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Diss Factsheets

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
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:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted 22 January 2001
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Germany)
Limit test:
no

Test material

Constituent 1
Reference substance name:
Reaction mass of 2-{[3-(trimethoxysilyl)propyl]amino}ethanaminium chloride and 2-{benzyl[3-(trimethoxysilyl)propyl]amino}ethanaminium chloride and N-[2-(benzylamino)ethyl]-3-(trimethoxysilyl)propan-1-aminium chloride and N-[2-(dibenzylamino)ethyl]-3-(trimethoxysilyl)propan-1-aminium chloride and N-benzyl-2-{benzyl[3-(trimethoxysilyl)propyl]amino}ethanaminium chloride and N-benzyl-N-[2-(dibenzylamino)ethyl]-3-(trimethoxysilyl)propan-1-aminium chloride.
Molecular formula:
Not applicable
IUPAC Name:
Reaction mass of 2-{[3-(trimethoxysilyl)propyl]amino}ethanaminium chloride and 2-{benzyl[3-(trimethoxysilyl)propyl]amino}ethanaminium chloride and N-[2-(benzylamino)ethyl]-3-(trimethoxysilyl)propan-1-aminium chloride and N-[2-(dibenzylamino)ethyl]-3-(trimethoxysilyl)propan-1-aminium chloride and N-benzyl-2-{benzyl[3-(trimethoxysilyl)propyl]amino}ethanaminium chloride and N-benzyl-N-[2-(dibenzylamino)ethyl]-3-(trimethoxysilyl)propan-1-aminium chloride.

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl: WI(Han) (Full Barrier)
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Age at study initiation: approx. 11-12 weeks old
- Weight at study initiation: males: 330 - 372 g; females: 196 - 250 g
- Housing: Individually housed in IVC cages on saw fiber bedding (except during the pre-mating period when females were kept in groups of two animals and during mating period when two females were paired with one male). During the pre-mating period and after mating, males were housed in groups (2 animals / cage) in IVC cages.
- Diet: Altromin 1324 maintenance diet provided ad libitum
- Water: Tap water, sulphur acidified to a pH of approximately 2.8 provided ad lititum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 10
- Air changes (per hr): 10
- Photoperiod: 12 hrs dark / 12 hrs light

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
DMSO
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test item formulation was prepared at least once every 7 days based on available stability data.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on the test item’s characteristics (hydrolysis in water) and poor miscibility with the standard vehicles corn oil, olive oil or paraffin oil.
- Concentration in vehicle: 100%
- Amount of vehicle (if gavage): 2 mL/kg bw
- Lot/batch no. (if required): SZBF0910V and SZBG1310
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
General homogeneity and stability of the test item in the vehicle was investigated before start of the study at Evonik Technology & Infrastructure GmbH, Paul-Baumann-Str. 1, 45772 Marl, Germany in standalone studies under GLP. According to the results, formulations of the test item in DMSO were shown to be homogenous at both concentrations tested (50 mg/mL and 500 mg/mL, taken from top, middle and bottom of prepared formulations) (Evonik study AN-ASB 0711). Furthermore, the test item was shown to be stable in DMSO at 50 mg/mL for at least 24 hours at room temperature and for at least one week at 2-8 °C. Formulation at 500 mg/mL was regarded as stable for at least 11 days at room temperature and at 2-8 °C (Evonik study AN-ASB 0707).

Samples for analysis of concentration of the test item in the vehicle will be taken in the first and last week of the study for all doses (8 samples in total). General homogeneity and stability of the test item in the vehicle was investigated before start of the study at Evonik Technology & Infrastructure GmbH, Paul-Baumann-Str. 1, 45772 Marl, Germany in standalone studies under GLP. According to the results, formulations of the test item in DMSO were shown to be homogenous at both concentrations tested (50 mg/mL and 500 mg/mL, taken from top, middle and bottom of prepared formulations) (Evonik study AN-ASB 0711). Furthermore, the test item was shown to be stable in DMSO at 50 mg/mL for at least 24 hours at room temperature and for at least one week at 2-8 °C. Formulation at 500 mg/mL was regarded as stable for at least 11 days at room temperature and at 2-8 °C (Evonik study AN-ASB 0707). Samples for analysis of concentration of the test item in the vehicle will be taken in the first and last week of the study for all doses (8 samples in total). The quantitative determination of the Si-content and the corresponding calculated concentration of the test item in the dosing formulations will be performed by means of ICP-OES analysis by Evonik Technology & Infrastructure GmbH in a separate standalone study in accordance with GLP.

