N-[3-(trimethoxysilyl)propyl-1,3-Benzenedimethanamine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro:
Gene mutation (Bacterial reverse mutation assay / Ames test): negative with and without metabolic activation in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 (equivalent to OECD 471) (BML Inc., 1997).

Cytogenicity in mammalian cells: negative with and without metabolic activation in Chinese hamster lung cells (OECD 473) (JBS Inc., 2007).

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:

12/08/1997 to 02/10/1997

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

The study was conducted according to a protocol that is similar to the appropriate OECD test guideline, with acceptable restrictions. The restrictions were that only duplicate plates were used.

Qualifier:

according to guideline

Guideline:

other: "Standards for Mutagenicity Tests using Microorganisms" (Notification No. 77, Ministry of Labour, Japan, September 1, 1988

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Only duplicate plates tested

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine (Salmonella strains) and tryptophan (E. coli strain)

Base-pair substitution type: Salmonella typhimurium TA100, TA1535, and Escherichia coli WP2 uvrA.
Frame-shift type: Salmonella typhimurium TA98, TA1537.

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital and 5,6-benzoflavene induced rat

Test concentrations with justification for top dose:

1.2, 4.9, 20, 78, 313, 1250 and 5000 µg/plate

Vehicle / solvent:

Test substance
- Vehicle(s)/solvent(s) used: DMSO.
- Justification for choice of solvent/vehicle: None given.

Positive controls
- AF-2, ICR-191, 2AA and B[a]P: DMSO.
- sodium azide: distilled water.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2)

Remarks:

-MA: TA 100, WP2 uvrA - 0.01 µg/plate; TA 98 - 0.1 µg/plate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

-MA: TA 1535 - 0.5 µg/plate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)aminopropylamino]acridine·2HCl (ICR-191)

Remarks:

-MA: TA 1537 - 1.0 µg/plate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

+MA: TA 1535 - 2.0 µg/plate; WP2 uvrA - 10.0 µg/plate

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

yes

Positive controls:

no

Positive control substance:

benzo(a)pyrene

Remarks:

+MA: TA 100, TA 98, TA 1537 - 5.0 µg/plate

Details on test system and experimental conditions:

ACTIVATION:
Composition of S9 mix (per 1 ml):
Water: 0.9 mL
S9: 0.1 mL
MgCl2: 8.0 µmol
KCl: 33.0 µmol
G-6-P: 5.0 µmol
NADPH: 4.0 µmol
NADH: 4.0 µmol
Na-phosphate buffer (pH 7.4): 100.0 µmol

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 minutes at 37°C
- Exposure duration: 48 hours at 37°C

SELECTION AGENT (mutation assays): minimal glucose agar

NUMBER OF REPLICATIONS: duplicate plates. An initial dose range-finding study was followed by two further independent experiments.

DETERMINATION OF CYTOTOXICITY: reduction in number of revertant colonies

Evaluation criteria:

If the number of revertant colonies on the test plates increased significantly in comparison with that on the control plates (based on twice as many as that of the negative control), and dose-response and reproducibility were also observed, the test substance was judged to be positive.

Statistics:

No statistical analysis done.

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING STUDY: Growth inhibition was determined for seven concentrations of test substance: 1.2, 4.9, 20, 78, 313, 1250 and 5000 µg/plate.

Growth inhibition was observed as follows:
≥4.9 µg/plate for TA1537 without S9.
≥20 µg/plate for TA100, TA1535 and TA98 without S9.
≥78 µg/plate for E.coli WP2 uvrA without metabolic activation.

Hence, the highest concentrations of test substance used in the main test were as follows:

S. typhimurium TA1537 without S9: 4.9 µg/plate.
S. typhimurium TA100, TA1535, TA98 without S9: 20 µg/plate.
E. coli WP2 uvrA without S9: 78g µg/plate.
All S. typhimurium strains with S9: 313 µg/plate.
E. coli WP2 uvrA with S9: 1250 µg/plate.

