Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
extended one-generation reproductive toxicity - with F2 generation (Cohorts 1A, and 1B with extension)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01-06-2018 to 09-2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Version / remarks:
2012
Deviations:
no
GLP compliance:
yes (incl. certificate)
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 the authorities

- Basis for dose level selection: based on NOAELs obtained in repeated dose toxicity studies

- Inclusion/exclusion of extension of Cohort 1B: in the course of the study an impairment of the female reproductive performance was observed which triggered the extension of Cohort 1B to produce the F2

- Termination time for F2: as given in the guideline (PND 4)

- Inclusion/exclusion of developmental neurotoxicity Cohorts 2A and 2B : not triggered by available data with the test item

- Inclusion/exclusion of developmental immunotoxicity Cohort 3: not triggered by available data with the test item

- Route of administration: gavage

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
Purity batch
> 99.0 % 7.32% active oxygenBatch 247408973
99.27 % 9407226339; 247408999
99.6 % 0410105225413
> 97 % 4289070300; 0419101226056
99.1 % 11600 0010-108
99.3 % 247413988
99.4 % 7.35 % active oxygen Batch 247409800
98.9 % 0707518256
> 99.1 % 247412384
98.5 % 0707603153
99.0 % 1703465433
99.5 % 150726; 247413309
99.3 % 000055055

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals 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: not older than 9 weeks

- Weight at study initiation: (P) Males: 256-309 g; Females: 151-184 g

- Fasting period before study: no

- Housing:
Before mating: 2 animals of the same sex/cage
Mating: 1 male and 1 female/cage
Pregnant females were housed individually.
Males after mating: 2 animals/cage
F1 offspring (after weaning): 2 animals of the same sex/cage

- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 13 days

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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: Sunflower oil (Helianthi annui oleum raffinatum)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

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: 20, 60, 200 mg/mL
- Treamtent volume: A constant treatment volume of 5 mL dose preparation/kg body weight was administered in all groups.
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

- Any other deviations from standard protocol: since 8 out of 24 females of the high dose group turned out to be non-pregnant (for all of them positive vaginal smear was obtained) corresponding 8 male partners were mated with untreated females in order to assess the reason of non-pregnancy, according to the guideline.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
A sufficient stability and homogeneity in the chosen vehicle was verified over the range of relevant concentrations at the appropriate frequency of preparation. Recovery from sunflower oil was 104 % and 98 % of nominal concentrations at ca. 2 mg/mL and ca. 500 mg/mL, respectively. The test item was stable at the intended concentrations for 24 hours at room temperature and for three days in a refrigerator (5 ± 3°C).
Duration of treatment / exposure:
P0 males: 153 - 156 days
P0 females: 114 - 129 days

F1
Cohort 1A: approx. 90 days (13 weeks)
Cohort 1B: approx. 120 days (17 weeks)
Frequency of treatment:
daily, 7 days/week
Details on study schedule:
- Selection of parents from F1 generation when pups were 21 days of age.
- Age at mating of the mated animals in the study: P0 animals first mating: 19 weeks; P0 males second mating (high dose and control): 20 weeks; P1/F1 animals: 13 weeks
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
vehicle control
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
P0: 24 / sex / dose

P1/F1: 20 / sex / dose
Control animals:
yes, concurrent vehicle
yes, historical
Details on study design:
- Rationale for animal assignment: random
Positive control:
not applicable

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: same as weighing

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 was be additionally weighed on gestation day 10 in order to give accurate treatment volumes, but these data were not evaluated statistically. Body weight data were reported individually for adult animals.

F1 animals selected for follow-up examinations were weighed on post-natal day 22, then twice a week during the two weeks following weaning, and once weekly thereafter.
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
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: daily by visual inspection

Oestrous cyclicity (parental animals):
Estrous cycle was monitored by examining vaginal smears from each parental female animal daily for two weeks before the mating started.
Vaginal smear was also prepared and estrous cycle was monitored daily during the mating period until evidence of copulation.
Vaginal smear was also 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 necropsy. Both numbers of motile and immotile sperms were recorded. Two samples were prepared from each animal. For the determination of sperm motility, the mean percentage of motile sperm was 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 was performed later.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 / F2 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

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: No
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals as soon as possible
- Maternal animals: All surviving animals after the litter was weaned (P0); at PND4 (F1, cohort 1B)

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 in high dose and control animals:

Parental animals and 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 unit (with their fluids)
- brain
- liver
- kidneys
- heart
- spleen
- thymus
- pituitary
- thyroid glands (post-fixation)
- adrenal glands

Animals of Cohort 1B:
- 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

Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals were sacrificed at 21 days of age, and all F2 offspring on PND 4 or shortly thereafter.
- These animals were subjected to postmortem examinations macroscopic and microscopic examination as follows:

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

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated above were prepared for microscopic examination and weighed, respectively.
Statistics:
The statistical evaluation of appropriate data was performed with the statistical program package SPSS PC+4.0.

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 assess the significance of inter-group 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. Frequency of toxic response, pathological and histopathological findings by sex and dose were calculated.
Reproductive indices:
Copulatory Index (Measure of animals ability to mate):
Males: Number of males with confirmed mating / Total number of males cohabited x 100
Females: Number of sperm positive females / Total number of females cohabited x 100

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

Gestation Index (Measure of pregnancy that provides at least one live pup):
Number of females with live born pups / Number of pregnant females x 100
Offspring viability indices:
Formulas for Calculation of Pup Mortality and Sex Ratio Indices:

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

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

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

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

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Adverse signs of systemic toxicity related to the test item treatment were not detected at any dose level at the daily clinical observations (100, 300 and 1000 mg/kg bw/day, parental male or female).

Salivation and nuzzling up the bedding material were detected in male and female animals at 300 and 1000 mg/kg bw/day with variable incidence and duration. These observations were related to the treatment/test item and were considered to be toxicologically not relevant because of the transient occurrence and short duration after the administration.

The parental male animals were normal in control group during the entire observation period.
Reddish colored hair on the forelimbs was noted for one parental male animal (1/24) at 100 mg/kg bw/day as individual findings between Days 63 and 75.
Salivation (10/24 at 300 mg/kg bw/day, 24/24 at 1000 mg/kg bw/day) and nuzzling up the bedding material (7/24 at 300 mg/kg bw/day, 24/24 at 1000 mg/kg bw/day) were observed in parental male animals with variable incidence and in a dose related manner shortly after the administration for some days/weeks.

Parental female animals in the control (24/24) and 100 mg/kg bw/day (24/24) groups were normal during the pre-mating, mating, post-mating and gestation periods. Alopecia on the abdomen was detected in one control dam (1/21) between lactation days 16 and 20.
Salivation and nuzzling up the bedding material were noted for parental female animal at 300 mg/kg bw/day (6/24) and at 1000 mg/kg bw/day (24/24) during the pre-mating period, as well as during the post-mating period (8/8) and gestation period (3/16 and 12/16, respectively) only at 1000 mg/kg bw/day.
In one dam at 1000 mg/kg bw/day, sanguineous vaginal orifice was observed on lactation day 1 probably as a late consequence of delivery.
Alopecia was also observed in some female animals at 1000 mg/kg bw/day as follows:
- in two dams (2/16) under the right ear then both ears from Day 35 up to lactation day 17 or on the forelimbs and base of the tail (1/16) between lactation days 5 and 21;
- in non-pregnant female animals (2/8) between the ears between Day 63 and 94 and on the abdomen on Days 98 and 99;
Alopecia on the skin is a species-specific finding, which is also observed in untreated experimental rats of this strain with similar age. These were individual findings with low incidence in animals of control or lower dose groups and were thus not considered related to the treatment.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
There was no test item related mortality in parental animals in 100, 300 or 1000 mg/kg bw/day groups (male or female) during the course of study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The body weight development was continuously reduced in parental male animals administered with 1000 mg/kg bw/day.

The mean body weight was comparable to the control in male animals at 100 and 300 mg/kg bw/day during the entire observation period (pre-mating, mating and post-mating periods). Some sporadic statistically significant difference with respect to the control was detected in the mean body weight gain of male animals at 100 and 300 mg/kg bw/day (lower or higher). However, these minor differences in the mean body weight gain had no influence on the mean absolute body weight of male animals.

Statistical significances were detected at the permanently lower mean body weight in male animals at 1000 mg/kg bw/day from Day 28 up to termination of the study (Day 152; up to -13% of the control). The mean body weight gain of male animals at 1000 mg/kg bw/day was lower than in the control group by weekly interval in the most cases during the observation period and if summarized for the whole study (between Days 0 and 152). The difference to the control reached statistical significance in several cases in male animals at 1000 mg/kg bw/day.

The mean body weight and body weight gain was comparable in the control and test item treated female animals at 100, 300 and 1000 mg/kg bw/day during the pre-mating, gestation and lactation periods. Statistical significance was only noted for the slightly higher mean body weight of dams at 1000 mg/kg bw/day on lactation day 21.
Similarly, some sporadic statistical significance was observed at the lower or higher mean body weight gain of female animals during the pre-mating period at 100, 300 or 1000 mg/kg bw/day and between lactation days 14 and 21 at 1000 mg/kg bw/day and if summarized for lactation period (between lactation days 0 and 21). These changes in body weight gain had no toxicologically relevance as there was no significant influence on the mean body weight.
Therefore, these minor statistically significant differences with respect to the control in male animals at 100 and 300 mg/kg bw/day and in female animals at 100, 300 and 1000 mg/kg bw/day had no biological significances during this study.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The food consumption was not adversely affected in parental male or female animals at 100, 300 and 1000 mg/kg bw/day.

Considering the body weight changes and food consumption of male animals at 1000 mg/kg bw/day, a slightly worse feed efficiency is presumed during the post-mating period. The mean daily food consumption of parental male animals was slightly lower than in the control group at 100 mg/kg bw/day between Days 104 and 118 and at 300 mg/kg bw/day between Days 11 and 118.

In the male animals at 1000 mg/kg bw/day, the mean daily food consumption was slightly higher than in the control group from week 10 (between Day 63-69) reaching statistical significances in most cases by weekly interval.

In the parental female animals, statistical significances were observed at the slightly higher mean daily food consumption at 100 mg/kg bw/day between days 7-14, 35-42, 63-69 and at 300 mg/kg bw/day between Day 7-14 during the pre-mating period.
In the parental female animals, statistical significance with respect to the control was observed at the lower mean daily food consumption between Days 0 and 7 and at the slightly higher mean daily food consumption between Days 7-14 and 63-69.
The mean daily food consumption was similar in the control and test item administered female animals during the gestation and lactation period except for dams at 1000 mg/kg bw/day during one week of lactation period. Statistical significance was noted for the lower mean daily food intake of dams at 1000 mg/kg bw/day between lactation days 7 and 14.
These slight differences with respect to the control were of low degree and without consistency during the treatment period. Therefore, these were not considered to be toxicologically relevant.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
There were no test item related adverse changes in the examined hematological parameters in parental male or female animals at 100, 300 or 1000 mg/kg bw/day.

