w./day) during the microscopic examination.
Nevertheless, one dead male of the high dose group was reported (no. 336). However, histomorphologically the direct cause(s) of animals morbidity was not established in male no. 336 of group 4 from F1 Generation Cohort 1A. The remaining microscopic findings of male no. 336 were stress-related changes or non-specific changes that are frequently observed in animals with deteriorated general conditions. Findings that could be specific to the test item treatment were not identified. However, due to the animal’s general conditions during the exposure with higher dose level, effects attributed to the test item can not be excluded.
In the previous study (OECD 422 study) in which the treatment in the high dose group was started at a dose of 300 mg/kg b.w./day (thereafter reduced to 240 mg/kg b.w./day during the treatment period), treatment-related microscopic changes were observed in the stomach and kidneys.
However, the histopathological examination of the present study revealed that the test item did not produce treatment-related microscopic changes not only in the stomach and kidneys but also in any other organs including male and female reproductive system of the animals treated with 160 mg/kg b.w./day in F1 Generation Cohort 1A.

Male reproductive organs
No histomorphological changes to be toxicologically concerned in the view of reproductive performance were found in the male reproductive organs including testes, epididymides, prostate glands, seminal vesicles and coagulating glands in cohort 1A.
For testes, the stages were checked on completeness of cell populations, completeness of stages and checked for degenerative changes, while taking also account into the interstitial cell stucture, for both generations. As a result, all testes examined showed completeness of stages and cell population.
All the other findings recorded in the male reproductive organs were within the range of normal background changes that may be observed in this type of study or animals of this strain and age, and hence, deemed not to be treatment-related.

Females
No test item-related changes were observed for the examined female animals of the high dose group (160 mg IPDA/kg b.w./day) during the microscopic examination.
The histopathological examination of the present study revealed that the test item did not produce treatment-related microscopic changes not only in the stomach and kidneys but also in any other organs including female reproductive system of the high dose animals in F1 generation Cohort 1A.

Female Reproductive Organs - including quantitative examination of ovaries
No histomorphological changes to be of toxicologically concerend in the view of reproductive performance were found in the female reproductive organs including ovaries, oviducts, uterus, uterine cervix and vagina in the F1 cohort 1A.
All findings recorded were within the range of normal background lesions or physiological alterations that may be observed in this type of study or animals of this strain and age.
For ovaries, the quantitative evaluation was performed in F1 generation Cohort 1A, in addition to qualitative histopathology examination.
No test item-related or statistically significant differences were noted in all parameters including primordial follicles, summed up primordial and growing follicles, antral follicles and corpora lutea.
Other effects:
no effects observed
Description (incidence and severity):
Cohort 1A:
Monitoring of estrous cycles - 2-week period
The stages of the estrous cycle of the cohort 1A animals were monitored on 14 test days between test days 49 and 62 of the F1 Generation Study.
No test item-related differences were noted for the mean length and the mean number of estrous cycles per female animal between the females of the control group and the treatment groups (25, 80 or 160 mg IPDA/kg b.w./day).
A slight, but statistically significantly elongated mean cycle length was noted at the high dose level (4.5 days compared to 4.12 days in the control group, p ≤ 0.05). However, the difference was only small and the mean cycle length of the individual females of the high dose group were in the range of the mean cycle length of the individual females of the control group. Secondly, the physiological cycle length of a rat lies between 4 and 5 days. Hence, the observed increase in the mean cycle length is still in physiological range. Therefore, this observation was considered to be spontaneous.

Examination of the sperm number, viability and morphology
No test item-related difference was noted between the rats of the control group and the rats treated with 25, 80 or 160 mg IPDA/kg b.w./day for the sperm number, the viability and morphology.

Cohort 1B: Stage of estrous cycle at necropsy
No test item-related differences were noted between the control group and the treatment groups (25, 80 or 160 mg IPDA/kg b.w./day) in the distribution of the stages of the estrous cycle evaluated from vaginal lavages that were taken at necropsy.

Details on results (F1)

F1 PUPS
Birth indices and post-implantation loss - F1 Pups
No test item-related differences were noted for the mean number of implantation sites per dam, the mean number of pups born (alive and dead) per dam and the mean number of live born pups per dam between the control group and the treatment groups (25, 80 or 240/160 mg IPDA/kg b.w./day).
Also the reproductive indices as the birth index, the live birth index and the percentage of post implantation loss revealed no test item-related differences between the control group and the treatment groups (25, 80 or 240/160 mg IPDA/kg b.w./day).
A slightly decreased birth index was noted between the control group (94.27 ± 8.38) and the treatment groups 25, 80 or 240/160 mg IPDA/kg b.w./day, 91.16 ± 10.31%, 93.10 ± 8.24 % and 89.46 ± 10.34%, respectively. However, as no dose response relationship was noted, these differences were considered to be spontaneous.
The decreased birth index was due to an increased number of resorptions in the treatment groups. Further on, as no dose response relationship was noted, these differences were considered to be spontaneous.
Furthermore, due to an increased number of stillbirths at the low and the intermediate dose group a slightly and statistically not significantly decreased live birth index was noted in these groups (97.25 ± 6.27 % or 97.85 ± 6.03 %) in comparison to the control group (99.68 ± 1.52 %).
In detail, 9 stillbirths (from 5 dams) were noted in the low dose group and 7 (from 4 dams) in the intermediate dose group in comparison to 1 stillbirths in the control group and 2 in the high dose group.
The increased number of resorptions in the treated groups as well as the increased number of stillbirths in the low and the intermediate dose led to an increased post-implantation loss (statistically not significant). In detail, the percentages of post-implantation loss that were noted in the treatment groups were 11.31 ± 11.91 % for low dose, 8.83 ± 10.38 % for intermediate dose and 11.20 ± 10.65 % for high dose in comparison to 6.05 ± 8.28 % for the control group. Additionally, the percentages of post-implantation loss per group in the treatment groups were within the Provivo background range.
Furthermore, as no dose response relationship was noted for the number of resorptions and the number of stillbirths, these differences were considered to be spontaneous

Number of live pups - F1 Pups
No test item-related or statistically significant differences were noted for the mean number of live pups per dam between the control group and the treatment groups (25, 80 or 240/160 mg IPDA/kg b.w./day) during the lactation period

Male to female ratio of the pups - F1 Pups
No test item-related influence on the male to female ratio was noted for all treatment groups (25, 80 or 240/160 mg IPDA/kg b.w./day).




F1 ADULTS
Cohort 1A
This part of the study reports the effects of the test item IPDA on the development of the F1 male and female animals of cohort 1A after weaning on lactation day 21 until necropsy. Necropsy was carried out between postnatal days 90 to 92 (males and females).
One male animal of the high dose group (160 mg IPDA/kg b.w./day) had to be prematurely sacrificed due to poor health conditions. The death is considered to be test item-related.
Adverse test item-related observations in the form of breathing sounds that were noted for several high dosed animals of the F0 Generation were markedly reduced for the animals of Cohort 1A.
No influence was noted for the male and female animals on body weight, food consumption, the haematological and biochemical parameters, lymphocyte typing in the spleen, urinalysis, the levels of the thyroid hormones, the sperm parameter and sexual maturation.
Nevertheless, a dose dependent decrease in the TSH values in the females of the intermediate and high dose below the detection limit were recorded. However, the histopathology observations did not report any histopathological changes in the pituitary gland compared to the control group. Furthermore, the observed differences in the TSH concentration were considered to be spontaneous, as they showed an opposite trend for the male and female animals.
Therefore, it was considered that the decreased TSH values were not toxicological relevant.
No test item-related changes were noted during the macroscopic examination, the examination of bone marrow and for the relative and absolute organ weights.
The histopathological examination of all organs did not reveal any test item-related changes or differences. Moreover, no histomorphological changes to be toxicologically concerned in the view of reproductive performance were found in the male reproductive organs (testes, epididymides, prostate glands, seminal vesicles and coagulating glands) and female reproductive organs (ovaries, oviducts, uterus, uterine cervix and vagina).


Cohort 1B
This part of the Study reports the effects of the test item IPDA on the development of the F1 male and female animals of cohort 1B after weaning on lactation day 21 until necropsy. Necropsy was carried out between postnatal days 98 to 102 (males and females).
No test item-related premature death was noted for the animals of Cohort 1B. Even so, that one male animal of the intermediate dose group was found dead. But this death was considered to result from an accident during the process of dosing.
Adverse test item-related observations in the form of breathing sounds and piloerection were noted for one or two male and female animals of the intermediate and high dose group of Cohort 1B.
No influence was noted for the male and female animals on food consumption and sexual maturation. For the female animals a slightly but statistically significantly reduced body weight was noted. Due to no correspondence with the body weight gain of the females of the treated groups this was considered to be spontaneous.
No test item-related changes were noted during the macroscopic examination and for the relative and absolute organ weight for the male and female animals. Nevertheless, an increase of the left ovary weight in the female animals of Cohort 1B was observed. However, no statistically significant changes were noted for the relative and absolute organ weights of the right ovary of the females from Cohort 1B and the left and the right ovary weights from the females of Cohort 1A. Additionally, no histomorphological changes of toxicologically concerned were found in the female reproductive organs, including ovaries of the F1 Cohort 1A. Hence, the increased weights of the left ovary that were noted for the females of Cohort 1B were considered to be spontaneous.

Effect levels (F1)

open allclose all
Key result
Dose descriptor:
NOAEL
Generation:
other: F1 pups
Effect level:
> 160 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: pre- and postnatal development
Key result
Dose descriptor:
NOAEL
Generation:
other: F1 adult (Cohort 1A & 1B)
Effect level:
80 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

Text Table 7‑1:     Reproductive outcome of the female animals.












































































































IPDA



Group 1


Control



Group 2


25 mg/kg



Group 3


80 mg/kg



Group 4


240/160 mg/kg



Females started dosing



24



24



24



24



Females deceased during pre-mating period



0



0



0



3


(nos. 182, 174, 178)



Females used for pairing


(placed together with a male at the beginning of the mating period)



24



24



24



21



Females with no verified copulation during the mating period of 14 days



0



1 (no. 95)



0



2 (nos. 177, 189)



 



- thereof non-pregnant



-



1 (no. 95)



-



2 (nos. 177, 189



 



- thereof pregnant



-



-



-



-



Females with verified copulation during the mating period of 14 days,



24



23



24



19



 



- thereof non-pregnant



1 (no. 28)



-



2


(nos. 137, 140)



-



 



- thereof pregnant



23



23



22



19



Fertility index


(Pregnant females with verified copulation related to females with verified copulation)



96 %


(23 of 24)



100 %


(23 of 23)



92 %


(22 of 24)



100 %


(19 of 19)



Females deceased during gestation period



1 (no. 40)



0



1 (no.126)



1 (no. 187)



Females with total resorption of implants



0



0



0



0



Females with live born pups



22



23



21



18



 


Text Table 7‑2:     Prematurely sacrificed animals for which no direct cause of death could be established.















































Prematurely sacrificed animals due to poor health condition


(most likely test item-related)



No.



Sex



Group



Test day #1



Number of doses



Type of fate



182



Female



4



24



7 #2



Premature sacrifice


 



178



Female



4



59



45



153



Male



4



73



59



#1:



Dose reduction on test day 51.



#2:



Dosing for animal no. 182 was stopped on test day 21 (see 2.13 ‘Study Plan deviations).



 


Text Table 7-3:     Observations that were noted for the prematurely sacrificed animal nos. 153, 178 and 182 during the daily cage side inspection on the days before sacrifice (common observations as salivation are not considered).























































Observations during the daily cage side inspections for the prematurely sacrificed animal nos. 153, 178 and 182 #1



Gr.



No,



Sex



Symptom



Observed on test day (day of death #2)



4



153



M



Gasping


Reduced motility


Breathing sounds


Laboured breathing



72, 73


73


Consistently from test day 60 until 73


72, 73 (day of death: 73)



178



F



Breathing sounds



58, 59 (59)



182



F



Breathing sounds



21 - 23



Laboured breathing



24



Haemorrhagic nose/snout



24 (day of death: 24)



 



Gr.: Group; M; F: Male; Female



#1:



See tables 3-1B ‘Clinical Signs – Prematurely Deceased Males - Individual Data’ and 3-2B ‘Clinical Signs – Prematurely Deceased Females – Premating/Mating - Individual Data - Females’



#2:



Day of death is given in brackets.



 


Text Table 7‑4A:   Prematurely deceased or sacrificed animals due to an accident during the process of dosing.

































































































Prematurely deceased or sacrificed animals - death caused by accident



No.



Sex



Group



Test day #1



Number of doses



Type of fate


 



Cause of death



164 #2



Male



4



16



1



Found dead



Accidental death



151



Male



4



49



35



Found dead



174



Female



4



49



35



Premature sacrifice



126 #3



Female



3



95 (GD6)



81



Found dead


 



187 #4



Female



4



106 (GD20)



92



Premature sacrifice



40 #5



Female



1



107 (GD19)



93



Found dead



#1:



Dose reduction on test day 51.



#2:



No. 164 was replaced by spare animal no. 193 (Amendment no. 2).



#3:



Female no. 126 was pregnant. During necropsy (including laparotomy) on gestation day 6, 15 implantation sites were noted.



#4:



Female no. 187 was pregnant. During necropsy (including laparotomy) on gestation day 20, 13 implantation sites were noted.



#5:



Female no. 40 was pregnant. During necropsy (including laparotomy) on gestation day 19, 14 implantation sites were noted.


        

 


Text Table 7-4B:   Observations that were noted during the daily cage side inspection on the days before sacrifice or death (common observations as salivation are not considered).















































































Observations during the daily cage side inspections #1


Prematurely deceased or sacrificed animals - death caused by accident



Gr.



No,



Sex



Symptom



Observed on test day (day of death #2)



1



40



F



No observations



- (GD 19)



3



126



F



No observations



- (GD 6)



4



151



M



Haemorrhagic nose/snout



47



Haemorrhagic canthus



47 (49)



164



M



No observations



- (16)



174



F



Breathing sounds



46 - 49 (174)



187



F



Breathing sounds



GD 18 - 20



Piloerection



GD 18



Haemorrhagic nose/snout



18 – 20 (GD 20)



 



Gr.: Group; M; F: Male; Female, GD = gestation day.



