tert-butyl 3,5,5-trimethylperoxyhexanoate

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

A reproduction/ developmental toxicity screening test was performed in 2010 with TBPIN according to OECD TG 421. The following NOAELs were obtained: 50 mg/kg bw/day for maternal toxicity (parent generation) and for offspring generation and for reproductive performance 400 mg/kg bw/day (male) and 50 mg/kg bw/day (female).

A modified study according OECD TG 421 in rats was performed in 2021 with TBPIN, in which the parent (P) generation was dosed 10 weeks prior to mating. For the peroxyesters TBPND (CAS 26748-41-4) and TBPPI (CAS 927-07-1) the same modified design was used in two additional OECD TG 421 studies (2021). The rational was to compare the three peroxyesters regarding effects on reproductive performance after 10 weeks pre-mating exposure and to conclude on the possibility to perform only one subsequent OECD TG 443 study for all three peroxyesters (grouping approach). This 10-weeks pre-mating exposure is recommended in the OECD TG 443 study design. All three substances did not show any adverse effects on the parameters of reproductive performance.

Based on this results, only one study according OECD TG 443 with TBPND was initiated for the peroxyester group. This substance was chosen since ECHA requested for TBPND to include Cohort 3 (immunotoxicity, CCH-D-2114493102-56-01/F). The study results from this OECD TG 443 study will be used in a read-across grouping approach for the peroxyester TBPIN to fulfil ECHAs requests on an EOGRT study (CCH-D-2114493103-54-01/F). This approach was also chosen because of animal welfare reasons.

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 adverse 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. However, this is considered secondary to maternal toxicity and as a nonspecific effect. 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

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

25 June 2018

Deviations:

no

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)

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

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

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

150 mg/kg bw/day (nominal)

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

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

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: 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.

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

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

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

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

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.

Behaviour (functional findings):

not examined

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.

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

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

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

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

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

Key result

Reproductive effects observed:

no

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

Reference 2

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2021-05-19 to 2021-10-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

29th July 2016

Deviations:

yes

Remarks:

10 weeks pre-treatment, no hematology and clinical biochemistry measurements, histopathology only performed for ovaries

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Han:WIST

Details on species / strain selection:

The rat is regarded as suitable species for toxicity and reproduction toxicity studies and the test guidelines were designed to use the rat. The Wistar rat was selected due to a wide range of experience with this strain of rat in toxicity and 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. 1103 Budapest, Hungary
- Females nulliparous and non-pregnant: yes
- Age at study initiation:
Male animals: 38 - 43 days old
Female animals: 33 - 36 days old
- Weight at study initiation:
Male animals: 159 – 178 g
Female animals: 102 – 131 g
- Fasting period before study: no
- Housing: Type III polypropylene/polycarbonate cages
Before mating: 2 animals of the same sex/cage
Mating hours: 1 male and 1 female/cage
Mated females were housed individually
Males after mating: 2 animals/cage
- Diet: ad libitum, ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice, Spezialdiäten GmbH, D-59494 Soest, Germany
- Water, ad libitum, tap water
- Acclimation period: 5 days

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

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 vehicle in concentrations of 50 mg/mL, 125 mg/mL and 175 mg/mL in the formulation laboratory of the Test Facility not longer than for three days before the use. Analysis of formulations was performed in the Analytical Laboratory of Test Facility three times during the treatment period.

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: 50, 125, 175 mg/mL
- Amount of vehicle: 2 mL

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: Females were cohabited with the same male until copulation occurred (10 days for all pairs)
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged: Sperm positive females were caged individually.
- Any other deviations from standard protocol: no

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of dosing formulations was between the range of 90 and 108 % of the nominal concentrations. A sufficient stability and homogeneity in the chosen vehicle were verified over the range of relevant concentrations at the appropriate frequency of preparation. TBPPI-75-AL in sunflower oil was stable at room temperature for 24 hours (recovery: 98 % at nominal concentrations of ca. 1 mg/mL and ca. 500 mg/mL, both) and in a refrigerator (5 ± 3 °C) for 3 days (recovery: 105 and 103 % at nominal concentrations of ca. 1 mg/mL and ca. 500 mg/mL, respectively).

Duration of treatment / exposure:

All animals of the parent (P) generation were dosed prior to mating (70 days) and throughout mating. In addition, males received the test item or vehicle after mating up to the day before the necropsy (altogether for 100 days). Dams were additionally exposed through the gestation period and up to lactation days 21-23 (altogether for 114, 115, 118 or 125 days). Non-pregnant female animals and dam not delivered were administered for 95, 97, 98 or 102 days.

Frequency of treatment:

daily

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose setting with 100, 250 and 350 mg/kg bw/day is based on results obtained in previous studies performed with TBPPI-75-AL (14-Day Oral Gavage Dose Range Finding Study with TBPPI-75-AL in the Rat; Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test with TBPPI-75-AL in the Rat).

Positive control:

none

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All parental animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each day).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a day, after the treatment at approximately the same time
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 was 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), twice a week for four weeks and weekly thereafter and on the day of the necropsy. Parental females were weighed on the first day of dosing (Day 0), twice a week for four weeks and weekly thereafter, then on gestation days 0, 7, 14 and 21 and on days 0 (within 24 hours after parturition), 4, 7, 14 and 21 post-partum. Additionally, female animals were weighed on gestational day 10 in order to give accurate treatment volumes

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- The food consumption of parent animals was determined weekly by weighing the given and non-consumed diet by weekly interval with an accuracy of 1 g during the treatment period except mating phase: during the premating period and during post-mating period for male animals; premating period, gestation days 0, 7, 14 and 21, lactation days 0, 7, 14 and 21 for female animals.

WATER CONSUMPTION AND COMPOUND INTAKE: No

DETERMINATION OF THYROID HORMONES:
Blood samples were collected for determination of serum levels of thyroid hormones (FT4, TSH) as follows:
- from all dams on lactation/ post-natal day 22;
- from all parent male animals at termination on Day 100;
- non-pregnant female animals and not delivered dam at termination, on Days 95-102;

Oestrous cyclicity (parental animals):

Estrous cycle was monitored by examining vaginal smears daily before the mating started from each animal for two weeks and from the beginning of the mating period until evidence of copulation. Vaginal smear was also prepared on the day of the necropsy. Vaginal smears were stained with 1 % aqueous methylene blue solution and examined with a light microscope after drying.

Sperm parameters (parental animals):

Parameters examined in male parental generation:
testis weight, epididymis weight, weight of prostate and seminal vesicles with coagulating glands, spermatogenesis, histopathology

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

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, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities

On the day of birth, pups found dead were subjected to a lung flotation test to differentiate pups died intrauterine (stillborn; negative lung flotation test) from pups died after the birth (dead pups; positive lung flotation test).

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: no

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no

DETERMINATION OF THYROID HORMONES:
Blood samples were collected for determination of serum levels of thyroid hormones (FT4, TSH) as
follows:
- from 2-5 pups per litter on post-natal day 4 (except for litters with 9 or less pups)
- from 3-6 pups per litter on lactation/ post-natal day 22;
Blood samples were collected from the surplus pups (at least two pups per litter), pooled and used for determination of serum FT4 and TSH levels on post-natal days 4 and 22.

OTHER:
One or two male and one or two female pups per litter were selected on post-natal day 21 and were identified by individual numbers written with a marker pen on the tail, were caged (2-3 pups/cage) and were administered (2 mL/kg bw/day) from postnatal day 22 up to and including post-natal day 35. Clinical signs were observed daily after the treatment and body weight were determined twice weekly and were recorded. The food consumption was determined on post-natal day 22, 29 and 35. All pups were subjected to macroscopic necropsy observation on post-natal day 36.

Postmortem examinations (parental animals):

SACRIFICE
Gross necropsy was performed on each parental animal one day after the last treatment. Animals were euthanized by exsanguination after verification of Isofluran-narcosis and were subjected to gross necropsy as follows:
- Parental male animals: on Day 100;
- Dams: on post-partum days 22, 23 or 24 (on Days 114-125);
- Non-pregnant (but mated) female animals and not delivered dam on Days 95, 97, 98 or 102

GROSS NECROPSY
- After examination of the external appearance, the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed, and any abnormality was recorded including details of the location, color, shape and size. Special attention was paid to the organs of the reproductive system.

HISTOPATHOLOGY / ORGAN WEIGHTS
At the time of termination, body weight, brain weight and weight of the testes and epididymides as well as prostate and seminal vesicles with coagulating glands as a whole of adult male animals were determined. Paired organs were weighed together. The thyroid weight was not determined because there were no test item related changes in the hypothalamic-pituitary-thyroid function at the thyroid hormone examinations. Histopathological processing and quantitative examinations of the ovaries were performed in female animals in the control and high dose groups. Follicular enumeration (primordial and small growing follicles – primary, secondary, tertiary follicles, corpora lutea, atresia) was performed in the largest surface section of the ovaries. The fixed ovaries 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.

Postmortem examinations (offspring):

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

HISTOPATHOLOGY / ORGAN WEIGTHS: Not determined

Statistics:

The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was 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 inter-group differences. Getting significant result 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:

See table in "Any other information on materials and methods".

Offspring viability indices:

See table in "Any other information on materials and methods".

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no adverse test item related clinical signs in any group, i.e. parental animals (male and female) exhibited normal behavior and physical condition with no abnormalities in the control and at 100, 250 or 350 mg/kg bw/day at the daily clinical observations.
Salivation and nuzzling up the bedding material were observed in all male and female animals at 250 and 350 mg/kg bw/day (12/12, each) from the second/ third week of the study. Both these signs appeared shortly after the treatment and ceased after short duration. Therefore, these signs were judged to be toxicologically not relevant. There were no clinical signs in male or female animals in the control and 100 mg/kg bw/day groups during the entire observation period. In the male animals, salivation and nuzzling up the bedding material were observed at 250 and 350 mg/kg bw/day with variable occurrence from Day 12 onward up to the termination of the study (Day 99). Similarly, salivation and nuzzling up the bedding material were noted for most of female animals at 250 and 350 mg/kg bw/day during the premating, mating, gestation and lactation periods with variable occurrence. Exophthalmos as individual sign was detected in one dam at 250 mg/kg bw/day from gestation day 2 up to the end of the study (L21).