In the male animals, statistical significances were detected at the slightly shorter mean prothrombin time (PT) at 1000 mg/kg bw/day when compared to the control. All other examined parameters were comparable in male animals in the control and 100, 300 and 1000 mg/kg bw/day.
Statistical significance was detected at the slightly higher mean percentage of reticulocytes (RET) in female animals at 100 and 1000 mg/kg bw/day and at the slightly higher mean hematocrit value (HCT) at 300 mg/kg bw/day when compared to the control. All other examined hematological and blood coagulation parameters were comparable in female animals in the control and 100, 300 and 1000 mg/kg bw/day groups.
The individual values PT, RET and HCT were well within the historical control range in male or female animals, where relevant. There were no related changes in other hematological parameters. tTherefore, the differences in these parameters were considered to have little or no toxicological relevance.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
The examined clinical chemistry parameters were not adversely affected in parental male or female animals at 100, 300 or 1000 mg/kg bw/day.
Clinical chemistry investigations revealed a slightly lower mean activity of aspartate aminotransferase (AST) at 300 mg/kg bw/day and lower mean concentration of total protein (TPROT) at 1000 mg/kg bw/day in the male animals.
In the female animals, statistically significant difference with respect to the control was detected at the lower mean concentration of creatinine (CREA) at 100 mg/kg bw/day.
All examined parameters were comparable with the control in the female animals at 300 mg/kg bw/day.
In the female animals at 1000 mg/kg bw/day, lower mean concentration of creatinine and higher mean concentrations of urea and cholesterol (CHOL) were observed when compared to the control.
The changes in AST, TPROT, CREA, UREA and CHOL were judged to be related with corresponding organ weight increases (liver and kidney) and presumed increased metabolic and excretory activity. Toxicological relevance due to the minor degree of changes in these parameters is, however, questionable.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
There were no test item related adverse changes in the examined urine parameters in parental male or female animals at 100, 300 or 1000 mg/kg bw/day.

Statistically significantly higher mean volume (male, +65 % and female, +32 %) and lower pH in males and females (without reaching statistical significance) of the urine with respect to their control were observed. Considering findings of increased kidney weight these observations might be related to test item treatment (e. g. increased metabolic and excretion functionality) despite the lack of changes in the related parameters (clinical chemistry, histopathology) at 1000 mg/kg bw/day. Therefore, these findings were considered to be indicative of metabolic and excretory activity and not about adverse effects on the kidneys.
In the parental male animals, no statistical significances were detected at the higher mean volume of the urine at 100 and 300 mg/kg bw/day.
At 1000 mg/kg bw/day, the volume of urine was higher and the pH of the urine was lower than in the control group. Positive sediment was detected in all male animals at 1000 mg/kg bw/day due to the presence of larger amount of crystals (uric acid and amorphous crystals).
The examined urine parameters were comparable in the parental female animals in the control, 100 and 300 mg/kg bw/day groups. Statistical significance was detected at the higher mean volume of the urine at 1000 mg/kg bw/day when compared to the control.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The weights of kidneys (absolute, relative to body and brain weights) were elevated in parental male animals at 100, 300 or 1000 mg/kg bw/day in a dose-related manner. The liver weights also exceeded the control value in parental male and female animals at 1000 mg/kg bw/day.
Based on the results of histological investigations, the high dose of the test item was a predisposing factor in the pathogenesis of renal lesions (chronic progressive nephropathy). In the lack of related histological alteration, liver weight changes were probably due to the enhanced activity, i. e. xenobiotic metabolism and excretion of the organ.
In the male animals at 100 mg/kg bw/day, statistical significance was detected at the slightly higher mean kidneys weights and epididymides weights – absolute and relative to brain weight for both organs.
At 300 mg/kg bw/day, statistical significance was noted for the higher mean liver weight relative to body weight, higher mean kidneys weights (absolute and relative to body and brain weights) and higher mean epididymides weight relative to brain weight in parental male animals.
In the male animals at 1000 mg/kg bw/day, statistical significances were observed at the absolute mean weights of liver, kidneys (higher both), brain, heart, thymus, prostate and fasted body weight (lower each) when compared to the control. Similarly, the organ weights relative to body weight were statistically significantly higher than in the control in case of brain, liver, kidneys, heart, spleen, testes, epididymides, seminal vesicle, adrenal glands, thyroid glands and pituitary. The differences in the organ weights relative to body weight were partly originated from the significantly lower fasted body weight of parental male animals at 1000 mg/kg bw/day and are thus not directly but only secondary associated with the test item treatment.
The organ weights relative to brain weight were significantly higher than in the control in case of liver, kidneys, testes and seminal vesicles while it was lower than in the control in case of prostate and fasted body weight relative to brain weight in parental male animals at 1000 mg/kg bw/day.
In the female animals at 100 mg/kg bw/day, brain weight relative to body weight was lower and the body weight relative to brain weight was higher than in the control group.
At 300 mg/kg bw/day a slightly lower mean brain weight relative to body weight and higher mean liver weights (absolute and relative to brain weight) and higher mean body weight relative to brain weight were detected in parental female animals when compared to the relevant control.
In the female animals at 1000 mg/kg bw/day, higher mean weights of liver (absolute, relative to body and brain weights), kidneys (relative to brain weight) and higher mean body weight relative to brain weight were observed comparing to their control. The brain weight (absolute and relative to body weight), heart and pituitary weights (both relative to body weight) and body weight relative to brain weight were lower than in the control group.
A test item influence was supposed in development of the higher mean weights of kidneys (male) and liver (male and female). Histological examinations revealed chronic progressive nephropathy in male animals at 1000 mg/kg bw/day.
Hepatocellular damage was not detected at the histopathological examination and hematology investigations as well as clinical chemistry parameters did not reveal test item related abnormalities. Therefore, liver weight changes were considered to be of an adaptive nature.
The statistically significant differences with respect to the control at several organs (brain, heart, spleen, testes, epididymides, seminal vesicle, prostate, thymus, adrenal glands, thyroid glands or pituitary) were judged to have little or no toxicological relevance due to the minor degree and in the lack of associated histopathological alterations.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic alterations related to the effect of the test item were not detected in male or female animals at 100, 300 or 1000 mg/kg bw/day at the necropsy.

In the parental male animals in control group, thymic hemorrhage (1/24), nutmeg-like patterned liver (1/24), renal pyelectasia (1/24, right side) smaller than normal seminal vesicle (1/24; one side) and hard tissue formation in the abdominal fatty tissue (1/24) were observed at the necropsy.
In the male animals at 100 mg/kg bw/day, hemorrhages in the thymus (1/24), congestive mucous membrane in the stomach (2/24), pyelectasia (1/24, left side) and hard tissue formation in the abdominal fatty tissue (3/24) were observed were detected at the necropsy.
At 300 mg/kg bw/day, thymic hemorrhage (3/24), brown black colored lungs (1/24), congestive mucous membrane in the stomach (1/24), and pyelectasia (4/24, right side or both sides) were noted for some male animals.
At 1000 mg/kg bw/day, hemorrhage in the thymus (1/24), congestive mucous membrane in the stomach (3/24) and pyelectasia (2/24, right side) were seen at the necropsy.
In control dams, congestive mucous membrane in the stomach (4/21), pyelectasia (1/21, both sides), hydrometra (5/21, slight, moderate or marked) and reddish colored mesenterial lymph nodes (1/21) were observed.
At 100 mg/kg bw/day, congestive mucous membrane in the stomach (10/23), pyelectasia (3/23, right or both sided), hydrometra (4/23, slight or moderate) were noted for dams.
In dams at 300 mg/kg bw/day, hemorrhage in the thymus (1/23), congestive mucous membrane in the stomach (8/23), pyelectasia (3/23, right or both sided) and hydrometra (3/23, marked or moderate) were observed.
At 1000 mg/kg bw/day, hemorrhage in the thymus (1/16), congestive mucous membrane in the stomach (3/16), pyelectasia (3/16, left or right side) and hydrometra (2/16, slight or marked) were detected.
In non-pregnant female animals, pyelectasia (1/1 at 100 and 200 mg/kg bw/day, both, right or both sided), ovarian cyst (1/8 at 1000 mg/kg bw/day), hydrometra (1/2 control, slight; 4/8 at 1000 mg/kg bw/day, slight or marked), hard knot at the cervix of uterus (1/1 at 300 mg/kg bw/day) and alopecia on the thorax (1/8 at 1000 mg/kg bw/day) were detected at the necropsy.
Not mated control female animal showed marked hydrometra.
Congestive mucous membrane (male animals) and hemorrhage in the stomach mucosa (female) were probably due to the local effect of the test item or treatment procedure. There was no dose response relationship in the incidence of these findings or related changes. Therefore, changes in the stomach mucosa were considered toxicologically not relevant.
Hydrometra (i.e. dilatation of uterine horns), related to the female sexual cycle, is a frequent observation in experimental rats. In the lack of related inflammatory or other pathological signs, it was judged to be toxicologically not relevant and not test item related as no dose response was noted.
Hard knot in the fatty tissue of abdominal cavity, ovarian cyst, hard knot at the cervix and alopecia on the skin are common macroscopic findings in experimental rats of this strain with similar age. These occurred with low incidence and were considered to be toxicologically not relevant.
Pyelectasia is frequently observed in this strain of experimental rats. Histological examination did not reveal degeneration, inflammation or fibrosis. Therefore, this finding was considered as slight individual lesion without toxicological significance.
Hemorrhages in the lungs or thymus are indicative of circulatory disturbance developing during the exsanguination.
Smaller than normal seminal vesicle, nutmeg-like patterned liver and reddish colored mesenterial lymph nodes are individual findings in control animals (male or female).
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The investigated organs of reproductive system (testes, epididymides, prostate seminal vesicles, coagulating glands) were histologically normal and characteristic onfor the sexually mature organism in all parental male animals in the control and 1000 mg/kg bw/day groups. Decreased number of developing follicles and increased number of follicular atresia were detected in female animals at 1000 mg/kg bw/day (pregnant or non-pregnant) compared with their control (on the actual level of section investigated). Histopathological investigations revealed chronic progressive nephropathy in a higher incidence of male animals at 1000 mg/kg bw/day with respect to the control. In the parental male animals in the control and 1000 mg/kg bw/day groups the investigated organs of reproductive system (testes, epididymides, prostate seminal vesicles, coagulating glands) were histologically normal and characteristic on the sexually mature organism in all cases. The various spermatogenic cells (the spermatogonia, the spermatocytes, the spermatids and spermatozoa) representing different phases in the development and differentiation of the spermatozoons and the interstitial cells were the same in quantity and morphologically in the testes of investigated control and high dose treated animals.
The histological picture of epididymides, prostate, seminal vesicles, and coagulating glands was normal in all cases as well, except for one control male animal (1/24). In this animal, decreased amount of secrete in the seminal vesicle (one side) was observed. This phenomenon, without inflammatory or degenerative lesion was considered as individual disorder, without toxicological significance.
In the female animals of the control and 1000 mg/kg bw/day groups, the ovaries, uterus, cervix, vagina 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. In addition, in non-pregnant female animals (8/8) at 1000 mg/kg bw/day decreased umber of developing follicles and increased number of follicular atresia were observed along with developing follicles and corpora lutea on the actual level of section.
In addition, the mean number of the developing follicles was lower and the mean number of follicular atresia was higher than in the control group at 1000 mg/kg bw/day at the examined level of histological section. This finding was more excessive in non-pregnant female animals at 1000 mg/kg bw/day (8/8).