#1:



See tables 3-1B ‘Clinical Signs – Prematurely Deceased Males – Individual Data’, 3-2B ‘Clinical Signs - Prematurely Deceased Females – Pre-mating/Mating – Individual Data’ and 3-3B ‘Clinical Signs - Prematurely Deceased Females – Gestation – Individual Data’



#2:



Day of death is given in brackets.



 


Text Table 7‑5:     Observations that were noted during the daily cage-side observations for the male animals of the low, the intermediate and the high dose level.







































































































Observations in group 2


(25 mg IPDA/kg b.w./day) #1



Observation (males)



Affected animals



First to last seen


(test days)


#2



Number of times recorded #3



Haemorrhagic nose / snout



1 of 24



98 (no. 71)



1



Water consumption increased



1 of 24



69 - 75 (no. 52)



7



Pultaceous faeces



1 of 24



75 (no. 64)



1



Observations in group 3


(80 mg IPDA/kg b.w./day)



Salivation



17 of 24



24 - 125



65



Water consumption increased



1 of 24



72 - 75 (no. 120)



4



Observations in group 4


(240/160 mg IPDA/kg b.w./day)



Salivation



22 of 22 #4



16 - 127



704



Breathing sounds



10 of 22



16 - 117



67



Piloerection



5 of 22



22 - 73



14



Haemorrhagic urine



1 of 22



1 (no. 159)



1



Water consumption increased



2 of 22



72 – 79


(nos. 146, 158)



11



Pultaceous faeces



1 of 22



103 -104 (no. 145)



2



#1:



See table 3-1A ‘Clinical Signs - Summary - Males - F0 Generation’ and table 3-5 ‘Clinical Signs - Individual Data - Males - F0 Generation’ for the number of the individual animals.



#2:



If only 1 or 2 animals were affected, the animals are listed in brackets.



#3:



Number of times the symptom was observed for the affected animals.



#4:



Group 4: The prematurely deceased animals nos. 151, 164 were excluded from the summary.



 


Text Table 7‑6:     Observations that were noted during the daily cage-side observations for the female animals during the pre-mating/mating period, the gestation period and the lactation period until necropsy.
























































































































































Observations in group 2


(25 mg IPDA/kg b.w./day)



 



Affected females per period



Number of times recorded



Observation


(females)



Pre-mating/


Mating #1



Gestation


#1



Lactation


#1



Pre-mating/


Mating



Gestation



Lactation



Salivation



n.d.



n.d.



1 of 23 #2



n.d.



n.d.



3



Observations in group 3


(80 mg IPDA/kg b.w./day)



Salivation



9 of 24



2 of 21 #3



2 of 21 #3



18



2


(no. 125, 134)



2


(no. 125, 135)



Observations in group 4


(240/160 mg IPDA/kg b.w./day)



Salivation



21 of 21 #4



12 of 18 #5



12 of 18 #5



440



26



32



Breathing sounds



10 of 21



4 of 18



1 of 18



159



19



7


(no. 186)



Piloerection



5 of 21



n.d.



1 of 18



31



n.d.



2


(no. 184)



Gasping



1 of 21



n.d.



n.d.



1 (no. 187)



n.d.



n.d.



Haemorrhagic nose-snout



1 of 21



n.d.



n.d.



3 (no. 187)



n.d.



n.d.



Increased water consumption



1 of 21



n.d.



n.d.



8 (no. 188)



n.d.



n.d.



Respiratory rate increased



1 of 21



n.d.



n.d.



1 (no. 181)



n.d.



n.d.



Laboured breathing



1 of 21



n.d.



n.d.



6 (no. 187)



n.d.



n.d.



Thickened Abdomen



1 of 21



n.d.



n.d.



1 (no. 187)



n.d.



n.d.



n.d.:



Not detected



#1:



See table 3-2A ‘Clinical Signs - Summary - Females - Pre-mating/Mating - F0 Generation’, table 3-3A ‘Clinical Signs - Summary - Females - Gestation - F0 Generation’ and table 3-4 ‘Clinical Signs - Summary - Females - Lactation - F0 Generation’.



#2:



Group 2: The non-mated and non-pregnant female no. 95 is not considered for the gestation and the lactation period.



#3:



Group 3: The non-pregnant female nos. 137, 140 and the prematurely deceased female no. 126 are missing in the gestation period and the lactation period.



#4:



Group 4: The prematurely deceased animals no. 174, 178, 182 (died during pre-mating period) are excluded from the summary.



#5:



Group 4: The prematurely deceased animal no. 187 (died during gestation period) and the non-mated and non-pregnant female nos. 177, 189 are excluded from the summaries in the gestation and the lactation periods.



 


Text Table 7‑7:     Start and duration of observations that were noted for the male animals.

























































































Start and duration of symptoms - Males #1



Symptom



Time frames in relation to application



Number of times recorded


(in all groups)



Appearing of  the symptom



Disappearing of the


symptom



Salivation



0 - 5 min



5 - 20 min



4



0 - 5 min



20 - 60 min



100



5 - 20 min



5 - 20 min



11



5 - 20 min



20 - 60 min



651



5 - 20 min



6 - 24 h



2



Consistently during the day



1



Breathing sounds



0 - 5 min



-



1 #2



0 - 5 min



2 - 6 h



1



0 - 5 min



6 - 24 h



2



5 - 20 min



20 - 60 min



1



Consistently during the day



62



#1:



See table 3-9-1 ‘Start and Duration of Symptoms - Summary - Males - F0 Generation’.



#2:



No endpoint was determined in one case.


      

 


Text Table 7‑8:     Start and duration of observations that were noted for the female animals.





































































































Start and duration of symptoms - Females #1



Symptom



Time frames in relation to application



Number of times recorded


(in all groups and periods)



Appearing of  the symptom



Disappearing of the


symptom



Salivation



0 - 5 min



5 - 20 min



2



0 - 5 min



20 - 60 min



39



5 - 20 min



-



1 #2



5 - 20 min



5 - 20 min



10



5 - 20 min



20 - 60 min



471



Breathing sounds



-



-



1 #3



0 - 5 min



-



2 #2



0 - 5 min



6  -24 h



3



5 - 20 min



1- 2 h



1



20 - 60 min



2 - 6 h



1



Consistently during the day



177



Laboured breathing



Consistently during the day



6



Gasping



Consistently during the day



1



Increased respiratory rate



Consistently during the day



1



#1:



See table 3-9-2 ‘ Start and Duration of Symptoms - Summary -  Females - Pre-mating / Mating - F0 Generation’, table 3-9-3 ‘Start and Duration of Symptoms - Summary -  Females - Gestation - F0 Generation’ and table 3-9-4 ‘Start and Duration of Symptoms - Summary -  Females - Lactation - F0 Generation’.



#2:



No endpoint was determined during the pre-mating / mating period in one case each for salivation and breathing sounds.



#3:



No start and no endpoint was determined in one case of breathing sounds during the pre-mating / mating period.



 


Text Table 7‑9:     Body weight gain of the male animals from test day 15 to test day 126.
























Males



Group 1


Control



Group 2


25 mg/kg



Group 3


80 mg/kg



Group 4


240/160 mg/kg



Body weight gain #


(test day 15 to test day 126)



+44.6%



+47.3%



+43.1%



+43.6%



#:



Values taken from table 6-1 'Body Weight Gain - Summary - Males - F0 Generation'



 


Text Table 7‑10:   Body weight gain of the female animals during the pre-mating period, the gestation period and the lactation period.


































Females



Group 1


Control



Group 2


25 mg/kg



Group 3


80 mg/kg



Group 4


240/160 mg/kg



Body weight gain (%)


(pre-mating period)


(test days 15 to 85) #1



+21.3%



+20.6%



+20.3%



+19.0%



Body weight gain (%)


(gestation period) #2



+50.3%



+47.7%



+47.0%



+49.1%



Body weight gain (%)


(lactation period) #3



+1.1%



+2.5%



+3.9%



+6.7%



 


Text Table 7-11:   Comparison of haematological parameters with the Provivo background data.































































































F0 Females


Parameter



Values from this study #2


Mean value per group ± SD


(range of the individual values (n = 10))


[number of individual values below or above the stated range]



Provivo Background Data #1


obtained from the control groups of 6 OECD 443 studies performed at Provivo from


2019 – 2021



WBC


[x10E3 cells/µL]



Group 1



9.292 ± 1.488


(6.20 – 11.75) [n=1]



 


5 to 95 % Percentile


 


4.65 - 11.38


 



Group 2



8.161 ± 1.965


(5.80 – 12.11) [n=1]



Group 3



6.761 ± 1.506*


(4.70 - 9.91)



Group 4



9.852 ± 3.564


(5.06 – 12.24) [n=2]



EOS


[x10E3 cells/µL]



Group 1



0.241 ± 0.136


(0.09 - 0.54) [n=1]



 


5 to 95 % Percentile


 


0.05 - 0.42


 



Group 2



0.115 ± 0.050**


(0.06 - 0.23)



Group 3



0.130 ± 0.062*


[n=1] (0.04 - 0.22)



Group 4



0.117 ± 0.036*


(0.05 - 0.16)



Cohort 1A


WBC


[x10E3 cells/µL]


 



Group 1



14.031 ± 22.068


(5.07 - 76.60) [n=1]



5 to 95 % Percentile


 


4.33 - 11.52



Group 2



7.167 ± 1.766


(4.47 - 8.93)



Group 3



8.677 ± 2.281


(6.05 - 12.96) [n=1]



Group 4



8.937 ± 2.091


(6.58 - 12.38) [n=2]



Baso


[x10E3 cells/µL]



Group 1



0.042 ± 0.018


(0.02 - 0.07)



 


5 to 95 % Percentile


 


0.00 - 0.07


 



Group 2



0.042 ± 0.020


(0.02 - 0.07)



Group 3



0.021 ± 0.009*


(0.01 - 0.04)



Group 4



0.037 ± 0.017


(0.01 - 0.07)



#1:



Data not audited by QAU



*/**:



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



#2:



Taken from table 8-2 'Haematological Parameters - Summary - Females' and table 8-4 'Haematological Parameters - Individual Data - Females'.



 


Text Table 7-12:   Statistically significant changes of the haematological parameters that were not considered to be test item-related.






































































































































 



 



Changes in comparison to control #1


[%]



Reason



Parameter #


(F0 Generation)



Sex



Group 1


Control



Group 2


25 mg/kg



Group 3


80 mg/kg



Group 4


240/160 mg/kg



 



WBC


[x10E3/µL]



Females #2



n.a.


(9.292)



-12.2


(8.161)



-27.2*


(6.761)



+6.0


(9.852)



A



WBC


[x10E3/µL]



Males  #3



n.a.


(9.609)



+16.4


(11.187)



- 9.0


(8.745)



- 2.4


(9.380)



D



WBC


[x10E3/µL]



Males #4


(Co1A)



n.a.


(9.340)



+1.1


(9.440)



+18.1


(11.033)



+27.6


(11.917)



D



WBC


[x10E3/µL]



Females  #5


(Co1A)



n.a.


(14.031)



- 48.9


(7.167)



- 38.2


(8.677)



- 36.3


(8.937)



D



WBC


[x10E3/µL]



Females  #6


(Co1A)



n.a.


(7.079)



+1.2


(7.167)



+22.6


(8.677)



+26.2


(8.937)



D



EOS


[x10E3/µL]



Females #1



n.a.


(0.241)



-52.3**


(0.115)



-46.1*


(0.130)



-51.5*


(0.117)



B



Baso


[x10E3/µL]



Females #1



n.a.


(0.042)



0.0


(0.042)



-50.0*


(0.021)



-11.9


(0.037)



C



n.a.:



Not applicable



*/**:



Statistically significant from control at p ≤ 0.05/0.01 (DUNNETT test)



A:



The statistically significantly decreased value at the intermediate dose level was considered to be spontaneous, as no dose response-relationship was noted. Furthermore, a comparison with the statistically significant changes that were noted for the F0 male animals and the statistically not significant changes that were noted for the F0 females and the male and female animals of Cohort 1A revealed no consistent tendencies.



B:



The similar reductions in the treatment groups were considered to be due to an increased value of the control group and considered as not relevant.



C;



No dose response-relationship was noted and all values were within the Provivo background range.



D:



Only for comparison.



#1:



The group mean values are given in the brackets.



#2:



Values taken from table 8-2 'Haematological Parameters - Summary - Females'.



#3:



Values taken from table 8-1 'Haematological Parameters - Summary - Males'.



#4:



Values taken from table 6-1-Co1A 'Haematological Parameters - Summary - Males'.



#5:



Values taken from table 6-2-Co1A 'Haematological Parameters - Summary - Females'.



#6:



Mean of the control group without animal no. 237 that had extremely increased numbers of white blood cells.



 


Text Table 7-12:   Comparison of haematological parameters with the Provivo background data.































































































F0 Females


Parameter



Values from this study #2


Mean value per group ± SD


(range of the individual values (n = 10))


[number of individual values below or above the stated range]



Provivo Background Data #1


obtained from the control groups of 6 OECD 443 studies performed at Provivo from


2019 – 2021



WBC


[x10E3 cells/µL]



Group 1



9.292 ± 1.488


(6.20 – 11.75) [n=1]



 


5 to 95 % Percentile


 


4.65 - 11.38


 



Group 2



8.161 ± 1.965


(5.80 – 12.11) [n=1]



Group 3



6.761 ± 1.506*


(4.70 - 9.91)



Group 4



9.852 ± 3.564


(5.06 – 12.24) [n=2]



Cohort 1A


WBC


[x10E3 cells/µL]


 



Group 1



14.031 ± 22.068


(5.07 - 76.60)  [n=1



5 to 95 % Percentile


 


4.33 - 11.52


 



Group 2



7.167 ± 1.766


(4.47 - 8.93)



Group 3



8.677 ± 2.281


(6.05 - 12.96)  [n=1]



Group 4



8.937 ± 2.091


(6.58 - 12.38)  [n=2]



EOS


[x10E3 cells/µL]



Group 1



0.241 ± 0.136


(0.09 - 0.54) [n=1]



 


5 to 95 % Percentile


 


0.05 - 0.42


 



Group 2



0.115 ± 0.050**


(0.06 - 0.23)



Group 3



0.130 ± 0.062*


[n=1] (0.04 - 0.22)



Group 4



0.117 ± 0.036*


(0.05 - 0.16)



Baso


[x10E3 cells/µL]



Group 1



0.042 ± 0.018


(0.02 - 0.07)



 


5 to 95 % Percentile


 


0.00 - 0.07


 



Group 2



0.042 ± 0.020


(0.02 - 0.07)



Group 3



0.021 ± 0.009*


(0.01 - 0.04)



Group 4



0.037 ± 0.017


(0.01 - 0.07)



#1:



Data not audited by QAU



*/**:



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



#2:



Taken from table 8-2 'Haematological Parameters - Summary - Females' and table 8-4 'Haematological Parameters - Individual Data - Females'.



