tert-butyl 3,5,5-trimethylperoxyhexanoate

Toxicity to reproduction

Administrative data

Endpoint:

extended one-generation reproductive toxicity - with developmental immunotoxicity (Cohorts 1A, 1B without extension, and 3)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-03-26 to 2022-05-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)

Version / remarks:

25 June 2018

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.56 (Extended One-Generation Reproductive Toxicity Study)

Version / remarks:

15 July 2014

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS
- Premating exposure duration for parental (P0) animals: 10 weeks, as requested by ECHA.
- Basis for dose level selection: Based on a pre-test conduced with TBPND (Modified OECD 421 with 10 weeks pre-mating treatment); furthermore, results from a OECD 422 toxicity study in rats was taken into consideration for dose level selection (see supporting information, RSS).
- Exclusion of extension of Cohort 1B: not requested by ECHA based on available data and not necessary based on study results
- Exclusion of developmental neurotoxicity Cohorts 2A and 2B: not request by ECHA based on available data
- Inclusion of developmental immunotoxicity Cohort 3, as requested by ECHA
- Route of administration: oral (gavage)

Test material

Test material information

Constituent 1

Reference substance name:

tert-butyl peroxyneodecanoate

EC Number:

247-955-1

EC Name:

tert-butyl peroxyneodecanoate

Cas Number:

26748-41-4

Molecular formula:

C14H28O3

IUPAC Name:

Reaction products of neodecanoic acid (or neodecanoyl chloride derived from neodecanoic acid) and tert-butyl hydroperoxide

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The rat is regarded as suitable species for reproduction studies and the test guideline is designed to use the rat. The Wistar rat was selected due to a wide range of experience with this strain of rat in reproduction toxicity studies and well-known fertility parameters.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Toxi-Coop Zrt. Cserkesz u. 90. H-1103 Budapest, Hungary
- Females nulliparous and non-pregnant: yes
- Age at study initiation: P0 males and females:
Male animals: 41 - 43 days (6 weeks) old
Female animals: 39 - 42 days (6 weeks) old
- Weight at study initiation:
(P) Male animals: 158 – 222 g
(P) Female animals:120 – 147 g
- Fasting period before study: no
- Housing: Type III polypropylene/polycarbonate
Before mating: 2 animals of the same sex/cage
Mating: 1 male and 1 female/cage
Pregnant females will be housed individually.
Males after mating: 2 animals/cage
F1 offspring (after weaning): 2 animals of the same sex/cage
- Diet: ad libitum, ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice
- Water: ad libitum, tap water
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3°C
- Humidity: 30-70 %
- Air changes: > 10 per hr
- Photoperiod: 12/12 hrs dark / hrs light

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

vegetable oil

Remarks:

Sunflower oil (Helianthi annui oleum raffinatum)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test item was formulated in the vehicle in concentrations of 25, 75 and 225 mg/mL. Formulations were prepared in the formulation laboratory of Test Facility not longer than for three days before the use. Analysis of formulations was performed in the Analytical Laboratory of Test Facility. Five samples were taken from different places from each concentration (Groups 2, 3 and 4) and measured on 5 occasions. Similarly, five samples were taken from the control solution (Group 1) from different places and analyzed.
VEHICLE
- Justification for use and choice of vehicle: The test item is not stable in water. Therefore, sunflower oil was used for preparing formulations appropriate for oral administration. Sunflower oil is a suitable vehicle to facilitate formulation analysis for the test item.
- Concentration in vehicle: 25, 75 and 225 mg/mL
- Amount of vehicle: 2 mL/kg bw

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: max. 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: single

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

A sufficient stability and homogeneity in the chosen vehicle have been verified over the range of relevant concentrations at the appropriate frequency of preparation. Recovery was 98 and 102 % of nominal concentrations at 1 and 500 mg/mL in sunflower oil, respectively. TBPND proved to be stable at room temperature for four hours (recovery was 105 % of starting concentration at 1 mg/mL and 100 % at 500 mg/mL) and at 5 ± 3°C for 3 days (recovery was 98 % of starting concentration at 1 mg/mL and 101 % at 500 mg/mL).

Duration of treatment / exposure:

Parental males were dosed for 70 days pre-mating and up to 14 days mating and until to the weaning of offspring. Overall treatment period is therefore up to 18 weeks for male parental animals.
Parental females were dosed for 70 days pre-mating, through up to 14 days mating period and throughout pregnancy and at least up to and including post-partum day 21 or up to the day before sacrifice. The day of birth (viz. when parturition is complete) is defined as day 0 post-partum. Overall treatment period is therefore up to 18 weeks for female parental animals.
Dosing of F1 offspring selected for follow-up examinations began on PND 22 up to post-natal day 90 (Cohort 1A) or at least up to post-natal day 97 (Cohort 1B).
F1 animals selected for determination of primary IgM antibody response to a T-cell dependent antigen in Cohort 3, were administered and observed individually up to and including the day before euthanasia on PND 61 ± 3.

Frequency of treatment:

daily, 7 days/week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

50 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

150 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

450 mg/kg bw/day (nominal)

No. of animals per sex per dose:

P0: 26
F1, Cohort 1A: 20
F1, Cohort 1B: 20
F1, Cohort 3: 20

Control animals:

yes, concurrent vehicle

yes, historical

Details on study design:

- Dose selection rationale:
The dose setting for the OECD TG 443 study is based on findings obtained in previous studies with TBPND in rats. The high dose was chosen with the aim of inducing toxic effects but no mortality or severe suffering of animals. The low dose was chosen to induce no toxic effect.
A reproduction/ developmental toxicity screening test according OECD TG 422 was performed in 2012 with TBPND using dosages of 60, 200 and 600 mg/kg bw/day. Here, TBPND caused salivation, changes in body weight and food consumption and clinical pathology parameters (lower hemoglobin concentration and hematocrit value, and elevated percent of reticulocytes in female animals, higher mean activity of alanine aminotransferase and urea concentration in male and female animals, higher mean serum levels of creatinine in male animals), and changes in organ pathology (enlarged and pale kidneys, higher kidney weights and hyaline droplet nephropathy of male rats, higher liver weights in male and female animals) following an oral administration at 600 mg/kg bw/day to Hsd.Brl.Han:Wistar rats. At 200 mg/kg bw/day, salivation, reduced body weight development, changes in clinical pathology parameters (elevated percent of reticulocytes, higher mean activity of alanine aminotransferase and higher mean serum levels of urea in females), and changes in organ pathology (higher kidney weights and hyaline droplet nephropathy of male rats, higher liver weights in female animals) were observed. At 60 mg/kg bw/day, salivation (male and female animals), higher percent of reticulocytes, slightly elevated mean activity of alanine aminotransferase and liver weight in female animals and hyaline droplet nephropathy of male rats were detected. Dam’s delivery was affected at 600 mg/kg bw/day as the number of dams with prolonged pregnancy was higher consequently the mean duration of pregnancy was longer than in the control group, and higher percentage and litter mean of post-implantation loss and stillborns were observed. At 600 mg/kg bw/day, the extra uterine mortality of offspring was higher in percentage and mean with respect to control and the offspring’s body weight development (for litter and pup’s weights) was depressed at 600 and 200 mg/kg bw/day. Based on these observations the following NOAELs were determined:
NOAEL systemic for male rats: 60 mg/kg bw/day
NOAEL systemic for female rats: 60 mg/kg bw/day
NOAEL for reproductive performance of the male and female rats: 200 mg/kg bw/day
NOAEL for F1 Offspring: 60 mg/kg bw/day
Based on these results a modified study according OECD TG 421 was performed in Hsd.Brl.Han:Wistar rats with TBPND in 2021, in which the parent (P) generation was dosed 10 weeks prior to mating. The highest dose was reduced to 300 mg/kg bw/day based on significant reductions in body weight/body weight gain during gestation and lactation in females observed in the OECD TG 422 study from 2012. In males, effects in kidney especially in the high dose of 600 mg/kg bw/day were the basis for high dose reduction to 300 mg/kg bw/day. Therefore, the dosage levels tested were 50, 150 and 300 mg/kg bw/day. This study was performed to conclude on the dose setting for the subsequent OECD TG 443 study and to investigate also the effect of longer pre-treatment period on systemic toxicity and fertility. At 300 mg/kg bw/day, the body weight development of dams was reduced during the gestation and lactation period. There were no signs of systemic toxicity at 50, 150 mg/kg bw/day (male and female) or 300 mg/kg bw/day (male). TBPND caused reduction of mean number of births (total, live and alive) after administration of 300 mg/kg bw/day by oral gavage to Han:WIST rats as far as investigated in this study. 50 or 150 mg/kg bw/day did not adversely influence the reproductive performance (gonad function, mating behaviour, conception, parturition) in parental male and female animals. The body weight development of the offspring was depressed between post-natal day 0 and 21 after repeated oral administration of dams at 150 or 300 mg/kg bw/day. The body weight development and food consumption of the F1 animals were reduced at 150 and 300 mg/kg bw/day during the 14-day post-weaning treatment. Based on these observations the NOAELs were determined as following:
NOAEL for systemic toxicity of male/ female rats: 300 mg/kg bw/day
NOAEL for reproductive performance of male/ female rats:150 mg/kg bw/day
NOAEL for F1 Offspring: 50 mg/kg bw/day
Since no significant systemic toxicity was observed at 300 mg/kg bw/day in the modified OECD 421 study, the highest dose for the OECD TG 443 study was elevated to 450 mg/kg bw/day. The doses which were chosen for the main study have been the following:
Group 1: 0 mg/kg bw/day
Group 2: 50 mg/kg bw/day
Group 3: 150 mg/kg bw/day
Group 4: 450 mg/kg bw/day

- Fasting period before blood sampling for clinical biochemistry: yes

Positive control:

not applicable

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Pertinent behavioral changes, signs of difficult or prolonged parturition and all signs of toxicity including mortality were recorded including onset, degree and duration of signs.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: More detailed examinations were made at the times of weekly weighing, prior to and during the mating and until necropsy. Detailed clinical observations were made on all animals outside the home cage in a standard arena once prior to the first exposure and once weekly thereafter. Observations were performed on the skin, fur, eyes and mucous membranes, autonomic activity (lachrymation, piloerection, pupil size, respiratory pattern, occurrence of secretions and excretions), circulatory and central nervous system, somatomotor activity and behavior pattern, changes in gait, posture and response to handling. Special attention will be directed towards the observation of tremors, convulsions, salivation, diarrhea, lethargy, sleep and coma.

BODY WEIGHT: Yes
- Time schedule for examinations: Parental males were weighed on the first day of dosing (Day 0) and weekly thereafter. Parental females were weighed on the first day of dosing (Day 0) then weekly, on gestation days 0, 7, 14 and 21 and on post-partum days 0 (within 24 hours after parturition), 4, 7, 14 and 21. Body weight of the female animals were additionally weighed on gestation day 10 in order to give accurate treatment volumes, but these data were not be evaluated statistically. F1 animals selected for follow-up examinations were weighed on post-natal day 22, then twice a week during the two weeks following weaning (on PND 25, 29, 32, 36, 39 and 42), and once weekly thereafter (Cohorts 1A, 1B and Cohort 3). For selected F1offspring, the body weight was recorded on the day when they attain puberty (completion of balano-preputial separation or vaginal patency). Fasted body weight was measured on the day of necropsy for all animals (P and F1)

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- The food consumption was determined weekly by reweighing the non consumed diet with a precision of 1 g during the treatment period except mating phase as follows:
- by weekly interval during premating and post-mating periods for P male animals and for F1 animals (Cohorts 1A, 1B and Cohort 3) after weaning;
- by weekly interval during premating period, on gestation days 0, 7, 14 and 21, on lactation days 0, 7, 14 and 21, then weekly if needed, for P female animals

WATER CONSUMPTION AND COMPOUND INTAKE: No

URINALYSIS: Yes
The following parameters were evaluated in selected test animals of P and F1 Cohort 1A generation:
Nitrite (NIT), pH, Glucose (GLUC), Urobilinogen (UBG), Bilirubin (BIL), Ketone (KET), Blood, Leucocytes (LEU), Specific Gravity (SG), Protein (PROT), Volume (VOL), Sediment (SED), Colour, Clarity

HEMATOLOGY/BLOOD COAGULATION
The following parameters were measured in all selected animals of P and F1 Cohort 1A generation:
White blood cell (leukocyte) count (WBC), Red blood cell (erythrocyte) count (RBC), Hemoglobin concentration (HGB), Hematocrit (HCT), Mean Corpuscular (erythrocyte) Volume (MCV), Mean Corpuscular (erythrocyte) hemoglobin (MCH), Mean Corpuscular (erythrocyte) hemoglobin concentration (MCHC), Platelet (thrombocyte) count (PLT), Reticulocytes (RET), Differential white blood cell count, Activated partial Thromboplastin Time (APTT), Prothrombin Time (PT).

