Morpholine, 4-[(triethoxysilyl)methyl]-

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

other: OECD 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-03-24 to 2012-10-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD test under GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)

Deviations:

yes

Remarks:

Additional fertility parameters were evaluated as was discussed and accepted during the ECHA test proposal procedure instead of conducting a separate reproductive screening study.

Principles of method if other than guideline:

not applicable

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

480-370-1

EC Name:

-

Cas Number:

21743-27-1

Molecular formula:

Hill formula: C11H25NO4Si CAS formula: C11H25NO4Si

IUPAC Name:

4-[(triethoxysilyl)methyl]morpholine

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Age at study initiation: 7-8 weeks
- Weight at study initiation: males: 188 - 251 g , females: 152 - 194 g
- Housing: full barrier in an air conditioned room
- Diet (e.g. ad libitum): at libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days

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

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

VEHICLE
- Justification for use and choice of vehicle (if other than water): The vehicle was selected as suggested by the sponsor based on the test item’s characteristics and testing guideline.

Details on mating procedure:

Not applicable for the test guideline followed.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Does verification was performed by quantification of the test item using AAS. The fortification level covered approximately 112, 306 and 1000 mg test item/ 5 ml corn oil.

Duration of treatment / exposure:

Test duration: 90 days.

Frequency of treatment:

Dosing regime: 7 days/week

Details on study schedule:

Not applicable for the test guideline followed.

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw/day (m/f)
Basis:
actual ingested

No. of animals per sex per dose:

Male: 15 animals at 0 mg/kg bw/day
Male: 10 animals at 100 mg/kg bw/day
Male: 10 animals at 300 mg/kg bw/day
Male: 15 animals at 1000 mg/kg bw/day
Female: 15 animals at 0 mg/kg bw/day
Female: 10 animals at 100 mg/kg bw/day
Female: 10 animals at 300 mg/kg bw/day
Female: 15 animals at 1000 mg/kg bw/day

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: results of a 28-day repeated dose oral toxicity study (BSL study no. 071114) and an oral prenatal developmental toxicity study which was performed at Harlan Laboratories Ltd
- Post-exposure recovery period in satellite groups: 5 animals per gender of the control and of the HD group (1000 mg/kg bw/day) served as recovery group

Positive control:

No.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS:
Detailed cage side 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
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 for weekends and public holidays when observations were made once daily.
Changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypies or bizarre behaviour were recorded.

BODY WEIGHT: 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 period.

FOOD CONSUMPTION:
Food consumption was measured weekly during the treatment and recovery period.

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

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

OPHTHALMOSCOPIC EXAMINATION: Ophthalmological examination, using an ophthalmoscope, were made on all animals 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.


HAEMATOLOGY: Yes
- Time schedule for collection of blood: day of necropsy
- Anaesthetic used for blood collection: ketamine, Bremer Pharma GmbH, lot no: 29116, expiry date: July 2012 and xylazin, Riemser Arzneimittel AG, lot no. 000660/1, expiry date: December 2011
- Animals fasted: Yes
- How many animals: all

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day of necropsy
- Animals fasted: Yes
- How many animals: all

URINALYSIS: Yes
- Time schedule for collection of urine: day of necropsy
- Metabolism cages used for collection of urine: No
- Animals fasted: Yes

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:Once before the first exposure and once during the last week of the treatment period multiple detailed behavioural observations were made outside the home cage using a functional observational battery of tests [8]. These tests were additionally performed in the 4th week after the end of the treatment period on the animals that were sacrificed at the end of the recovery period.
- Dose groups that were examined: 0, 100, 300, 1000 mg/kg bw/day

Oestrous cyclicity (parental animals):

Daily over a period of 8 days, the estrous cycle of female animals, excluding the animals to be evaluated at the end of the recovery period, were examined at the beginning of the second month of treatment, (study week 5), at the beginning of the third month of treatment (study week 9) and at the end of the treatment period (study week 13). In the recovery animals the estrous cycle was examined during the last week of the recovery period (study week 17).

Sperm parameters (parental animals):

At necropsy at the end of the treatment period and at the end of the recovery period (see 11.18), one epididymis of each male animal was separated and immediately used for evaluation of epididymal sperm motility. At necropsy also one testis was separated and deep frozen in liquid nitrogen and stored at <-70°C until evaluation of testicular sperm count. All sperm parameters were analyzed using Hamilton Thorn Sperm Analyser (TOX IVOS Version 12.3).

