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Toxicological information

Developmental toxicity / teratogenicity

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Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15th November 2016 to 16th January 2017
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 guideline
Qualifier:
according to guideline
Guideline:
other: OECD Guidelines for Testing of Chemicals, Section 4, No. 422, “Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test”
Version / remarks:
1996
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
N,N',N''-tricyclohexyl-1-methylsilanetriamine
EC Number:
240-040-8
EC Name:
N,N',N''-tricyclohexyl-1-methylsilanetriamine
Cas Number:
15901-40-3
Molecular formula:
C19H39N3Si
IUPAC Name:
N,N',N''-tricyclohexyl-1-methylsilanetriamine
Test material form:
liquid
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature protected from light and moisture- Stability under test conditions: Stable in paraffin oil due to its aliphatic nature.
- Solubility and stability of the test substance in the solvent/vehicle: The test item formulation was prepared with paraffin oil.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: The test item showed acceptable stability in paraffin oil due to its aliphatic nature (80% recovery after 2 hours). Therefore, paraffin oil was selected based on these test item’s characteristics and as it is an acceptable and suitable vehicle for oral administration of rats in toxicity studies.
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was weighed into a tared glass vial on a suitable precision balance and the vehicle was added to give the appropriate final concentration of the test item and further vortexing it for 2-3 minutes. The test item formulation was prepared freshly daily before the administration to animals.
- Preliminary purification step (if any): none

Test animals

Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: males: 10-11 weeks old, females: 10-11 weeks old
- Weight at study initiation: males: 255 – 289 g; females: 170 - 197 g
- Fasting period before study: none
- Housing: Animals were housed in groups of 2 animals / sex / cage in IVC cages during the premating period for both males and females and during post-mating period for males depending on the mating status. During mating period males and females were housed together in ratio 1:1 (male to female). After the confirmation of mating, females were kept individually during gestation/lactation period and males were returned to its original cage. Animals of the recovery groups were housed in groups of 2 animals / sex / cage. In each cage Altromin saw fibre were used as bedding.
- Diet: food, ad libitum
- Water: water, ad libitum
- Acclimation period: 5 days
DETAILS OF FOOD AND WATER QUALITY: Certificates of food, water and bedding are filed for two years at BSL Munich and afterwards archived at Eurofins Munich.

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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
paraffin oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test item was weighed into a tared glass vial on a sui table precision balance and the vehicle was added to give the appropriate final concentration of the test item and further vortexing it for 2-3 minutes. To protect from hydrolysis by humidity, the test item container and formulation vials were purged with argon after the preparation and before closing. The test item formulation was prepared freshly daily before the administration to animals.
VEHICLE
- Justification for use and choice of vehicle (if other than water): According to the results by GC analytical method (see Appendix 4), the test item was shown to be unstable in dried corn oil. In dried corn oil, the recovery of the compound in the vehicle was down to 30% recovery after 2 hours. However, the test item showed acceptable stability in paraffin oil due to its aliphatic nature (80% recovery after 2 hours). Therefore, paraffin oil was selected based on these test item’s characteristics and as it is an acceptable and suit able vehicle for oral administration of rats in toxicity studies.
- Concentration in vehicle: not specified
- Amount of vehicle (if gavage): not specified
- Lot/batch no. (if required): not specified
- Purity: not specified
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
During the study samples were collected for the investigation of homogeneity and nominal substance concentration.
Samples for homogeneity and concentration verification analysis were taken from the top, middle and bottom of the high dose and the low dose formulation in study week 1 (first day of pre-mating period), 3 (first week of mating), 5 (gestation) and in the last week of the study (gestation/lactation) from all groups (40 samples).
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility: Not specified
- Further matings after two unsuccessful attempts: not specified
- After successful mating each pregnant female was caged (how): After the confirmation of mating, females were kept individually during gestation/lactation period and males were returned to its original cage.
- Any other deviations from standard protocol: none
Duration of treatment / exposure:
males: 28 days females: 39 to 52 days
recovery group males: treated for 28 days and were subjected to necropsy 14 days after the last administration
recovery group females: treated up to the first scheduled necropsy of dams and were subjected to necropsy 14 days thereafter
Frequency of treatment:
7 days a week
Duration of test:
Up to 52 days
Doses / concentrationsopen allclose all
Dose / conc.:
25 mg/kg bw/day (nominal)
Dose / conc.:
75 mg/kg bw/day (nominal)
Dose / conc.:
150 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Animals of the high dose group were dosed with 150 mg/kg bw on the first day of application but reduced to 100 mg/kg bw from study day 2 and treated until the end of the treatment period. One male died and severe clinical symptoms were observed in animals at 150 mg/kg bw/day.
No. of animals per sex per dose:
104 animals (52 males and 52 females) were included in the study. This included the recovery animals (12 males and 12 females).
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose selection was based on results from a dose-range finding study.
- Rationale for animal assignment (if not random): random
- Rationale for selecting satellite groups:
- Post-exposure recovery period in satellite groups: 14 days
- Section schedule rationale (if not random): random