CLINICAL CHEMISTRY
The following parameters were measured in all selected animals of P and F1 Cohort 1A generation:
Alanine Aminotransferase activity (ALT), Aspartate Aminotransferase activity (AST), Total Bilirubin concentration (TBIL), Creatinine concentration (CREA), Urea concentration (UREA), Glucose concentration (GLUC), Cholesterol concentration (CHOL), Sodium concentration (Na+), Potassium concentration (K+), Albumin concentration (ALB), Total protein concentration (TPROT)

DETERMINATION OF SERUM LEVELS OF THYROID HORMONES
Blood samples were collected for determination of serum levels of thyroid hormones (FT3, FT4, TSH) as follows:
- from 10 parent animals/sex/ group

Oestrous cyclicity (parental animals):

Estrous cycle was monitored by examining vaginal smears from each parental female animal daily for two weeks before the mating starts.Vaginal smears were also prepared and estrous cycle was monitored daily during the mating period until evidence of copulation. Vaginal smear was prepared on the day of the necropsy of parental animals.Vaginal smears were examined for all F1 Cohort 1A females selected for follow-up examinations after the onset of vaginal patency until the first cornified smear is recorded thus determining the time interval between these events. Estrous cycle of F1 adult female animals was examined for a period of two weeks commencing on PND77 and PND84 in Cohort 1A and Cohort 1B, respectively, including necropsy days. Vaginal smears were stained with 1 % aqueous methylene blue solution. After drying, the smears were examined with a light microscope.

Sperm parameters (parental animals):

Sperm parameters were measured in all control and high dose male animals in P generation and in F1 generation in Cohort 1A. The one-side testes and epididymides were used for examinations. The weights of one-side testes and epididymides were determined and recorded. Sperm from the ductus deferens was collected for evaluation of sperm motility and morphology at the necropsy. Both numbers of motile and immotile sperms were recorded. Two samples were prepared from each animal. For the determination of the sperm motility, the mean percentage of motile sperms were determined. A morphological evaluation of ductus deferens sperms sample was performed from the same animals. Sperm was examined as fixed, wet preparations and classified as either normal or abnormal (isolated heads, misshapen heads and/or tails). The epididymis was used for enumeration of cauda epididymis sperm reserves. The total number of sperm in homogenization was enumerated. The testis and epididymidis were frozen and enumeration were performed later.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- maximum of 10 pups/litter (5/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups on PND13, FT3, FT4 and TSH in surplus pups at PND 4 (pooled by litters) and in pups not selected for Cohorts on post-natal day 22.

Sexual maturity of selected F1 animals was examined by observing balano-preputial separation (between post-natal days 25 and 35) or vaginal patency (between post-natal days 28 and 40; or until PND45). The body weight was determined on the day when balanopreputial separation or vaginal patency is completed

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: Yes, Cohort 3

Animals of Cohort 3 were administered daily from weaning up to the day before the termination. The following observations will be conducted:
- Checking of mortality/ morbidity – twice daily;
- General clinical observations – daily;
- Detailed clinical observations were made outside the home cage in a standard arena once prior to the first exposure and once weekly thereafter.
- Body weight measurement – on PND 22, then twice a week during the two weeks following weaning and once weekly thereafter;
- Food consumption – on PND 22 and weekly thereafter
- Blood sampling for T-cell dependent antibody response assay (TDAR) on PND 56±3 and PND 61±3;
On PND 61±3 the primary IgM antibody response to a T-cell dependent antigen – keyhole limpet hemocyanin (KLH) – was assessed after subcutaneous immunization of all Cohort 3 animals with KLH (0.2 mL at a concentration of 1.5 mg/mL on PND56±3; prior dosing with test-item). Anti-KLH IgM antigen concentrations in serum was measured before immunization and 5 days after immunization using a validated ELISA method (Rat Anti-KLH IgM ELISA kit, Life Diagnostics, Inc.). Responses typically peak five days after immunization.

DETERMINATION OF SERUM LEVELS OF THYROID HORMONES
Blood samples were collected for determination of serum levels of thyroid hormones (FT3, FT4, TSH) as follows:
- in surplus pups at PND 4 (pooled by litters)
- from 10 adult F1 male and female animals/group (Cohort 1A ) at termination
- from 10 F1 pups/group not selected for cohorts on PND22

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: after mating period
- Maternal animals: on PND 22.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following tissues were prepared for microscopic examination and weighed, respectively, for high dose and control animals:

Organ weights (all parental (P) animal and all adult F1 animals of Cohort 1A):
- uterus (with oviducts and cervix)
- ovaries
- testes
- epididymides
- prostate (dorsolateral and ventral parts combined)
- seminal vesicles with coagulating glands as one units (with their fluids)
- brain
- liver
- kidneys
- heart
- spleen
- thymus
- pituitary
- thyroid glands (post-fixation)
- adrenal glands

The organs were fixed in 4% buffered formaldehyde solution. Paired organs were weighed together except for organs with macroscopically visible difference in size between the two organs. Absolute organ weight was recorded and reported.

Full histological examinations was performed on the above listed organs and tissues of control and high dose treated parental animals. Reproductive organs were examined in all animals suspected of reduced fertility (not mated, non pregnant or not delivered) in the low and mid dose group.

SPLENIC LYMPHOCYTE SUBPOPULATION ANALYSIS
At termination, weighing of the lymph nodes and splenic lymphocyte subpopulation analysis was performed in 10 male and 10 female Cohort 1A animals from each group (1 male or 1 female per litter; all litters represented by at least 1 pup; randomly selected) as follows:
- weighing of the lymph nodes associated with and distant from the route of exposure (submandibular and popliteal lymph nodes);
- splenic lymphocyte subpopulation analysis: CD4+ (Helper T cells) and CD8+ (Cytotoxic T cells) T lymphocytes, B lymphocytes and natural killer (NK) cells were identified by a validated flow cytometry method. Immuno-staining and immunophenotyping of spleen lymphocytes were carried out after preparation of single cell suspensions from one half of each provided spleen. For further information please refer to the attached background material.

Postmortem examinations (offspring):

SACRIFICE
- Offspring selected for Cohort 1A and Cohort 1B: on post-natal days 91 and 98, respectively
- Offspring selected for Cohort 3 on PND 61±3
- Offspring not selected: on post-natal day 22

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGTHS
Organ weights ( all adult F1 animals of Cohort 1A):
- uterus (with oviducts and cervix)
- ovaries
- testes
- epididymides
- prostate (dorsolateral and ventral parts combined)
- seminal vesicles with coagulating glands as one units (with their fluids)
- brain
- liver
- kidneys
- heart
- spleen
- thymus
- pituitary
- thyroid glands (post-fixation)
- adrenal glands

In animals of Cohort 1B, the weight of following organs was determined:
- uterus (with oviducts and cervix)
- ovaries
- testes
- epididymides
- prostate (dorsolateral and ventral parts combined)
- seminal vesicles with coagulating glands as one units (with their fluids)
- brain,
- pituitary

For 10 male and 10 female pups per group, not selected for Cohorts, – from as many litters as possible – brain, spleen and thymus was weighed.

In animals of Cohort 3, the final body weight was determined.

Full histological examinations was performed on the above listed organs and tissues of control and high dose treated adult F1 animals in Cohort 1A. In adult F1 animals in Cohort 1B, the uterus (with oviducts and cervix), ovaries, testes, epididymides, prostate (dorsolateral and ventral parts combined), seminal vesicles with coagulating glands as one unit (with their fluids), brain, pituitary will be processed to the block stage. In the ovaries of F1 adult females of control and high dose groups, a quantitative evaluation of primordial and small growing follicles, as well as corpora lutea was performed.

Detailed histological examination of testis was conducted with special emphasis on stages of spermatogenesis in the F1 male gonads and histopathology of interstitial testicular cell structure. Rete testis – where feasible – caput, corpus, and cauda of the epididymides were examined. All gross lesions were examined histologically. The fixed tissues were trimmed, processed (dehydrated), embedded in paraffin, sectioned with a microtome (at a thickness of 2-4 µm), placed on glass microscope slides, stained with hematoxylin and eosin and examined by light microscopy.

Statistics:

The homogeneity of variance between groups was checked by Bartlett’s
homogeneity of variance test. Where no significant heterogeneity is detected, a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant, Duncan Multiple Range test was used to access the significance of intergroup differences. Getting significant results at Bartlett’s test the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed if feasible.

Reproductive indices:

Copulatory Index (Measure of animals ability to mate):
Males: Number of males with confirmed mating / Total number of males cohabited x 100
Females: Number of sperm positive females / Total number of females cohabited x 100

Fertility Index (Measure of male’s ability to produce sperm that can fertilize eggs and measure of female’s ability to become pregnant):
Males: Number of males impregnating a females / Total number of males with confirmed mating x 100
Females: Number of pregnant females / Number of sperm positive females x 100

Gestation Index (Measure of pregnancy that provides at least one live pup):
Number of females with live born pups / Number of pregnant females x 100

Offspring viability indices:

Formulas for Calculation of Pup Mortality and Sex Ratio Indices:

Post-implantation mortality: Number of implantations – Number of liveborns / Number of implantation x 100

Post-natal mortality: Number of liveborns – Number of live pups on PND 13 / Number of liveborns x 100

Survival Index: Number of live pups on PND 13 / Number of liveborns x 100

Sex ratio: Number of pups examined – Number of pups males (females) / Number of pups examined x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Clinical signs of systemic toxicity related to the test item were not detected at any dose level at the daily clinical observations (50, 150 or 450 mg/kg bw/day).
Salivation was observed in several male and in some female animals at 450 mg/kg bw/day with variable incidence and with short duration immediately after the administration. Therefore, salivation was judged to be toxicologically not relevant in this study. There were no preceding clinical signs in dead animal at the daily or at the detailed weekly clinical observations. The death occurred after working hours as animal was found dead at the morning checking.
Clinical signs of systemic toxicity related to the test item were not detected at any dose level at the daily clinical observations (50, 150 or 450 mg/kg bw/day).
Salivation was observed in several male and in some female animals at 450 mg/kg bw/day with variable incidence and with short duration immediately after the administration. Therefore, salivation was judged to be toxicologically not relevant in this study.
There were no preceding clinical signs in dead animal at the daily or at the detailed weekly clinical observations. The death occurred after working hours as animal was found dead at the morning checking.
The behavior and physical condition of the animals was not impaired at any dose level (50, 150 or 450 mg/kg bw/day) during the entire treatment period.
In the male animals, slight salivation was observed at 450 mg/kg bw/day with variable onset and duration from Day 13 (9/26).
In the female animals at 450 mg/kg bw/day, salivation was detected during the pre-mating (5/26), gestation and lactation (4/24, both) periods.
Additionally, the following clinical signs were seen in some animals:
-Reddish colored hairs:
-around the right eye – 1/26 control male from Day 14 up to termination
-around the neck: from Day 87 to 98, 1/26 female at 150 mg/kg bw/day
-Scar:
-behind left ear and on right side scapula from Day 7 up to Day 27 or 34, 1/26 male at 50 mg/kg bw/day
-on right side scapula between Day 21 and 27, 1/26 female at 50 mg/kg bw/day
-Alopecia in female animals:
-1/24 control, 1/26 at 50 mg/kg bw/day, 2/23 at 150 mg/kg bw/day, 1/24 at 450 mg/kg bw/day during the gestation period and 2/24 control, 4/23 at 150 mg/kg bw/day and 1/24 at 450 mg/kg bw/day during the lactation period
-Soft swelling on pudenda 1/26 at 150 mg/kg bw/day from Day 76 to 82
These dermal changes and discoloration of hair are common findings in experimental rats of this strain with similar age. These were detected independently from doses in this study. Swelling at pudenda was an individual alteration occurring also in untreated rats and was due to the congenital absence of vaginal orifice (introitus vaginae).

