tert-butyl 2-ethylperoxyhexanoate

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

Under the conditions of an EOGRTS, according to OECD 443, 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

Link to relevant study records

Reference

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:

2018-06-01 to 2020-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS:

- Premating exposure duration for parental (P0) animals : 10 weeks as requested by 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

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Toxi-Coop Zrt. Cserkesz u. 90. H-1103 Budapest, Hungary
- Females nulliparous and non-pregnant: yes
- Age at study initiation: P0 males and females: 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

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

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

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.

A determination of spleenic subpopulation analysis was considered not required. A detailed justification is attached as background material.

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

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 chest 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 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; -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 toxicological 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 reduced 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) until the end of the study reaching statistical significances in most cases by weekly interval.
In the parental female animals, slightly higher mean daily food consumption was statistically significant 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 at 1000 mg/kg bw/day, 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. The lower mean daily food intake of dams at 1000 mg/kg bw/day reached statistical significance between lactation days 7 and 14.

These slight differences with respect to the control were of low degree and not consistent 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 significance was 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 to the control in male animals at 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. 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):

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, a 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 at 1000 mg/kg bw/day. 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 enhanced renal activity and not of adverse effects on the kidneys.
In the parental male animals, the lower mean pH of the urine at 100 and 300 mg/kg bw/day were statistically significant.
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 for the higher mean volume of the urine of female animals 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

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 for 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). This finding was supported by the results of quantitative examinations of the ovaries
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 morphology 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 number of developing follicles and increased number of follicular atresia were observed along with developing follicles and corpora lutea on the actual level of section by qualitative histological examination.
At the quantitative examinations, the mean number of secondary and tertiary follicles and corpora lutea slightly exceeded the control value in dams at 100 and 300 mg/kg bw/day reaching statistical significance only in the mid dose treated animals.
At 1000 mg/kg bw/day, the mean number of secondary and tertiary follicles was lower and the mean number of follicular atresia was higher than in the control group in dam 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), as indicated in table 10.
Statistical significance was also observed in delivered female animals at 1000 mg/kg bw/day at the slightly lower mean number of primordial and primary follicles.

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. (Suttie, A.W., et al: Boorman’s Pathology of the rat. Reference and Atlas. Second Edition. Academic Press, Elsevier, London, San Diego, Cambridge, Oxford, 2018.)
In our study the follicular atresia affected only partly the ovarian functions, however 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; 4/24 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 (11/24). 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. Chronic progressive nephropathy was not detected in the kidneys of male animals at 100 or 300 mg/kg bw/day (24/24, both groups).
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, dysnea 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:
The thyroid hormone (FT3, FT4 and TSH) levels were not adversely influenced in the parental male or female animals or in PND22 F1 offspring at any dose levels.
Slight, statistically significant difference was detected at the lower mean FT3 and FT4 concentrations in male animals at 1000 mg/kg bw/day. In the lack of related elevation of TSH level, changes in organ weight or histopathology, these findings were considered to be toxicologically not relevant.
There were no statistically significant differences with respect to their control in the FT3, FT4 and TSH concentrations in the parental female animals or in PND 22 F1 offspring at any dose levels.

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 last weeks of an overall of 10 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 reveal 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 reproduction 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 reproduction ability 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 (reproduction 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.

Delivery data of dams was not adversely affected at 100, 300 or 1000 mg/kg bw/day dose levels.
The lower number of females delivering in the 1000 mg/kg bw/day group as compared to the control group was due to the lower number of pregnant females. All pregnant females delivered and there were no significant differences in most of the examined parameters with respect to the control. The mean number of implantation sites, the mean number of post-implantation loss, the mean number of total births, mean number of viable pups and live borns and the live birth index (live pups/total birth) were comparable in all groups.
The slightly longer mean duration of pregnancy of dams at 1000 mg/kg bw/day was statistically significant. This minor difference was considered to be of no toxicological relevance as values were within the historical control range.

Please also refer to summary tables attached to this RSS.

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

Key result

Critical effects observed:

no

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: 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.

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

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

Key result

Critical effects observed:

no

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.

A determination of spleenic subpopulation analysis was considered not required. A detailed justification is attached as background material.

Behaviour (functional findings):

no effects observed

Developmental immunotoxicity:

no effects observed

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

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

Key result

Critical effects observed:

not specified

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.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

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

Key result

Critical effects observed:

no

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

Please refer to summary tables attached.

Table 1 Quantitative evaluation of ovaries in parent (P) females. Differentiation of pregnant and non-pregnant (NP) females of the control and high dose (1000 mg/kg bw/day).

Animal’s status		Pregnant		Non-pregnant
Group (mg/kg bw/day)(n)		Control (21)	1000 (16)	Control (3)	1000 (8)
Follicular atresia	Mean	5.2	11.4	5.7	16.6
	Range	2 - 9	7 – 16	5-7	13 - 19
Primordial and primary follicles	Mean	31.2	28.7	34.3	30.6
	Range	9 - 41	21 - 34	31-39	23 - 36
Developing follicles (secondary, tertiary)	Mean	12.3	8.1	14.3	7.6
	Range	4 - 20	6 - 13	13-16	5 - 15

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 the reproductive toxicity of tert. butylperoxy-2-ethylhexanoate 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 post-natal effects of the test item 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 the test item 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 100-129 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.

As the number of pregnancies was low in the high dose group, a second mating of the P males with untreated females (untreated group; n= 8 naïve females) was performed to clarify if the male fertility of the high group was impaired. Dams and offspring in the untreated group were terminated on post-partum/ PND (post-natal 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).

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

Dosing of F1 offspring selected for follow-up examinations (Cohort 1A and Cohort 1B) begun on post-natal 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 exposure – on PND 91-97.

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 PND 5-8.

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 PND 4, from 1-2 pups/10 litters on PND 22, from 10 dams (P)/group and from 10 parental (P) male animals/group at termination, from 10 male animals/ group and from 10 female animals in Cohort 1A, from all male and female animals in Cohort 1B 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 PND22 F1 offsprings.

All adult animals (P, F1 Cohort 1A and Cohort 1B) were subjected to gross pathology with complete tissue preservation one day after the last treatment. Brain, spleen, thymus and mammary tissues were preserved for 10 male and 10 female pups per group – where feasible – in F1 offspring not selected for Cohorts on PND22 or shortly thereafter 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 organ weights 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 emphasis on sexual organs and tissues.

Reproductive organs were also processed and examined histologically in non-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. The kidneys of male animals at 100 and 300 mg/kg bw/day were processed and evaluated histologically based on the organ weight data and histological observations at 1000 mg/kg bw/day.

Based on the low reproduction 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.

Clinical observation

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.

Salivation and nuzzling up the bedding material were noted for parental (P) male and female animals at 300 and 1000 mg/kg bw/day shortly after the administration with variable incidence and duration in a dose related manner.

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 last weeks of the ten 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, a second mating showed that the male animals of the parental (P) 1000 mg/kg bw/day group – which did not fertilize their partners of main group – mated successfully with non-treated females (n= 8 naïve females).

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). Slightly elevated urine volume and lowered pH levels were apparent in both sexes and in (P) generation and in male animals in F1 Cohort 1B at 1000 mg/kg bw/day and were considered to be indicative of enhanced renal activity and not related to adverse effects on the kidneys in absence of alterations in the related parameters (clinical chemistry, histopathology).

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).

There were no test item related adverse changes in the serum thyroid hormone levels (FT3, FT4, TSH) at any dose (male and female parental (P), F1 Cohort 1A and F1 Cohort 1B, F1 offspring PND22). Slightly lower FT3 and FT4 levels in parental (P) and F1 Cohort 1A male animals at 1000 mg/kg bw/day as compared to the control was probably due to difference in the activity of thyroid glands.

There were no accompanying alterations in the TSH levels, organ weight or histology of thyroid glands. Therefore, these minor changes in thyroid hormone levels had low or no biological importance.

 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 functions of these organs and are thus not considered adverse.

Sperm examinations did not reveal any test item related influence on the sperm cell morphology and 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 of 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. The same effects were 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 (CPN) in a higher incidence of parental (P) male animals at 1000 mg/kg bw/day but not in parental (P) male animals at 100 or 300 mg/kg bw/day or in male animals in F1 Cohort 1A or Cohort 1B.

A slightly higher mortality (F1 and F2), lower survival index on PND4 (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.

