1,3-bis(trimethylsilyl)urea

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:

19 Jun - 17 Jul 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted in 1997

Deviations:

no

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted in 2020

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

adopted in 2008

GLP compliance:

yes (incl. QA statement)

Remarks:

Hessisches Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

1,3-bis(trimethylsilyl)urea

EC Number:

242-177-9

EC Name:

1,3-bis(trimethylsilyl)urea

Cas Number:

18297-63-7

Molecular formula:

C7H20N2OSi2

IUPAC Name:

1,3-bis(trimethylsilyl)urea

Method

Target gene:

His operon (S. typhimurium strains) and trp operon (E. coli strain)

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system: Cofactor supplemented post-mitochondrial fraction (S9 mix).
- method of preparation of S9 mix : S9 mix was prepared from the livers of rats that were induced with phenobarbital / beta-naphthoflavone.
- quality controls of S9: The enzymatic activity of each S9 batch was routinely tested for its capability to activate the known mutagens benzo[a]pyrene and 2-aminoanthracene in the Ames test.

Test concentrations with justification for top dose:

Pre-experiment / Experiment 1: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate
Experiment 2: 33, 100, 333, 1000, 2500 and 5000 µg/plate
Justification for top dose: In the pre-experiment the test item concentration range was 3 - 5000 µg/plate. As no cytotoxicity was observed, 5000 µg/plate was selected as highest dose and the pre-experiment were designated as experiment 1.

Vehicle / solvent:

- Vehicle/solvent used: DMSO

- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine (4-NOPD), -S9, 10 µg/plate in DMSO for TA98 and 50 µg/plate for TA1537; 2-aminoanthracene (2-AA), +S9, 2.5 µg/plate in DMSO for TA98, TA100, TA1535 and TA1537 and 10.0 µg/plate for WP2uvrA

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicates
- Number of independent experiments: 2

METHOD OF APPLICATION: in agar (plate incorporation, Experiment 1) and pre-incubation (Experiment 2)

TREATMENT AND HARVEST SCHEDULE:
- Pre-incubation period: 60 min
- Exposure duration: 48 h

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Toxicity of the test item results in a reduction in the number of spontaneous revertants (below a factor of 0.5) or a clearing of the bacterial background lawn.

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants of two-fold or above (strains TA98, TA100, and WP2uvrA) or three-fold or above (strains TA1535 and TA1537) the spontaneous mutation rate of the corresponding solvent control is observed. A dose dependent increase is considered biologically relevant if the threshold is reached or exceeded at more than one concentration.
An increase of revertant colonies equal or above the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

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

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

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

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

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

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

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

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

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

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: There was no precipitation of the test item observed in the overlay agar at any concentration, neither with nor without S9 mix.

CYTOTOXICITY:
There was no cytotoxicity observed up to and including the highest concentration of 5000 µg/plate in all strains, with and without metabolic activation.

RANGE-FINDING/SCREENING STUDIES: A pre-experiment was performed in S. typhimurium strains TA98, TA100, TA1535 and TA1537 and E. coli strain WP2uvrA according to the plate-incorporation method. Test item concentrations ranging from 3 - 5000 µg/plate were tested in all strains in the presence and absence of metabolic acitivation. There was no cytotoxicity observed for any strain, neither in the presence nor absence of S9 mix. As all acceptability criteria were met, the pre-experiment was reported as experiment 1 of the main mutation assay.

STUDY RESULTS : Please refer to Tables No. 1 and 2 under "Any other information on results incl. tables".

HISTORICAL CONTROL DATA: Please refer to Table No. 3 under "Any other information on results incl. tables".

Any other information on results incl. tables

Table 1: Results of the pre-experiment / Experiment 1 (plate incorporation test)

Metabolic activation	Compound	Concentration (µg/plate)	TA1535	TA1537	TA98	TA100	WP2uvrA
Without S9 mix	DMSO	/	13 ± 4	14 ± 5	31 ± 3	111 ± 16	54 ± 10
	Untreated	/	12 ± 1	13 ± 2	24 ± 8	114 ± 6	57 ± 3
	Test item	3	13 ± 2	15 ± 6	25 ± 3	104 ± 18	54 ± 6
		10	15 ± 6	18 ± 3	26 ± 5	123 ± 9	62 ± 8
		33	15 ± 6	16 ± 6	30 ± 3	105 ± 12	51 ± 6
		100	12 ± 5	16 ± 5	27 ± 5	119 ± 6	54 ± 9
		333	14 ± 5	14 ± 3	24 ± 7	107 ± 11	59 ± 12
		1000	14 ± 4	15 ± 4	23 ± 3	106 ± 10	55 ± 10
		2500	11 ± 2	16 ± 3	27 ± 5	105 ± 23	56 ± 5
		5000	11 ± 3	11 ± 1	30 ± 5	99 ± 17	49 ± 2
	NaN3	10	1112 ± 103	/	/	1574 ± 127	/
	4-NOPD	10	/	/	341 ± 37	/	/
	4-NOPD	50	/	76 ± 6	/	/	/
	MMS	2.0 µL/plate	/	/	/	/	930 ± 66
With S9 mix	DMSO	/	16 ± 2	18 ± 6	45 ± 8	120 ± 2	66 ± 12
	Untreated	/	16 ± 4	19 ± 3	39 ± 3	113 ± 27	60 ± 7
	Test item	3	12 ± 4	13 ± 3	36 ± 8	99 ± 18	63 ± 1
		10	14 ± 3	17 ± 6	40 ± 5	118 ± 8	61 ± 8
		33	14 ± 4	19 ± 2	40 ± 9	117 ± 4	68 ± 15
		100	11 ± 4	19 ± 6	39 ± 3	111 ± 12	63 ± 8
		333	11 ± 5	18 ± 2	34 ± 5	104 ± 6	58 ± 5
		1000	14 ± 2	17 ± 5	35 ± 7	108 ± 8	67 ± 8
		2500	13 ± 1	13 ± 4	41 ± 9	91 ± 5	57 ± 14
		5000	11 ± 1	14 ± 4	36 ± 4	92 ± 16	60 ± 2
	2-AA	2.5	315 ± 25	509 ± 16	3858 ± 202	3870 ± 271	/
	2-AA	10	/	/	/	/	391 ± 35
NaN3 sodium azide; 2-AA 2-aminoanthracene; 4-NOPD 4-nitro-o-phenylene-diamine; MMS methyl methane sulfonate