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There was no test item related fatal death at any dose level (50, 150 or 450 mg/kg bw/day) during the course of the observation period. One male animal at 450 mg/kg bw/day was found dead on Day 64. Based on necropsy and histological findings, this animal died as a consequence of suffocation and shock.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The body weight development was depressed in a dose related manner in parental male animals at 150 and 450 mg/kg bw/day during the entire treatment period.
Slightly reduced mean body weight gain resulted in only minor changes in the mean body weight in male animals at 150 mg/kg bw/day (less than 5 % relative to control). Therefore, these findings were considered to have little or no toxicological relevance. There was no significant difference between the control and 50 mg/kg bw/day groups in the mean body weight of male animals during the entire observation period.
The mean body weight gain was lower than in the control at 50 mg/kg bw/day during the first week (Day 0-7). However, the summarized body weight gain was comparable to the control in male animals at 50 mg/kg bw/day.
The mean body weight and body weight gain were slightly lowered in male animals at 150 mg/kg bw/day comparing to the control during the observation period. Statistical significances with respect to the control were detected at the lower mean body weight (-4, -5 %) on Days 63, 69, 83, 97, 104 and 111, as well as at the lower mean body weight gain at 150 mg/kg bw/day between Days 0-7, 14-21, 28-35, 49-56, 97-104 and for the study overall (0-111) in male animals.
At 450 mg/kg bw/day, the mean body weight of male animals was significantly lower than in the control from Day 21 onwards (-5, -13 %) in consequence of the lower mean body weight gain during the entire observation period with statistical significance on week 1, 3, 4, 5, 8, 9, 10, 15, 16 and for the study overall. The mean body weight gain slightly exceeded the control in male animals at 450 mg/kg bw/day on week 11.
In the female animals at 50 and 150 mg/kg bw/day, the mean body weight was predominantly comparable with the control during the entire observation period. Statistically significant difference with respect to the control was observed occasionally at the lower mean body weight of female animals at 50 mg/kg bw/day on lactation day 14 and at 150 mg/ kg bw/day on gestation day 21 and on lactation day 0 when compared to the control.
At 450 mg/kg bw/day, the mean body weight of female animals exceeded the control (4-6 %) from Day 14 up to the end of pre-mating period and was lower than in the control on gestation day 21, lactation days 0, 4, 14 and 21 (up to -6 %),
Statistical significances were detected at the lower or higher mean body weight gain of female animals when compared to the control as follows:
-at 50 mg/kg bw/day: lowered mean body weight gain on week 6 and between lactation days 7-14
-at 150 mg/kg bw/day: higher mean body weight gain on weeks 1, 2 and 7, as well as lower mean body weight gain on week 6 and during 3rd week of gestation and for the entire gestation period (0-21, ca. 10%)
-at 450 mg/kg bw/day: higher mean body weight gain on weeks 1, 2 and for pre-mating period overall (Days 0-69) and lower mean body weight gain between gestation days 14 and 21 and during the entire gestation period (0-21, ca. 22%)

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

The mean daily food consumption was not adversely affected in parental male or female animals at 50, 150 or 450 mg/kg bw/day.
Slight, toxicologically not relevant changes in the mean daily food intake were in accordance with the body weight variations in male and female animals at each dose level and were not strictly dose related or consistent.
Statistical significance with respect to the control was detected at the lower mean daily food consumption of parental male animals at 50 mg/kg bw/day on weeks 1 and 3 (-4, -5%) and at 150 mg/kg bw/day on weeks 1, 3, 4 and 10 (-5, -19 %).
At 450 mg/kg bw/day, the mean daily food consumption was lower than in the control group in parental male animals on weeks 1, 2, 3, 7 and 10 (-6, -10 %).
In the female animals the mean daily food intake was comparable to the control at 50 mg/kg bw/day during pre-mating, gestation and lactation periods.
Higher mean daily food consumption was detected in female animals at 150 mg/kg bw/day on week 2 (6 %) and at 450 mg/kg bw/day on weeks 2, 5 and 8 (8, 13 %) when compared to the control.
Statistical significance with respect to the control was observed at the lower mean daily food consumption in the female animals at 150 mg/kg bw/day and at 450 mg/kg bw/day on gestation week 3 (-9 %, both) and between lactation day 4-7, on lactation weeks 2 and 3 (-8, -16 %).
These slight differences with respect to the control were of low degree and not consistent during the treatment period. Therefore, these were considered to be of low or no toxicologically relevance.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related adverse changes in the examined hematological or blood coagulation parameters in parental male or female animals at 50, 150 or 450 mg/kg bw/day. The examined hematological and blood coagulation parameters were comparable in the control, 50 mg/kg bw/day (female) and 150 mg/kg bw/day (male). Statistical significance with respect to the control was observed at lower mean percentage of eosinophil granulocytes (EOS) at 50 and 450 mg/kg bw/day and at the shorter mean prothrombin time (PT) at 450 mg/kg bw/day in male animals.
In female animals, statistical significance was detected at the higher mean percentage of neutrophil granulocytes (NEU) along with lowered mean percentage of lymphocytes (LYM) at 150 mg/kg bw/day and at the elevated mean percentage of reticulocytes (RET) at 450 mg/kg bw/day when compared to the control. The differences with respect to the control group reached statistical significance at the mentioned parameters. However, there were no dose relevance and the individual values met the historical control range (i.e., were within or close to the range) in male and female animals.
Therefore, the differences in these hematological parameters were considered to have little or no toxicological relevance.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Clinical chemistry investigations revealed elevated activity of alanine aminotransferase (male and female), creatinine (male), urea (male and female) and cholesterol (female) at 450 mg/kg bw/day. Slight alterations in creatinine (male), cholesterol (female) indicated test item influence at 150 mg/kg bw/day in a lesser degree.
The examined clinical chemistry parameters were comparable with their control in male and female animals at 50 mg/kg bw/day.
Statistically significant differences with respect to the control were detected at the higher mean creatinine (CREA) and sodium (Na+) levels at 150 mg/kg bw/day and at the elevated mean activity of alanine aminotransferase (ALT), creatinine, urea and mean lowered mean concentrations of cholesterol, sodium and potassium (K+) at 450 mg/kg bw/day in male animals.
In the female animals at 150 mg/kg bw/day, the mean concentration of CREA and cholesterol (CHOL) was higher and the concentration of glucose (GLUC) was lower than in the control group.
At 450 mg/kg bw/day, higher mean activity of alanine aminotransferase, higher mean level of urea, cholesterol and potassium, lower mean concentration of total bilirubin (TBIL), creatinine and glucose were observed when compared to their control in female animals.
Although findings were of low degree, test item influence in the high dose animals is presumed. Elevated levels of creatinine and urea in the males may be based on renal lesions, as there was an indication for renal alterations in the same sex noted at the histopathological examinations. The increase in alanine aminotransferase in both sexes and in cholesterol of the females may indicate liver function alterations.
The differences to the control of the above-mentioned parameters reached statistical significance, the values of most of these parameters corresponded well to the historical control values – Na +, K+ in male animals, TBIL, CREA, GLUC, K+ in female animals.

Endocrine findings:

effects observed, non-treatment-related

Description (incidence and severity):

The thyroid hormone (FT3, FT4 and TSH) levels were not adversely influenced in the parental male or female animals at any dose levels.
Neither a dose-dependent effect nor statistically significant differences were detected between the control and test item administered animals in the serum levels of FT3 and FT4. The FT3 and FT4 levels were comparable in male and female parental animals in the control, 50, 150 and 450 mg/kg bw/day groups.
Statistically significant difference was detected at the higher mean TSH level at 450 mg/kg bw/ in parental male animals. The degree of changes was minor and individual and mean values corresponds to the historical control ranges. In the absence of related changes in reproductive performance, organ weight or histopathology observations, these findings in female animals were considered to be toxicologically not relevant.
The cytomorphology of endocrine glands were the same in the control and treated animals.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

The pH of urine was slightly lowered, protein and ketone were present in urine in male animals at 450 mg/kg bw/day presumably in accordance with renal damage revealed by histological investigations.
Statistical significances were detected at the lower mean pH of the urine in male animals at 450 mg/kg bw/day and in female animals at 150 mg/kg bw/day.
Ketone test was positive in 3/10 male animals at 150 mg/kg bw/day and 8/10 male animals at 450 mg/kg bw/day groups. Normally, ketones are totally resorbed by renal tubules. Elevated level in the urine may refer to tubular damage or exceedance of the tubular resorption threshold.
Proteinuria is well known in experimental rats; however, protein was detected with high incidence (100 %) in male animals at 450 mg/kg bw/day.

Behaviour (functional findings):

not examined

Immunological findings:

effects observed, non-treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathology investigation did not reveal toxic or other test item-related lesions of investigated genital organs of the experimental male and female parental animals at 50, 150 or 450 mg/kg bw/day doses.

Renal changes – distended tubules with hyaline casts, tubular basophilia, lymphocytic and histiocytic infiltration, characteristic on the chronic progressive nephropathy (CPN) in the rat – were detected only in male animals at 150 and 450 mg/kg bw/day (50 and 100% of examined animals, respectively). CPN is a spontaneous renal disease of laboratory rat with similar age, therefore 150 and 450 mg/kg bw/day doses of test item were assumed to be a predisposing factor in the development of pathogenesis of CPN in male animals.
In dead animal at 450 mg/kg bw/day (1/26 male), histological examination revealed acute alveolar emphysema and edema in the lungs (probably in connection with suffocation and shock), distended tubules with hyaline cats in the kidneys. No degenerative or other toxic lesions were observed in the investigated organs of this animal.
The investigated organs of the male reproductive system (testes, epididymides, prostate seminal vesicles, coagulating glands) were histologically normal and characteristic for the sexually mature organism in all parental male animals in the control and 450 mg/kg bw/day groups (26/26 for both groups, including dead animal) as well as in not mated male (1/1) and in males not impregnated females (2/2) at 150 mg/kg bw/day. Sperm granuloma in one side epididymis (1/26 at 450 mg/kg bw/day, including dead animal) is a spontaneous individual disorder occurring in laboratory rats.
In the female animals at 450 mg/kg bw/day and control groups and in not mated (1/1), non-pregnant (2/2) female animals at 150 mg/kg bw/day of parental generation, the ovaries had a normal structure characteristic of the species, age and phase of the active sexual cycle. The cortical region of ovaries contained primary, secondary and tertiary follicles and corpora lutea, indicating the active maturation of oocytes, and ovulation. The epithelial capsule and ovarian stroma were normal in all cases as well.
Dilatation of uterine horns was detected in some female animals in each group: 5/26, 6/6, 2/4 and 2/26, respectively to groups of control, 50, 150 and 450 mg/kg bw/day. Dilatation of uterine horns is a slight neuro-hormonal phenomenon in connection with sexual function – pro-estrous phase – of the inner genital organs.
Some incidental individual findings – occurring commonly in experimental rats – were also detected histologically in female reproductive organs: cyst at the oviducts (1/26 at 450 mg/kg bw/day) or in the vagina (1/4 at 150 mg/kg bw/day), hemangioma in the uterus (1/26 at 450 mg/kg bw/day) and endometritis (1/26 at 450 mg/kg bw/day).
Tubular basophilia and lymphocytic infiltration were detected in one female animal at 450 mg/kg bw/day (1/26).
One or both sided renal pyelectasia occurred in each group of male and female animals independently from doses as follows:
-male 6/26 control, 3/26 at 50 mg/kg bw/day, 3/26 at 150 mg/kg bw/day, 9/26 at 450 mg/kg bw/day
-female: 2/26 control, 4/4 at 50 mg/kg bw/day, 2/2 at 150 mg/kg bw/day, 1/26 at 450 mg/kg bw/day
Pyelectasia without significant histological lesions (inflammation, degeneration, fibrosis etc.) is common in laboratory rats of this strain and is considered to be an individual disorder without pathological significance.
Histopathology investigation also revealed spontaneous individual disorders, which are well-known lesions and commonly observed in experimental rats of this strain and not related to the test item:
-hepatic granuloma 1/3 male at 150 mg/kg bw/day
-vacuolation of hepatocytes in the liver 1/3 male at 150 mg/kg bw/day
-focal hyperplasia in the spleen 1/26 female at 450 mg/kg bw/day
-focal fibrosis in the spleen 1/1 female at 150 mg/kg bw/day
Focal fibrosis in the Glisson’s capsule of the liver develops due to the diaphragmatic hernia because of mechanical irritation (2/26 male control, 1/1 female at 50 mg/kg bw/day, 1/3 male and 1/1 female at 150 mg/kg bw/day,
The focal hemorrhage (1/26, 2/5, 3/6) and erosion (2/26, 2/5, 3/6, respectively to groups control, 50 and 150 mg/kg bw/day) in the stomach of female animals could be in connection with mechanical origin (gastric tube) and may be considered as individual disorder.
The atrophy of hair follicles (1/26 female control, 1/1 female at 150 mg/kg bw/day) is common finding in laboratory rats in accordance with dermal alopecia.
There was no morphological evidence of test item-related acute or subacute injury (degeneration, inflammation, necrosis etc.) in the small and large intestines, liver, pancreas, cardiovascular system, respiratory system, immune system, hematopoietic system, skeleton, muscular system, central, or peripheral nervous system, eyes, integumentary system.
The cytomorphology of endocrine glands were the same in the control and treated animals.

