N-[3-(dimethoxymethylsilyl)propyl]ethylenediamine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Information is available from three bacterial mutagenicity studies on N-[3-(dimethoxymethylsilyl)propyl]ethylenediamine (CAS No. 3069-29-2, EC No. 221-336-6). No further information is available for the registered substance, however, reliable data are available for the closely related substance, N-(3-(trimethoxysilyl)propyl)ethylenediamine (CAS No. 1760-24-3, EC No. 217-164-6).

Gene mutation (Bacterial reverse mutation assay / Ames test): negative with and without metabolic activation in Salmonella typhimurium strains TA 98, 100, 102, 1535, 1537 (OECD Test Guideline 471) (LPT, 2002).
Mutagenicity in mammalian cells: read-across from closely related substance N-(3-(trimethoxysilyl)propyl)ethylenediamine (CAS 1760-24-3): negative with and without metabolic activation in Chinese hamster ovary cells (similar to OECD Test Guideline 476) (BRRC, 1988a).

Link to relevant study records

Reference

Reference 1

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

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

Target gene:

Histidine operon

Species / strain / cell type:

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

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

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

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

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

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

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)

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.

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

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

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

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

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.

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

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.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1988-01-13 to 1988-03-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Qualifier:

according to guideline

Guideline:

other: EPA Health Effects Test Guidelines HG-Gene Muta-Somatic Cells (1983)

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

HGPRT (hypoxanthine-guanine phosphoribosyl transferase)

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: Ham's Modified F12 Medium
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: no

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-1254 induced rat liver S9

Test concentrations with justification for top dose:

without metabolic activation: 2.5, 3.0, 3.25, 3.5 and 4.0 mg/ml, with metabolic activation: 2.0, 2.5, 3.0, 3.5, 4.0 and 4.5 mg/ml

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Remarks:

cell culture medium

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Untreated negative controls:

yes

Remarks:

cell culture medium

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-dimethylnitrosamine

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 5 hours (with and without metabolic activation)
- Expression time (cells in growth medium): 9-12 days


SELECTION AGENT (mutation assays): TG selective medium

NUMBER OF REPLICATIONS: Duplicate cultures. Surviving fraction was determined at 18 and 24 hours after removal of chemical using 4 plates per culture and 100 cells per plate; mutant frequency was determined using 5 plates per culture. The test was repeated.



DETERMINATION OF CYTOTOXICITY
- Method: plating efficiency

ACTIVATION: S9 mix contained glucose-6-phosphate and NADP as co-factors, and 5% of S9. 1.0 ml of S9 mix was added to 4 ml of culture medium, giving a final concentration of 1% S9.

Evaluation criteria:

The criteria for interpretation of the test results as a positive or negative response depend upon both the level of statistical significance from the concurrent control and the evidence of a dose-response effect following treatment. When a definite dose-response relationship is not evident, but one or more marginally significant values are obtained, a careful examination of the data from the concurrent positive and negative controls and comparisons to historical control data may be used to evaluate the probable biological significance of the responses.

Statistics:

Box-Cox transformation

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

4 mg/ml with metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

CHO Mutation Assay: with and without metabolic activation

Results on evaluation of plating efficiency and mutant colonies - Experiment 1

	Mutant colonies		Plating efficiency
Test chemical	(Total of 2 plates)		% of Combined Solvent Controls
	- S9	+S9	-S9	+S9
Negative control	0	18	106.3	105.9
Solvent control A	20	2	93.7	102.3
Solvent control B	0	0	106.3	97.8
2.0A	-	6	-	120.5
2.0B	-	13	-	104.4
2.5A	6	35	103.4	107.5
2.5B	1	15	110.0	100.5
3.0A	5	4	104.6	91.0
3.0B	2	0	124.4	109.2
3.25A	5	-	101.1	-
3.25B	5	-	96.0	-
3.5A	0	0	89.4	116.0
3.5B	1	0	103.7	107.7
4.0A	0	-**	114.7	-**
4.0B	5	-**	94.0	-**
Positive control	143	55	115.7	113.2

 

**= Cytotoxic, cultures did not grow

Results on evaluation of plating efficiency and mutant colonies - Experiment 2

Test chemical	Mutant colonies (Total of 2 plates)	Plating efficiency
	+S9
Negative control	13	88.2
Solvent control A	0	104.1
Solvent control B	1	95.8
2.25A	12	118.7
2.25B	0	99.7
2.5A	0	106.8
2.5B	0	111.1
2.75A	0	101.9
2.75B	0	96.6
Positive control	63	92.6

Conclusions:

N-(3-trimethoxysilyl)propyl)ethylenediamine has been tested in a reliable study according to an appropriate US EPA test guideline that is equivalent to OECD 476 and in compliance with GLP. An increase in mutant frequency was observed in one of two duplicate cultures with metabolic activation at one concentration in the initial experiment. No other increase in mutant frequency was observed at any concentration with and without activation (5 hours treatment). The experiment was repeated (with metabolic activation): no increase in mutant frequency was observed. The appropriate solvent and positive controls were included and gave expected results. It is concluded that the test substance is negative for the induction of mutations in CHO cells under the conditions of the test.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