Mortality:

no mortality observed

Description (incidence):

There was no test item related mortality at 100, 250 or 350 mg/kg bw/day
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 was reduced in male animals at 250 and 350 mg/kg bw/day. The difference with respect to the control was less than 10 % at 250 mg/kg bw/day therefore, this slight change was judged to be toxicologically not relevant. The mean body weight was comparable in the male animals in the control and 100 mg/kg bw/day groups on each measuring day from Day 0 up to
and including Day 97. Statistical significance with respect to the control was observed at the lower mean body weight gain of low dose treated male animals between Days 76 and 83. At 250 mg/kg bw/day, statistical significance with respect to the control was detected at the slightly lower mean body weight on Days 56, 76, 83, 90, and 97, as well as at the lower mean body weight gain between Days 24-28, 35-42, 42-49, 49-56 and for the study overall (between Days 0 and 97) in male animals. Compared to their control, statistical significance was detected at the lower mean body weight on Days 28, 35 and from Day 49 up to the termination of the study (Day 97) and at the lower mean body weight gain between Days 28-35, 42-49, 49-56, 56-63, 76-83 and for the study overall in male
animals at 350 mg/kg bw/day. In the female animals at 100 mg/kg bw/day, the mean body weight exceeded the control during the pre-mating, gestation and lactation periods (with statistical significance on Day 14 and from Day 21 onwards by weekly interval during pre-mating period and during the entire gestation and lactation periods). In the female animals at 250 and 350 mg/kg bw/day, the mean body weight and body weight gain were comparable to their control during the entire study except for the slightly lower mean body weight gain at 250 mg/kg bw/day between Day 35 and 42. The statistically significant differences with respect to the control (in male and female animals at 100 mg/kg bw/day and in female animals at 250 mg/kg bw/day) were considered to be indication of the biological variation as similar findings were not detected at the highest dose group.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item related toxic changes in the mean daily food consumption of male or female animals at 100, 250 or 350 mg/kg bw/day. In accordance with the body weight development, the mean daily food consumption was slightly reduced in male animals at 350 mg/kg bw/day and was elevated in female animals at 100 mg/kg bw/day during the premating and gestation periods. The mean daily food consumption was similar in male animals in the control and test item treated groups at 100 and 250 mg/kg bw/day during the pre-mating and post-mating periods. Statistical significances with respect to the control was observed at the slightly lower mean daily food consumption of male animals at 350 mg/kg bw/day on weeks 1, 6, 8 and 13. However, the mean food consumption was lower than in the control at each measurement day. In the female animals at 100 mg/kg bw/day, statistical significances were detected at the slightly higher mean daily food consumption on week 2 and from week 4 up to the end of gestation period when compared to control. The mean daily food consumption was comparable in the female animals in the control and 250 and 350 mg/kg bw/day groups during the pre-mating, gestation ad lactation periods.
These changes in the mean daily food consumption of male and female animals were considered to be toxicologically not relevant because of the minor degree.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Quantitative examinations of ovaries did not reveal toxicologically relevant differences between the control and 350 mg/kg bw/day group. The mean number of primordial, primary, secondary and tertiary follicle were similar in the control and 350 mg/kg bw/day. Statistical significance with respect to the control was observed at the lower mean number of corpora lutea and follicular atresia, which were probably indicative of biological variation.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

The FT4 and TSH levels were not adversely affected in the parental male animals at any dose levels. The FT4 levels were similar in parental male animals in the control and 100and 250 mg/kg bw/day group and was slightly lowered at 350 mg/kg bw/day. This minor change was considered to be toxicologically not relevant as the TSH level was comparable in the control and high dose groups.

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

The estrous cycle length was slightly elongated with respect to the control at 250 and 350 mg/kg bw/day. However, this slight change was judged to be toxicologically not relevant. There were no significant differences between the control and test item treated groups in the number or percentage of animals with regular/ irregular cycles, in the mean number of cycles, mean number of days in pro-estrus or estrous or diestrous, in the number or percentage of animals in prolonged diestrous during pre-mating period.

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

The examined parameters of reproductive performance were not affected by the treatment with the test item in male or female animals at 100, 250 or 350 mg/kg bw/day. The copulatory and fertility indices were comparable in male animal in the control and test item administered groups. Statistical significances with respect to the control were detected at the slightly lower mean percentage of dams delivered (gestation index) at 350 mg/kg bw/day because one pregnant female animal did not give birth (not-delivered). This minor difference was considered to be toxicologically not relevant and values met the historical control.

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive performance

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed up to highest dose tested

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no adverse effects observed up to highest dose tested

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

Key result

Critical effects observed:

no

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Offspring:
There were no adverse test item related clinical signs in the offspring from post-natal day 0 to 22. Slightly higher percentage of cold pups were observed at 350 mg/kg bw/day (28 %) with respect to the control.The percentage of not suckled pups (no milk in the stomach) was slightly higher (25 %) than in the control group in 250 mg/kg bw/day groups. Alopecia was observed on pups in one litter at 250 mg/kg bw/day (8 %). Some pup was pale (2 % at 100 mg/kg bw/day) or was smaller than others (1 % at 250 mg/kg bw/day). Cold and not suckled pups (no milk in the stomach) were observed with the highest incidence mainly on postnatal day 0 or during the first few post-natal days. These signs were considered to be toxicologically not relevant as the signs were transient and were with low incidence or met the historical control.
F1 generation: There were no clinical signs in male or female animals of F1 generation in the control and 100, 250 or 350 mg/kg bw/day during the entire observation period (from post-natal day 22 up to and including post-natal day 35).

Mortality / viability:

no mortality observed

Description (incidence and severity):

There was no test item related effect on offspring’s extra uterine mortality.The extrauterine mortality was comparable in the control, 100, 250 and 350 mg/kg bw/day on post-natal day 0 and from birth to post-natal day 21. The mean number of live births per litter and number of viable pups per litter were comparable in the control and 100, 250 and 350 mg/kg bw/day groups on post-natal days 0, 4 and 21. There was no significant difference between the control and test item treated (100, 250 and 350 mg/kg bw/day) groups in the survival indices on post-natal day 4.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Offspring:
The body weight development of the offspring was not adversely affected by the test item at 100, 250 and 350 mg/kg bw/day from post-natal day 0 up to postnatal day 21. Higher mean litter weight and higher mean litter weight gain were detected at 100 mg/kg bw/day (on post-natal day 14 and between post-natal days 0-4 and 7-14, respectively). At 250 mg/kg bw/day, statistical significance with respect to the control was observed at the lower mean litter weight on post-natal day 7 and at the lower mean litter weight gain between post-natal days 4 and 7. The mean litter weight and litter weight gain were similar in the control and 350 mg/kg bw/day from post-natal day 0 up to termination. The mean body weight exceeded the control in offspring at 100 mg/kg bw/day from post-natal day 4 up to and including post-natal 21. The mean body weight gain was also higher than in the control in pups at 100 mg/kg bw/day during the observation period reaching statistical significance between post-natal days 0-4, 7-14 and 0-21. At 250 mg/kg bw/day, the mean body weight of offspring was comparable with the control although, the mean body weight gain was slightly lower than in the control between post-natal days 4 and 7. The mean body weight and body weight gain were comparable in the control and 350 mg/kg bw/day from post-natal day 0 up to termination. In accordance with the litter weight and offspring’s mean body weight, the mean body weight of male and female offspring – evaluated by gender – was higher than in the control group at 100 mg/kg bw/day on post-natal day 4. The mean body weight of female offspring was lower with respect to the control at 250 mg/kg bw/day. The mean body weight was similar to the control male and female offspring at 350 mg/kg bw/day (evaluated by gender) on post-natal day 4. The toxicological relevance of the enhanced body weight development of offspring at the low dose is questionable as similar finding was not observed at the higher doses.
F1 generation:
The body weight development of the F1 animals was not adversely affected at 100, 250 or 350 mg/kg bw/day from post-natal day 22 up to post-natal day 35.
Minor changes at 100 mg/kg bw/day (female) and at 250 mg/kg bw/day (male and female) were probably indicative of biological variation as similar findings were not detected at the high dose. The body weight and body weight gain were similar in male animals in the control and 100 mg/kg bw/day administered groups during the 14-day observation period. Statistical significance with respect to the control was detected at the lower mean body weight of F1 male animals at 250 mg/kg bw/day from postnatal day 25 up to termination and at the lower mean body weight gain between post-natal days 22-25 and 22-35. The mean body weight was similar in male animals in the control and 350 mg/kg bw/day groups although, the mean body weight gain of these animals was lowered between post-natal days 22-25, 25-29 and 22-35. In the F1 female animals, statistical significances with respect to the control were observed at the higher mean body weight at 100 mg/kg bw/day during the entire observation period and at the lower mean body weight at 250 mg/kg bw/day on post-natal days 25, 32 and 35. The mean body weight gain was comparable in F1 female animals in the
control and at 100, 250 and 350 mg/kg bw/day between post-natal days 22 and 35.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

F1 generation: The mean daily food consumption was not affected in the control and test item administered male and female animals of F1 generation during the two weeks observation period. Compared to the control, statistical significance was detected at the lowered mean daily food consumption in F1 male animals at 250 mg/kg bw/day during the two weeks observation. The mean daily food consumption was similar to their control in male animals at 100 and 350 mg/kg bw/day and in female animals at 100, 250 and 350 mg/kg bw/day.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Description (incidence and severity):

The sex distribution of male and female pups (mean percentage of pups per litter) was comparable in all groups on post-natal days 0, 4 and 21.

Anogenital distance (AGD):

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item related changes in the anogenital distances at 100, 250 or 350 mg/kg bw/day in male or female offspring. The absolute and normalized anogenital distance were similar in male offspring in the control and test item administered groups. Statistical significances were noted for the slightly shorter mean normalized anogenital distance in female offspring at 100 mg/kg bw/day.
Similar finding was not observed at the higher doses therefore, this difference was considered to be of no toxicological relevance.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

Nipples/areoles were not seen in any of the examined male offspring in the control or 100, 250 or 350 mg/kg bw/day groups on post-natal day 13.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Offspring: Test item related macroscopic alterations were not found in stillborn offspring subjected to gross pathological examination on post-natal day 0. There were no macroscopic findings in organs or tissues in stillborn pups in the control (4/4) and in 250 mg/kg bw/day (2/2) groups. Slight autolysis and missing head (due to cannibalism) was noted for stillborn pup (1/1) at 350 mg/kg bw/day).
F1 generation: Macroscopic alterations in connection with the test item were not detected in male or female animals of F1 generation at the necropsy. Species specific findings – pyelectasia and hydrometra - were observed in all groups as follows:
pyelectasia (one or both sides):
male: 3/11 control; 7/12 at 100 mg/kg bw/day; 4/12 at 250 mg/kg bw/day; 5/14 at 350 mg/kg bw/day;
female: 1/11 control; 4/12 at 100 mg/kg bw/day; 2/12 at 250 mg/kg bw/day; 4/14 at 350 mg/kg bw/day; hydrometra (slight, moderate or marked): 3/11 control; 2/12 at 100 mg/kg bw/day; 1/12 at 250 mg/kg bw/day; 2/14 at 350 mg/kg bw/day.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

The FT4 and TSH levels were not adversely affected in PN22 offspring at any dose levels.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed up to highest dose tested

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Conclusions:

Under the conditions of the present study, the test substancewas administered at 100, 250 or 350 mg/kg bw/day oral gavage did not adversely influence the reproductive performance (gonad function, mating behavior, conception, parturition) in parental male and female Han:WIST rats as far as investigated in this study. The NOAEL for systemic toxicity of male rats is 250 mg/kg bw/day, the NOAEL for systemic toxicity of female rats is 350 mg/kg bw/day NOAEL for reproductive performance of male/ female rats is 350 mg/kg bw/day and the NOAEL for F1 is 350 mg/kg bw/day.