 

In conclusion, under the conditions of the present study, the test item caused body weight reduction (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, slight changes in liver and kidneys weights (not adverse, 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, indicated by higher mortality (F1 and F2), lower survival index (F2), clinical signs (F1), reduced body weight (F1 and F2) and slower development of reflexes (F1) were observed. 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

 

 

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The study was conducted in accordance with GLP regulation and the respective OECD/EU guideline and is considered acceptable for the assessment of effects on fertility of the test substance.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

OECD 421

Tert.-Butylperoxy- 2-ethylhexanoat was tested in a reproduction/developmental toxicity screening test according OECD guideline 421. In addition, it provided information on possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of the conceptus and parturition. Tert.-Butylperoxy- 2-ethylhexanoat was administered orally (by gavage) to Wistar rats at repeated doses of 0, 100, 300 and 1000 mg/kg body weight/day, for 14 days during the pre-pairing period, and the pairing, the gestation and the lactation periods until 3 post partum. Treatment at 1000 mg/kg was associated with decreased food consumption in male and female animals during the first week of the pre-pairing period. Some dams were noted with ruffled fur after parturition. No test item-related effects were noted during necropsy and for macroscopic findings. For reproduction parameters, treatment at 1000 mg/kg was associated with an increase of pre-implantation-, post-implantation-, and post-natal loss, and a reduction of live pups. The mean body weight of pups also was reduced at this systemic maternal toxic dose. Based on these data, it can be concluded that the NOEL was at 300 mg/kg body weight/day for the parental generation as well as for the offspring.

OECD 443, rat

The subject of this study was to investigate the reproductive toxicity of tert. butylperoxy-2-ethylhexanoate 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 post-natal effects of the test item 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 the test item 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 100-129 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.

As the number of pregnancies was low in the high dose group, a second mating of the P males with untreated females (untreated group; n= 8 naïve females) was performed to clarify if the male fertility of the high group was impaired. Dams and offspring in the untreated group were terminated on post-partum/ PND (post-natal 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).

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

Dosing of F1 offspring selected for follow-up examinations (Cohort 1A and Cohort 1B) begun on post-natal 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 exposure – on PND 91-97.

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 PND 5-8.

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 PND 4, from 1-2 pups/10 litters on PND 22, from 10 dams (P)/group and from 10 parental (P) male animals/group at termination, from 10 male animals/ group and from 10 female animals in Cohort 1A, from all male and female animals in Cohort 1B 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 PND22 F1 offsprings.

All adult animals (P, F1 Cohort 1A and Cohort 1B) were subjected to gross pathology with complete tissue preservation one day after the last treatment. Brain, spleen, thymus and mammary tissues were preserved for 10 male and 10 female pups per group – where feasible – in F1 offspring not selected for Cohorts on PND22 or shortly thereafter 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 organ weights 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 emphasis on sexual organs and tissues.

Reproductive organs were also processed and examined histologically in non-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. The kidneys of male animals at 100 and 300 mg/kg bw/day were processed and evaluated histologically based on the organ weight data and histological observations at 1000 mg/kg bw/day.

Based on the low reproduction 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.

Clinical observation

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.

Salivation and nuzzling up the bedding material were noted for parental (P) male and female animals at 300 and 1000 mg/kg bw/day shortly after the administration with variable incidence and duration in a dose related manner.

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 last weeks of the ten 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, a second mating showed that the male animals of the parental (P) 1000 mg/kg bw/day group – which did not fertilize their partners of main group – mated successfully with non-treated females (n= 8 naïve females).

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). Slightly elevated urine volume and lowered pH levels were apparent in both sexes and in (P) generation and in male animals in F1 Cohort 1B at 1000 mg/kg bw/day and were considered to be indicative of enhanced renal activity and not related to adverse effects on the kidneys in absence of alterations in the related parameters (clinical chemistry, histopathology).

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).

There were no test item related adverse changes in the serum thyroid hormone levels (FT3, FT4, TSH) at any dose (male and female parental (P), F1 Cohort 1A and F1 Cohort 1B, F1 offspring PND22). Slightly lower FT3 and FT4 levels in parental (P) and F1 Cohort 1A male animals at 1000 mg/kg bw/day as compared to the control was probably due to difference in the activity of thyroid glands.

There were no accompanying alterations in the TSH levels, organ weight or histology of thyroid glands. Therefore, these minor changes in thyroid hormone levels had low or no biological importance.

 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 functions of these organs and are thus not considered adverse.

Sperm examinations did not reveal any test item related influence on the sperm cell morphology and 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 of 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. The same effects were 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 (CPN) in a higher incidence of parental (P) male animals at 1000 mg/kg bw/day but not in parental (P) male animals at 100 or 300 mg/kg bw/day or in male animals in F1 Cohort 1A or Cohort 1B.

A slightly higher mortality (F1 and F2), lower survival index on PND4 (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.

 

In conclusion, under the conditions of the present study, the test item caused body weight reduction (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, slight changes in liver and kidneys weights (not adverse, 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, indicated by higher mortality (F1 and F2), lower survival index (F2), clinical signs (F1), reduced body weight (F1 and F2) and slower development of reflexes (F1) were observed. 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

 

 

 

Effects on developmental toxicity

Description of key information

OECD 414, rat

Oral treatment of pregnant Hsd. Brl. Han: WISTAR rats from gestation day 5 up to day 19 with the test item at the dose levels of 200, 400 and 1000 mg/kg bw/day did not cause death and necropsy findings. Tert.-Butylperoxy- 2-ethylhexanoat did not reveal any adverse effect on the pregnancy and the intrauterine mortality of the conceptuses, the number of viable fetuses and their sex distribution. Further the test substance did not increase significantly the incidence of external and visceral variations, and caused no skeletal malformations. The slight delay in ossification in fetuses of the 1000 mg/kg bw/day dose group is considered to be non-adverse.
Based on these observations the No Observed Adverse Effect Level (NOAEL) was determined as follows:
NOAEL maternal toxicity: 1000 mg/kg bw/day
NOAEL developmental toxicity: 1000 mg/kg bw/day
Based on these observations the No Observed Effect Level (NOEL) was determined as follows:
NOEL maternal toxicity:: 400 mg/kg bw/day
NOEL developmental toxicity: 400 mg/kg bw/day

OECD 414, rabbit

Oral treatment of inseminated New Zealand White rabbits with TBPEH at dose levels of 30, 100 and 300 mg/kg bw/day respectively from day 6 up to and including day 27 post insemination revealed pronounced maternal toxicity in the high and mid dose. Four does got into moribund state at 300 mg/kg bw/day after the food consumption was extremely reduced and weight loss, reduced activity, lying, weakness was observed as clear indication of severe toxicity. In association with the treatment eight does aborted in the 300 mg/kg bw/day group and two in the 100 mg/kg bw/day group. Increase of early embryonic death and post-implantation loss/total intrauterine mortality as well as decrease of mean number of viable fetuses was recorded in the 300 mg/kg bw/day dose group. Also, the incidence of females with total post-implantation loss was higher in the high dose group. There was no significant difference in incidence of overall fetal malformations among the experimental groups. Significantly lower fetal weight and crownrump length as well as increase of growth retarded fetuses were observed in the 300 mg/kg bw/day dose group as well as increasing skeletal variations mainly due to delayed ossification (e.g. phalanges) associated to the lower body weights. Multiple malformations of ribs and vertebrae in three fetuses were considered as likely to be secondary due to the severe maternal toxicity. The visceral development of the fetuses was not affected.

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

NOAEL (maternal toxicity): 30 mg/kg bw/day

NOAEL (developmental toxicity): 100 mg/kg bw/day

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-10-17 to 2013-02-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

22 January 2001

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:Toxi-Coop Zrt. 1103 Budapest Cserkesz u. 90. Hungary
- Age at study initiation: Females: Young adult and nulliparous females, 10-11 weeks of age at start of the mating period. Males: experienced males 35-37 weeks of age at start of the mating period.
- Fasting period before study: no
- Housing: Before mating: 1-3 females per cage, 1-2 males per cage. Mating: 1 male and 1-3 females / cage. During gestation: 2-3 sperm positive females per cage, if not possible 1 sperm positive female per cage.
- Diet: ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and maintenance" ad libitum
- Water: tap water ad libitum
- Acclimation period: 20 days for females and 181 days for males

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-22
- Humidity (%): 36-46
- Air changes (per hr): 8-12
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2012-10-17 To: 2012-11-13

Route of administration:

oral: gavage

Vehicle:

other: Helianthy Annui Oleum Raffinatum / Sunflower oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Formulations were prepared in the laboratory of Toxi-Coop Zrt. daily or according to the stability data of the formulations (based on previous analytical measurements performed in the Laboratory of Toxi-Coop Zrt).