In three female animals at 1000 mg/kg bw/day, one or both sided follicular cyst (3/24) was detected in the ovaries. The mucous membrane of uterus, cervix and vagina was normal in these female animals similar to that in the control group.
The effect of high dose of test item could be considered in the development of decrease in the number of developing follicles, and the increase in the number of follicular atresia and the follicular cyst forming, in the high dose treated female animals.
Follicular atresia is a normal, physiological process in the ovary, to regulate the number of follicles in the developing pool and increase in follicular atresia can be observed secondary to xenobiotic administration. Since the development of small parental follicles is gonadotropin independent, an increase in atresia in these follicles is typically seen with direct-acting cytotoxic compounds, heavy metals or radiation.
In our study the follicular atresia affected only partly the ovarian functions, but absence of corpora lutea (lack of ovulation) or total ovarian atrophy was not detectable.
The 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.
In some cases, dilatation of uterine horns was observed (7/24 control; 5/24 at 100 mg/kg bw/day; 4/24 at 300 g/kg bw/day; 7/24 at 1000 mg/kg bw/day). This finding – without inflammatory or other pathological lesions – is a slight neuro-hormonal phenomenon and is in connection with the normal sexual cycle (proestrus phase) of uterus without pathological significance.
Histological examination revealed the earliest stage of chronic progressive nephropathy (CPN) in a proportion of male animals in control group (5/24) and in the 1000 mg/kg bw/day group (12/24). Scattered tubular dilatation, hyaline casts, tubular basophilia, lymphocytic and histiocytic infiltrations were observed. CPN is a spontaneous renal disease of the commonly used strains of laboratory male rat. In this study, CPN was seen with a higher incidence in male animals at 1000 mg/kg bw/ comparing to the control, Therefore, it is presumed, that the high dose of test item was a predisposing factor in the pathogenesis of this renal lesions.
Damage of mucous membrane of the stomach was observed in several parental male and female animals as follows:
Erosion and congestion:
male animals: 1/2 at 100 mg/kg bw/day, 1/1 at 300 mg/kg bw/day; 3/24 at 1000 mg/kg bw/day;
female animals: 3/10 at 100 mg/kg bw/day; 8/8 at 300 mg/kg bw/day; 3/24 at 1000 mg/kg bw/day;
Congestion:
male animals: 1/2 at 100 mg/kg bw/day
female animals: 2/24 control; 7/10 female at 100 mg/kg bw/day;
Mucosal damage in the stomach is a frequent observation in animals administered by gavage. Although, there were no dose response relationship in the incidence of findings in this study, erosion was detected only in the test item treated animals. A local effect of the test item might be supposed.
Pyelectasia occurred in parental male and female animals: 2/24, 1/1, 4/4, 2/24 in male animals and 0/24, 4/4, 4/4, 3/24 in female animals, respectively to groups of control, 100, 300 and 1000 mg/kg bw/day. Pyelectasia without other histopathological lesions (for example degeneration, inflammation, fibrosis etc.) is considered as an individual disorder without toxicological significance. No histological signs of chronic progressive nephropathy (CPN) were observed in these kidney samples except for one male animal.
Alveolar emphysema (minimal degree) in the lungs (1/24 male control; 1/24 female control), and acute hemorrhage in the thymus (1/1 male at 100 mg/kg bw/day, 3/3 male at 300 mg/kg bw/day; 1/24 male at 1000 mg/kg bw/day; 1/1 female at 300 mg/kg bw/day) occurred sporadically and are considered as consequence of hypoxia, dyspnea and circulatory disturbance developed during exsanguinations.
Hyperplasia of bronchus associated lymphoid tissue (BALT) in some control and treated animals (2/24 male and 1/24 female control; 1/24 male and 1/24 female at 1000 mg/kg bw/day) is an immuno-morphological phenomenon, without toxicological significance.
Alveolar histiocytosis accompanied with congestion in the lungs in one male animal at 300 mg/kg bw/day (1/1) is a common incidental finding in elder rats and consists of small focal intra-alveolar collections of alveolar macrophages with abundant foamy (lipid-containing) cytoplasm.
Lipoma in the abdominal cavity (3/3 male at 100 mg/kg bw/day) and the abscess in the wall of uterus (1/5 female at 300 mg/kg bw/day) are sporadically observed in experimental rats of this strain and these findings were considered as individual disorder, without toxicological significance.
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:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Ovary Follicle Count:
Quantitative examinations of ovaries revealed test item related decrease in the number of developing follicles and increase in the number of follicular atresia in parental male animals at 1000 mg/kg bw/day.
The mean number of secondary and tertiary follicles slightly exceeded the control value in female animals at 100 and 300 mg/kg bw/day.
At 1000 mg/kg bw/day, statistical significance was observed in female animals at the slightly lower mean number of primordial and primary follicles and secondary and tertiary follicles as well as at the higher mean number of follicular atresia. Please also refer to summary tables attached to this RSS.

Thyroid Hormone measurements:
T3 and T4 levels were statistically significantly reduced in males animals of the high dose group (-25 and -19 %) when compared to the control. This finding is consistent with other cohorts (F1 cohort 1A and 1B).

Since the above values still remained within historical control ranges and due to the fact that no histopathological findings in corresponding organs of the hypothalamus-pituitary-thyroid- axis were seen, their toxicilogocal meaning is considered unclear.
Thyroid hormone levels are very sensitive to circadian changes, fasting and/or general stress level of respective animals. A short and transient but daily repeated irritating effect of the test item shortly after administration is anticipated based on the clinical signs of the animals at the high and mid dose. This could reflect such a stressor that might have influenced stress level of animals at these dose groups.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
The estrous cycle was irregular in several parental female animals at 1000 mg/kg bw/day during the two weeks pre-mating period.
The examined parameters of the estrous cycle were comparable in the control and 100 and 300 mg/kg bw/day groups.
Statistical significance was noted for the lower percentage of female animals with regular cycle and for the lower mean number of days in pre-estrous at 1000 mg/kg bw/day. The number of female animals in prolonged estrous was also higher than in the control group at 1000 mg/kg bw/day.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Sperm examinations did not point out any test item related influence on the sperm cells at 1000 mg/kg bw/day.
Statistical or biological significances were not detected at the mean percentage of motile sperm cells or mean percentage of immotile sperms in parental male animals at 1000 mg/kg bw/day.
The total sperm count and sperms with not normal morphology (separated head and tail) were similar in the 1000 mg/kg bw/day and in the control groups.
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
Significantly lower fertility indices were observed in female animals at 1000 mg/kg bw/day with respect to their control. Mating of male animals – which did not fertilize their partners of main group – with not treated female animals provided clear evidence of fertility of these male animals.

The examined parameters of reproductive performance were not affected by the treatment with the test item in male or female animals at 100 or 300 mg/kg bw/day.
The copulatory index was higher than in the control group in all test item administered groups (100, 300 and 1000 mg/kg bw/day) as one control pair failed to mate.
The percentage of pregnant females (fertility index) was statistically significantly lower and percentage of non-pregnant female animals was statistically significantly higher with respect to their control group at 1000 mg/kg bw/day which was associated with the test item treatment.

Statistical significance was observed at the slightly higher mean number of conceiving days in female animals at 1000 mg/kg bw/day. This minor difference was mainly due to the late mating of one single female animal (conceiving days=15), which showed vaginal smears referring to pseudo-pregnancy on the most of mating days. Therefore, this difference in the mean number conceiving days was considered to be toxicologically not relevant.

Please also refer to summary tables attached to this RSS.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Remarks:
systemic and fertility
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
organ weights and organ / body weight ratios
histopathology: non-neoplastic
reproductive performance