Executive summary:

A modified OECD 421 study in rats was performed. The purpose of this study was to obtain information on the possible effects of the test item on reproduction and development when repeatedly administered orally (by gavage) to rats at doses of 100, 250 and 350 mg/kg bw/day compared to control animals receiving vehicle only. Four groups of Han:WIST rats (n=12/sex/group) were administered with the test item orally (by gavage) once a day at 0 (vehicle), 100, 250 and 350 mg/kg bw/day doses corresponding to concentrations of 0, 50 mg/mL, 125 mg/mL and 175 mg/mL. The application volume was 2 mL/kg bw. Control animals received the vehicle, sunflower oil. The suitability of the vehicle at the intended concentrations of the test item was analytically verified up front (concentration and homogeneity). The concentration of the test item in the dosing formulations used for animal’s treatment was checked three times during the study. Concentration of TBPPI-75-AL in the dosing formulations varied between the range of 90 % and 108 % of the nominal values and formulations were homogenous thereby confirming the proper dosing. All animals of the parent (P) generation were dosed prior to mating (70 days) and throughout mating. In addition, males received the test item or vehicle after mating up to the day before the necropsy (altogether for 100 days). Dams were additionally exposed through the gestation period and up to lactation days 21-23 (altogether for 114, 115, 118 or 125 days). Non-pregnant female animals and dam not delivered were administered for 95, 97, 98 or 102 days. Observations included mortality, clinical signs, body weight, food consumption, mating, pregnancy and delivery process, as well as development of offspring. Estrous cycle was monitored by examining vaginal smears for two weeks before the start of mating period and during the mating period until evidence of mating and on the day of the necropsy. The dams were allowed to litter and rear their offspring up to day 21 - 23 post-partum then were subjected to necropsy on post-partum day 22 - 24. Litters were weighed and offspring were observed for possible abnormalities. One or two male and one or two female pups per litter were selected on post-natal day 21 and were separated and administered (2 mL/kg bw/day) from postnatal day 22 up to and including post-natal day 35. Clinical signs were observed daily after the treatment and body weight was determined twice weekly and were recorded. The food consumption was determined by weekly interval. All selected pups were subjected to macroscopic necropsy observation on post-natal day 36. Remaining pups were euthanized on post-natal day 22. Blood samples were collected for determination of serum levels of thyroid hormones (FT4, TSH) from 2-5 pups per litter (except for litters with 9 or less pups) on post-natal day 4, from all dams and from 3-6 pups per litter on post-partum/post-natal day 22 and from all parent male animals at termination. Non-pregnant female animals and dam not giving birth were sampled for thyroid hormone determination at termination on the day of the necropsy (Days 95, 97, 98 or 102). All parental animals were subjected to gross pathology one day after the last treatment. The ovaries, uterus with oviduct and cervix, vagina, testes, epididymides (total and cauda), prostate, and seminal vesicles with coagulating glands and thyroid glands of all adult animals were preserved. In addition, organs with macroscopic findings were also preserved for a possible histological examination. A quantitative examination of the ovaries was performed in all female animals in the control and high dose groups. The body weight, brain weight, weight of the testes, epididymides and prostate and seminal vesicles with coagulating glands as a whole of all adult male animals were determined.

There was no mortality at any dose level (100, 250 or 350 mg/kg bw/day). Clinical signs of systemic toxicity related to the test item were not detected at any dose level at the daily clinical observations. The behavior and physical condition of the animals was not impaired at any dose level (100, 250 or 350 mg/kg bw/day) during the entire treatment period. Salivation and nuzzling up the bedding material observed at 250 and 350 mg/kg bw/day were with short duration after the administration of the test item therefore these signs were considered to be toxicologically not relevant.

The body weight development was depressed in male animals at 250 and 350 mg/kg bw/day during the pre-mating, mating and post-mating periods. The difference with respect to the control was lower than 10 % at 250 mg/kg bw/day therefore was judged to be toxicologically not relevant.

The mean daily food consumption was slightly reduced in male animals at 350 mg/kg bw/day in accordance with the body weight changes during the entire treatment period. Estrous cycle The examined parameters of the estrous cycle were not adversely affected in any group (control, 100, 250 and 350 mg/kg bw/day). Delivery data of dams There were no significant or dose related differences between the control and test item treated female animals in the examined parameters of the delivery (100, 250 and 350 mg/kg bw/day).

A test item influence on the examined parameters of reproductive performance was not found at any dose level. Serum thyroid hormones The FT4 and TSH levels were not adversely affected in the parental male animals or in PN22 offspring at any dose levels.

Necropsy examinations did not reveal specific macroscopic alterations related to the effect of the test item in male or female animals at 100, 250 or 350 mg/kg bw/day.

The weights of examined organs (absolute, relative to body and brain weights) were not affected by the test item in male animals at 100, 250 or 350 mg/kg bw/day.

There were no toxicologically relevant differences in the mean number of primordial, primary, secondary, tertiary follicles, atresia or corpora lutea at 350 mg/kg bw/day at the quantitative examination of ovaries.

The offspring’s development was not adversely influenced at any dose level. No adverse effects on mortality, clinical signs, body weight development, anogenital distance (male and female) or nipple retention (male) were detected. There were no clinical signs, changes in body weight or mean daily food consumption or necropsy findings in F1 generation (male and female) after the 14 days administration of 100, 250 or 350 mg/kg bw/day.

Based on these observations the NOAELs were determined as follows:

NOAEL for systemic toxicity of male rats: 250 mg/kg bw/day

NOAEL for systemic toxicity of female rats: 350 mg/kg bw/day

NOAEL for reproductive performance of male/ female rats: 350 mg/kg bw/day

NOAEL for F1 Offspring (PN 0-21): 350 mg/kg bw/day

NOAEL for F1 generation (PN 22-35): 350 mg/kg bw/day

Reference 3

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Justification for type of information:

Please refer to Section 13 for the Read-Across Justification-

Reason / purpose for cross-reference:

read-across source

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no adverse effects observed up to highest dose tested

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive performance

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed up to highest dose tested

Key result

Critical effects observed:

no

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed up to highest dose tested

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Reference 4

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Justification for type of information:

Please refer to Section 13 for Read-Across Justification.

Reason / purpose for cross-reference:

read-across source

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed up to highest dose tested

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: reduction of mean number of births (total, live and alive) a

Key result

Critical effects observed:

no

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Reference 5

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2021-06-15 to 2021-06-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

29th July 2016

Deviations:

yes

Remarks:

10 weeks pre-treatment, no hematology and clinical biochemistry measurements, histopathology only performed for ovaries

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Han:WIST

Details on species / strain selection:

The rat is regarded as suitable species for toxicity and reproduction toxicity studies and the test guidelines were designed to use the rat. The Wistar rat was selected due to a wide range of experience with this strain of rat in toxicity and 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. 1103 Budapest, Hungary
- Females nulliparous and non-pregnant: yes
- Age at study initiation:
Male animals: 46- 50 days old
Female animals: 41 - 46 days old
- Weight at study initiation:
Male animals: 200 – 240 g
Female animals: 126 – 156 g
- Fasting period before study: no
- Housing: Type III polypropylene/polycarbonate cages
Before mating: 2 animals of the same sex/cage
Mating hours: 1 male and 1 female/cage
Mated females were housed individually
Males after mating: 2 animals/cage
- Diet: ad libitum, ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice, Spezialdiäten GmbH, D-59494 Soest, Germany
- Water, ad libitum, tap water
- Acclimation period: 13 days

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

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 vehicle in concentrations of 25 mg/mL, 75 mg/mL and 150 mg/mL in the formulation laboratory of the Test Facility not longer than for three days before the use. Analysis of formulations was performed in the Analytical Laboratory of Test Facility three times during the treatment period.

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, 150 mg/mL
- Amount of vehicle: 2 mL

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: Females were cohabited with the same male until copulation occurred
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged: Sperm positive females were caged individually.
- Any other deviations from standard protocol: no

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of dosing formulations was between the range of 95 % to 109 % of the nominal 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 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 female animals were dosed for 70 days pre-mating, through up to 1-13 days mating period and throughout pregnancy and up to and including lactation days 21-23 and then were sacrificed and subjected to a detailed necropsy. Overall treatment period was therefore 114-127 days for dams. Non-pregnant female animals were administered for 96 days.

Frequency of treatment:

daily

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose setting was based on results obtained in previous studies performed with TBPND in the rat: 14-Day Oral Gavage Dose Range Finding Study with TBPND in the Rat andCombined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test with TBPND in the Rat.

Positive control:

none

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All parental animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each day).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a day, after the treatment at approximately the same time
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 was 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), twice a week for four weeks and weekly thereafter and on the day of the necropsy. Parental females were weighed on the first day of dosing (Day 0), twice a week for four weeks and weekly thereafter, then on gestation days 0, 7, 14 and 21 and on days 0 (within 24 hours after parturition), 4, 7, 14 and 21 post-partum. Additionally, female animals were weighed on gestational day 10 in order to give accurate treatment volumes

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- The food consumption of parent animals was determined weekly by weighing the given and non-consumed diet by weekly interval with an accuracy of 1 g during the treatment period except mating phase: during the premating period and during post-mating period for male animals; premating period, gestation days 0, 7, 14 and 21, lactation days 0, 7, 14 and 21 for female animals.