VEHICLE
- Justification for use and choice of vehicle: The test item is not soluble and not stable in water therefore oleum helianthy was used for preparing formulations appropriate for oral administration. Oleum helianthy /sunflower oil is a suitable vehicle to facilitate formulation analysis for the test item.
- Concentration in vehicle: 100, 200, 500 mg/mL
- Amount of vehicle: 2 mL/kg bw
- Lot/batch no.: 19T3

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical control (concentration, homogeneity) of dosing solutions was performed in the Laboratory of Toxi-Coop Zrt. on the first and last treatment weeks.
The suitability of the chosen vehicle for the test item at the intended concentrations was analytically verified up front. A sufficient stability and homogeneity in the chosen vehicle was verified over the range of relevant concentrations at the appropriate frequency of preparation. The test item concentrations in the samples were found to be 94 – 106 % in comparison to the nominal values.

Analytical method:
- HPLC-UV
- Detector: 210 nm
- Column: HyperPrep HS C18, 250 x 4.6 mm, 8 μm,
- Mobil Phase: Acetonitrile: Water = 9 : 1 (v/v)
- Flow: 1.2 mL/min
- Injection volume: 50 μL
- Temperature: 25 °C
- Retention time: 5.3 min ± 10 %

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/3
- Length of cohabitation: in the mornings for two to four hours
- Further matings after two unsuccessful attempts: no
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

GD 5 to GD 19

Frequency of treatment:

The test item was administered in a single dose by oral gavage (stomach tube) on a 7 days/week basis every day at similar time.

Duration of test:

GD 5 to GD 19

Dose / conc.:

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

Dose / conc.:

400 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:

24 Females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The doses have been chosen by the Sponsor on the basis of a previous study (GLP OECD 421 Reproduction/Developmental Toxicity Screening Study of Tert.-Butylperoxy- 2-ethylhexanoat in the Wistar Rat).
- Rationale for animal assignment: The sperm positive females were allocated to each experimental group on each mating day in such a way that the group averages of the body weight were as similar as possible on the first day of gestation. If possible, females paired with the same male were allocated to different groups on the same mating day.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Observations for signs of morbidity and mortality were made twice daily, at the beginning of the working period and in the afternoon.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once a day, after treatment at approximately the same time. Individual observation included the check of behavior and general condition.
Duration and severity of the clinical signs were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: Gestation days 0, 3, 5, 8, 11, 14, 17 and 20

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Between gestation days 0 to 3, 3 to 5, 5 to 8, 8 to 11, 11 to 14, 14 to 17 and 17 to 20

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined:
the uterus with cervix and the left ovary were removed and weighed. The right ovary was placed into a Petri dish after removal. After removing the uterus gross pathology of dams' viscera was performed. The number of corpora lutea in each ovary and implantation sites in each uterine horn, live fetuses, early and late embryonic death and fetal death were counted. Animals, in which unambiguous implantation sites, but not fetuses have been found, were considered as pregnant.

EXAMINATION OF PLACENTAL SIGNS:
All sperm positive animals were examined for vaginal bleeding (placental sign of gestation) on the 13th gestational day. If negative on day 13, the examination was repeated on day 14 of gestation.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter

Statistics:

The statistical evaluation of data was performed with the 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’s Multiple Range test was used to access the significance of inter-group differences. If significance was the result of the Bartlett’s test, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test.
Dams or litters were excluded from the data evaluation in cases of:
- Any disease or death of the dam unrelated to the treatment (total exclusion)
- Non pregnant females or dams with 3 or less implantations independent of their viability (total exclusion)
Although these animals were excluded from the data evaluation the final report contains all data of these animals, too.
A male/female fetus was considered as retarded in body weight, when its weight is below the average minus twofold standard deviation of the control male/female fetuses.

Indices:

- Number of corpora lutea
- Number of implantations
- Number and percent of live fetuses
- Pre-implantation loss (%):
(Number of corpora lutea - Number of implantation) / Number of corpora lutea x 100
- Post-implantation loss (%):
(Number of implantations - Number of live foetuses) / Number of implantations x 100
- Sex distribution (%):
Number of Male (Female) foetuses / Number of foetuses x 100
- External abnormalities per litter (%)
Number of fetuses with abnormality / Number of fetuses x 100
- Visceral abnormalities per litter (%)
Number of fetuses with abnormality / Number of foetuses examined x 100
- Skeletal abnormalities per litter (%)
Number of fetuses with abnormality / Number of foetuses examined x 100

Historical control data:

Historical contral data are available and were used for evaluation of study results.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Alopecia was found sporadically without a dose response in the females. Salivation was recorded in association the treatment in nine of 23 females in the 400 mg/kg bw/day group and in all of the dams of the 1000 mg/kg bw/day dose group, directly after treatment. This was judged to be in relationship with the taste of the test item.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One pregnant female in the control group died in the course of the study on gestational day 20. The death was considered to be due to the intrauterine autolyzing of dead embryos and fetuses. This dam had no clinical signs before death but lost weight.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There was no indication of an effect of the test item on body weight development of the dams in the 200 and 400 mg/kg bw/day dose groups.
The body weight gain was statistically significant (p<0.01) reduced on the first three days of treatment in the 1000 mg/kg bw/day group. This finding was considered as test item related but not adverse. Between gestational days 8 and 11 it turned to an increased body weight gain with a statistical significance (p<0.05).
There were no dose related differences in the corrected body weight and corrected body weight gain of the dams in the experimental groups.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

There was no indication of an effect of the test item on the food consumption of the dams in the 200 and 400 mg/kg bw/day dose groups.
There was a statistically significantly (p<0.01) reduced food consumption on the first six days of treatment in the 1000 mg/kg bw/day dose group related to the treatment with the test item. Statistical significant increases (p<0.05) were indicated in two occasions (once before the treatment period and once at the beginning of it) in the food consumption of the animals in the 200 mg/kg bw/day dose group which are not associated with the test item.

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

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no macroscopic alterations recorded for the dams during necropsy.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

not examined

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

The mean fetal weight was similar in the control, 200 and 400 mg/kg bw/day groups. The slight but statistically significant (p<0.01) reduction in the mean body weight of the male and female fetuses in the 1000 mg/kg bw/day group might be attributed to the statistically significant reduced body weight gain and statistically significant lower food consumption of the dams between gestational day 5 and 8 and 5 and 11 respectively in this dose group. Although a statistical significance in the fetal weight in the 1000 mg/kg bw/day group was noted, the value was in the range of the historical control data and therefore considered to be non-adverse. Placental weight was similar in all experimental groups. There was a statistically significant increase indicated in the relative placental weight in the 1000 mg/kg bw/day dose group (p<0.05), however it was below the historical control level.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

There was no dose related significant difference in the intrauterine mortality of the conceptuses, the number of implantations, viable fetuses and their sex distribution. The number of late embryonic death increased slightly but statistically significantly (p<0.05) in the 400 mg/kg bw/day dose group and without a statistical significance in the 1000 mg/kg bw/day group. No dose response was indicated and the values are in the range of the historical control data. There was no statistical significance indicated in the mean percentage value of the late embryonic death in the experimental groups.

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

The number of evaluated fetuses was 228, 190, 239 and 192 at external and 114, 96, 120 and 97 at visceral examination in the control, 200, 400 and 1000 mg/kg bw/day groups, respectively.
The incidence of visceral abnormalities (malformations and variations) was statistically significant (p<0.05) higher in the 1000 mg/kg bw/day dose group. However, the number of affected fetuses is well within the historical control range and therefore considered to be of no biological relevance.

- Malformations
Umbilical hernia was found in one fetus as a malformation at external and visceral examination in the 1000 mg/kg bw/day dose group which was neither proven nor closed out to be in relationship with an effect of the test item. According to the experience of this laboratory and the scientific literature umbilical hernia is a rather common finding in the tester strain used. Therefore, this single event is not considered to be an indication for a test substance related effect.

- Variation
There was no increased incidence of external and visceral variations in the test item treated groups. Visceral variations such as bilateral hydroureter or hydroureter with dilated renal pelvis occurred with a very low incidence without significant difference among the experimental groups, including control.

