N-[3-(dimethoxymethylsilyl)propyl]ethylenediamine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2002-04-10 to 2002-06-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

N-[3-(dimethoxymethylsilyl)propyl]ethylenediamine

EC Number:

221-336-6

EC Name:

N-[3-(dimethoxymethylsilyl)propyl]ethylenediamine

Cas Number:

3069-29-2

Molecular formula:

C8H22N2O2Si

IUPAC Name:

N-[3-[dimethoxy(methyl)silyl]propyl]ethane-1,2-diamine

Method

Target gene:

Histidine operon

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 102

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9

Test concentrations with justification for top dose:

31.6, 100, 316, 1000 and 3160 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Ethylene glycol dimethylether

- Justification for choice of solvent/vehicle: Solubility properties and relative non-toxicity to bacteria

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA 1535, TA 100 (without metabolic activation): 10 µg/plate

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

TA 98 (without metabolic activation): 10 µg/plate

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA 1537 (without metabolic activation): 100 µg/plate

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

TA 102 (without metabolic activation): 1300 µg/plate

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-anthracene amide 2 µg/plate

Remarks:

TA 98, TA 102, TA 1537 (with metabolic activation)

Controls 6

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

TA 100, TA 1535 (with metabolic activation): 1500 µg/plate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 60 minutes
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: 3 plates for each test concentration x 2

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth


METHOD OF APPLICATION in agar (plate incorporation)

ACTIVATION: S9 mix contained glucose-6-phosphate and NADP as co-factors. 0.5 ml S9 mix was added to 2 ml top agar, 0.1 ml test solution, 0.1 ml salmonella cell suspension giving a final concentration of 0.9% S9.

DURATION
- Preincubation period: 1 hour (second experiment)
- Exposure duration: 48 hours



NUMBER OF REPLICATIONS: 3 replicates per concentration, experiment repeated


DETERMINATION OF CYTOTOXICITY
- Method: other: condition of background lawn

Evaluation criteria:

A result is positive if the number of revertants is significantly increased compared with the solvent control to at least 2-fold of the solvent control for TA 98, TA 100 and TA 102 and 3-fold of the solvent control for TA 1535 and TA 1537 in both experiments.

Positive results have to be reproducible and the histidine independence of the revertants has to be confirmed by streaking on histidine-free agar plates.

Cytotoxicity is defined as a reduction in the number of colonies by >50% compared with the solvent control and/or a sparse background lawn.

Statistics:

MANN and WHITNEY and Spearman’s rank.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

background lawn reduced in preincubation experiment at 100 µg/plate without metabolic activation and at 316 µg/plate with metabolic activation. Top dose limited to 3160 µg/plate based on cytotoxicity observed in preliminary test test.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

background lawn reduced in preincubation experiment at 316 µg/plate without metabolic activation and at 1000 µg/plate with metabolic activation. Top dose limited to 3160 µg/plate based on cytotoxicity observed in preliminary test.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

background lawn reduced in preincubation experiment at 3160 µg/plate without metabolic activation and at 316 µg/plate with metabolic activation. Top dose limited to 3160 µg/plate based on cytoxicity observed in preliminary test.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

background lawn reduced at 3160 µg/plate in plate incorporation assay and at 316 µg/plate in preincubation experiment

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

background lawn reduced in preincubation experiment at 316 µg/plate without metabolic activation and at 31.6 µg/plate with metabolic activation. Top dose limited to 3160 µg/plate based on cytotoxicity observed in preliminary test.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

COMPARISON WITH HISTORICAL CONTROL DATA: Controls were within range of historical controls.

Cytotoxicity: In the plate incorporation test slight cytotoxicity was noted in the test substance in TA 100, with and without metabolic activation.

In the preincubation test without metabolic activation pronounced cytotoxicity was noted at 3160 µg/plate in TA98, from 316 µg/plate to 3160 µg/plate in TA 100, TA 102 and TA 1537, and from 100 µg/plate up to 3160 µg/plate in TA 1535.

With metabolic activation, pronounced cytotoxicity was noted at concentrations 316 µg/plate – 3160 µg/plate in TA 98 and TA 1535, at 1000 µg/plate and 3160 µg/plate in TA 100 and TA 1537 and from 100 µg/plate – 3160 µg/plate in TA 102.

