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.

Toxicity to reproduction

Administrative data

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the extended one-generation reproductive toxicity study does not need to be conducted because there are no results from available repeated dose toxicity studies that indicate adverse effects on reproductive organs or tissues, or reveal other concerns in relation with reproductive toxicity

Cross-reference

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Expected completion date: 29 September 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

adopted 21 September 1998

Deviations:

yes

Remarks:

Additional reproductive endpoints are covered.

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Geshundheit und Lebensmittelsicherheit

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Crl: WI(Han) (Full Barrier)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 7-8 weeks old
- Housing: caged in IVC cages (type IV, polysulphone cages) on Altromin saw fibre bedding
- Diet: free access to Altromin 1324 maintenance diet for rats and mice
- Water: free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- Acclimation period: 5 days


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

Route of administration:

oral: gavage

Vehicle:

DMSO

Details on oral 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 the start of the study at Evonik Technology & Infrastructure GmbH, Paul-Baumann-Str. 1, 45772 Marl, Germany. Samples for homogeneity analysis were taken from the top, middle and bottom of a low dose (50 mg/mL) and a high dose formulation (500 mg/mL). For determination of the concentration of test item in dosing formulations, samples will be retained from all groups in weeks 1, 5, 9 and in the last week of treatment (16 samples in total). In weeks 1, 5 and in the last week of treatment, samples for the testing of homogeneity will be taken from the top, middle and bottom of the freshly prepared low and high dose formulations (18 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 ISP-OES analysis by Evonik Technology & Infrastructure GmbH in a separate stand-alone study in accordance with GLP.

Duration of treatment / exposure:

90 days

Frequency of treatment:

daily

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:

10 animals/sex for each dose group.
second control group (C2) and recovery groups: 5 animals/sex

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: According to the results of a previous dose range finding study and in consultation with the sponsor the following doses 100, 300, and 1000 mg/kg bw/day were selected for the 3 dose groups (LD = low dose, MD = medium dose, HD = high dose).
- Rationale for selecting satellite groups: Two control groups were used in this study. C1, the vehicle control group was administered daily with 100 % DMSO. To control for possible vehicle-related effects a second control group was used. C2 is a second control group treated daily with aqua ad injectionem. As 100 % DMSO is not a standard vehicle in toxicity studies and as no historical control data exists for this vehicle, it could not be excluded that vehicle-related effects could be induced for parameters such as body weight, food consumption, blood parameters, organ weights or microscopic organ findings. A negative control group treated with aqua ad injectionem was used to better allow for discrimination between possible vehicle-induced effects or toxicologically relevant effects related to the test item in the dose groups. Therefore, for evaluation of data, results of both control groups were compared in the first place to check for statistically and biologically significant differences. Without relevant differences, statistical assessment of the results of the different parameters of the dose groups was only related to the vehicle control group C1. The animals in the vehicle control group (C1) were handled in an identical manner to the test group subjects and received 100 % DMSO using the same volume as used for the dose groups. The animals in the control group C2 were handled in an identical manner to the test group subjects and received aqua ad injectionem at an application volume of 2 mL/kg bw.
- Post-exposure recovery period in satellite groups: 28 days for control (C1) and high dose group

Positive control:

not applicable

Observations and examinations performed and frequency:

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: spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes
- Time schedule for examinations: body weight was recorded once before the assignment to the experimental groups, on the first day of administration and weekly during the treatment and recovery period.

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

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before the first administration and in the last week of the treatment period as well as at the end of the recovery period in the recovery animals
- Dose groups that were examined: all animals


HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the treatment and recovery period prior to or as part of the sacrifice of the animals
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Yes for treatment groups, No for recovery groups
- Parameters checked in tables 1 and 2 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the treatment and recovery period prior to or as part of the sacrifice of the animals
- Animals fasted: Yes for treatment groups, no for recovery groups
- Parameters checked in table 3 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: prior to or as part of the sacrifice of the animals at the end of the treatment and the recovery period
- Animals fasted: Not specified
- Parameters checked in table 4 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: once before the first exposure and once in the last week of exposure as well as in the last week of the recovery period
- Dose groups that were examined: all dose groups
- Battery of functions tested: Sensory reactivity to different modalities, grip strength and motor activity assessments and other behavioral observations as well as rearing supported and not supported, urination, defecation, startle/ auditory response, equilibrium reflex, positional passivity, visual placing, fore and hind limb grip strength, tail pinch response, toe pinch reflex, extensor thrust/limb tone, hind limb reflex, righting reflex on the ground, air righting reflex, pupil response, body temperature and ophthalmoscopy (anterior chamber of the eye and fundus of eye).