Skeletal malformations:

no effects observed

Description (incidence and severity):

The number of examined fetuses was 114, 94, 119 and 95 in the control, 200, 400 and 1000 mg/kg bw/day respectively.
The incidence of skeletal abnormalities (malformations and variations) increased with a statistical significance (p<0.01) due to the increase in the variations (p<0.01) in the 1000 mg/kg bw/day dose group.

-Malformation
Malformations were recorded such as a bipartite thoracic centrum and dumb-bell shaped cartilage of thoracic centrum in two fetuses in the control and in one in the 1000 mg/kg bw/day dose group without a relationship with the test item.

- Variation
Incomplete ossification of the skull, bipartite supraoccipital, incompletely ossified or misaligned sternebrae, wavy ribs, dumb-bell shaped or bipartite vertebral centra, incomplete or asymmetric ossification of sacral arches and asymmetric or incomplete ossification of metacarpal or metatarsal, were evaluated as variations during the skeletal examination. There was a slightly but statistically significant (p<0.01) increase in the incidence of fetuses with incomplete ossification of the skull-bones and metacarpal/metatarsal in the 1000 mg/kg bw/day dose group. At this dose level, reduced body weight and food consumption of the dams might explain this slight delay in ossification. Therefore, this variation is not considered to be an indication for developmental toxicity. This assumption is supported by Mylchreest et al. (2005), who stated that retarded skull bone ossification is a relatively common observation and may not be a reliable indicator for developmental toxicity.

Visceral malformations:

no effects observed

Description (incidence and severity):

The number of evaluated fetuses was 228, 190, 239 and 192 at external and 114, 96, 120 and 97 at visceral examination in the control, 200, 400 and 1000 mg/kg bw/day groups, respectively.
The incidence of visceral abnormalities (malformations and variations) was statistically significant (p<0.05) higher in the 1000 mg/kg bw/day dose group. However, the number of affected fetuses is well within the historical control range and therefore considered to be of no biological relevance.

- Malformations
Umbilical hernia was found in one fetus as a malformation at external and visceral examination in the 1000 mg/kg bw/day dose group which was neither proven nor closed out to be in relationship with an effect of the test item. According to the experience of this laboratory and the scientific literature umbilical hernia is a rather common finding in the tester strain used. Therefore, this single event is not considered to be an indication for a test substance related effect.

- Variation
There was no increased incidence of external and visceral variations in the test item treated groups. Visceral variations such as bilateral hydroureter or hydroureter with dilated renal pelvis occurred with a very low incidence without significant difference among the experimental groups, including control.

Other effects:

no effects observed

Details on embryotoxic / teratogenic effects:

References
- Mylchreest, E., Munley, S.M., and Kennedy Jr., G.L. (2005) Evaluation of the Developmental Toxicity of 8-2 Telomer B Alcohol. Drug and Chemical Toxicology, 28:315-328.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

Oral treatment of pregnant Hsd. Brl. Han: WISTAR rats from gestation day 5 up to day 19 with Tert.-Butylperoxy- 2-ethylhexanoat at the dose levels of 200, 400 and 1000 mg/kg bw/day did not cause death and necropsy findings. The test item did not reveal any adverse effect on the pregnancy and the intrauterine mortality of the conceptuses, the number of viable fetuses and their sex distribution. Further the test substance did not increase significantly the incidence of external and visceral variations, and caused no skeletal malformations. The slight delay in ossification in fetuses of the 1000 mg/kg bw/day dose group is considered to be non-adverse.
Based on these observations the No Observed Adverse Effect Level (NOAEL) was determined as follows:

NOAEL maternal toxicity: 1000 mg/kg bw/day
NOAEL developmental toxicity: 1000 mg/kg bw/day

Executive summary:

Groups of 24 sperm-positive female Hsd. Brl. Han: WISTAR rats were treated with Tert.-Butylperoxy- 2-ethylhexanoat by oral administration daily at three dose levels of 200, 400 and 1000 mg/kg bw/day from day 5 up to and including day 19 post coitum. A control group of 24 sperm positive females was included and the animals were given the vehicle sunflower oil. The treatment volume was 2 mL/kg bw.

During the study, mortality was checked for and clinical observations were performed. Body weight and food consumption of the dams were also recorded. The day when sperm was detected in the vaginal smear was regarded as day 0 of gestation. A Caesarean section and gross pathology were performed on gestational day 20. The number of implantations, early and late resorptions, live and dead fetuses in each uterine horn and the number of corpora lutea were recorded. Each fetus was weighed and examined for sex and gross external abnormalities. The placentas were weighed and examined externally. About half of each litter was preserved for visceral examination and the other half of the litters were preserved for skeletal evaluation. At visceral examination the bodies were micro dissected by means of a dissecting microscope. The heads were examined by Wilson's free-hand razor blade method.

After cartilage-bone staining the skeletons were examined by means of a dissecting microscope. All abnormalities found during the fetal examinations were recorded.

In total, there were 21, 19, 23 and 19 evaluated litters in the control, 200, 400 and 1000 mg/kg groups, respectively. One pregnant female died in the course of the study in the control group which was found dead on gestational day 20 due to total intrauterine death.

No other clinical signs than alopecia in a few females unrelated to the treatment and salivation in the 400 and 1000 mg/kg bw/day dams immediately after treatment were observed. This was attributed to be an effect of the treatment, however as non-adverse.

There were no findings observed at necropsy.

There was no indication of an effect of the test item on body weight development and food consumption of the dams in the 200 and 400 mg/kg bw/day dose groups. The statistically significantly (p<0.01) reduced body weight gain on the first three days of treatment and the statistically significantly (p<0.01) reduced food consumption in the first week of treatment in the 1000 mg/kg bw/day dose were considered as test item related but not adverse.

There was no dose related significant difference in the intrauterine mortality of the conceptuses, the number of implantations, viable fetuses and their sex distribution. The number of late embryonic death increased slightly but statistically significant (p<0.05) in the 400 mg/kg bw/day dose group (and without a statistical significance in the 1000 mg/kg bw/day group) and was around the historical control level. There was no statistical significance indicated in the mean percentage value of the late embryonic death in the experimental groups. The statistically significant (p<0.01) reduction in the body weight of the male and female fetuses in the 1000 mg/kg bw/day group, which was in the range of historical control data, might be a consequence of the statistically significant reduction of the food consumption between gestation day 5 and 11 and lower mean body weight gain of the dams between gestation day 5 and 8. Placental weight was similar in all experimental groups. There was a statistically significant increase indicated in the relative placental weight in the 1000 mg/kg bw/day dose group, however it was below the historical control level. The distribution of external and visceral variations was homogenous in the test item treated groups, however the incidence of visceral abnormalities (variations and malformation) increased statistically significant. The occurrence of single type of variations was in the historical control range. Umbilical hernia as a malformation occurred in one fetus in the 1000 mg/kg bw/day dose group which was neither proven nor closed out to be in relationship with an effect of the test item. According to the experience of the formar laboratory of this facility and the scientific literature umbilical hernia is a rather common finding in the rat strain used. There was no test item related effect indicated at skeletal examination of the fetuses in the 200 and 400 mg/kg bw/day dose group. The incidence of the fetuses with skeletal variations increased significantly (p<0.01) in the 1000 mg/kg bw/day dose group due to the higher incidence of the delayed ossification of skull and metacarpal/metatarsal. These findings might be attributed to the effects on body weight gain and food consumption of the dams observed in the 1000 mg/kg bw/day dose group. Based on these observations, and the assumptions in the international literature that retarded skull bone ossification is a relatively common observation and may not be a reliable indicator for developmental toxicity (Mylchreest et al. (2005)), the slight delay in ossification is considered to be non-adverse. Skeletal malformations were found only in the control and 1000 mg/kg bw/day dose group with an incidence of 2 and 1 respectively, thus the test item did not induced skeletal malformations.

