N-[3-(trimethoxysilyl)propylcyclohexylamine]

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 Jul - 24 Nov 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

(2014)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

(2008)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Schwabach, Germany

Type of assay:

other: in vitro mammalian chromosome aberration test (migrated information)

Test material

Test material information

Constituent 1

Reference substance name:

N-[3-(trimethoxysilyl)propylcyclohexylamine]

EC Number:

221-329-8

EC Name:

N-[3-(trimethoxysilyl)propylcyclohexylamine]

Cas Number:

3068-78-8

Molecular formula:

C12H27NO3Si

IUPAC Name:

N-[3-(trimethoxysilyl)propyl]cyclohexanamine

Details on test material:

- Name of test material (as cited in study report): 3-(Cyclohexylamino)propyl trimethoxysilane
- Physical state: yellowish liquid
- Expiration date of the lot/batch: 06 Feb 2017
- Storage condition of test material: at room temperature, protected from light and moisture

Method

Target gene:

Not applicable

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Type and identity of media (culture medium for long-term treatment and post incubation):
- MEM supplemented with 10% (v/v) fetal bovine serum, 100 U/100 µg/ml penicillin/streptomycin solution, 2 mM L-glutamine, 2.5 µg/ml amphotericin and 25 mM HEPES
Type and identity of media (culture medium for short-term treatment and post incubation):
- MEM supplemented with 100 U/100 µg/ml penicillin/streptomycin solution, 2 mM L-glutamine, 2.5 µg/ml amphotericin and 25 mM HEPES

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital/β-naphtholflavone induced rat liver S9-mix

Test concentrations with justification for top dose:

Preliminary experiment:
- 8, 16, 32, 63, 125, 250, 500, 1000, 1500 and 2000 µg/mL (4 h treatment, with and without metabolic activation)

Experiment I:
- 250, 500*, 1000, 1500* and 2000* μg/mL (4 h treatment, without metabolic activation)
- 250, 500*, 1000*, 1500* and 2000 μg/mL (4 h treatment, with metabolic activation)

Experiment II:
- 100, 250, 500*, 1000*, 1500* and 2000 μg/mL (21 h treatment, without metabolic activation)
* cells evaluated for chromosomal aberrations

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: A solubility test was performed with different solvents and vehicles up to the maximum recommended concentration of 2 mg/mL. According to the results of the solubility test the test item was dissolved in DMSO and diluted prior to treatment.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Remarks:

-S9-mix: ethylmethanesulfonate (EMS): 600 µg/mL; +S9-mix: cyclophosphamide (CPA): 1.5 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 and 21 h
- Fixation time (start of exposure up to fixation or harvest of cells): 4 h treatment: 21 h; 21 h treatment: 21 h

SPINDLE INHIBITOR: colcemid (0.2 µg/mL culture medium)

STAIN: Giemsa

NUMBER OF REPLICATIONS: duplicate cultures for each test concentration and negative, solvent and positive control

NUMBER OF CELLS EVALUATED: 150 well-spread metaphases per culture (300 per test concentration)

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; relative increase in cell count (RICC)

OTHER EXAMINATIONS:
- Determination of polyploidy: yes

Evaluation criteria:

There are several criteria for determining a positive result: a clear and dose-related increase in the number of cells with aberrations and a biologically relevant response for at least one of the dose groups, which is higher than the laboratory negative control range. According to the OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results. A statistical evaluation could be used as an aid for interpretation of the results; a statistical evaluation of the results is nor regarded necessary. However, for the interpretation of the data, both biological and optionally evaluated statistical significance should be considered together. A test item is considered to be negative if there is no biologically relevant increase in the percentages of aberrant cells above concurrent control levels, at any dose group. Although most experiments give clearly positive or negative results, in some cases the data set precludes making a definitive judgement about the activity of the test substance.

Statistics:

Statistical significance at the 5% level (p <0.05) was evaluated by the Fischer´s exact test. The p value was used as a limit in judging for significance levels in comparison with the corresponding negative control. Aberrant cells without gaps were only used for the calculation. Gaps are recorded separately and reported but generally not included in the total aberration frequency calculation according to the guideline.

Results and discussion

Test results

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

≥1500 µg/mLwithout metabolic activation (experiment I)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: In experiment I, precipitation of the test item was noted without metabolic activation at a concentration of 2000 µg/mL and with metabolic activation at a concentration of 1500 µg/mL. In experiment II, precipitation of the test item was seen without metabolic activation at a concentration of 1500 µg/mL.

RANGE-FINDING/SCREENING STUDIES
According to the guideline the highest recommended concentration was 2 mg/mL. The test item was dissolved in DMSO. No precipitation of the test item was noted. The highest dose group evaluated in the pre-experiment was 2 mg/mL. The relative mitotic index and the relative increase in cell count (RICC) were used as parameter for toxicity. The concentrations evaluated in the main experiment are based on the results obtained in the pre-experiment.