WATER CONSUMPTION AND COMPOUND INTAKE: No

DETERMINATION OF THYROID HORMONES:
Blood samples were collected for determination of serum levels of thyroid hormones (FT4, TSH) as follows:
- from all dams on lactation/ post-natal day 22;
- from all parent male animals at termination on Day 99;
- non-pregnant female animals on Day 96;

Oestrous cyclicity (parental animals):

Estrous cycle was monitored by examining vaginal smears daily before the mating started from each animal for two weeks and from the beginning of the mating period until evidence of copulation. Vaginal smear was also prepared on the day of the necropsy. Vaginal smears were stained with 1 % aqueous methylene blue solution and examined with a light microscope after drying.

Sperm parameters (parental animals):

Parameters examined in male parental generation:
testis weight, epididymis weight, weight of prostate and seminal vesicles with coagulating glands, spermatogenesis, histopathology

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

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, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities

On the day of birth, pups found dead were subjected to a lung flotation test to differentiate pups died intrauterine (stillborn; negative lung flotation test) from pups died after the birth (dead pups; positive lung flotation test).

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: no

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no

DETERMINATION OF THYROID HORMONES:
Blood samples were collected for determination of serum levels of thyroid hormones (FT4, TSH) as
follows:
- from 2-7 pups per litter on post-natal day 4 (except for litters with 9 or less pups)
- from 3-8 pups per litter on lactation/ post-natal day 22;
Blood samples were collected from the surplus pups (at least two pups per litter), pooled and used for determination of serum FT4 and TSH levels on post-natal days 4 and 22.

OTHER:
One or two male and one or two female pups per litter were selected on post-natal day 21 and were identified by individual numbers written with a marker pen on the tail, were caged (2-3 pups/cage) and were administered (2 mL/kg bw/day) from postnatal day 22 up to and including post-natal day 35. Clinical signs were observed daily after the treatment and body weight were determined twice weekly and were recorded. The food consumption was determined on post-natal day 22, 29 and 35. All pups were subjected to macroscopic necropsy observation on post-natal day 36.

Postmortem examinations (parental animals):

SACRIFICE
Gross necropsy was performed on each parental animal one day after the last treatment. Animals were euthanized by exsanguination after verification of Isofluran-narcosis and were subjected to gross necropsy as follows:
- Parental male animals: on Day 99;
- Dams: on post-partum days 22, 23 or 24 (on Days 114-127);
- Non-pregnant (but mated) female animals on Day 96

GROSS NECROPSY
- After examination of the external appearance, the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed, and any abnormality was recorded including details of the location, color, shape and size. Special attention was paid to the organs of the reproductive system.

HISTOPATHOLOGY / ORGAN WEIGHTS
At the time of termination, body weight, brain weight and weight of the testes and epididymides as well as prostate and seminal vesicles with coagulating glands as a whole of adult male animals were determined. Paired organs were weighed together. The thyroid weight was not determined because there were no test item related changes in the hypothalamic-pituitary-thyroid function at the thyroid hormone examinations. Histopathological processing and quantitative examinations of the ovaries were performed in female animals in the control and high dose groups. Follicular enumeration (primordial and small growing follicles – primary, secondary, tertiary follicles, corpora lutea, atresia) was performed in the largest surface section of the ovaries. The fixed ovaries 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.

Postmortem examinations (offspring):

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

HISTOPATHOLOGY / ORGAN WEIGTHS: Not determined

Statistics:

The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was 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 inter-group differences. Getting significant result 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:

See table in "Any other information on materials and methods".

Offspring viability indices:

See table in "Any other information on materials and methods".

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item related adverse clinical signs in any group (50, 150 or 300 mg/kg bw/day). Some male animal at 300 mg/kg bw/day showed salivation shortly after the administration for some minutes. Although the behavior and physical condition of these animals were also normal and therefore, salivation was considered to be not adverse. Salivation was noted for some male animal (7/12) at 300 mg/kg bw/day from Day 32 up to and including 65 or 98. Dermal changes were detected in one male animal at 300 mg/kg bw/day (1/12; scar at the neck region, left side, 0.5-1 cm in diameter, in slight, moderate or marked degree between Days 56 and 97) and in one female animal at 50 mg/kg bw/day (1/11 dam; alopecia on the fore limbs between lactation days 8 and 22). Similarly, reddish discoloration of the hair around eye is frequently observed in non-treated animals of this strain of experimental rats with similar age. This finding was detected in one dam from Day 33 up to and including lactation day 21. Scar, alopecia and reddish discoloration around the eye are common findings occurring also in non-treated animals of this strain of experimental rats with similar age. Therefore, these have no toxicological relevance.

Mortality:

no mortality observed

Description (incidence):

There was no mortality in control, 50, 150 or 300 mg/kg bw/day groups during the course of study (male and female).

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The body weight development was not influenced by the test item at 50, 150 or 300 mg/kg bw/day during the pre-mating period (male or female animals) and during the post mating period (male). However, the mean body weight was depressed in dams at 300 mg/kg bw/day during the course of gestation and lactation periods. The mean body weight gain was also reduced in dams at 300 mg/kg bw/day during the gestation period and during the first few days of lactation period.The mean body weight was comparable in the male animals in the control and all test item administered groups on each measuring day from Day 0 up to and including Day 97. Statistical significance with respect to the control was observed at the higher mean body weight gain of male animals at 150 and 300 mg/kg bw/day between Days 0-3 and at the lower mean body
weight gain of male animals at 300 mg/kg bw/day between Days 90-97. These changes in the body weight gain did not influence the mean body weight values of male animals. In the female animals, the mean body weight was comparable in the control and all dosed groups during the entire pre-mating period in-spite of the lower mean body weight gain at 300 mg/kg bw/day between Days 28-35,
56-63 and 0-69. At 150 mg/kg bw/day, the mean body weight gain exceeded the control between lactation day 7-14 in female animals.During the course of the gestation and lactation periods, statistical significance with respect to the control was observed at the lowered mean body weight and body weight gain of dams at 300 mg/kg bw/day as follows:
- lower mean body weight on gestation days 14 and 21 and lower mean body weight gain between gestation days 7-14, 14-21 and 0-21;
- lower mean body weight on lactation days 0, 4, 7, 14 and lower mean body weight gain between lactation days 0-4 and 7-14;
This change in the mean body weight was presumably in accordance with the lower number of fetuses of high dose treated dams.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item related adverse changes in the mean daily food consumption of male or female animals at 50, 150 or 300 mg/kg bw/day. The mean daily food consumption was comparable in male animals in the control and test item treated groups at 50, 150 and 300 mg/kg bw/day during the pre-mating and post-mating periods except for week 13 (between Days 83-90), where statistical significance with respect to the control was observed at the slightly higher mean daily food consumption of male animals at 300 mg/kg bw/day. In the female animals, statistical significances were detected at the slightly lower mean daily food consumption at 50 and 300 mg/kg bw/day on week 9 of pre-mating period and at 300 mg/kg bw/day between gestation days 14 and 21.
These changes in the mean daily food consumption were considered to be toxicologically not relevant because of the minor degree and transient occurrence.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

effects observed, non-treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Quantitative examinations of ovaries did not reveal toxicologically relevant differences between the control and 300 mg/kg bw/day group. The mean number of primordial, primary, secondary and tertiary follicle, corpora lutea were similar in the control and 300 mg/kg bw/day. Statistical significance with respect to the control was observed at the higher mean number of follicular atresia. Follicular atresia is a normal - physiological - process in the ovary to regulate the number of follicles in the developing pool. Increase in follicular atresia can be observed secondary due to different causes. Since the number of primary, secondary and tertiary follicles was not changed compared to the control the increased follicular atresia can be judged as a biological variation.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item related adverse changes in the serum thyroid hormone (FT4 and TSH) levels at any dose in the parental male animals. The FT4 levels were slightly lower than in the control group in parental male animals at 150 and 300 mg/kg bw/day groups. The TSH concentration was higher than in the control group in parent male animals at 150 and 300 mg/kg bw/day. These minor changes were considered to be of no toxicological relevance as values met well the historical control in the most animals. The changes in FT4 were considered to be toxicologically not relevant considering the lack of significant relevant changes in TSH values.

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

The examined parameters of the estrous cycle were not affected by the test item at 50, 150 or 300 mg/kg bw/day. There were no significant differences between the control and test item treated groups in the number or percentage of animals with regular/ irregular cycles, in the mean number or length of cycles, mean number of days in proestrus or estrous or diestrous during pre-mating period. The number or percentage of animals in prolonged diestrous was slightly higher in the control group than in the test item treated groups as an indication of biological variation.

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

The examined parameters of reproductive performance were not adversely affected by the treatment with the test item in male or female animals at 50, 150 or 300 mg/kg bw/day. Statistical significances with respect to the control were detected at the lower mean percentage of fertile male and female animals (fertility indices) at 50 and 300 mg/kg bw/day as a consequence of infertile mating of one pair (1/12) at 50 mg/kg bw/day and two pairs (2/12) at 300 mg/kg bw/day. The values were within the historical control range. The copulatory index and gestation indices were 100 % in all groups.

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed up to highest dose tested

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: reduction of mean number of births (total, live and alive)

Key result

Critical effects observed:

no

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Offspring: There were no adverse test item related clinical signs in the offspring from post-natal day 0 to 22. Slightly higher percentage of cold pups were detected at 150 and 300 mg/kg bw/day (16 and 24 %, respectively) with respect to the control. Not suckled pups were observed with the highest incidence at 150 and 300 mg/kg bw/day (35 and 29 %, respectively). Alopecia was observed on the whole body of pups in one litter (6 %) at 50 mg/kg bw/day. Additionally, smaller than normal, pale pups, hemorrhage (on the nose, abdomen), bite mark on the abdomen were noted for some pup. These signs were considered to be toxicologically not relevant as the signs were transient.
F1: There were no clinical signs in male or female animals of F1 generation in the control and 50, 150 or 300 mg/kg bw/day groups during the entire observation period (from post-natal day 22 up to and including postnatal day 35).

