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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 Mar 2016 - 20 Oct 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
2001
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
1998
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
2008
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: harles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: 10-12 weeks
- Weight at study initiation: 141.0 – 190.8 g.
- Fasting period before study: no
- Housing: individually in Polycarbonate cages type III
- Diet: round Kliba maintenance diet mouse/rat “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland, ad libitum
- Water: potable tap water, ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70%
- Air changes (per hr): 15 times per hour
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.5% Carboxymethylcellulose in drinking water (with 5 mg/100 mL Tween 80)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The aqueous test substance preparations were prepared at the beginning of the administration period and thereafter at intervals, which took into account the period of established stability. The preparations were kept at room temperature. For the test substance preparations, the specific amount of test substance was weighed, topped up with 0.5% Carboxymethylcellulose suspension in drinking water (with 5 mg/100 mL Tween 80) in a calibrated beaker and intensely mixed with a homogenizer. During administration, the preparations were kept homogeneous with a magnetic stirrer.

VEHICLE
- Concentration in vehicle: 1, 3 and 10 g/100 ml
- Amount of vehicle (if gavage): 10 ml/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability of the test substance in 0.5% Carboxymethylcellulose suspension in drinking water (with 5 mg/100 mL Tween 80) at room temperature over a period of 7 days had been verified prior to the start of the study. Samples of the test substance preparations were sent to the analytical laboratory once during the study period (at the beginning of administration) for verification of the concentrations. The samples were also used to verify the homogeneity of the low and the high concentrations (100 and 1000 mg/kg bw/d). Three samples (one from the top, middle and bottom in each case) were taken from the flask with a magnetic stirrer running.
Results:
The stability of the test substance in 0.5% Carboxymethylcellulose in drinking water with 5 mg/100 mL Tween 80 over a period of 7 days at ambient temperature was demonstrated.
The homogeneous distribution of the test substance in the vehicle (0.5% Carboxymethylcellulose in drinking water with Tween 80 (5 mg/100 mL)) was demonstrated.
The results of the analysis of the aqueous test substance preparations confirmed the correctness of the prepared concentrations. The analytical values of the samples corresponded to the expected values within the limits of the analytical method, i.e. were always above 90% and below 110% of the nominal concentrations.
Details on mating procedure:
The animals were paired by the breeder (“time-mated”); the day of evidence of mating (= detection of vaginal plug/sperm) was referred to as GD 0. The animals arrived on the same day (GD 0) at the experimental laboratory. The following day was designated as “GD 1".
Duration of treatment / exposure:
from implantation to one day prior to the expected day of parturition (GD 6-19).
Frequency of treatment:
daily
Duration of test:
On GD 20, the females were sacrificed in a randomized order and examined macroscopically. The fetuses were removed from the uterus and investigated.
Doses / concentrationsopen allclose all
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:
25
Control animals:
yes, concurrent vehicle

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
A check was made twice a day on working days or once a day on Saturdays, Sundays or on public holidays (GD 0-20).

DETAILED CLINICAL OBSERVATIONS: Yes
A cage-side examination was conducted at least once daily before and after treatment period (GD 0-5 and 20). During treatment period (GD 6-19) all rats were checked daily for any signs of morbidity, pertinent behavioral changes and/or signs of overt toxicity before administration as well as within 2 hours and within 5 hours after administration.

BODY WEIGHT: Yes
All animals were weighed on GD 0, 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20. The body weight change of the animals was calculated based on the obtained results. Furthermore, the corrected body weight gain was calculated after terminal sacrifice (terminal body weight on GD 20 minus weight of the unopened uterus minus body weight on GD 6).

FOOD CONSUMPTION: Yes
The consumption of food was recorded for the intervals GD 0-1, 1-3, 3-6, 6-8, 8-10, 10-13, 13-15, 15-17, 17-19 and 19-20.

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
After the dams had been sacrificed, they were necropsied and assessed for gross pathology, in randomized order.
Ovaries and uterine content:
The uteri and the ovaries were removed and the following data were recorded:
- Weight of the unopened uterus
- Number of corpora lutea
- Number and distribution of implantation sites
• Live fetuses
• Dead implantations:
a) Early resorptions (only decidual or placental tissues visible or according to SALEWSKI from uteri from apparently non-pregnant animals and the empty uterushorn in the case of single horn pregnancy)
b) Late resorptions (embryonic or fetal tissue in addition to placental tissue visible)
c) Dead fetuses (hypoxemic fetuses which did not breathe spontaneously after the uterus had been opened)
After the weight of the uterus had been determined, all subsequent evaluations of the dams and the gestational parameters were conducted by technicians unaware of treatment group in order to minimize bias. For this purpose animal numbers were encoded. classified as:
Fetal examinations:
All fetal analyses were conducted by technicians unaware of the treatment group, in order to minimize bias.