 


Text Table 7‑13:   Statistically significant changes of the biochemical parameters that were not considered to be test item-related.


















































































Parameter #


F0 Gemeration



 



Changes in comparison to control


[%]



Reason



Sex



Group 2


25 mg/kg



Group 3


80 mg/kg



Group 4


240/160 mg/kg



 



 



Bilirubin (total)


[µmol/L]



Females



+15.1*



+0.3



-3.4



A



Glucose [mmol/L]



Females



+0.2



-8.0



-18.7*



B



Glucose [mmol/L]



Males



- 1.2



- 4.0



- 2.2



C



Glucose [mmol/L]



Males


Co1A



+5.3



+8.6



+6.8



C



Glucose [mmol/L]



Females


Co1A



- 12.2



+5.5



- 5.0



C



*/**:



Statistically significant from control at p ≤ 0.05/0.01 (DUNNETT test)



A:



No dose response relationship was noted and all values of the low dose group were within the Provivo background range.



B:



No changes were noted for the F0 males and the males and females of Cohort 1A.



C:



Only for comparison.



#:



Values taken from table 9-1 'Biochemical Parameters - Summary - Males', table 9-2 'Biochemical Parameters - Summary - Females', table 7-1-Co1A 'Biochemical Parameters - Summary - Females' and from table 7-2-Co1A 'Biochemical Parameters - Summary - Females'.



 


Text Table 7-14:   Comparison of biochemical parameters with the Provivo background data.



























































F0 Females


Parameter



Values from this study #2


Mean value per group ± SD


(range of the individual values (n = 10))


[number of individual values below or above the stated range]



Provivo Background Data #1


obtained from the control groups of 6 OECD 443 studies performed at Provivo from 2019 – 2021



Bilirubin (total)


[µmol/L]



Group 1



2.98 ± 0.31


(2.5 – 3.4)



 


5 to 95 % Percentile


2.4 – 4.5



Group 2



3.43 ± 0.53*


(2.5 – 4.4)



Group 3



2.99 ± 0.31


(2.5 – 3.6)



Group 4



2.88 ± 0.37


[n=1] (2.2 – 3.4)



Glucose


[mmol/L]



Group 1



7.651 ± 0.878


(6.69 – 9.60)



5 to 95 % Percentile


5.88 – 9.72



Group 2



7.666 ± 1.737


(5.94 –10.89) [n=2]



Group 3



7.036 ± 0.943


[n=1] (5.82 – 8.10)



Group 4



6.223 ± 0.845*


[n=5] (5.43 – 7.90)



#1:



Data not audited by QAU



*/**:



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



#2:



Taken from table 9-2 'Biochemical Parameters - Summary - Females' and table 9-4 'Biochemical Parameters - Individual Data - Females'.



 


Text Table 7‑15:   Changes of the TSH concentration that were not considered to be test item-related.















































 



 



Changes in comparison to control


[%]



Reason



Parameter #1



Sex



Group 2


25 mg/kg



Group 3


80 mg/kg



Group 4


240/160 mg/kg



 



 



TSH


[ng/mL]



males



-51.5



-38.7



-14.2



A



TSH


[ng/mL]



females



-7.0



+39.4



+30.4



A



#1:



Values taken from table 11-1 ' Thyroid Hormone Level Analysis - Summary - Males ' and table 11-2 ' Thyroid Hormone Level Analysis - Summary - Females'



A:



The observed differences in the TSH concentration were not considered to be test item-related, as they were without statistical significance and no dose-response relationship was noted.



 


Text Table 7-16A: Comparison of TSH concentration of the male and female animals with the Provivo background data.






























































Parameter


(F0)


 



Values from this study #2


Mean value per group ± SD


(Range of the individual values (n = 10))


[number of individual values below or above the stated range]



Provivo Background Data #1


obtained from the control groups of 6 OECD 443 studies performed at Provivo


from 2019 – 2021



TSH


[ng/mL]



Group 1


(males)



3.3264 ± 1.6885


(1.545 - 7.507)  [n=1]



 


5% to 95% Percentile


 


0.081 – 6.964


 


 


25% to 95% Percentile


 


1.506 #3 – 6.964



Group 2


(males)



1.6145 ± 0.7953


[n=3 #3]  (0.081 - 2.667)



Group 3


(males)



2.0380 ± 1.9448


[n=4]  (0.081 - 6.157)



Group 4


(males)



2.8549 ± 3.4244


[n=5]  (0.373 - 9.500)  [n=2]



Group 1


(females)



2.1299 ± 1.5930


[n=1]  (0.081 - 6.017)  [n=1]



5% to 95% Percentile


 


0.081 – 4.123


 


 


25% to 95% Percentile


 


0.238 #3 – 4.123



Group 2


(females)



1.9813 ± 1.0509


(0.917 - 4.029)



Group 3


(females)



2.9693 ± 1.5341


(1.472 - 6.391)  [n=2]



Group 4


(females)



2.7774 ± 1.4983


(1.241 - 6.161)  [n=1]



#1:



Data not audited by QAU



*/**:



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



#2:



See tables 11-1 and 11-3 'Thyroid Hormone Level Analysis - Summary and Individual Data - Males', 11-2 and 11-4 'Thyroid Hormone Level Analysis - Summary and Individual Data - Females'.



#3



The 5 % percentile is at the lower level of detection (LOD = 0.081). Hence, for the comparison with the study values the 25 % percentile was used.



 


Text Table 7-16B: Comparison of T4 concentration of the male and female animals with the Provivo background data.


























































Parameter


(F0)


 



Values from this study #2


Mean value per group ± SD


(Range of the individual values (n = 10))


[number of individual values below or above the stated range]



Provivo Background Data #1


obtained from the control groups of 6 OECD 443 studies performed at Provivo


from 2019 – 2021



T4


[nmol/L]



Group 1


(males)



53.6940 ± 8.0417


(44.155 – 67.412)  [n=1]



 


5% to 95% Percentile


 


33.618 – 66.057



Group 2


(males)



53.1960 ± 6.5840


(46.028 – 64.072)



Group 3


(males)



53.6609 ± 7.0554


(41.450 – 64.077)



Group 4


(males)



53.2547 ± 5.2753


(45.532 – 60.183)



Group 1


(females)



38.1577 ± 9.351


(24.134 – 51.513)  [n=1]



5% to 95% Percentile


 


18.464 – 49.518



Group 2


(females)



38.3400 ± 6.9228


(27.011 – 49.735)  [n=1]



Group 3


(females)



40.4866 ± 7.4368


(30.140 – 50.574)  [n=1]



Group 4


(females)



38.3674 ± 8.1366


(19.662 – 47.310)



#1:



Data not audited by QAU



*/**:



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



#2:



See tables 11-1 and 11-3 'Thyroid Hormone Level Analysis - Summary and Individual Data - Males', 11-2 and 11-4 'Thyroid Hormone Level Analysis - Summary and Individual Data - Females'.



 


Text Table 7-17:   Changes of the sperm motility that were not considered to be test item-related.







































Percentage of motile spermatozoa in the cauda epididymis (%)



Parameter #1



Group 1


control



Group 2


25mg/kg



Group 3


80mg/kg



Group 4


240/160mg/kg



Percentage of motile spermatozoa



71.3



71.5



75.8* #2



68.5



#1:



Taken from table 12-1 'Sperm Count and Motility - Summary - F0 Generation'.



#2:



Not considered to be test item-related, as the difference between group 1 and group 3 was only small, no dose-response relationship was noted and an increased number of motile spermatozoa is not an adverse effect.


      

 


Text Table 7-18:   Comparison of sperm motility with the Provivo background data.





































F0 Males


Parameter



Values from this study #2


Mean value per group ± SD


(range of the individual values (n = 24/22))


[number of individual values below or above the stated range]



Provivo Background Data #1


obtained from the control groups of 6 OECD 443 studies performed at Provivo from 2019 – 2021



Sperm motility


[%]



Group 1



71.3 ± 6.9


(61 – 79)



 


5 to 95 % Percentile


 


51 – 86



Group 2



71.5 ± 8.0


(62 – 85)



Group 3



75.8 ± 5.5


(66 – 87)  [n=1]



Group 4



68.5 ± 9.0


(62 – 80)



#1:



Data not audited by QAU



#2:



Taken from table 12-1 'Sperm Count and Motility - Summary - F0 Generation' and table 12-3 'Sperm Motility – Individual Data - F0 Generation'



 


Text Table 7-19:   Pairs without verified copulation.

































Pairs without verified copulation



Group



Male partner



Female partner



2



72



95



4



152



177



4



163



189



#1:



See table 1 ‘Pairing List - F0 Generation’.



 


Text Table 7-20A: ------------------------------------------------------------------------------------------- Macroscopic findings that were noted for the male animals at necropsy (only the surviving animals were considered).





















































Observations at necropsy for the female animals



Organ



Observation



Group 1


Control


(n = 24 #1)



Group 2


25 mg/kg


(n = 24)



Group 3


80 mg/kg


(n = 24)



Group 4


240/160 mg/kg


(n = 22)



Liver



petechial bleeding



1 / 24


(22)



-



-



-



Trachea



tissue enlargement



-



1 / 24


(57)



-



-



Lymph node,


mesenteric



dark-red discoloured



-



1 / 24


(62)



-



-



#1:



Number of animals (only the surviving female animals are included). For the macroscopic post-mortem findings of the prematurely deceased animals refer to Appendix 5 section 3.1 'Mortality' of the histopathology phase report.



…/…:



Affected animals / Animals examined



(…):



The number of the affected animal is written in brackets.



 


Text Table 7-20B: -- Macroscopic findings that were noted for the female animals at necropsy (only surviving animals were considered).























































































































































Observations at necropsy for the female animals #



Organ



Observation



Group 1


Control


(n = 23)



Group 2


25 mg/kg


(n = 24)



Group 3


80 mg/kg


(n = 23)



Group 4


240/160 mg/kg


(n = 20]



Duodenum



mucosa reddened



2 / 23


(29, 33)



4 / 24


(82, 83, 91, 93)



7 / 23


(129, 131, 134-136, 143-144)



4 / 20


(170, 171, 175, 180)



dilated



1 / 23


(33)



-



-



-



thickened



-



-



1 / 23


(135)



1 / 20


(180)



Stomach



haemorrhagic foci



1 / 23


(35)



1 / 24


(87)



1 / 23


(133)



2 / 20


(176, 190)



mucosa black discoloured



-



1 / 24


(92)



-



-



Uterus



uterus horn: thickened



1 / 23


(34)



1 / 24


(93)



-



1 / 20


(186)



dilated, filled with liquid



-



1 / 24


(91)



2 / 23


(137, 138)



-



Lymph node


(cervical)



enlarged and indurated



1 / 23


(32, right))



-



-



-



Axilla



tissue enlargement



1 / 23


(32, right)



-



-



-



abscess



-



-



1 / 23


(128, right)



-



Thymus



reduced in size



1 / 23


(45)



-



-



-



Reddened (extremely)



 



 



 



1 / 24


(175)



Pituitary gland



enlarged (slightly)



-



-



1 / 23


(131)



-



Heart, lungs



adhered with thoracic wall



-



-



1 / 23


(134)



-



Lungs



petechial bleeding



-



-



-



1 / 24


(175)



Kidney



Tissue enlargement



-



-



-



1 / 24


(172, left)



#:



Number of animals (only the surviving female animals are included). For the macroscopic post-mortem findings of the prematurely deceased animals refer to Appendix 5 section 3.1 'Mortality' of the histopathology phase report.



…/…:



Affected animals / Animals examined



(…):



The number of the affected animal is written in brackets.



 


Text Table 7-21:        Stage of the estrous cycle at necropsy. The stage of the estrous cycle was evaluated from vaginal lavages that were taken at necropsy and during the microscopic examination of the vagina (only group 1 and 4).

























































































Stage of estrous cycle


at necropsy


(F0 Generation) #1



Group 1


Control #2



Group 2


25 mg/kg #3



Group 3


80 mg/kg #4



Group 4


240/160 mg/kg



N



H



N



N



N



H



Proestrus



2 of 22



6 of 20



2 of 23



1 of 22



2 of 24



5 of 20



Estrus



4 of 22



2 of 20



2 of 23



3 of 22



6 of 24



2 of 20



Metestrus



6 of 22



-



4 of 23



10 of 22



5 of 24



1 of 20



Diestrus



10 of 22



12 of 20 #5



15 of 23



8 of 22



11 of 24



12 of 20 #5



-:



not detected.



#1:



The values are taken from table 15 ‘Stage of Estrous Cycle at Necropsy - Individual Data - F0 Generation’.



N:



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



H:



Stage of the estrous cycle was determined during the microscopic examination of the vagina (see Appendix 5 'Histopathological Phase Report').



#2:



The vaginal lavages of female nos. 36 and 40 were not evaluable.



#3:



The vaginal lavage of female no. 92 was not evaluable.



#4:



The vaginal lavages of female nos. 126 and 143 were not evaluable.



#5:



Termed as lactational diestrus in the histopathology report.



