Registration Dossier

Administrative data

Description of key information

RDT oral (OECD 408): NOAEL = 100 mg/kg bw/day for females; NOAEL for males could not be established

RDT inhalation: no data available
RDT dermal: no data available

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-11-09 to 2019-10-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
25 June 2018
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Bayrisches Landesamt für Gesundheit und Lebensmittelsicherheit
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approx. 7-8 weeks old
- Weight at study initiation: males: 133 – 178 g; females: 115 – 154 g
- Housing: Housed in groups of 5 animals / sex / group / cage in IVC cages
- Diet: Altromin 1324 maintenance diet for rats and mice provided ad libitum
- Water: tap water, sulphur acidified to a pH of approximately 2.8 provided ad libitum
- Acclimation period: at least 5 days

DETAILS OF FOOD AND WATER QUALITY: Drinking water, municipal residue control, microbiological controls at regular intervals.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 10
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 hours light, 12 hours dark
Route of administration:
oral: gavage
Details on route of administration:
The test item formulation or vehicle were administered as a single dose to the animals by oral gavage at an application volume of 4 mL/kg bw.
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The dose formulations were prepared by adding the required volume of corn oil to give the appropriate final concentration of the test item and were vortexed and/or stirred until visual homogeneity was achieved.

VEHICLE
- Concentration in vehicle: undiluted
- Amount of vehicle (if gavage): The test item formulation or vehicle were administered as a single dose to the animals by oral gavage at an application volume of 4 mL/kg bw.
- Lot/batch no. (if required): MKCD1021; MKCG3257
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of formulation samples was performed at three concentrations, 25 mg/mL, 75 mg/mL and 250 mg/mL in study weeks 1, 5, 9 and in the last week of the study. The mean recoveries observed for the low-dose group were between 94.7% and 110.7% of the nominal value, between 94.0% and 104.8% for the mid-dose group and between 94.7% and 109.0% of the nominal value for the high-dose group. The mean recoveries observed in the low-, mid-, and high-dose groups were 102.5%, 99.6%, and 101.0% of the nominal concentration, respectively.

Nominal concentrations were confirmed for all dose groups, as measured concentrations were within acceptance criterion of 10%.

The high-dose formulation sample measured in week 9 was not valid. Therefore, a new high-dose sample was processed and measured. Until confirmation of the formulation concentration in week 9, all high-dose group animals were treated for 4 study days in week 9 with a measured mean formulation concentration of 164.4 mg/mL.
Duration of treatment / exposure:
90 days exposure; 28 days recovery for control and high-dose groups
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:
dose groups: 10
recovery groups: 5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: According to the results of a previous dose range finding study (BSL Munich Study No. 183769, non-GLP) in which no significant adverse effects were observed in male and female animals (3 animals / sex / dose) dosed at 100, 300 and 1000 mg/kg body weight (bw)/day for 14 consecutive days.
- Rationale for animal assignment: Randomisation was performed with IDBS Workbook 10.1.2 software.
- Fasting period before blood sampling for clinical biochemistry: yes
Positive control:
None
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 and considering the peak period of anticipated effects after dosing. 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
- Time schedule: Once before the first administration and at least once a week thereafter

BODY WEIGHT: Yes
- Time schedule for examinations: The 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 periods.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

FOOD EFFICIENCY: No

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

OPHTHALMOSCOPIC 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 multiple detailed behavioural observations were made outside the home cage using a functional observational battery of tests.
- Dose groups that were examined: All dose groups were evaluated.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the treatment and recovery periods as part of the sacrifice of the animals
- Anaesthetic used for blood collection: Yes; ketamine and xylazin
- Animals fasted: Yes; fasted overnight
- How many animals: all surviving animals
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the treatment and recovery periods as part of the sacrifice of the animals. For an evaluation of test item-related effects on the pituitary-thyroid axis and thyroid hormones, serum samples of all animals were retained at the end of treatment (80 animals) and recovery (20 animals) periods. T3, T4 and TSH serum levels were determined for the main study and recovery animals (100 animals).
- Animals fasted: Yes; ketamine and xylazin
- How many animals: all surviving animals
- Parameters checked in table 2 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: A urinalysis was performed with samples collected from all animals as part of the sacrifice of the animals.
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Not specified
- Parameters checked in table 3 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 multiple detailed behavioural observations were made outside the home cage using a functional observational battery of tests.
- Dose groups that were examined: All dose groups were evaluated.
- Sensory reactivity to different modalities, grip strength and motor activity assessments and other behavioural 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 using an ophthalmoscope (anterior chamber of the eye and fundus of eye), were assessed.