At necropsy each fetus was weighed, sexed, and external tissues and all orifices were examined macroscopically. The sex was determined by observing the distance between the anus and the base of the genitalia. Furthermore, the viability of the fetuses and the condition of placentae, umbilical cords, fetal membranes, and fluids were examined. The placentas were weighed and their individual weights were recorded. Thereafter, the fetuses were sacrificed by a subcutaneous injection of pentobarbital (Narcoren®; dose: 0.1 mL/fetus). After these examinations, approximately one half of the fetuses per dam were eviscerated, skinned and fixed in ethanol; the other half were placed in Harrison’s fluid for fixation.

The fetuses fixed in Harrison’s fluid were examined for any visceral findings according to the method of BARROW and TAYLOR. After this examination these fetuses were discarded.
The skeletons of the fetuses fixed in ethanol were stained according to a modified method of KIMMEL and TRAMMELL. Thereafter, the skeletons of these fetuses were examined under a stereomicroscope. After this examination the stained fetal skeletons were archived individually.
In the present study the glossary of WISE et al. (1997) and its updated version of MAKRIS et al. (2009) was essentially used to describe findings in fetal morphology. Classification of these findings was based on the terms and definitions proposed by CHAHOUD et al.
(1999) and SOLECKI et al. (2001, 2003):
- Malformation: A permanent structural change that is likely to adversely affect the survival or health.
- Variation: A change that also occurs in the fetuses of control animals and/or is unlikely to adversely affect the survival or health. This includes delays in growth or morphogenesis that have otherwise followed a normal pattern of development.
The term "unclassified observation" was used for those fetal findings, which could not be classified as malformations or variations. All fetal findings were listed in tables according to these classifications.
Statistics:
DUNNETT-test (two-sided): Food consumptiona), body weight, body weight change, corrected body weight gain (net maternal body weight change), carcass weight, weight of unopened uterus, number of corpora lutea, number of implantations, number of resorptions, number of live fetuses, proportions of preimplantation loss, proportions of postimplantation loss, proportions of resorptions, proportion of live fetuses in each litter, litter mean fetal body weight, litter mean placental weight
FISHER'S EXACT test (onesided): Female mortality, females pregnant at terminal sacrifice, number of litters with fetal findings
WILCOXON-test (one-sided): Proportions of fetuses with malformations, variations and/or unclassified observations in each litter
Indices:
- conception rate
- preimplantation loss
- postimplantation loss

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any female at dose levels of 100, 300 or 1000 mg/kg bw/d and the control during the entire study period.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
There were no substance-related or spontaneous mortalities in any females of all test groups including the control group (0, 100, 300 or 1000 mg/kg bw/d).
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The mean body weights and the average body weight gain of the low-, mid- and high-dose dams (100, 300 and 1000 mg/kg bw/d) were generally comparable to the concurrent control group throughout the entire study period.
The corrected body weight gain of test groups 1-3 (100, 300 and 1000 mg/kg bw/d) revealed no difference of any biological relevance to the concurrent control group. Moreover, mean carcass weights remained also unaffected by the treatment.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The mean food consumption of the dams in test groups 1-3 (100, 300 and 1000 mg/kg bw/d) was generally comparable to the concurrent control throughout the entire study period.
Food efficiency:
not specified
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:
no effects observed
Description (incidence and severity):
The mean gravid uterus weights of the animals of test groups 1-3 (100, 300 and 1000 mg/kg bw/d) were not influenced by the test substance. The differences between these groups and the concurrent control groups revealed no dose-dependency and were assessed to be without biological relevance.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No necropsy findings which could be attributed to the test substance were seen in any dam. There occurred two spontaneous findings:
• a diaphragmatic hernia in dam No. 77 of test group 3 (1000 mg/kg bw/d),
• a dilated renal pelvis in dam No. 32 of test group 1 (100 mg/kg bw/d).
These findings were detected in single animals and were, therefore, assessed as incidental.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
There were no test substance-related and/or biologically relevant differences between the test groups 0-3 in conception rate, in the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and the postimplantation losses, the number of resorptions and viable fetuses. All observed differences are considered to reflect the normal range of fluctuations for animals of this strain and age.
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
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined
Changes in number of pregnant:
no effects observed

Effect levels (maternal animals)

