N-[(triethoxysilyl)methyl]cyclohexylamine

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2004

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

454-780-6

EC Name:

-

Cas Number:

27445-54-1

Molecular formula:

Hill formula: C12H27NO2Si CAS formula: C12H27NO2Si

IUPAC Name:

Cyclohexyl-((diethoxy-methyl-silanyl)-methyl)-amine

Test material form:

liquid

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

Species: Hsd:Wistar rats (HsdBr1:WH,Full-Barrier)
Supplier: Harlan Winkelmann GmbH, D-33178 Borchen. The animals were derived from a controlled full barrier maintained breeding system (spi).
Health status: No special findings during the adaptation period; females nulliparous, non-pregnant.
Body weight at the commencement of the study: Females: 172 - 210 g, (mean: 182 g,:f: 20%= 146-219 g), Males: 202 - 233 g, (mean: 215 g,:f: 20%= 172-258 g)
Age at the commencement of the study: 7-9 weeks

The animals were barrier maintained (semi-barrier) in an air conditioned room:
Temperature: 22: +/-3°C
ReI. humidity: 55 +/- 10%
Artificial light sequence being 12 hours light, 12 hours dark
Air change: 10x / hour
Feeding ad libitum, Altromin 1324 maintenance diet for rats and mice, totally-pathogen- free (TPF)
Free access to tap water (drinking water, municipal residue control, microbiol. controlled periodically)
The animals were kept in Macrolon cages on Altromin saw fiber bedding

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

The test item was suspended in Com Oil, (SIGMA-ALDRICH CHEMIE GmbH, D-82041 Deisenhofen; Lot 122K0131). The vehicle was chosen due to its non-toxic characteristics and the chemical properties of the test item. The single dosages (vigorously shaken) were prepared on every application
day in a clean bench in the following ratios:
Low Dose (25 mg/kg BW): The test item was suspended in Com Oil at a ratio of 0.1 g to 20 mL
Medium Dose (150 mg/kg BW): The test item was suspended in Com Oil at a ratio of 0.6 g to 20 mL
High Dose (750 mg/kg BW): The test item was suspended in Com Oil at a ratio of3.0 g to 20 mL
The homogeneity of the preparations was visually checked. During the application procedure no obvious separation was detected.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of Stability and Homogeneity was performed at the GLP-certified contract laboratory Institut flir Biologische Analytik und Consulting IBACON GmbH., Arheilger Weg 17, 64380 Rossdorf; Germany.

Frequency of treatment:

The animals were dosed with the test item on 7 days per week fora period of 28 days by gavage, using a stomach tube. Totally 28 doses were administered per animal and group.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

25 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

150 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

750 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:

Body Weight Development
The animals were weighed prior to first application (day 0) and once a week thereafter (day 7, 14,21,27). The total and weekly mean weight gain for each group was calculated. The weights determined on the day of sacrifice were used for the calculation of the relative organ weights.

Food Consumption Food consumption was calculated weekly (day 7, 14, 21, 27) with determination of food supply and residual. The total mean and daily food consumption was determined for each group.

Clinical Observation
The observation period was 28 days. 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. At least twice daily, all animals were observed for morbidity and mortality. Cageside observations included spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoe, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size. Once before the first exposure and in the final week, detailed clinical observations were made in all animals with specific emphasis on locomotion and behaviour. These observations were made outside the home cage at the same time.