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:
no effects observed
Description (incidence and severity):
Adverse signs of systemic toxicity related to the test item were not detected at any dose level in F1 Cohort 1B animals (male or female) at 100, 300 and 1000 mg/kg bw/day at the daily clinical observations.
The behavior and physical state of all animals were normal during the entire observation period.
Alopecia was observed in one control male animal (1/20) on the fore limbs and right side of the abdomen from PN 106 up to PN155.
There were no clinical signs in male animals at 100, 300 or 1000 mg/kg bw/day.
Female animals were also symptom-free in the control and 1000 mg/kg bw/day during the entire observation period.
Alopecia was noted for two female animals at 100 mg/kg bw/day during the pre-mating and gestation period (2/20, on the chest; on the fore limbs and hind limbs or abdomen) and for one of them during the lactation period (1/20; forelimbs, hind limbs, abdomen). In one female animal at 300 mg/kg bw/day (1/16), alopecia was detected on the right side of abdomen during the gestation period and on the fore limbs, hind limbs and right side of the abdomen during the lactation period.
Alopecia on the skin is a species-specific finding, which are also observed in untreated experimental rats of this strain with similar age. These were individual findings with low incidence in animals of control, low and mid dose groups and were not related to the treatment.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There was no test item related mortality in F1 Cohort 1B animals in 100, 300 or 1000 mg/kg bw/day groups (male or female) during the course of the observation period. One control dam (1/19) was found dead on gestation day 22. There were no preceding clinical signs. Sanguineous vaginal orifice and 4 dead fetuses were observed at the necropsy. Histological investigation revealed metritis, pulmonary congestion and edema as individual lesions and cause of the death.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The body weight development was reduced in F1 Cohort 1B male animals administered with 1000 mg/kg bw/day. The lower mean body weight of female animals at 1000 mg/kg bw/day during the first two weeks observation period recovered during the remaining days of observation period.
In the male animals at 100 mg/kg bw/day, the mean body weight was slightly higher than in the control group on PND70, 77, 84 and was comparable to their control on the preceding and following days. Statistical significance with respect to the control was detected at the slightly higher mean body weight gain of low dose males between PND49 and PND56.
In the male animals at 300 mg/kg bw/day, the body weight was similar to their control during pre-mating, mating and post-mating periods. Statistical significance was only noted for the slightly higher mean body weight gain between PND22-PND25 and PND39-PND42.
Statistical significance with respect to the control was detected at the lower mean body weight of F1 Cohort 1B male animals at 1000 mg/kg bw/day from PND22 up to the termination of the observation period (PND154). The body weight gain of these animals was also lower than in the control in the most cases during the entire observation period reaching statistical significances in several cases by weekly interval and also for the summarized body weight gain (between PND22 and PND154).
The mean body weight and body weight gain was comparable in the control and test item treated F1 Cohort 1B female animals at 100 and 300 mg/kg bw/day during observation periods. The mean body weight gain was slightly higher than in the control in female animals at 300 mg/kg bw/day between PND98 and PND105. This minor and transient change in body weight gain was considered to be toxicologically not relevant.
The mean body weight of F1 Cohort 1B female animals at 1000 mg/kg bw/day was statistically significantly lower than in the control from PND22 up to PND36 and it was comparable with the control from PND39 to PND112, as well as during the gestation and lactation periods. The mean body weight gain was also similar in all F1 Cohort 1B female groups (control 100, 300 and 1000 mg/kg bw/day) during the observation period. Although, sporadic statistical significance with respect to the control was noted for F1 Cohort 1B female animals at 1000 mg/kg bw/day at the lower mean body weight gain between PND25 and PND29 and at the higher mean body weight gain between PND70-PND77 and PND112-PND119. The summarized mean body weight gain of F1 Cohort 1B female animals was comparable in all groups between PND22 and PND112.
There were no toxicologically significant differences between the control and test item treated groups (100, 300 and 1000 mg/kg bw/day) in the body weight or body weight gain of F1 Cohort 1B female animals during the gestation or lactation period. Statistical significance was only noted for the higher mean body weight gain of female animals at 1000 mg/kg bw/day between gestation days 0 and 7 as well as between lactation days 0 and 4.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The food consumption was not affected in F1 Cohort 1B animals at 100, 300 and 1000 mg/kg bw/day during the pre-mating and post-mating periods (male) or during the pre-mating, gestation or lactation periods (female).
Statistical significance with respect to the control was detected at the slightly higher mean daily food consumption of male animals at 100 mg/kg bw/day between PND70 and PND77 and at 1000 mg/kg bw/day between PND133 and PND147.
In the female animals, the mean daily food consumption was slightly higher than in the control at 1000 mg/kg bw/day between PND22 and PND29 as well as at 300 and 1000 mg/kg bw/day between gestation days 0 and7.
These sporadic and minor differences in the mean daily food intake of male and female animals in F1 Cohort 1A were considered to be toxicologically not relevant.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
no effects observed
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The weights of kidneys (absolute, or relative to body and brain weights) were elevated in F1 Cohort 1B male and female animals at 300 or 1000 mg/kg bw/day. In the lack of related macroscopic and histological alteration, changes in kidneys weight were probably due to the enhanced function of the organ.
In the male animals at 100 mg/kg bw/day, statistical significance was detected at the slightly higher mean kidneys weights relative to brain weight.
At 300 mg/kg bw/day, statistical significance was noted for the higher mean kidneys weight (relative to body and brain weight), higher mean weights of prostate (absolute and relative to brain weight) and higher mean epididymides weight relative to brain weight in male animals.
In the male animals at 1000 mg/kg bw/day, the fasted mean body weight was significantly lower than in the control group. Statistical significance with respect to the control was detected at the lower mean brain weight and at the at the higher mean brain weight relative to body weight, higher mean weights of kidneys and testes (absolute and relative to body and brain weight), lower mean prostate weight, higher mean weights of epididymides and seminal vesicles (both relative to body weight).
In the female animals the mean kidney weights slightly exceeded the control at 100 mg/kg bw/day.
At 300 mg/kg bw/day, higher mean kidneys weight (absolute and relative to body and brain weights) and higher mean weight of ovaries were detected in female animals when compared to their control.
In the female animals at 1000 mg/kg bw/day, statistical significance was observed at the lower mean brain weights (absolute and relative to body weight), at the higher mean kidneys weight (absolute and relative to body and brain weights) and higher mean body weight relative to brain weigh.
A test item influence was supposed in development of the higher mean weights of kidneys (male and female). Histological examinations revealed no morphological changes in the renal tissue. Hematology investigations as well as clinical chemistry parameters did not reveal test item related abnormalities. Therefore, kidney weight changes were considered to be adaptive ones and to be toxicologically not relevant.
The statistically significant differences with respect to the control at several organs (brain, testes, prostate, epididymides, seminal vesicle or ovary) were judged to have little or no toxicological relevance due to the minor degree and in the lack of associated histopathological alterations.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic alterations related to the effect of the test item were not detected in F1 Cohort 1B male or female animals at 100, 300 or 1000 mg/kg bw/day at the necropsy.
In the male animals in control group, Hernia diaphragmatica (1/20), right side pyelectasia (2/20), smaller than normal seminal vesicle on the right side (1/20) and alopecia on the skin of forelimb and abdomen (1/20) were observed.
There were no macroscopic changes in the organs or tissues in male animals at 100 mg/kg bw/day.
At 300 mg/kg bw/day, right side pyelectasia (2/20) was noted for two male animals at the necropsy.
Dark red small liver lobe (1/20) and right or both sided pyelectasia (5/20) were detected at the necropsy.
In dead F1 Cohort 1B dam, sanguineous vaginal orifice and four dead fetuses in the left uterine horn were observed.
In control F1 Cohort 1B dams, dark red and hard small liver lobe (1/18), and right-side pyelectasia (1/18) was seen.
In non-pregnant female animals, the organs and tissues were normal.
Thymic hemorrhage (1/20) and alopecia on the skin of fore limbs, hind limbs and abdomen (1/20) were observed in single animals at 100 mg/kg bw/day.
At 300 mg/kg bw/day, right side pyelectasia (1/16) and rudimental right uterine horn (1/16) was noted for dams.
In non-pregnant female animals at 300 mg/kg bw/day, hemorrhage in the submandibular lymph node (1/4) and slight, moderate or marked hydrometra (3/4) were detected.
In dams at 1000 mg/kg bw/day, hemorrhage one or both sided pyelectasia (2/10) was observed
In non-pregnant female animals at 1000 mg/kg bw/day, Hernia diaphragmatica (1/9) and marked hydrometra (2/9) were seen.
Not mated female animal at 1000 mg/kg bw/day showed moderate hydrometra (1/1).
Rudimental uterine horn was a developmental disorder in female animal at 300 mg/kg bw/day.
Pyelectasia is frequently observed in this strain of experimental rats. Histological examination did not reveal degeneration, inflammation or fibrosis. Therefore, this finding was considered as slight individual lesion without toxicological significance.
Hydrometra (i.e. dilatation of uterine horns), related to the female sexual cycle, is a frequent observation in experimental rats. In the lack of related inflammatory or other pathological signs, it was judged to be toxicologically not relevant and not test item related as no dose response was noted.
Dark liver lobe, smaller than normal seminal vesicle, alopecia on the skin, Hernia diaphragmatica are common macroscopic findings in experimental rats of this strain with similar age. These occurred with low incidence and were considered to be toxicologically not relevant.
Hemorrhages in the lungs or thymus or lymph nodes are indicative of circulatory disturbance developing during the exsanguination.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Histological examinations did not reveal pathologic alterations in the organs or tissues of F1 Cohort 1B male or female animals at 1000 mg/kg bw/day.

In dead female control animal, metritis – in connection with 4 dead embryos – occurred in the uterus. This finding is considered as individual disease.
In the F1 Cohort 1B male animals in the control (20/20), and 1000 mg/kg bw/day (20/20) groups the investigated organs of reproductive system (testes, epididymides, prostate seminal vesicles, coagulating glands) were histologically normal and characteristic on the sexually mature organism in all cases, including animals, which did not fertilize their partners at 300 mg/kgbw/day (4/4). The various spermatogenic cells (spermatogonia, spermatocytes, spermatids and spermatozoa) representing different phases in the development and differentiation of the spermatozoons and the interstitial cells were the same in quantity and morphologically in the testes of investigated control and high dose treated animals.
The histological picture of epididymides, prostate, seminal vesicles, and coagulating glands was normal in all cases except for one control male animal (1/20), in which decreased amount of secrete in the seminal vesicle (one side) was observed. This phenomenon, without inflammatory or degenerative lesion is considered as individual disorder, without toxicological significance.
In the F1 Cohort 1B female animals in the control (20/20) and 1000 mg/kg bw/day (20/20, including not mated female animal) groups and in non-pregnant female animals at 300 mg/kg bw/day (4/4), the ovaries, uterus, cervix, vagina 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.
The 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.
In two female animals at 300 mg/kg bw/day (2/4), dilatation of uterine horns was observed. This finding – without inflammatory or other pathological lesions – is a slight neuro-hormonal phenomenon and is in connection with the normal sexual cycle (proestrus phase) of uterus without pathological significance.
One side pyelectasia occurred in F1 Cohort 1B male and female animals: 2/2 control, 2/2 at 300 mg/kg bw/day, 5/5 at 1000 mg/kg bw/day in male animals; 1/1 control, 1/1 at 300 mg/kg bw/day, 1/1 at 1000 mg/kg bw/day in female animals.
Pyelectasia without other histopathological lesions (for example degeneration, inflammation, fibrosis etc.) is considered as an individual disorder without toxicological significance.
Focal fibrosis in the Glisson’s capsule of the liver (1/1 male and 1/1 female control; 1/1 female at 1000 mg/kg bw/day) is in connection with the mechanical irritation due to diaphragmatic hernia.
Acute hemorrhage in the submandibular lymph nodes (1/1 female at 300 mg/kg bw/day), in the thymus (1/1 female at 100 mg/kg bw/day) and congestive liver (1/1 male at 1000 mg/kg bw/day, 1/1 female control) occurred sporadically and may be considered as consequence of hypoxia, dyspnea and circulatory disturbance developed during exsanguination procedures.
The atrophy of hair follicles in connection with focal alopecia (1/1 male control and 1/1 female at 100 mg/kg bw/day) without inflammatory lesions or fungal and parasite bodies is in connection with trophy disorder of affected skin area.
There was no morphological evidence of test item related acute or subacute injury (degeneration, inflammation, necrosis etc.) in the stomach, 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:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Ovary Follicle Count:
Quantitative examinations of ovaries did not reveal test item related alterations in F1 Cohort 1B female animals. The mean number of primordial and primary follicles, secondary and tertiary follicles, corpora lutea and follicular atresia were comparable in the control, 100, 300 and 1000 mg/kg bw/day.
Please also refer to summary tables attached to this RSS.

Thyroid Hormone measurements:
At 1000 mg/kg bw/d levels of T3 as well as T4 were reduced in mals animals (-11 % and -15 %, respectifvely). T4 values were also reduced in female animals at this dose level (-10 %) when compared to the control. The reduced T3 and T4 levels are consistend with findings in Cohort 1A and P0 generation male animals. However, since these values still remained within historical control ranges and the fact, that no histopathological findings in corresponding organs of the hypothalamus-pituitary-thyroid- axis were seen, the toxicilogocal meaning is considered unclear. Thyroid hormone levels are very sensitive to circadian changes, fasting and/or general stress level of respective animals. A short and transient but daily repeated irritating effect of the test item shortly after administration is anticipated based on the clinical signs of the animals at the high and mid dose. This could reflect such a stressor that might have influenced stress level of animals at these dose groups.

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Cohort 1A (representative for cohort 1B):
The examined parameters of the estrous cycle were comparable in the F1 Cohort 1A female animals in the control and 100, 300 and 1000 mg/kg bw/day groups.
The estrous cycle was irregular in several female animals in the control, 100, 300 and 1000 mg/kg bw/day groups during the two weeks observation period.
The percentage of female animals with regular cycle, mean length of cycle, mean number of days in pre-estrous, estrous, diestrous and number of female animals in prolonged diestrous were similar in all groups (control, 100, 300 and 1000 mg/kg bw/day). The number and percentage of female animals in prolonged estrous was slightly higher than in the control group at 1000 mg/kg bw/day (N=3, 15 %). However, this minor difference with respect to the control was considered to be toxicologically not relevant as values met the historical control ranges (N= 0 – 5; 0 – 42 %; mean: 4 %).
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Sperm examinations did not point out any test item related influence on the sperm cells at 1000 mg/kg bw/day.
Statistical or biological significances were not detected at the mean percentage of motile sperm cells or mean percentage of immotile sperms in parental male animals at 1000 mg/kg bw/day.
The total sperm count and sperms with not normal morphology (separated head and tail) were similar in the 1000 mg/kg bw/day and in the control groups.
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
The reproductive performance was reduced in F1 Cohort 1B animals at 300 and 1000 mg/kg bw/day (male and female) based on the lower fertility indices.

The examined parameters of reproductive performance were not affected by the treatment with the test item in male or female animals at 100 mg/kg bw/day. In this dose group, all pairs mated successfully and males fertilized respective females, therefore fertility index even exceeded the control value.