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: no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
The mean fetal weights of test groups 1, 2 and 3 (100, 300 and 1000 mg/kg bw/d) were not influenced by the test substance and did not show any biologically relevant differences in comparison to the concurrent control group.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The sex distribution of the fetuses in test groups 1-3 (100, 300 and 1000 mg/kg bw/d) was comparable to the concurrent control fetuses.
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
One fetus, each, in test groups 2 and 3 (300 and 1000 mg/kg bw/d) had external malformations. Both fetuses had associated soft tissue or skeletal malformations. The total incidence of external malformations in treated animals did not differ significantly from the concurrent control group and the occurred findings were also observed in the historical control data. Therefore, it was not assessed as treatment-related.
No external variations were recorded.
No external unclassified observations were recorded.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
A number of skeletal malformations were detected in fetuses of test groups 0, 1 and 3 (0, 100 and 1000 mg/kg bw/d) affecting the vertebral column, forelimbs and ribs. One fetus of test group 3 (94-03) showed two skeletal malformations (absent sacral and caudal vertebra) and also associated external findings. Two other high-dose fetuses of one litter showed bent ribs. The appearance in two fetuses in one single litter indicates that this was a sporadic finding. It is not considered as treatment-related. All other
skeletal malformations (and also external findings of the fetus 94-03) were observed in the historical control data and were single cases, without a relation to dosing. Therefore, the findings were not considered to be treatment-related.
For all test groups, skeletal variations of different bone structures were observed, with or without effects on corresponding cartilages. The observed skeletal variations were related to several parts of fetal skeleton and appeared without a relation to dosing. The
overall incidences of skeletal variations were covered by the historical control data.
Additionally, some isolated cartilage findings without impact on the respective bone structures, which were designated as unclassified cartilage observations, occurred in all test groups. The observed unclassified cartilage findings were related to the skull, the vertebral column, the sternum and ribs and did not show any relation to dosing.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
One fetus, each, in test groups 1, 2 and 3 (100, 300 and 1000 mg/kg bw/d) had soft tissue malformations. Male high-dose fetus No. 80-05 had multiple visceral malformations, i.e. situs inversus (thoracic cavity, heart and great vessels), enlarged ventricular chambers, a partly thin ventricular septum and an abnormal lung lobation. The total incidence of soft tissue malformations in treated animals did not differ significantly from the concurrent control group and was covered by the historical control data.
Three soft tissue variations were detected, i.e. interrupted palatal rugae, dilated renal pelvis and dilated ureter. In all cases, the incidences were neither statistically significantly nor dosedependently changed in comparison to the concurrent control group. However, the number of these variations adds up to a statistically significantly higher value for the total soft tissue variation rate in test group 1. As the total incidences were not related to dose and the respective values of test group 1 are inside the range of the historical control data (HCD: mean of 3.2%, range of 0.0 - 6.9, they were not assessed as treatment-related.
No unclassified soft tissue observations were recorded.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
The mean placental weights were comparable between the control and test groups 1-3 (0, 100, 300 and 1000 mg/kg bw/d). Biologically relevant differences were not observed. The significantly higher mean placental weight (0.52 g) of all viable fetuses in test group 2 was
marginally above the other dose groups and also within the range of the historical control data. Therefore, the increase was considered to be incidental and not treatment-related.

Effect levels (fetuses)

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 effects observed

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Assessment of all fetal external, soft tissue and skeletal observations

There were noted external, soft tissue and skeletal malformations in all test groups (0, 100, 300 and 1000 mg/kg bw/d). The distribution of total malformations throughout the groups was not related to dose. Five fetuses were multiple-malformed. Female fetus No. 17-07 (control group) had a severely malformed vertebral column and ribs (in the region of thoracic, lumbar and sacral vertebrae, e.g. branched or short rib, absent or misshapen vertebra), while male fetus No. 19-05 (control group) had a thoracic hemivertebra and a branched rib. For female fetus No. 67-08 (300 mg/kg bw/d) gastroschisis and short intestines were recorded. Furthermore, male fetus No. 80-05 (1000 mg/kg bw/d) had multiple visceral malformations affecting the region of thoracic cavity, i.e. situs inversus (thoracic cavity, heart and great vessels), enlarged ventricular chambers, a partly thin ventricular septum and an abnormal lung lobation (three lobes on each side). The findings in male fetus No. 94-03 (1000 mg/kg bw/d) consisted of a thread-like tail, comprising absent sacral and caudal vertebrae during skeletal examination. No ontogenetic pattern is recognizable for these individual malformations and the highest incidence was noted in the control group. Most individual malformations can be found in the historical control data. An association of these findings to the treatment is therefore not assumed. Other observed malformations, such as situs inversus, misshapen thoracic vertebra, shortened scapula, split scapula, and shortened humerus in test groups 0-3 are common for this rat strain and most of them can also be found in the historical control data at a comparable or higher frequency. An association of these findings to the treatment is not assumed. Bent ribs were noted in one high-dose litter. The term “bent ribs” is often used as synonym to wavy ribs, at least the differentiation of these two findings is really only based on the morphological appearance and not on the underlying etiology. Wavy ribs represent a common finding in rodent prenatal toxicity studies. Numerous studies have been conducted to study the reversibility of wavy ribs during postnatal development. They have shown that wavy ribs represent a reversible finding. The appearance in two fetuses in one single litter indicates that this was a sporadic finding. It is not considered as treatment-related and as of toxicological concern.