Nominal concentrations were confirmed for all dose groups, as measured concentrations were within acceptance criterion of 10%. All samples were homogenous, as the coefficient of variation between the different sampling locations (top, middle and bottom) was below 10%.
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1:2 (male to female) ratio
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- Any other deviations from standard protocol: Due to an increased number of non-pregnant females and/or mortality in the groups, after getting 100 sperm positive females, the remaining females were also mated and the two more obtained sperm positive females were assigned to the control group (female no. 101) and the LD group (female no. 102) in order to obtain about 20 pregnant females per group.
Duration of treatment / exposure:
Gestation days 5 to 19
Frequency of treatment:
daily
Duration of test:
Gestation days 5 to 19
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:
26 female Wistar rats in the control group and the low dose group and of each 25 female Wistar rats in the medium dose group and the high dose group.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Doses were selected based on the results of the dose range finding study (BSL Munich study no. 152905, non-GLP) and in consultation with the sponsor.
- Rationale for animal assignment: Mated females were assigned in an unbiased manner to the control and treatment groups ensuring that group mean body weights were comparable with each other. Each animal was assigned a unique identification number.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: General clinical observations were made at least once a day, preferably at the same time each day. The health condition of the animals was recorded. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily.
- Cage side observations included: check for spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnea, asphyxia, vocalisation, diarrhea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size. Changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypes, difficult or prolonged parturition or bizarre behavior were recorded if present

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed once before initiation of pairing to ensure that the body weights are within ±20% variation. The sperm positive females were weighed during gestations days 0, 5, 8, 11, 14, 17 and 20. Males were not weighed in this study except once before initiation of pairing.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION AND COMPOUND INTAKE: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: all gross lesions, lungs, trachea, larynx, esophagus, brain, head with paranasal sinuses (after flushing with fixative) and stomach.
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 per litter
- Soft tissue examinations: Yes, half per litter
- Skeletal examinations: Yes, half per litter
- Head examinations: Yes, the first 20 litters per group
Statistics:
A statistical assessment of the results of the body weight, food consumption and litter data was performed by comparing values of dosed with control animals using a one-way ANOVA and a post-hoc Dunnett Test. Foetal evaluation parameters like external, visceral, craniofacial and skeletal parameters were analysed using Fisher’s exact test. The litter was used as the unit for data analysis. The statistics were performed with GraphPad Prism V.6.01 software (p<0.05 was considered as statistically significant).
Historical control data:
Wistar rat historical control data from prenatal developmental toxicity studies (years 2011-2015) were provided.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Clinical signs are summarized in Table 1. Abnormal breathing was noted without dose-dependency in 1/26 females of the control group, 5/26 of the LD group, 5/25 of the MD group and 2/25 of the HD group during the treatment period of this study. Abnormal breathing was linked to regurgitation. Regurgitation was observed on single days of treatment directly after dose application without dose-dependency in 1/26 females of the control group, 2/26 females of the LD group, 3/25 females of the MD group and 1/25 females of the HD group. Salivation was noted dose-dependently in 4/26 females of the LD group, 15/25 females of the MD group and 19/25 females of the HD group on several days of treatment. Furthermore, moving the bedding was observed dose-dependently in 6/26 females of the LD group, 21/25 females of the MD group and 23/25 females of the HD group on several days of treatment. Moving the bedding and salivation were seen transiently in timely relation to dose administration and were considered as slight clinical signs elicited by local effects of the test item formulation and/or attributed to discomfort of the animals due to the oral administration, but not systemic toxicity. All other clinical signs were not considered treatment-related and incidental in nature.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Mortality was observed in all dose groups without clear dose-response. Therefore these effects are not considered to be related to systemic toxicity. These results are illustrated in Table 2. 2/26 females of the LD group were found dead (no. 31 on GD 17, no. 37 on GD 12), 1/25 females of the MD group (no. 72 on GD 18) and 3/25 females of the HD group (no. 86 on GD 8, no. 87 on GD 18 and no. 98 on GD 7).
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no biologically or statistically significant effects on body weight and body weight development in the dose groups when compared to the control group.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no biologically or statistically significant effects in the dose groups when compared to the control group.
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):
Mean gravid uterus weights of treated animals were comparable to the control group.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
There were no toxicologically relevant gross pathological findings at necropsy; however, necropsy results are provided on Table 3 below. At necropsy, discolored dark lung was noted in both intercurrently dead females of the LD group (no. 31 and 37), the dead female of the MD group (no. 72) and in 1/3 intercurrently dead females of the HD group (no. 86). Furthermore, intestines were noted to be gas filled in one intercurrently dead female of the HD group (no. 98), in both dead females of the LD group and also discolored (dark) in one of them (no. 37). The heart was noted to be discolored (light) in the dead female no. 72 of the MD group.