See attachment to Section 13 for justification of read across

Reason / purpose for cross-reference:

read-across source

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

4 mg/ml with metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Micronucleus assay in mouse (ip administration): read-across from closely related substance N-(3-(trimethoxysilyl)propyl)ethylenediamine (CAS 1760-24-3): negative in peripheral lymphocytes (similar to OECD Test Guideline 474) (BRRC, 1988)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1988-03-15 to 1988-04-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA Health Effect T G, EPA Report 560/6-83-001

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

Swiss Webster

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Portage, MI
- Age at study initiation: 5 weeks
- Weight at study initiation: male - 22.5 g - 28.0 g. female - 19.8 g - 21.7 g
- Assigned to test groups randomly: randomised separately by sex
- Fasting period before study: no
- Housing: 5 mice/sex/cage in shoe-box type plastic cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): controlled but not specified
- Humidity (%): controlled but not specified
- Air changes (per hr): not known
- Photoperiod (hrs dark / hrs light): 12 hr light/dark cycle

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: incompatible with water thus corn oil was used
- Concentration of test material in vehicle: 200 mg/kg

Duration of treatment / exposure:

single i p dose

Frequency of treatment:

One treatment only

Post exposure period:

30, 48 and 72 hours

Dose / conc.:

87.5 mg/kg bw/day

Dose / conc.:

175 mg/kg bw/day

Dose / conc.:

280 mg/kg bw/day

No. of animals per sex per dose:

5

Control animals:

yes

Positive control(s):

Triethylenemelamine
- Justification for choice of positive control(s): known to demonstrate the sensitivity and responsiveness of the animals in the definitive test.
- Route of administration: i p
- Doses / concentrations: 0.3 mg/kg bw

Tissues and cell types examined:

Blood was collected by nicking the tail of each animal with a scalpel and slides prepared for each animal per sampling time. Polychromatic erythrocytes (PCE's) were examined.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Determination of the PCE/NCE ratio for the groups of animals with partial mortality was used to evaluate the possibility of bone marrow cytotoxicity from the test chemical. Three dose levels of approximately 80%, 50% and 25% of the LD50 value were evaluated for effects upon the incidence of micronuclei.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): i.p. injection followed by sampling times of 30, 48 and 72 hours

DETAILS OF SLIDE PREPARATION: Slides were stained with Gurr's R-66 Giemsa diluted in phosphate buffer. Slides were coded by animal number only and read blindly.

METHOD OF ANALYSIS: A minimum of 1000 PCE's were examined microscopically for each animal per sample time. The PCE/NCE ratio for approximately 1000 total cells was calculated and recorded. The number of micronucleated PCE/1000 NCE was recorded.

Evaluation criteria:

A test result is considered to be negative if no statistically significant or dose related increases are apparent between the vehicle control and groups of animals treated with Organofunctional Silane A-1120.

Statistics:

Fisher's Exact Test (Sokal and Rohlf, 1981)

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

LD50 354 mg/kg bw and above. PCE/NCE ratio was reduced at highest concentration, 48 h exposure and at all doses, 72 h exposure.

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 125 mg/kg bw - 2000 mg/kg b
- Solubility: Incompatible with water thus corn oil was used
- Clinical signs of toxicity in test animals: All mice dosed at 1000 mg/kg bw and 2000 mg/kg bw died. 500 mg/kg bw was lethal to 4 out of 5 females and all the male mice. One female tested at 250 mg/kg bw died.
- Evidence of cytotoxicity in tissue analyzed: a decrease in the PCE/NCE ratio to 80% of the control value was observed at the 48-hour treatment interval. At 72 hours the PCE/NCE ratios had increased.
- Harvest times: 48 hours


RESULTS OF DEFINITIVE STUDY

- Induction of micronuclei (for Micronucleus assay): negative.
- Ratio of PCE/NCE (for Micronucleus assay): PCE/NCE ratio was slightly decreased at 72 hour treatment interval.
- Appropriateness of dose levels and route: Both dose levels and route were appropriate.
- Statistical evaluation: Analysis of variance indicated no sex-related differences in the incidence of micronuclei. No statistically significant (p = 0.01) or treatment related increases in the numbers of micronuclei were observed at any dose or treatment interval.