Table 2: Results of Experiment 2 (pre-incubation test)

Metabolic activation	Compound	Concentration (µg/plate)	TA1535	TA1537	TA98	TA100	WP2uvrA
Without S9 mix	DMSO	/	13 ± 2	13 ± 2	30 ± 11	95 ± 7	43 ± 6
	Untreated	/	15 ± 5	13 ± 8	29 ± 3	115 ± 10	52 ± 6
	Test item	33	9 ± 1	14 ± 2	35 ± 2	105 ± 1	45 ± 9
		100	12 ± 3	14 ± 2	33 ± 8	93 ± 12	40 ± 2
		333	12 ± 6	9 ± 1	27 ± 5	103 ± 10	49 ± 4
		1000	13 ± 4	10 ± 3	26 ± 4	90 ± 9	42 ± 9
		2500	11 ± 4	10 ± 5	25 ± 6	92 ± 16	43 ± 7
		5000	13 ± 1	11 ± 3	22 ± 6	88 ± 19	43 ± 5
	NaN3	10	1212 ± 36	/	/	1830 ± 144	/
	4-NOPD	10	/	/	485 ± 26	/	/
	4-NOPD	50	/	88 ± 3	/	/	/
	MMS	2.0 µL/plate	/	/	/	/	716 ± 25
With S9 mix	DMSO	/	15 ± 1	11 ± 2	41 ± 5	97 ± 19	58 ± 7
	Untreated	/	13 ± 3	10 ± 3	36 ± 4	111 ± 8	59 ± 7
	Test item	33	15 ± 3	11 ± 6	38 ± 11	96 ± 8	49 ± 3
		100	15 ± 6	12 ± 1	38 ± 11	109 ± 9	57 ± 5
		333	12 ± 3	13 ± 2	40 ± 7	101 ± 3	59 ± 11
		1000	16 ± 5	13 ± 3	39 ± 6	102 ± 8	63 ± 6
		2500	9 ± 6	9 ± 4	38 ± 13	96 ± 3	47 ± 6
		5000	13 ± 3	8 ± 3	35 ± 6	80 ± 9	48 ± 12
	2-AA	2.5	416 ± 18	281 ± 20	3358 ± 561	3662 ± 645	/
	2-AA	10	/	/	/	/	404 ± 26
NaN3 sodium azide, 2-AA 2-aminoanthracene, 4-NOPD 4-nitro-o-phenylene-diamine, MMS methyl methane sulfonate

Table 3: Historical control data generated in the testing facility from Nov 2016 - Aug 2018 representing approx. 600 experiments (WP2uvrA historical control data are based on approx. 400 experiments)

Strain	Compound	without S9 mix		with S9 mix
		Mean ± SD	Range	Mean ± SD	Range
TA 1535	Solvent control	11 ± 2.3	6 - 22	12 ± 2.3	7 - 22
	Untreated control	11 ± 2.9	6 - 28	12 ± 2.8	7 - 23
	Positive control	1245 ± 161.4	367 - 1791	398 ± 61	183 - 613
TA 1537	Solvent control	10 ± 2.2	6 - 19	13 ± 3.2	7 - 30
	Untreated control	10 ± 2.7	5 - 21	14 ± 3.6	6 - 29
	Positive control	94 ± 30	48 - 231	170 ± 64.8	81 - 421
TA 98	Solvent control	26 ± 4.2	13 - 43	36 ± 6.1	12 - 56
	Untreated control	27 ± 4.7	14 - 40	39 ± 6.4	12 - 59
	Positive control	421 ± 176.8	196 - 2068	3908 ± 815	223 - 5918
TA 100	Solvent control	160 ± 29.3	79 - 214	148 ± 31.3	76 - 216
	Untreated control	181 ± 26.1	80 - 235	171 ± 27.7	87 - 218
	Positive control	2074 ± 262.7	511 - 2850	3626 ± 981.9	553 - 5860
WP2 uvrA	Solvent control	39 ± 6.4	26 - 60	48 ± 7.3	28 - 69
	Untreated control	40 ± 5.8	22 - 61	51 ± 7.4	32 - 87
	Positive control	865 ± 340.9	346 - 4732	426 ± 111.2	184 - 1265

SD = standard deviation

Applicant's summary and conclusion

Conclusions:

Under the conditions of the test, the test item was not mutagenic in S. typhimuirum strains TA98, TA100, TA1535 and TA1537 and in E. coli strain WP2uvrA with and without metabolic activation.