N-[3-(dimethoxymethylsilyl)propyl]ethylenediamine

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27-07-2020 to 13-01-2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

25 June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

N-[3-(dimethoxymethylsilyl)propyl]ethylenediamine

EC Number:

221-336-6

EC Name:

N-[3-(dimethoxymethylsilyl)propyl]ethylenediamine

Cas Number:

3069-29-2

Molecular formula:

C8H22N2O2Si

IUPAC Name:

N-[3-[dimethoxy(methyl)silyl]propyl]ethane-1,2-diamine

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl: WI(Han)

Details on species / strain selection:

Animal model: healthy Wistar rats, 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 (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 5-6 weeks old
- Weight at study initiation: 185 g to 216 g (males); 135 g to 169 g (females)
- Fasting period before study: No
- Housing: The animals were kept in groups of 5 animals / sex / group / cage in IVC cages (type IV, polysulphone cages) on Altromin saw fibre bedding
- Diet: Altromin 1324 maintenance diet, ad libitum
- Water: Tap water, sulphur acidified to a pH of approximately 2.8, ad libitum
- Acclimation period: At least 5 days

DETAILS OF FOOD AND WATER QUALITY:
Municipal residue control and microbiological controls are done for the drinking water, at regular intervals.

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

IN-LIFE DATES: From: July 2020 To: 01 December 2020

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The test item was dissolved in dried and de-acidified corn oil. 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. The formulation was alternately vortexed and/or stirred until visual homogeneity was achieved.
Based on the results of stability testing, the test item formulations were prepared fresh on each administration day before the administration procedure. The prepared formulations were stored protected from light and at room temperature. Formulates were kept under magnetic stirring during the daily administration. The vehicle was also used as the control item.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The vehicle was selected based on the test substance’s characteristics and the subsequent relevant OECD testing guidelines.
- Concentration in vehicle: 0, 25, 25, 75, 250/187.5/125* mg/mL
*The high dose level was reduced due to the irritating nature of the test substance and mortality at 1000 mg/kg bw/day starting on the 27th of August 2020, and at 750 mg/kg bw/day starting on the 11th of September 2020)
- Amount of vehicle (if gavage): 4 mL/kg body weight
- Lot/batch no. (if required): MKCK6411
- Purity: not specified

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples were collected for concentration analysis in weeks 1, 5, 9 and in the last week of treatment for all doses (16 samples in total). Each sample taken during the study was retained in duplicate (sample A, sample B, each of at least 3 mL). The A-samples were analysed at Eurofins Munich and until then stored under appropriate conditions based on available stability data. The B-samples are retained at below 15 °C at BSL Munich (test facility) and discarded after completion of the final study report.
Before beginning of the treatment period, formulation samples were prepared and analysed in order to obtain knowledge about stability and homogeneity of the test item in the selected vehicle at Eurofins Munich as part of a separate GLP study.

Duration of treatment / exposure:

90-day exposure

Frequency of treatment:

Once daily, 7 days per week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Negative control group; Group 1

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

Group 2

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Remarks:

Group 3

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

Group 4 [reduced to 750 and 500 mg/kg bw/day during the course of the study]

No. of animals per sex per dose:

10 animals per sex per dose. To determine reversibility of potential effects of the test item, additional recovery animals (5/sex in Group 4) were included in the study. Additional recovery animals (5/sex in Group 1 i.e. at dose level 0 mg/kg bw/day) were required for comparison purposes.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Doses for this study were selected based on the results of a 28-day dose range finding study (BSL Munich Study No STUGC19AA1217-1, non-GLP) in rats orally dosed daily at 50, 100, 250, 500 and 750 mg/kg bw/day for up to 4 weeks. Beginning on Day 15, females dosed at =600 mg/kg bw//day exhibited neurological clinical signs of abnormal gait, hindlimb splayed, impaired locomotion, flattened posture and/or decreased activity. There were no clinical signs, mortality, effects on food consumption, changes in haematological parameters, gross macroscopic lesions or toxicologically relevant differences in organ weights which could clearly be related to treatment with the test item up to 750 mg/kg bw/day. While there was a reduction in body weight gain in males at 500 (statistically significant) and in males and females at 750 (not statistically significant in either sex), overall the test item had no effect on body weight development (i.e. the mean body weight increased during the observation period in the control group and in all male and female treated groups). The clinical biochemistry parameters ALAT and ASAT were significantly increased in males at 750 mg/kg bw/day compared to the control group and may be dose-dependent. Based upon the absence of adverse findings in this dose range finding study and in keeping with the principle that “the highest dose level should be chosen with the aim to induce toxicity but not death or severe suffering”, the HD was increased in the subsequent main OECD 408 study to 1000 mg/kg bw/day. However, due to increased mortality in the HD group during this 90-day study, the HD was subsequently reduced from 1000 mg/kg bw/day to 750 mg/kg bw/day on 27 August 2020 (Day 24 of treatment: animal nos. 1-5, 11-15, 21-25, 31-35, Day 23 of treatment: animal nos. 6-10, 41-45, 91-95, 16-20, 26-30, 36-40, 46-50, 96-100, Day 22 of treatment: animal nos. 51-55, 61-65, 71-75, 81-85, Day 21 of treatment: animal nos. 56-60, 66-70, 76-80, 86-90) and then reduced again to 500 mg/kg bw/day on 11 September 2020 (Day 39 of treatment: animal nos. 1-5, 11-15, 21-25, 31-35, Day 38 of treatment: animal nos. 6-10, 41-45, 91-95, 16-20, 26-30, 36-40, 46-50, 96-100, Day 37 of treatment: animal nos. 51-55, 61-65, 71-75, 81-85, Day 36 of treatment: animal nos. 56-60, 66-70, 76-80, 86-90).