Histopathological findings: neoplastic:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

The estrous cycle was not influenced by TBPND at 50, 150 or 450 mg/kg bw/day.
There were no statistically or biologically significant differences between the control and test item treated groups (50, 150 or 450 mg/kg bw/day) in the examined parameters of estrous cycle: the percentage of animals with regular cycles, the mean number of cycles, the mean length of cycles, mean number of days pro-estrous, estrous or diestrus were similar in all groups and there were no animals with prolonged estrous during the pre-mating period.

Reproductive function: sperm measures:

effects observed, non-treatment-related

Description (incidence and severity):

Sperm examinations did not reveal any test item-related influence on the sperm cells at 450 mg/kg bw/day.
Statistical significances were detected at the lowered mean percentage of immotile sperm cells at 450 mg/kg bw/day.
The mean percentage of sperms with normal morphology was similar in the control and 450 mg/kg bw/day groups.
Differences in the percentage of immotile sperm cells was considered to be indicative of biological variation and normal function of male genital organs. Since no test-item related influence on sperm examinations was detected at the highest dose the lower dose groups have not been examined.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

The reproductive performance of male and female animals was not affected by the treatment at 50, 150 or 450 mg/kg bw/day with respect to their control.
Statistical significances detected at some parameters (copulatory, fertility and gestation indices, percentage of pregnant/ non-pregnant female animals, mean number of conceiving days) were indicative of biological variation and not related to the test item.
In the male animals, statistical significance with respect to the control was detected at the higher copulatory index (higher percentage of mated male animals) at 50 and 150 mg/kg bw/day and at the higher fertility index (higher percentage of fertile male and lower percentage of infertile animals) at 50 mg/kg bw/day.
In the female animals, the copulatory index (higher percentage of mated animals) exceeded the control in each group, while the fertility index (percentage of fertile animals and lower percentage of infertile animals) was above the control only at 50 mg/kg bw/day and the gestation index (percentage of dams delivered) was below the control at 450 mg/kg bw/day.
Statistical significance was also detected at the higher percentage of pregnant female animals at 50 mg/kg bw/day and lower mean number of conceiving days at 450 mg/kg bw/day when compared to their control.
The differences between the control and test item treated groups are without any biological relevance and lay well within the biological range of variation.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive performance

Effect level:

450 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest dose tested

2

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

150 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

clinical biochemistry

organ weights and organ / body weight ratios

3

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

50 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

clinical biochemistry

urinalysis

gross pathology

histopathology: non-neoplastic

Target system / organ toxicity (P0)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

150 mg/kg bw/day (actual dose received)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no adverse clinical signs in the F1 offspring from post-natal day 0 to 21. The percentage of offspring showing signs (no milk in the stomach, cold, missing) was higher in litters of dams treated with 450 mg/kg bw/day than in the control. Most of these observations were detected on the day of delivery and corresponded with the findings of inadequate nursing behavior of the respective dams. Some other sporadic clinical signs were detected with similar incidence (smaller than normal, pale, hemorrhage, found dead) in the control and 50, 150 or 450 mg/kg bw/day dose groups.

There were no clinical signs in male or female animals in F1 Cohort 1A generation in 50, 150 or 450 mg/kg bw/day groups during the entire observation period (from post-natal day 22 up to and including post-natal day 91-97). The behavior and physical condition of all F1 Cohort 1A animals was normal at each dose level (50, 150 or 450 mg/kg bw/day) based on the weekly detailed clinical observations during the entire treatment period.

There were no test item related clinical signs in male or female animals in F1 Cohort 1B generation in 50, 150 or 450 mg/kg bw/day groups during the entire observation period (from post-natal day 22 up to and including post-natal day 96-105). Most of the male and female animals were normal in the control, 50, 150 and 450 mg/kg bw/day during the observation period. Alopecia was noted on the fore-limbs for only one female animal at 150 mg/kg bw/day from post-natal day 98 up to post-natal day 105. Alopecia on the skin is a species-specific finding detectable in non-treated experimental rats of this strain. Animals in the F1 Cohort 1B showed normal behavior and physical condition at each dose level (control, 50, 150 or 450 mg/kg bw/day) based on the weekly detailed clinical observations during the entire treatment period. Alopecia was detected on single female animal at 150 mg/kg bw/day on post-natal days 98 and 105 at the detailed weekly clinical observation, too.

Clinical signs were not detected in male or female animals in F1 Cohort 3 in control, 50, 150 or 450 mg/kg bw/day groups.
All animals in the F1 Cohort 3 showed normal behavior and physical condition at each dose level (control, 50, 150 or 450 mg/kg bw/day) based on the daily general and weekly detailed clinical observations during the entire treatment period.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

There was no test item-related effect on offspring’s extra uterine mortality. The extrauterine mortality was low and comparable in the control, 50, 150 and 450 mg/kg bw/day group on post-natal day 0 and from birth to post-natal day 21.

There was no mortality in F1 Cohort 1A animals in control, 50, 150 or 450 mg/kg bw/day groups during the course of study (male and female).

There was no test item related mortality in F1 Cohort 1B animals in 50, 150 or 450 mg/kg bw/day groups during the course of study (male and female).
One female animal at 150 mg/kg bw/day (1/20) died immediately after the daily administration on post-natal day 76 presumably due to a probably mis-gavage during the administration with gastric tube. No preceding clinical observations or body weight loss were observed.

No animal died in F1 Cohort 3 in the control, 50, 150 or 450 mg/kg bw/day groups during the course of study (male and female).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The body weight development of the F1 offspring was depressed at 50, 150 and 450 mg/kg bw/day. The reduction in body weight gain and body weight was with low degree but was consistent at 50 and 150 mg/kg bw/day and was therefore considered to be related to the test item. However, the values for 50 mg/kg bw/day were within the historical control ranges and therefore it was considered to have low or no toxicological relevance. The dose of 450 mg/kg bw/day significantly and consistently lowered the body weight and body weight gain of offspring from birth to post-natal day 21.Statistical significances with respect to the control were observed at the lower mean body weight and body weight gain of offspring predominantly at each measuring day at 50, 150 and 450 mg/kg bw from birth up to post-natal day 21. An exception is 50 mg/kg bw/day group, where the mean body weight exceeded the control on post-natal days 0 and 4 and the mean body weight gain was similar to the control between PND0 and 4. Similarly, the mean body weight was comparable with the control at 150 mg/kg bw/day at birth (PND0).When evaluating the body weight separately by gender, lowered mean body weight was detected at 150 mg/kg bw/day (male and female pups) and at 450 mg/kg bw/day (male and female pups) on PND4, when compared to their control. The mean litter weight and litter weight gain were comparable with control and 50 mg/kg bw/day groups during the 21-day observation period.
Statistical significance with respect to the control was detected at the lower mean litter weight at 150 mg/kg bw/day from post-natal day 7 up to PND21 (-8, -10 %) and lower mean litter weight gain from post-natal day 0 up to PND14 (-10, -16 %) and if summarized between post-natal day 0 and 21 (-9%). At 450 mg/kg bw/day, the mean litter weight and litter weight gain were significantly lower than in the control at each measuring occasion.

The body weight development was depressed in F1 Cohort 1A (male and female animals) at 450 mg/kg bw/day during the entire treatment period. Similar change was observed in the body weight at 150 mg/kg bw/day, however in a lesser degree however., Here, the difference from the control was lower than 10 % the and therefore, body weight changes in the mid dose group was considered to have little or no toxicological relevance.
Statistically significant difference with respect to the control were detected in the body weight and body weight gain per dose as follows:
Male animals
- 50 mg/kg bw/day: lower mean body weight on post-natal day 22 (-7 %), higher mean body weight gain between post-natal days 25-29
- 150 mg/kg bw/day: lower mean body weight on post-natal days 22, 25, 29, 36, 39, 63, 70, 77, 84, 91 (4-9 %), lower mean summarized body weight gain (between postnatal days 22 and 91)
- 450 mg/kg bw/day: lower mean body weight from post-natal day 22 up to and including post-natal day 91 (14-24 %) and lower mean body weight gain between post-natal days 22-36, 42-70, 77-91 and if summarized
-The changes in the body weight gain at 450 mg/kg bw/day resulted in significant changes in the body weight of male animals, and therefore, were considered to be toxicologically relevant.
Female animals:
-50 mg/kg bw/day: lower mean body weight on post-natal day 22 (-6 %);
-150 mg/kg bw/day: lower mean body weight on post-natal days 22, 25, (8-10 %),
-450 mg/kg bw/day: lower mean body weight from post-natal day 22 up to and including post-natal day 91 (9-27 %) lower mean body weight gain between post-natal days 22-25, 25-29 and higher mean body weight gain between post-natal days 36-39
These minor changes in the body weight gain observed for females resulted in significant changes in the body weight, however, were not consistent and therefore, were considered to be toxicologically not relevant.

