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Effects on fertility

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
310 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
GLP and guideline study without restrictions.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

The purpose of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental toxicity screening test was to provide initial information concerning the toxic potential of TBPPI and on its possible effects on male and female reproductive performance such as gonadal function, mating behavior, conception, pregnancy, parturition as well as on development of the F1 offspring from conception to day 4 post-partum associated with oral administration to rats at repeated doses. Four groups of Hsd.Brl.Han:Wist rats (n=12/sex/group) were administered orally (by gavage) once a day at 0 (vehicle only), 50, 150 and 310 mg/kg bw/day at concentrations of 25 mg/mL, 75 mg/mL and 155 mg/mL, corresponding to 2 mL/kg bw dose volume. The suitability of the chosen vehicle for the test item at the intended concentrations was analytically verified up front. TBPPI was stable at room temperature for 24 hours and in a refrigerator (5 ± 3 °C) for 3 days. Concentration of the test item in the dosing formulations varied in the range of 98 % to 104 % in comparison to the nominal values, thereby confirming proper dosing. All animals of the parent (P) generation received test item or vehicle prior to mating (14 days) and throughout mating. Test item or vehicle was administered to male animals post mating up to the day before the necropsy. For females with living pups, test item was administered through the gestation period and up to lactation days 3 – 6, i.e. up to the day before the necropsy. Observations included mortality, clinical signs, body weight, food consumption, mating, pregnancy and delivery process, as well as development of pups. Five dams and males cohabited with were selected from each group for further toxicity examinations such as functional observations, hematology, clinical chemistry, gross necropsy, organ weighing and histopathology. The dams were allowed to litter, and rear their young up to termination on days 4 – 7 postpartum. Pups were weighed and observed for possible abnormalities. All parental animals were subjected to gross pathology one day after the last treatment and offspring were euthanized. Selected organs were weighed. Full histopathology was performed in the selected animals of control and high dose groups. Histopathology examination was performed on reproductive organs and pituitary of the remaining animals in the control and high dose groups. The reproductive organs and pituitary of non-pregnant female animals and males cohabited with in the low and mid dose groups were also processed and evaluated histologically. The results were interpreted comparing treatment groups with respect to controls, which were treated concurrently with vehicle (sunflower oil) only.

Results

Mortality: There was no test item related mortality at any dose level (310, 150 and 50 mg/kg bw/day).

Clinical observation: Test item related salivation appeared in male and female animals administered with 310 and 150 mg/kg bw/day groups with a dose related onset and incidence. No toxic signs related to the test item were found at the detailed weekly and terminal functional observations. The behavior and physical condition of animals were normal during the entire observation period (pre-mating, mating, post-mating, gestation and lactation periods).

Body weight and body weight gain: The body weight gain was reduced with respect to controls at 310 mg/kg bw/day in male animals during the entire observation period (with statistical significances on weeks 1, 2, 3 and 6 and if summarized) and in dams during lactation period. These changes resulted in a slightly less body weight in the range of -5 to -7 % in male animals with respect to control from day 20 up to the termination and -13 % in dams on lactation day 4.

Food consumption: The mean daily food consumption was slightly less comparing to the control group at 310 and 150 mg/kg bw/day doses during first week of premating period (male), during the premating (female) and at 310 mg/kg bw/day between lactation days 0 and 4.

Hematology: Hematology examinations did not reveal any test item related changes in the evaluated hematological parameters at any dose level (310, 150 and 50 mg/kg bw/day).

Clinical chemistry: No test item-related changes were observed in investigated clinical chemistry parameters for the male animals. In the female animals treated with 310 mg/kg bw/day, the glucose (GLUC) concentration was slightly but significantly below the value of control and historical control ranges in this treatment group and a test item related effect cannot be ruled out. However, as the reduction in GLUC was very slight, the finding was considered to be of no toxicological concern.

Necropsy: Specific macroscopic alterations related to the test item were not found during the necropsy.

Organ weight: A test item influence on renal function was observed as mean weights of kidneys (absolute, and relative to body and brain weights) were slightly higher with respect to the controls and above the historical control ranges in male animals at 310 mg/kg bw/day dose. The increases in absolute, relative to body, and brain weights were determined to be 26, 31 and 23%, respectively.