Table 3: Summary of micronucleus results

	30 hours				48 hours				72 hours
Treatment groups mg/kg bw	Number of PCE’s with micronuclei per 1000 PCE’s		Mean PCE’s/1000 NCE (SD)		Number of PCE’s with micronuclei per 1000 PCE’s		Mean PCE’s/1000 NCE (SD)		Number of PCE’s with micronuclei per 1000 PCE’s		Mean PCE’s/1000 NCE (SD)
	Male	Female	Group mean	SD	Male	Female	Group mean	SD	Male	Female	Group mean	SD
Vehicle control	4.0 (2.00)	1.6 (0.89)	33.7	5.52	2.4 (2.70)	3.6 (1.14)	36.4	4.24	3.8 (2.05)	3.8 (2.68)	42.9	3.25
87.5	5.0 (2.55)	3.6 (1.52)	31.8	6.79	3.0 (1.73)	2.2 (0.84)	36.0	0.28	6.4 (2.19)	2.8 (1.64)	33.9	2.12
175	2.6 (1.52)	4.0 (1.22)	34.9	8.06	3.2 (1.10)	3.8 (1.64)	37.2	7.92	2.3 (0.96)	5.2 (3.96)	35.5	6.68
280	3.4 (2.19)	3.6 (2.19)	35.4	4.81	3.4 (3.29)	3.0 (1.22)	30.4	3.39	3.8 (1.92)	1.2 (0.84)	27.9	1.27
Positive control	36.2 (16.84)	28.6 (10.31)	27.4	9.05	Not evaluated		Not evaluated		Not evaluated		Not evaluated

Table 4 Summary of micronucleus frequency and statistical differences from controls (Fisher's exact test, one-tailed)

Treatment/dose mg/kg bw	No. PCE observed	PCE with micronuclei	Statistical significance
	30 hour sample
Vehicle control	10 000	28	NS
87.5	10 000	43	0.05>p>0.01*
175	10 000	33	NS
280	10 000	35	NS
Positive control	10 000	324	p<  0.001
	48 hour sample
Vehicle control	10 000	30	NS
87.5	10 000	26	NS
175	10 000	35	NS
280	10 000	32	NS
	72 hour sample
Vehicle control	10 000	38	NS
87.5	10 000	46	NS
175	10 000	35	NS
280	10 000	25	NS

Results from sexes are combined as no sex-related differences were shown.

* not considered of biological significance

Conclusions:

N-(3-(trimethoxysilyl)propyl)ethylenediamine has been tested in a reliable study according to EPA Health Effect T G, Report 560/6-83-001, which is similar to OECD 474, and in compliance with GLP conditions. No treatment related increases in numbers of micronuclei in PCE's of Swiss-Webster mice were observed. Relatively high dose levels of the test compound were tested up to 80% of the LD50 with no indication of significant induction of micronuclei. The PCE/NCE ratio was reduced at the highest concentration, 48 h exposure and at all doses, 72 h exposure, indicating exposure of the target tissue to the test substance. The test compound is considered to be negative for the induction of micronuclei under the conditions of this test.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

See attachment to Section 13 for justification of read-across.

Reason / purpose for cross-reference:

read-across source

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

LD50 354 mg/kg bw and above. PCE/NCE ratio was reduced at highest concentration, 48 h exposure and at all doses, 72 h exposure.

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Information is available from three bacterial mutagenicity studies on N-[3-(dimethoxymethylsilyl)propyl]ethylenediamine. No further information is available for the registered substance, however, reliable data are available for the closely related substance, N-[3-(trimethoxysilyl)propyl]ethylenediamine.

N-[3-(dimethoxymethylsilyl)propyl]ethylenediamine has been tested in a reliable bacterial mutagenicity study conducted according to OECD Test Guideline 471 and in compliance with GLP. 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 (plate incorporation) and in all strains in the pre-incubation assay. Appropriate solvent and positive controls were included and gave expected results (LPT, 2002).

Two additional bacterial mutagenicity studies also gave negative results (Microtest Research Ltd., 1988 and Huntingdon Life Sciences, 1999). The most reliable study was chosen as key study.

In vitro cytogenicity testing is not required as there is an in vivo cytogenicity study is available for the analogue substance.

The analogue substance, N-(3-trimethoxysilyl)propyl)ethylenediamine, has been tested in a reliable study according to an appropriate US EPA test guideline that is equivalent to OECD Test Guideline 476 and in compliance with GLP. An increase in mutant frequency was observed in one of two duplicate cultures with metabolic activation at one concentration in the initial experiment. No other increase in mutant frequency was observed at any concentration with or without metabolic activation (5 hours treatment). The experiment was repeated (with metabolic activation): no increase in mutant frequency was observed. The appropriate solvent and positive controls were included and gave expected results. It is concluded that the test substance is negative for the induction of mutations in CHO cells under the conditions of the test (BRRC, 1988a).

The analogue substance, N-(3-(trimethoxysilyl)propyl)ethylenediamine, has been tested in a reliable in vivo micronucleus study according to EPA Health Effect T G, Report 560/6-83-001, which is similar to OECD Test Guideline 474, and in compliance with GLP conditions. No treatment related increases in numbers of micronuclei in PCE's of Swiss-Webster mice were observed. Relatively high dose levels of the test compound were tested up to 80% of the LD50 with no indication of significant induction of micronuclei. The PCE/NCE ratio was reduced at the highest concentration, 48 h exposure and at all doses, 72 h exposure, indicating exposure of the target tissue to the test substance. The test compound is considered to be negative for the induction of micronuclei under the conditions of this test (BRRC, 1988b).

Justification for classification or non-classification

Based on the available in vitro and in vivo genotoxicity data, N-[3-(dimethoxymethylsilyl)propyl]ethylenediamine is not classified for mutagenicity according to Regulation (EC) No. 1272/2008.