COMPARISON WITH HISTORICAL CONTROL DATA
In experiment I without metabolic activation the aberration rate of the negative control (3%), the solvent control (2%) and two of three dose groups treated with the test item 3.7% (500 µg/mL) and 3.3% (1500 µg/mL) were within the historical control data of the testing facility (0 - 4%). An increase of aberrant cells was noted at a concentration of 2000 µg/mL (9%). With metabolic activation, the aberration rates of the negative control (2.3%), the solvent control (1.3%) and two of three dose groups treated with the test item 2% (500 µg/mL) and 1% (1000 µg/mL) were within the historical control data of the testing facility (0 - 4.3%). An increase of aberrant cells was noted at a concentration of 1500 µg/mL (11%). The increase of aberrant cells in the highest concentration without and with metabolic activation is regarded as not biologically relevant, due to precipitation in these dose groups. Precipitation might lead to artefactual effects as mentioned in the OECD guideline 473. In experiment II without metabolic activation the aberration rate of the negative control (1.3%), the solvent control (1%) and all dose groups treated with the test item (1.7% at 500, 1000 and 1500 µg/mL) were within the historical control data of the testing facility (0 - 4%). The number of aberrant cells found in the dose groups treated with the test item did not show a biologically relevant increase compared to the corresponding negative control. In addition, no dose-response relationship was observed. Moreover, no biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test item.

ADDITIONAL INFORMATION ON CYTOTOXICITY
No cytotoxic effects of the test item were noted in experiment I with metabolic activation and in experiment II without metabolic activation in all dose groups evaluated (considering relative mitotic index and RICC). In experiment I without metabolic activation, no cytotoxic effects of the test item were noted considering the relative mitotic index. However, considering the RICC, cytotoxic effects were observed at 1500 µg/mL and higher.

ACCEPTABILITY OF THE TEST
The chromosomal aberration assay is considered acceptable if it meets the following criteria: the number of aberration found in the negative control and/or solvent controls falls within the range of historical laboratory control data; concurrent positive controls should induce responses that are comparable with those generated in the historical positive control data base and produce a statistically significant increase compared with the concurrent negative control; the proliferation rate of the solvent control should be similar to the corresponding negative control value.

Any other information on results incl. tables

Table 1: Preliminary experiment - Cytotoxicity data

Dose Group [µg/ml]	Relative Mitotic Index [%]	RICC [%]
Without metabolic activation
Negative control	109	102
Solvent	100	100
8	110	109
16	95	122
32	112	93
63	122	122
125	124	105
250	103	105
500	101	120
1000	106	131
1500	113	129
2000	96	106
With metabolic activation
Negative control	110	123
Solvent	100	100
8	124	101
16	126	95
32	99	123
63	106	105
125	132	94
250	125	89
500	106	93
1000	106	84
1500	131	95
2000	135	85

The mitotic index was determined in 1000 cells/culture of each test group.

RICC: Relative Increase in Cell Count

Table 2: Chromosome aberrations in Chinese hamster V79 cells with and without metabolic activation - Experiment I + II

Test item	Concentration [µg/ml]	Relative Mitotic Index [%]	RICC [%]	Aberrant Cells [%]
				with gaps	without gaps
Exposure period 4 h, fixation time 21 h, without S9-mix
Negative control	0	110	133	5.7	3.0
Solvent	0	100	100	4.0	2.0
EMS	600	105	70	15.7	11.7
Test item	500	90	59	5.7	3.7
	1500	75	50	5.0	3.3
	2000 P	72	67	11.2	9.0
Exposure period 4 h, fixation time 21 h, with S9-mix
Negative control	0	105	93	2.7	2.3
Solvent	0	100	100	2.7	1.3
CPA	1.5	112	113	11.7	7.0
Test item	500	89	91	3.3	2.0
	1000	87	86	2.7	1.0
	1500 P	94	81	13.7	11.0
Exposure period 21 h, fixation time 21 h, without S9-mix
Negative control	0	86	96	2.3	1.3
Solvent	0	100	100	2.7	1.0
EMS	600	74	62	21.3	20.9
Test item	500	110	94	3.0	1.7
	1000	99	74	3.0	1.7
	1500 P	92	85	3.7	1.7

P: precipitation

CPA: cyclophosphamide

EMS: ethylmethanesulfonate

Relative Mitotic Index: the relative mitotic index is related to the solvent controls and determined in 1000 cells/culture of each test group

RICC: relative increase in cell count, calculated by the increase in cell number of the test groups compared to the control groups.

Applicant's summary and conclusion

Conclusions:

In conclusion, it can be stated that during the described in vitro chromosome aberration test performed according to OECD guideline 473 and under the experimental conditions reported, that the test item did only induce structural chromosome aberrations in the V79 Chinese hamster cell line in the highest concentration with and without metabolic activation after a short term treatment of 4 h. However, these increased aberration rates are considered as not biologically relevant, due to precipitation. Therefore, the test item is considered to be non-clastogenic in this chromosome aberration test.