The mean body weight was reduced in F1 Cohort 1B at 150 mg/kg bw/day (male) and at 450 mg/kg bw/day (male and female) during the entire treatment period (from post-natal day 22 up to post-natal day 98). The difference from the control remained consistent in male animals and lowered in female animals by the end of treatment period. The difference from the control was lower than 10 % at 150 mg/kg bw/day and therefore, body weight changes in the mid dose group were considered to have little or no toxicological relevance. The mean body weight was comparable in male and female animals in the control and 50 mg/kg bw/day body during the entire observation period. Statistical significances with respect to the control was detected at the lower mean body weight of male animals at 150 and 450 mg/kg bw/day in a dose related manner from weaning up to the termination of the treatment period. The body weight gain was also reduced in male animals at 150 and 450 mg/kg bw/day (-8% and -19% between PND22 and 98, respectively) and was comparable with control in female animals at 450 mg/kg bw/day, if summarized (between PND22 and 98).
Changes in the mean body weight gain comparing to the control showed sporadic occurrence in male animals as follows:
-50 mg/kg bw/day: lowered between PND49-56, elevated between PND56-63
-150 mg/kg bw/day. lowered between PND49-56 and if summarized (between PND22-98)
-450 mg/kg bw/day: lowered during the entire observation period reaching statistical significances between PND22-36, each occasion, between 42-56, 63-70 and if summarized
In the female animals, the mean body weight was statistically significantly lower than in the control group at 150 mg/kg bw/day on PND22 and at 450 mg/kg bw/day at each occasion between PND22 and 98.
The following statistically significant differences with respect to the control were detected in the mean body weight gain in the female animals:
-50 mg/kg bw/day: higher mean body weight gain between PND56-63
-150 mg/kg bw/day: lower mean body weight gain between PND49-56
-450 mg/kg bw/day: lowered between PND22-29, elevated between PND39-42 and between PND 91-98

The mean body weight was reduced in F1 Cohort 3 at 450 mg/kg bw/day (male and female) during the entire treatment period (from post-natal day 22 up to post-natal day 56). The mean body weight was mostly comparable in male animals in the control, 50 and 150 mg/kg bw/day body during the entire observation period. The mean body weight was lower than in the control in male animals at 50 mg/kg bw/day on starting day (PND22) and the mean body weight gain exceeded the control at 150 mg/kg bw/day between post-natal day 25-29.
Statistical significances with respect to the control was detected at the lower mean body weight of male animals at 450 mg/kg bw/day from weaning up to the termination on post-natal day 56. The mean body weight gain of these animals was also lowered when compared to control between post-natal days 22-25, 25-29, 29-32, 32-36, 42-49, 49-56 and if summarized between post-natal days 22-56. In the female animals, the mean body weight was similar in the control and at 50 mg/kg bw/day during the observation period and the mean body weight gain was higher than in the control between post-natal days 36-39. At 150 mg/kg bw/day, the mean body weight was lowered comparing to the control in female animals on post-natal days 22, 25 and the mean body weight gain was comparable with that of the control group during the entire observation period. At 450 mg/kg bw/day, the mean body weight remained below the control between post-natal day 22-56 and the body weight gain was lower than in the control between post-natal day 22-25, 25-29 and was higher than in the control between post-natal days 32-36, 36-39 and 39-42. Thus, the summarized body weight gain was comparable with the control in female animals administered with the high dose of the test item.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The mean daily food consumption of F1 Cohort A male animals was reduced at 450 mg/kg bw/day in accordance of body weight changes during the observation period. The mean daily food consumption was comparable with the control in all treated female animals and in male animals at 50 and 150 mg/kg bw/day during the entire observation period.At 450 mg/kg bw/day, statistically significant difference with respect to the control was detected at the lower mean daily food consumption in male animals by weekly interval between post-natal days 22-91 (-7, -29 %, no statistical significance between PND70-77) and in female animals on weeks 1 and 2 (-30, -15 %, respectively). Transient difference with respect to the control in female animals at 450 mg/kg bw/day was judged to be toxicologically not relevant.

The mean daily food consumption was reduced in male animals at 150 and 450 mg/kg bw/day during the entire treatment period and in female animals at 450 mg/kg bw/day during the course of the first two weeks of the treatment in F1 Cohort 1B. The difference from the control was -9 up to -33% in male animals at 450 mg/kg bw/day. The difference from the control was minor (≤11 %) in male animals at 150 mg/kg bw/day and was transient in female animals therefore these were considered to have little or no toxicological significance. The mean daily food consumption was similar to their control in male animals of F1 Cohort 1B at 50 mg/kg bw/day during the entire observation period (between PND22 and PND98). A lowered mean daily food consumption was observed compared to control in male animals at 150 and 450 mg/kg bw/day during the entire study with statistical significance between PND22-29, at each occasion between PND42-77 and PND84-91 in mid dose group and at each occasion between PND22-77 and PND91-98 in high dose treated animals. In the female animals, the mean daily food consumption was higher than in the control group at 50 mg/kg bw/day between PND56-63 and PND 91-98 and at 150 mg/kg bw/day between PND91 and 98. At 450 mg/kg bw/day, the mean daily food consumption of female animals was lower than in the control group between PND22-29 and PND29-36 and was higher than in the control between PND91-98.

The mean daily food consumption was lowered at 450 mg/kg bw/day during the first two weeks of the treatment in female animals and during the entire treatment period in female animals. The mean daily food consumption was mostly similar to their control in male and female animals of F1 Cohort 3 at 50 and 150 mg/kg bw/day during the entire observation period (between PND22 and PN56). Statistical significance with respect to the control was only detected at the higher mean daily food intake of female animals at 150 mg/kg bw/day between post-natal 36-42. At 450 mg/kg bw/day, the mean daily food consumption remained below the control between post-natal days 22 and 56 in male animal and between post-natal days 22 and 36 in female animals.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related adverse changes in the examined hematological and blood coagulation parameters in male or female animals at 50, 150 or 450 mg/kg bw/day in F1 Cohort 1A. Statistical significance with respect to the control was detected at the elevated percentage of neutrophil granulocytes (NEU) along with lowered percentage of lymphocytes (LYM) and lower mean hemoglobin concentration (HGB) in male animals at 50 mg/kg bw/day. At 150 mg/kg bw/day, the mean hemoglobin concentration and hematocrit (HCT) were lower than in the control in male animals. In the female animals, the mean white blood cell count was higher at 50, 150 or 450 mg/kg bw/day and the mean prothrombin time (PT) and activated partial thromboplastin time (APTT) were slightly shorter when compared to the control. The minor changes noted in the hematological and blood coagulation parameters were considered to have little or no toxicological relevance. Statistical significances at NEU, LYM, HGB and HCT were detected at the lower dose group but not at high dose treated animals. White blood cell count was relatively low in the female control group resulting statistical significances in all treated groups. Decreased in blood coagulation times has no toxicologic meaning. All individual values were either close to or within the historical control range.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Pathologic alterations were not detected at the evaluation of clinical chemistry parameters in F1 Cohort 1A male or female animals at 50, 150 or 450 mg/kg bw/day.
An elevated mean concentration of creatinine may be associated with the renal changes in male animals at 450 mg/kg bw/day.
In the male animals, higher mean activity of alanine aminotransferase (ALT) was detected in all treatment groups being statistically significant at 150 and 450 mg/kg bw/day compared to the control. The concentrations of creatinine (CREA) and UREA exceeded the control in male animals at 450 mg/kg bw/day.
The examined clinical chemistry parameters were comparable in the female animals in the control, 50 and 150 mg/kg bw/day groups.
Statistical significance with respect to the control was detected at the higher mean activity of alanine aminotransferase and higher mean concentration of total bilirubin (TBIL) and urea in female animals at 450 mg/kg bw/day.
The individual values of most of these parameters corresponded well to the historical control values – ALT, UREA, TBIL – in male or female animals. Elevation in alanine aminotransferase activity may indicate liver function alterations in both sexes and has no toxicological relevance in the absence of histological changes.
Higher concentration of creatinine and urea in the males may be based on renal lesions, as there was an indication for renal alterations detected at the histopathological examinations.

The thyroid hormone (FT3, FT4 and TSH) levels were not adversely affected in male or female animals in the F1 Cohort 1A at 50, 150 or 450 mg/kg bw/day.
In the male animals, the thyroid hormone concentrations were mostly comparable with their control at each dose level. Statistical significance was only detected at the slightly lower mean FT4 level at 450 mg/kg bw/day. Individual values were close or within the historical control of FT4 .
In the female animals, statistical significances with respect to the control were detected at the higher mean concentrations of FT3 at 50 and 150 mg/kg bw/day and of FT4 at 50, 150 and 450 mg/kg bw/day. In the lack of dose relevancy and related findings (macroscopic or microscopic changes in pituitary or thyroid gland, changes on estrous cycle) a relation to treatment is unlikely.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

The examined urine parameters were predominantly comparable in the F1 Cohort 1A male and female animals in the control and 50, 150 and 450 mg/kg bw/day groups. However, the pH of urine was slightly but statistical significant lowered and ketone was present in urine in male animals at 150 and 450 mg/kg bw/day presumably in accordance with renal damage revealed by histological investigations.
Ketone test was positive in 7/10 male animals at 150 mg/kg bw/day and 9/10 male animals and 1/10 female at 450 mg/kg bw/day groups. Normally, ketones are totally resorbed by renal tubules. Elevated level in the urine may refer to tubular damage or exceedance of the tubular resorption threshold.
These minor changes were considered to have little or no toxicological significance as vales of pH in male animals remained within the control ranges.

Sexual maturation:

effects observed, non-treatment-related

Description (incidence and severity):

The sexual maturity was not adversely affected in F1 male or female animals at 50, 150 or 450 mg/kg bw/day (Cohort 1A, Cohort 1B, Cohort 3, N=60).

Anogenital distance (AGD):

effects observed, non-treatment-related

Nipple retention in male pups:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Test item related changes were observed in the weights of kidneys in male animals and in the liver in female animals at 450 mg/kg bw/day in F1 Cohort 1A. The changes in kidneys weights were supported by histological findings (chronic progressive nephropathy) in male animals at 150 and 450 mg/kg bw/day. However, there were no accompanying histological lesions in the kidneys at the low dose administered male animals or in female animals at any dose level, therefore, elevation in the kidneys or liver weights may be interpreted as an adaptational phenomenon.
In the male animals at 50 mg/kg bw/day, the mean weights of kidneys exceeded the control and the mean weights of submandibular lymph nodes were lower than in the control (absolute and relative to body and brain weights, for both organs).
At 150 mg/kg bw/day, statistically significant difference with respect to the control was observed at the lower mean fasted body weight in males (absolute and relative to brain weight), at the higher mean weights of kidneys (relative to body and brain weights) and at the lower mean pituitary weights (absolute and relative to brain weight). The weight of thymus (absolute) was lower, while the weights of several organs relative to body weight was higher than in the control (brain, liver, spleen, testes, epididymides, thyroid glands) in consequence of the lowered mean body fasted weight of animals in this group.
At 450 mg/kg bw/day, lower mean fasted body weight (absolute and relative to brain weight), higher mean weights of kidneys (absolute and relative to body and brain weights) and liver (relative to body and brain weights), lower mean thymus weights (absolute and relative to brain weight) were observed in males when compared to their control.
In relation to the lower mean fasted body weight in male animals at 450 mg/kg bw/day, statistical significances with respect to the control was detected at the lower mean absolute organ weights (seminal vesicles, prostate, pituitary), at lower mean absolute weights along with higher body weight related mean organ weights (brain, heart, testes, epididymides) at higher mean organ weight relative to brain and body weights (heart, spleen, thyroid gland, popliteal lymph nodes and adrenal glands).
In the female animals at 50 mg/kg bw/day, the mean weights of liver (absolute and relative to body and brain weights) were elevated with respect to their control.
At 150 mg/kg bw/day, statistical significance with respect to the control was observed at lower mean fasted body weight, higher mean liver and heart weights (relative to body and brain weights, both organs), higher mean kidney weights relative to body weight and at the lower mean weight of ovaries (absolute).
At 450 mg/kg bw/day, statistical significance with respect to the control was observed at lower mean fasted body weight and brain weight (absolute for both organs), at higher mean liver and heart weights (absolute and relative to body and brain weights, both organs), at the lower mean weight of thymus and pituitary (absolute, both) in the female animals. The mean thyroid weight (relative to body weight), the mean weights of kidneys, ovaries, spleen and adrenal glands (relative to body and brain weight, each) also exceeded the control value in female animals at 450 mg/kg bw/day.
The most of statistically significant differences with respect to the control were due to the lowered body weight of male animals at 150 mg/kg bw/day and in male and female animals at 450 mg/kg bw/day as well as to the lowered brain weight in male and female animals at 450 mg/kg bw/day. Accompanying morphological changes were not detected at the histopathological examination and hematology investigations. Clinical chemistry parameters did not reveal test item-related abnormalities. Therefore, changes in these organ weights were judged to have little or no toxicological relevance due to the minor degree and in the lack of dose dependency (where relevant) or associated histopathological alterations.