IMMUNOLOGY: No

OTHER: Daily over a period of 8 days, the estrous cycle of all female animals was examined in study week 5, 9 and the last week of treatment. In the recovery animals the estrous cycle was also examined during the last week of the recovery period.
At necropsy (one day after the last administration) and at the end of the recovery period, left epididymis and left testis were separated and used for evaluation of sperm parameters. Epididymal sperm motility and testicular sperm count were evaluated in all male animals using Hamilton Thorne Sperm Analyser (TOX IVOS Version 13.0).

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 5)

HISTOPATHOLOGY: Yes (see table 6)

Statistics:

A statistical assessment of the results of the body weight, food consumption, parameters of hematology, blood coagulation and clinical biochemistry and absolute and relative organ weights and sperm analysis will be 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 may be performed with a Student’s t-Test or Mann-Whitney U-Test when appropriate. These statistics will be performed with Ascentos 1.1.3 software or GraphPad Prism V.6.01 software (p<0.05 is considered as statistically significant).

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Abnormal breathing was noted in 1/15 males and 1/15 females of the vehicle control group C1, 1/10 males and 3/10 females of the LD group, 4/10 males and 5/10 females of the MD group and 7/15 males and 1/15 females of the HD group during the treatment period. There was no clear dose-dependent pattern. In some animals this clinical sign was only noted for some days of treatment whereas others were affected up to terminal sacrifice or until inter-current death or sacrifice. In some cases occurrence of abnormal breathing lead to reduced health condition with clinical signs such as reduced spontaneous activity. Abnormal breathing did not persist throughout the recovery period. As animals from all groups (including controls) were affected by abnormal breathing, the sudden appearance of this clinical sign at various time points during the treatment period was considered to be related to local effects of the vehicle DMSO and/or its formulation with the test item based on e.g. regurgitation and incidental aspiration after dose administration. Therefore, abnormal breathing was not considered to be related to a systemic toxic effect of the test item.

During the treatment period of the study, dose-dependently increased salivation was noted transiently in some weeks and some males and females mainly of the MD and the HD group when compared to the respective controls. Statistical significance was achieved in week 2 (female main HD group), week 3 (male main HD group, female recovery HD group), week 4 (male main HD group), week 5 (male and female main HD group, male recovery HD group), week 6 (male main HD group) and week 10 (female main HD group).

Spontaneous activity was noted to be slightly, dose-dependently lower in the male and female MD and HD group when compared to the respective controls C1. Statistical significance was achieved in week 2 (male and female main HD group), week 3 (male MD group, male main HD group and male recovery HD group), week 4 (male main HD group), week 5 (male main HD group), week 6 (male main HD group) and week 8 (female MD group). As this was only seen transiently in some weeks of the treatment period without relevant impact on other parameters like food consumption and as there were no statistically or biologically significant differences during the recovery period, it was not considered as an adverse effect.

Statistical but not biological significance was achieved for the female LD group for reduced reaction to finger approach in week 9 and for the female recovery HD group for reduced response to handling in week 8 when compared to the respective controls C1. Further slight but statistically significant differences between the groups for sleeping or moving in the cage during cage-side observation in few weeks irrespective of the treatment group were not considered as toxicologically relevant but incidental.