Gross necropsy:
After weighing, one testis and one epididymis was separated and used for the evaluation of sperm parameters (11.13).

Histopathology:
For testis, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle at evaluation of additional hematoxylin-PAS (Periodic Acid Schiff) stained slides.

Litter observations:

Not applicable for the test guideline followed.

Postmortem examinations (parental animals):

GROSS PATHOLOGY: Yes
All animals in the study were subjected to a full, detailed gross necropsy which included careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavaties and their contents.

HISTOPATHOLOGY: Yes
Full histopathology was carried out on the preserved organs and tissues of all animals in the Control (0 mg/kg bw/day) and High Dose (1000 mg/kg bw/day) groups. Additionally, kidneys, lungs, bone marrow (sterum) and adrenal glands were examinated histopathologically in male animals of the 100 mg/kg bw/day and 300 mg/kg bw/day groups. A full histopathological evaluation was also performed on animal no. 23 of the 300 mg/kg bw/day group that had died during the treatment period

Postmortem examinations (offspring):

Not applicable for the test guideline followed.

Statistics:

Not applicable.

Reproductive indices:

Not applicable for the test guideline followed.

Offspring viability indices:

Not examined.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

s. below under

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight : s. below under

; food consumption: no effects

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Body weight : s. below under

; food consumption: no effects

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

s. below under

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

not examined

Description (incidence and severity):

Not applicable for the test guideline followed.

Details on results (P0)

CLINICAL SIGNS AND MORTALITY
One male animal of the MD group died on study day 85 of the treatment period, due to severe renal inflammation and scarring and a subsequent systemic inflammatory state with secondary myocarditis. Prior to death slightly reduced spontaneous activity, moderate to severe piloerection and slight body weight loss were observed in this animal.
Besides, no test item related clinical signs were noted during this study

BODY WEIGHT AND WEIGHT GAIN
Neither in male nor in female animals where there any statistically significant or biologically relevant differences between the dose groups and the control group in body weight development or food consumption. Mean daily body weight gain of male animals of the HD group was slightly and statistically significantly attenuated when compared to controls at the end of the treatment period.


ORGAN WEIGHTS
Kidneys of male animals of the HD group were moderately higher in weight than control animals. This was most prominent in animals showing histopathological alterations (see below). The effect on kidney weight of male animals was completely reversible at the end of the recovery period. Kidney weight in female animals - although slightly lower than in the respective controls, is not assumed to be affected by (N-Morpholinomethyl)triethoxysilan.
Adrenal glands in female animals of all dose groups were slightly lower in weight than in control animals. At the end of the recovery period no significant difference in adrenal gland weight of the female animals was found between HD groups and controls. As no associated histopathological findings were noted, a relation to the test item is unclear.
Slightly – but statistically significantly lower absolute spleen weight was seen in female – but not male animals, mainly in MD and HD group. At the end of the recovery period no significant difference in spleen weight of the female animals was found between HD groups and controls.

GROSS PATHOLOGY
At the end of the treatment period macroscopic alteration in the kidney were observed in 3/10 male animals of the HD group: A slightly abnormally shaped or an enlarged and yellow mottled kidney or multiple yellowish pin-point foci. Besides, no gross test item related alterations were observed at the end of the treatment or recovery period of this study.

HISTOPATHOLOGY: NON-NEOPLASTIC
Treatment at 300 mg/kg/day led to the spontaneous death of one male rat towards the end of the treatment period, caused by severe renal inflammation and scarring and a subsequent systemic inflammatory state with secondary myocarditis.
In five males treated at 1000 mg/kg/day, a multifocal interstitial nephritis/scarring was observed, its severity ranging from mild to severe. It was associated with multifocal dilated cortical tubules, and with an increase in basophilic tubules and mononuclear cell infiltrates, when compared to control males. Minor lesions of the pelvic urothelium were also noted, comprising suburothelial mononuclear cell infiltrates and a multifocal to diffuse urothelial hyperplasia. At 300 mg/kg/day, a mild interstitial nephritis/scarring, associated with mild numbers of basophilic tubules and mild mononuclear cell infiltrates, was observed in one single male and was also considered test item-related. The mechanism of the renal changes could not be elucidated in this study.
In the bone marrow, a minimally increased myeloid:erythroid ratio was seen in two males treated at 1000 mg/kg/day and was considered to be a borderline reaction to the renal inflammation observed in the same animals. It was therefore not considered to be directly test item-related.
In the lung of male rats treated at 1000 mg/kg/day, there was a tendency towards higher severity grades of multifocal alveolar macrophages, perivascular/peribronchial mixed cell infiltrates and foci of pneumonitis, when compared to the background level seen in controls. A multifocal minimal epithelialization was seen in three males. In view of their low incidence and degree, the significance of the pulmonary findings is not clear, but a direct toxic effect of the test item on the lung cannot be fully excluded.
After the 28-day recovery period, renal lesions were found to be partially and lung lesions were found to be completely reversible. Bone marrow changes had not yet resolved.