Remarks on result:

other: all strains/cell types tested

Conclusions:

The submission substance has been tested for mutagenicity to bacteria in a study conducted according to a protocol that is similar to OECD 471. No evidence for a test substance induced increase in the number of revertants was observed when tested up to cytotoxic concentrations with and without metabolic activation in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 using the preincubation method in the initial and the repeat experiment. Appropriate positive and solvent controls were included and gave expected results. It is concluded that the test substance is negative for mutagenicity under the conditions of the test.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05/04/2007 to 12/07/2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

1997

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

N/A

Species / strain / cell type:

other: CHO/IU

Details on mammalian cell type (if applicable):

- Type and identity of media: liquid medium of Eagle's MEM supplemented with 10% of dehydrated HPLC grade dimethylsulfoxide
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

S9

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: None given.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

mitomycin C

Details on test system and experimental conditions:

ACTIVATION:
S9 mix included 30% S9 fraction, MgCl2, KCl, glucose-6-phosphate and NADP. The final concentration of S9 was 5%

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 6 h with and without metabolic activation; 24 hours without metabolic activation
- Expression time (cells in growth medium): 18 hours (following 6 h treatment)
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hours

SPINDLE INHIBITOR (cytogenetic assays): colcemid
STAIN (for cytogenetic assays): Giesma

NUMBER OF REPLICATIONS: The initial pulse treatment with and without metabolic activation was followed by 24 and 28 hour exposure without metabolic activation. Duplicate cultures do not appear to have been used.

NUMBER OF CELLS EVALUATED: 100 metaphases per slide (200 at each dose level) evaluated for chromosome aberrations;

DETERMINATION OF CYTOTOXICITY
- Method: other: cell viability

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Evaluation criteria:

The test substance was judged positive for clastogenicity when a clear dose-related increase in the frequency of the aberrant cells and 10% or more increase in the frequency of the aberrant cells were both observed.

Statistics:

No statistical analysis was done.

Key result

Species / strain:

mammalian cell line, other: Chinese hamster lung cell line CHL/IU.

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

50% viability occurred between 0.74 and 1.11 mg/ml -MA and 1.11 and 1.67 mg/mg +MA, 6 hour treatment

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH:
- Effects of osmolality:
- Evaporation from medium:
- Water solubility:
- Precipitation: precipitation occurred but did not interfere.
- Other confounding effects: pH was raised, but not to a level which caused an increase in chromosome aberrations

RANGE-FINDING/SCREENING STUDIES:

COMPARISON WITH HISTORICAL CONTROL DATA:

ADDITIONAL INFORMATION ON CYTOTOXICITY:

Table 1: Results of chromosome aberration test - Treatment condition 6-18 with and without metabolic activation

Dose (mg/ml)	Number of structural aberrations		Viability of cells (%)		Number of numerical aberrations
	-MA	+MA	-MA	+MA	-MA	+MA
Vehicle control	5	1	100	100	0	1
0.33	4	-	93	-	4	-
0.49	4	2	81	97	2	3
0.74	4	4	82	86	3	0
1.11	*	3	35	88	*	3
1.67	*	*	19	30	*	*
2.50	*	*	0	0	*	*
Positive control	39	117	92	74	5	0

*TOX: metaphase cells were not observed

Table 2: Results of chromosome aberration test - Treatment condition 24-0 without metabolic activation

Dose (mg/ml)	Number of structural aberrations	Viability of cells (%)	Number of numerical aberrations
Vehicle control	4	100	1
0.33	5	93	3
0.49	5	85	1
0.74	3	71	0
1.11	*	31	*
1.67	*	0	*
Positive control	28	99	2

*TOX: metaphase cells were not observed

Conclusions:

The submission substance has been tested in an in vitro cytogenicity assay conducted in accordance with OECD 473 (1997) and in compliance with GLP. No test substance induced increase in the frequency of structural or numerical aberrations was observed when the substance was tested in the presence and absence of metabolic activation up to cytotoxic concentrations in Chinese hamster lung cells. Appropriate positive and solvent controls were included and gave expected results. It is concluded that the test substance is negative for the induction of chromosome aberrations under the conditions of the test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The submission substance has been tested for mutagenicity to bacteria in a study conducted according to a protocol that is similar to OECD 471 (BML Inc., 1997). No evidence for a test substance induced increase in the number of revertants was observed when tested up to cytotoxic concentrations with and without metabolic activation in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 using the preincubation method in the initial and the repeat experiment. Appropriate positive and solvent controls were included and gave expected results. It is concluded that the test substance is negative for mutagenicity under the conditions of the test.

The submission substance has been tested in an in vitro cytogenicity assay conducted in accordance with OECD 473 (1997) and in compliance with GLP (JBS Inc., 2007). No test substance induced increase in the frequency of structural or numerical aberrations was observed when the substance was tested in the presence and absence of metabolic activation up to cytotoxic concentrations in Chinese hamster lung cells. Appropriate positive and solvent controls were included and gave expected results. It is concluded that the test substance is negative for the induction of chromosome aberrations under the conditions of the test.

Justification for classification or non-classification

Based on the available in vitro genotoxicity data, the submission substance does not require classification for mutagenicity according to Regulation (EC) No 1272/2008.