The copulatory index was lower than in the control group at 1000 mg/kg bw/day as two male animals failed to mate (male #758 and #760). Regarding male#760, corresponding female partners (female#771 and #790) did either not mate even with exchanged males (#771), or mated but did not achieve pregnancy (#790).
One female partner of male #758, where no mating could be observed, was also female #771. The other female partner of male#758 (female#780) mated successfully achieving pregnancy with an exchanged partner male.
Overall, the decreased copulatory index as a consequence of impaired mating behaviour by male animals must be treated with caution, as it cannot be completely excluded that this finding originates from the corresponding female partner.


Statistical significance was observed at the lower percentage of fertile male animals (i.e. fertility index) and higher percentage of infertile male animals at 300 and 1000 mg/kg bw/day. This is due to the not achieved pregnancies of respective females. However, due to findings in the P0 generation, where mating of male animals – which did not fertilize their partners of main group – with not treated female animals provided clear evidence of fertility of these male animals, the males of the F1 generation are also not considered infertile, but only their respective female partner animal.

The percentage of pregnant females (fertility index) was lower and percentage of non-pregnant female animals was higher with respect to their control group at 300 and 1000 mg/kg bw/day. This finding is consistent with observations in the P0 generation and considered test item related. Please also refer to summary tables attached to this RSS.

One control female animal died on gestation day 22 – not delivered – while all pregnant animals delivered at 100, 300 and 1000 mg/kg bw/day.

Statistical significance was observed at the slightly higher mean number of conceiving days in female animals at 300 mg/kg bw/day. Similar finding was not detected at the high dose, therefore, this difference in the mean number conceiving days at 300 mg/kg bw/day was considered to be toxicologically not relevant.

Details on results (P1)

Results of Cohort 1B only (PND22 to approx. PND140).

Effect levels (P1)

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Key result
Dose descriptor:
NOAEL
Remarks:
fertility
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
reproductive performance
Remarks on result:
other: Cohort 1B
Key result
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
organ weights and organ / body weight ratios
Remarks on result:
other: Cohort 1B

Target system / organ toxicity (P1)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
Pups:
There were no adverse clinical signs in the F1 offspring from post-natal day 0 to 21. However, the percentage of offspring showing signs (no milk in the stomach, cold, found dead, missing, alopecia) was higher at 1000 mg/kg bw/day compared to the control. These observations correspond with the findings of inadequate nursing behaviour of the respective dams. The percentage of offspring, which were cold, did not suckle were similar in the control and 100 or 300 mg/kg bw/day groups. Some other sporadic clinical signs were also observed in the control and 100 or 300 mg/kg bw/day dose groups (cachexia, found dead, missing, wound). At 1000 mg/kg bw/day, wound, hemorrhage, exophthalmos missing or atrophied limb were noted for single pups

Cohort 1A:
There were no clinical signs in male or female animals in F1 Cohort 1A generation in control, 100, 300 or 1000 mg/kg bw/day. The behavior and physical condition of all animals were normal during the entire observation period.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
Pups:
The extra uterine mortality of F1 offspring exceeded the control at 1000 mg/kg bw/day on post-natal day 0 and between postnatal days 0 and 21. The extrauterine mortality was low and comparable in the control, 100, and 300 mg/kg bw/day from birth to post-natal day 21.

Cohort 1A:
There was no mortality in F1 Cohort 1A animals in control, 100, 300 or 1000 mg/kg bw/day groups (male or female) during the course of study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Pups:
Offspring of the high dose animals had a slightly reduced body weight on postnatal day 0 (-6.5 %). Body weight development between PND0 - PND21was also slightly depressed when compared to control group offspring (- 8.6 %). Body weight of the offspring of the low and mid dose group was unremarkable and comparable to the control group.

The body weight and weight development of the F1 offspring was slightly reduced at 1000 mg/kg bw/day.
The mean litter weight was comparable with control and at 100, 300 mg/kg bw/day during the 21-day observation period.
The mean litter weight remained below the control at 1000 mg/kg bw/day on post-natal days 0, 4, 7, 14 and 21.
The mean litter weight gain was similar in the control and at 100 and 300 groups by interval of the measurements and between post-natal days 0 and 21. The mean litter weight gain was slightly lower than in the control group at 1000 mg/kg bw/day.
Statistical significance with respect to the control was detected at the higher mean body weight of pups (male and female) at 100 mg/kg bw/day on post-natal days 4 and 7 and at the lower mean body weight of pups at 300 mg/kg bw/day on post-natal days 0 and 14. The terminal body weight (post-natal day 21) was comparable with the control in these groups, therefore, the minor differences were considered to be toxicologically not relevant. The mean body weight of offspring remained below the control at 1000 mg/kg bw/day on post-natal days 0, 4, 7, 14 and 21 being always statistically significantly lower.
The body weight of male pups and female pups at 100 and 300 mg/kg bw/day – evaluating separately the two genders – was similar to the control between post-natal days 0 and 21. Although, some statistical significances with respect to the control were detected at the slightly lower or higher mean body weight of male or female pups at 100 and 300 mg/kg bw/day during the first week after birth, the terminal body weight was similar to the control. The mean body weight was statistically significantly lower than in their control both in male and in female offspring on postnatal days 0, 4, 7, 14 and 21 at 1000 mg/kg bw/d.
Statistical significance was detected with respect to the control at the slightly higher mean pup weight gain between postnatal days 0 and 4 and at the slightly lower mean body weight gain of pups between post-natal days 7 and 14 at 100 and 300 mg/kg bw/day. The summarized body weight gain (between post-natal days 0 and 21) was comparable with the control in both of these groups. Therefore, these minor changes in body weight gain of offspring (male and female) were considered to be toxicologically not relevant at 100 and 300 mg/kg bw/day. The mean body weight gain was slightly reduced in offspring at 1000 mg/kg bw/day when compared to the control by intervals of measurement and if summarized.
The mean body weight gain of male and female offspring – evaluated separately – was comparable to their control at 100 and 300 mg/kg bw/day in-spite of the sporadic statistically significant differences during the first week after birth. The mean body weight gain remained below the control in male and in female offspring at 1000 mg/kg bw/day by intervals of measurement and if summarized.

Cohort 1A:
The body weight development was reduced in F1 Cohort 1A male animals administered with 1000 mg/kg bw/day. The lower mean body weight of female animals at 1000 mg/kg bw/day during the first half of the observation period recovered during the second half of the observation period. The mean body weight was comparable to their control in F1 Cohort 1A male animals at 100 and 300 mg/kg bw/day during the entire observation period. Statistically significant difference with respect to the control was detected at the lower mean body weight gain of male animals at 100 mg/kg bw/day between PN42 and PN49 and at 300 mg/kg bw/day between PN32 and PN36. However, these minor differences in the mean body weight gain had no influence on the mean body weight of these male animals.
In F1 Cohort 1A male animals at 1000 mg/kg bw/day, the mean body weight was significantly lower than in the control during the entire observation period (from PND22 up to and including PND90). The body weight gain of these animals was also lower than in the control during the entire observation period reaching statistical significances in several cases by weekly interval and also for the summarized body weight gain (between PND22 and PND90).
The mean body weight and body weight gain was comparable in the control and test item treated F1 Cohort 1A female animals at 100 and 300 mg/kg bw/day during observation periods. Statistical significance at the slightly higher mean body weight of female animals at 100 mg/kg bw/day was considered to be toxicologically not relevant.
The mean body weight of F1 Cohort 1A female animals at 1000 mg/kg bw/day was statistically significantly lower than in the control from PND22 up to PND42 and it was comparable with the control between PN49 and 90. The mean body weight gain was similar in all F1 Cohort 1A female groups (control 100, 300 and 1000 mg/kg bw/day) during the observation period. Although, statistical significance with respect to the control was noted for F1 Cohort 1A female animals at 1000 mg/kg bw/day at the lower mean body weight gain between PND22 and PND29 and at the higher mean body weight gain between PND42 and PND49. The summarized mean body weight gains of F1 Cohort 1A female animals were comparable in all groups between PND22 and PND90.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Pups: not applicable

Cohort 1A:
The food consumption was not affected in F1 Cohort 1A male or female animals at 100, 300 and 1000 mg/kg bw/day.
The mean daily food consumption of F1 Cohort 1A male or female animals was similar in the control and test item treated groups (100, 300 and 1000 mg/kg bw/day) during the observation period (between PND22 and PND90).
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
Pups: not examined

Cohort 1A:
There were no test item related adverse changes in the examined hematological parameters in F1 Cohort 1A male or female animals at 100, 300 or 1000 mg/kg bw/day.
In the male animals at 100 mg/kg bw/day, statistical significances were detected at the slightly higher mean percentage of neutrophil granulocytes (NEU) and reticulocytes (RET) and at the slightly shorter mean prothrombin time (PT) when compared to the control.
At 300 mg/kg bw/day, higher mean percentage of neutrophil granulocytes and monocytes (MONO) and lower mean percentage of lymphocytes (LYM) were observed in male animals when compared to the control.
At 1000 mg/kg bw/day, statistical significances were detected at the slightly higher mean percentage of neutrophil granulocytes, lower mean percentage of lymphocytes, eosinophil granulocytes (EOS)
In the female animals at 100 mg/kg bw/day, the mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) were slightly lower than in the control group.
All examined parameters were comparable with the control in female animals at 300 mg/kg bw/day.
Statistical significance was detected with respect to the control at the slightly higher mean percentage of monocytes (MONO), at the lower mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration and at the slightly shorter mean prothrombin time (PT) in F1 Cohort1A female animals at 1000 mg/kg bw/day.
The changes noted in these hematology or blood coagulation parameters were considered to have little or no toxicological relevance. Slight elevation in NEU in male animals were mainly due to the relative low control value. Higher mean percentage of monocytes in female animals were not accompanied by related signs of inflammation, therefore were considered to be toxicologically not relevant.
The mean values of LYM, EOS, MCHC, RET, PT, MCH and MCHC were well within the historical control range in male or female animals, where relevant. There were no related changes in other hematological parameters therefore, the differences in these parameters were considered to have little or no toxicological relevance.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Pups: not examined

Cohort 1A:
Pathologic alterations were not detected at the evaluation of clinical chemistry parameters in F1 Cohort 1A male or female animals at 100, 300 or 1000 mg/kg bw/day.
The examined clinical chemistry parameters were comparable in male animals in the control and 100 and 300 mg/kg bw/day groups. In the male animals at 1000 mg/kg bw/day, statistical significance was noted for the slightly lower mean concentration of total protein (TPROT)
In the F1 Cohort 1A female animals, statistically significant difference with respect to the control was detected at the lower mean activity of alanine aminotransferase (ALT) at 100 mg/kg bw/day.
All examined parameters were comparable with the control in the female animals at 300 mg/kg bw/day.
In the female animals at 1000 mg/kg bw/day, higher mean activity of alanine aminotransferase, higher mean concentrations of total bilirubin (TBIL) and cholesterol (CHOL) were observed when compared to the control.
The statistically significant changes of some clinical chemistry parameters were considered to be of little or no biological significance as the mean values correlated well with the historical control values (total protein, ALT, TBIL, CHOL) or the profile of change has no biological significance (ALT in low dose female animals).
Significantly elevated activity of ALT and aspartate aminotransferase (AST) in one female animal at 1000 mg/kg bw/day (no. 728) was considered to be individual alteration. There were no supporting histological findings in this female animal. Therefore changes in enzyme activities might be indicative of functional alteration.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Pups: not examined

Cohort 1A:
There were no test item related adverse changes in the examined urine parameters in F1 Cohort 1A animals (male or female) at 100, 300 or 1000 mg/kg bw/day.
Most of the examined urine parameters were comparable in the control and 100, 300 or 1000 mg/kg bw/day groups (male and female).
Slightly but statistically significantly higher volume of the urine with respect to their control was detected in male animals at 1000 mg/kg bw/day.
In the female animals, statistical significance was noted for the slightly lower pH of urine at 300 and 1000 mg/kg bw/day and the sediment was positive in four rats (4/10) at 1000 mg/kg bw/day due the moderate amount of amorphous crystals.
Sexual maturation:
no effects observed
Description (incidence and severity):
Pups:
The F1 offspring’s development (surface righting reflex, pinna detachment, eye opening) no clear test item influence was observerd.
There were no toxicologically relevant differences in the offspring’s development between the control and 100 or 300 mg/kg bw/day groups. Some statistical significance indicates higher percentage of pups with positive response or lower percentage of pups with negative response at 100 mg/kg bw/day (pinna detachment, eye opening) or at 300 mg/kg bw/day (eye opening). At 1000 mg/kg bw/day, the percentage of pups with positive response was lower and the percentage of pups with negative response was higher than in the control group at surface righting reflex, pinna detachment and eye opening.