Mortality / viability:

no mortality observed

Description (incidence and severity):

There was no test item related effect on offspring’s extra uterine mortality. The extrauterine mortality was comparable in the control, 50, 150 and 300 mg/kg bw/day on post-natal day 0 and from birth to post-natal day 21.
The survival of offspring were not affected by the test item at 50, 150 or 300 mg/kg bw/day on post-natal days 0, 4 and 21. Statistical significances with respect to the control was detected at 300 mg/kg bw/day at the lower mean number of pups (live and alive) on post-natal day 0 and mean number of pups per litter on post-natal day 4. Although, the extrauterine mortality was low and similar in each group. There was no significant difference between the control and test item treated (50, 150 and 300 mg/kg bw/day) groups in the survival indices on post-natal day 4. The survival index was higher at 300 mg/kg bw/day than in the control group on post-natal day 21 due to the lower number of offspring euthanized on postnatal day 4.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Offspring: The body weight development of the offspring was depressed at 150 and 300 mg/kg bw/day between post-natal day 0 up to postnatal day 21. The mean litter weight, litter weigh gain, body weight and body weight gain were comparable in the control and 50 mg/kg bw/day after birth up to and including post-natal day 21. At 150 mg/kg bw/day, statistical significance with respect to the control was detected at the lower mean litter weight gain between post-natal days 0 and 4 however the mean litter weight was similar to the control. The mean pup weight was lower than in the control at 150 mg/kg bw/day on post-natal
days 4, 7 and 14 as a consequence of the lower mean body weight gain between post-natal days 0-4 and 4-7. The mean litter weight and mean litter weight gain were significantly lower than in the control in offspring at 300 mg/kg bw/day from post-natal day 0 up to and including post-natal day 21 reaching statistical significances in the most cases. Compared to the control, statistically significant difference was observed at the lower mean body weight of pups at 300 mg/kg bw/day on post-natal days 7, 14 and 21 as well as at lower mean body weight gain of pups between postnatal days 4-7 and 7-14, as well as between post-natal days 0-21. Considering the offspring’s body weight in males and females separately, statistically significant difference with respect to the control was detected at the slightly lower mean weight of male pups at 50, 150 and 300 mg/kg bw/day and of female pups at 150 mg/kg bw/day on post-natal day 4.
F1: The body weight development of the F1 animals was reduced at 150 and
300 mg/kg bw/day from post-natal day 22 up to post-natal day 35. The difference with respect to the control was lower than 10 % at 150 mg/kg bw/day (male and female) and in female animals at 300 mg/kg bw/day. The reduction of body weight below 10 % is considered to be toxicologically not relevant. The body weight and body weight gain were similar in the control and 50 mg/kg bw/day administered male animals during the observation period. In the male animals at 150 mg/kg bw/day, the mean body weight was lower than in the control group during the two weeks observation period with statistical significance on post-natal days 22, 25, 29. The mean body weight gain for these animals was comparable to their control. At 300 mg/kg bw/day, the mean body weight and body weight gain of male animals were significantly lower (up to 17% lower body weight) than in the control reaching statistical significances in the most cases.
The body weight and body weight gain were comparable to the control in
female animals at 50 mg/kg bw/day during the observation period. At 150 mg/kg bw/day, the mean body weight of female animals was lower than in the control group during the two weeks observation period with statistical significance on each measuring days. Statistical significance with respect to the control was detected at the lower mean body weight gain for these animals between post-natal days 25 and 29. In the female animals at 300 mg/kg bw/day, the mean body weight and body weight gain were significantly lower than in the control reaching statistical significances for body weight on each measuring days and for body weight gain between postnatal days 22-25 and 25-29.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

F1: The mean daily food consumption was lowered at 150 and 300 mg/kg bw/day in F1 male and female animals in accordance with the body weight
changes. The mean daily food consumption was similar in the control and 50 mg/kg bw/day groups (male and female) during the 14-day observation period.
Statistical significance with respect to the control was detected at the lower mean daily food consumption of male animals at 150 mg/kg bw/day between post-natal days 22-29 and at 300 mg/kg bw/day between post-natal days 22-29 and 29-35. In the female animals, lower mean daily food consumption was observed at 150 mg/kg bw/day between post-natal days 29-35 and at 300 mg/kg bw/day between post-natal days 22-29 and 29-35.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Description (incidence and severity):

The sex distribution of male and female pups (mean percentage of pups per litter) was comparable in all groups on post-natal days 0, 4 and 21.

Anogenital distance (AGD):

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item related changes in the anogenital distances at 50, 150 or 300 mg/kg bw/day in male or female offspring. Statistical significance was observed at the slightly shorter mean absolute anogenital distance in female pups at 150 and 300 mg/kg bw/day. The mean normalized anogenital distance exceeded the control in male pups at 300 mg/kg bw/day and was below the control in female pups at 300 mg/kg bw/day. These differences were with minor degree and within the range of the historical control data. Therefore, were considered to have no toxicological relevance.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

Nipples/areoles were not seen in any of the examined male offspring in the control or 50, 150 or 300 mg/kg bw/day groups on post-natal day 13.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Offspring: Test item related macroscopic alterations were not found in offspring subjected to gross pathological examination (stillborn or dead pups on postnatal day 4). In dead pup in the control group (1/1 male), empty stomach and slightly
autolyzed visceral organs were observed. At 50 mg/kg bw/day, there were no macroscopic findings in one dead pup (1/1). In one stillborn pup at 150 mg/kg bw/day (1/7), malformations – missing orifices of eyes, nose and mouth – were observed. There were no macroscopic findings in organs or tissues in stillborn pups: 1/1 at 50 mg/kg bw/day, 6/7 at 150 mg/kg bw/day and 3/3 at 300 mg/kg
bw/day.
F1: Macroscopic alterations in connection with the test item were not detected in male or female animals of F1 generation at the necropsy. Species specific macroscopic findings – renal changes (male and female) and hydrometra (female) – were observed. In male animals, pale kidneys (1/12 control, 1/11 at 300 mg/kg bw/day) and right side pyelectasia (3/12 at 150 mg/kg bw/day; 1/11 at 300 mg/kg bw/day) were observed. In female animals, slight, moderate or marked hydrometra was detected as follows: 1/11 in control group; 1/11 at 50 mg/kg bw/day; 3/14 at 150 mg/kg bw/day). Additionally, pale kidneys (2/11 at 50 mg/kg bw/day; 1/14 at 150 mg/kg bw/day; 1/10 at 300 mg/kg bw/day) and one or both sided pyelectasia (1/11 at 50 mg/kg bw/day; 2/10 at 300 mg/kg bw/day) were seen. Pyelectasia and hydrometra are common macroscopic findings in experimental rats of this strain with similar age.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

The FT4 and TSH levels were not adversely affected in PN22 offspring at any dose levels.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Conclusions:

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. 50 or 150 mg/kg bw/day did not adversely influence the reproductive performance (gonad function, mating behavior, conception, parturition) in parental male and female animals. 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). 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 NOAEL for systemic toxicity of male/ female rats is 300 mg/kg bw/day, the NOAEL for reproductive performance of male/ female rats is 150 mg/kg bw/day and the NOAEL for F1 Offspring is 50 mg/kg bw/day.

Executive summary:

A modified OECD 421 study in rats was performed. The purpose of this study was to determine the dose levels for the main study (OECD 443) and obtain information on the possible effects of the test item on reproduction and development when repeatedly administered orally (by gavage) to rats at doses of 50, 150 and 300 mg/kg bw/day compared to control animals receiving vehicle only. Four groups of Han:WIST rats (n=12/sex/group) were administered with the test item orally (by gavage) once a day at 0 (vehicle), 50, 150 and 300 mg/kg bw/day doses corresponding to concentrations of 0, 25, 75 and 150 mg/mL. The application volume was 2 mL/kg bw. Control animals received the vehicle, sunflower oil. The suitability of the vehicle at the intended concentrations of the test item was analytically verified up front (concentration and homogeneity). The concentration of the test item in the dosing formulations used for animal’s treatment was checked three times during the study. Concentration of TBPND in the dosing formulations varied between the range of 95 % and 109 % of the nominal values and formulations were homogenous thereby confirming the proper dosing. All animals of the parent (P) generation were dosed prior to mating (70 days) and throughout mating. In addition, males received the test item or vehicle after mating up to the day before the necropsy (altogether for 99 days). Dams were additionally exposed through the gestation period and up to lactation days 21-23 (altogether for 114-127 days). Non-pregnant female animals were administered for 96 days. Observations included mortality, clinical signs, body weight, food consumption, mating, pregnancy and delivery process, as well as development of offspring. Estrous cycle was monitored by examining vaginal smears for two weeks before the start of mating period and during the mating period until evidence of mating. Vaginal smears were also prepared and investigated on the day of the necropsy for each dam. The dams were allowed to litter and rear their offspring up to days 21 post-partum then were subjected to necropsy on post-partum days 22 - 24. Litters were weighed and offspring were observed for possible abnormalities. One or two male and one or two female pups per litter were selected on post-natal day 21 and were separated and administered (2 mL/kg bw/day) from postnatal day 22 up to and including post-natal day 35. Clinical signs were observed daily after the treatment and body weight was determined twice weekly and were recorded. The food consumption was determined by weekly interval. All selected pups were subjected to macroscopic necropsy observation on post-natal day 36. Remaining pups were euthanized on post-natal day 22. Blood samples were collected for possible determination of serum levels of thyroid hormones (FT4, TSH) from 2-7 pups per litter (except for litters with 9 or less pups) on post-natal day 4, from all dams and from 3-8 pups per litter on post-partum/post-natal day 22 and from all parent male animals at termination. Non-pregnant female animals were sampled for thyroid hormone determination at termination, on Day 96.

All parental animals were subjected to gross pathology one day after the last treatment. The ovaries, uterus with oviduct and cervix, vagina, testes, epididymides (total and cauda), prostate, and seminal vesicles with coagulating glands, thyroid glands of all adult animals were preserved. In addition, based on macroscopic observations, skin of one female animal at 50 mg/kg bw/day, liver, spleen, heart, lungs in one dam at 150 mg/kg bw/day as well as the kidneys of some animal were also preserved for a possible histological examination. The body weight, brain weight, weight of the testes, epididymides and prostate and seminal vesicles with coagulating glands as a whole of all adult male animals were determined. A quantitative examination of ovaries was performed in all female animals in the control and high dose groups.

There was no test item related mortality at any dose level (50, 150 or 300 mg/kg bw/day). Clinical signs of systemic toxicity related to the test item were not detected at any dose level at the daily clinical observations. The behavior and physical condition of the animals was not impaired at any dose level (50, 150 or 300 mg/kg bw/day) during the entire treatment period. Salivation observed in male animals at 300 mg/kg bw/day was judged to be toxicologically not relevant because of the short duration immediately after the administration. The body weight development was not influenced by the test item at 50, 150 or 300 mg/kg bw/day during the pre-mating period (male or female animals) and during the post mating period (male). However, the mean body weight was depressed in dams at 300 mg/kg bw/day during the course of the last two weeks of gestation and during the lactation period. This change in the mean body weight was presumably in accordance with the lower number of fetuses of high dose treated dams. The mean daily food consumption was not adversely affected in male or female animals at 50, 150 and 300 mg/kg bw/day during the entire treatment period.