IMMUNOLOGY: No

OTHER: Daily over a period of 14 days, the estrous cycle of all female animals in the treatment period groups was monitored during the last two weeks of treatment. In the recovery animals the estrous cycle was also monitored during the last week of the recovery period.

At necropsy (one day after the last administration) and at the end of the recovery period (day 29), the 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 a Hamilton Thorne Sperm Analyser (TOX IVOS Version 13.0).
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 4)

HISTOPATHOLOGY: Yes (see table 5)
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 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 1.3.4 software or GraphPad Prism V.6.01 software (p<0.05 is considered as statistically significant).
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
The findings in the male low-dose group (reduced spontaneous activity), male mid-dose group (moderate piloerection and abnormal breathing) or male and female high-dose groups (slight to moderate piloerection, diarrhoea, ataxia, analgesia, apathy, recumbency, slightly to severely reduced spontaneous activity, hypotonia, half eyelid-closure, cough/sneezing) were seen on an occasional base in single animals on single or several days during the treatment period and are therefore not considered to be an adverse effect of the test item.

Clinical signs like slight to moderate salivation and moving the bedding materials were observed immediately after the dose administration in the dose groups and are therefore considered to be a sign of a local reaction to the test item rather than a systemic adverse effect and without toxicological relevance. The occurrence on the first day of the recovery phase is considered to be incidental.

Other findings like scratch/cut at the head, wound at the right neck, crust, alopecia at the neck were considered to be incidental findings and were seen in single animals of the control or dose groups.
Mortality:
mortality observed, treatment-related
Description (incidence):
Test item-related mortality was found for one male animal in the high-dose recovery group (1000 mg/kg bw/day) on study day 97. The cause of death was associated with presence of precipitates within the ureteral lumen leading to an obstructive nephropathy, a subsequent severe acute urinary bladder dilatation, marked hydronephrosis and severe secondary metastatic calcification due to renal failure and hypercalcaemia in multiple organs.

One male control group animal was found dead on study day 57 of the treatment period. No clinical signs were noted during treatment. The death was caused accidentally during oral gavage after treatment with vehicle.

No further mortalities were observed during the treatment and recovery phases.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Statistical significance was seen for a moderate increase of body weight change in week 12 of the treatment phase in the male mid-dose group.

During the recovery phase, mean body weight of the female high-dose group was decreased when compared to control group (between 5.52% below control on day 90 and 7.95% below control on day 118) and body weight change was statistically significantly decreased in the last week before necropsy in the female high-dose group. Mean body weight was additionally decreased when compared to control in the male high-dose group during the recovery period (up to 4.47% below control on day 104). Mean body weight change calculated between day 90 to 118 showed a decrease of 7.05% in the male group and a statistically significant weight loss in the female group. Overall, mean body weight showed an increase of 3.11% in males and a slight decrease of 0.64% in females on day 118 when compared to day 90.

The differences in body weight development between the male and female dose groups compared to control group during the treatment phase or recovery phase were not considered to be related to an adverse effect of the test item, as they were only slight at the end of treatment or recovery periods and no dose dependency was observed. Overall, mean values for body weight and body weight gain were comparable to control group for all dose groups.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Statistical significance was seen for a slightly decreased food consumption in the male high-dose group in week 4 and 5 (7.3% and 8.5% below control) and for the female high-dose group in weeks 4 to 6 (13.3%, 11.5% and 7.6% below control) of the study.

Weekly food consumption of all male and female dose groups was comparable to control groups and no differences were seen during the treatment period and recovery period. Therefore, the statistical significances were not considered to be an effect of test item treatment. As the significances were slight and were seen in single weeks, they are deemed to be incidental.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
At the end of the treatment period, statistical significance was noted for a slightly decreased mean value of the parameter glucose (25.45% below control) in the male high-dose group and a slight increase of Na in the female low-dose group (2.33% above control). In females, crreatinine was slightly and statistically significantly decreased in the high-dose group when compared to control (22.3% below control) at the end of recovery.

Differences between test item-treated males or females and their respective controls showed no dose-dependency or consistency at the end of the treatment or recovery periods and therefore, a toxicologically relevant effect on parameters of clinical biochemistry of males and females in any of the test item-treated groups is not considered.