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: General clinical observations were made once a day approximately at the same time after dosing. 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 checked included: The health condition of the animals was recorded.
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before the first exposure, and weekly thereafter, detailed clinical observations were made in all animals of the main groups and the recovery groups outside the home cage in a standard arena. Clinical observations include spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, 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 bizar re behaviour were recorded.
BODY WEIGHT: Yes
- Time schedule for examinations: The animals were weighed once before the assignment to the experimental groups, on the first day of dosing and weekly thereafter as well as at the end of the study. During pregnancy, females were weighed on gestation days (GD) 0, 7, 14 and 20 and within 24 hours of parturition (day 0 post-partum) as well as on day 4 post-partum along with the pups. FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): not applicable
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not applicable
POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # : All surviving animals were sacrificed on the respective post-natal day 4 by using anaesthesia (e.g. ketamine/xylazine).
- Organs examined: See table 1
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
- Soft tissue examinations: Yes
- Skeletal examinations: Yes
- Head examinations: Yes
Statistics:
A statistical assessment of the results of the body weight, food consumption, parameters of haematology, blood coagulation and clinical biochemistry and absolute and relative organ weights were performed for each gender by comparing values of dosed with control animals using either a parametric one-way ANOVA and a post-hoc Dunnett Test or a non-parametric Kruskal-Wallis Test and a post-hoc Dunn’s Test, based on the results of homogeneity and normality tests. Furthermore, statistical comparisons of data acquired during the recovery period were performed with a Student’s t-Test or Mann-Whitney UTest when appropriate. These statistics were performed with Ascentos 1.1.3 software or GraphPad Prism V.6.01 software (p<0.05 is considered as statistically significant).
Indices:
Copulation, fertility, delivery and viability indices