 

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

NOAEL maternal toxicity: 1000 mg/kg bw/day

NOAEL developmental toxicity: 1000 mg/kg bw/day

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-10-26 to 2018-05-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

2001

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD Guidance No. 43 on Mammalian Reproductive Toxicity Testing and Assessment, 24th July 2008

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: S & K-LAP Kft. Császár út 135 2173 Kartal HUNGARY
- Age at study initiation: young, healthy and breeding mature rabbits Females were nulliparous before first insemination at study initiation
- Weight at study initiation (insemination): 3566-4484 g
- Fasting period before study: no
- Housing: individually
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature: 15 - 21°C
- Humidity: 29 - 62 %
- Air changes: 8 - 12 per hr
- Photoperiod: 12 / 12 hrs dark / hrs light

Route of administration:

oral: gavage

Vehicle:

other: sunflower oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in the vehicle in concentrations of 15 mg/mL, 50 mg/mL and 150 mg/mL in the laboratory of Toxi-Coop Zrt. from daily to every three days and stored in at 5 ± 3°C.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test item is not stable in water therefore sunflower oil was used for preparing formulations appropriate for oral administration. Oleum helianthy/sunflower oil is a suitable vehicle to facilitate formulation analysis for the test item.
- Concentration in vehicle: 15, 50 and 150 mg/mL
- Treatment volume: A constant treatment volume of 2 mL dose preparation/kg body weight was administered in all groups. The individual volume of the treatment was based on the most recent individual body weight of the animals (which was determined at least every three days).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The suitability of the chosen vehicle for the test item was analytically proven. TPBEH was proved to be stable in sunflower oil formulations at ~2 and ~500 mg/mL concentration levels at least for 24 hours at room temperature and at least 3 days in refrigerator (5 ± 3°C) according to the partial analytical method validation at Toxi-Coop Zrt. Recovery of TBPEH from sunflower oil formulations at two concentration levels (~2 and ~500 mg/mL) was 104 % and 98 %. The dosing solutions were stored according to these results. Analytical control of dosing solutions (control of test item concentration) was performed in the Analytical Laboratory of Test Facility twice during the study. The mean of the test item concentrations of the test item in the dosing formulations varied in the acceptable range between 94 and 108 % of nominal concentrations at both analytical occasions confirming proper dosing.

Details on mating procedure:

- Impregnation procedure: artificial insemination
Day of insemination was regarded as day 0 of gestation. Synchronization of the cycle was completed 48 hours prior to insemination by administering PMSG (gonadotropin) hormone subcutaneously into the neck region. The insemination procedure was performed at the test facility by the breeder.

Duration of treatment / exposure:

From GD 6 to 27

Frequency of treatment:

The test item was administered in a single dose by oral gavage (stomach tube) on a 7 days/week basis every day at similar time. Control animals were treated concurrently with the vehicle only. Animals were not treated on the day of gross pathology.

Duration of test:

21 days

Dose / conc.:

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

Remarks:

Vehicle control

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

26 - 27 (inseminated)

Control animals:

yes, concurrent vehicle

yes, historical

Details on study design:

- Dose selection rationale: The dose setting was based on findings obtained in the non GLP preliminary study. In this dose range finding study the administration of TBPEH caused 100% mortality at 1000 mg/kg bw/day, no death but a single event of severe and reversible clinical signs in the 300 mg/kg bw/day dose group as well as increased early embryonic death at the dose level of 100 mg/kg bw/day.
- Rationale for animal assignment: random

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day
- Cage side observations checked in table were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a day, after treatment

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weight was recorded on gestation days 0, 3, 6, 9, 12, 15, 18, 21, 24, 27 and 28 (accuracy 1 g).
The corrected body weight was calculated for the 28th day of pregnancy (body weight on day 28 minus the weight of the gravid uterus).

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes; The food consumption was measured between gestation days 0 to 3, 3 to 6, 6 to 9, 9 to 12, 12 to 15, 15 to 18, 18 to 21, 21 to 24, 24 to 27 and 27 to 28 by re-weighing the non-consumed diet (accuracy 1 g).

WATER CONSUMPTION AND COMPOUND INTAKE: Yes, visual inspection

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 28
- Organs examined: The organs of neck, thorax and abdomen of the does were examined macroscopically. Organs pathological changes which could not be diagnosed macroscopically were fixed in 4 % neutral formaldehyde solution. Corresponding organs from control animals were kept for comparison. Histological examination on organs was not performed. The ovaries and uterus were removed and the uterus (including cervix) of the pregnant females was weighed (accuracy 1 gram). Uterus of each female was examined for early, late embryonic and fetal death and for the number of live fetuses.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: half per litter

Statistics:

Data were individually recorded on data sheets, transferred, and compiled by computer or compiled manually.
The statistical evaluation of data was performed with the 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 is detected a one-way analysis of variance (ANOVA) is carried out. If the obtained result is significant Duncan’s Multiple Range test was used to assess the significance of intergroup differences. If significance is the result of the Bartlett’s test, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test.

Does or litters were excluded from the data evaluation in cases of:
- A disease or death of the doe unrelated to the treatment (total exclusion)

- Non pregnant females i.e. females with no implantation and no corpora lutea (total exclusion)

- Body weight, body weight gain, food consumption, clinical signs and necropsy findings of females with no implantation but corpora lutea i.e. total preimplantation loss (only the intrauterine parameters were evaluated, partial exclusion) - Circumstances unrelated to the test item which are considered to be reason for exclusion, at the discretion of the Study Director Although these animals were excluded from the data evaluation the study report contains all data. A male/female fetus was considered as retarded in body weight/crown-rump length, when its weight/length was below the average minus twofold standard deviation of the control male/female fetuses.

Indices:

Pre-Implantation loss
Number of corpora lutea - Number of implantations / Number of corpora lutea x 100 (%, group mean)

Post-implantation loss
Number of implantation loss – Number of live fetuses / Number of implantations x 100 (%, group mean)


Fetuses

Sex distribution
Number of Male (Female) fetuses / Number of fetuses x 100 (%, group mean)

External abnormalities/litter
Number of fetuses with abnormality / Number of fetuses x 100 (%, group mean)

Visceral abnormalities/litter
Number of fetuses with abnormality / Number of fetuses examined x 100 (%, group mean)

Skeletal abnormalities/litter
Number of fetuses with abnormality / Number of fetuses examined x 100 (%, group mean)

Historical control data:

yes

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

The occurrence of abortions was 0, 1, 2 and 8 in the control, 30, 100 and 300 mg/kg bw/day groups, respectively hence it increased significantly in the 300 mg/kg bw/day dose group. The two cases in the 100 mg/kg bw/day group were attributed to the treatment. The single case of abortion at 30 mg/kg bw/day was not proved to be due to the treatment since abortion may occur unrelated to treatment according to the experience in the laboratory and to published historical control data of other laboratories (Makoto Ema et al., Congenital Anomalies 2012; 52, 158). Early delivery (g.d. 27 or 28 (before or in the morning of scheduled necropsy)) occurred in case of 2 does both in the control and 100 mg/kg bw/day groups, this was not attributed to the treatment. The increase of gastro-intestinal tract related observations like absence or decreased amount (observed in all groups but increased significantly in the high dose) or weak consistency of faeces (observed also in one doe each in the 30 and 100 mg/kg bw/day dose group however only in one occasion and judged to be not adverse) was attributed to the treatment at 300 mg/kg bw/day.
Bleeding from the vagina (associated to abortion or postimplantation loss), was attributed to the treatment at 300 mg/kg bw/day. Blood in the bedding was recorded for one rabbit each in the 30 and 100 mg/kg bw/day groups, both aborted. Orange discoloration in the bedding was observed in all groups and it was significantly more frequent in the 300 mg/kg bw/day dose group which was attributed to the treatment however judged as likely not adverse since the reddish colour of the urine of rabbits is considered to be normal according to specialist literature. “Red urine is a descriptive term for the condition where a rabbit's urine varies in color from the normal pale yellow to dark yellow, carrot orange, brown, or bright red”. Red urine is not a medical problem (Harkness and Wagner, 1988.) Based on this, red discoloration of the bedding in case of one animal at 30 mg/kg bw/day was not attributed to the treatment. “Lying or/and weakness and reduced activity was recorded for all moribund animals (four, one also aborted) after significant weight loss. Also, noisy breath was observed in one of these animals. Sneezing was recorded in the groups without a dose relationship.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