The examined parameters of the estrous cycle were comparable in all groups (control, 50, 150 and 300 mg/kg bw/day). The mean number of total birth, live born and alive pups was reduced at 300 mg/kg bw/day when compared to the actual control and historical control. A test item influence on the examined parameters of reproductive performance was not found at any dose level.

The FT4 and TSH levels were not adversely affected in the parental male animals or in PN22 offspring at any dose levels. Macroscopic alterations related to the effect of the test item were not detected at 50, 150 or 300 mg/kg bw/day (male or female) at the necropsy examinations. The weights of examined organs (absolute, relative to body and brain weights) were not affected by the test item in male animals at 50, 150 or 300 mg/kg bw/day. Quantitative examinations of ovaries revealed enhanced follicular atresia at 300 mg/kg bw/day with respect to their control, but no effects in primary, secondary or tertiary follicle counts.

The body weight development of the offspring was depressed at 150 and 300 mg/kg bw/day between post-natal day 0 and 21. No adverse effects on extrauterine mortality, clinical signs, anogenital distance (male and female) or nipple retention (male) were detected. F1 generation (post-natal days 22-35) The body weight development and food consumption of the F1 animals was reduced at 150 and 300 mg/kg bw/day from post-natal day 22 up to post-natal day 35. The difference with respect to the control was lower than 10 % at 150 mg/kg bw/day (male and female) and in female animals at 300 mg/kg bw/day. The reduction of body weight below 10 % is considered to be toxicologically not relevant. There were no clinical signs or necropsy findings in F1 generation (male and female) after the 14 days administration of 50, 150 or 300 mg/kg bw/day.

Based on these observations the NOAELs were determined as follows:

NOAEL for systemic toxicity of male/ female rats: 300 mg/kg bw/day

NOAEL for overall reproductive performance of male/ female rats: 150 mg/kg bw/day

NOAEL for F1 Offspring: 50 mg/kg bw/day

Reference 6

Endpoint:

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

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Based on the results from the modified OECD 421 studies for the three peroxyesters only one study according OECD TG 443 with TBPND (CAS 26748-41-4) was initiated for the peroxyester group. This substance was chosen since ECHA requested for TBPND to include Cohort 3 (immuntoxicity, CCH-D-2114493102-56-01/F). The study results from this OECD TG 443 study are used in a read-across grouping approach for the peroxyesters TBPIN and TBPPI to fulfil ECHAs requests on EOGRTS with these peroxyesters (CCH-D-2114493103-54-01/F and CCH-D-2114493105-50-01/F, respectively). This approach was also chosen because of animal welfare reasons.

Reason / purpose for cross-reference:

read-across source

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

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

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

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

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

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

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

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

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

Key result

Reproductive effects observed:

no

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

450 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

OECD TG 443/GLP

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

OECD 421 (2010)

A Reproduction/Developmental toxicity screening test with TBPIN was performed according to OECD 421. The purpose of this study was to obtain initial information on the possible effects of the test item on reproduction and development when administered orally (by gavage) to rats at repeated doses of 50, 160 and 500/400 mg/kg bw/day compared to control animals. As a screening test, it was intended to provide initial information on possible effects on male and female reproductive performance such as gonad function, mating behaviour, conception, pregnancy, parturition as well as on development of the F1 offspring from conception to day 4 post-partum associated with administration of repeated doses. In this study, 12 animals/sex/dose were involved with exception of the 500/400 mg/kg bw group (13 males) and the 160 mg/kg bw/day group (13 females). All animals of the P generation were treated prior to mating (14 days) and throughout mating (16 days). Male animals were treated 17 days post mating. For females, treatment was continued through the gestation period and up to lactation day 3 or shortly thereafter, i.e. up to the day before the necropsy. Observations included mortality, clinical symptoms, body weight, food consumption, estrous cycle, mating, and pregnancy and delivery process.

TBPIN caused death of pregnant Hsd.Brl.HAN Wistar rats at 160 and 400 mg/kg bw/day at the end of pregnancy and at early lactation due to acute-subacute tubular damage in the kidneys and related pulmonary alterations.

Reproductive performance of males was unaffected by treatment. Reproductive performance of female animals was affected by the treatment at 160 and 400 mg/kg bw/day through the parturition and nourishing and limited to the systemic toxicity at those doses. In the F1 generation, the higher mortality, clinical signs and depressed body weight development was considered to be the consequence of maternal toxicity at 160 and 400 mg/kg bw/day. Effects on reproduction and/or developmental of pups were thus only at doses pronounced in concentration with maternal toxicity and are in conclusion not regarded as relevant for indication for a specific hazard.

The NOAEL for parental effects was 50 mg/kg bw /day. For reproduction parameters, the NOAELs were 400 mg/kg bw/day for male rats and 50 mg/kg bw/day for female rats. For F1 offspring, the NOAEL was 50 mg/kg bw/day.

Modified OECD 421 (2021)

A modified study according OECD 421 in rats was performed with TBPIN, in which the parent (P) generation was dosed 10 weeks prior to mating. For the peroxyesters TBPND (CAS 26748-41-4) and TBPPI (CAS 927-07-1) the same modified design was used in two additional OECD 421 studies. The rational was to compare the three peroxyesters regarding effects on reproductive performance after 10 weeks pre-mating exposure and to conclude on the possibility to perform only one subsequent OECD 443 study for all three peroxyesters (grouping approach). This 10-weeks pre-mating exposure is recommended in the OECD 443 study design. The results of all three modified OECD 421 studies are presented below:

TBPIN

A modified OECD 421 study in rats was performed. Males received the test item or vehicle after mating up to the day before the necropsy (altogether for 98 days). Dams were additionally exposed through the gestation period and up to lactation days 21-22 (altogether for 114-122 days). Furthermore, one or two male and one or two female pups per litter were selected on post-natal day 21 and were separated and administered from postnatal day 22 up to and including post-natal day 36.

Four groups of Han:WIST rats (n=12/sex/group) were administered with the test item orally (by gavage) once a day at 0 (vehicle), 50, 100 and 160 mg/kg bw/day doses corresponding to concentrations of 0, 10, 20 and 32 mg/mL. The application volume was 5 mL/kg bw. The doses were selected based on the OECD TG 421 study presented above and the OECD TG 408 study. Control animals received the vehicle, sunflower oil. The suitability of the vehicle at the intended concentrations of the test item was analytically verified up front (concentration and homogeneity). The concentration of the test item in the dosing formulations used for animal’s treatment was checked three times during the study. Concentration of test substance in the dosing formulations varied between the range of 99 % and 107 % of the nominal values and formulations were homogenous thereby confirming the proper dosing. Observations included mortality, clinical signs, body weight, food consumption, mating, pregnancy and delivery process, as well as development of offspring. Estrous cycle was monitored by examining vaginal smears for two weeks before the start of mating period and during the mating period until evidence of mating and on the day of the necropsy. The dams were allowed to litter and rear their offspring up to day 21 or 22 post-partum then were subjected to necropsy on post-partum day 22 or 23. Litters were weighed and offspring were observed for possible abnormalities. Clinical signs were observed daily after the treatment and body weight was determined twice weekly and were recorded. The food consumption was determined by weekly interval. All selected pups were subjected to macroscopic necropsy observation on post-natal day 37. Remaining pups were euthanized on post-natal day 22. Blood samples were collected for possible determination of serum levels of thyroid hormones (FT4, TSH) from 2-8 pups per litter (except for litters with 9 or less pups) on post-natal day 4, from all dams and from 2-6 pups per litter on post-partum/post-natal day 22 and from all parent male animals at termination. Not mated, non-pregnant female animals and dams for which no living pups remained were sampled for thyroid hormone determination at termination, on Day 96 or 98.

All parental animals were subjected to gross pathology one day after the last treatment. The ovaries, uterus with oviduct and cervix, vagina, testes, epididymides (total and cauda), prostate, and seminal vesicles with coagulating glands of all adult animals were preserved. In addition, based on macroscopic observations, skin of one male animal at 100 mg/kg bw/day as well as the liver and kidneys of all animals were also preserved for a possible histological examination. Dam euthanized early in moribund state was subjected to necropsy on post-partum day 3 and all organs and tissues were preserved. The body weight, brain weight, weight of the testes, epididymides and prostate and seminal vesicles with coagulating glands as a whole of all adult male animals were determined.

There was no test item related mortality at any dose level (50, 100 or 160 mg/kg bw/day). One dam at 100 mg/kg bw/day was euthanized in moribund state on lactation day 3. Based on clinical observations and necropsy findings, large amount of blood loss at the parturition caused the poor condition of this dam. There were no similar findings at the higher dose, therefore the moribund state of this dam was considered to be a consequence of individual (not treatment related) disorder. Clinical signs of systemic toxicity related to the test item were not detected at any dose level at the daily clinical observations. The behavior and physical condition of the animals was not impaired at any dose level (50, 100 or 160 mg/kg bw/day) during the entire treatment period.

The body weight development was similar in male and female animals in the control and at 50, 100 and 160 mg/kg bw/day during the entire treatment period (pre-mating, mating and post-mating period for male animals; pre-mating, mating, gestation and lactation periods for female animals). Slight and transient changes in body weight gain at 160 mg/kg bw/day did not result in significant changes in the mean body weight in male or female animals during the pre-mating period (weeks 1 and 2).

The mean daily food consumption was not adversely affected in male or female animals at 50, 100 and 160 mg/kg bw/day during the entire treatment period. The examined parameters of the estrous cycle were comparable in all groups (control, 50, 100 and 160 mg/kg bw/day).

There were no significant or dose related differences between the control and test item treated female animals in the examined parameters of the delivery (50, 100 and 160 mg/kg bw/day).

A test item influence on the examined parameters of reproductive performance was not found at any dose level. The FT4 and TSH levels were not adversely affected in the parental male animals or in PN22 offspring at any dose levels.

Necropsy examinations revealed macroscopic alterations presumably related to the effect of the test item in the liver (pale or yellowish-brown color, nut-meg-like pattern) in male animals at 100 or 160 mg/kg bw/day and in the kidneys (paleness and enlargement) in male animals at 160 mg/kg bw/day. The weights of examined organs (absolute, relative to body and brain weights) were not affected by the test item in male animals at 50, 100 or 160 mg/kg bw/day.