Total fetal malformations:

    Test group 0
0 mg/kg bw/d
Test group 1
100 mg/kg bw/d
Test group 2
300 mg/kg bw/d
Test group 3
1000 mg/kg bw/d
Litter Fetuses

N

N

25
240
24
227
24
224
25
227
Fetal incidence N (%) 5 (2.1) 2 (0.9) 1 (0.4) 4 (1.8)
Litter incidence N (%) 4 (16) 2 (8.3) 1 (4.2) 3 (12)
Affected fetuses/litter Mean% 2.3 0.8 0.5 2.0

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

External variations did not occur in any of the fetuses in this study. Some soft tissue variations and a range of skeletal variations were noted in all test groups including the controls. None of the incidences showed a relation to dosing. The skeletal variations are equally distributed about the different test groups, if normal biological variation is taken into account, and can be found in the historical control data at a comparable frequency.

Total fetal variations:

    Test group 0
0 mg/kg bw/d
Test group 1
100 mg/kg bw/d
Test group 2
300 mg/kg bw/d
Test group 3
1000 mg/kg bw/d
Litter Fetuses

N

N

25
240
24
227
24
224
25
227
Fetal incidence N (%) 126 (53) 125 (55) 114 (51) 123 (54)
Litter incidence N (%) 25 (100) 24 (100) 24 (100) 25 (100)
Affected fetuses/litter Mean% 53.4 56.1 50.8 54.6

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Occurrence of statistically significantly increased fetal skeletal variations (expressed as mean percentage of affected fetuses/litter):

Finding

Test group 0
0 mg/kg bw/d
Test group 1
100 mg/kg bw/d
Test group 2
300 mg/kg bw/d
Test group 3
1000 mg/kg bw/d
HCD
Mean % (range)
Incomplete ossification of parietal;
unchanged cartilage
3.1 11.3* 7.6 5.7 10.3
(2.0 – 17.1)
Supernumerary thoracic vertebra 4.0 3.0 11.0* 7.1 4.0
(0.8 – 11.0)

mg/kg bw/d = milligram per kilogram body weight per day; HCD = Historical control data; % = per cent * = p ≤ 0.05 (Wilcoxon-test [one-sided]) ** = p ≤ 0.01 (Wilcoxon-test [one-sided])

No unclassified external and unclassified soft tissue observations were recorded for any of the fetuses in this study. A spontaneous origin was assumed for the unclassified skeletal cartilage observations which were observed in several fetuses of all test groups (0, 100, 300 and 1000 mg/kg bw/d). The distribution and type of these findings do not suggest any relation to treatment. Finally, fetal examinations revealed that there is no effect of the compound on the respective morphological structures up to the highest tested dose (1000 mg/kg bw/d).

Applicant's summary and conclusion

Conclusions:
The no observed adverse effect level (NOAEL) for maternal and prenatal developmental toxicity is the highest tested dose of 1000 mg/kg bw/d.
Executive summary:

The test item was tested for its prenatal developmental toxicity in Wistar rats. The test substance was administered as an aqueous preparation to groups of 25 time-mated female Wistar rats by gavage at doses of 100, 300 and 1000 mg/kg body weight/day (mg/kg bw/d) on gestation days (GD) 6 through 19. One control group, consisting of 25 females, was dosed with the vehicle (0.5% Carboxymethylcellulose in drinking water (with 5 mg/100 mL Tween 80)) in parallel. A standard dose volume of 10 mL/kg body weight was used for each test group. At terminal sacrifice on GD 20, 24 - 25 females per group had implantation sites. Food consumption and body weights of the animals were recorded regularly throughout the study period. The state of health of the animals was checked each day. On GD 20, all females were sacrificed by decapitation (under isoflurane anesthesia) and assessed by gross pathology (including weight determinations of the unopened uterus and the placentae). For each dam, corpora lutea were counted and number and distribution of implantation sites (differentiated between resorptions, live and dead fetuses) were determined. The fetuses were removed from the uterus, sexed, weighed and further investigated for external findings. Thereafter, one half of the fetuses of each litter were examined for soft tissue findings and the remaining fetuses for skeletal (inclusive cartilage) findings. The stability of the test substance preparations was demonstrated over a period of 7 days at room temperature. The homogeneous distribution of the test substance in the vehicle (drinking water) was confirmed. The correctness of the prepared concentrations was shown. No test substance-related adverse effects on dams, gestational parameters or fetuses were observed in any of the dose groups tested. In conclusion, under the conditions of this prenatal developmental toxicity study, the oral administration of the test item to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-19) at doses as high as 1000 mg/kg bw/d caused neither evidence of maternal nor developmental toxicity. The no observed adverse effect level (NOAEL) for maternal and prenatal developmental toxicity is the highest tested dose of 1000 mg/kg bw per day.