One of these females aborted before g.d. 25 each in the 30 and 100 mg/kg bw/day group and five (one also in moribund state) in the 300 mg/kg bw/day group and was killed before scheduled necropsy. There were three additional moribund animals in the high dose which were euthanized before scheduled necropsy. The number of animals died due to a technical reason (misgavage) was one in the control, and two each in the 100 and 300 mg/kg bw/day dose group. One female died (diarrhea, inflammation of the intestines, and lungs) as well as one was moribund and euthanized in the control group (lying unmoved, weak, purulent inflammation of the lungs and trachea and abnormal pattern of the liver was recorded at necropsy). One female with a litter of live fetuses was excluded from the evaluation because of a large, purulent abscess in the abdomen at 100 mg/kg bw/day. In total, on gestation day 28 there were 22, 24, 20 and 17 does with implantation sites (including the animals aborted from g. d. 25) and 20, 23, 16 and 10 evaluated litters in the control, 30, 100 and 300 mg/kg bw/day group respectively.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The mean body weight of the animals in the high dose group was lower from after the first treatment in the whole in-life phase (p<0.01 from g.d. 9 to 21 and p<0.05 on g.d. 24) at 300 mg/kg bw/day. The average of body weight gain was negative in all groups on the first three days of treatment, but dose relationship was indicated only in the 300 mg/kg bw/day dose group. Weight loss was observed up to day 18 (statistical significance: p<0.01 from g.d. 6 to 12 and 15 to 18) in the 300 mg/kg bw/day dose group. From GD 12 to 15 also weight loss was indicated without a statistical significance. From g.d. 15 to 18 a statistically significantly lower body weight gain (p<0.05) was observed in the 100 mg/kg bw/day dose group. From gestation day 18 there were no dose related reductions observed in the groups. The corrected body weight gain was negative in all groups and more expressed in the high dose than the other groups where no dose relationship was observed. The significant reductions of the body weight in the 300 mg/kg bw/day dose group were attributed to the test item and considered to be adverse. The lower value of body weight gain in the 100 mg/kg bw/day dose group between GD 15 and 18 was attributed to the treatment.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Significantly reduced food consumption was observed from start of the treatment up to gestation day 21 in the 300 mg/kg bw/day dose group (p<0.01 from g.d. 6 to 18 and p<0.05 from g.d. 18 to 21). The majority of the animals which aborted, were moribund or had total post-implantation loss at 300 mg/kg bw/day had minimal or zero food consumption sporadically during the in-life phase. These reductions were attributed to the treatment. Between gestation day 9 and 12 a slight reduction (p<0.05) was seen also at 100 mg/kg bw/day. This was attributed to the treatment and judged as adverse based on the individual food consumption of the two aborted females which was markedly lower on more days before abortion.

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

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:

not examined

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Darker or brighter bulges, larger brownish areas, dark discoloration and dark points in the lungs as well as black points in the stomach wall, markedly filled gall bladder with bloody content, markedly full stomach (in ten does at 300 mg/kg bw/day) filled with food or with dry content, empty intestines and distended gall bladder with bloody content both in two does were attributed to the treatment at the 300 mg/kg bw/day dose group. Stomach filled to distention was also recorded for one doe in the 30 mg/kg bw/day group, however just one with the slighter observation “fuller stomach than usual” in the 100 mg/kg bw/day group hence based on the lack of dose response these changes were not attributed to the treatment in these groups. The same female at 30 mg/kg bw/day had bloody fluid in the abdomen as a single observation and in the low dose hence not attributed to the treatment. Pinhead-sized or point-like haemorrhages or/and brownish discoloration, reddish mottled lungs, pale liver, pinched spleen were seen in the groups unrelated to the treatment. Gas filled intestines in one doe at the high dose could be in association with the treatment/reduced food consumption. The observations “vaginal orifice tainted with blood or anal region tainted with faeces” was in association with abortion in the 100 and 300 mg/kg bw/day group or weak faeces in one female in the 300 mg/kg bw/day group.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Does or litters listed below were excluded from the data evaluation:

A disease or death of the doe unrelated to the treatment (total exclusion):
- Control: No.: 40124 (disease -weakness, lying, moribund state, yellowish, greyish purulent plexuses in the lungs, pur in the trachea, nutmeg like pattern in the liver), 40097 (technical reason, died), 40078 (disease (diarrhea, reduced activity, died, inflammation of the intestines, greyish discoloration of the lungs,)
- 100 mg/kg bw/day: 40119 (technical reason, died), 40028 (technical reason, died), 60043 (disease revealed at necropsy (ca. 10 cm diameter white round formation full with pur), live litter)
- 300 mg/kg bw/day: 60048 (technical reason, died), 40186 (technical reason, died)

Non pregnant females i.e. females with no implantation and no corpora lutea (total exclusion):
- 30 mg/kg bw/day: 60027
- 100 mg/kg bw/day: 40019, 40070, 60089

Number of abortions:

effects observed, treatment-related

Description (incidence and severity):

The number of aborted females was one, two and eight in the 30, 100 and 300 mg/kg bw/day groups respectively. One of these females aborted before g.d. 25 each in the 30 and 100 mg/kg bw/day group and five (one also in moribund state) in the 300 mg/kg bw/day group and was killed before scheduled necropsy.

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

Increase of early embryonic death and post-implantation loss (statistically significant if the number and percent of resorptions evaluated and not statistically significant if the mean number and SD calculated, probably due to the high standard deviation) as well as slight decrease (without a statistical significance) of mean number of viable fetuses was indicated. The number of does with total post-implantation loss was four in the 300 and none in the other groups and judged to be due an effect of the test item. No treatment related adverse effect was indicated in the pre- implantation loss, the mean number of implantations, late embryonic death, dead fetuses and the sex distribution in the dose groups. Moreover the total intrauterine mortality (sum, %) and pre-implantation loss was statistically significantly lower (p<0.01) at 100 mg/kg bw/day, which is not considered to be of biological relevance.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Placental weight
There was no significant difference in the mean placental and relative placental weight.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

mortality

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Significantly lower fetal weight (p<0.01 if the sexes evaluated together, p<0.05 for females and lower but statistically not significant for males) and crown-rump
length were observed in the 300 mg/kg bw/day dose group which might be due to the severe maternal toxicity. One litter was completely affected (i.e. 10/10 fetuses). The maternal animal (# 031740146) of this litter revealed clinical signs such as reduced food consumption, body weight loss and no or reduced feces during gestation. This is indicative for the effects observed in the high dose group and supports the assumption that fetal retardations observed are likely linked to maternal distress and malnutrition.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

The number of evaluated litters/fetuses was 20/191, 23/216, 16/164 and 10/91 in the control, 30, 100 and 300 mg/kg bw/day groups, respectively. There was no significant difference in the litter incidence (11 (55%), 8 (35%), 7 (44%) and 6 (60%)) in the control, 30, 100 and 300 mg/kg bw/day groups respectively regarding the all over fetal malformation.

Malformation
The number of the affected litters was one both in the control and the 100 mg/kg bw/day group, respectively. A fetus was found with multiply malformed head (skull bones and facial bones absent ca. from the line of the oral orifice and upper line of ears, ca. 1/3 part of brain present, brain not covered by meninges and skull bones, tongue present, anophthalmia bilateral, maxilla absent) in the 100 mg/kg bw/day dose group.
The placenta of this fetus was fused with the next late resorption which could cause a disturbance in the placenta functioning and evolving of this malformation. Considering this and the fact that it was a single case in the test item treated groups this malformation was not considered to be a consequence of the treatment. In addition, cleft palate was found in one control fetus.

Variations
The incidence of abnormalities increased significantly (p<0.01) in the 300 mg/kg bw/day group due to growth retardation (body weight below 22.43 g for males and 21.79 g for females or crown-rump length below 77.48 mm for males and 76.71 mm for females) which were evaluated as external variations.

Placentas
The placenta of the malformed fetus was fused with the next implantation (late resorption) and paler lobe of a placenta was found in the 100 mg/kg bw/day dose group as well as two placentas were fused with each other in the 30 mg/kg bw/day dose group. These placenta changes were not attributed to the treatment considering the low incidence and different type.

Skeletal malformations:

no effects observed

Description (incidence and severity):

The incidence of fetuses with skeletal abnormalities (variations and malformations) increased in the 300 mg/kg bw/day dose group (p<0.05) but with a similar litter incidence. The litter incidence of malformations was similar in the groups (9 (45%), 7 (30%), 7 (44%), 5 (50%), while the incidence of variations was higher (not statistically significant) at 300 mg/kg bw/day.