Single further macroscopic findings in surviving animals were noted without dose-dependency and were considered as incidental. Non-pregnant female no. 8 of the control group was noted with fluid filled uterus and oviducts. In female no. 84 of the HD group, discoloration of the liver and the fat tissue next to kidneys was noted.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
None of the females showed signs of abortion or premature delivery prior to the scheduled sacrifice.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
Pre- and post-implantation loss were unaffected in all dose groups when compared to the control group.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
There were no treatment-related changes in the total litter losses by resorption for all dose groups when compared to the control group.
Early or late resorptions:
no effects observed
Description (incidence and severity):
There were no treatment-related changes in the number of early and/or late resorptions for all dose groups when compared to the control group.
Dead fetuses:
no effects observed
Description (incidence and severity):
There were no treatment-related changes in the number of dead fetuses found for all dose groups when compared to the control group. One dead fetus was found in the MD group and was considered as incidental.
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
Successful mating resulted in 23/26 pregnancies in the LD group, 25/25 in the MD group and 21/25 in the HD group when compared to 22/26 pregnancies in the control group.
Other effects:
not examined

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: No adverse effects observed up to the highest dose tested.

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
Litter data is presented in Table 4 below. There were no treatment-related changes to fetal body weight at any dose level when compared to the control group; however, mean fetus weight was marginally but statistically significantly higher in the LD and the MD group but not the HD group when compared to the control group (3 and 4 %, respectively).
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
There were no treatment-related changes to the number of live offspring at any dose level when compared to the control group.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
There were no treatment-related changes to the sex ratio at any dose level when compared to the control group.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
There were no treatment-related changes in litter size and weight at any dose level when compared to the control group.
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
There were no external abnormalities considered to be of toxicological relevance in any of the dose groups. Statistical analysis of litter incidences showed no significant differences to the control group.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Selected skeletal changes are presented in Table 5 below. Although skeletal changes were observed (some statistically significant), none were considered treatment related. Slightly but not statistically significantly increased litter incidence of unossified 5th sternebra was seen in the MD (20.00%) and the HD group (21.05%) when compared to the control group (5.00%). Statistical analysis showed no significant differences to the control group except for a higher litter incidence of increased ossification of calcaneus in the HD group (21.05%) compared to 0.00% in controls, higher litter incidence of unossified 5th metacarpal in the HD group (52.63%) compared to 15.00% in controls and lower litter incidence of incomplete ossification of parietal bones in the HD group (10.53%) compared to 45.00% in controls. Additionally, the variation of wavy ribs was seen in moderately less fetuses of the HD group when compared to the control group with achieving statistical significance (litter incidence 5.26% compared to 35.00% in controls). This was considered as incidental. One fetus (no. 5) of dam no. 60 of the MD group was affected with multiple rare abnormalities including wide 1st sternebra and misaligned sternebra (classified as grey zones) and malformations such as proximal branched rib, proximal fused ribs, absent rib, fused and misshapen cartilage of atlas and axis, fused cervical arches, fused cartilage of 1st cervical arch with cartilage of exoccipitals, malpositioned or absent thoracic centrum and absent thoracic arch. As only one single fetus of an intermediate dose group was affected, these findings were considered as incidental in nature without relation to the treatment with the test item.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Observed findings were either minor variations and/or due to a lack of dose-dependency and consistency, no toxicological significance can be attributed to these findings and they were considered to be spontaneous in nature. Incidences of observed findings were well within historical control data.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Craniofacial examination revealed few findings (dilated third ventricle, dilated lateral ventricle, retinal fold and small pituitary gland) at low frequencies generally comparable to or in some cases slightly higher or lower in frequency compared to controls without any dose-dependent trend. Statistical analysis of the data revealed no significant effect. These findings were considered to be spontaneous in nature and not related to the treatment with the test item.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effect up to the highest dose tested

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
no

Any other information on results incl. tables

Table 1:  Clinical Observations - Females

Clinical Finding

C

LD

MD

HD

Total number of animals examined

26

26

25

25

abnormal breathing

1

5

5

2

alopecia

1

2

0

1

increased salivation

0

4

15

19

moving the bedding

0

6

21

23

piloerection

1

1

1

0

vocalisation

0

1

0

0

regurgitation of formulation

1

2

3

1

Table 2:  Mortality Summary

Group

Gender

Day and Cause of Death

Female

C

0/26

-

LD

2/26

Found dead: no. 31 (GD 17)
Found dead: no. 37 (GD 12)

MD

1/25

Found dead: no. 72 (GD 18)

HD

3/25

Found dead: no. 86 (GD 8)
Found dead: no. 87 (GD 18)
Found dead: no. 98 (GD 7)

Animals affected / total number of animals

Given in brackets: study day of death, GD = gestation day

 