Histopathology: Histopathology investigation did not detect any microscopic changes related to the test item effect. Reproduction There were no differences between the control and test item treated groups in the reproductive performance of male and female animals and in delivery data of dams.

Reproduction: There were no differences between the control and test item treated groups in the reproductive performance of male and female animals and in delivery data of dams.

Offspring: A test item effect on the offspring development was observed in the significantly higher number and percentage of extra uterine mortality in 310 mg/kg bw/day group between postnatal days 0 and 4, and in the significantly less litter weight and litter weight gain and mean pup’s weight and weight gain at 310 mg/kg bw/day. These effects were probably a consequence of the observed maternal systemic effect (reduced body weight and food consumption during lactation period).

Conclusion

Under the conditions of the present study, TBPPI caused salivation (male and female), changes in body weight (male and female), food consumption (male and female), slightly reduced glucose concentration (female) and elevated kidney weights (male) following an oral administration at 310 mg/kg bw/day to Hsd.Brl.Han:Wistar rats during the Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test. At 150 mg/kg bw/day, salivation and slightly reduced food consumption were observed in male and female animals. At 50 mg/kg bw/day, no test item related adverse effects were observed. Based on these observations the No Observed Adverse Effect Levels (NOAEL) were determined as follows:

NOAEL for male and female rats: 150 mg/kg bw/day

NOAEL for reproductive performance of the male and female rats: 310 mg/kg bw/day

NOAEL for F1 Offspring: 150 mg/kg bw/day


Short description of key information:
The subacute toxicity oral of TBPPI were assessed in a study performed according to OECD Guideline 422 in rats. Based on these observations the No Observed Adverse Effect Level (NOAEL) were determined as follows:
NOAEL for reproductive performance of the male and female rats: 310 mg/kg bw/day
NOAEL for male and female rats: 150 mg/kg bw/day
NOAEL for F1 Offspring: 150 mg/kg bw/day

Justification for selection of Effect on fertility via oral route:
Only one study available.

Effects on developmental toxicity

Description of key information
In a study conducted according to OECD Guideline No 414, the NOAEL value for developmental toxicity was determined to be 150 mg/kg bw/day. The NOAEL for maternal toxicity was found to be 50 mg/kg bw/day.
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-03-20 to 2015-07-29
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and OECD Guideline conform study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
2001
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
1998
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OECD Guidance No. 43 on Mammalian Reproductive Toxicity Testing and Assessment (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: Young adult and nulliparous females, 12- 13 weeks; males: 18-21 weeks
-Body weight at study initiation: mean ca. 200 g
- Housing: Before mating: 1-3 females per cage, 1-2 males per cage; during mating: 1 male and 1-3 females / cage; during gestation: 2 sperm positive females per cage, if not possible 1 sperm positive female per cage; Type II polypropylene/polycarbonate
- Diet: ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest, Germany, ad libitum
- Water: tap water ad libitum
- Acclimation period: 14 days females, 7 days males

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-22
- Humidity (%): 30 - 36
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:
oral: gavage
Vehicle:
other: sunflower oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations were prepared in the formulation laboratory of the Test Facility not longer than for 3 days before the administration.
Analytical control of dosing solutions (control of concentration) was performed in the Analytical Laboratory of the Test Facility twice during the study. Concentrations of the test item in the dosing formulations varied in the acceptable range between 98 and 109 % of nominal concentrations at both analytical occasions confirming proper dosing. A sufficient stability and homogeneity in the chosen vehicle were verified over the range of relevant concentrations at the appropriate frequency of preparation.

VEHICLE
- Justification for use and choice of vehicle: The test item is not soluble in water therefore sunflower oil was used for preparing formulations appropriate for oral administration. Sunflower oil was a suitable vehicle to facilitate formulation analysis for the test item.
- Concentration in vehicle: 0, 25, 75, 225 mg/mL
- Amount of vehicle: treatment volume: 2 mL
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical control of dosing solutions (control of concentration) was performed in the Analytical Laboratory of Test Facility twice during the study. Concentrations of the test item in the dosing formulations varied in the acceptable range between 98 and 109 % of nominal concentrations at both analytical occasions confirming proper dosing. A sufficient stability and homogeneity in the chosen vehicle were verified over the range of relevant concentrations at the appropriate frequency of preparation.
The test item was analyzed using reverse phase HPLC method with UV detection. Five samples were taken from different places of each test concentration and from the vehicle. The samples were stored at 5 ± 3 °C until the analysis.
HPLC system: Hitachi LaChrom Elite,
L-2000 Organizer, No.: 18E32-083
L-2130 HPLC pump, No.: 18E17-015
L-2200 Autosampler, No.: 19E40-005
L-2400 UV Detector, No.: 18E33-030
L-2350 Column Oven, No.: 1831-023