Behaviour/Functional Observation
Once before the first exposure and in the fourth exposure week sensory reactivity to stimuli of different types (e.g. auditory, visual and proprioceptive stimuli, and motor activity assessment) was conducted. The functional observational battery was oriented according to the method described by Moser et aI., 1991 (Moser V.c., K.L. McDaniel, P.M. Phillips, Rat Strain and Stock Comparisons Using a Functional Observational Battery, Toxicol. Appl. Pharmacol. 108,267-283, 1991). The examination began with a brief horne cage observation, describing the posture, palpebral closure, and the presence of convulsions or tremors. The rat's reactivity to removal from the cage and handling were then rated and the observer noted presence of salivation, lacrimation, and piloerection. Next the rat was placed on an open field (on a table surrounded by a 10 cm cardboard rim) for 3 min. during which time the arousal level (alertness) and gait characteristics were recorded. The number of rears, supported (using the cardboard rim as support) and unsupported (unassisted), were counted separately. In addition, fecal boluses and pools of urine were counted. The presence of convulsions and tremors was again noted. Reactions to the approach of a pencil, touch on the rump, and tail pinch using forceps were noted. The pupillary response was tested with the aid of a red light using a penlight stimulus. Flexion reflex, equilibrium, grasping reflex (together with grip strength), righting reflexes and auditory startle were tested. Finally the rectal body temperature was taken. Blood Sampling The withdrawal of blood was performed by puncture of the abdominal aorta of the anaesthesized animals after overnight fasting. This was part of the procedure of killing the animals on the day of necropsy. The blood was collected into small tubes containing EDTA for haematology, citrat for clotting tests and plain tubes for clinical biochemistry. Urine Sampling The urine was collected into plain tubes by puncture of the urine bladder as a part of the necropsy.

Blood Sampling
The withdrawal of blood was performed by puncture of the abdominal aorta of the anaesthesized animals after overnight fasting. This was part of the procedure of killing the animals on the day of necropsy. The blood was collected into small tubes containing EDTA for haematology, citrat for clotting tests and plain tubes for clinical biochemistry.

Urine Sampling
The urine was collected into plain tubes by puncture of the urine bladder as a part of the necropsy.

Haematology and Clinical Biochemistry
Haematocrit (Hct)
Haemoglobin (Hb)
Erythrocyte count (RBC)
Total leucocyte count (WBC)
Platelet count
APTT/PTT
Differential leucocyte count
AST (GOT) aspartate aminotransferase
ALT (GPT) alanine aminotransferase
Alkaline Phosphatase (AP)
Cholesterol (Chol)
Total Protein (TP)
Glucose (GLU)
Urea
Creatinine (CREA)
Albumin (ALB)
Na
K

Sacrifice and pathology:

Pathology
All animals in the study were subj ected 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 cavities and their contents.

Organ Weight
The wet weight was taken from the following tissues immediately after preparation:
liver, kidneys, adrenals, testes, epididymides, thymus, spleen, brain, heart

Calculation of the Relative Organ Weight
The relative organ weight was calculated in relation to the bodyweights determined on the day of sacrifice and was expressed in %.

Organ Preparation
The following tissues were preserved in buffered (sodium dihydrogen orthophosphate/
disodium hydrogen orthophosphate) 10 % formalin solution from
all animals of all groups. It is the most appropriate fixation medium for both
the type of tissue and the intended subsequent histopathological examination:
all gross lesions
brain (representative regions including cerebrum, cerebellum and pons), spinal cord, liver, kidneys, adrenals, stomach, small and large intestines (including Peyer's patches, Lymphnodes acc., to application), thymus, thyroid, spleen, lung and trachea, heart, gonads, accessory sex organs (e.g. uterus, prostate, vesicula seminalis), urinary bladder, lymph nodes (Lymphocentrum mandibulare; Lnn axillares), peripheral nerve, bone marrow

Histopathology
Full histopathology was carried out on the preserved organs and tissues of all animals in the Control and High Dose groups. Additionally Thyroid was evaluated from all animals in LD and MD.

Statistics:

For statistical analysis one-way analysis of variance (ANOVA) was carried out, followed by Student's t-test to reveal any differences between controland test groups

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The mean weight gain in all groups was less as expected according to the standard growth curve for this strain (see table 3, 4 and 5). These diminished weight gains are due to the daily treatment associated with mechanical manipulation and therefore with increased stress for the animals. Reduced weight gain was observed for the female and male High Dose groups (esp. in the first application week) as compared to the Control group, reaching statistical significance for the females. Compound relation and toxicological relevance of this finding is given. Dose dependency is observed.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Reduced food consumption was observed for the female and male High Dose groups (esp. in the first application week) as compared to the Control group, reaching statistical significance for the males. Compound relation and toxicological relevance of this finding can be concluded. Remaining animals showed normal food intake and no further significant reduction in food consumption was found.