 


Text Table 7-22:        Statistically significant changes in organ weights, not considered to be test item-related.



















































































F0 Generation



 



Changes in comparison to control


[%]



Reason



Parameter



Sex



Group 2



Group 3



Group 4



 



 



Thymus


(absolute)



Male


#1



- 9.4



- 14.1



- 19.1**



A



Thymus


(relative)



Male


#2



- 11.5



- 12.9



- 18.5**



A



Thyroid / Parathyroid


(left, absolute)



Female


#3



+30.1*



+6.1



+12.8



B



Thyroid / Parathyroid


(left, relative)



Female


#4



+30.8



+6.1



+16.8



B



*/**:



Statistically significant from control at p ≤ 0.05/0.01 (DUNNETT test)



#1:



Taken from table 18-1 ‘Absolute Organ Weights - Summary - Males - F0’.



#2:



Taken from table 17-1 ‘Relative Organ Weights - Summary - Males - F0’.



#3:



Taken from table 18-2 ‘Absolute Organ Weights - Summary - Females - F0’.



#4:



Taken from table 17-2 ‘Relative Organ Weights - Summary - Females - F0’.



A:



The observation was considered to be spontaneous, as no correlation with the histopathology results was noted.



B:



The observation was considered to be spontaneous, as no dose response relationship and no correlation with the histopathology results were noted.



 


Text Table 7-23:   Comparison of organ weights with the Provivo background data.





































































































Parameter



Values from this study #2


Mean value per group ± SD


(range of the individual values (n= up to 24))


[number of individual values below or above the stated range]



Provivo Background Data #1


obtained from the control groups of 6 OECD 443 studies performed at Provivo from 2019 – 2021



males



Thymus


[g]



Group 1



0.349 ± 0.079


[n=1] (0.20 – 0.48)



 


5 to 95 % Percentile


0.22 – 0.48



Group 2



0.316 ± 0.070


(0.22 – 0.53) [n=1]



Group 3



0.300 ± 0.086


[n=4] (0.16 – 0.49) [n=1]



Group 4



0.282 ± 0.054**


[n=2] (0.19 – 0.41)



Thymus


[g/kg b.w.]



Group 1



0.580 ± 0.125


[n=2] (0.35 – 0.87) [n=1]



5 to 95 % Percentile


0.41 – 0.85



Group 2



0.513 ± 0.094


[n=1] (0.36 – 0.75)



Group 3



0.505 ± 0.133


[n=6] (0.27 – 0.83)



Group 4



0.473 ± 0.080**


[n=3] (0.34 – 0.68)



females



Thyr./Parathyr.


[g]



Group 1



0.0106 ± 0.0024


(0.007 – 0.017)



5 to 95 % Percentile


0.007 – 0.017



Group 2



0.0138 ± 0.0045*


(0.007 – 0.027) [n=4]



Group 3



0.0112 ± 0.0037


(0.007 – 0.019) [n=1]



Group 4



0.0119 ± 0.0033


[n=1] (0.006 – 0.017)



Thyr./Parathyr.


[g/kg b.w.]



Group 1



0.0341 ± 0.0084


[n=3] (0.029 – 0.049)



5 to 95 % Percentile


0.023 – 0.056



Group 2



0.0446 ± 0.0170


[n=1] (0.027 – 0.055) [n=4]



Group 3



0.0361 ± 0.0134


[n=1] (0.023 – 0.051) [n=2]



Group 4



0.0398 ± 0.0120


[n=1] (0.025 – 0.052) [n=2]



#1:



Data not audited by QAU



*/**:



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



#2:



Taken from table 17-1 to table 18-4.



 


Text Table 7-24:   Mean length and mean number of estrous cycles.



































































Parameter


F0



Group 1


Control



Group 2


25 mg/kg



Group 3


80 mg/kg



Group 4


240/160 mg/kg



Pre-mating: Test day 15 (start of treatment) until pairing (test day 85) #1



Mean cycle length (days)



4.50



4.72



4.52



5.05*



Number of cycles



15.0



14.3



14.8



13.3



Pre-mating: Test day 85 (start of pairing) until verification of copulation #1



Mean cycle length (days)



#3



#3



#3



4.00 #2



Number of cycles



#3



#3



#3



3



#1:



Values taken from table 19-1 'Estrous Cycle Data – Start of Dosing until Mating - Summary - F0 Generation'.



#2:



Female no. 189 revealed 3 complete estrous cycles during her mating period of 15 test days. No positive mating sign was noted during the mating period and the non-pregnancy status was confirmed by Salewski Staining at laparotomy at the end of the pseudo-gestation period.



#3:



No rats with complete estrous cycles were noted.


      

 


 


Text Table 7-25A: Range of mean cycle length.































Parameter #1



Group 1


Control



Group 2


25 mg/kg



Group 3


80 mg/kg



Group 4


240/160 mg/kg



Pre-mating: Test day 15 (start of treatment) until pairing (test day 85) #



Range of mean cycle length of the individual females (days)



4.0 - 6.8



4.0 - 6.6



3.9 - 6.1



4.1 -  6.7 +


7.7 #2



#1:



Values taken from table 19-2 'Estrous Cycle Data – Start of Dosing until Mating - Individual Data - F0 Generation'.



#2:



No. 187 with a mean cycle length of 7.7 days revealed 2 largely elongated estrous cycles of 16 and 14 test days. However, such elongated estrous cycles were also noted for females of the control group (nos. 39 and 48 with estrous cycles of 19 or 28 test days; mean cycle length 4.0 or 5.4).



 


Text Table 7-25B: Comparison of mean cycle length with the Provivo background data.





































F0 Females


Parameter



Values from this study #2


Mean value per group ± SD


(range of the individual values (n = 24))


[number of individual values below or above the stated range]



Provivo Background Data #1


obtained from the control groups of 6 OECD 443 studies performed at Provivo from 2019 – 2021



Mean cycle length


(pre-mating period)


[days]



Group 1



4.50 ± 0.78


(4.0 – 6.8)  [n=1]



 


5 to 95 % Percentile


 


3.8 – 6.4



Group 2



4.72 ± 0.86


(4.0 – 6.6)  [n=1]



Group 3



4.52 ± 0.67


(3.9 – 6.1)



Group 4



5.05 ± 0.98*


(4.1 – 7.7)  [n=2]



#1:



Data not audited by QAU



#2:



Taken from table 19-1 'Estrous Cycle Data – Start of Dosing until Mating - Summary - F0 Generation' and table 19-2 'Estrous Cycle Data – Start of Dosing until Mating – Individual Data - F0 Generation'



 


Text Table 7-26:   Non-pregnant females.






































Parameter #1



Group 1


Control



Group 2


25 mg/kg



Group 3


80 mg/kg



Group 4


240/160 mg/kg



Non-pregnant females



Non-pregnant female with verified copulation (sperm detection)



no. 28


(no. 3 #2)



 



nos. 137, 140


(nos. 114, 115)



 



Non-pregnant female without verified copulation (no sperm detection)



 



no. 95


(no. 72)



 



nos. 177, 189


(nos. 152, 163)



#1:



See table 21-2 ‘Reproductive Outcomes - Individual Data - Females - Fertility and Gestation Indices - F0 Generation’.



#2:



The numbers of the male partners are given in brackets (see table 1 ‘Pairing List’. No changes were noted for the sperm parameters of their male partners (see section 8.12 ‘Examination of the sperm number, viability and morphology).



 


Text Table 7-27:   Fertility indices per group.



















































Group / Dose level



Fertility index


% #1



Pregnant females with verified copulation /


Females with verified copulation



Group 1


(Control)



96



23 / 24 #2



Group 2


(25 mg/kg)



100



23 / 23 #3



Group 3


(80 mg/kg)



92



22 / 24 #4



Group 4


(240/160 mg/kg)



100



21 / 21 #5



#1:



See table 21-1 ‘Reproductive Outcomes and Indices per Group - Females - Fertility and Gestation Indices - F0 Generation’.



#2:



Group 1: One non-pregnant animal with a verified copulation (no. 28) was noted.



#3:



Group 2: One non pregnant female with no verified copulation was noted (no. 95).



#4:



Group 3: Two non-pregnant animals with verified copulation were noted (nos. 137. 140).



#5:



Group 4: Two non-pregnant animals with no verified copulation were noted (nos. 177, 189).


Three animals deceased during the pre-mating period, hence only 21 animals were left for pairing.



 


Text Table 7-28:   Gestation indices per group.















































Group / Dose level



Gestation index


% #1



Dams with live pups / Pregnant rats



Group 1


(Control)



100



22 / 22 #2



Group 2


(25 mg/kg)



100



23 / 23



Group 3


(80 mg/kg)



100



21 / 21 #3



Group 4


(240/160 mg/kg)



100



18 / 18 #4



#1:



See table 21-1 ‘Reproductive Outcomes and Indices per Group - Females - Fertility and Gestation Indices - F0 Generation’.



#2:



Group 1: Female animal no. 40 which died during the gestation period by accident was excluded.



#3:



Group 3: Female animal no. 126 which died during the gestation period by accident was excluded.



#4:



Group 4: Female animal no. 187 was prematurely sacrificed during the gestation period and excluded.



 


Text Table 7-29A: Comparison of the percentages of post-implantation loss per group with the Provivo background data.





































F0 Females


Parameter



Values from this study #2


Percent of post-implantation loss per group



Provivo Background Data #1


obtained from the control groups of 6 OECD 443 studies performed at Provivo from 2019 – 2021



Post-implantation loss


(percent per group)



Group 1



6.29 %



 


Range of the post-implantation loss from 6 control groups


 


6.14 – 14.86 %



Group 2



11.90 %



Group 3



9.01 %



Group 4



11.45 %



#1:



Data not audited by QAU



#2:



Taken from table 22 'Number of Pups and Indices Overview per Group'.



 


Text Table 7-29B: Overview of the reproductive data.






























































































































Parental females


(F0 Generation)



Reproductive data



Group 1


(Control)



Group 2


25 mg/kg



Group 3


80 mg/kg



Group 4


240/160 mg/kg



Number of implantation sites and pups



Implantation sites #1



15.0 ± 2.7



15.1 ± 3.0



15.9 ± 2.0



16.2 ± 2.5



Pups #1


(born alive and dead)



14.3 ± 2.7



13.9 ± 2.7



15.2 ± 1.9



14.7 ± 2.7



Pups born alive #1



14.2 ± 2.7



13.5 ± 2.7



14.9 ± 2.1



14.6 ± 2.7



Reproductive indices [%]



Birth index



per dam (group mean) #1


per group #2



94.27 ± 27


94.01



91.16 ± 10.31


90.65



93.10 ± 8.24


93.02



89.46 ± 10.34


89.23



Live birth index



per dam (group mean) #1


per group #2



99.68 ± 1.52


99.68



97.25 ± 6.27


97.19



97.85 ± 6.03


97.81



99.25 ± 2.25


99.25



Post-implantation loss



per dam (group mean) #1


per group #2



6.05 ± 8.28


6.29



11.31 ± 11.91


11.90



8.83 ± 10.38


9.01



11.20 ± 10.65


11.45



Twins (number of shared placentae)



per dam (group mean)


per group



1.0 ± 0.0


3



1.7 ± 1.2


5



2.5 ± 1.3


10



2.0 ± 1.0


6



Resorptions and stillbirths



Sum of resorptions and stillbirths


(difference between number of implantation sites and stillbirths)



per dam (group mean)


per group



1.0 ± 1.3


21



1.8 ± 2.0


42



1.5 ± 1.8


31



1.9 ± 1.8


34



Number of stillbirths



1



9



7



2



Number of resorptions



20



33



24



32



#1:



Statistical calculation was performed by ANOVA / DUNNETT


(*/**: p ≤ 0.05 / p ≤ 0.01).



#2:



No statistical evaluation was performed for the group values.



#3:



See table 23-1 ‘Birth Indices and Post-implantation Loss - Values per Dam - Summary’.



#4:



See table 22 ‘Number of Pups and Indices - Overview per Group’.



 


Text Table 7‑30:   Viability indices and prematurely deceased pups during the pre-cull period.










































Pre-cull period



Parameter #



Group 1


(Control)



Group 2


25 mg/kg



Group 3


80 mg/kg



Group 4


240/160 mg/kg



Prematurely deceased pups between lactation days 0/1 to 4


 /


Total number of live born pups



2 / 313



6 / 311



8 / 313



3 / 263



Viability index (group values)


[%]



99.36



98.07



97.44



98.86



#:



See table 22 ‘Number of Pups and Indices - Overview per Group’.


      

 


Text Table 7‑31:   Dams with prematurely deceased pups during the pre-cull period.



















































































 


Number of prematurely deceased pups per dam (pre-cull period) #



Group 1


(Control)



Group 2


25 mg/kg



Group 3


80 mg/kg



Group 4


240/160 mg/kg



dam no.



deceased pups



dam no.



deceased pups



dam no.



deceased pups



dam no.



deceased pups



29



1



77



1



123



1



171



1



35



1



79



1



124



1



172



1



 



93



4



127



1



192



1



 



134



1



 



135



3



144



1



#:



See table 24-2 ‘Dead Pups per Dam and Viability Index - Individual Data - F1 Pups’.


         

 


 


 


Text Table 7‑32:   Viability indices and prematurely deceased pups during the post-cull period.










































Post-cull period



Parameter #1



Group 1


(Control)



Group 2


25 mg/kg



Group 3


80 mg/kg



Group 4


240/160 mg/kg



Prematurely deceased pups between lactation days 5 and 21


/


Number of pups alive on lactation day 4 after culling



0 / 217



0 / 226



0 / 210


(1 / 210) #2



1 / 180



Viability index (group values)


[%]



100.00



100.00



100.00


(99.52) #2



99.44



#1:



See table 22 ‘Number of Pups and Indices - Overview per Group’.



#2:



Pup 133-13 died on lactation day 20 but due to technical problems, it could not be written as dead to the databank (see Table A ‘Individual Pup Data - Dam no. 133’).



 



 



 


Text Table 7‑33:   Male to female ratios on lactation days 1 and 4.







































Male / Female ratio of the pups #



 



Time point



Group 1


(Control)



Group 2


25 mg/kg



Group 3


80 mg/kg



Group 4


240/160 mg/kg



 



Lactation day 1



0.80



0.98



1.00



0.74



 



Lactation day 4



0.81



0.98



1.02



0.73



 



 



#:



See table 22 ‘Number of Pups and Indices - Overview per Group’.