The weights of the examined organs were not adversely affected in male and female animals at 50, 150 or 450 mg/kg bw/day in F1 Cohort 1B.

In the male animals, the weights of all examined organs were comparable with the control at 50 mg/kg bw/day. At 150 mg/kg bw/day, the mean fasted body weight and the mean thymus weight (absolute and relative to brain weight, both) were lower and the mean brain weight relative to body weight was higher than in the control. In the male animals at 450 mg/kg bw/day, lower mean fasted body weight (absolute and relative to brain weight), lower mean weights (absolute and relative to brain weight, each) of thymus, prostate and pituitary were observed when compared to the control. The mean weights (absolute) of brain, testes, epididymides, seminal vesicles were below the control and was higher than in the control if referred to body weight in case of brain, spleen and testes in male animals administered with high dose. In the female animals at 50 mg/kg bw/day, statistical significances with respect to the control was detected at the higher mean weights of uterus (absolute and relative to body and brain weights) and ovaries (absolute and relative to brain weight). At 150 mg/kg bw/day, elevated mean weights of spleen and ovaries both relative to body and brain weights were observed when compared to the control. Statistical significances with respect to the control was detected at the lower mean fasted body weight, at lower mean weights of brain and pituitary (absolute), at higher mean spleen weights (relative to body and brain weights) and higher mean weights of ovaries (absolute and relative to body and brain weights) in female animals at 450 mg/kg bw/day.
Minor changes in thymus weights in male animals at 150 and 450 mg/kg bw/day may be due to a slightly enhanced involution of the organ or to body and brain weight changes. The lowered fasted body weight and brain weight may result in statistical significances in the absolute and relative organ weights both in male and female animals (brain, spleen, testes, epididymides, seminal vesicles, prostate, pituitary, ovary, uterus). The values were close or within the historical control ranges. Therefore, these minor changes were considered to be toxicologically not relevant.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

There were no test item related specific macroscopic findings in offspring subjected to early necropsy (before weaning) or terminally (at the weaning).
Before the weaning, pups were subjected to necropsy because of death (stillborn and pups found dead) or after euthanasia on PND4 (surplus pups). Gas content (1/35) or milk (2/35) in the stomach at 50 mg/kg bw/day, autolyzed organs (2/43) at 150 mg/kg bw/day, pale body (4/47) and pale lungs (1/47), not cleaned pup with umbilical cord (1/47) at 450 mg/kg bw/day were detected. In offspring subjected to necropsy at weaning, both sided pyelectasia was observed in single pup at 150 mg/kg bw/day (1/69). Pyelectasia is a common finding in this strain of experimental rat with similar age.

Macroscopic alterations related to the effect of the test item were detected in the kidneys in male animals at 450 mg/kg bw/day in Cohort 1A (paleness, white area between the cortex and medulla) at the necropsy.
A test item effect may be supposed but cannot be proved with the results of this study in development of yellowish deposition on serous membrane of several organs in the thoracic cavity in male or female animals at 50, 150 or 450 mg/kg bw/day.
These necropsy observations were in full accordance with histopathological findings.
Male
-control: right or both sided pyelectasia (9/20)
-50 mg/kg bw/day: right or both sided pyelectasia (10/20), pale kidneys (1/20) yellowish deposition at bifurcation of the lungs (1/20), on lower part or right side of thymus (2/20) or on the muscle on larynx/ trachea (1/20)
-150 mg/kg bw/day: right or both sided pyelectasia (7/20); thickened bifurcation in lungs (1/20), brownish-red colored thymus (1/20), hard knot in the thymic tissue (1/20), hernia diaphragmatica with small part of liver (1/20 two peppers-sized) and yellow lentil sized subcutaneous formula at sternum (1/20);
-450 mg/kg bw/day: right or left sided pyelectasia (10/20), pale kidneys (5/20), white area between renal cortex and medulla (4/20), yellowish deposition on the lungs (1/20), thymus (1/20) and on ribs under lungs (1/20), adhesion of lungs to the diaphragm (1/20), hard knot in the thymic tissue (1/20), larger than normal kidneys (1/20)
Female
-control: right or both sided pyelectasia (5/20), slight, moderate or marked hydrometra (6/20);
-50 mg/kg bw/day: one or both sided pyelectasia (6/20), slight, moderate or marked hydrometra (7/20), adhesion of lungs to ribs and white granules on the surface (1/20), yellow deposit on the right upper side of thymus (1/20), white granules on the surface of diaphragm (1/20)
-150 mg/kg bw/day: right or both sided pyelectasia (10/20), slight, moderate or marked hydrometra (8/20), yellowish thick deposit on the lungs (1/20), brownish red colored thymus (1/20), greyish-yellow hard formation in the thymus (1/20), yellowish thick deposit on the wall of heart (1/20)
-450 mg/kg bw/day: right or both sided pyelectasia (4/20), slight, moderate or marked hydrometra (6/20), yellowish deposition in one (1/20, each) or two animals on the following organs: esophagus, trachea, thoracic aorta, thorax, thymus (2/20), adhesion of thoracic aorta to spine (1/20), adhesion of lungs to ribs (1/20) and diaphragm (1/20), brownish-red colored thymus (1/20), thickened spleen with rounded margins (1/20) and thickened thymus (left side, 1/20)
Renal alterations (paleness, white area between the cortex and medulla) in male animals and yellowish deposition on several organs in the thoracic cavity in male and female animals were in full accordance with histopathological findings. Some findings (adhesions, thickening, knots or different formations) on thoracic organs were consequences of accumulation of yellow or grayish yellow tissue. Regarding the incidence of these findings in groups, a test item influence cannot be verified or excluded with the results of this study. Yellowish deposition was only detected in test item administered animals independently from doses.
Pyelectasia and hydrometra are common macroscopic findings in experimental rats of this strain with similar age. Histological examination did not reveal degeneration, inflammation or fibrosis. Therefore, these findings were considered as individual lesion without toxicological significance. Hernia diaphragmatica were individual alterations and are considered to be also species-specific change and not treatment-related.
Splenic signs, brownish-red thymus and cyst near to sternum in individual animals were considered to be incidental individual findings.

Necropsy observations revealed test item-related alterations in the kidneys in male animals at 150 and 450 mg/kg bw/day in Cohort 1B. Yellow deposition and adhesion of organs mainly in the thoracic cavity were observed only in single animals at 50 and 450 mg/kg bw/day.
Dead animal
Brownish-red colored thymus and pea-sized erosion on the fundic part of the stomach were observed in dead female animal (1/1) at 150 mg/kg bw/day.
Surviving animals
In the male animals, necropsy observations revealed the following findings:
-control group: brownish-red thymus (1/20), right or both sided pyelectasia in the kidneys (8/20) and
-50 mg/kg bw/day: one or both sided pyelectasia in the kidneys (9/20)
-150 mg/kg bw/day: one or both sided pyelectasia in the kidneys (10/20), pale color of kidneys and white area between the renal cortex and medulla (1/20);
-450 mg/kg bw/day: right or both sided pyelectasia in the kidneys (10/20); pale color of kidneys (7/20), white area between the renal cortex and medulla (2/20) and swelling of kidneys (1/20), adhesion of right lung-lobe to heart and thymus by a yellowish deposition and thickened pericardium and adhesion to heart muscle (1/20), Hernia diaphragmatica with a part of liver (1/20), smaller than normal testes and epididymides (1/20)
In the female animals, necropsy observations revealed the following findings:
-control group: brownish-red thymus (1/20), one or both sided pyelectasia in kidneys, (4/20), slight, moderate or marked hydrometra (4/20)
-50 mg/kg bw/day: right or both sided pyelectasia in the kidneys (7/20), moderate or marked hydrometra (9/20), yellowish deposition on left lobe of thymus, on lungs, liver, spine and adhesion of liver to diaphragm in one animal (1/20)
-150 mg/kg bw/day: brownish-red thymus (1/19), both sided pyelectasia in the kidneys (6/19), slight, moderate or marked hydrometra (7/19), alopecia on the fore-limbs (1/19), pea-sized erosion on the fundic part of the stomach (1/19)
-450 mg/kg bw/day: right or both sided pyelectasia in the kidneys (5/20), slight, moderate or marked hydrometra (10/20), Hernia diaphragmatica with a part of liver (1/20),
Yellow deposition caused adhesion of organs in the thoracic cavity in one male animal at 450 mg/kg bw/day and in one female animal at 50 mg/kg/bw/day the latter extending to surface of liver.
Pyelectasia, hydrometra and alopecia are common findings in experimental rats of this strain and age. Hydrometra (i.e., dilatation of uterine horns with clear liquid content) related to the female sexual cycle, is a physiological phenomenon. In the lack of related inflammatory or other pathological signs, these findings were judged to be toxicologically not relevant and not test item-related as no dose response was noted.
Brownish-red colored thymus is also frequently observed in experimental rats in connection with the exsanguination procedure.
Hernia diaphragmatica is a developmental disorder occurring commonly in untreated experimental rats, too.
Erosion of the stomach mucosa and smaller than normal testes and epididymides (supported by organ weights) were considered to individual findings.

Histopathological findings:

effects observed, treatment-related

Description (incidence and severity):