HPLC Conditions:
Detector: 210 nm
Column: HyperPrep HS C18, 250 x 4.6 mm, 8 μm, No.: 10190790
Mobil Phase: Acetonitrile : water = 70 : 30 (v/v)
Flow Rate: 1 mL/min
Injection volume: 50 μL
Temperature: 25 °C
Retention time: 7 min ± 5 %
Run time: 10 minutes
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1 male and 1-3 females / cage
- Length of cohabitation: The females were paired to males in the mornings for two to four hours (one male: one to three females) until the number of sperm positive females per group achieves at least twenty four.
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
The sperm positive females were treated from gestational day 5 to 19.
Frequency of treatment:
daily, 7 days/week
Duration of test:
gestational day 5 to 19
Remarks:
Doses / Concentrations:
0, 50, 150, 450 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
24 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose setting based on findings obtained in a Non GLP 14-Day Oral Gavage Dose Range Finding Study with TBPPI in the Rat.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

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

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: uterus with cervix, ovaries

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 live fetuses per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter
Statistics:
The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test.
Where no significant heterogeneity is detected a one-way analysis of variance (ANOVA) is carried out. If the obtained result is significant Duncan’s Multiple Range test was used to access the significance of inter-group 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.
Historical control data:
The results were compared to the laboratory's historical control data.
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Reduced activity, piloerection, hypotonicity and hyperventilation of the animals as well as reduced food consumption, weight loss and lower body weight in the 450 mg/kg bw/day group as well as reduced body weight- and corrected body weight gain in the 150 mg/kg bw/day dose group was in association with the test item.
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Moderately increased incidence of split sternum (7 of 108 fetuses) in the 450 mg/kg bw/day dose group might be a consequence of the treatment. Split sternum occurs however with low incidence also in control fetuses according to the Summary of Laboratory’s historical control data. The increased late embryonic death, lower mean body weight and the increased incidence of fetuses with delayed ossification of skeletons or bipartite ossification of sternebra in the 450 mg/kg bw/day group might be attributed to the clinical signs, the statistically significant reduced body weight from gestation day 11 up to the end of in-life phase, the weight loss on the first three days of treatment and the reduced body weight gain between gestation days 8 and 11 and 17 to 20 as well as the affected food intake in the entire treatment period at this dose level. The delayed ossification of the skull bones of the fetuses in the 150 mg/kg bw/day group might be attributed to the statistically significant reduced body weight gain between gestational day 5 and 8 and a lower corrected body weight gain of the dams at this dose level. Based on the maternal toxicity observed, 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, 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.), the delay in ossification is considered to be non-adverse.
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
Based on the observations the No Observed Adverse Effect Level (NOAEL) was determined as follows:
NOAEL (maternal toxicity): 50 mg/kg bw/day
NOAEL (developmental toxicity): 150 mg/kg bw/day

Executive summary:

TBPPI was examined for its possible prenatal developmental toxicity. Groups of 24 sperm-positive female Hsd. Brl. Han: Wistar rats were treated with TBPPI by oral administration daily at three dose levels of 50, 150 and 450 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 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. 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.Reduced activity, piloerection, hypotonicity and hyperventilation of the animals as well as reduced food consumption, weight loss and lower body weight in the 450 mg/kg bw/day group as well as reduced body weight- and corrected body weight gain in the 150 mg/kg bw/day dose group was in association with the test item. 

TBPPI did not reveal any adverse effect on the preimplantation loss, early embryonic- and fetal death, number of implantation and the sex distribution of the fetuses moreover did not decrease the number of viable fetuses significantly. There was a scattered occurrence of few brain malformations (dilated lateral ventricles, deficient or misshapen brain tissue and enlarged perimeningeal space) in three fetuses, each one fetus in one litter, in the 450 mg/kg bw/day group. However, a test item relationship can neither be concluded nor excluded with certainty as according to the experience with this species and to the international literature similar findings occur spontaneously with low incidence in the rat strain used. Therefore it is finally considered as more likely that these single brain alterations in isolated fetuses are not caused by the treatment with the test item but as incidental in nature.