No adverse test item-related effect on T3, T4 and TSH measurement after the end of treatment and recovery period was observed. Statistical significance of TSH in the female low-dose group at the end of treatment was not considered to be an adverse toxicological effect. No dose-dependent increase was noted for this parameter and additionally, histopathological evaluation showed no adverse test item-related effects on thyroid gland.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
High protein levels were found in the urine of males and females in all groups including the control group at the end of treatment and recovery periods, which were more prominent in male dose groups. Values of up to 100 mg/dL were measured with exception of one high- and one mid-dose male at the end of treatment or recovery periods. For these animals a protein level of 500 mg/dL was measured. Although protein was found in control groups, a test item-related effect for the increased levels observed in dose groups cannot be excluded as histopathological effects were seen in the urinary system of males and females.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
In the week before treatment, statistically significant mean values were noted in the male mid-dose group for a decrease in animal sleeps and increase of moving in cage, in the male high-dose group for a slight increase of response to handling, fear and decrease in rearing supported, a slight increase for mean body temperature in the male mid-dose group. For the last week of treatment slightly increased mean values were seen for rearing supported in the male low-dose group, rearing not supported in the mid-dose group, defecation in the male low-dose and high-dose groups, equilibrium reflex in the male high-dose group and body temperature in the male high-dose group.

In females, head touch was statistically significantly decreased in in the mid-dose group, startle response increased in the female low-dose group and slightly increased for body temperature in the high-dose group in the week before treatment. Female groups showed statistical significance for an increase in response to handling, fear, rearing not supported for the female low-dose group in the last week of treatment.

No statistical significances were seen in the male and female recovery groups.

The statistically significant differences were seen without dose dependency or before start of treatment and are therefore not considered to be related to treatment with test item.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
No statistical significant changes were noted for absolute and relative weight of all organs measured at the end of treatment period in all male and female dose groups when compared to control with exception of mean organ weight-to-brain ratio for testes in the male low-dose group. The relative mean weight was slightly decreased (8.31 % below control). A slight decrease was seen in all other male dose groups but without dose-dependency. Therefore, the statistical significance for testes was considered to be of no toxicological relevance.

Statistical significance was found for a slightly increased mean relative kidney weight-to-body weight ratio in the females of the high-dose recovery group (15.44% above control). No statistically significant changes for kidney were seen after end of treatment in both genders for all dose groups and at the end of recovery for males.

Further statistical significances in the female recovery high-dose group were a slight increase for relative mean weight of brain-to-body weight ratio (15.66% above control) and mean weight of ovaries-to-body weight ratio (27.73% above control). They were not associated with histopathological alterations and no toxicological changes were noted at the end of treatment for brain in males and females. The slightly decreased absolute (up to 18.48% below control in the low-dose group) and relative weight of ovaries at the end of treatment (up to 17.18% below control in the low-dose group) was seen without dose-dependency, did not coincide with histopathological findings and therefore, the statistical significances at the end of the recovery period are not considered to be of toxicological relevance.

Absolute organ weight of thymus was found in high-dose group males with a decrease of 15.83% below control and 16.06% below control relative to brain at the end of treatment. In females, mean absolute weight was seen with a decrease of 12.70% below control and relative to brain to be 12.90% below control in the mid-dose group. At the end of recovery, the thymus weight was found in females to be 15.57% below control (absolute weight) and 16.21% below control (relative weight to brain). No dose-dependent effect was noted at the end of treatment.