- Rationale for animal assignment (if not random): random

- Fasting period before blood sampling for clinical biochemistry: yes, animals were fasted overnight prior to each blood collection interval.

Positive control:

No

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations considering 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 were made outside the home cage in a standard arena once before the first administration and at least once a week thereafter.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: General clinical observations were made at least once a day, preferably at the same time each day and considering the peak period of anticipated effects after dosing. Inadvertently, the clinical observation was not recorded for the following animals on a single study day:
Day 9: animal no. 86
Day 10: animal nos. 81,83, 84, 85
Day 11: animal nos. 28, 40, 47, 96-99
Day 12: animal nos. 21,24,31, 34, 35


BODY WEIGHT: Yes
- Time schedule for examinations: Once before assignment to the experimental groups, on the first day of administration and weekly during the treatment and recovery periods.

FOOD CONSUMPTION AND COMPOUND INTAKE
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes. Food consumption was measured (weighed) weekly, during the treatment and recovery periods for each cage.


FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: once 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 (surviving) animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at termination of dosing and at the end of recovery prior to or as part of the sacrifice of the animals
- Anaesthetic used for blood collection: Yes (ketamine/xylazin)
- Animals fasted: Yes
- How many animals: all surviving animals of the treatment/recovery period
- Parameters checked in table were examined. : yes, see Table 1

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at termination of dosing and at the end of recovery
- Animals fasted: Yes (ketamine/xylazin)
- How many animals: all suriviving animals of the treatment/recovery
- Parameters checked in table were examined: yes, see Table 1


URINALYSIS: Yes
- Time schedule for collection of urine: prior to or as part of the sacrifice of the animals
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Not specified
- Parameters examined: qualitative parameters: urine colour and appearance; quantitative indicators: specific gravity, nitrite, pH-value, protein, glucose


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: once before the first exposure and once towards the end of exposure but not earlier than in week 11, as well as in the last week of the recovery period
- Dose groups that were examined: all animals
- Battery of functions tested: Functional observational battery (FOB) assessment was performed and the following parameters were observed: home cage evaluations (sleeps, piloerection, vocalizations, motor movements, grooming); examinations before transfer in open field/standard arena (salivation, lachrymation, changes in skin, cyanosis, exophthalmos, eyes opening, respiration, response to handling); examination immediately after transfer in open field (arousal, fear, finger approach, head touch); open field evaluations (body position, spontaneous locomotor activity, ataxic gait, hypotonic gait, twitches, tremors, seizures, unusual behaviour, stereotypical behaviour, rearing, urination, defaecation, faeces consistency, abnormal vocalisation, aggressiveness/irritability, grooming, startle response); examination while removing from open field and manipulating (equilibrium reflex, positional passivity, visual placing, grip strength, sensitivity to pinching the tail, toe pinch reflex, extensor thrust/limb tone, hind limb reflex, righting reflex on the ground, air righting reflex, pupil reflex, body temperature, anterior chamber of eye, fundus of eye)

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see Table 2)

HISTOPATHOLOGY: Yes (see Table 2)

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 was performed for each gender by comparing values of treated animals 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 was performed with a Student’s t-Test or Mann-Whitney U-Test when appropriate. These statistics were performed with Ascentos 1.3.4 software or GraphPad Prism V.6.01 software (p<0.05 is considered as statistically significant).