Any other information on results incl. tables

Table 2: Dose range-finding study Number of revertants per plate (2 plates)

	TA100
Conc. (µg/plate)	Plate 1	Plate 2	Cytotoxic (yes/no)
0*	145	138	No
0.316	145	141	No
1	128	148	No
3.16	145	135	No
10	174	139	No
31.6	137	141	No
100	148	146	No
316	163	140	No
1000	156	150	No
3160	0	0	Yes
5000	0	0	Yes

*solvent control with ethylene glycol dimethyl ether

Table 3: Experiment 1 Plate incorporation Number of revertants per plate (mean of 3 plates)

	TA98			TA100			TA102
Conc. (µg/plate)	— MA	+ MA	Cytotoxic (yes/no)	— MA	+ MA	Cytotoxic (yes/no)	— MA	+ MA	Cytotoxic (yes/no)
0*	22.7	27.7	No	133.3	138.7	No	264.7	268	No
31.6	25	21.7	No	117	156	No	296.3	285.7	No
100	23.3	30.7	No	123.7	136.7	No	268	269.7	No
316	28.7	33.3	No	110.3	141.7	No	267	274.7	No
1000	26.3	29	No	125	124.3	No	275	264	No
3160	25	27.3	No	114.7	150.3	Yes**	249	244	No
Positive control	1026.3	1066	No	1161	1175.3	No	1191	1170.7	No

*solvent control with ethylene glycol dimethyl ether

** scarce background bacterial lawn

Table 3: Experiment 1 Plate incorporation Number of revertants per plate (mean of 3 plates)

	TA1535			TA1537
Conc. (µg/plate)	— MA	+ MA	Cytotoxic (yes/no)	— MA	+ MA	Cytotoxic (yes/no)
0*	16.3	411.7	No	11.3	13.3	No
31.6	14	18	No	12	14.3	No
100	15.3	21.7	No	13.7	17.3	No
316	17.3	22.7	No	12.7	16.3	No
1000	17.3	19.7	No	14.7	12.3	No
3160	17.7	18.7	No	14.7	11.7	No
Positive control	401	411.7	No	974.3	945.7	No

*solvent control with ethylene glycol dimethyl ether

Table 4: Experiment 2 Preincubation Number of revertants per plate (mean of 3 plates)

	TA98			TA100			TA102
Conc. (µg/plate)	— MA	+ MA	Cytotoxic (yes/no)	— MA	+ MA	Cytotoxic (yes/no)	— MA	+ MA	Cytotoxic (yes/no)
0*	42.7	38.7	No	128.3	155.7	No	291.7	287.3	No
3.16	27.3	32	No	119	147.3	No	254.7	300	No
10	29	34.3	No	128.7	151	No	258.3	277.3	No
31.6	34	30.3	No	120.7	140.7	No	252.7	290.3	No
100	22.7	27	No	126	153.7	No	257	0	Yes
316	24.7	0	Yes	0	170.7	Yes	0	0	Yes
1000	24.3	0	Yes	0	0	Yes	0	0	Yes
3160	0	0	Yes	0	0	Yes	0	0	Yes
Positive control	983.3	1085.3	No	1471	1443.3	No	1488.3	1445.3	No

*solvent control with ethylene glycol dimethyl ether

Table 4: Experiment 2 Preincubation Number of revertants per plate (mean of 3 plates)

	TA1535			TA1537
Conc. (µg/plate)	— MA	+ MA	Cytotoxic (yes/no)	— MA	+ MA	Cytotoxic (yes/no)
0*	15.3	15.3	No	9.7	14	No
3.16	13	17	No	8.3	9	No
10	13.3	15	No	8	11.7	No
31.6	13	16.3	No	8.7	10.7	No
100	0	13	Yes	9	10.7	No
316	0	0	Yes	0	10.3	Yes
1000	0	0	Yes	0	0	Yes
3160	0	0	Yes	0	0	Yes
Positive control	684.3	680.3	No	529.3	540.3	No

*solvent control with ethylene glycol dimethyl ether

Applicant's summary and conclusion

Conclusions:

N-[3-(dimethoxymethylsilyl)propyl]ethylenediamine has been tested in a reliable bacterial mutagenicity study conducted according to OECD 471, and in compliance with GLP conditions. No increase in revertant frequency was observed with or without metabolic activation when the substance was tested up to a concentration of 3160 µg/plate in Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 in either the initial plate incorporation assay or the repeat experiment using the pre-incubation method. Cytotoxicity was observed at the highest concentration tested in strain TA 100 in the plate incorporation test, and in all strains in the pre-incubation assay. Appropriate solvent and positive controls were included and gave expected results. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test.