Mortality:

mortality observed, treatment-related

Description (incidence):

Mortality was observed in all groups except the water control group C2 and during recovery phase. Most deaths occurred in the higher dose groups though no clear pattern in terms of dose-dependency or duration of treatment was observed.
vehicle control: 1 female (no 103) found dead (sudden death related to misgavaging)
low dose: 2 males (no 020 and 017) found dead (related to inadvertent misgavaging), 1 female (no 078) was intercurrently sacrificed on day 80 for animal welfare reasons based on a tissue mass of approx. 2.5 cm in diameter on its left cheek which prove to be a spontaneous adenocarcinoma at histopathology
mid dose: female no. 085 was found dead on day 42 after showing abnormal breathing for few days as well as slight loss of body weight. Furthermore, female no. 081, 087 and 089 were euthanised on the respective treatment day 15, 43 and 55 in a moribund condition with clinical signs such as abnormal breathing, reduced spontaneous activity and piloerection. Female no. 089 was additionally seen with a bloated belly (related to a gas filled stomach) and dehydration at sacrifice.
high dose: Two males were found dead without any preceding signs of morbidity (no. 036 on day 47 and no. 055 on day 62). Furthermore, male no. 044 was found dead on day 64 after moderate loss of body weight and male no. 037 was found dead on day 40 after showing abnormal breathing and piloerection one day before. Male no. 039 was euthanised on day 45 in a moribund condition due to gasping. Two females of the HD group were also found dead without any preceding signs of morbidity (no. 094 on day 69 and no. 110 on day 81) and female no. 095 was found dead on day 15 after moderate loss of body weight.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Marginal but statistically significant differences in mean body weight between the male control groups C1 and C2 were seen on treatment day 15, 22, 29 and 43 (4 to 7% above group C1). Due to the transiency and the marginal difference this was not considered as biologically relevant. Accordingly slightly but statistically significantly higher body weight gain was noted in the 5th week of treatment for the male C2 group. No such effects were seen in the female control groups except for statistically significantly higher body weight gain in the very first week in the female C2 group when compared to C1 which was considered as incidental.

Few slight but statistically significant differences for body weight change between the dose groups and the respective control group C1 in single weeks of treatment and recovery without consistency were considered as incidental in nature (males: slightly higher body weight gain in week 2 and 5 of the MD group, in week 5 of the main HD group and in week 9 of the recovery HD group; marginal body weight loss in week 16 of the recovery HD group; females: slightly higher body weight gain in week 3, week 7 and week 10 of the female recovery HD group when compared to the respective controls.)

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Single statistically significant effects were only seen in either males or females and were considered as incidental as values were within the normal range of variation (males: percentage lymphocytes 8% below controls C1, percentage neutrophils 32% above controls C1; females: MCV 4% below controls C1, MCHC 3% above controls C1, percentage monocytes 62% above controls C1). At the end of the recovery period, no toxicologically relevant effect on parameters of hematology was observed. The isolated but statistically significant change of higher percentage of eosinophils in the male HD group (138% above controls) and higher percentage of large unstained cells in the male HD group (34% above controls) was not observed in females or at the end of the treatment period and was considered as incidental.

Blood coagulation was not affected by the test item. Marginally but statistically significantly higher value for prothrombin time in the male HD group at the end of the treatment period (13% above controls C1) was not seen in females or at the end of the recovery period and was considered as incidental with values within the normal range of variation.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant changes were observed in the male MD group when compared to the respective controls C1 which did not follow a dose-dependent pattern (ASAT 16%, albumin 6% and TBA 44% below controls C1). Mean value for alkaline phosphatase was statistically significantly below controls in the male HD group (43% below controls C1) and the female HD group (43% below controls C1) but within the normal range of variation. Furthermore, slightly but statistically significantly lower mean values for creatinine (25% below controls) and for urease (19% below controls) were seen in the female HD group at the end of the treatment period when compared to the respective control group C1. No such finding was seen in males. Though statistically significant, these single findings in males and/or females at the end of the treatment period within the normal range of variation and without histopathological correlate in the related organs (except adaptive liver hypertrophy in the male HD group) were not assumed to be adverse in nature.