 


Text Table 7-34:   Changes of pup body weight in comparison to the control group for the male and female pups combined. Not considered to be test item-related.

























































Pup body weight



Changes in comparison to the control group


[%] #



Group 2


(25 mg/kg)



Group 3


(80 mg/kg)



Group 4


(240/160 mg/kg)



 



Lactation day 1



males + females


combined



- 0.2



- 2.6



+1.2



Lactation day 4



males + females


combined



- 0.7



- 2.4



- 0.1



Lactation day 7



males + females


combined



- 3.1



- 3.2



- 1.8



Lactation day 14



males + females


combined



- 4.1



- 3.7



- 4.5



Lactation day 21



males + females


combined



- 3.2



- 1.5



- 2.2



#:



Values are taken from table 26-1 'Mean Body Weight of the Pups per Dam - Summary'.



 


Text Table 7-35:   Changes in litter weight that were not considered to be test item-related.


















































































 



Changes in comparison to control


[%] #



Reason



Parameter


Sex



Control



Group 2



Group 3



Group 4



 



 



Litter weight


(male pups on lactation day 14)



n.c.



4.6



10.7



- 1.2



-



Litter weight


(female pups on lactation day 14)



n.c.



- 12.2*



- 13.3*



- 5.5



A



Litter weight


(male and female pups combined on lactation day 14)



n.c.



- 4.4



- 2.2



- 3.5



-



Pup body weight


(female pups on lactation day 14)



n.c.



- 4.8*



- 4.4



- 5.1*



-



Mean number of female pups per dam on lactation day 14



5.4



4.9



4.9



5.3



-



*/**:



Statistically significant from control at p ≤ 0.05/0.01 (DUNNETT test)



A:



Not considered to be test item-related as the differences in litter weight were due to small and spontaneous differences in pup body weight and pup number on lactation day 14.



n.c.:



Not calculable



-:



Only for clarification.



#:



Values taken from table 27-1 'Liter Weight per Dam - Summary'



 


Text Table 7-36:   Ano-genital distance of the pups. Changes in comparison to the control value. Not considered to be test item-related.
























































Ano-genital Distance #



 



Changes in comparison to control


[%]



Sex



Group 2


25 mg/kg



Group 3


80 mg/kg



Group 4


240/160 mg/kg



 



Ocular units


(absolute)



Male



-1.5



-4.8*



-3.6



Ocular units


(absolute)



Female



-0.4



-4.8



-5.6



Ocular units / g b.w.


(relative)



Male



-1,4



-4.2**



-3.9*



Ocular units / g b.w.


(relative)



Female



+0.3



-3.8



-5.3*



*/**:


 


 



Statistically significant from control at p ≤ 0.05/0.01 (DUNNETT test)


Ocular units/g b.w.: Ano-genital distance was normalized to cube root of pup body weight.



#:



Values taken from table 28-1 ‘Ano-genital Distance of the Pups - Mean Values per Dam - Summary’.



 


Text Table 7‑37:   Overview of the pups with nipple retention.






































































































































































































































Pups with nipples #1



Group 1


(Control)



Group 2


25 mg/kg



Group 3


80 mg/kg



Group 4


240/160 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)



25



25-03 (1)



77



77-1 (4)



123



123-3 (1)



169



169-4 (1)



26



26-05 (1)



 



77-2 (1)



127



127-5 (1)



 



169-5 (1)



29



29-06 (1)



 



77-3 (2)



130



130-1 (1)



171



171-1 (2)



32



32-06 (1)



 



77-4 (4)



132



132-7 (1)



172



172-2 (2)



34



34-01 (1)



 



77-5 (4)



 



132-8 (2)



 



172-4 (3)



42



42-07 (3)



79



79-2 (1)



133



133-3 (1)



 



172-5 (1)



46



46-07 (1)



 



79-3 (1)



134



134-2 (1)



173



173-7 (1)



48



48-01 (1)



81



81-2 (1)



135



135-2 (1)



 



173-9 (1)



 



48-02 (1)



 



81-3 (1)



141



141-3 (1)



179



179-1 (3)



 



 



82



82-3 (4)



144



144-5 (2)



183



183-3 (1)



 



 



84



84-7 (1)



 



 



 



183-5 (1)



 



 



96



96-3 (2)



 



 



184



184-4 (4)



 



 



 



96-4 (1)



 



 



188



188-5 (1)



Summary



8



9 Pups



6



13 Pups



9



10 Pups



8



13 Pups



Number of nipples per pup



1 Nip



8 Pups



1 Nip



7 Pups



1 Nip



8 Pups



1 Nip



8 pups



2 Nip



-



2 Nip



2 Pups



2 Nip



2 Pups



2 Nip



2 Pups



3 Nip



1 Pup



3 Nip



-



3 Nip



-



3 Nip



2 Pup



 



 



4 Nip



4 Pups



4 Nip



-



4 Nip



1 Pup



#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 7-38A: Changes of T4 level in comparison to the control group for the male and female pups alone or combined.




























































T4 level


(nmol/L)



Changes in comparison to the control group


[%] #



Group 2


25 mg/kg



Group 3


80 mg/kg



Group 4


240/160 mg/kg



 



Lactation day (PND) 4



males



+27.1**



+17.3



+31.6*



females



+15.3



+12.1



+20.9*



males + females


combined



+18.6*



+16.5



+26.0**



Lactation day (PND) 21/22



males



- 4.0



- 15.4**



- 10.0



females



- 11.5*



- 16.7**



- 9.7



males + females


combined



- 7.4



- 15.7**



- 9.6*



#:



Values are taken from table 30-1 'Thyroid Levels of the Pups on Lactation Day 4 - Mean Values per Dam - Summary' and table 30-2 ‘Thyroid Levels of the Pups on Lactation Days 21/22 - Mean Values per Dam - Summary’.



 


Text Table 7-38B: Comparison of thyroid hormone levels with the Provivo background data.



























































Parameter



Values from this study #2


Mean value per group ± SD


(Range of the individual values)


[number of individual values below or above the stated range]



Provivo Background Data #1


obtained from the control groups of 6 OECD 443 studies performed at Provivo


from 2019 – 2021



T4


Males + Females combined


PND 4


(nmol/L)



Group 1



20.5283 ± 4.5130


(14.115 – 30.476)  [n=2]



5% to 95% Percentile


 


11.437 – 26.191



Group 2



24.3513 ± 3.6228*


(17.623 – 33.460)  [n=6]



Group 3



23.9188 ± 11.4526


(10.953 – 51.619)  [n=4]



Group 4



25.8694 ± 5.2237**


(18.943 – 37.331)  [n=8]



T4


Males + Females combined


PND 21/22


(nmol/L)



Group 1



52.0715 ± 6.8407


(36.019 – 64.788)  [n=4]



5% to 95% Percentile


 


29.035 – 59.099



Group 2



48.2146 ± 6.4779


(35.380 – 57.225)



Group 3



43.8786 ± 6.7427**


(32.621 – 55.705)



Group 4



47.0810 ± 4.6636*


(37.765 – 56.833)



#1:



Data not audited by QAU



*/**:



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



#2:



Taken from tables 30-3 ‘Thyroid Levels of the Pups on Lactation Day 4 – Mean Values per Dam – Individual Data’ and 30-4 'Thyroid Levels of the Pups on Lactation Days 21/22 – Mean Values per Dam – Individual Data'.



Text Table 7-39A: Changes of TSH levels in comparison to the control group for the male and female pups alone or combined.









































TSH level


(ng/mL)



Changes in comparison to the control group


[%] #



Group 2


25 mg/kg



Group 3


80 mg/kg



Group 4


240/160 mg/kg



 



Lactation day (PND) 21/22



males



- 11.8



+10.1



- 27.9



females



- 9.5



+50.6



- 2.6



males + females


combined



- 11.2



+29.9



- 16.2



#:



Values are taken from table 30-2 ‘Thyroid Levels of the Pups on Lactation Days 21/22 - Mean Values per Dam - Summary’.



Text Table 7-39B: Comparison of thyroid hormone levels with the Provivo background data.













































Parameter



Values from this study #2


Mean value per group ± SD


(Range of the individual values)


[number of individual values below or above the stated range]



Provivo Background Data #1


obtained from the control groups of 6 OECD 443 studies performed at Provivo


from 2019 – 2021



TSH


Males + Females combined


PND 21/22


(ng/mL)



Group 1



0.7054 ± 0.2389


[n=4 #3]  (0.406 – 1.255)



5% to 95% Percentile


 


0.081 #3 – 2.576


 


 


25% to 95% Percentile


 


0.460 – 2.576



Group 2



0.6267 ± 0.3018


[n=8]  (0.235 – 1.356)



Group 3



0.9162 ± 0.4584


[n=3]  (0.245 – 2.120)



Group 4



0.5912 ± 0.5466


[n=8]  (0.081 – 1.748)



#1:



Data not audited by QAU



*/**:



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



#2:



Taken from tables 30-3 ‘Thyroid Levels of the Pups on Lactation Day 4 – Mean Values per Dam – Individual Data’ and 30-4 'Thyroid Levels of the Pups on Lactation Days 21/22 – Mean Values per Dam – Individual Data'.



#3:



The 5 % percentile is at the lower level of detection (LOD = 0.081). Hence, for the comparison with the study values the 25 % percentile was used.



Text Table 8-2:     Observations that were noted for the prematurely deceased animal no. 336 during the daily cage side inspection on the days before death (common observations as salivation are not considered).























Observations during the daily cage side inspections for the prematurely deceased


animal no. 336 #



Gr.



No.



Sex



Symptom



Observed on PND (day of death)



4



336



M



Piloerection


Respiratory rate decreased


Breathing sounds


Laboured breathing



50 - 53


52, 53


51


53 (54)



Text Table 8-3:     Observations that were noted during the daily cage-side observations for the male animals of the low, the intermediate and the high dose level.






















































Observations in group 3 #1


(80 mg IPDA/kg b.w./day)



Observation (males)



Affected animals



First to last seen


(postnatal days) #2



Number of times recorded



Salivation



3 of 20



42 - 77



5



Observations in group 4


(160 mg IPDA/kg b.w./day)



Salivation



11 of 19 #3



30 - 77



26



Breathing sounds



2 of 19



54  -77 (325, 326)



23



Hunched



1 of 19



63 - 74 (326)



12



Piloerection



1 of 19



54 - 74 (326)



18



Laboured breathing



1 of 19



63 - 73 (326)



11


 



Text Table 8-4:     Observations that were noted during the daily cage-side observations for the female animals of the low, the intermediate and the high dose level.



























Observations in group 4 #1


(160 mg IPDA/kg b.w./day)



Observation (females)



Affected animals



First to last seen


(postnatal days) #2



Number of times recorded



Salivation



15 of 20



29 - 76



27



Breathing sounds



2 of 20



33 - 58 (351, 342)



5



Text Table 8‑7:     Differences in body weight in comparison to control on postnatal day 26 and on postnatal day 88 / 89.













































Cohort 1A


Body Weight


 



Changes in comparison to control [%]



Group 2


25 mg/kg



Group 3


80 mg/kg



Group 4


160 mg/kg



Males #1



PND 26 #3



- 1.5



- 2.5



- 1.3



PND 88 #4



+0.5



+0.1



+0.4



Females #2



PND 26 #3



- 1.6



- 1.4



- 4.3



PND 89 #4



- 0.2



- 3.7



- 4.5



Text Table 8‑8:     Body weight gain of the male and female animals from postnatal day 26 to postnatal day 88.



























Body weight gain



Group 1


Control



Group 2


25 mg/kg



Group 3


80 mg/kg



Group 4


160 mg/kg



Males #1


(postnatal day 26 to 88)



+495.2%



+508.1%



+510.3%



+504.4%



Females 2


(postnatal day 26 to 89)



+259.6%



+266.4%



+250.7%



+258.4%



Text Table 8-10A: Statistically significant changes of the haematological parameters that were not considered to be test item-related.






















































































Parameter #


(Co1A)



Sex



Changes in comparison to control [%]



Reason



Group 2


25 mg/kg



Group 3


80 mg/kg



Group 4


160 mg/kg



 



 



Platelets (PLT)


[x103/µl]



Males #1



+9.5



+13.7



+28.3**



A



Platelets (PLT)


[x103/µl]



Females #2



- 9.3



+8.6



+7.6



C



MCV


[fL]



Females #2



- 0.7



0.0



- 2.6*



B



MCV


[fL]



Males #1



+0.5



+1.5



- 0.6



C



*/**:



Statistically significant from control at p ≤ 0.05/0.01 (DUNNETT test)



#1:



Values taken from table 6-1-Co1A 'Haematological Parameters - Summary - Males'.



#2:



Values taken from table 6-2-Co1A 'Haematological Parameters - Summary - Females'.



A:



The moderate increase is considered to be spontaneous, as no statistically significant changes were noted for the female animals and only 2 individual values were above the Provivo background data.



B:



The decreased MCV value was considered to be spontaneous. Only one individual value of the high dosed females was below the Provivo background range and no changes were noted for the male animals.



C:



Only for comparison.


       

Text Table 8-10B: Comparison of haematological parameters with the Provivo background data.



























































Parameter


Co1A



Values from this study #2


Mean value per group ± SD


(range of the individual values (n = 10))


[number of individual values below or above the stated range]



Provivo Background Data #1


obtained from the control groups of 6 OECD 443 studies performed at Provivo from


2019 – 2021



Platelets (PLT)


[x103/µl]


(males)



Group 1



913.0 ± 119.4


(744 – 1049)



 


5 to 95 % Percentile


 


562 - 1268


 



Group 2



999.6 ± 137.3


(810 – 1166)



Group 3



1038.2 ± 108.1


(906 - 1191)



Group 4



1171.6 ± 237.2**


(876 – 1727) [n=2]



MCV


[fL]


(females)



Group 1



53.21 ± 1.74


[n=1]  (50.4 – 55.5)



 


5 to 95 % Percentile


 


50.7 – 55.8


 



Group 2



52.85 ± 0.56


(52.0 – 53.9)



Group 3



53.20 ± 1.07


(52.0 – 55.0)



Group 4



51.83 ± 1.33*


[n=1]  (49.9 – 54.3)



#1:



Data not audited by QAU



*/**:



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



#2:



Taken from table 6-3-Co1A 'Haematological Parameters – Individual Data - Males' and table 6-4-Co1A 'Haematological Parameters - Individual Data - Females'.