Cohort 1A:
The sexual maturity was not adversely affected in F1 Cohort 1A male or female animals at 100, 300 or 1000 mg/kg bw/day.
The balano-preputional separation was completed in all F1 Cohort 1A male animals – control, 100, 300 and 1000 mg/kg bw/day – on post-natal day 35, although, the mean body weight was slightly lower with respect to the control in male animals at 1000 mg/kg bw/day on PND35 as described above.
In the F1 Cohort 1A female animals, statistical significance was noted for the longer period of vaginal patency at 1000 mg/kg bw/day and at the longer period of appearance of the first cornified vaginal smear at 100 and 1000 mg/kg bw/day. The interval between days of vaginal patency and first cornified smear were similar in all groups (control, 100, 300 and 1000 mg/kg bw/day).
These differences in the female sexual maturity were considered to be toxicologically not relevant because of the minor degree and in the absence of dose response relationship. The slightly delayed vaginal patency and appearance of first cornified vaginal smear was similar in the low and high dose treated animals.
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
Pups:
The anogenital distances were not adversely affected by the test item in male or female offspring at 100, 300 and 1000 mg/kg bw/day. Statistical significance was detected at the shorter absolute anogenital distance of male and female pups at 1000 mg/kg bw/day. However, the normalized anogenital distances were comparable with the control both in male and female offspring at 1000 mg/kg bw/day. Slightly shorter normalized anogenital distance at 100 mg/kg bw/day was judged to be toxicologically not relevant due to the minor degree and in the lack of dose response relationship.

Cohort 1A: not applicable
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
Pups:
Nipples/areoles were not visible in any of the examined male offspring in the control or 100, 300 or 1000 mg/kg bw/day groups on post-natal day 13.

Cohort 1A: not applicable
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Pups:
There were no test item related changes in the weights of examined organs (absolute and relative to body and brain weights) in male and female F1 offspring (necropsy at weaning).
The examined organ weights were comparable in selected male offspring at the weaning.
Statistical significances with respect to the control were detected at the slightly higher mean thymus weights (absolute and relative to brain weight) at 100 mg/kg bw/day and at the lower mean body weight and brain weight at 1000 mg/kg bw/day in female pups.
The minor changes of thymus weights were considered to be independent from the treatment as similar findings was not detected at the higher doses.
The lower mean brain weight was probably related to the lower mean body weight as it exceeded the control value – no statistical significance – if related to the body weight.

Cohort 1A:
The weights of the examined organs were not adversely affected in F1 Cohort 1A male and female animals at 100, 300 or 1000 mg/kg bw/day.
Slight elevation in the weights of liver and kidneys at 300 mg/kg bw/day (female) and at 1000 mg/kg bw/day (male and female) were indicative of the enhanced function of these organs. There were no supporting histopathological alterations in the liver or kidneys therefore, test item effect on the weight of these organs were considered to be not adverse.
Slight reduction of thymus weights (absolute and relative to body and brain weights) in F1 Cohort 1A male animals at 1000 mg/kg bw/day dose might be related to the test item influence. Nevertheless, in the lack of related histopathological findings and changes in immune system it was considered to be sign of a normal but enhanced involution. Moreover, absolute as well as relative weight values range within the historical control values.
In the male animals at 100 mg/kg bw/day, statistical significance with respect to the control was detected at the slightly lower mean thymus weight and higher mean testes weight relative to body and brain weights.
At 300 mg/kg bw/day, the mean thymus weight was slightly lower and the kidney weight relative to body weight was slightly higher than in the control group in F1 Cohort 1A male animals.
In the male animals at 1000 mg/kg bw/day, the mean fasted body weight was significantly lower than in the control group resulting in lower mean body weight relative to brain weight, lower mean weights of some organ and higher mean weights of some organ referred to body weight.
Statistical significance was detected at the lower mean brain weight, higher mean brain weight relative to body weight, higher mean liver and kidneys weight (absolute and relative to the body and brain weights, both), at the lower mean weights of heart, thymus (absolute and relative to the body and brain weights), prostate and pituitary in male animals at 1000 mg/kg bw/day when compared to the control. The weights of testes (relative to body and brain weights), epididymides (relative to body weight) and adrenal glands (relative to body and brain weight) exceeded the control value in male animals administered with the high dose.
In the female animals at 100 mg/kg bw/day, statistical significance with respect to the control was observed at the slightly higher mean body weight relative to brain weight and lower mean brain weight relative to body weight, at the higher mean weights of liver and thyroid glands both relative to brain weight.
In female animals at 300 mg/kg bw/day, the body weight relative to brain weight was higher, the brain weights (absolute and relative to body weight) were lower than in the control group. Higher mean weights of liver and kidneys (absolute and relative to body and brain weights), thyroid glands (absolute and relative to brain weight), adrenal gland relative to brain weight.
At 1000 mg/kg bw/day, statistical significances with respect to the control were observed at the higher mean body weight relative to brain weight, at the lower mean brain weights (absolute and relative to body weight), at the higher mean weights of liver and kidneys (absolute and relative to body and brain weights), adrenal gland relative to body and brain weights and thyroid glands relative to brain weight in the F1 Cohort 1A female animals.
A test item influence was supposed in development of the higher mean weights of kidneys and liver (male and female). Morphological changes were not detected at the histopathological examination and hematology investigations as well as clinical chemistry parameters did not reveal test item related abnormalities. Therefore, changes in liver and kidneys weights were considered to be adaptive ones and to be toxicologically not relevant.
The statistically significant differences with respect to the control at several organs (brain, heart, prostate, testes, epididymides, adrenal glands, thyroid glands or pituitary) were judged to have little or no toxicological relevance due to the minor degree and in the lack of associated histopathological alterations.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Pups:
Specific macroscopic alterations were not found in F1 offspring subjected to gross pathological examination before the weaning or at the weaning.
Some common sporadic necropsy findings were detected in pups necropsied before the weaning: wound around anus (1/23 male at 300 mg/kg bw/day), partially cannibalized (1/14 male and 4/23 female at 100 mg/kg bw/day; 1/24 female at 300 mg/kg bw/day; 1/7 male at 1000 mg/kg bw/day). The organs and tissues were free of morphological changes in dead or stillborn offspring – empty stomach or autolysis in visceral organs were only detected. At weaning, one or both side pyelectasia was noted for several male and female pups at each dose level except for female pups at 1000 mg/kg bw/day. In the lack of dose response relationship and inflammatory or other pathological changes pyelectasia was considered to be a species specific alteration and not related to the test item.
Some individual findings without relation to the test item treatment were also detected as follows:
- red granulous spot (1/70 male at 100 mg/kg bw/day) or hemorrhage (1/62 male at 300 mg/kg bw/day) in the stomach;
- scar on the tail (1/70 female control);
- alopecia (3/28 male and 3/35 female at 1000 mg/kg bw/day);
- exophthalmos (1/35 male at 1000 mg/kg bw/day);
- damage of forelimb (1/35 female at 1000 mg/kg bw/day);

Cohort 1A:
Macroscopic alterations related to the effect of the test item were not detected in F1 Cohort 1A male or female animals at 100, 300 or 1000 mg/kg bw/day at the necropsy.
Hemorrhage in the stomach (1/20), Hernia diaphragmatica (1/20) and renal pyelectasia (4/20, right side, each) were observed in the control male animals.
In the male animals at 100 mg/kg bw/day, right side pyelectasia was detected in some animals (3/20).
At 300 mg/kg bw/day, hemorrhage in the stomach (1/20) and right side pyelectasia (1/20) were noted for single male animals.
In the male animals at 1000 mg/kg bw/day, hemorrhage in the lungs (1/20) and stomach (1/20) and pyelectasia (8/20, right or both sided) were seen at the necropsy.
In control female animals necropsy observations revealed the following findings: right or both sided pyelectasia (3/20); slight, moderate or marked hydrometra (5/20); soft formation in the left horn of uterus (1/20) and ovarian cyst (1/20).
One side pyelectasia (1/20) and slight, moderate or marked hydrometra (8/20) were noted for some female animals at 100 mg/kg bw/day.
At 300 mg/kg bw/day, pyelectasia (2/20, right or both sided), hydrometra (3/20, slight, moderate or marked) and ovarian cyst (1/20) were observed in female animals.
In female animals at 1000 mg/kg bw/day, thymic hemorrhage (1/20) one or both sided pyelectasia (5/20) and slight or moderate hydrometra (3/20) were
These macroscopic findings are common in experimental rats of this strain and age.
Pyelectasia is frequently observed in this strain of experimental rats. Histological examination did not reveal degeneration, inflammation or fibrosis. Therefore, this finding was considered as slight individual lesion without toxicological significance.
Hydrometra (i.e. dilatation of uterine horns), related to the female sexual cycle, is a frequent observation in experimental rats. In the lack of related inflammatory or other pathological signs, it was judged to be toxicologically not relevant and not test item related as no dose response was noted.
Hemorrhage in the thymus and lungs were related to exsanguination procedure. Hemorrhage in the stomach mucosa was probably related to the treatment procedure. Hernia diaphragmatica, ovarian cyst and soft formation in the uterine horn are also species-specific changes occurring in not treated animals.
Histopathological findings:
no effects observed
Description (incidence and severity):
Pups:
Histological investigation did not reveal test item related pathologic changes in the examined organs in F1 offspring.
Renal pyelectasia was observed in examined offspring – with macroscopic findings: 6/6 control; 13/13 at 100 mg/kg bw/day; 14/14 at 300 mg/kg bw/day; 2/2 at 1000 mg/kg bw/day. Pyelectasia without signs of inflammation or other pathological findings is considered as a species-specific alteration. There was no dose relevancy in the incidence therefore renal pyelectasia was judged to be toxicologically not relevant in this study.
Congestion in the stomach mucosa was detected in two offspring (1/1 female at 100 mg/kg bw/day and 1/1 male at 300 mg/kg bw/day) as individual alteration. This finding was not seen in offspring in the high dose group therefore test item effect was excluded.