Malformation
Absent skull bones and short mandible was found in one fetus at 100 mg/kgbw/day and were not judged to be related to the treatment (discussed at external examination). Sternebral (split xiphoid or other sternal cartilage, slightly wider sternum), rib (fused), and vertebral (absent cervical arch, asymmetric thoracic centrum including cartilage, dumb-bell shaped cartilage of thoracic centrum, fused lumbar arches, multiple malformed vertebrae) malformations occurred without a dose response or a statistical significance. If the incidence of fused sternum is summarized (3, 6, 9, 5 in the control, 30, 100 and 300 mg/kg bw/day groups respectively), a statistically significant increase was seen in the 100 mg/kg bw/day (p<0.05) group and not at 300 mg/kg bw/day. Considering that this malformation was observed also inthe control group (3 (15%) litter and 4 (2%) fetal incidence), and that this malformation occurs also in control fetuses according to the Background pregnancy and fetal data, the appearance was not attributed to the treatment. Multiply malformed ribs and vertebrae were found in one fetus in the 30 and in three in the 300 mg/kg bw/day, latter statistically significantly increased. Fused ribs however were seen only in the control (four fetuses) and 30 mg/kg bw/day (one fetus) groups. Multiply malformed vertebrae may occur in fetuses of does treated with inactive substances according to the Background pregnancy and fetal data.
The increased incidence of fused sternum and multiply malformed ribs and vertebrae were suspected to be secondary to the maternal toxicity in the 300 mg/kg bw/day dose group. One fetus (the same as with malformation at external examination) had besides cleft palate slightly shorter mandible, misshapen skull and facial bones, fused sternebra and bent scapula unilateral.

Variations
Enlarged anterior or/and posterior fontanel, irregular shape of anteriorfontanel, slightly shorter maxilla, incomplete ossification of sternum, misaligned, bipartite, dumb-bell shaped sternebra, misshapen ossification of one sternebra, slightly pinched sternal cartilage, supernumerary ossificationpoint in sternum, fusing tendency of sternebra, lack of sternal connection of 7th rib, bent and or interrupted 13th rib, dumb-bell shaped, bipartite or/and asymmetric ossification thoracic centrum, bipartite, dumb-bell shaped or and asymmetric coccygeal, less than 14 ossified coccygeal, unossified pubic, talus, pollex, less than 3.5 ossified metacarpal, less than 7/7 ossified proximal and middle phalanges were recorded as skeletal variations.

Visceral malformations:

no effects observed

Description (incidence and severity):

There were no test item related adverse effects on the visceral development of fetuses.

Malformations
There was no significant difference in the number of malformed fetuses (2, 1 and 1 in the control, 30 and 300 mg/kg bw/day dose group respectively).
Partial deficiency in the thalamus tissue was found in one fetus in the 300 mg/kg bw/day group. One fetus was found with an absence of one kidney at 30 mg/kg bw/day. Considering the low incidence, these malformations were not attributed to an effect due to the test item. Two fetuses had malformation of the great arteries/heart in the control group (enlarged right ventricle, thickened arteria pulmonalis and thin aortic arch originating directly next to arteria pulmonalis from the right ventricle).

Variations
Slightly or moderately enlarged space between cerebral hemisphere and thalamus or skull, slightly or moderately dilated IIIrd brain ventricle, convoluted- or hydroureter, malpositioned testis were found with single cases or low incidences and without a dose response, hence not attributed to the treatment.

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

Based on the observations in an OECD 414 compliant study with the test item, the No Observed Adverse Effect Level (NOAEL) was determined to be 30 mg/kg bw/day (maternal toxicity) and 100 mg/kg bw/day (developmental toxicity).

Executive summary:

The test item was examined for its possible prenatal developmental toxicity. Groups of 26 (low and mid dose) and 27 (high dose) inseminated New Zealand White rabbits were treated with TBPEH by oral (gavage) administration daily at three dose levels of 30, 100 and 300 mg/kg bw/day respectively from day 6 up to and including day 27 post insemination. A control group of 26 inseminated females was included and the animals were given the vehicle sunflower oil. The treatment volume was 2 mL/kg bw. The test item was proved to be stable in sunflower oil formulations at ~2 and ~500 mg/mL concentration levels at least for 24 hours at room temperature and at least 3 days in refrigerator (5 ± 3°C) according to the partial analytical method validation (Study no.: 552.102.3877 and 552.958.2097) at Toxi-Coop Zrt. The suitability of the chosen vehicle for the test item was analytically proven. Analytical control of dosing solutions was performed during the first and last week of treatment. The mean of the test item concentrations of the test item in the dosing formulations varied in the acceptable range between 94 and 108 % of nominal concentrations at both analytical occasions confirming proper dosing.

During the study, mortality was checked and clinical observations were performed. Moribund animals or animals aborted before gestation day 25 were euthanized by an overdose pentobarbitat solution. Animals aborted on or after gestation day 25 were necropsied at scheduled Caesarean section. Body weight and food consumption of the does were recorded. The day of insemination was regarded as day 0 of gestation. Caesarean section and gross pathology were performed on gestational day 28. The number of implantations, early and late resorptions, live and dead fetuses in each uterine horn and the number of corpora lutea were recorded. Each fetus was weighed, measured and examined for gross external abnormalities. The placentas were weighed and examined externally. Fresh visceral examination of each fetus was performed including the determination of the gender. Head of about 50 % of each litter was removed, fixed in modified Sanomiya solution and washed in 90 % isopropanol. Examination of the heads was done by Wilson's free-hand razor blade method. All skeletons were examined by means of a dissecting microscope after cartilage-bone staining.

Four does got into moribund state at 300 mg/kg bw/day between gestation days 17 and 25 after the food consumption was extremely reduced and weight loss, reduced activity, lying, weakness was observed as clear indication of severe toxicity. In association with the treatment eight does aborted in the 300 mg/kg bw/day group and two in the 100 mg/kg bw/day group. In total, on gestation day 28 there were 22, 24, 20 and 17 does with implantation sites (including the animals aborted from g. d. 25) and 20, 23, 16 and 10 evaluated litters in the control, 30, 100 and 300 mg/kg bw/day group respectively. Absence of faces, orange discoloration or blood in the bedding as well as bleeding from the vagina (associated to abortion or post-implantation loss), lying and reduced activity (moribund animals) was attributed to the treatment at 300 mg/kg bw/day. Darker or brighter bulges, larger brownish areas, dark discoloration and dark points in the lungs as well as black points in the stomach wall as well as distended stomach filled with food or dry content were attributed to the treatment at the 300 mg/kg bw/day dose group. Bloody fluid filled, distended gall bladder, empty or gas filled intestines each in one of this dose group doe were considered likely to be attributed to treatment. Significantly reduced food consumption was observed at 300 mg/kg bw/day and a slight reduction was indicated at 100 mg/kg bw/day (individually marked reduction i.e. in the two females before abortion). Lower body weight and weight loss was seen at 300 mg/kg bw/day as well as a slightly lower mean body weight gain at 100 mg/kg bw/day.

Increase of early embryonic death and post-implantation loss/total intrauterine mortality as well as decrease of mean number of viable fetuses was recorded in the 300 mg/kg bw/day dose group. Also, the incidence of females with total post-implantation loss was higher in the high dose group.

There was no significant difference in incidence of overall fetal malformations among the experimental groups. Significantly lower fetal weight and crownrump length as well as increase of growth retarded fetuses were observed in the 300 mg/kg bw/day dose group as well as increasing skeletal variations mainly due to delayed ossification (e.g. phalanges) associated to the lower body weights. Multiple malformations of ribs and vertebrae in three fetuses were considered as likely to be secondary due to the severe maternal toxicity. The visceral development of the fetuses was not affected.

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

NOAEL (maternal toxicity): 30 mg/kg bw/day

NOAEL (developmental toxicity): 100 mg/kg bw/day

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subacute

Species:

rabbit

Quality of whole database:

Two studies according to GLP and OECD 414 guideline are available in the rat and in the rabbit.Based on NOAELs obtained, the rabbit being more sensitive than the rat is considered to reflect the worst case.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

OECD 414, rat

Groups of 24 sperm-positive female Hsd. Brl. Han: WISTAR rats were treated with tert.-Butylperoxy- 2-ethylhexanoat by oral administration daily at three dose levels of 200, 400 and 1000 mg/kg bw/day from day 5 up to and including day 19 post coitum. A control group of 24 sperm positive females was included and the animals were given the vehicle sunflower oil. The treatment volume was 2 ml/kg bw.

During the study, mortality was checked for and clinical observations were performed. Body weight and food consumption of the dams were also recorded. The day when sperm was detected in the vaginal smear was regarded as day 0 of gestation. A Caesarean section and gross pathology were performed on gestational day 20. The number of implantations, early and late resorptions, live and dead fetuses in each uterine horn and the number of corpora lutea were recorded. Each fetus was weighed and examined for sex and gross external abnormalities. The placentas were weighed and examined externally. About half of each litter was preserved for visceral examination and the other half of the litters were preserved for skeletal evaluation. At visceral examination the bodies were micro dissected by means of a dissecting microscope. The heads were examined by Wilson's free-hand razor blade method.