Histopathological examinations revealed signs of chronic progressive nephropathy (distended tubules with hyaline casts, tubular basophilia or lymphocytic and histiocytic infiltration) in male animals at 150 and 450 mg/kg bw/day and accumulation of brown fatty tissue with mineral deposits in male and female animals at 50, 150 and 450 mg/kg bw/day in Cohort 1A.
The investigated organs of reproductive system (testes, epididymides, prostate seminal vesicles, coagulating glands, ovaries, uterus, vagina) were histologically normal and characteristic for the sexually mature organism in all of examined male and female animals in Cohort 1A.
Quantitative analysis of ovaries verified normal structure and function of the organ (primordial, primary, secondary and tertiary follicles, follicular atresia and corpora lutea) in the control and high dose treated animals in F1 Cohort 1A.
The following histological findings were detected in male and female animals per group:
Male animals:
-control group: one or both sided pyelectasia (9/20)
-50 mg/kg bw/day: one or both sided pyelectasia (8/20), accumulation of brown fatty tissue with mineral deposits (2/2)
-150 mg/kg bw/day: distended renal tubules with hyaline casts (6/20), one or both sided pyelectasia (7/20), accumulation of brown fatty tissue with mineral deposits (1/1), dermoid cyst at sternum region (1/1)
-450 mg/kg bw/day: distended renal tubules with hyaline casts (18/20), tubular basophilia (16/20), lymphocytic and histiocytic infiltration (14/20), one or both sided pyelectasia (11/20), accumulation of brown fatty tissue with mineral deposits (2/2)
Female animals:
-control group: one or both sided pyelectasia (6/20), dilatation of uterine horns (6/20)
-50 mg/kg bw/day: one or both sided pyelectasia (5/5), accumulation of brown fatty tissue with mineral deposits (2/2), dilatation of uterine horns (6/6)
-150 mg/kg bw/day: one or both sided pyelectasia (10/10), accumulation of brown fatty tissue with mineral deposits (2/2), dilatation of uterine horns (8/8)
-450 mg/kg bw/day: one or both sided pyelectasia (3/20), accumulation of brown fatty tissue with mineral deposits (3/3), splenic hyperplasia (1/20), thymic congestion (1/20), dilatation of uterine horns (6/20)
Quantitative analysis of ovaries in animals of F1 Cohort 1A verified normal structure and function of the organ in the control and high dose treated animals. The mean number of primordial and primary follicles and corpora lutea was similar in the control and at 450 mg/kg bw/day. Slight but statistically significant difference with respect to the control was detected at the slightly lowered mean number of secondary and tertiary follicles and follicular atresia at 450 mg/kg bw/day.
These findings were considered to be variation and not related to the test item. Individual values were comparable in the control and high dose treated animals.
theThe histological structure and the cellularity of pituitary with special attention on the cytomorphology and proportion of acidophilic and basophilic cells in the adenohypophysis were the same in the control and treated male and female animals.
The severity and incidence of renal lesions (distended tubules with hyaline casts, tubular basophilia, lymphocytic and histiocytic infiltration, characteristic on the chronic progressive nephropathy (CPN) in the rat) were related to doses of 150 and 450 mg/kg bw/day in male animals and were not present in male animals in control or low dose group or in female animals at any dose level in F1 Cohort 1A. CPN is a spontaneous renal disease of laboratory rat with similar age, therefore 150 and 450 mg/kg bw/day doses of test item were assumed to be a predisposing/ accelerating factor in the development of pathogenesis of CPN in male animals. Accumulation of brown fatty tissue with mineral deposits were detected in some male and female animals in each dose group independently from doses with different localization, mainly on the surface of serous membranes of thoracic organs (thymus, lung, pharynx, thoracic pleura, sternum, diaphragm, bifurcation region of the lungs). The appearance was demarcated from the surrounding organs or tissues, and the accumulated adipose tissue was demarcated with serous membrane as well. The structure was built by brown fat cells, smaller than white fat cells, and the intercellular connective tissue consisted of fibrocytes, collagen and reticular fibers and capillaries. Scattered basophilic mineral deposits occurred in the intercellular connective tissue. Mononuclear cells and separately giant cells were seen around the deposits. No necrosis, fibrosis, abscess formation or malignant tumor cells with high mitotic activity were observed. The focal accumulation of brown adipose tissue with mineral deposits – regarding its macroscopic appearance, and the presence of mineral deposits, the mononuclear cell infiltrates and the giant cells, - is considered as a pathophysiologic phenomenon, in contrast with the normal brown adipose tissue. The pathogenesis of this phenomenon may be accidental, but the predisposing role of test item cannot be excluded in the development of this lesion. Dilatation of uterine horns is a slight neuro-hormonal phenomenon in connection with sexual function – pro-estrous phase – of the inner genital organs. Pyelectasia without significant histological lesions (inflammation, degeneration, fibrosis etc.) is common in laboratory rats of this strain and is considered to be an individual disorder without pathological significance.
Some spontaneous individual disorders were also detected at the histopathology investigation – splenic hyperplasia, thymic congestion – which are well-known lesions and commonly observed in experimental rats of this strain and not related to the test item: There was no morphological evidence of test item-related acute or subacute injury (degeneration, inflammation, necrosis etc.) in the small and large intestines, liver, pancreas, cardiovascular system, respiratory system, immune system, hematopoietic system, skeleton, muscular system, central, or peripheral nervous system, eyes, integumentary system. The cytomorphology of endocrine glands were the same in the control and treated animals.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Estrous Cycle: The estrous cycle was not affected in the F1 Cohort 1A female animals at 50, 150 or 450 mg/kg bw/day.The examined parameters of the estrous cycle – percentage of animals with regular or irregular cycles, number of cycles, length of cycles, number of days in pro-estrous, percentage of animals in prolonged estrous/ diestrus – were comparable in the control and 50, 150 or 450 mg/kg bw/day groups. The mean number of irregular cycles was slightly higher in the 450 mg/kg bw/day group but this value lies within the historical control data. The mean number of days in estrous was slightly lower and the mean number of days in diestrus was slightly higher than in the control group at 450 mg/kg bw/day. These minor differences from control were judged to be biological variation and lie in the range of the historical control data and independent from the test item.
The estrous cycle was not adversely affected in the F1 Cohort 1B female animals at 50, 150 or 450 mg/kg bw/day.Statistical significances were observed at the lower mean number of estrous cycle at 150 and 450 mg/kg bw/day and at the lower mean number of days in estrous in female animals 150 mg/kg bw/day when compared to control. These minor differences with respect to the control in the mid and high dose groups were independent from doses and were considered to be indicative of biological variation as values were close or within the historical control. Therefore, these changes have no toxicological relevance.

Sperm Examinations: Sperm examinations did not reveal any test item-related influence on the sperm cells at 450 mg/kg bw/day.
The mean percentage of motile, immotile sperms, sperms with normal morphology and separated head and tail were comparable in the control and 450 mg/kg bw/day groups. Since no effects have been observed, the low and mid dose groups were not analyzed.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

effects observed, non-treatment-related

Description (incidence and severity):

Splenic Lymphocyte Subpopulation Analysis: There were no test item-related changes in the splenic lymphocyte subpopulations in male or female animals in F1 Cohort 1A animals in 50, 150 or 450 mg/kg bw/day groups. The percentage of T lymphocytes (T cells), B lymphocytes (B cells), Natural killer (NK) cells, helper T cells and cytotoxic cells were predominantly comparable in male and female animals in the control, 50, 150 and 450 mg/kg bw/day groups. Statistical significance with respect to the control was only detected at the slightly elevated mean number of NK cells in male animals at 450 g/kg bw/day.
This minor change was considered to be indicative of biological variation as all mean values met well or were close to the historical control ranges. Some outlier individual vales (1-3 animals/ group) were probably individual variations.

Cohorte 3: No anti-IgM antigen peak response after immunization with KLH (Keyhole limpet hemocyanin) was detected on day 6 in the control animals due to technical reasons. A validation study was performed for the ELISA Assay (Study number 392-504-6274), which showed that the assay as such detected standard rat KLH IgM according to the established protocol. The same ELISA protocol was followed for the rat samples in the main study. In a pre-liminary study the antibody peak was observed 6 days after subcutaneous immunization in Han:WIST rats (no report available). A different batch of KLH was used for immunization in the main study for Cohort 3 animals, which might be the reason for the non-response which
was observed.

Details on results (F1)

The F1 offspring’s development (pinna detachment, eye opening, body weight on PND4) was depressed at 450 mg/kg bw/day.

Effect levels (F1)

1

Key result

Dose descriptor:

NOAEL

Remarks:

development

Generation:

F1

Effect level:

150 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

other: pinna detachment

2

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Generation:

F1

Effect level:

150 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

3

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Generation:

F1

Effect level:

50 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Target system / organ toxicity (F1)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

150 mg/kg bw/day (actual dose received)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

The main study according to OECD TG 443 was performed in Han:Wist rats including Cohort 3 (ECHA request, CCH-D-2114493102-56-01/F). Based on the results of the pre-study the parent (P) generation was dosed 10 weeks prior to mating with dosages of 50, 150 or 450 mg/kg bw/day. The reproductive performance was not impaired in male or female rats after administration of TBPND via oral gavage. No treatment-related changes were noted in any of the reproductive parameters investigated (i.e. mating and fertility indices, sperm parameters, precoital time, number of implantations, estrous cycle). Test-item related systemic effects in the body weight, food consumption and in the kidneys- related parameters (urine and clinical chemistry, organ weights, macroscopic and microscopic findings) were observed in high dose parental male animals. Signs of systemic effect manifested in changes and of clinical chemistry parameters and increased liver weight in parental female animals in the high dose group.
In F1 offspring (pre-weaning), depressed development (pinna detachment, eye opening, body weight) were observed at 450 mg/kg bw/day. No treatment-related effect on anogenital distance, areola/nipple retention, surface righting reflex and thyroid hormone levels (FT4 and TSH in PND 22 pups) were detected in F1 offspring.
In F1 adult (F1 Cohort 1A, Cohort 1B, Cohort 3, post-weaning) at 450 mg/kg bw/day reduction in body weight and food consumption (Cohorts 1A, 1B and 3) in male or female animals and kidneys related parameters (urine and clinical chemistry parameters, organ weight, macroscopic and microscopic findings) in male animals of F1 generation (Cohorts 1A) was detected. No treatment-related effects were recorded for developmental parameters in F1-animals, including balano-preputial separation, vaginal opening, occurrence of first estrus, sperm parameters, ovarian follicle counts and histopathology of reproductive organs.
At 150 mg/kg bw/day of male animals in F1 generation (Cohorts 1A and B), body weight and kidney-related findings were observed in a lesser degree. No test item-related adverse effects were detected at 50 mg/kg bw/day in F1 adults (Cohorts 1A, 1B and 3).
No treatment-related immunotoxic effects have been detected since no histopathology findings in the lymphoid organs, no changes in hematological parameters and no test item-related changes in splenic lymphocyte subpopulations (Cohort 1A) were noted.
Based on these observations the NOAELs were determined as follows:
NOAEL for systemic toxicity (P/ F1 adult male rats): 50 mg/kg bw/day
NOAEL for systemic toxicity (P/ F1 adult female rats): 150 mg/kg bw/day
NOAEL for reproductive performance (male and female rats): 450 mg/kg bw/day
NOAEL for F1 Offspring development: 150 mg/kg bw/day

Executive summary:

The subject of this study was to investigate the reproductive toxicity of TBPND by conducting the Extended One-Generation Reproduction Toxicity Study in the rat according to OECD 443 as requested by the European Chemical Agency (ECHA). The guideline is designed for using the rat, which is the preferred rodent species for reproduction toxicity testing. The aim of the extended one-generation reproduction toxicity study was to provide an evaluation of the pre- and post-natal effects of the test item TBPND on development as well as a thorough evaluation of systemic toxicity in male and female animals, in pregnant and lactating females and in young and adult offspring when repeatedly administered via oral gavage to animals at doses of 50, 150 and 450 mg/kg bw/day compared to control animals. The effect of the test item on the male and female reproductive performance, such as gonadal function, estrous cycle, mating behavior, conception, parturition, gestation, lactation and weaning (P generation) was investigated. Furthermore, the effect on offspring viability, neonatal health and mortality, growth and development of the offspring (F1 generation; Cohort 1A, 1B and Cohort 3) to adulthood following oral (by gavage) administration was the main focus of this study. Four groups of Han:WIST rats (n= 26/sex/group) were administered with the test item via oral gavage once a day at 0 (vehicle), 50, 150 and 450 mg/kg bw/day doses corresponding to concentrations of 0, 25, 75 and 225 mg/mL in parental generation in a volume of 2 mL. Control animals received only vehicle, sunflower oil, in an identical manner. The suitability of the vehicle at the intended concentrations of the test item was analytically verified up front (concentration and homogeneity). Analysis of formulations used for administration were performed six times during the study. TBPND concentrations in the formulations varied within the range of 98 % and 109 % in comparison to the nominal values and samples were homogenous.

All animals of the parent (P) generation were dosed for 10 weeks prior to mating and throughout mating. In addition, males received the test item or vehicle after mating up to the day before the necropsy (altogether for 112, 113 or 114 days). Dams were exposed through the mating and gestation periods and at least up to lactation day 21 (altogether for 114-125 days). Not delivered, not mated and non-pregnant females were administered for 99 or 104 days.

Clinical observations (clinical signs, body weight, food consumption, estrous cycle) and pathology (clinical and organ pathology) examinations were performed on parental (P) animals for signs of toxicity, with special emphasis on the integrity and performance of the male and female reproductive systems. Clinical pathology examinations - urinalysis, hematology, blood coagulation, clinical chemistry - were conducted in randomly selected ten male and ten female animals from each group (parental animals and Cohort 1A). Estrous cycle was monitored in parental animals by examining vaginal smears before the mating for two weeks and during the mating period until evidence of copulation and on the day of the necropsy. The dams were allowed to litter and rear their offspring up to day 21 post-partum. All F1 offspring were observed individually for health, growth, development and function up to and including post-natal day 21 (clinical signs, body weight, surface righting reflex, pinna detachment, eye opening, anogenital distance, nipple retention). Twenty F1 animals/sex/group were randomly selected for Cohort 1A, Cohort 1B and Cohort 3 in the control, low, mid and high dose groups on post-natal day 21 for follow-up examinations. Dosing of F1 offspring selected for follow-up examinations begun on post-natal day 22 and treatment was continued up to the day before the necropsy. F1 adult animals in Cohort 1A and Cohort 1B were observed identically to parental animals – clinical signs, body weight, food consumption, estrous cycle, clinical pathology (Cohort 1A only) and organ pathology. Sexual maturity of offspring was investigated by observation of balano-preputial separation, vaginal patency (Cohort 1A, Cohort 1B, Cohort 3) and appearance of first cornified vaginal smear (Cohort 1A).