Cohort 1A:
Histological examinations did not reveal pathologic alterations in the organs or tissues of F1 Cohort 1A male or female animals at 1000 mg/kg bw/day.
The investigated organs of reproductive system (testes, epididymides, prostate seminal vesicles, coagulating glands) were histologically normal and characteristic for the sexually mature organism in all F1 Cohort 1A male animals in the control and 1000 mg/kg bw/day groups.
In the F1 Cohort 1A male animals in the control and 1000 mg/kg bw/day groups the investigated organs of reproductive system (testes, epididymides, prostate seminal vesicles, coagulating glands) were histologically normal and characteristic for the sexually mature organism in all cases. The various spermatogenic cells (the spermatogonia, the spermatocytes, the spermatids and spermatozoa) representing different phases in the development and differentiation of the spermatozoons and the interstitial cells were the same in quantity and morphologically in the testes of investigated control and high dose treated animals.
The histological picture of epididymides, prostate, seminal vesicles, and coagulating glands was normal in all cases as well.
In the F1 Cohort 1A female animals of the control and 1000 mg/kg bw/day groups, the ovaries, uterus, cervix, vagina 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.
The 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.
In some cases, dilatation of uterine horns was observed (5/20 control; 8/8 at 100 mg/kg bw/day; 3/3 at 300 g/kg bw/day; 3/20 at 1000 mg/kg bw/day). This finding – without inflammatory or other pathological lesions – is a slight neuro-hormonal phenomenon and is in connection with the normal sexual cycle (proestrus phase) of uterus without pathological significance. In one control female animal (1/20) adenoma was observed in the uterine horn as an individual disease.
One or both sided pyelectasia was seen in several F1 Cohort 1A male and female animals: 4/20 male and 3/20 female control; 3/3 male at 100 mg/kg bw/day; 1/1 male and 2/2 female at 300 mg/kg bw/day; 8/20 male and 5/20 female at 1000 mg/kg bw/day. Pyelectasia without other histopathological lesions (for example degeneration, inflammation, fibrosis etc.) is considered as an individual disorder without toxicological significance.
Alveolar emphysema (minimal or moderate degree) in the lungs (2/20 male 1/20 female control; 1/20 male and 2/20 female at 1000 mg/kg bw/day) and acute hemorrhage in the lungs (1/20 male at 1000 mg/kg bw/day) and in the thymus (1/20 female at 1000 mg/kg bw/day) occurred sporadically and are considered as consequence of hypoxia, dyspnea and circulatory disturbance developed during exsanguinations.
Hyperplasia of bronchus associated lymphoid tissue (BALT) in some control and treated animals (1/20 male and 1/20 female control; 2/20 female at 1000 mg/kg bw/day) is an immuno-morphological phenomenon, without toxicological significance.
Erosion and congestion in the mucous membrane of stomach was observed in some male animal (1/20 control, 1/1 at 300 mg/kg bw/day, 1/20 at 1000 mg/kg bw/day) presumably due to the gavage administration of the vehicle or test item.
The focal interstitial fibrosis in the Glisson’s capsule of the liver in one control male animal (1/20) was in connection with the mechanical irritation due to diaphragmatic hernia.
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:
no effects observed
Description (incidence and severity):
Sperm parameters:
Cohort 1A:
Sperm examinations did not point out any test item related influence on the sperm cells at 1000 mg/kg bw/day.
Statistical or biological significances were not detected at the mean percentage of motile sperm cells or mean percentage of immotile sperms in animals of 1000 mg/kg bw/day.
The total sperm count and sperms with not normal morphology (separated head and tail) were similar in the 1000 mg/kg bw/day and in the control groups.

Ovary Follicle Count:
Quantitative examinations of ovaries did not reveal test item related changes in the number of developing follicles, corpora lutea or in the number of follicular atresia at the examined level of section of F1 Cohort 1A female animals at 1000 mg/kg bw/day.
The mean number of primordial and primary follicles were slightly higher than in the control group in F1 Cohort 1A female animals at 100, 300 and 1000 mg/kg bw/day. The mean number of secondary and tertiary follicles, corpora lutea and follicular atresia was similar in the control and at 100, 300 and 1000 mg/kg bw/day.
Please also refer to summary tables attached to this RSS.


Thyroid Hormone measurements:
F1 Pups (PND22):
Mean values of thyroid hormones (T3 and T4) from pooled samples per litter of thyroid hormones were not changed when compared to the control.

Cohort 1A (at necropsy, approx. PND90):
T3 and T4 levels were statistically significantly reduced in males animals of the high dose group (-22 and -25 %) when compared to the control. This finding is consistent with other cohorts (1B and P0 generation).

Since the above values still remained within historical control ranges and due to the fact that no histopathological findings in corresponding organs of the hypothalamus-pituitary-thyroid- axis were seen, their toxicilogocal meaning is considered unclear.
Thyroid hormone levels are very sensitive to circadian changes, fasting and/or general stress level of respective animals. A short and transient but daily repeated irritating effect of the test item shortly after administration is anticipated based on the clinical signs of the animals at the high and mid dose. This could reflect such a stressor that might have influenced stress level of animals at these dose groups.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
no effects observed

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
no effects observed

Details on results (F1)

Results of F1 (PND0 to PND21) and Cohort 1A (PND22 to approx. PND90).

Effect levels (F1)

open allclose all
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
body weight and weight gain
Remarks on result:
other: F1 Offspring of P0; PND0 to PND21
Key result
Dose descriptor:
NOAEL
Generation:
F1 (cohort 1A)
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
organ weights and organ / body weight ratios
Remarks on result:
other: Cohort 1A at approx. PND 92

Target system / organ toxicity (F1)

Key result
Critical effects observed:
not specified

Results: F2 generation

General toxicity (F2)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no adverse clinical signs in the F2 offspring at 100, 300 or 1000 mg/kg bw/day from post-natal day 0 to 4.
The percentage of offspring showing signs (no milk in the stomach, cold, missing, pale) was similar in all groups.
The percentage of dead F2 offspring was higher than in the control group at 1000 mg/kg bw/day.
Some other clinical signs were detected in single animals as follows: damaged tail in the control and 1000 mg/kg bw/day; darker than normal eye at 300 mg/kg bw/day
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
The extra uterine mortality of F2 offspring was higher than in the control group at 1000 mg/kg bw/day on post-natal day 0 and between postnatal days 0 and 4.
The extrauterine mortality was low and comparable in the control, 100, and 300 mg/kg bw/day from birth to post-natal day 4.
Statistical significance with respect to the control was noted for the higher mean number of dead pups on postnatal day 0 and between postnatal days 0 and 4 at 1000 mg/kg bw/day.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The body weight development of the F2 offspring was slightly reduced at 1000 mg/kg bw/day.
Statistical significance with respect to the control was detected at the higher mean body weight of pups (male and female) at 100 mg/kg bw/day on post-natal day 0 and at the lower mean body weight of pups at 300 and 1000 mg/kg bw/day on post-natal days 0 and 4. The mean pup weight gain was slightly lower than in the control between PND0 and PND4.
The mean litter weight and litter weight gain were similar in the control and in 100 and 300 mg/kg bw/day groups between PND0 and PND4.
The mean litter weight on PND4 and the mean litter weight gain between PND0 and PND4 were slightly lower than in the control at 1000 mg/kg bw/day.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Description (incidence and severity):
The sex distribution of F2 offspring were not affected by the test item on post-natal days 0 and 4.
The survival index was slightly lower at 1000 mg/kg bw/day with respect to the control at 1000 mg/kg bw/day.
The mean number of live births per litter, and mean number of viable pups per litter were comparable in the control and 100, 300 and 1000 mg/kg bw/day groups on post-natal days 0 and 4.
The sex distribution (mean percentage of male and female pups per litter) was comparable in the control and 300 and 1000 mg/kg bw/day groups on post-natal days 0 and 4. Statistical significance with respect to the control was detected at the lower mean percentage of male pups and higher mean percentage of female pups at 100 mg/kg bw/day on post-natal days 0 and 4. Similar findings was not observed at the higher dose groups, therefore this difference was considered to be indicative of biological variation and not related to the treatment
Anogenital distance (AGD):
not examined
Nipple retention in male pups:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no test item related changes in the weights of examined organs (absolute and relative to body and brain weights) in male or female F2 offspring at 100, 300 and 1000 mg/kg bw/day.
The weights of examined organs (brain, spleen, thymus; absolute and relative to body and brain weights) were comparable in selected male and female F2 offspring.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Specific macroscopic alterations were not found in F2 offspring subjected to gross pathological examination after PND4.
In dead F2 offspring, undernourishment, underdevelopment, empty stomach and autolysis were detected at 300 mg/kg bw/day (1/1, each).
At 1000 mg/kg bw/day, empty stomach (9/14), autolysis (2/14) yellow foamy intestinal content (1/14) or gas filled intestines (1/14) were observed.

In surviving offspring, there were no macroscopic findings in the control (192/192) and 300 mg/kg bw/day (191/191) at the necropsy.
Undernourishment, underdevelopment and empty stomach (1/220) and wounds on the back by bite (1/220) were observed at 100 mg/kg bw/day.
At 1000 mg/kg bw/day, undernourishment, underdevelopment (2/95) and damaged tail (1/95) were detected.
These findings were considered to be toxicologically not relevant as these occurred with low incidence and were not related to doses.
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Development:
The F2 offspring’s development (surface righting reflex, pinna detachment, anogenital distance) was undisturbed at 100, 300 and 1000 mg/kg bw/day.
There were no toxicologically relevant differences in the offspring’s development between the control and 100, 300 or 1000 mg/kg bw/day groups.
Statistical significance with respect to the control was detected at the lower percentage of pups with positive response and higher percentage of pups with negative response in pinna detachment at 300 mg/kg bw/day.
The absolute anogenital distance was slightly shorter than in the control group in male pups at 1000 mg/kg bw/day but the normalized anogenital distance was similar in the control and 1000 mg/kg bw/day male pups.
Statistical significance was detected at the slightly longer normalized anogenital distance of male pups at 100 and 300 mg/kg bw/day. This minor difference with respect to the control was judged to be toxicologically not relevant.

Developmental neurotoxicity (F2)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:
not examined

Effect levels (F2)

Key result
Dose descriptor:
NOAEL
Generation:
F2
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
body weight and weight gain

Target system / organ toxicity (F2)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects in the absence of other toxic effects
Dose response relationship:
yes
Relevant for humans:
not specified

Any other information on results incl. tables

Please refer to summary tables attached.

Applicant's summary and conclusion

Conclusions:
Under the conditions of this EOGRTS, TBPEH caused reduced body weight (P, F1 Cohort 1A, Cohort 1B male animals), irregular estrous cycle (P female), disturbances in reproductive performance (P, F1 Cohort 1B), changes in kidneys in parental (P) male animals (elevated weight, chronic progressive nephropathy), changes in liver and kidneys weight (parental (P) male and female, F1 Cohort 1A, Cohort 1B) in Han:WIST rats at 1000 mg/kg bw/day by oral gavage as investigated in this study.

At 300 mg/kg bw/day, changes in liver and kidneys weights (F1 Cohort 1A or Cohort 1B) and disturbed reproductive ability (F1 Cohort 1B) were observed.

At 100 mg/kg bw/day, there were no signs of systemic toxicity or influence on the reproductive performance (male or female animals, parental (P), F1 Cohort 1A, F1 Cohort1B).

