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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:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

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

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
Details on test material:
Expiry date 26/07/2038

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: About 11-13 weeks
- Fasting period before study: no
- Housing: single
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: From GD 0 (day of supply) to the beginning of administration (GD 6), the animals will be accustomed to the environmental
conditions and to the diet.

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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Details on exposure:
Route of administration: Orally by gavage
Reason for the selection of the route of administration: The oral administration of a test substance has been proven useful worldwide in numerous studies for discovering a potential toxicological profile.
Frequency of administration/number of administrations: Once daily (GD 6-19)/14
Volume to be administered: 10 ml/kg body weight; the body weight determined most recently will be used to calculate the administration volume.
Preparation: For the test substance preparations, the specific amount of test substance will be weighed, topped up with 0.5% Carboxymethylcellulose suspension in drinking water in a calibrated beaker and intensely mixed with a homogenizer. Before and during administration, the preparations will be kept homogeneous with a magnetic stirrer.
Preparation frequency: daily
Storage conditions of the preparations: RT
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical investigations of the test substance preparations will be carried out as a
separate study at the test facility Competence Center Analytics of BASF SE, 67056
Ludwigshafen, Germany, under the responsibility of the study director of this test facility.
The study will be carried out in compliance with the Principles of Good Laboratory
Practice.
Details on mating procedure:
The animals are paired by the breeder and will be supplied at noon on the day of evidence of mating; this day is referred to as GD O and the following day as GD 1.
Duration of treatment / exposure:
The day of evidence of mating is referred to as GD O and the following day as GD 1.
The test substance will be administered to the animals orally by gavage from GD 6 through GD 19
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on information of the combined reprotox / repeated dose study and the 90d study, 100, 300 and 1000 mg/kg bw were chose.

The random distribution of the animals to the individual groups will be carried out on the day of supply (= GD 0) by randomly removing the animals from the transport boxes.

On GD 20, all surviving dams will be sacrificed and examined. All evaluations except for the gross-pathological evaluation and the determination of the uterus weights will be carried out by technicians unaware of the treatment groups using coded animal numbers. The fetuses will be removed from the uterus, which has been opened before and examined.

Examinations

Maternal examinations:
Mortality
A check for moribund and dead animals will be made twice daily from Mondays to Fridays and once daily on Saturdays, Sundays and public holidays (GD 0 to 20).

Clinical signs
A cageside examination will be conducted at least once daily for any signs of morbidity, pertinent behavioral changes and/or signs of overt toxicity.
During the administration period (GD 6 - 19) all animals will be checked daily for any abnormal clinically signs before the administration as well as within 2 hours and within 5 hours after the administration. Abnormalities and changes will be documented for each animal.

Food consumption
Food consumption will be recorded for GD 0-1, 1-3, 3-6, 6-8, 8-10, 10-13, 13-15, 15-17, 17-19 and 19-20.

Body weight
Body weights will be recorded on GD 0, 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20.

POST-MORTEM EXAMINATIONS
On GD 20, the surviving dams will be anesthetized with isoflurane, sacrificed by cervical dislocation in a randomized sequence and examined. The fetuses will be removed from the uterus, which has been opened before and examined.
Moribund animals and animals that die intercurrently will be examined if possible (with the exception of the uterus weight).
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes / No / No data
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
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
Statistics:
Means and standard deviations will be calculated. In addition, the following statistical analyses will be carried out:

Food consumption, body weight, body weight change, corrected body weight gain, carcass weight, weight of the unopened uterus, weight of the placentas and
fetuses, corpora lutea, implantations, pre- and postimplantation losses, resorptions and live fetuses --> DUNNETT's test

Number of pregnant animals at the end of the study, mortality rate (of the dams) and number of litters with fetal findings --> FISHER's exact test

Proportion of fetuses with findings per litter --> WILCOXON test
Indices:
conception rate (in %) = (number of pregnant animals / number of fertilized animals) x 100
preimplantation loss (in %) for each individual pregnant animal which underwent scheduled sacrifice = ((number of corpora lutea – number of implantations)/number of corpora lutea) x 100
The postimplantation loss (in %) for each individual pregnant animal which underwent scheduled sacrifice = ((number of implantations – number of live fetuses) / number of implantations) x 100
Historical control data:
OECD 414 studies in wistar rats between 2012 - 2016 (n=34)

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 during the entire study period.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
There were no test substance-related or spontaneous mortalities in any female of all test groups (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 average body weight gain of the low-, mid- and high-dose dams (100, 300 or 1000 mg/kg bw/d) were in general comparable to the concurrent control group throughout the entire study period.
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, 2 and 3 (100, 300 and 1000 mg/kg bw/d) was comparable to the concurrent control throughout the entire study period.
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:
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 control group revealed no dose-dependency and were assessed to be without biological relevance.
Gross pathological findings:
no effects observed
Description (incidence and severity):
A yellow discolored content of the stomach was recorded in 5 out of 25 mid-dose females (20%) and in 6 high-dose females (24%). This yellow discoloration mirrors presence of the test substance (or its metabolites) in the gastrointestinal tract. It is not considered as an adverse, toxic effect by itself.

No further necropsy findings which could be attributed to the test substance were seen in any dam.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined

Maternal developmental toxicity

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:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
The conception rate reached 92% in the mid-dose group (300 mg/kg bw/d), 96% in the low- and high-dose groups (100 and 1000 mg/kg bw/d) and 100% in the control group (0 mg/kg bw/d). With these rates, a sufficient number of pregnant females were available for the purpose of the study.
Details on maternal toxic effects:
There were no test substance-related and/or biologically relevant differences between 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 fluc- tuations for animals of this strain and age.