After cartilage-bone staining the skeletons were examined by means of a dissecting microscope. All abnormalities found during the fetal examinations were recorded.

In total, there were 21, 19, 23 and 19 evaluated litters in the control, 200, 400 and 1000 mg/kg groups, respectively.

One pregnant female died in the course of the study in the control group which was found dead on gestational day 20 due to total intrauterine death.

No other clinical signs than alopecia in a few females unrelated to the treatment and salivation in the 400 and 1000 mg/kg bw/day dams immediately after treatment were observed. This was attributed to be an effect of the treatment, however as non-adverse.

There were no findings observed at necropsy.

There was no indication of an effect of the test item on body weight development and food consumption of the dams in the 200 and 400 mg/kg bw/day dose groups. The statistically significantly (p<0.01) reduced body weight gain on the first three days of treatment and the statistically significantly (p<0.01) reduced food consumption in the first week of treatment in the 1000 mg/kg bw/day dose were considered as test item related but not adverse.

There was no dose related significant difference in the intrauterine mortality of the conceptuses, the number of implantations, viable fetuses and their sex distribution.

The number of late embryonic death increased slightly but statistically significant (p<0.05) in the 400 mg/kg bw/day dose group (and without a statistical significance in the 1000 mg/kg bw/day group) and was around the historical control level. There was no statistical significance indicated in the mean percentage value of the late embryonic death in the experimental groups.

The statistically significant (p<0.01) reduction in the body weight of the male and female fetuses in the 1000 mg/kg bw/day group, which was in the range of historical control data, might be a consequence of the statistically significant reduction of the food consumption between gestation day 5 and 11 and lower mean body weight gain of the dams between gestation day 5 and 8. Placental weight was similar in all experimental groups. There was a statistically significant increase indicated in the relative placental weight in the 1000 mg/kg bw/day dose group, however it was below the historical control level.

The distribution of external and visceral variations was homogenous in the test item treated groups, however the incidence of visceral abnormalities (variations and malformation) increased statistically significant. The occurrence of single type of variations was in the historical control range. Umbilical hernia as a malformation occurred in one fetus in the 1000 mg/kg bw/day dose group which was neither proven nor closed out to be in relationship with an effect of the test item. According to the experience of the formar laboratory of this facility and the scientific literature umbilical hernia is a rather common finding in the rat strain used.

There was no test item related effect indicated at skeletal examination of the fetuses in the 200 and 400 mg/kg bw/day dose group. The incidence of the fetuses with skeletal variations increased significantly (p<0.01) in the 1000 mg/kg bw/day dose group due to the higher incidence of the delayed ossification of skull and metacarpal/metatarsal. These findings might be attributed to the effects on body weight gain and food consumption of the dams observed in the 1000 mg/kg bw/day dose group.

Based on these observations, and the assumptions in the international literature that retarded skull bone ossification is a relatively common observation and may not be a reliable indicator for developmental toxicity (Mylchreest et al. (2005)), the slight delay in ossification is considered to be non-adverse.

Skeletal malformations were found only in the control and 1000 mg/kg bw/day dose group with an incidence of 2 and 1 respectively, thus the test item did not induced skeletal malformations.

 

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

NOAEL maternal toxicity: 1000 mg/kg bw/day

NOAEL developmental toxicity: 1000 mg/kg bw/day

 

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

NOEL maternal toxicity: 400 mg/kg bw/day

NOEL developmental toxicity: 400 mg/kg bw/day

 

OECD 414, rabbit

The test item was examined for its possible prenatal developmental toxicity. Groups of 26 (low and mid dose) and 27 (high dose) inseminated New Zealand White rabbits were treated with TBPEH by oral (gavage) administration daily at three dose levels of 30, 100 and 300 mg/kg bw/day respectively from day 6 up to and including day 27 post insemination. A control group of 26 inseminated females was included and the animals were given the vehicle sunflower oil. The treatment volume was 2 mL/kg bw. The test item was proved to be stable in sunflower oil formulations at ~2 and ~500 mg/mL concentration levels at least for 24 hours at room temperature and at least 3 days in refrigerator (5 ± 3°C) according to the partial analytical method validation (Study no.: 552.102.3877 and 552.958.2097) at Toxi-Coop Zrt. The suitability of the chosen vehicle for the test item was analytically proven. Analytical control of dosing solutions was performed during the first and last week of treatment. The mean of the test item concentrations of the test item in the dosing formulations varied in the acceptable range between 94 and 108 % of nominal concentrations at both analytical occasions confirming proper dosing.

During the study, mortality was checked and clinical observations were performed. Moribund animals or animals aborted before gestation day 25 were euthanized by an overdose pentobarbitat solution. Animals aborted on or after gestation day 25 were necropsied at scheduled Caesarean section. Body weight and food consumption of the does were recorded. The day of insemination was regarded as day 0 of gestation. Caesarean section and gross pathology were performed on gestational day 28. The number of implantations, early and late resorptions, live and dead fetuses in each uterine horn and the number of corpora lutea were recorded. Each fetus was weighed, measured and examined for gross external abnormalities. The placentas were weighed and examined externally. Fresh visceral examination of each fetus was performed including the determination of the gender. Head of about 50 % of each litter was removed, fixed in modified Sanomiya solution and washed in 90 % isopropanol. Examination of the heads was done by Wilson's free-hand razor blade method. All skeletons were examined by means of a dissecting microscope after cartilage-bone staining.

Four does got into moribund state at 300 mg/kg bw/day between gestation days 17 and 25 after the food consumption was extremely reduced and weight loss, reduced activity, lying, weakness was observed as clear indication of severe toxicity. In association with the treatment eight does aborted in the 300 mg/kg bw/day group and two in the 100 mg/kg bw/day group. In total, on gestation day 28 there were 22, 24, 20 and 17 does with implantation sites (including the animals aborted from g. d. 25) and 20, 23, 16 and 10 evaluated litters in the control, 30, 100 and 300 mg/kg bw/day group respectively. Absence of faces, orange discoloration or blood in the bedding as well as bleeding from the vagina (associated to abortion or post-implantation loss), lying and reduced activity (moribund animals) was attributed to the treatment at 300 mg/kg bw/day. Darker or brighter bulges, larger brownish areas, dark discoloration and dark points in the lungs as well as black points in the stomach wall as well as distended stomach filled with food or dry content were attributed to the treatment at the 300 mg/kg bw/day dose group. Bloody fluid filled, distended gall bladder, empty or gas filled intestines each in one of this dose group doe were considered likely to be attributed to treatment. Significantly reduced food consumption was observed at 300 mg/kg bw/day and a slight reduction was indicated at 100 mg/kg bw/day (individually marked reduction i.e. in the two females before abortion). Lower body weight and weight loss was seen at 300 mg/kg bw/day as well as a slightly lower mean body weight gain at 100 mg/kg bw/day.

Increase of early embryonic death and post-implantation loss/total intrauterine mortality as well as decrease of mean number of viable fetuses was recorded in the 300 mg/kg bw/day dose group. Also, the incidence of females with total post-implantation loss was higher in the high dose group. There was no significant difference in incidence of overall fetal malformations among the experimental groups. Significantly lower fetal weight and crownrump length as well as increase of growth retarded fetuses were observed in the 300 mg/kg bw/day dose group as well as increasing skeletal variations mainly due to delayed ossification (e.g. phalanges) associated to the lower body weights. Multiple malformations of ribs and vertebrae in three fetuses were considered as likely to be secondary due to the severe maternal toxicity. The visceral development of the fetuses was not affected.

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

NOAEL (maternal toxicity): 30 mg/kg bw/day

NOAEL (developmental toxicity): 100 mg/kg bw/day

Justification for classification or non-classification

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

The available experimental test data are not yet complete to allow a final conclusion on classification under Regulation (EC) No 1272/2008.

Based on the results of an EOGRTS, revealing an effect of the test item on female rat fertility, a hazard to humans cannot be excluded. Therefore, the test item is classified as Repro. Cat. 1B (H360F: “May damage fertility”) according to CLP Regulation.

Available data on toxicity to prenatal developmental toxicity / teratogenicity in two species, rat and rabbit, are sufficient and reliable for classification purposes. Based on the available data, the test item does not require classification being a prenatal developmental toxicant or teratogen.