The development of the F1 offspring was slightly impaired at 1000 mg/kg bw/day – higher mortality (F1 and F2), lower survival index (F2) and reduced body weight (F1 and F2).
Based on these observations the No Observed Adverse Effect Levels (NOAEL) were determined as follows:
NOAEL for systemic toxicity: 300 mg/kg bw/day
NOAEL for reproductive performance 100 mg/kg bw/day
NOAEL for F1, F2 Offspring: 300 mg/kg bw/day
Executive summary:

The subject of this study was to investigate reproductive toxicity of TBPEH 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 use with the rat, which is the preferred rodent species for reproduction toxicity testing.

The aim of the one-generation reproduction toxicity study was to provide an evaluation of the pre- and postnatal effects of the test item TBPEH on development as well as a thorough evaluation of systemic toxicity in male and female animals, in pregnant and lactating females and young and adult offspring when repeatedly administered orally (by gavage) to parental animals at doses of 100, 300 and 1000 mg/kg bw/day compared to control animals.

The effect of the test item was examined on the male and female reproductive performance, such as gonadal function, estrous cycle, mating behavior, conception, parturition, gestation, lactation and weaning (P and F1 Cohort 1B generation) and on the offspring viability, neonatal health and mortality, growth and development of the offspring (F1 generation; Cohort 1A and Cohort 1B later producing F2 generation) to adulthood following oral (by gavage) administration.

Four groups of Han:WIST rats (n=24/sex/group) were administered with the test item orally (by gavage) once a day at 0 (vehicle), 100, 300 and 1000 mg/kg bw/day doses corresponding to concentrations of 0, 20, 60 and 200 mg/mL. The application volume was 5 mL/kg bw. Control animals received the vehicle, sunflower oil, only.

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 six times during the study. Concentration of TBPEH in the dosing formulations varied in the acceptable range between 92 % and 107 % 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 (10 weeks) and throughout mating. In addition, males received the test item or vehicle after mating up to the day before the necropsy (altogether for 153-156 days). Dams were additionally exposed through the mating and gestation periods and up to lactation days 21-23 (altogether for 114-129 days). Not mated and non-pregnant females and dams without living pups were administered for 100 or 103 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. Estrous cycle was monitored by examining vaginal smears before the mating for two weeks 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 post-partum. A second mating of the P males with untreated females (untreated group; n= 8 naïve females) was performed to clarify findings in fertility of the high dose male animals due to the low number of P females achieving pregnancy in the high dose group. Dams and offspring in the untreated group were terminated on post-partal/ postnatal days 5-8.

All F1 offspring were observed individually for the 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).

20 F1 animals/sex/group for Cohort 1A and 20 F1 animals/sex/group for Cohort 1B were randomly selected on postnatal day 21 for follow-up examinations.

Dosing of F1 offspring selected for follow-up examinations (Cohort 1A and Cohort 1B) begun on postnatal day 22 and treatment was continued up to the day before the necropsy.

F1 offspring (Cohort 1A and Cohort 1B) were observed identically to parental animals – clinical signs, body weight, food consumption, estrous cycle, clinical pathology and organ pathology. Sexual maturity of offspring (Cohort 1A and Cohort 1B) was investigated by observation of balano-preputional separation, vaginal patency and appearance of first cornified vaginal smear.

Cohort 1A animals were subjected to necropsy, organ weighing and sperm analysis – one day after the termination of the exposition – on post-natal days 91-96.

Cohort 1B animals were mated to produce a second (F2) generation after at least 90-day pre-mating period and were observed identically to parental (P) animals. F2 offspring were observed and subjected to necropsy up to postnatal day 6-8.

Blood samples were collected for determination of serum levels of thyroid hormones (FT3, FT4 and TSH) from 3-5 F1 pups per litter (where it was feasible) on post-natal day 4, from 1-2 pups/10 litters on postnatal day 22, from 10 dams (P)/ groups and from 10 parental (P) male animals/ groups at termination, from 10 male animals/ groups and from 10 female animals in Cohort 1A, from all male and female animals in Cohort 1B (n=20/ sex / groups) at termination and from F2 pups on post-natal day 5 or shortly thereafter. Thyroid hormone levels were determined in adult animals (P, F1 Cohort 1A, Cohort 1B) and in PN22 F1 offspring.

All adult animals (P, F1 Cohort 1A and Cohort 1B) were subjected to gross pathology with complete tissue reservation 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 and in F2 offspring on PND5 or shortly thereafter.

Special attention was paid to the organs and tissues of the reproductive system for P, F1 or F2 animals. Selected organs were determined in adult animals (P, F1) and in offspring (PND22 or shortly thereafter and in F2 offspring on PND5).

Sperm parameters were determined in all control and high dose male animals in P generation and in F1 generation (Cohort 1A and Cohort 1B). Full histopathology examinations were performed on the organs and tissues of adult animals (P, F1 Cohort 1A and Cohort 1B) in control and high dose groups with special emphasize on sexual organs and tissues. Reproductive organs were also processed and examined histologically in not mated and non-pregnant female animals and their mating partners (P and Cohort 1B) in the low and mid dose groups. 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 and Cohort 1B) and in F1 offspring.

Based on the low fertility index and histopathological findings in parental (P) female animals at 1000 mg/kg bw/day, a quantitative evaluation of primordial, small growing, secondary and tertiary follicles, as well as corpora lutea was performed in all adult female animals (P, F1 Cohort 1A and Cohort 1B) in the control, 100, 300 and 1000 mg/kg bw/day. The results were interpreted comparing treatment groups with respect to controls, which were treated concurrently with vehicle (sunflower oil) only.

 

There was no test item related mortality in 100, 300 or 1000 mg/kg bw/day groups (male or female, P, F1 Cohort 1A and F1 Cohort 1B) during the course of study. One control dam in F1 Cohort 1B died due to an individual disease (metritis, pulmonary edema and congestion) on gestation day 22. Adverse signs of systemic toxicity related to the test item were not detected at any dose level (100, 300 and 1000 mg/kg bw/day, male or female, P, F1 Cohort 1A and Cohort 1B) at the daily or detailed weekly clinical observations or at the terminal functional observations. Salivation and nuzzling up the bedding material were noted for parental (P) male and female animals shortly after the administration with variable incidence and duration (predominantly in the high dose and to a lower extend in the mid dose group, but not in the low dose group). The mean body weight gain and mean body weight were slightly reduced in male animals at 1000 mg/kg bw/day (parental (P), F1 Cohort 1A and F1 Cohort 1B). The body weight of female animals in F1 Cohort 1A and Cohort 1B at 1000 mg/kg bw/day was slightly lower during the first weeks of the treatment period and the difference with respect to the control recovered during the following weeks. The food consumption was not adversely affected in male or female animals (parental (P), F1 Cohort 1A and F1 Cohort 1B) at 100, 300 and 1000 mg/kg bw/day.

The estrous cycle was irregular in several parental (P) female animals at 1000 mg/kg bw/day during the two weeks pre-mating period.

A test item influence on the estrous cycle was not detected in F1 Cohort 1A female animals at any dose level. The reproductive performance was reduced in parental (P) animals at 1000 mg/kg bw/day and in F1 Cohort 1B animals at 300 and 1000 mg/kg bw/day. However, results of mating of parental (P) male animals – which did not fertilize their partners of main group – with not treated female animals proved fertility of these male animals. Delivery data of dams was not adversely affected at 100, 300 or 1000 mg/kg bw/day dose level (parental (P) and F1 Cohort 1B). The examined urine parameters were not adversely affected in male and female animals at 100, 300 or 1000 mg/kg bw/day (parental (P) and F1 Cohort 1A). However, a statistically significantly increased urine volume with lower pH levels was apparent in both sexes and generations. There were no test item related adverse changes in the examined clinical pathology parameters in male or female animals at 100, 300 or 1000 mg/kg bw/day (parental (P) and F1 Cohort 1A). Specific macroscopic alterations related to the effect of the test item were not detected in male or female animals at 100, 300 or 1000 mg/kg bw/day at the terminal necropsy (parental (P), F1 Cohort 1A and F1 Cohort 1B). Elevated weights of kidneys were indicative of the test item effect in parental (P) male animals at 1000 mg/kg bw/day and in accordance with histopathological findings.Elevated liver weights in male and female parental (P) animals at 1000 mg/kg bw/day, elevated liver and kidney weights in male and female animals at 300 and 1000 mg/kg bw/day (F1 Cohort 1A and Cohort 1B) were considered to be related to the test item treatment . However, in the lack of supporting histological alterations, these organ weight changes were probably caused by the enhanced metabolic and excretion functions of these organs and are thus not considered adverse. Sperm examinations did not reveal any test item related influence on the sperm cells in regards to morphology, motility and total sperm count at 1000 mg/kg bw/day (parental (P), F1 Cohort 1A and F1 Cohort 1B). The investigated organs of reproductive system (testes, epididymides, prostate, seminal vesicles, coagulating glands) were histologically normal and characteristic on the sexually mature organism in all parental (P), F1 Cohort 1A and Cohort 1B male animals at 1000 mg/kg bw/day. Decreased number of developing follicles and increased number of follicular atresia were detected by qualitative histological as well as quantitative evaluation in parental (P) female animals at 1000 mg/kg bw/day compared with their control. Based on qualitative evaluation, a decreased developing follicle count as well as increased atresia was seen in the females not achieving pregnancy. Similar findings were not detected in female animals in F1 Cohort 1A and Cohort 1B. Histopathological investigations revealed chronic progressive nephropathy in a higher incidence of parental (P) male animals at 1000 mg/kg bw/day but not in F1 Cohort 1A or Cohort 1B.

The offspring’s development was slightly influenced at 1000 mg/kg bw/day. A slightly higher mortality (F1 and F2), lower survival index on PN4 (F2) and reduced body weight (F1 and F2) were observed in offspring at 1000 mg/kg bw/day. This is associated to an increased inadequate nursing behaviour of respective dams observed during lactation period and correspondingly increased findings such as cold and unfed pups (no milk in stomach) at the high dose level.

The sexual maturity of offspring (F1 cohort 1A and Cohort 1B) was not adversely affected at any dose level.

 

Conclusion

Under the conditions of the present study, TBPEH caused reduced body weight (P, F1 Cohort 1A, Cohort 1B male animals), irregular estrus cycle (P female), disturbances in reproductive performance (P, F1 Cohort 1B), changes in kidneys in parental (P) male animals (elevated weight, chronic progressive nephropathy), and changes in liver and kidney weights (parental (P) male and female, F1 Cohort 1A, Cohort 1B) in Han:WIST rats at 1000 mg/kg bw/day by oral gavage as investigated in this study.

At 300 mg/kg bw/day, changes in liver and kidneys weights (F1 Cohort 1A or Cohort 1B) and disturbed reproductive ability (F1 Cohort 1B) were observed. At 100 mg/kg bw/day, there were no signs of systemic toxicity or influence on the reproductive performance (male or female animals, parental (P), F1 Cohort 1A, F1 Cohort1B). The development of the F1 offspring was slightly impaired at 1000 mg/kg bw/day – higher mortality (F1 and F2), lower survival index (F2) and reduced body weight (F1 and F2).

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

NOAEL for systemic toxicity:          300 mg/kg bw/day

NOAEL for reproductive performance         100 mg/kg bw/day

NOAEL for F1, F2 Offspring:           300 mg/kg bw/day