Food efficiency:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Individual deviations in the platelet count are found without clear dose dependency. With the exception of one animal for the whole male HD group high platelet values were found indicating on a possible compound relation. As platelet determination in rats is known to be burdened with the possibility of error due to the small size of platelets the toxicological relevance of these individual findings cannot be concluded. However, accuracy is usually sufficient to be useful in detecting severe alterations.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

With the exception of the female LD-group ( 3 high borderline values) and two individual high borderline values in the female MD and HD group, all mean and all individual ALB values are within the expected range. Significant differences (slight increase) are found for the male MD and HD and the female LD group. Toxicological relevance for this finding cannot clearly be concluded. In combination with the findings concerning the TP values compound relation may be assumed.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Upon the assessment of organ weights the most relevant findings were made for the spleen and thymus. Although no marked findings, and not confirmed by other findings (e.g. histopathological evaluation of these organs) compound relation may be assumed. The mean relative and absolute liver weight in the female and male MD groups was slightly higher than the liver weight in the corresponding Control groups, reaching statistical significance for these groups, as well as the relative liver weight for the female HD group. These results may be interpretated as compensatory raise in liver weight (commonly seen in toxicological stu dies), but normally without morphological correlation (no histopathological findings) and without correlation in clinical biochemistry. The mean absolute heart weight in the male HD group was slightly lower than the heart weight in the corresponding Control group, reaching statistical significance. The mean relative adrenals weight in the male LD group was slightly lower than the weight in the corresponding Control group, reaching statistical significance. Toxicological relevance of these organ weight findings is questionable. Dose dependency was not observed and compound relation cannot be concluded. Beside these findings no significant results in relative and absolute organ weights for both sexes and any of the groups concerning the other organs have been found.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The sole treatment-related change was a dosage related thyroid follicular epithelial hypertrophy in the two greater dosage groups. Thyroid follicular epithelial hypertrophy was present in 2/5 male rats in HD group (750 mg/kg) at a mild degree and once at a minimal level. 4/5 male rats of MD group (150 mg/kg) were affected with a minimal change, as were single male rats
in LD group (25 mg/kg) and the Control. There was a limited response in female rats in that 2/5 female rats in the HD (750 mg/kg) were affected at a minimal level. The degree of slight variability in control animals is usual in a blind read such as this, as is the fact that with many goitrogenic substances, male rats are more affected than female rats.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Effect levels

1

Dose descriptor:

NOEL

Effect level:

25 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other:

Remarks:

NOEL in the histo report but NOAEL in the study report

2

Dose descriptor:

NOAEL

Remarks on result:

not determinable because of methodological limitations

Remarks:

in the absence of data on the reversibility and/or T3 and T4 levels adversity of effects is unclear

Target system / organ toxicity

Critical effects observed:

no

Lowest effective dose / conc.:

150 mg/kg bw/day (nominal)

System:

other: thyroid system

Organ:

thyroid gland

Any other information on results incl. tables

Incidences and severity of thyroid follicular epithelial hypertrophy

	Control	LD	MD	HD
Female
Thyroids examined	5	5	5	5
Minimal	0	1	1	2
Male
Thyroids examined	5	5	5	5
Minimal	1	1	4	1
Mild	0	0	0	2

Applicant's summary and conclusion

Conclusions:

Due to mild effects in males of the MD (150 mg/kg bw) and HD (750 mg/kg bw) on the thyroid gland the NOEL is set to LD of 25 mg/kg b.w.. Major functional changes in other organ systems with any consistent changes in clinical biochemistry or haematology indicating severe organ damage or indicating an association with the thyroidal effects were not observed. Considering the large dose increment between mid and high dose the corresponding negligabel increase in severity with the concomitant decrease in the incidences a specific adverse interference with either the released hormones or with the gland epithelial cells is unlikely.
In general, rodents are far more sensitive for effects on the the thyroidal system as binding proteins are less effective. As a consequece changes in T3/T4 requirements are more rapidly compensated by adaptive growth of epithelial tissue of the adrenal gland.
Without any information on the hormone status and/or the reversibility of the effects a conlusion on the NOAEL is not possilbe.