N-[3-(trimethoxysilyl)propyl]butylamine

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:

29 March 2000 to 19 December 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

(1998)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(2000)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

HESSISCHES MINISTERIUM FÜR UMWELT, ENREGIE, JUGEND, FAMILIE UND GESUNDHEIT, Wiesbaden, Germany

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

157923-74-5

EC Number:

605-117-6

Cas Number:

157923-74-5

IUPAC Name:

157923-74-5

Details on test material:

- Name of test material (as cited in study report): Y-11637
- Physical state: colourless to pale yellow liquid
- Lot/batch No.: 3198-35
- Stability in solvent: not indicated by the sponsor
- Storage condition of test material: room temperature, light protected, moisture protected, stored in the exsiccator under nitrogen
- Expiration date: 28 February 2002

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:
complete medium (for maintainance and exposure without metabolic activation): minimal essential medium, supplemented with 10% foetal calf serum
serum free medium (for exposure with metabolic activation): minimal essential medium
- Properly maintained: yes

Metabolic activation:

with and without

Metabolic activation system:

S9 liver microsomal fraction from male rats

Test concentrations with justification for top dose:

- Range-Finder (cytotoxicity testing): 19.6, 39.1, 78.1, 156.3, 312.5, 625, 1250, and 2500 ml/ml
- Main study (Experiment I, 18 h preparation period and 4 h exposure; -S9): 156.3, 312.5, 625, 1250, 2500, and 5000 nl/ml
- Main study (Experiment II, 18 h preparation period and 18 h exposure; -S9): 312.5, 625, 1250, 2500, 3750, and 5000 nl/ml
- Main study (Experiment II, 28 h preparation period and 28 h exposure; -S9): 1250, 2500, 3750, and 5000 nl/ml
- Main study (Experiment I, 18 h preparation period and 4 h exposure; +S9): 156.3, 312.5, 625, 1250, 2500, and 5000 nl/ml
- Main study (Experiment II, 28 h preparation period and 4 h exposure; +S9): 312.5, 625, 1250, 2500, 3750, and 5000 nl/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (E. MERCK, Darmstadt, Germany; purity: 99.5%); the top concentration was applied undiluted

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation system; 600 µg/ml=4.8 mM (continous exposure) and 1000 µg/ml=8.0 mM (4 h exposure)

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation system; 0.7 µg/ml=2.5 µM (4 h exposure, 18 h preparation interval) and 1.0 µg/ml=3.5 µM (4 h exposure, 28 h preparation interval)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4, 18, and 28 h
- Expression time (cells in growth medium): 14 and 24 h
- Fixation time (start of exposure up to fixation or harvest of cells):

SPINDLE INHIBITOR (cytogenetic assays): Colcemid (0.2 µg/ml culture medium) for 2 h
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2 cultures in 2 independent experiments

NUMBER OF CELLS EVALUATED: 100/culture

DETERMINATION OF CYTOTOXICITY
- Ranger-finder
Method: growth inhibition and and evaluation of cell morphology after treatment with the test item for 4 and 24 h
- Main study
Method: (1) Additional samples treated with the test item in parallel to the samples for the cytogenicity testing, but without addition of Colcemid, were stained and microscopically evaluated for the cell number. The toxicity is given as reduction of % cells as compared to the solvent control. (2) Mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes

Evaluation criteria:

The chromosome aberration assay is considered acceptable if it meets the following criteria:
- The number of structural aberrations found in the negative and/or solvent controls falls within the range of the historical laboratory control data: 0.0%-4.0%
- The positive control substances should produce significant increases in the number of cells with structural chromosome aberrations, which are within the range of the laboratories historical control data: EMS (600 µg/ml) 9.0-39.0; CPA (0.47-0.93 µg/ml) 7.5-49.5

A test item is classified as non-clastogenic if:
- the number of induced structural chromosome aberrations in all evaluated dose groups are in the range of the historical control data and/or
- no significant increase in the number of structural chromosome aberrations is observed.
A test ietm is classified as clastogenic if:
- the number of induced structural chromosome aberrations are not in the range of the historical control data and
- either a concentration-related or a significant increase in the number of structural chromosome aberrations is observed.

Statistics:

Statistical significance was confirmed by means of Fischer's exact test (p<0.05).

Results and discussion

Test results

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: at treatment concentrations of 1250 and 2500 µg/ml the pH in culture medium containing the test item was > 7.5 and therefore had to be adjusted to about 7.4 with 1 N HCl
- Effects of osmolality: none (solvent control 397 mOsm versus 381 mOsm at 2500 ml/ml in the main study, experiment I)
- Precipitation: none observed up to the limit concentration

RANGE-FINDING/SCREENING STUDIES:
Using reduced cell numbers as an indicator for toxicity in the pre-test, no strong toxic effects were observed after treatment with the test item.

