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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
2001
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
1998
Deviations:
no
Qualifier:
according to
Guideline:
other: OECD Guidance No. 43 on Mammalian Reproductive Toxicity Testing and Assessment (2008)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid

Test animals

Species:
rat
Strain:
Wistar
Details on test animals 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

Administration / exposure

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
Doses / concentrations
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.

Examinations

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.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

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.

Effect levels (maternal animals)

open allclose all
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

Results (fetuses)

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.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

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