Cohort 1A animals were subjected to necropsy, organ weighing, sperm analysis and immunotoxic examinations – one day after the termination of the exposure – on PND92-98.

Cohort 1B animals were subjected to necropsy and organ weighing on PND97-106.

Cohort 3 animals selected for determination of primary IgM antibody response to a T-cell dependent antigen were administered and observed up to and including the day before blood sampling and euthanasia on PND61. Blood samples were collected for determination of serum levels of thyroid hormones (FT3, FT4 and TSH) from 10 parent (P, male and female) animals/sex/ group at termination, in surplus pups at PND4 (pooled by litters), from F1 pups not selected for cohorts on PND22 and from 10 adult F1 Cohort 1A male and female animals/group at termination. Thyroid hormone levels were determined  in adult animals (P, F1 Cohort 1A) and in PND22 F1 offspring. All adult animals (P, F1 Cohort 1A and Cohort 1B) were subjected to gross pathology with complete tissue preservation one day after the last treatment. Brain, spleen, thymus and mammary tissues were preserved for 10 male and 10 female pups per group – where feasible – in F1 offspring not selected for Cohorts on PND22 or shortly thereafter. Special attention was paid to the organs and tissues of the reproductive system for P or F1 animals at necropsy. Selected organ weights were determined in adult animals (P, F1). Sperm parameters were determined in all control and high dose male animals in P generation and in all control and high dose male animals F1 generation (Cohort 1A). At termination, weighing of the lymph nodes and splenic lymphocyte subpopulation analysis was performed in 10 male and 10 female Cohort 1A animals from each group. Full histopathology examinations were performed on the organs and tissues of adult animals (P, F1 Cohort 1A) in control and high dose groups with special emphasis on sexual organs and tissues. The kidneys were also examined histologically in all parental and F1 Cohort 1A male animals (control, low, mid and high dose group) on the basis of necropsy observation. In addition, organs showing macroscopic changes were also processed and examined histologically in adult animals in low or mid dose groups (P, F1 Cohort 1A).Reproductive organs were also processed and examined histologically in non-mated and non-pregnant female animals and their mating partners in the low and mid dose groups. A quantitative evaluation of follicles (primordial, primary, secondary and tertiary follicles, follicular atresia) as well as corpora lutea was performed in F1 Cohort 1A female animals in the control and 450 mg/kg bw/day groups. The results were interpreted comparing treatment groups with respect to controls, which were treated concurrently with vehicle (sunflower oil) only.

 

Results

Analytical control of dosing formulations

TBPND concentrations in the samples varied within the range of 98 % to 109 % in comparison to the nominal values and formulations were homogenous thereby confirming the proper dosing of parental animals (P) and F1 generations.

Parental (P) generation

Mortality

There was no test item related fatal death in parental animals at any dose level (50, 150 or 450 mg/kg bw/day) during the course of the observation period.

One male animal at 450 mg/kg bw/day was found dead presumably due to suffocation and shock on Day 64.

Clinical observation

Clinical signs of systemic toxicity related to the test item were not detected at any dose level at the daily clinical observations (50, 150 or 450 mg/kg bw/day). Salivation followed test item administration was observed in several male and in some female animals at 450 mg/kg bw/day with variable incidence and with short duration. Therefore, salivation was judged to be toxicologically not relevant in this study.

The behavior and physical condition of all male and female animals was normal in each group at the detailed weekly observations.

Body weight and body weight gain

The body weight development was depressed in a dose related manner in parental male animals at 150 and 450 mg/kg bw/day during the entire treatment period. The body weight reduction was less than 5 % relative to control in male animals at 150 mg/kg bw/day, therefore, was judged to be toxicologically not relevant.

Food consumption

The mean daily food consumption was not adversely affected in parental male or female animals at 50, 150 or 450 mg/kg bw/day during the entire treatment period.

Estrous cycle

The estrous cycle was similar in the control and 50, 150 and 450 mg/kg bw/day groups during the two weeks observation period.

Delivery data of dams

There were no toxicologically relevant differences in the evaluated parameters of pregnancy and delivery in female animals between the control and 50, 150 or 450 mg/kg bw/day groups.

Reproductive performance

The reproductive performance of male and female animals was normal (the same as in not treated animals of this strain of experimental animals) in control, 50, 150 and 450 mg/kg bw/day groups.

Urinalysis

Urinalysis revealed changes in male urine (pH, protein and ketone body), which were probably related to renal tubular changes in male animals at 150 and 450 mg/kg bw/day.

Hematology and blood coagulation

There were no toxic changes in the examined hematology and blood coagulation or clinical pathology parameters in parental male or female animals at 50, 150 or 450 mg/kg bw/day.

Clinical chemistry

Minor changes in creatinine (male) and cholesterol (female) concentrations at 150 mg/kg as well as elevated activity of alanine aminotransferase (male and female), higher concentrations in creatinine (male), cholesterol (female) and urea (male and female) at 450 mg/kg bw/day referred to a test item influence on renal and/or hepatic function.

Thyroid hormones

The serum thyroid hormone (FT3, FT4 and TSH) levels were not adversely influenced in the parental male or female animals or in PND22 F1 offspring at any dose levels.

Necropsy

Gross necropsy observations revealed test item related changes in the kidneys (pale, enlarged, soft) in majority of parental male animals at 450 mg/kg bw/day.

The findings of dead animal refer to systemic changes caused by suffocation and acute stress supported by histopathological findings.

Organ weight

Elevated weights of kidneys in male animals refer to test item influence in accordance with macroscopic or histopathology findings at 450 mg/kg bw/day and in a lesser degree at 150 mg/kg bw/day. Test item related changes were observed in the elevated weights of the liver in female animals at 450 mg/kg bw/day.

Sperm examinations

Sperm examinations did not reveal any test item-related damage in the sperm cell morphology and motility, and total sperm count in parental male animals at 450 mg/kg bw/day.

Histopathology

The cell morphology of examined reproductive organs (testes, epididymides, prostate, seminal vesicles, coagulating glands, ovaries, oviduct, uterus cervix, vagina) were histologically normal and characteristic of the sexually mature organism in all parental animals in control and 450 mg/kg bw/day including not mated, non-pregnant and not delivered female animals and their mating partners and in mid dose, too. Test item related renal changes – distended tubules with hyaline casts, tubular basophilia, lymphocytic and histiocytic infiltration, characteristic on the chronic progressive nephropathy (CPN) in the rat – were detected only in male animals at 150 and 450 mg/kg bw/day (50 and 100% of examined animals, respectively) in a dose related manner.

Offspring

The F1 offspring’s development (pinna detachment, eye opening, body weight) was depressed at 450 mg/kg bw/day while the mortality, sex distribution, clinical signs, surface righting reflex, anogenital distance, necropsy findings and organ weight were comparable to their control at 50, 150 and 450 mg/kg bw/day.

F1 adult generation (Cohort 1A, Cohort 1B and Cohort 3)

Mortality

There was no test item related mortality in F1 Cohort 1A, Cohort 1B or Cohort 3 animals in control, 50, 150 or 450 mg/kg bw/day groups during the course of study (male and female).

In F1 Cohort 1B, one female animal at 150 mg/kg bw/day (1/20) died due to a probably mis-gavage during the administration with gastric tube on post-natal day 76.

Clinical observation

The behavior and physical condition of all animals was normal at each dose level (50, 150 or 450 mg/kg bw/day) in F1 Cohort 1A, Cohort 1B and Cohort 3 based on the daily and weekly detailed clinical observations during the entire treatment period.

Body weight and body weight gain

The body weight development was depressed in F1 Cohort 1A, Cohort 1B and Cohort 3 (male and female animals) at 450 mg/kg bw/day during the entire treatment period.

Food consumption

The mean daily food consumption of male animals was reduced at 450 mg/kg bw/day F1 Cohort 1A, Cohort 1B Cohort 3.

Sexual maturity

The sexual maturity was similar to control in male or female animals in F1 Cohort 1A, 1B and Cohort 3 at 50, 150 and 450 mg/kg bw/day.

Estrous cycle

The estrous cycle was comparable with their control in the F1 Cohort 1A, and Cohort 1B female animals at 50, 150 and 450 mg/kg bw/day.

Urinalysis

The pH of urine was slightly lowered and ketone bodies were present in urine in male animals at 150 and 450 mg/kg bw/day presumably in accordance with renal damage revealed by histological investigations in F1 Cohort 1A.

Hematology and blood coagulation, clinical chemistry

Pathologic alterations were not detected at the evaluation of hematology and blood coagulation parameters in F1 Cohort 1A male or female animals at 50, 150 or 450 mg/kg bw/day.

Clinical chemistry

A slightly higher mean creatinine level may be related to the renal changes in male animals at 450 mg/kg bw/day in F1 Cohort 1A.

Thyroid hormones

The thyroid hormone (FT3, FT4 and TSH) levels were not adversely affected in male or female animals in the F1 Cohort 1A at 50, 150 or 450 mg/kg bw/day.

Necropsy

Macroscopic alterations related to the effect of the test item were detected in the kidneys in male animals at 450 mg/kg bw/day in Cohort 1A, Cohort 1B (paleness, white area between the cortex and medulla) at the necropsy.

A test item effect may be supposed in development of yellowish deposition on serous membrane of several organs in the thoracic cavity in some male or female animals at 50, 150 or 450 mg/kg bw/day in F1 Cohort 1A and Cohort 1B.

Organ weight

Test item related changes were observed in the weights of kidneys in male animals and in the liver in female animals at 450 mg/kg bw/day in F1 Cohort 1A. The weights of the examined organs were not adversely affected in male or female animals at any dose level in F1 Cohort 1B.

Sperm examinations

Sperm morphology and motility, and total sperm count were similar to their control in male animals at 450 mg/kg bw/day in F1 Cohort 1A.

Histopathology

Histopathological examinations revealed chronic progressive nephropathy (distended tubules with hyaline casts, tubular basophilia or lymphocytic and histiocytic infiltration) in male animals at 150 and 450 mg/kg bw/day and accumulation of brown fatty tissue with mineral deposits in male and female animals at 50, 150 and 450 mg/kg bw/day in Cohort 1A.

Quantitative analysis of ovaries in animals of F1 Cohort 1A verified normal structure and function of the organ in the control and high dose treated animals.

Splenic lymphocyte subpopulation analysis

There were no test item-related changes in the splenic lymphocyte subpopulations in male or female animals in F1 Cohort 1A animals in 50, 150 or 450 mg/kg bw/day groups.

Developmental immunotoxicity testing

No anti-IgM antigen peak response after immunization was detected on day 6 in the control animals due to technical reasons. A validation study was performed for the ELISA Assay, which showed that the assay as such detected standard rat KLH IgM according to the established protocol. The same ELISA protocol was followed for the rat samples in the main study. In a pre-liminary study the antibody peak was observed 6 days after subcutaneous immunization in Han:WIST rats (no report available). A different batch of KLH was used for immunization in the main study for Cohort 3 animals, which might be the reason for the non-response which was observed. No treatment-related immunotoxic effects have been detected since no histopathology findings in the lymphoid organs, no changes in hematological parameters and no test item-related changes in splenic lymphocyte subpopulations (Cohort 1A) were noted.

Based on these observations the NOAELs were determined as follows:
NOAEL for systemic toxicity (P/ F1 adult male rats): 50 mg/kg bw/day
NOAEL for systemic toxicity (P/ F1 adult female rats): 150 mg/kg bw/day
NOAEL for reproductive performance (male and female rats): 450 mg/kg bw/day
NOAEL for F1 Offspring development: 150 mg/kg bw/day