COMPARISON WITH HISTORICAL CONTROL DATA:

ADDITIONAL INFORMATION ON CYTOTOXICITY:

Any other information on results incl. tables

Tab. 2: Cytotoxicity of the test item to cultures of Chinese hamster cell line V79

Concentration [nl/ml]	- S9 mix, 4 h exposure		+ S9 mix, 4 h exposure		- S9 mix, 24 h exposure
	Number of cells	% of solvent control	Number of cells	% of solvent control	Number of cells	% of solvent control
Solvent control	491	100	372	100	336	100
19.6	445	91	381	102	461	137
39.1	269	55	430	116	504	150
78.1	378	77	280	75	465	138
156.3	326	66	293	79	389	16
312.5	474	97	324	87	530	158
625.0	389	79	259	70	367	109
1250.0	361	73	287	77	461	137
2500.0	258	53	220	59	412	123

Tab. 3: Experiment I: Number of polyploid cells and mitotic index: preparation interval 18 h with and without S9 mix

Treatment group	Concentration per ml	S9 mix	Exposure period/Recovery	Polyploidy cells *				Mitotic indices **				Aberrant cells
				Culture		total	%	absolute		mean	% ***	incl. gaps	excl. gaps
				1	2			1	2
Negative control		-	4/14 h	29	26	55	5.5	14.2	12.1	13.2	100.0	0	0
Solvent control #	0.5%	-	4/14 h	18	11	29	2.9	14.5	13.6	14.1	100.0	1	0.5
Positive control ##	1000 µg	-	4/14 h	16	12	28	2.8	9.5	10.7	10.1	76.8	13.5	13.5
Test item	1250 nl	-	4/14 h	18	27	45	4.5	12.9	12.4	12.7	90.0	1.5	1.5
	2500 nl	-	4/14 h	16	10	26	2.6	15.3	14.9	15.1	107.5	2.5	1.5
	5000 nl	-	4/14 h	14	13	27	2.7	14.3	15.0	14.7	104.3	1.0	0.5
Negative control		+	4/14 h	11	34	45	4.5	13.1	13.2	13.2	100.0	1.5	0.5
Solvent control #	0.5%	+	4/14 h	2	7	9	0.9	13.0	12.3	12.7	100.0	0	0
Positive control ###	0.7 µg	+	4/14 h	18	2	20	2.0	10.1	11.9	11.0	83.7	14.5	14.0
Test item	1250 nl	+	4/14 h	3	31	34	3.4	19.2	17.0	18.1	143.1	2.5	2.5
	2500 nl	+	4/14 h	6	22	28	2.8	19.0	18.0	18.5	146.2	7.5	7.5
	5000 nl	+	4/14 h	7	5	12	1.2	17.3	16.9	17.1	135.2	3.5	3.0

Tab. 4: Experiment II: Number of polyploid cells and mitotic index; preparation interval 18 and 28 h without S9 mix ; preparation interval 28 h with S9 mix

Treatment group	Concentration per ml	S9 mix	Exposure period/Recovery	Polyploidy cells *				Mitotic indices **				% Aberrant cells
				Culture		total	%	absolute		mean	% ***	incl. gaps	excl. gaps
				1	2			1	2
Negative control		-	18/- h	8	15	23	2.3	9.7	10.0	9.9	100.0	1.0	0.5
Solvent control #	0.5%	-	18/- h	14	11	25	2.5	10.6	11.5	11.1	100.0	1.0	0.5
Positive control ##	600 µg	-	18/- h	11	12	23	2.3	5.4	6.7	6.1	61.4	18.5	18.5
Test item	1250 nl	-	18/- h	5	9	14	1.4	12.5	18.7	15.6	141.2	0.5	0.5
	2500 nl	-	18/- h	8	15	23	2.3	12.8	11.7	12.3	110.9	1.0	0.5
	5000 nl	-	18/- h	7	4	11	1.1	9.0	8.7	8.9	80.1	1.5	1.5
Negative control		-	28/- h	12	14	26	2.6	19.0	19.1	19.1	100.0	0.5	0.5
Solvent control #	0.5%	-	28/- h	6	14	20	2.0	14.2	16.2	15.2	100.0	0.5	0.5
Positive control ##	600 µg	-	28/- h	77	6	13	1.3	12.7	13.9	13.3	69.8	19.5	19.5
Test item	5000 µl	-	28/- h	15	14	29	2.9	14.0	18.1	16.1	105.6	1.0	1.0
Negative control		+	4/24 h	4	10	14	1.4	10.0	12.1	11.1	100.0	0	0
Solvent control #	0.5%	+	4/24 h	12	11	23	2.3	15.2	11.8	13.5	100.0	0.5	0.5
Positive control ###	1 µg	+	4/24 h	10	8	18	1.8	12.4	12.2	12.3	111.3	11.0	10.5
Test item	1250 nl	+	4/24 h	7	7	14	1.4	11.7	14.8	13.3	98.1	3.5	1.5
	2500 nl	+	4/24 h	8	6	14	1.4	16.8	19.9	18.4	135.9	2.0	1.5
	5000 nl	+	4/24 h	6	9	15	1.5	18.2	18.3	18.3	135.2	2.0	2.0

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative

In a chromosome aberration assay according to OECD 473 and GLP, no clastogenic effect was observed for the test substance tested up to the top dose of 5000 µg/ml in any of the independent experiments without and with metabolic activation. The test substance is not clastogenic under the applied conditions.