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no findings of toxicological relevance observed during the weekly detailed clinical observations during the treatment and recovery periods. No statistically significant differences were found between all treated groups and the respective control group.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Mortality was observed during the treatment period in the MD group (1/10 males, 2/10 females) and the HD group (9/15 males, 8/15 females), and additionally in one LD group female. No mortality occurred during the recovery period for the remaining HD group animals and the animals of the control group. Based on the histopathological evaluation, necrotic and/or inflammatory lesions were observed in the respiratory tract, as well as in the pleural region and/or oesophagus in decedents of the LD and HD groups, and were most likely involved in the animals’ death and morbidity. These lesions were considered to be due to incidental influx or gavage error during administration of the test item formulation with its irritating nature, and were deemed not to be due to a systemic effect of the test item. Furthermore, in decedents of the HD group, there were also necrotic and/or inflammatory lesions in the stomach. These stomach lesions were considered to be a local tissue injury associated with the irritating nature of the test item and were also likely to be involved in the animals’ death and morbidity.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Treatment with the test item showed no clear concentration-related statistically significant effect on body weight development in male and female animals of all treated groups. With the exception of a statistically significant increase in body weight of 40.86% in LD group males compared to the controls in the first week of the treatment period, there were no statistically significant differences in the male treated groups during the treatment and recovery periods and additionally, no statistically significant differences occurred during the treatment period in females. A single statistically significant reduction in body weight (6.65% below the control) was observed in the HD group females on day 118 during the recovery period and body weight change from day 97 to day 104 in these females was also reduced compared to the control (123.44% below the control); these changes were considered to be incidental and not related to treatment with the test item.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There was no test item-related effect on food consumption of all male and female treated groups during the treatment and recovery periods.

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):

The test item had no toxicologically relevant effect on haematological and coagulation parameters in male and female animals analysed at the end of the treatment and recovery periods.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

The test item had no toxicologically relevant effect on clinical biochemical parameters and hormone analysis in male and female animals analysed at the end of the treatment and recovery periods.

Urinalysis findings:

no effects observed

Description (incidence and severity):

The test item had no toxicologically relevant effect on urinary parameters analysed in male and female animals at the end of the treatment and recovery periods.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

The statistically significant differences at the end of the treatment or recovery period were seen in only one gender, without dose dependency or only at the end of the recovery period. Therefore, no toxicological relevance was attributed to these observations.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The treatment with the test item showed an effect on the absolute and relative weight of the kidney to body weight and brain in the HD group, which was more prominent in males than in females. The absolute and relative (to body weight/brain) weight of kidney showed a statistically significant increase for HD group males, but the increase in HD females was lower than in males and showed no statistical significance. No statistical significance was noted for the differences in absolute and relative kidney weight compared to the control in LD and MD males and females. At the end of the recovery phase, the kidney weight recovered in HD males and the decrease in the female HD recovery group was not statistically significant below the control.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Histopathologically, most gross findings were considered to be a local tissue injury (due to incidental or accidental influx of the test item to the respiratory tract) associated with the irritating nature of the test item, or non-specific changes including agonal and postmortem changes that are frequently observed in dead or moribund animals. These findings included fluid filling or abnormal content in thoracic cavity, abnormal content in pericardium, alimentary tract filled with gas, abnormal colour and shape in several organs and tissues and failure to collapse in lungs. Thus, there were no gross lesions that could be due to a systemic effect of the test item in decedents.
The macroscopic findings which were observed for the surviving animals at scheduled necropsy were a dilated uterus in the control group and in several treated groups. Single findings in the LD group were white content in the urinary bladder and yellow fluid in the thoracic cavity. Based on the histopathological evaluation, there were no macroscopic findings in any survivors of the treated groups that could be attributed to treatment with the test item.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopic changes that could be attributed to a systemic effect of the test item were observed in the kidney of both decedents and survivors. These included proximal tubular cell vacuolation, proximal tubular cell necrosis, and increased incidence and/or severity of tubular basophilia and tubular dilatation. These renal lesions were considered to correlate with a significant increase or increased tendency in kidney weights of the HD group. In animals necropsied at the end of the recovery period, proximal tubular vacuolation and necrosis were not found in any HD animals, and there were no qualitative differences in the other renal findings between the control and HD groups. There were also no differences in kidney weights in both sexes. Thus, the histopathological examination revealed that the treatment- related renal changes completely subsided during the recovery phase of this study.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not specified

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

500 mg/kg bw/day (actual dose received)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

no

Relevant for humans:

not specified

Applicant's summary and conclusion

Conclusions:

In a 90-day oral repeated dose toxicity study with the registered substance N-[3-(dimethoxymethylsilyl)-propyl]ethylenediamine, conducted according to OECD Test Guideline 408 and in compliance with GLP, the NOAEL for systemic effects was concluded to be 300 mg/kg bw/day based on renal lesions.