 


Text Table 8-11A: Statistically significant changes of the biochemical parameters that were not considered to be test item-related.


































































Parameter


Co1A



Sex



Changes in comparison to control [%]



Reason



Group 2


25 mg/kg



Group 3


80 mg/kg



Group 4


160 mg/kg



 



 



Sodium


(mmol/L)



Females #1



- 0.2



- 1.1*



- 0.7



A



Sodium


(mmol/L)



Males #2



- 0.4



- 0.7



- 0.7



B



*/**:



Statistically significant from control at p ≤ 0.05/0.01 (DUNNETT test)



#1:



Values taken from table 7-2-Co1A ‘Biochemical Parameters - Summary - Females'.



#2:



Values taken from table 7-1-Co1A 'Biochemical Parameters - Summary - Males'.



A:



The decrease at the intermediate dose level is considered to be spontaneous. No dose response-relationship was noted and no decrease was noted for the male animals.



B:



Only for comparison


       

Text Table 8-11B: Comparison of biochemical parameters with the Provivo background data.





























F1 Females


Parameter



Values from this study #2


Mean value per group ± SD


(range of the individual values (n = 10))


[number of individual values below or above the stated range]



Provivo Background Data #1


obtained from the control groups of 6 OECD 443 studies performed at Provivo from 2019 – 2021



Sodium


[mmol/L]



Group 1



137.3 ± 1.3


[n=1]  (135 – 139)



 


5 to 95 % Percentile


136 – 141



Group 2



137.0 ± 0.9*


[n=1]  (135 – 138)



Group 3



135.8 ± 1.0


[n=4]  (134 – 137)



Group 4



136.3 ± 1.6


[n=2] (134 – 139)



 


Text Table 8-12A: Statistically significant changes of the thyroid hormone TSH (not considered to be test item-related).

















































Parameter #1


(Cohort 1A)



Sex



Changes in comparison to control [%]



Reason



Group 2


25 mg/kg



Group 3


80 mg/kg



Group 4


160 mg/kg



 



 



TSH


[ng/mL]



males



+4.7



+50.0



+80.8



A



TSH


[ng/mL]



females



- 31.5



- 66.6



- 79.5*



B



#1:



Values taken from table 10-1-Co1A 'Thyroid Hormone Level Analysis - Summary - Males' and from table 10-2-Co1A ‘Thyroid Hormone Level Analysis - Summary - Females'.



A:



The increased TSH concentrations that were noted at the intermediate and the high dose group were considered to be spontaneous. No individual value from the treatment groups was above the upper range of the background data and the individual values were highly variable.


Finally an opposite trend was noted for the female animals.



B:



The decreased TSH concentrations were not accompanied by histomorphological changes of the pituitary gland.



 


Text Table 8-12B: Comparison of TSH concentration of the male and female animals with the Provivo background data.


































































Parameter


(Co1A)


 



Values from this study #2


Mean value per group ± SD


(Range of the individual values (n = 10))


[number of individual values below or above the stated range]



Provivo Background Data #1


obtained from the control groups of 6 OECD 443 studies performed at Provivo


from 2019 – 2021



TSH


[ng/mL]



Group 1


(males)



0.8242 ± 0.6144


[n=3 #3]  (0.081 – 1.530)



 


5% to 95% Percentile


 


0.081 #3 – 5.699


 


 


25% to 95% Percentile


 


0.142 – 5.699



Group 2


(males)



0.8627 ± 0.8861


[n=4]  (0.081 - 2.589)



Group 3


(males)



1.2363 ± 1.4994


[n=2]  (0.081 – 5.126)



Group 4


(males)



1.4902 ± 1.2938


[n=1]  (0.081 – 4.043)



Group 1


(females)



1.0719 ± 0.9819


(0.081 – 2.765)  [n=2]



5% to 95% Percentile


 


0.081 #3 – 2.523


 


 


25% to 95% Percentile


 


0.081 #4 – 2.523



Group 2


(females)



0.7345 ± 0.5231


(0.081 – 1.620)



Group 3


(females)



0.3585 ± 0.4497


(0.081 – 1.128)



Group 4


(females)



0.2197 ± 0.2818*


(0.081 – 0.962)



#1:



Data not audited by QAU



*/**:



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



#2:



See tables 10-1-Co1A and 10-3-Co1A 'Thyroid Hormone Level Analysis - Summary and Individual Data - Males', 10-2-Co1A and 10-4-Co1A 'Thyroid Hormone Level Analysis - Summary and Individual Data - Females'.



#3:



The 5 % percentile is at the lower level of detection (LOD = 0.081). Hence, for the comparison with the study values the 25 % percentile was used.



#4:



Also the 25 % percentile is at the lower level of detection.



 


Text Table 8-12C: Comparison of T4 concentration of the male and female animals with the Provivo background data.














































Parameter


(Co1A)


 



Values from this study #2


Mean value per group ± SD


(Range of the individual values (n = 10))


[number of individual values below or above the stated range]



Provivo Background Data #1


obtained from the control groups of 6 OECD 443 studies performed at Provivo


from 2019 – 2021



T4


[nmol/L]



Group 1


(males)



64.6330 ± 5.9232


(55.943 – 73.694)



 


5% to 95% Percentile


 


42.528 – 76.219



Group 2


(males)



66.1330 ± 18.4558


(32.569 – 93.925)  [n=4)



Group 3


(males)



66.3058 ± 9.4665


(46.540 – 78.571)  [n=1]



Group 4


(males)



64.5040 ± 7.4090


(51.423 – 76.457)  [n=1]



Group 1


(females)



38.1076 ± 12.4532


[n=1]  (19.673 – 60.465)  [n=1]



5% to 95% Percentile


 


21.861 – 59.502



Group 2


(females)



40.3537 ± 11.5878


(24.899 – 65.859)  [n=1]



Group 3


(females)



31.8250 ± 6.4195


[n=1]  (19.395 – 41.300)



Group 4


(females)



29.8639 ± 5.2173


(25.313 – 42.504)



Text Table 8-13:   Time points (postnatal day) of balano preputial separation





























Parameter


(Co1A and Co1B combined)



Values from this study #2


Mean value per group ± SD


(range of the individual values (n= up to 40)


[number of individual values below or above the stated range]



Provivo Background Data #1


obtained from the control groups of 6 OECD 443 studies performed at Provivo from 2019 – 2021



Day balano preputial separation


(postnatal day)



Group 1



22.6 ± 0.7


(22 – 24)  [n=4]



 


5 to 95 % Percentile


22.0 – 23.0



Group 2



22.5 ± 0.6


(22 – 24)  [n=2]



Group 3



22.2 ± 0.5*


(22 – 24)  [n=1]



Group 4



22.2 ± 0.5**


(22 – 24)  [n=2]



Text Table 8-14A: Time points (postnatal day) of vaginal opening.





































Parameter



Group 1


Control



Group 2


25 mg/kg



Group 3


80 mg/kg



Group 4


160 mg/kg



Cohort 1A and 1B combined



Day of vaginal opening (PND) #1



33.1 ± 2.5



34.2 ± 2.8



33.1 ± 1.9



34.6 ± 2.3**



Day of vaginal opening (PND) (Range of individual animals) #2



30 - 43



30 - 44



30 - 37



30 - 43



Body weight on the day of vaginal opening (% changes in comparison to control) #1



-



+4.3



- 0.7



+3.9



Text Table 8-14B: Day of vaginal opening - comparison with the Provivo background data.





























Parameter


(Co1A and Co1B combined)



Values from this study #2


Mean value per group ± SD


(range of the individual values (n= up to 40)


[number of individual values below or above the stated range]



Provivo Background Data #1


obtained from the control groups of 6 OECD 443 studies performed at Provivo from 2019 – 2021



Day of vaginal opening


(postnatal day)



Group 1



33.1 ± 2.5


(30 – 43)  [n=1]



 


5 to 95 % Percentile


30 – 37



Group 2



34.2 ± 2.8


(30 – 44)  [n=2]



Group 3



33.1 ± 1.9


(30 – 37)



Group 4



34.6 ± 2.3**


(30 – 43)  [n=1]



Text Table 8-15:   Sexual maturation of female animals.








































Parameters #


(Co1A)



Parameters of sexual maturation


Mean values per group



Group 1


(Control)



Group 2


25 mg/kg



Group 3


80 mg/kg



Group 4


160 mg/kg



-



day of vaginal opening (PND) (Co1A only)



33.0 ± 3.0



34.5 ± 3.4



33.1 ± 1.7



34.9 ± 2.7*



-



day of appearance of cornified cells (PND)



34.9 ± 3.5



36.3 ± 4.0



34.8 ± 3.1



35.6 ± 3.7



-



period between day of vaginal opening and day of appearance of cornified cells (test days)



1.9 ± 1.7



1.8 ± 2.0



1.7 ± 2.3



0.7 ± 1.3



Text Table 8-16:   Range of mean cycle length.












































Parameter


(Cohort 1A)



Group 1


Control



Group 2


25 mg/kg



Group 3


80 mg/kg



Group 4


160 mg/kg



From test day 49 until test day 62



Number of cycles #1



1.9 ± 1.0



1.7 ± 0.8



2.0 ± 0.6



1.9 ± 0.6



Mean cycle length per group #1



4.12 ± 0.33



4.29 ± 0.56



4.08 ± 0.30



4.50 ± 0.58*



Range of mean cycle length of the individual females (days) #2



4.0 - 5.0



4.0 - 6.0



3.5 - 5.0



4.0 -  6.0



Number of females with no complete estrous cycle #2



3


(nos. 229, 231, 236)



3


(nos. 264, 270, 271)



1


(no. 303)



1


(no. 360)



Text Table 8-17:   Macroscopic findings noted for the females of the control group from Cohort 1A.














































Sex



Gr.



No.



Macroscopic findings #



Affected Organ



Finding



F



1



223



Uterus



Dilated and filled with clear liquid.



230



Uterus



Dilated and filled with clear liquid.



235



Thymus



Reddened left lobe.



237



Lymph node



Enlarged mesenteric lymph node



Spleen



Enlarged



Intestines



Thickened mucosa



Text Table 8-18:        Stage of the estrous cycle at necropsy. The stage of the estrous cycle was evaluated from vaginal lavages that were taken at necropsy and during the microscopic examination of the vagina (only group 1 and 4).

































































Stage of estrus at necropsy


(Cohort 1A females)



Group 1


Control



Group 2


25 mg/kg



Group 3


80 mg/kg


#3



Group 4


160 mg/kg



N #1



H #2



N



N



N



H



Proestrus



-



5 of 20



3 of 20



-



-



5 of 20



Estrus



6 of 20



6 of 20



3 of 20



4 of 20



4 of 20



5 of 20



Metestrus



5 of 20



1 of 20



8 of 20



5 of 20



9 of 20



3 of 20



Diestrus



9 of 20



8 of 20



6 of 20



10 of 20



7 of 20



7 of 20



- :



not detected



 



N = Necropsy; H = Histopathological examination of the vagina



Text Table 8-19A:      Changes in organ weights of the male animals of Cohort 1A, unrelated to the test item.





















































































Parameter


Male


(Co1A)



Cohort



Changes in comparison to control


[%]



Reason



Group 2


25 mg/kg



Group 3


80 mg/kg



Group 4


160 mg/kg



 



 



Prostate #1


(relative)



Cohort 1A



- 0.5



- 9.2



- 13.9*



A



Prostate #2


(relative)



Cohort 1B



- 9.4



- 9.6



- 5.5



B



Prostate #5


(relative)



Cohort 1A + 1B



- 5.2



- 9.5*



- 9.2*



B



Prostate #3


(absolute)



Cohort 1A



- 4.4



- 8.9



- 8.4



A



Prostate #4


(absolute)



Cohort 1B



- 7.1



- 9.1



- 4.3



B



Prostate #6


(absolute)



Cohort 1A + 1B



- 4.4



- 8.9



- 8.4



B



Epididymis, r. #5


(relative)



Cohort 1A + 1B



- 7.7*



- 4.5



- 4.1



C



Epididymis, r. #6


(absolute)



Cohort 1A + 1B



- 6.5



- 4.3



- 3.0



C



Text Table 8-19B: Comparison of organ weights with the Provivo background data.





























Parameter


Males


(Co1A)



Values from this study #2


Mean value per group ± SD


(range of the individual values (n= up to 20))


[number of individual values below or above the stated range]



Provivo Background Data #1


obtained from the control groups of 6 OECD 443 studies performed at Provivo from 2019 – 2021



Prostate


[g/kg b.w.]



Group 1



2.7998 ± 0.4325


[n=2] (1.851 – 3.472)



 


5 to 95 % Percentile


2.337 – 4.294



Group 2



2.7851 ± 0.5185


[n=5] (2.230 – 4.173)



Group 3



2.5428 ± 0.3962


[n=6] (1.901 – 3.159)



Group 4



2.4107 ± 0.4434*


[n=11] (1.814 – 3.659)



Text Table 9-2:     Observations that were noted for the prematurely deceased animal no. 443 during the daily cage side inspection on the days before death (common observations as salivation are not considered).



























Observations during the daily cage side inspections for the prematurely deceased animal no. 443 #



Gr.



No.



Sex



Symptom



Observed on postnatal day (day of death)



3



443



M



Piloerection


Pale, whole body


Pale ears


Exopthalmus, eyes



75, 76; 78 - 83


75 - 83


61


61 (day of death: 84)



 



Gr.: Group; M; F: Male; Female



Text Table 9-3:     Observations that were noted during the daily cage-side observations for the male animals of the low, the intermediate and the high dose level.











































































Observations in group 2


(25 mg IPDA/kg b.w./day)



Observation (males) #1


(Cohort 1B)



Affected animals



First to last seen


 (postnatal days) #2



Number of times recorded



Salivation



1 of 20



63 (406)



1



Observations in group 3


(80 mg IPDA/kg b.w./day) #3



Salivation



1 of 19



45 (452)



1



Piloerection



1 of 19



100 (456)



1



Haemorrhagic urine



1 of 19



100 (460)



1



Observations in group 4


(160 mg IPDA/kg b.w./day)



Salivation



16 of 20



28 - 100



38



Breathing sounds



2 of 20



48 - 75


(492, 493)



2



Haemorrhagic urine



1 of 20



96 (488)



1



#1:



Taken from table 2-1A-Co1B ' Clinical Signs - Summary - Males'.



