Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2007-08-02 to 2007-10-23
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD Guideline under GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
2000/32/EG, B.12
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann GmbH D-33178 Borchen
- Age at Start of Acclimatisation: 8 - 10 weeks
- Weight at Start of Treatment: males mean value 35.5 g (SD ± 1.8 g), females mean value 28.0 g (SD ± 1.3 g)
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: single
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 day


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/-3 °C
- Humidity (%): 30-78%
- Air changes (per hr): not given in the report
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: unspecified
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
On the day of the experiment, the test item was formulated in corn oil. The vehicle was chosen to its relative non-toxicity for the animals. All animals received a single standard volume of 10 mL/kg body weight orally.
About 50 % of the test item N-(Morpholinomethyl)triethoxysilane was formed into ethanol by hydrolysis within the gastrointestinal tract of the animals. In order to evaluate the influence of this amount of ethanol on the frequency of micronuclei an additional test group was performed using ethanol formulated in corn oil at a final concentration of 50% of the high dose, i.e. if the high dose was 2000 mg/kg b.w. the ethanol control group was treated with 1000 mg/kg b.w. ethanol.

The animals of all dose groups were examined for acute toxic symptoms at intervals of around 1 h, 2 - 4 h, 6 h, 24 h and 48 h after administration of the test item.
Duration of treatment / exposure:
The animals received the test item, the vehicle, the ethanol control or the positive control substance once.
Frequency of treatment:
The animals received the test item, the vehicle, the ethanol control or the positive control substance once.
Post exposure period:
24/48h
Doses / concentrations
Remarks:
Doses / Concentrations:
500, 1000, 2000 mg/kg b.w.
Basis:
nominal conc.
No. of animals per sex per dose:
Twelve animals, six males and six females, were treated per dose group and sampling time.
Control animals:
yes, concurrent vehicle
other: ethanol control ( 1000 mg/kg b.w. ethanol).
Positive control(s):
Cyclophosphamide
- Justification for choice of positive control(s): historical control data
- Route of administration: oral
- Doses / concentrations: 40 mg/kg b.w.

Examinations

Tissues and cell types examined:
erythrocytes from bone marrow of the femora
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
It is generally recommended to use the maximum tolerated dose or the highest dose that can be formulated and administered reproducibly or 2000 mg/kg as the upper limit for non-toxic test items.
The maximum tolerated dose level is determined to be the dose that causes toxic reactions without having major effects on survival within 48 hours.
The volume to be administered should be compatible with physiological space available.
Three adequately spaced dose levels spaced by a factor of 2 were administered, and samples were collected at the central sampling interval 24 h after treatment.


TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
All concentrations at 24 hours
High dose additionally at 48 h

DETAILS OF SLIDE PREPARATION:
The animals were sacrificed using CO2 followed by bleeding. The femora were removed, the epiphyses were cut off and the marrow was flushed out with foetal calf serum using a syringe. The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 minutes and the supernatant was discarded. A small drop of the re-suspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald (Merck, D-64293 Darmstadt)/Giemsa (Merck, D-64293 Darmstadt). Cover slips were mounted with EUKITT (Kindler, D-79110 Freiburg). At least one slide was made from each bone marrow sample.


METHOD OF ANALYSIS:
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. At least 2000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in polychromatic erythrocytes per 2000 erythrocytes. The analysis was performed with coded slides.
Ten animals (5 males, 5 females) per test group were evaluated as described. The remaining 6th animal of each sex in the respective test group test group is usually evaluated in case an animal dies in its test group spontaneously.
Evaluation criteria:
The study was considered valid as the following criteria are met:
- the negative controls are in the range of our historical control data.
- the positive controls are in the range of our historical control data.
- at least 4 animals per group and sex can be evaluated
- PCE to erythrocyte ratio should not be less than 20 % of the negative control.
A test item is classified as mutagenic if it induces either a dose-related increase or a clear increase in the number of micronucleated polychromatic erythrocytes in a single dose group. Statistical methods (nonparametric Mann-Whitney test (8)) will be used as an aid in evaluating the results. However, the primary point of consideration is the biological relevance of the results.
A test item that fails to produce a biological relevant increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in this system.
Statistics:
Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
The animals treated with 2000 mg/kg b.w. did not express any toxic reactions.

RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay): micronuclei
- Induction of micronuclei (for Micronucleus assay):The percentage of micronucleated PCEs after treatment of the animals with the high dose of the test item was statistically significantly higher than the vehicle control at both preparation intervals. The obtained values (0.110% micronucleated PCEs), however, are well within the historical vehicle control range (up to 0.160%). Thus, the observed statistical significance does not bear a biological relevance. The mean values of micronuclei observed after treatment with the two lower doses N-(Morpholinomethyl)triethoxysilane were near to the value of the vehicle control group. The treatment with 1000 mg/kg b.w. ethanol also gave a value (0.140%), which were significantly higher than the vehicle control value (0.065%). However, also in this case the obtained value was within the historical control range. Thus, the observed effect at the tested dose can not be attributed to a genotoxic effect.
- Appropriateness of dose levels and route: As estimated by a pre-experiment 2000 mg N-(Morpholinomethyl)triethoxysilane per kg b.w. (the maximum guideline-recommended dose) was suitable.
- Ratio of PCE/NCE (for Micronucleus assay): The mean number of polychromatic erythrocytes was not decreased after treatment with the test item as compared to the mean value of PCEs of the vehicle control indicating that N-(Morpholinomethyl)triethoxysilane did not have any cytotoxic properties in the bone marrow.

Any other information on results incl. tables

Table 1: Summary of results of the micronuclei test

test group

dose mg/kg b.w.

sampling time (h)

PCEs with MN(%)

range

of MN

PCE per 2000 erythocytes

vehicle

0

24

0.065

0 - 3

1181

test item

500

24

0.090

0 - 5

1203

test item

1000

24

0.105

0 - 7

1167

test item

2000

24

0.110

1 - 4

1204

ethanol control

1000

24

0.140

1 - 6

1160

positive control

40

24

2.490

25 -68

1133

test item

2000

48

0.110

1 - 4

1121

MN: Micronuclei; PCE Polychromatic erythrocytes

Additional information:

In a separate study the bioavailabilty of N-(Morpholinomethyl)triethoxysilane

was examined. Twelve male and twelve female NMRI mice were treated once orally with 14C- N-(Morpholinomethyl)triethoxysilane at a nominal dose of 2000 mg/kg. The animals were sacrificed after one (three males and three females), after four (three males and three females) and after twentyfour hours (six males and six females). The concentrations of total radioactive residues were determined by Liquid Scintillation Counting (LSC) in blood, plasma, femur, stomach, large intestine, small intestine, GI tract contents, liver and kidney. 24 hours after treatment total radioactivity was additionally determined in faeces and urine.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.
Executive summary:

(N-Morpholinomethyl)triethoxysilan was investigated for its potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrrow of the mouse.

In comparison to the corresponding vehicle controls there was not a bilogically relevant enhancement in the frequency of the deteced micronuclei at any preparation interval after administration of the test item and with any dose level used. The positve control worked as expected. In a separate study the bioavailabilty of (N-Morpholinomethyl)triethoxysilan was examined. Overall significant mean levels of the test item were found in blood and plasma as early as 1 hour after application. This indicates that after oral administration the test item was rapidly absorbed in significant amounts.

Dose and formulation of the radiolabeled test item in this study were comparable to this study. Therefore, an adequate exposure of plasma and bone marrow to (N-Morpholinomethyl)triethoxysilan and/or its metabolites in the micronucleus assay can be assumed. In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test item did not induce micronuclei.