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
There were no clinical symptoms up to MD group. There were clinical signs in the HD group males and females. In males and females, moving the bedding and piloerection were more often in HD group. The reduced spontaneous activity in HD males was seen in week 1 of treatment, which lessened with the study progress and is considered to have prevailed after treatment on day 1 at 150 mg/kg body weight. Slightly increased salivation was in single male (no. 51) of HD group on treatment day 1. The increased salivation was transiently seen in 5/10 HD females on few occasions.
Mortality:
no mortality observed
Description (incidence):
There was no mortality up to HD group of females.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In females, the body weight was lower in MD and HD groups compared to control attaining statistical significance on GD 20 in MD (-6%) and HD (-7%) groups and lactation day 4 in HD group (-8%). The body weight gain was statistically significantly lower during gestation period and lactation days 0-4 in MD and HD groups. In recovery females, the body weight and body weight gain were not affected during the treatment or the recovery period.
Thus, the effect on body weight development of male and the gravid female animals of MD and HD groups was considered to be related to test item treatment.
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
In females, there was no effect on haematology and blood coagulations parameters at the end of treatment and recovery period. However, in recovery females mean RBC, Hb and HCT values were statistically significantly higher and mean reticulocyte count was statistically significantly lower in HD group compared to the corresponding control. As there were no effects at the end of the treatment, the findings at the end of recovery had no relevance to toxicity.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
In both males and females including recovery groups, there were no effects on clinical biochemistry. The mean glucose in male LD group and the mean Chol values in female MD and HD group were statistically significantly lower at the end of treatment. These findings were not of toxicological relevance.
Urinalysis findings:
no effects observed
Description (incidence and severity):
There was no significant effect on urine parameters of males and females (main and recovery groups) in the test item groups compared to corresponding control.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
The functional neurological assessment revealed no significant differences between the control and test item group animals. In females, the “rearing not supported” was statistically significantly lower in HD recovery group (0.2) in the last week of recovery compared to corresponding control (2.7), but considering the baseline value (0.2) the finding was not of toxicological relevance.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
In females, the organ weights were not affected. There were no statistically significant differences in organ weights between the control and the test item groups. Other observed changes in organ weight did not attain statistical significance.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
In female, at the end of treatment there was no macroscopic findings. At the end of recovery there was dilated ureters in female no. 95 and dilated uterus in female no. 104.
Neuropathological findings:
not examined
Description (incidence and severity):
The main test item related histopathological change was forestomach ulceration. This finding was present in males from the MD and the HD group. In females ulceration was present in the HD group. Based on its incidence and severity in both decedents and survivors the forestomach ulceration was considered adverse, although reversibility occurred during the recovery period.
Epithelial cell (keratinocytes) vacuolization was recorded in both males and females from the HD group and in males from the MD group. It was dose dependent in males and showed partial reversibility during recovery period. In females, the vacuolization fully recovered during the treatment free period. This finding was mainly associated with the forestomach ulceration and was considered a milder change than the ulceration/necrosis. However, a progression towards ulceration cannot be fully excluded, thus this change was considered adverse.
Submucosal hemorrhages were observed only in decedent animals and were considered an incidental finding related to the forestomach ulceration and the blood accumulation in the stomach. However, severe bleeding contributed to the morbidity and the hemorrhages could be considered adverse.
Mixed inflammatory cell infiltrates were observed in both males and females and were considered most likely related to the presence of forestomach ulceration. Dose dependent increase in incidence and severity of the mixed inflammatory cell infiltrates in males and partial resolution in recovery animals were noticed. Based on the distribution, incidence, severity of the lesions and the partial resolution the presence of mixed inflammatory cell infiltrates was considered a non-adverse finding.
Squamous hyperplasia and hyperkeratosis were observed in both males and females and were considered a reactive/regenerative process. In recovery animals from the high dose group the incidence of the severity of both findings increased while the severity decreased revealing that regenerative processes occurred. Thus, partial recovery occurred and these findings wer considered non-adverse.
Submucosal edema was present only in the animal that died at day 1 of treatment. This finding was considered a component of the acute inflammation that preceded the test item induced ulceration and was considered non-adverse.
In the kidneys from high dose group male rats simple tubular dilatation of proximal tubules and hyaline droplets were observed. The simple tubular dilatation may occur secondary to renal ischemia or from prolonged diuresis related to drug administration. Based on the low severity and this finding was considered non-adverse. Hyaline droplets represent an accumulation of secondary lysosomes within the cytoplasm and contain alpha-2u-globulin that reversibly bind to the inducing xenobiotics and/or metabolites. Hyaline droplet deposition is a male rat specific event, and it is of no human relevance.
In the adrenal gland vacuolization of cortical cells of Zona fasciculata was observed in male animals for the high dose group only. This histopathological change can occur secondary to stress and after test item administration. Based on the low incidence the finding was considered non-adverse.
In the white matter of the cerebellum, vacuolization was observed in animals from the high dose group and in control animals. The main causes of white matter vacuolization are edema and demyelination or the vacuolization represents an artifact of fixation. These aforementioned potential causes of white matter vacuolization are challenging to be differentiated at light microscopic level and may require further investigation. However, based on the presence of this finding in control animals and the low severity in animals from the high dose group the white matter vacuolation was considered as most likely incidental and not adverse.
Minimal focal randomly distributed hepatocytes necrosis was observed in two decedent male animals (no. 31 and 33) and was considered an incidental finding most likely not test item related. Further, agonal changes were observed in thymus of two decedents (mild multifocal hemorrhages) and the lung of one decedent (aveolar emphysema and edema).
Minimal tubular degeneration was observed in 1/10 males (animal no. 35) from the high dose group, whereas in the control animals no tubular degeneration was noticed. The tubular degeneration was characterized by the presence of single seminiferous tubules containing with few germ cells undergoing degeneration/necrosis, without spermatogenesis impairment and was deemed incidental.
Mild cystic endometrial hyperplasia was observed in 1/10 females from the high dose group and in 1/10 control animals. This finding was considered as related to the female reproductive cycle and therefore incidental.
All other microscopic findings recorded were within the range of normal background lesions which may be recorded in this study type and animals of this strain and age.
The sperm stages were checked on completeness of cell populations, completeness of stages and degenerative changes. The treatment with the test item did not reveal effects on the completeness of stages or cell populations.
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
In females, the food consumption was statistically significantly lower in week 2 of premating in MD and HD groups compared to the control. Lower food consumption was also in MD and HD groups during gestation and lactation days attaining statistical significance during GD 7-14 and Lactation days 0-4. In recovery female group, slightly lower food consumption during the treatment period. No effect during the recovery period.
The lower food consumption in female animals (main and recovery groups) of MD and HD groups corroborated lower body weights in male and/or female animals.