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 at any dose level

Maternal abnormalities

Key result
Abnormalities:
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 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 control fetuses.
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Description (incidence and severity):
No external malformations were recorded.
No external variations were recorded.
No external unclassified observations were recorded.
Skeletal malformations:
no effects observed
Description (incidence and severity):
Skeletal malformations were detected exclusively for one fetus of the control group (0 mg/kg bw/d). Therefore, these findings are considered as incidental and not treat- ment-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 skeletons and appeared without a relation to dosing. The overall incidences of skeletal variations were comparable between treated groups and concurrent control as well as with the historical control . As can be seen from the corresponding table, the affected fetuses per litter incidences of ‘misshapen sternebra (unchanged cartilage)’ in test group 2 and 3 were well below the mean value of historical control data. Thus, it is not considered as an adverse event. The finding ‘incomplete ossification of supraoccipital; unchanged cartilage’ was statistically sig- nificantly increased in test group 1 (litter incidence: n=19*, 79%). However, the increase was not dose-related and the value was well within the range of the historical control data (mean 79%, range of 50 - 100%). Therefore, it is not assessed as being treatment-related.
Additionally, some isolated cartilage findings without impact on the respective bony structures, which were designated as unclassified cartilage observations, occurred in all test groups. The observed unclassified cartilage findings were related to the skull, the ribs and the sternum.
The incidence of ‘bipartite processus xiphoideus’ was statistically significantly increased in test group 2 (300 mg/kg bw/d). As a consequence of this occasional increase, the incidence of total fetal skeletal unclassified cartilage observations was statistically significantly increased in this test group. Since there is no dose-response relationship and the finding can be found in the historical control data at a higher frequency, an association to the treatment and a toxicological relevance is not assumed.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No soft tissue malformations were recorded.
Two soft tissue variations were detected in all test groups including the control (0, 100, 300 or 1000 mg/kg bw/d), i.e. dilated renal pelvis and dilated ureter. The incidences of these variations were neither statistically significantly different from control nor dose-dependent and, therefore, not considered biologically relevant. All of them can be found in the historical control data at comparable incidences.
No soft tissue unclassified observations were recorded.
Other effects:
no effects observed

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 at any dose level

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

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

NN

25

238

24

242

23

242

24

226

 

Fetal incidence

 

N (%)

 

1 (0.4)

 

0.0

 

0.0

 

0.0

 

Litter incidence

 

N (%)

 

1 (4.0)

 

0.0

 

0.0

 

0.0

Affected fetuses / litter

 

Mean%

 

0.6

 

0.0

 

0.0

 

0.0

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

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

LitterFetuses

NN

25

238

24

242

23

242

24

226

 

Fetal incidence

 

N (%)

 

129 (54)

 

130 (54)

 

133 (55)

 

121 (54)

 

Litter incidence

 

N (%)

 

25 (100)

 

24 (100)

 

23 (100)

 

24 (100)

Affected fetuses/litter

 

Mean%

 

55.8

 

55.4

 

55.0

 

54.3

Total soft tissue 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

LitterFetuses

N N

24

111

23

113

23

114

24

107

 

Fetal incidence

 

N (%)

 

5 (4.5)

 

1 (0.9)

 

5 (4.4)

 

3 (2.8)

 

Litter incidence

 

N (%)

 

4 (17)

 

1 (4.3)

 

4 (17)

 

3 (13)

Affectedfetuses/litter

 

Mean%

 

4.0

 

0.7

 

4.1

 

2.5

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

Total skeletal 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

LitterFetuses

NN

25

127

24

129

23

128

24

119

 

Fetal incidence

 

N (%)

 

1 (0.8)

 

0.0

 

0.0

 

0.0

 

Litter incidence

 

N (%)

 

1 (4.0)

 

0.0

 

0.0

 

0.0

Affectedfetuses/litter

 

Mean%

 

1.0

 

0.0

 

0.0

 

0.0

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

Total fetal skeletal 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

LitterFetuses

N N

25

127

24

129

23

128

24

119

 

Fetal incidence

 

N (%)

 

124 (98)

 

129 (100)

 

128 (100)

 

118 (99)

 

Litter incidence

 

N (%)

 

25 (100)

 

24 (100)

 

23 (100)

 

24 (100)

Affectedfetuses/litter

 

Mean%

 

98.1

 

100.0

 

100.0

 

99.4

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

Occurrence of statistically significantly increased 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)

 

Misshapen sternebra; unchanged cartilage

 

20.5

 

23.8

 

30.5*

 

32.6*

 

46.6

(20.0 - 76.9)

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])

Total unclassified cartilage observations

 

 

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

LitterFetuses

N N

25

127

24

129

23

128

24

119

 

Fetal incidence

 

N (%)

 

72 (57)

 

85 (66)

 

93 (73)

 

81 (68)

 

Litter incidence

 

N (%)

 

24 (96)

 

23 (96)

 

23 (100)

 

23 (96)

Affectedfetuses/litter

 

Mean%

 

57.8

 

63.8

 

73.5*

 

66.4

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

* = p ≤ 0.05 (Wilcoxon-test [one-sided])

Applicant's summary and conclusion

Conclusions:
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 a dose of 1000 mg/kg bw/d caused neither evidence of maternal nor developmental toxicity.
In conclusion, the no observed adverse effect level (NOAEL) for maternal and prenatal developmental toxicology is 1000 mg/kg bw/d.