The decreased absolute weight and weight relative to brain or body weight observed for thyroid/parathyroid in both genders for high-dose and mid-dose groups at the end of treatment (up to 26.00% below control for absolute weight in high-dose group males, absolute weight up to 25.11% below control for high-dose group females) was not considered to be of toxicological relevance. No statistical significance was found for all male and female dose groups when compared to control and histopathological assessment showed no test item-related changes.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
One high-dose male was found dead on study day 97 during the recovery phase. Macroscopic findings for the animal were dark red coloured lung, gas filled intestinal organs, dilated kidneys and ureters, dilated and red coloured urinary bladder and red coloured thymus. The findings observed for the urogenital tract correlate to histopathological findings. All other macroscopic findings in dose groups were seen occasionally in single animals without dose dependency and histopathological evaluation showed no test item-related findings and, therefore, were not considered treatment-related effects.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
At histopathological evaluation, test-item related lesions in the lower urinary tract were found in males at ≥ 100 mg/kg bw/day and females at ≥ 300 mg/kg bw/day in the main test and recovery groups, associated with urolithiasis being findings dose-related, and more prominent and frequent in males than females. The main induced findings in the urinary system were urothelial hyperplasia of the lower urinary tract (renal pelvis, urinary bladder, ureter and/or urethra) in males at ≥ 100 mg/kg bw/day and females at ≥ 300 mg/kg bw/day, precipitates in the urine in males at ≥ 100 mg/kg bw/day and females at ≥ 300 mg/kg bw/day, hyaline droplets in the renal cortex of males at ≥ 100 mg/kg bw/day and lipid-laden cell accumulations were detected within the renal medulla or bladder submucosa from both genders at 1000 mg/kg bw/day. Thymic atrophy in males at ≥ 100 mg/kg bw/day was considered mainly a stress-related finding, indirectly linked to the treatment with the test item.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Evaluation of the oestrous cycle in the last two weeks of the treatment and at the end of recovery period showed no toxicologically relevant changes. There were no statistical significances for mean sperm count and mean sperm motility for all dose groups in the treatment and recovery period. No treatment-related effects on the mean testis weight, mean sperm motility and mean testis sperm count in both treatment and recovery periods were found.
Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
LOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (nominal)
System:
urinary
Organ:
bladder
ureter
urethra
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Conclusions:
The test item was examined for toxicity after repeated exposure in a subchronic study with rats according to OECD 408 and in compliance with GLP. Based on the treatment related findings and effects of toxicological relevance the NOAEL is 100 mg/kg bw/day in females and the NOAEL for males could not be established.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The available information comprises an adequate and reliable study (Klimisch score 1), and is thus sufficient to fulfil the standard information requirements set out in Annex VII, 8.7, of Regulation (EC) No 1907/2006.
System:
urinary
Organ:
bladder
ureter
urethra

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

One subchronic repeated dose toxicity study on methyl-N-[3-(trimethoxysilyl)propyl]carbamate (CAS 23432-62-4) is available.

 

In a repeated dose toxicity study that adhered to OECD 408 test guideline and was GLP compliant, the test material was administered daily to 4 dose groups for 90 days. Dose groups consisted of a control group administered corn oil, a low-dose group administered 100 mg/kg bw/day, a mid-dose group administered 300 mg/kg bw/day, and a high-dose group administered 1000 mg/kg bw/day. The four groups comprised of 10 male and 10 female Wistar rats. In addition, animals in a recovery group were observed for a period of 28 days following the last administration (high dose and control groups only, each group consisting of 5 male and 5 female Wistar rats).

 

Test item-related mortality was found for one male animal in the high-dose recovery group (1000 mg/kg bw/day). The cause of death was associated with presence of precipitates within the ureteral lumen leading to an obstructive nephropathy, a subsequent severe acute urinary bladder dilatation, marked hydronephrosis and severe secondary metastatic calcification due to renal failure and hypercalcaemia in multiple organs.

 

No adverse effects of test item were found on male and female clinical observations, functional observations, body weight development, food consumption, thyroid hormone analysis, sperm parameters, haematology, coagulation, and clinical biochemistry and organ weights.

 

Test-item related lesions in the lower urinary tract were found in males at ≥ 100 mg/kg bw/day and females at ≥ 300 mg/kg bw/day in the main test and recovery groups, associated with urolithiasis.

 

The main induced findings in the urinary system were urothelial hyperplasia of the lower urinary tract (renal pelvis, urinary bladder, ureter and/or urethra) in males at ≥ 100 mg/kg bw/day and females at ≥ 300 mg/kg bw/day, precipitates in the urine in males at ≥ 100 mg/kg bw/day and females at ≥ 300 mg/kg bw/day, hyaline droplets in the renal cortex of males at ≥ 100 mg/kg bw/day and lipid-laden cell accumulations were detected within the renal medulla or bladder submucosa from both genders at 1000 mg/kg bw/day. The changes in the urinary tract correlate to macroscopic findings at necropsy (granular sticky, yellow abnormal content in the urinary bladder).

 

Thymic atrophy in males at ≥ 100 mg/kg bw/day was considered mainly a stress-related finding, indirectly linked to the treatment with the test item and associated with an organ weight decrease compared to control group. No morphological toxicologically relevant effects in the reproductive organs were found.

 

The no observed adverse effect level (NOAEL) of methyl [3-(trimethoxysilyl)propyl]carbamate (CAS 23432-62-4) in this study is considered to be at 100 mg/kg bw/day in females; the NOAEL for males could not be established.

Justification for classification or non-classification

The available data on methyl-N-[3 (trimethoxysilyl)propyl]carbamate meet the criteria for classification according to Regulation (EC) 1272/2008, and therefore the substance is classified as STOT RE2.