Quantitative examinations of ovaries did not reveal toxicologically relevant differences between the control and 160 mg/kg bw/day group.

The offspring’s development was not adversely influenced at any dose level. No adverse effects on mortality, clinical signs, body weight development, anogenital distance (male and female) or nipple retention (male) were detected.

Under the conditions of the present study, TBPIN administered at 50, 100 or 160 mg/kg bw/day by oral gavage did not adversely influence the reproductive performance (gonad function, mating behavior, conception, parturition) in parental male and female Han:WIST rats as far as investigated in this study. There were no signs of systemic toxicity in male or female animals at 50, 100 or 160 mg/kg bw/day. The development of the F1 offspring was not impaired from birth to post-natal day 21 as far as investigated in this study after repeated oral administration of dams at 50, 100 or 160 mg/kg bw/day. There were no signs of systemic toxicity in male or female F1 animals at 50, 100 or 160 mg/kg bw/day after 14-day post-weaning treatment. Based on these observations the NOAEL were determined as follows:

NOAEL for systemic toxicity of male/ female rats: 160 mg/kg bw/day

NOAEL for reproductive performance of male/ female rats: 160 mg/kg bw/day

NOAEL for F1 Offspring: 160 mg/kg bw/day

TBPND

A modified OECD 421 study in rats was performed with TBPND. Rats were administered orally (by gavage) at doses of 50, 150 and 300 mg/kg bw/day and compared to control animals receiving vehicle only. Four groups of Han:WIST rats (n=12/sex/group) were administered with the test item orally (by gavage) once a day at 0 (vehicle), 50, 150 and 300 mg/kg bw/day doses corresponding to concentrations of 0, 25, 75 and 150 mg/mL. The application volume was 2 mL/kg bw. Control animals received the vehicle, sunflower oil. The suitability of the vehicle at the intended concentrations of the test item was analytically verified up front (concentration and homogeneity). The concentration of the test item in the dosing formulations used for animal’s treatment was checked three times during the study. Concentration of TBPND in the dosing formulations varied between the range of 95 % and 109 % of the nominal values and formulations were homogenous thereby confirming the proper dosing. All animals of the parent (P) generation were dosed prior to mating (70 days) and throughout mating. In addition, males received the test item or vehicle after mating up to the day before the necropsy (altogether for 99 days). Dams were additionally exposed through the gestation period and up to lactation days 21-23 (altogether for 114-127 days). Non-pregnant female animals were administered for 96 days. Observations included mortality, clinical signs, body weight, food consumption, mating, pregnancy and delivery process, as well as development of offspring. Estrous cycle was monitored by examining vaginal smears for two weeks before the start of mating period and during the mating period until evidence of mating. Vaginal smears were also prepared and investigated on the day of the necropsy for each dam. The dams were allowed to litter and rear their offspring up to days 21 post-partum then were subjected to necropsy on post-partum days 22 - 24. Litters were weighed and offspring were observed for possible abnormalities. One or two male and one or two female pups per litter were selected on post-natal day 21 and were separated and administered (2 mL/kg bw/day) from postnatal day 22 up to and including post-natal day 35. Clinical signs were observed daily after the treatment and body weight was determined twice weekly and were recorded. The food consumption was determined by weekly interval. All selected pups were subjected to macroscopic necropsy observation on post-natal day 36. Remaining pups were euthanized on post-natal day 22. Blood samples were collected for possible determination of serum levels of thyroid hormones (FT4, TSH) from 2-7 pups per litter (except for litters with 9 or less pups) on post-natal day 4, from all dams and from 3-8 pups per litter on post-partum/post-natal day 22 and from all parent male animals at termination. Non-pregnant female animals were sampled for thyroid hormone determination at termination, on Day 96.

All parental animals were subjected to gross pathology one day after the last treatment. The ovaries, uterus with oviduct and cervix, vagina, testes, epididymides (total and cauda), prostate, and seminal vesicles with coagulating glands, thyroid glands of all adult animals were preserved. In addition, based on macroscopic observations, skin of one female animal at 50 mg/kg bw/day, liver, spleen, heart, lungs in one dam at 150 mg/kg bw/day as well as the kidneys of some animal were also preserved for a possible histological examination. The body weight, brain weight, weight of the testes, epididymides and prostate and seminal vesicles with coagulating glands as a whole of all adult male animals were determined. A quantitative examination of ovaries was performed in all female animals in the control and high dose groups.

There was no test item related mortality at any dose level (50, 150 or 300 mg/kg bw/day). Clinical signs of systemic toxicity related to the test item were not detected at any dose level at the daily clinical observations. The behavior and physical condition of the animals was not impaired at any dose level (50, 150 or 300 mg/kg bw/day) during the entire treatment period. Salivation observed in male animals at 300 mg/kg bw/day was judged to be toxicologically not relevant because of the short duration immediately after the administration. The body weight development was not influenced by the test item at 50, 150 or 300 mg/kg bw/day during the pre-mating period (male or female animals) and during the post mating period (male). However, the mean body weight was depressed in dams at 300 mg/kg bw/day during the course of the last two weeks of gestation and during the lactation period. This change in the mean body weight was presumably in accordance with the lower number of fetuses of high dose treated dams. The mean daily food consumption was not adversely affected in male or female animals at 50, 150 and 300 mg/kg bw/day during the entire treatment period.

The examined parameters of the estrous cycle were comparable in all groups (control, 50, 150 and 300 mg/kg bw/day). The mean number of total birth, live born and alive pups was reduced at 300 mg/kg bw/day when compared to the actual control and historical control. A test item influence on the examined parameters of reproductive performance was not found at any dose level.

The FT4 and TSH levels were not adversely affected in the parental male animals or in PN22 offspring at any dose levels. Macroscopic alterations related to the effect of the test item were not detected at 50, 150 or 300 mg/kg bw/day (male or female) at the necropsy examinations. The weights of examined organs (absolute, relative to body and brain weights) were not affected by the test item in male animals at 50, 150 or 300 mg/kg bw/day. Quantitative examinations of ovaries revealed enhanced follicular atresia at 300 mg/kg bw/day with respect to their control, but no effects in primary, secondary or tertiary follicle counts.

The body weight development of the offspring was depressed at 150 and 300 mg/kg bw/day between post-natal day 0 and 21. No adverse effects on extrauterine mortality, clinical signs, anogenital distance (male and female) or nipple retention (male) were detected. F1 generation (post-natal days 22-35) The body weight development and food consumption of the F1 animals was reduced at 150 and 300 mg/kg bw/day from post-natal day 22 up to post-natal day 35. The difference with respect to the control was lower than 10 % at 150 mg/kg bw/day (male and female) and in female animals at 300 mg/kg bw/day. The reduction of body weight below 10 % is considered to be toxicologically not relevant. There were no clinical signs or necropsy findings in F1 generation (male and female) after the 14 days administration of 50, 150 or 300 mg/kg bw/day.

Based on these observations the NOAELs were determined as follows:

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

TBPPI

A modified OECD 421 study in rats was performed with the grouping substance TBPPI (CAS 927-07-1). The purpose of this study was to obtain information on the possible effects of the test item on reproduction and development when repeatedly administered orally (by gavage) to rats at doses of 100, 250 and 350 mg/kg bw/day compared to control animals receiving vehicle only. Four groups of Han:WIST rats (n=12/sex/group) were administered with the test item orally (by gavage) once a day at 0 (vehicle), 100, 250 and 350 mg/kg bw/day doses corresponding to concentrations of 0, 50 mg/mL, 125 mg/mL and 175 mg/mL. The application volume was 2 mL/kg bw. Control animals received the vehicle, sunflower oil. The suitability of the vehicle at the intended concentrations of the test item was analytically verified up front (concentration and homogeneity). The concentration of the test item in the dosing formulations used for animal’s treatment was checked three times during the study. Concentration of TBPPI-75-AL in the dosing formulations varied between the range of 90 % and 108 % of the nominal values and formulations were homogenous thereby confirming the proper dosing. All animals of the parent (P) generation were dosed prior to mating (70 days) and throughout mating. In addition, males received the test item or vehicle after mating up to the day before the necropsy (altogether for 100 days). Dams were additionally exposed through the gestation period and up to lactation days 21-23 (altogether for 114, 115, 118 or 125 days). Non-pregnant female animals and dam not delivered were administered for 95, 97, 98 or 102 days. Observations included mortality, clinical signs, body weight, food consumption, mating, pregnancy and delivery process, as well as development of offspring. Estrous cycle was monitored by examining vaginal smears for two weeks before the start of mating period and during the mating period until evidence of mating and on the day of the necropsy. The dams were allowed to litter and rear their offspring up to day 21 - 23 post-partum then were subjected to necropsy on post-partum day 22 - 24. Litters were weighed and offspring were observed for possible abnormalities. One or two male and one or two female pups per litter were selected on post-natal day 21 and were separated and administered (2 mL/kg bw/day) from postnatal day 22 up to and including post-natal day 35. Clinical signs were observed daily after the treatment and body weight was determined twice weekly and were recorded. The food consumption was determined by weekly interval. All selected pups were subjected to macroscopic necropsy observation on post-natal day 36. Remaining pups were euthanized on post-natal day 22. Blood samples were collected for determination of serum levels of thyroid hormones (FT4, TSH) from 2-5 pups per litter (except for litters with 9 or less pups) on post-natal day 4, from all dams and from 3-6 pups per litter on post-partum/post-natal day 22 and from all parent male animals at termination. Non-pregnant female animals and dam not giving birth were sampled for thyroid hormone determination at termination on the day of the necropsy (Days 95, 97, 98 or 102). All parental animals were subjected to gross pathology one day after the last treatment. The ovaries, uterus with oviduct and cervix, vagina, testes, epididymides (total and cauda), prostate, and seminal vesicles with coagulating glands and thyroid glands of all adult animals were preserved. In addition, organs with macroscopic findings were also preserved for a possible histological examination. A quantitative examination of the ovaries was performed in all female animals in the control and high dose groups. The body weight, brain weight, weight of the testes, epididymides and prostate and seminal vesicles with coagulating glands as a whole of all adult male animals were determined.

There was no mortality at any dose level (100, 250 or 350 mg/kg bw/day). Clinical signs of systemic toxicity related to the test item were not detected at any dose level at the daily clinical observations. The behavior and physical condition of the animals was not impaired at any dose level (100, 250 or 350 mg/kg bw/day) during the entire treatment period. Salivation and nuzzling up the bedding material observed at 250 and 350 mg/kg bw/day were with short duration after the administration of the test item therefore these signs were considered to be toxicologically not relevant.