Maternal developmental toxicity

Number of abortions:
not examined
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
The number of corpora lutea and the number of implantation sites were not affected in test item groups compared to the control. The percentage of pre-implantation loss was higher in MD (13.5%) and HD (11.93%) groups compared to corresponding control (5.07%) but without dose-response relationship. There was no dose-related increase in the percentage of post-implantation loss. However, the percentage of post-implantation was higher in LD and MD groups compared to the control, but because there was no dose-response relationship, the finding was not considered adverse.
Total litter losses by resorption:
not examined
Early or late resorptions:
not examined
Dead fetuses:
no effects observed
Description (incidence and severity):
The number of live pups on PND 0 and 4 were not affected.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
The precoital and the duration of gestation were not affected due to test item treatment.
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): The precoital and the duration of gestation were not affected due to test item treatment.
Changes in number of pregnant:
not examined

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
>= 100 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: No adverse effects observed.

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
The pup mean weight in HD group was lower on PND 0 and 4 attaining statistical significance on PND 4. The total litter weight was lower in test item groups on PND 0 and 4 attaining the statistical significance in MD and HD groups, but because there was no dose-response relationship, the finding was not considered adverse.
Male and female litter weight on PND 0 and 4 were slightly lower in test item groups compared to the control, but were not statistically significantly different.

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): The pup mean weight in HD group was lower on PND 0 and 4 attaining statistical significance on PND 4. The total litter weight was lower in test item groups on PND 0 and 4 attaining the statistical significance in MD and HD groups, but because there was no dose-response relationship, the finding was not considered adverse.
Male and female litter weight on PND 0 and 4 were slightly lower in test item groups compared to the control, but were not statistically significantly different.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
the number of live pups on PND 0 and 4 were not affected.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The number of male and female pups, sex-ratio (No. of male pups/No. of female pups) on PND 0 and 4 were not affected.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
The pup mean weight in HD group was lower on PND 0 and 4 attaining statistical significance on PND 4. The total litter weight was lower in test item groups on PND 0 and 4 attaining the statistical significance in MD and HD groups, but because there was no dose-response relationship, the finding was not considered adverse.
Male and female litter weight on PND 0 and 4 were slightly lower in test item groups compared to the control, but were not statistically significantly different.
Changes in postnatal survival:
no effects observed
Description (incidence and severity):
A higher percentage of pup mortality was in LD (0.91%) and MD (6.67%) groups, but none in HD group. Thus, the pup mortality in LD and MD groups was not considered to be of toxicological relevance.
External malformations:
no effects observed
Description (incidence and severity):
There were no adverse gross external findings in pups of test item groups compared to the control. However, dark snout was noted in pup no. 7 (dam no. 82 of MD), pups 1, 2, 6 (dam no. 83 of HD) and pup no. 6 (dam no. 92 of HD group). There was also a small, hypothermic and weak pup (pup no. 6 of dam no. 86 of HD group). These findings were in isolated pups and were considered incidental.
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
>= 100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed.

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
In the Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening test, the reported developmental toxicity NOAEL for N,N’,N’’-tricyclohexyl-1-methylsilanetriamine was at least 100 mg/kg bw/day based on there being no adverse developmental effects observed at any dose tested.

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