Moderately increased incidence of split sternum (7 of 108 fetuses) in the 450 mg/kg bw/day dose group is considered to be a consequence of the treatment. Split sternum occurs, however, with low incidence also in control fetuses according to the historical control data.

The increased late embryonic death, lower mean body weight and the increased incidence of fetuses with delayed ossification of skeletons or bipartite ossification of sternebra in the 450 mg/kg bw/day group are considered to be attributed to maternal toxicity, which occurred in the form of clinical signs, statistically significant reduced body weight from gestation day 11 up to the end of in-life phase, weight loss on the first three days of treatment and reduced body weight gain between gestation days 8 and 11 and 17 to 20 as well as affected food intake in the entire treatment period at this dose level. The delayed ossification of the skull bones of the fetuses in the 150 mg/kg bw/day group might be attributed to the statistically significant reduced body weight gain between gestational day 5 and 8 and a lower corrected body weight gain of the dams at this dose level. Based on the maternal toxicity observed, and the hind in the peer reviewed international literature that retarded skull bone ossification is a relatively common observation and may not be a reliable indicator for developmental toxicity, the delay in ossification is finally considered to be non-adverse.

 

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

NOAEL (maternal toxicity): 50 mg/kg bw/day

NOAEL (developmental toxicity):150  mg/kg bw/day

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
150 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
GLP and OECD Guideline conform study, reliable sufficient for assessment.
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

TBPPI was examined for its possible prenatal developmental toxicity. Groups of 24 sperm-positive female Hsd. Brl. Han: Wistar rats were treated with TBPPI by oral administration daily at three dose levels of 50, 150 and 450 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 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. 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.Reduced activity, piloerection, hypotonicity and hyperventilation of the animals as well as reduced food consumption, weight loss and lower body weight in the 450 mg/kg bw/day group as well as reduced body weight- and corrected body weight gain in the 150 mg/kg bw/day dose group was in association with the test item. 

TBPPI did not reveal any adverse effect on the preimplantation loss, early embryonic- and fetal death, number of implantation and the sex distribution of the fetuses moreover did not decrease the number of viable fetuses significantly. There was a scattered occurrence of few brain malformations (dilated lateral ventricles, deficient or misshapen brain tissue and enlarged perimeningeal space) in three fetuses, each one fetus in one litter, in the 450 mg/kg bw/day group. However, a test item relationship can neither be concluded nor excluded with certainty as according to the experience with this species and to the international literature similar findings occur spontaneously with low incidence in the rat strain used. Therefore it is finally considered as more likely that these single brain alterations in isolated fetuses are not caused by the treatment with the test item but as incidental in nature.

Moderately increased incidence of split sternum (7 of 108 fetuses) in the 450 mg/kg bw/day dose group is considered to be a consequence of the treatment. Split sternum occurs, however, with low incidence also in control fetuses according to the historical control data.

The increased late embryonic death, lower mean body weight and the increased incidence of fetuses with delayed ossification of skeletons or bipartite ossification of sternebra in the 450 mg/kg bw/day group are considered to be attributed to maternal toxicity, which occurred in the form of clinical signs, statistically significant reduced body weight from gestation day 11 up to the end of in-life phase, weight loss on the first three days of treatment and reduced body weight gain between gestation days 8 and 11 and 17 to 20 as well as affected food intake in the entire treatment period at this dose level. The delayed ossification of the skull bones of the fetuses in the 150 mg/kg bw/day group might be attributed to the statistically significant reduced body weight gain between gestational day 5 and 8 and a lower corrected body weight gain of the dams at this dose level. Based on the maternal toxicity observed, and the hind in the peer reviewed international literature that retarded skull bone ossification is a relatively common observation and may not be a reliable indicator for developmental toxicity, the delay in ossification is finally considered to be non-adverse.

 

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

NOAEL (maternal toxicity): 50 mg/kg bw/day

NOAEL (developmental toxicity):150  mg/kg bw/day


Justification for selection of Effect on developmental toxicity: via oral route:
only one study available

Justification for classification or non-classification

Based on the results of the reproduction and developmental toxicity screening test, the test item was not classified and labelled according to Regulation (EC) No 1272/2008 (CLP).

Additional information