The body weight development was depressed in male animals at 250 and 350 mg/kg bw/day during the pre-mating, mating and post-mating periods. The difference with respect to the control was lower than 10 % at 250 mg/kg bw/day and therefore was judged to be toxicologically not relevant.

The mean daily food consumption was slightly reduced in male animals at 350 mg/kg bw/day in accordance with the body weight changes during the entire treatment period. Estrous cycle The examined parameters of the estrous cycle were not adversely affected in any group (control, 100, 250 and 350 mg/kg bw/day). Delivery data of dams There were no significant or dose related differences between the control and test item treated female animals in the examined parameters of the delivery (100, 250 and 350 mg/kg bw/day).

A test item influence on the examined parameters of reproductive performance was not found at any dose level. Serum thyroid hormones FT4 and TSH levels were not adversely affected in the parental male animals or in PN22 offspring at any dose levels.

Necropsy examinations did not reveal specific macroscopic alterations related to the effect of the test item in male or female animals at 100, 250 or 350 mg/kg bw/day.

The weights of examined organs (absolute, relative to body and brain weights) were not affected by the test item in male animals at 100, 250 or 350 mg/kg bw/day.

There were no toxicologically relevant differences in the mean number of primordial, primary, secondary, tertiary follicles, atresia or corpora lutea at 350 mg/kg bw/day at the quantitative examination of ovaries.

The offspring’s development was not adversely influenced at any dose level. No adverse effects on mortality, clinical signs, body weight development, anogenital distance (male and female) or nipple retention (male) were detected. There were no clinical signs, changes in body weight or mean daily food consumption or necropsy findings in F1 generation (male and female) after the 14 days administration of 100, 250 or 350 mg/kg bw/day.

Based on these observations the NOAELs were determined as follows:

NOAEL for systemic toxicity of male rats: 250 mg/kg bw/day

NOAEL for systemic toxicity of female rats: 350 mg/kg bw/day

NOAEL for reproductive performance of male/ female rats: 350 mg/kg bw/day

NOAEL for F1 Offspring (PN 0-21): 350 mg/kg bw/day

NOAEL for F1 generation (PN 22-35): 350 mg/kg bw/day

Overall Conclusion

In the OECD TG 421 study performed in 2010, higher mortality, clinical signs and depressed body weight development for F1 offspring was considered to be the consequence of maternal toxicity, which was observed in this study at 160 and 400 mg/kg bw/day.

The 10-week pre-mating exposure to TBPIN in a modified OECD TG 421 study did not adversely affect reproductive performance and fertility of male and females rats up to the highest dose of 160 mg/kg bw/day. For the peroxyesters TBPPI and TBPND the results for the modified. i.e. extended 10-weeks premating exposure OECD 421 studies also show no adverse effects on the parameters of reproductive performance of male and female rats. The applied 10-weeks pre-mating exposure is recommended in the OECD 443 study design. For all three peroxyesters, no adverse effects on estrous cycle and ovaries (follicle counts) have been observed in the modified OECD 421 studies. The development of the F1 was not adversely effected after exposure to TPBIN and TBPPI in the modified OECD 421 studies up to the highest doses tested. For TBPND the body weight development of the offspring was depressed up to PND 36 at mid and high dose.

Based on the results from the modified OECD 421 studies for the three peroxyesters only one study according OECD 443 with TBPND was initiated for the peroxyester group. This substance was chosen since some effects on F1 offspring have been reported for TPBND, which was not the case for the two other peroxyesters. Therefore, read-across to the initiated OECD 443 study performed with TBPND will be a worst case approach. Furthermore,  ECHA requested for TBPND to include Cohort 3 (immunotoxicity, CCH-D-2114493102-56-01/F). The study results from this OECD TG 443 study are used in a read-across grouping approach for the peroxyesters TBPIN and TBPPI to fulfil ECHAs requests on EOGRT studies with these peroxyesters (CCH-D-2114493103-54-01/F and CCH-D-2114493105-50-01/F, respectively). This approach was also chosen because of animal welfare reasons.

OECD TG 443 (2022)

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

In F1 offspring pre-weaning, depressed development (pinna detachment, eye opening, body weight) were observed at 450 mg/kg bw/day. However, this is considered secondary to maternal toxicity and as a nonspecific effect. 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.

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 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, 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. 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.

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

Effects on developmental toxicity

Description of key information

Under the conditions of a guideline and GLP compliant study, the no observed effect level (NOEL) for tert-Butylperoxy-3,5,5-trimethylhexanoat for maternal toxicity was 150 mg/kg bw /day and for developmental toxicity was 20 mg/kg bw/day for male and female rats. The no observed adverse effect level (NOAEL) for maternal and developmental toxicity was 150 mg/kg bw/day for male and female rats.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

150 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The study is considered acceptable without restrictions as it was conducted according to GLP regulations and OECD/EU guideline.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

OECD 414 rat

Groups of 24 sperm-positive female Hsd. Brl. Han: WISTAR rats were treated with TBPIN by oral administration daily at three dose levels of 20, 50 and 150 mg/kg bw/day from day 6 up to and including day 19 post coitum. A control group of 25 sperm positive females was included and the animals were given the vehicle sunflower oil. The treatment volume was 2 ml/kg/bw. During the study, mortality was checked for and clinical observations were performed. Body weight and food consumption of the dams were also recorded. The day when sperm was detected in the vaginal smear was regarded as day 0 of gestation. A Caesarean section and gross pathology were performed on gestational day 20. The number of implantations, early and late resorptions, live and dead fetuses in each uterine horn and the number of corpora lutea were recorded. Each fetus was weighed and examined for sex and gross external abnormalities. The placentas were weighed and examined externally. About half of each litter was preserved for visceral examination and the other half of the litters were preserved for skeletal evaluation. At visceral examination the bodies were micro dissected by means of a dissecting microscope. The heads were examined by Wilson's free-hand razor blade method. After cartilage-bone staining the skeletons were examined by means of a dissecting microscope. All abnormalities found during the fetal examinations were recorded. In total, there were 19, 20, 21 and 22 evaluated litters in the control, 20, 50 and 150 mg/kg groups, respectively. Two pregnant females died in the course of the study, one female in the 50 mg/kg bw/day group on gestational day 17 and one in the 150 mg/kg bw/day dose group on gestational day 9. The death and clinical signs such as reduced body tone and reduced activity shortly before death of the female in the mid dose group and the death and necropsy findings (brownish-yellowish discolouration in the whole lung tissue and dark areas in the liver) of the dam at 150 mg/kg bw/day were considered to be likely without a relationship with an effect of the test item. There were no clinical signs and necropsy findings recorded for the survived dams in the experimental groups. There was no indication of an effect of the test item on body weight, corrected body weight and food consumption of the dams in the 20, 50, 150 mg/kg bw/day dose groups. There was no effect indicated related to the administration of TBPIN in the intrauterine mortality of the conceptuses, the number of implantations, viable fetuses and their sex distribution. The mean fetal weight, was similar in the control and 20 mg/kg bw/day groups. The slight but statistically significant reduction in the bodyweight of the fetuses in the 50 mg/kg bw/day and 150 mg/kg bw/day dose groups might be attributed to an effect of the test item. Since fetal weights were still within the range of the laboratory´s historical control data the weight reduction was considered as non-adverse. Relative placental weight was similar in all experimental groups.

The increased incidence of external variations i.e. growth retarded fetuses in the 150 mg/kg bw/day dose group was within the laboratory´s historical control range and thus, considered as non-adverse.

The presence of the fetal malformations found at external examination (one in the control and one in the high dose group) was considered to be incidental.

The visceral malformations were considered to be incidental and were not attributed to the administration of the test item to the dams. The incidence of visceral variations was similar to the vehicle control level.

Skeletal malformations in two fetuses of the low dose group and in one fetus of the control group were without a relationship to the test item. Statistically significant skeletal variations were seen in the 20 and in the 150 mg/kg bw/day dose group when the ossification of sternum (3 or less ossified sternebra) was evaluated. Considering that there was no dose response indicated between the low and mid dose group and that the mean percent of the high dose group was slightly above the laboratory´s historical control level, this variation was judged to be in the biological variations.

Incomplete ossification of the skull-bones (marked and less marked) was evaluated as variation during skeletal examination. Incomplete ossification of the skull bones correlated with the slightly lower mean fetal weight observed at 50 and 150 mg/kg bw/day dose groups, which was below the mean value seen in the laboratory´s historical control data. However, the data pool of laboratory´s historical control data comprises only a few studies and thus, it is not considered that the whole range of this rat strain is reflected. In general, retarded skull bone ossification is a relatively common observation and may not be a reliable indicator for developmental toxicity (Mylchreest et al., 2005). The maximum incidence of incomplete ossification of 8% observed in this study is within the normal range reported for rats (e.g. Mylchreest et al., 2005, RMS France, 2007). Moreover, since the incomplete ossification was observed in skull bones and not in bones that are normally well ossified the effect is classified as a low significant finding which may not be adverse according to Carney and Kimmel (2007). Overall, incomplete ossification of the skull bones (marked and less marked) seen at 50 mg/kg bw/day and at 150 mg/kg bw/day is considered to be non adverse.

 

Based on these observations the No Observed Effect Level (NOEL) was determined as follows:

NOELmaternal toxicity: 150 mg/kg bw/day

NOELdevelopmental toxicity: 20 mg/kg bw/day

 

Based on these observations the No Observed Adverse Effect Level (NOAEL) was determined as follows:

NOAELmaternal toxicity: 150 mg/kg bw/day

NOAELdevelopmental toxicity: 150 mg/kg bw/day

 

REFERENCES

- Carney, E.W. and C.A. Kimmel (2007): Interpretation of skeletal variations for human risk assessment: delayed ossification and wavy ribs. Birth Defects Res., 80:473-496

- Mylchreest, E.et al. (2005): Evaluation of the developmental toxicity of 8-2 Telomer B Alcohol.Drug and Chemical Toxicology., 28:315-328

- Rapportour Member State (RMS) France (2007): Draft Assessment Report, Initial risk assessment provided by the rapporteur Member State France for the existing active substance Fluazifop-P-Butyl of the third stage (part A) of the review programme referred to the Article 8(2) of Council Directive 91/414/EEC.Volume 3, Annex B, part 4, B.8

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on reproduction and developmental toxicity the test item is not classified according to Regulation (EC) No 1272/2008 (CLP), as amended for the eighteenth time in Regulation (EU) 2022/692.