#2:



If only 1 or 2 animals were affected, the animals are listed in brackets.



#3:



Group 3: The prematurely deceased male no. 443 was excluded from the summary.



Text Table 9-4:     Observations that were noted during the daily cage-side observations for the female animals of the low, the intermediate and the high dose level.










































Observations in group 3


(80 mg IPDA/kg b.w./day)



Observation (females) #1


(Cohort 1B)



Affected animals



First to last seen


(postnatal days) #2



Number of times recorded



Salivation



2 of 20



45 - 76


(463, 466)



2



Observations in group 4


(160 mg IPDA/kg b.w./day)



Salivation



20 of 20



26 - 99



62



Breathing sounds



1 of 20



46 - 70 (502)



16



Piloerection



1 of 20



28 (506)



1



Text Table 9‑6:     Start and duration of observations that were noted for the female animals.



































Start and duration of symptoms - Females



Symptom (Cohort 1B) #



Time frames in relation to application



Frequency observed


(in all groups)



Appearing of  the symptom



Disappearing of the


symptom



Salivation



0 - 5 min



5 - 20 min



17



 



20 - 60 min



47



Breathing sounds



0 - 5 min



6 - 24 h



16



Text Table 9‑7A:   Differences in body weight in comparison to control on postnatal day 26 and on postnatal day 98.










































Cohort 1B


Body Weight


 



Changes in comparison to control [%]



Group 2


25 mg/kg



Group 3


80 mg/kg



Group 4


160 mg/kg



Males #1



PND 26 #2



- 1.9



+1.0



- 0.5



PND 98 #3



+1.5



+0.7



+0.9



#1:



See table 4-1-Co1B ‘Body Weight - Summary - Males’.



#2:



At start of the F1 Study, the transferred pups were between 22 and 26 days old. Hence, the first day of age, for which a body weight was available for all transferred pups was postnatal day 26.



#3:



Before removal of the male animals of cohort 1A.



 


Text Table 9‑7B:   Body weight gain of the male animals of Cohort 1B from postnatal day 26 to postnatal day 98.




















Body weight gain



Group 1


Control



Group 2


25 mg/kg



Group 3


80 mg/kg



Group 4


160 mg/kg



Males


(postnatal day 26 to 98)



+543.5%



+564.3%



+543.1%



+552.8%



Text Table 9-8A:   Differences in body weight in comparison to control between postnatal days 26 and 98.


































































Cohort 1B


Body Weight


 



Changes in comparison to control [%]



Group 2


25 mg/kg



Group 3


80 mg/kg



Group 4


160 mg/kg



Females #1



PND 26 #2



- 4.1



- 2.3



- 4.5



PND 40



- 3.6



-  5.9**



- 8.2**



PND 47



- 4.6*



- 8.2**



- 8.4**



PND 54



- 5.4*



- 6.0*



- 7.2**



PND 61



- 5.4*



- 5.9*



- 6.8**



PND 68



- 5.2



- 6.2*



- 6.4*



PND 96



- 4.4



- 5.8*



- 5.3



PND 98



- 3.2



- 5.0



- 5.3



Text Table 9‑8B:   Body weight gain of the female animals of Cohort 1B from postnatal day 26 to postnatal day 98.




















Body weight gain



Group 1


Control



Group 2


25 mg/kg



Group 3


80 mg/kg



Group 4


160 mg/kg



Females


(postnatal day 26 to 98)



+285.2%



+289.0%



+274.5%



+282.3%



Text Table 9‑9:     Statistically significant differences in food consumption that were considered to be spontaneous.
























Parameter


(male)



Ref. Table no.



Difference to control [%]



Group / sex



 Test days



Statistical significance



Reason



Relative food consumption



6-1-Co1B



- 5.0



4 m



57 - 64



p £ 0.05



A



A:         The reduction was only slight and temporary


Text Table 9-10:   Time points (postnatal day) of balano-preputial separation.















































Parameter



Group 1


Control



Group 2


25 mg/kg



Group 3


80 mg/kg



Group 4


160 mg/kg



Cohort 1A



Day of balano-preputial separation (PND) #1



22.6 ± 0.7



22.5 ± 0.7



22.2 ± 0.4



22.3 ± 0.7



Cohort 1B



Day of balano-preputial separation (PND) #2



22.6 ± 0.7



22.4 ± 0.5



22.3 ± 0.6



22.1 ± 0.2*



Cohort 1A and 1B combined



Day of balano-preputial separation (PND) #3



22.6 ±  0.7



22.5 ± 0.6



22.2 ± 0.5*



22.2 ± 0.5**



*/**:



Statistically significant from control at p ≤ 0.05/0.01 (DUNNETT test)



Text Table 9-11:        Stage of the estrous cycle at necropsy. The stage of the estrous cycle was evaluated from vaginal lavages that were taken at necropsy.













































Stage of estrus at necropsy


(Cohort 1B females) #



Group 1


Control



Group 2


25 mg/kg



Group 3


80 mg/kg



Group 4


160 mg/kg



Proestrus



-



2 of 20



1 of 20



2 of 20



Estrus



7 of 20



9 of 20



4 of 20



8 of 20



Metestrus



2 of 20



3 of 20



6 of 20



3 of 20



Diestrus



11 of 20



6 of 20



9 of 20



7 of 20



- :



not detected



Text Table 9-12:        Statistically significant changes in organ weights unrelated to the test item for the male animals of Cohort 1B (the values of Cohort 1A are given for comparison).







































































Parameter


Cohort 1B (males)



Cohort



Changes in comparison to control


[%]



Reason



Group 2


25 mg/kg



Group 3


80 mg/kg



Group 4


160 mg/kg



 



 



Pituitary #1


(relative)



Cohort 1B



+5.6



- 2.4



+21.9**



A



Cohort 1A



- 11.1



- 4.1



- 4.2



B



Pituitary #2


(absolute)



Cohort 1B



+8.0



- 1.3



+22.6*



A



Cohort 1A



- 1.5



- 2.3



+9.4



B



*/**:



Statistically significant from control at p ≤ 0.05 (ANOVA / DUNNETT test)



#1:



Values taken from table 11-1-Co1B 'Relative Organ Weights - Summary - Males'.



#2:



Values taken from table 12-1-Co1B 'Absolute Organ Weights - Summary - Males'



A:



The increased weights that were noted at the low dose level and the high dose level for the weight of the pituitary gland were considered to be spontaneous, as no dose response relationship and no such changes were noted in Cohort 1A.



B:



Only for comparison.



Text Table 9-13:        Changes in the relative and absolute weight of the right adrenal gland and the relative and absolute weight of the left ovary (not considered to be test item-related). The values form the left adrenal gland, the right ovary and the respective values from Cohort 1A are given for comparison.






































































































































































Cohort 1B (females)



Cohort



Changes in comparison to control


[%]



Reason



Parameter



Group 2


25 mg/kg



Group 3


80 mg/kg



Group 4


160 mg/kg



 



 



Adrenal gland; left


(relative) #1



Cohort 1B



- 5.0



- 0.2



- 5.8



C



Cohort 1A



- 1.4



- 5.7



- 0.7



C



Adrenal gland, right


(relative) #1



Cohort 1B



- 12.4



- 1.9



- 11.5



A



Cohort 1A



- 0.4



- 2.9



- 2.2



C



Adrenal gland; left


(absolute) #2



Cohort 1B



- 6.8



- 3.4



- 9.7



C



Cohort 1A



- 0.7



- 8.0



- 3.5



C



Adrenal gland; right


(absolute) #2



Cohort 1B



- 14.5*



- 5.0



- 15.4**



A



Cohort 1A



- 0.3



- 5.6



- 5.3



C



Ovary; left


(relative) #1



Cohort 1B



+16.6*



+20.1**



+20.8*



B



Cohort 1A



- 0.4



+5.9



+10.6



C



Ovary; right


(relative) #1



Cohort 1B



+5.5



+14.8



+14.3



C



Cohort 1A



+2.5



- 2.0



+8.7



C



Ovary; left


(absolute) #2



Cohort 1B



+13.8



+15.2



+15.0



B



Cohort 1A



+1.4



+3.6



+8.2



C



Ovary; right


(absolute) #2



Cohort 1B



+2.7



+9.6



+8.7



C



Cohort 1A



+4.2



- 4.2



+5.8



C



*/**:



Statistically significant from control at p ≤ 0.05/0.01 (ANOVA / DUNNETT test)



#1:



Values taken from table 18-Co1A 'Relative Organ Weights – Cohort 1A' and table 11-Co1B 'Relative Organ Weights – Cohort 1B'



#2:



Values taken from table 19-Co1A 'Absolute Organ Weights – Cohort 1A' and table 12-Co1B 'Absolute Organ Weights – Cohort 1B'



A:



The reduced absolute and relative organ weights of the right adrenal gland were considered to be spontaneous. No statistically significant changes were noted for the left adrenal gland from the females of Cohort 1B and the left and right adrenal gland from the females of Cohort 1A.



B:



The increased absolute and relative organ weights of the left ovary were considered to be spontaneous. No statistically significant changes were noted for the right ovary from the animals of Cohort 1B and the left and right ovaries from the females of Cohort 1A. In addition, the histopathological examination of the reproductive organs from the high dosed females of Cohort 1A revealed no relevant toxicological changes.



C:



For comparison only.



Text Table 10-1:      Test item formulation analysis.









































Parameter



Sampling / Dealing



Percent of nominal concentration [%]



Concentration



F0 Generation: At the end of the pre-mating period, before administration to the last animals (test day 84 of the F0 Study)



105.7 % - 107.8 %



F0 Generation: At termination of the F0 Generation, before administration of the last animals (test day 128 of the F0 Study).



104.7 % - 108.4 %



F1 Generation: At termination of the F1 Generation, before administration of the last animal (test day 66 of the F1 Study



101.6 % - 103.1 %



Homogeneity



F0 Generation (at start of dosing on test day 15):


At start of administration, during administration and before administration to the last animal per group.



102.0 % - 105.7 %



F0 Generation (at dose reduction of the high dose level on test day 51):


At start of administration, during administration and before administration to the last animal per group.



103.4 % - 104.7 %



F1 Generation (test day 1 of the F1 Study):


At start of administration, during administration and before administration to the last animal per group.



106.0 % - 109.6 %



 



Taken from table 1-F0/F1 ‘Test Item Formulation Analysis'.



 

Applicant's summary and conclusion

Conclusions:
The oral exposure to 25, 80, and 240/160 mg test item/kg bw (male and female Sprague-Dawley rats; OECD 443; Provivo, 2021) in the extended one generation reproductive toxicity study led to no detectable changes in fertility and reproductive performance of the parental animals.
The pre- and post-development of the pups and the postnatal development of the pups was also not affected by the test item. No malformations or variations were noted during the macroscopic external and internal examinations of the pups at necropsy.
No test item-related influence was noted on the development of the reproductive system of eighter sex.

However, a test item-related effect was noted at 240/160 mg test item/kg b.w./day for the male and female animals in the form of 3 test item-related premature deaths from in total 48 high dose animals. Further, breathing sounds and piloerection were noted at the high dose level. The mortality rate of the high dose correlates with the observations made in the OECD 422 study (Provivo , 2021). One test item-related death was noted in the adult F1 animals at the high dose of 160 mg/kg b.w./day. The animals of the F1 Generation showed no further signs of general toxicity (body weight, food consumption thyroid hormone levels). Breathing sounds and piloerection were only noted for a few animals of the F1 Generation at 160 mg test item/kg/d.

The No Observed Adverse Effect Level (NOAEL) for reproduction was therefore considered to be above 240 mg test item/kg/day.
The NOEAL for pre- and postnatal development was above 160 mg IPDA/kg b.w./day.

The No Observed Adverse Effect Level (NOAEL) for systemic toxicity in the F0 and in the adult animals of Cohort 1A and 1B was determined to be 80 mg/kg/day.
Executive summary:

The aim of the study was to evaluate the effects of the test item IPDA at dose levels of 25, 80 or 240/160 mg/kg b.w./day on the general and reproductive toxicity of the F0 Parents and of the developmental toxicity of the F1 Generation from weaning until adulthood (OECD 443). The high dose level was reduced from 240 to 160 mg/kg b.w./day on test day 51 (after the administration of 240 mg/kg for 37 days) due to changes in behaviour and the external appearance and one test item-related premature death. For this reason, the high dose animals of the F1 Generation also only received a dose level of 160 mg/kg.


General and reproductive toxicity (F0 Generation and F1 Pups)


General toxicity


A test item-related effect was noted at 240/160 mg IPDA/kg b.w./day for the male and female animals in the form of 3 test item-related premature deaths from in total 48 high dose animals. Further, breathing sounds and piloerection were noted at the high dose level. The mortality rate of the high dose correlates with the observations made in the OECD 422 study.


No influence was noted on body weight, food consumption, the laboratory examinations, during necropsy and the histopathological examination.


Reproductive toxicity


No test item-related influence was noted on the reproductive performance of the parental animals (number and length of estrous cycles, fertility and gestation index, pre-coital time and gestation length).


The prenatal development of the pups (number of resorptions, stillbirths and live born pups) and the postnatal development of the pups (pup body weight, viability index, ano-genital distance, nipple retention, thyroid hormone levels, pup organ weights) was also not effected by the test item. No malformations or variations were noted during the macroscopic external and internal examinations of the pups at necropsy.


 


Developmental toxicity (F1 - Cohorts 1A and 1B)


During their post-weaning development one test item-related death was noted at the high dose of 160 mg/kg b.w./day. The animals of the F1 Generation showed no further signs of general toxicity (body weight, food consumption thyroid hormone levels). Breathing sounds and piloerection that were noted as adverse and test item-related observations for several animals of the F0 Generation at 240/160 mg IPDA/kg were only noted for a few animals of the F1 Generation at 160 mg IPDA/kg. Due to the low incidences of breathing sounds and piloerection at the F1 Generation, these observations were not considered as an adverse effect for the developmental toxicity of the F1 Generation.