Table 3: Macroscopic Findings - Females

Organ

Finding

C

LD

MD

HD

Total number of animals examined

26

26

25

25

brain

autolytic

0

1

0

0

caecum

gas filled

0

2

0

1

dark red

0

1

0

0

cervix

fluid filled

1

0

0

0

colon

gas filled

0

1

0

0

duodenum

gas filled

0

2

0

0

dark red

0

1

0

0

heart

discoloured light

0

0

1

0

ileum/ Peyer's patches

gas filled

0

2

0

0

dark red

0

1

0

0

jejunum/ Peyer's patches

gas filled

0

2

0

0

dark red

0

1

0

0

kidney

fat next to kidneys (B)

0

0

0

1

discoloured yellow

0

0

0

1

liver

autolytic

0

1

0

0

focus discoloured yellow diffuse on right lateral lobe

0

0

0

1

fatty

0

0

0

1

lung

discoloured dark

0

2

1

1

oviduct

fluid filled

1

0

0

0

stomach

dark red

0

1

0

0

gas filled

0

1

0

1

uterus

fluid filled

1

0

0

0

Table 4:Litter Data

Group

 

Number of Males
(Litter Basis)

Number of Females
(Litter Basis)

Total No. of Foetuses
(Litter Basis)

Mean Foetus Weight (g)
(Individual Basis)

Male Foetus Weight (g)
(Individual Basis)

Female Foetus Weight (g)
(Individual Basis)

Mean Foetus Weight (g)
(Litter Basis)

Total Litter Weight (g)
(Litter Basis)

Male Litter Weight (g)
(Litter Basis)

Female Litter Weight (g)
(Litter Basis)

C

Mean

6.59

5.27

11.86

3.41

3.49

3.32

3.39

40.47

22.97

17.50

SD

1.97

1.70

2.19

0.31

0.33

0.26

0.18

8.61

7.50

5.91

N

22

22

22

261

145

116

22

22

22

22

LD

Mean

5.38

5.38

10.76

3.53**

3.61*

3.44*

3.55

37.96

19.44

18.51

SD

2.67

2.25

3.37

0.42

0.39

0.43

0.35

11.18

9.24

7.58

N

21

21

21

226

113

113

21

21

21

21

MD

Mean

5.08

5.75

10.83

3.54**

3.62*

3.46*

3.59

38.30

18.41

19.89

SD

1.86

1.98

2.68

0.44

0.42

0.43

0.42

8.88

6.36

6.66

N

24

24

24

260

122

138

24

24

24

24

HD

Mean

5.58

6.16

11.74

3.46

3.58

3.36

3.48

40.66

19.99

20.67

SD

2.46

2.14

2.16

0.44

0.47

0.37

0.37

7.44

9.03

7.09

N

19

19

19

223

106

117

19

19

19

19

Asterisks indicate statistically significant differences to reference item group, with* p<0.05, ** p<0.01 and *** p<0.001.

 

 

Table 5:Fetal Skeletal Findings

Skeletal Finding

Group

C

LD

MD

HD

Skull parietal (B) incomplete ossification

Classification: V

No of Incidences

12

8

13

3

Total No of Observed Foetuses

121

110

114

117

% Fetal Incidence

9.92

7.27

11.40

2.56

No of Litters with at least 1 Incidence

9

6

7

2*

Total No of Observed Litters

20

20

20

19

% Litter Incidence

45.00

30.00

35.00

10.53

Ribs wavy

Classification: V

No of Incidences

12

10

8

1

Total No of Observed Foetuses

121

110

114

117

% Fetal Incidence

9.92

9.09

7.02

0.85

No of Litters with at least 1 Incidence

7

7

6

1*

Total No of Observed Litters

20

20

20

19

% Litter Incidence

35.00

35.00

30.00

5.26

Forelimb metacarpal(s) unossified

Classification: V

No of Incidences

4

15

18

18

Total No of Observed Foetuses

121

110

114

117

% Fetal Incidence

3.31

13.64

15.79

15.38

No of Litters with at least 1 Incidence

3

7

8

10*

Total No of Observed Litters

20

20

20

19

% Litter Incidence

15.00

35.00

40.00

52.63

Hindlimb calcaneus(i) increased ossification

Classification: V

No of Incidences

0

0

5

4

Total No of Observed Foetuses

121

110

114

117

% Fetal Incidence

0.00

0.00

4.39

3.42

No of Litters with at least 1 Incidence

0

0

4

4*

Total No of Observed Litters

20

20

20

19

% Litter Incidence

0.00

0.00

20.00

21.05

 Asterisks indicate statistically significant differences to reference item group, with* p<0.05, ** p<0.01 and *** p<0.001.

Applicant's summary and conclusion