Effect levels (P0)

Key result

Dose descriptor:

NOEL

Remarks:

for reproductive effects

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: A reproduction toxicological effect of (N-Morpholinomethyl)triethoxysilan was not found in this study.

Results: F1 generation

General toxicity (F1)

Clinical signs:

not examined

Description (incidence and severity):

Not applicable for the test guideline followed.

Mortality / viability:

not examined

Description (incidence and severity):

Not applicable for the test guideline followed.

Body weight and weight changes:

not examined

Description (incidence and severity):

Not applicable for the test guideline followed.

Sexual maturation:

not examined

Description (incidence and severity):

Not applicable for the test guideline followed.

Organ weight findings including organ / body weight ratios:

not examined

Description (incidence and severity):

Not applicable for the test guideline followed.

Gross pathological findings:

not examined

Description (incidence and severity):

Not applicable for the test guideline followed.

Histopathological findings:

not examined

Description (incidence and severity):

Not applicable for the test guideline followed.

Details on results (F1)

Not applicable to the test guideline followed.

Effect levels (F1)

Remarks on result:

other: no offspring generation created in this study

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Fertility Parameters

(N-Morpholinomethyl)triethoxysilan had no effect on epididymal sperm motility or testicular sperm count analyzed at the end of the treatment or recovery period of this study. Neither in the percentage of motile, static or rapidly moving epididymal sperms nor in testicular number of sperms/g testis was there any statistically significance between control and any of the dose groups.

(N-Morpholinomethyl)triethoxysilan had also no effect on the oestrus cycle analyzed in study weeks 5 or 9 or at the end of the treatment or recovery period. Considering individual values, there was no considerable difference in cycle length or sequence of cycle stages between dose groups and the control group.

Applicant's summary and conclusion

Conclusions:

In a reliable 90-day repeated dose toxicity study with (N-Morpholinomethyl)triethoxysilan, conducted according to OECD test guideline 408 and in compliance with GLP, there were no effects on any of the reproductive organs examined in male or females (testes, epididymis, ovaries, uterus), at any dose level up to the highest test concentration of 1000 mg/kg bw.day. Furthermore, there were no effects on any of the additional fertility parameters investigated as well.

Executive summary:

In a 90-day repeated oral dose toxicity study with the test item (N-Morpholinomethyl)triethoxysilan groups of 10 male and female Wistar rats were exposed to 0, 100, 300 and 1000 mg/kg bw/day by gavage with corn oil as the vehicle.

Histopathological findings directly attributed to the test item were seen in the kidney of males treated at 1000 and 300 mg/kg/day.

At 300 mg/kg/day, one surviving male rat showed also kidney lesions considered test item-related, and another male died spontaneously of severe renal pathology with secondary myocarditis. Renal lesions were partially reversible after 28 days. A minimally increased myeloid:erythroid ratio of the bone marrow was considered indicative of increased myelopoiesis in individuals presenting prominent renal lesions, and was therefore considered indirectly test item-related. It was not reversible after 28 days. The significance of some minor pulmonary lesions seen in males at 1000 mg/kg/day could not be elucidated, a test item relationship cannot be excluded. Pulmonary lesions had resolved after 28 days without treatment.

Further associated findings in the HD group were: macroscopic findings in the kidney, slightly higher kidney weight and increase in blood urea and creatinine in male animals. Finally, overall body weight gain was slightly attenuated in male animals.

On the basis of these findings, the kidney can be considered the major target organ of (N-Morpholinomethyl)triethoxysilan.

A reproduction toxicological effect of (N-Morpholinomethyl)triethoxysilan was not found in this study.

A slightly lower weight in adrenal glands of female animals of all dose groups and a slightly higher spleen weight in female animals of the MD and HD group cannot not be elucidated but was absent at the end of the recovery period. A relation to the test item is unclear.

The dose level of 100 mg/kg/day marks the NOAEL for systemic toxicity in this study.

As no reproduction toxicological effect was found in this study, the dose level of 1000 mg/kg/day marks the NOEL

for reproductive effects.