No test item-related influence was noted on the development of the reproductive system (time points of sexual maturations, number and length of estrous cycles, sperm parameter, detailed histopathological examination of testis and epididymides, number of primordial and growing follicles and number of corpora lutea in the ovaries).


 


The following no-observed-adverse-effect levels (NOAEL) were established for the parental animals of the F0 Generation and the F1 Generation:


F0 Generation:


General toxicity


NOAEL                                                            80 mg IPDA/kg b.w./day


Based on the premature death of 3 animals (for which a test item-related cause of death cannot be excluded) and clinical signs observed in animals treated with 240/160 mg IPDA/kg b.w./day.


 


Reproductive toxicity


NOAEL                                       above 240/160 mg IPDA/kg b.w./day


 


 


F1 Generation:


Developmental toxicity


Adverse effects on pre- and postnatal development



  1. Adverse effects on prenatal development (conceptus to birth)


NOAEL                                   above 160 mg IPDA/kg b.w./day



  1. Adverse effects on postnatal development (pup)


NOAEL                                   above 160 mg IPDA/kg b.w./day


 


General toxicity (Cohorts 1A and 1B)


NOAEL                                                            80 mg IPDA/kg b.w./day


 


Based on one premature death observed in animals treated with 160 mg IPDA/kg b.w./day, for which a test item-related premature death cannot be excluded.


1.1.1         Findings - F0 Generation and F1 Pups













































































Mortality


 



Males


None of the male animals treated with 25 or 80 mg IPDA/kg b.w./day died prematurely.


At (240/160 mg IPDA/kg b.w./day) one premature death was noted. Male no. 153 was prematurely sacrificed on test day 73 due to poor health condition. The histopathological examination revealed no specific test item-related findings for the animal that was prematurely sacrificed due to moribund conditions. Also no signs of misgavage, accidental influx or incidental regurgitation of the test item-formulation into the respiratory tract were noted.


Females


None of the female animals treated with 25 or 80 mg IPDA/kg b.w./day died prematurely.


At (240/160 mg IPDA/kg b.w./day) two females (no. 182 and no. 178) were prematurely sacrificed due to poor health condition on test days 24 or 59, respectively.


The histopathological examination revealed no specific test item-related findings for the 2 animals that were prematurely sacrificed due to moribund conditions. No signs of misgavage or an accidental influx or incidental regurgitation of the test item-formulation into the respiratory tract were noted for these 2 animals.


 



 



 




Clinical signs



Males and females


Salivation was noted for several male and female animals at 80 mg IPDA/kg b.w./day.


At the high dose level (240/160 mg IPDA/kg b.w./day) test item-related changes were noted for all or several male and female animals in the form of salivation, breathing sounds and piloerection.


The other observations were considered to be spontaneous, due to their low incidence.


 


Start and duration:


In nearly all cases salivation was a short lasting post‑dosing symptom, whereas breathing sounds were often noted for several hours after dosing or for the whole day.


Detailed clinical observations:


No further observations in addtion to those made during the daily cage side observations were noted for the male and female animals of the control and the treatment groups.



 



 



Body weight and


body weight gain



 


Males and females


No test item-related differences were noted.



 



 



Food consumption



Males and females


No test item-related differences were noted.



 


Haematology


(test days 127 to 133,


at necropsy)



 


 


 


Males and females


No test item-related changes were noted.


Nevertheless, for the female animals a decrease in the number of white blood cells was observed, which was statistically significant for the 25 and 80 mg IPDA/kg b.w./day. However, these changes were considered spontaneous as no dose response-relationship was noted.



 



 




Clinical biochemistry


(test days 127 to 133,


at necropsy)


 



 


 


Males and females


No test item-related changes were noted.



 



 



Urinalysis


(test days 127 to 133,


at necropsy)


 



 


 


Males and females


No test item-related changes were noted.



 



 



Thyroid hormone levels


(test days 127 to 133,


at necropsy)


 


 


 



 


 


Males and females


No test item-related changes were noted for the examined thyroid hormones T4 and TSH.


Differences between the control group and all treatment groups were noted for the TSH concentration of the male and female animals. However, as the differences were not statistically significant and nearly all individual values were within the Provivo background data range and no dose-response relationship was noted, they were not considered to be test item-related.


 



 



 



Sperm parameter


(test days 127, 128, 129,


at necropsy)


 


 



 


 


Males


No test item-related changes were noted in sperm number, motility and morphology.


   

 














































Necropsy


(test days 127, 128, 129 for the males,


test days 129 to 134 for the females)



Macroscopic post-mortem


findings



 


Males and females


No toxicologically relevant macroscopic changes that could be associated with the test item were noted for the surviving male and female animals that were dosed with 25, 80 or 240/160 mg IPDA/kg b.w./day.



 



 



Body weight at autopsy



Males and females


No test item-related changes were noted.



 



 



Estrous stage at necropsy



No test item-related differences were noted.



 



 



Organ weights



Males and females


No test item-related differences were noted.


Nevertheless, in the males the relative and absolute thymus weight showed a decrease. This observation was considered to be spontaneous, as no correlation with the histopathology results was noted.


The thyroid of the female animals showed a non‑dose dependent weight increase with no correlation in histopathology. Hence, the thyroid weight increase was also considered to be spontaneous and of no toxicological relevance.



 



 



Bone marrow examination



Males and females


No test item-related differences were noted.



 










Histopathological examinations


(Groups 1 and 4)



 


 


Males and females


No test item-related observations were noted for the examined male and female animals of group 4.


Additionally, previously (OECD 422, Provivo study no. 37482, 2021) observed microscopic changes in the kidney and stomach at the high dose animals (300/240 mg IPDA/kg b.w./day) were not observed at this study at 240/160 mg IPDA/kg b.w./day.


 


 


Examination of reproductive organs


No test item-related observations were noted for the examined reproductive organs of the male and female animals of group 4.


All testes examined showed completeness of stages and cell population.



 


















































































































Reproductive toxicity


Reproductive parameters of the parental females



Estrous cycle data


(test day15 until evidence of


copulation)



 


No test item-related influence was noted on the mean number and the mean length of the estrous cycles.



Fertility index



No test item-related influence was noted.



Gestation index



No test item-related influence was noted.



Pre-coital time



No test item-related influence was noted.



Gestation length



No test item-related influence was noted.



 



 



F1 Pups - Pre- and postnatal development



- Prenatal development (from conceptus to birth)



Reproductive parameters



No test item-related influence was noted on the birth index, the live birth index, and the percentage of post-implantation loss.



 



 



- Postnatal development (pup)



Mortality (Viability index)



Pre- and post-cull period


No test item-related influence was noted.



 



 



Pup body weight



No test item-related influence was noted.


Nevertheless, a significant reduced body weight was noted at LD14, which resolved at LD21. Therefore, these observations were considered to be spontaneous.



 



 



Ano-genital distance



No test item-related influence was noted.


However, a slight reduction was observed at both sexes, which probably resulted from the developmental delay at LD4, which had normalised until LD21. Hence, these slight differences were considered to be spontaneous and have no toxicological relevance.



 



 



Count of male nipples


(nipple retention)



 


No test item-related influence was noted.



 



 



F1 Pups - Examination at necropsy (surplus pups; not used for F1 generation)



External and


internal examination



 


No malformations or variations were noted.



 



 



T4 determination


(lactation day 4; culled pups)



 


No test item-related difference was noted.



 



 



T4, TSH determination


(lactation day 21/22)



 


No test item-related difference was noted.



 



 



Pup organ weights



No test item-related difference was noted.



1.1.1         Findings - Cohort 1A



























































Mortality



Males and females


One male animal (no. 336) of the high dose group (160 mg IPDA/kg b.w./day) was prematurely sacrificed on test day 73 due to poor health condition. No specific test item-related findings were noted during the histopathological examination. No signs of misgavage, accidental influx or incidental regurgitation of the test item-formulation into the respiratory tract were noted.



 



 



Clinical signs


 



Males and females


Salivation was noted for several male animals at 80 mg IPDA/kg b.w./day.


At the high dose level (240/160 mg IPDA/kg b.w./day) test item-related changes were noted for half the male and female animals in the form of salivation. Breathing sounds, piloerection, hunched position and laboured breathing were only noted for 1 or 2 animals per sex.



 



 



Body weight and


body weight gain



 


Males and females


No test item-related differences were noted.



 



 



Food consumption



Males and females


No test item-related changes were noted. A transient increase in the food consumption was noted for the high dose male animals between TD 43-57.



 



 



Haematology


(postnatal days 90 to 92,


at necropsy)



 


 


Males and females


No test item-related changes were noted.



 



 




Clinical biochemistry


(postnatal days 90 to 92,


at necropsy)



 


 


Males and females


No test item-related changes were noted.



 



 


   

 


 

































































































Lymphocyte typing (spleen)


(postnatal days 90 to 92,


at necropsy)



 


 


Males and females


No test item-related changes were noted.



 



 



Urinalysis


(postnatal days 90 to 92,


at necropsy)


 



 


 


Males and females


No test item-related changes were noted.



 



 



Thyroid hormone levels


(postnatal days 90 to 92,


at necropsy)


 



 


 


Males and females


No test item-related changes were noted for the examined thyroid hormones T4 and TSH.


However, a dose dependent decrease in the TSH values in the intermediate and high dose below the detection limit were recorded for the female animals of the treatment groups. Nevertheless, the histopathology observations did not report any histopathological changes in the pituitary gland compared to the control group. Furthermore, the differences in the TSH values showed an opposite trend between the male (increased values in comparison to the control) and the female animals (decreased values in comparison to the control). Due to this opposite trend and the high variability between the individual values, the differences that were noted between the control group and the treatment groups were considered to be spontaneous and toxicological not relevant.



 



 



Sexual Maturation



Males


No test item-related influence was noted on the day of balanopreputial separation and the body weight on the day of balanopreputial separation.


 


Females


No test item-related influence was noted on the day of vaginal opening, the body weight on the day of vaginal opening, the day of the appearance of cornified cells in the vaginal smear and the period between the day of vaginal opening and the appearance of cornified cells in the vaginal smear.



 



 



Estrous cycle data



Females


No test item-related influence on the number and length of the estrous cycles was noted during the examination of a 2-week period between test days 50 and 63.



Sperm parameter


(PND 90 to 92; at necropsy)


 



 


Males


No test item-related changes were noted.



 



 




Necropsy


(PND 90 to 92 for the males and females)



Macroscopic post-mortem


findings



 


Males and females


No test item-related observations were noted.


In the female animals dilated uterus, a one lobe reddened thymus, enlarged mesenteric lymph node and a thickened intestinal mucus was observed in one or two animals of the control group.



 



 



Body weight at autopsy



Males and females


No test item-related changes were noted.



 



 



Estrous stage at necropsy



No test item-related influence was noted on the number of the different stages between the control group and the treatment groups.



 



 



Organ weights



Males and females


No test item-related differences were noted.


However, a slight decrease in the relative prostate weight was observed in the male animals. Nevertheless, observed differences were considered to be spontaneous and not test item-related.



 



 



Bone marrow examination



Males and females


No test item-related differences were noted.



 



 



Histopathology


(groups 1 and 4)



 


Males and females


No test item-related observation were noted for the examined male and female animals of group 4.


Additionally, previously (OECD 422, Provivo study no. 37482, 2021) observed microscopic changes in the kidney and stomach at the high dose animals (300/240 mg IPDA/kg b.w./day) were not observed at this study at 160 mg IPDA/kg b.w./day.


Examination of reproductive organs


No test item-related observation were noted for the examined reproductive organs of the male and female animals of group 4.


All testes examined showed completeness of stages and cell population.


 


Quantitative evaluation of primordial and small growing follicles and corpora lutea


No test item-related differences were noted between the females of the control group and the females of the high dose group in the number of follicles and corpora lutea.



1.1.2         Findings - Cohort 1B






























Mortality


 



Males and females


No test item-related death was noted for all treatment groups (25, 80 or 160 mg IPDA/kg b.w./day).



 



 



Clinical signs



Males and females


Salivation was noted for 1 to 2 animals of the low and intermediated dose group.


At the high dose level (160 mg IPDA/kg b.w./day) test item-related changes were noted for more than half of the male and female animals in the form of salivation. Breathing sounds, haemorrhagic urine and piloerection were only noted for 1 or 2 animals per sex at the intermediate and high dose.


 


Detailed clinical observations:


Males and females


No further observations were noted during the weekly detailed clinical observations for the surviving male animals.


 


Females


Breathing sounds were noted for one female (no. 502) of the high dose group in two test weeks.



 



 



Body weight and


body weight gain



 


Males and females


No test item-related differences were noted.


However, marginal reductions in body weight were noted for the females of the three treatment groups compared to the control group animals from postnatal day 26 on until the end of the study on postnatal day 98. These marginal differences were considered to be non-adverse, as there was no correspondence in the body weight gain noticed.


 



 



 



 


 





















































Food consumption



Males and females


No test item-related changes were noted. Even so, a significant difference in food consumption was noted for the males of the high dose group between test days 57-64.




 



 



Sexual maturation


 


 



Males and females


No test item-related differences were noted for the time points of balanopreputial gland cleavage and vaginal opening.


Also, no test item-related differences were noted for the body weight at the time points of balanopreputial gland cleavage and vaginal opening.



 



 




Necropsy



Macroscopic post-mortem


findings



 


Males and females


No test item-related observations were noted.



 



 



Body weight at autopsy



Males and females


No test item-related changes were noted.



 



 



Estrous stage at necropsy



No test item-related influence was noted.



 



 



Organ weights



Males and females


No test item-related differences were noted.


However, a statistically significantly increase was noted for the relative organ weights of the left ovary at all dose levels of the females of cohort 1B. Nevertheless, no histomorphological changes were found in the female reproductive organs including ovaries in the F1 cohort 1A. Therefore, it was considered that there is no toxicological significance in the weight changes detected in these organs.



 


 


1.1.3         Analysis of test item-formulations










Test item-formulation analysis



The measured concentrations of IPDA in the test item-formulations were between 101.6 % and 109.6 % of the nominal concentration, indicating correctly prepared and homogeneous test item-formulations.