At the end of the recovery period, slight to moderate, statistically significant effects were seen in the male HD group (lower ALAT, AP, creatinine, total protein and albumin when compared to controls C1). As no such effect was seen in females and values were within the normal range of variation, these statistically significant differences were not considered as toxicologically relevant. There was no histopathological correlate. No statistically significant changes were noted in females at the end of the recovery period.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

In the functional observation battery, slight differences between the groups before initiation of treatment were considered as incidental in nature such as slightly but statistically significantly higher number of defecation in the male main HD group and marginally but statistically significantly lower body temperature in the male LD and MD group.

As seen during the clinical observations, increased salivation was also noted dose-dependently in males and females in the functional observation battery at the end of the treatment period with achieving statistical significance in the male main HD group and the male recovery HD group when compared to the respective controls C1. This was not observed during the recovery period.

The isolated findings of statistically significantly less number of females moving in the cage in all main dose groups during cage-side observation and statistically significantly lower number of non-supported rearing (but not of supported rearing) compared to controls C1 at the end of the treatment period was considered as incidental. Furthermore, marginally but statistically significantly lower body temperature of the female main dose groups at the end of the treatment period was considered as incidental and was within the normal range of variation. No such differences to controls were seen at the end of the recovery period.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

At the end of the treatment period, no effects of toxicological relevance were observed on organ weights. For males and females, there were no statistically significant differences between the dose groups and the control group C1 except slightly and not dose-dependently lower ovaries weight in the MD group (absolute weight 38% below controls C1). With values being in the normal range of variation, this was considered as incidental.

At the end of the recovery period, no effects of toxicological relevance were observed on organ weights. For males and females, there were no statistically significant differences between the dose groups and the control group C1 except marginally lower absolute testes weight (11% below controls C1) in the HD group. Without an effect at the end of the treatment period and without related effects on fertility parameters at the end of the treatment and at the end of the recovery period, this marginal weight difference was not considered as toxicologically relevant.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Few gross pathological changes were recorded at necropsy at the end of the treatment and the recovery period without following a dose-dependent pattern. Some more findings were seen in intercurrently sacrificed or found dead animals.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Under the conditions of this study, the test item caused minor systemic effects. They consisted of hepatocellular hypertrophy in single males of the LD, MD and HD group. There were no further findings recorded in the livers. No hepatocellular hypertrophy was observed at the end of the recovery period. Therefore, this finding was not considered to be of adverse but adaptive nature.

In three animals (no. 017, 020 and 103), there were morphological indications for misgavage consisting of necrotizing inflammation in the trachea and/or larynx. In further five animals (no. 037, 039, 085, 087 and 089), inflammatory secretion and acute inflammation in the nasal cavities indicated regurgitation. All these cases are deemed to be related to the study methods and/or the consistency of the vehicle (100 % DMSO) and the test item.
In one case, there was a finding of an adenocarcinoma in the mandibular gland of a female. Such findings are rare spontaneous incidents, however have been reported previously.
In male no. 055, there were hemorrhage in the heart (moderate) and thymus (slight), associated with a chronic peri-/arteritis (severe) in the stomach. The peri-/arteritis may be of considered of spontaneous background. The reason for the aforementioned hemorrhages is unknown but may be related to the peri-/arteritis. Such lesions may be encountered in control rats as spontaneous background lesions.
The cause of morbidity/death could not be established in two females (no. 081 and 110) based on the morphological evaluation. Furthermore, no diagnose was made due to cannibalism and/or autolysis in two females (no. 094 and 095) and two males (no. 036 and 044). However, based on the observed sudden spontaneous death without preceding morbidity in these animals, these mortalities were also assumed to be related to oral gavaging of formulations with DMSO and its acute local effects after possible regurgitation and incidental aspiration.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

There were no biologically or statistically significant differences for epididymal sperm motility and testicular sperm count between the treated and control animals.

Reaction product of trimethoxysilylpropylethylenediamine and benzylchloride had no statistically or biologically significant effect on the estrous cycle evaluated 4, 8 and 12 weeks after the first administration and in the last week of the recovery period. There were no considerable, consistent differences in the length or sequence of cycle stages between the dose groups and the control group.

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 adverse effects observed up to highest dose tested

Critical effects observed:

no

Results and discussion

Overall reproductive toxicity

Reproductive effects observed:

not specified