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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:
10.04.2013 - 11.10.2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2014

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline

Test animals

Species:
rat
Strain:
other: CD / Crl: CD (SD)
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 8 - 9 weeks (on day 0 of pregnancy)
- Weight at study initiation: 178.6 - 235.3 g (on day 0 of pregnancy)
- Housing: individually
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C +/- 3°C
- Humidity (%): 55% +/- 15%
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
peanut oil
Details on exposure:
The test item was dissolved in the vehicle to the appropriate concentrations. The test item-formulations were freshly prepared on each administration day.
The test item was administered orally at a constant volume (4 mL/kg b.w./day) once daily. The dose of the test item was adjusted to the animal’s body weight daily.
The control animals received the vehicle (peanut oil) at a constant volume of 4 mL/kg b.w. orally once daily in the same way.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analysis of TAC concentration revealed correctly prepared test item formulations. The actual concentrations of the test item within the
formulations were within the measured range of approx. 98% and 103% of the nominal TAC concentrations. No TAC could be detected in any of
the control samples.
Details on mating procedure:
Sexually mature ('proved') male rats of the same breed served as partners. The female breeding partners were randomly chosen.
Mating was monogamous: 1 male and 1 female animal were placed together in one cage during the dark period. Each morning a vaginal smear was
taken to check for the presence of sperm. If findings were negative, mating was repeated with the same partner. The day on which sperm was found was considered as the day of conception (day 0 of pregnancy). This procedure was repeated until enough pregnant dams were available for all
groups. Rats which did not become pregnant were excluded from the analysis of the results and replaced by other animals. A post-mortem negative staining according to SALEWSKI was carried out in the replaced animals in order to confirm the non-pregnancy status.
Duration of treatment / exposure:
Day 6 to 19 of gestation
Frequency of treatment:
Once daily
Duration of test:
23.04.2013 - 16.05.2013
No. of animals per sex per dose:
20
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale: The dose levels were selected based on the results of a 28-day dose-range-finding study in rats dosed with 100 or 200 mg TAC/kg b.w./day.

Examinations

Maternal examinations:
- Clinical signs: Individual animals were observed daily for behaviour, external appearance and nature of the faeces. Dated and signed records of appearance, change and disappearance of clinical signs were maintained on clinical history sheets for individual animals.
- Viability: Checks were made to look for dead or moribund animals twice daily.
- Body weight: The weight of each rat was recorded on day 0 of gestation (the day of detection of a positive mating sign), followed by daily weighings
always at the same time of the day. The body weight gain was also calculated in intervals (i.e. day 0-3, 3-6, 6-9, 9-12, 12-15, 15-18 and 18-20).
- Food and drinking water consumption: The quantity of food consumed by each rat was recorded daily. Food intake per rat (g/rat/day) was calculated using the total amount of food given to and left by each rat in each group on completion of a treatment day. Daily monitoring by visual appraisal of the drinking water bottles was maintained throughout the study.
Ovaries and uterine content:
- Macroscopic inspection (gross evaluation) of the placentae for example for focal indurations.
- Number and size of resorptions.
- Corpora lutea in the ovaries, implantations and location of fetuses in the uterus.
- Gravid uterus weight.
Fetal examinations:
- Number of fetuses (alive and dead) and placentae.
- Sex and viability of fetuses.
- Weights of fetuses and weights of the placentae (fetuses were considered as runts if their weight was less than 70% of the mean litter weight).
- External inspection of fetuses for damages, especially for malformations. 50% of the number of fetuses in each litter were examined for skeletal anomalies. The remaining 50% of the number of fetuses in each litter were examined for soft tissue anomalies.

Statistics:
For all numerical values homogeneity of variances was tested by using the BARTLETT chi-square test. If the variances were homogeneous, the DUNNETT test (p ≤ 0.05 and p ≤ 0.01) was used to compare the experimental groups with the control group.
In case of heterogeneity of variances, the STUDENT's t-test was carried out; limit of significance was p ≤ 0.05 and p ≤ 0.01.
For the comparison of classification measurements (for example malformation-, resorption-, retardation- and variation rate) the FISHER's exact test (n < 100) or chi2-test with Yates' correction for continuity (n ≥ 100; p ≤ 0.05 and p ≤ 0.01) was employed.
Indices:
- Resorption rate
- Malformation rate
- Variation rate
- Retardation rate
- Pre-implantation loss
- Post-implantation loss
Historical control data:
Summarized results from 53 teratogenicity studies performed with Sprague-Dawley rats in the years 2000 - 2013 were used as background data.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Under the present test conditions, the no-observed-adverse-effect level (NOAEL) was 30 mg TAC/kg b.w./day for the dams.
120 mg TAC/kg b.w./day were in the lethal range, 3 out of 25 dams were found dead on gestation day 7, all of them showed reduced motility and prone position after the first treatment on gestation day 6. Observation of the 3 prematurely deceased dams at necropsy revealed haemorrhagic eyes and changes in the lungs and the stomach. Systemic intolerance reactions in the form of reduced motility, prone position, piloerection, slight to moderate salivation and haemorrhagic eyes were noted in all or a few of the surviving high dose dams. In addition, reductions were noted for the net body weight change from gestation day 6 until laparotomy and for the food consumption.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
The no-observed-adverse effect level (NOAEL) for the fetal organism was 30 mg TAC/kg b.w./day.
Embryotoxic properties in the form of developmental delays such as reduced fetal and placental weights, increased fetal incidences noted for retarded skeletal ossification (of metacarpalia and metatarsalia, the skull, the sternebra(e) (not ossified), the thoracic vertebral bodies (dumbbell-shaped or reduced in size), the pelvic and caudal vertebral bodies, os ischii and os pubis) and increased soft tissue variations (dilated 4th cerebral ventricle) were noted at the materno-toxic/lethal dose level of 120 mg TAC/kg b.w./day. Skeletal retardations and soft tissue variations were only noted at the materno-toxic dose level, and, hence, all changes are considered as secondary toxicity caused by adverse maternal toxicity. No test item-related increase was noted in the incidence of malformations, not even at the materno-toxic dose level.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: teratogenicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Maternal findings:

Mortality: Three of 25 high dose dams (120 mg TAC/kg b.w./day) were found dead on gestation day 7, one day after the first administration. All 3 animals showed reduced motility and prone position after the first treatment on test day 6. External observation at autopsy revealed haemorrhagic eyes in all 3 animals. In addition, dark-red discoloured lungs and diffusely scattered haemorrhagic foci in the stomach were noted in 2 animals, but not in the third animal. All three premature deaths were considered as test item-related.

Clinical signs: In the high dose group (120 mg TAC/kg b.w./day) all evaluated surviving animals showed reduced motility and prone position daily during the whole treatment period. In addition, piloerection was noted in 6 animals for 4 to 6 consecutive test days, predominantly during the first treatment days. A slight to moderate salivation was noted in 4 further animals for one up to 5 consecutive test days, starting between gestation days 12 and 15. Additionally to the 3 prematurely deceased rats, haemorrhagic eyes were noted in 3 of the surviving dams for one up to 7 days.

Body weight and body weight gain: In the high dose group (120 mg TAC/kg b.w./day), a slight but statistically significant reduction in body weight was noted on gestation days 19 and 20 (by max. 6.9%; p ≤ 0.05). More pronounced reductions were found for the absolute body weight change between gestation day 6 and the day of laparotomy (by 28%; p ≤ 0.01) and the net weight change from gestation day 6 until the day of laparotomy (by 38%; p ≤ 0.01).

Food consumption: At 120 mg TAC/kg b.w./day, the relative food intake of the dams was reduced during the treatment period, being 58% below the control on gestation day 7 and between 18% and 28% below the control on gestation days 8 to 13. Statistically significant reductions (at p ≤ 0.01 or p ≤ 0.05) were noted on gestation days 7 to 17.

Necropsy findings: The macroscopic changes in the lungs and the stomach and the haemorrhagic eyes noted in the prematurely deceased animals are considered as test item-related.

Uterus and carcass weights: At 120 mg TAC/kg b.w./day, significant reductions (at p ≤ 0.01) were noted for the mean gravid uterus weight (23% below the control) predominantly due to the severely reduced mean fetal weights at the high dose level.

Fetal findings:

No test item-related influence was detected on the prenatal fetal development at 10, 30 or 120 mg TAC/kg b.w./day with respect to the number of resorptions, number of live fetuses, the values calculated for the post-implantation loss and the sex distribution of fetuses.

Mortality: No dead fetuses were noted at laparotomy.

Fetal and placental weights: At 120 mg TAC/kg b.w./day, statistically significant reductions (p ≤ 0.01) compared to the control were noted for the mean fetal body weights (by 22%) and the mean placental weights (by 23%).

Malformations: No test item-related malformations were noted at any tested dose level during external / internal examination, skeletal examination and during soft tissue evaluation.

Variations: No test item-related variations were noted in the fetuses during external / internal examination or skeletal examination. At 120 mg TAC/kg b.w./day, soft tissue examination revealed a statistically significantly increased (p ≤ 0.01) fetal incidence for a dilatation of the 4th cerebral ventricle. No increase was noted for total soft tissue variations.

Retardations: At 120 mg TAC/kg b.w./day, statistically significant fetal incidences (at p ≤ 0.05 or p ≤ 0.01) were noted for a retarded ossification of metacarpalia and metatarsalia, the skull, the sternebra(e) (not ossified), the thoracic vertebral bodies (dumbbell-shaped or reduced in size), the pelvic and caudal vertebral bodies, os ischii and os pubis. However, no increase was noted for the incidence of total skeletal retardations. The retardations were only noted at the materno-toxic dose level, and, thus, considered as secondary toxicity caused by adverse maternal toxicity.

Applicant's summary and conclusion

Conclusions:
In this teratogenicity study the test item TAC was administered to female rats at dose levels of 10, 30 or 120 mg/kg b.w./day. The no-observed-adverse effect level (NOAEL) for the dams as well as for the fetal organism was 30 mg TAC/kg b.w./day.
No test item-related influence was detected on the prenatal fetal development at 10, 30 or 120 mg TAC/kg b.w./day with respect to the number of resorptions, number of live fetuses, the values calculated for the post-implantation loss and the sex distribution of fetuses. Embryotoxic properties in the form of developmental delays such as reduced fetal and placental weights, increased fetal incidences noted for retarded skeletal ossification and increased soft tissue variations were noted at the materno-toxic/lethal dose level of 120 mg TAC/kg b.w./day only. Thus, all changes are considered as secondary toxicity caused by adverse maternal toxicity. No test item-related increase was noted in the incidence of malformations, not even at the materno-toxic dose level of 120 mg TAC/kg b.w./day.