1,3,5-tris[3-(trimethoxysilyl)propyl]-1,3,5-triazine-2,4,6(1H,3H,5H)-trione

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial reverse mutation assay: the substance, 1,3,5-tris[3-(trimethoxysilyl)propyl]-1,3,5-triazinane-2,4,6-trione, was negative in S. typhimurium strains TA 98, TA100, TA1535, TA1537 and E. coli WP2 uvr A (Microbiological Associates, 1996). The study was conducted according to an appropriate OECD test guideline and in compliance with GLP.

In vitro cytogenicity assays:

The substance, 1,3,5-tris[3-(trimethoxysilyl)propyl]-1,3,5-triazinane-2,4,6-trione, was considered negative with and without metabolic activation after 4-hour exposure, and equivocal in the absence of metabolic activation after 24-hour exposure in peripheral human lymphocytes (Eurofins, 2018a). The study was conducted according to OECD TG 473 and in compliance with GLP.

The substance, 1,3,5-tris[3-(trimethoxysilyl)propyl]-1,3,5-triazinane-2,4,6-trione, was considered negative with and without metabolic activation after 4-hour exposure in peripheral human lymphocytes (Eurofins, 2019). The study was conducted according to OECD TG 487 and in compliance with GLP.

In vitro mammalian mutagenicity assays:

In vitro mammalian cell gene mutation assay: the substance, 1,3,5-tris[3-(trimethoxysilyl)propyl]-1,3,5-triazinane-2,4,6-trione, was negative in mouse lymphoma L5178Y cell in the presence and absence of metabolic activation (Eurofins, 2018b). The study was conducted according to OECD TG 490 and in compliance with GLP.

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:

1996-08-13 to 1996-09-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Aroclor induced rat liver S9

Test concentrations with justification for top dose:

Pre-test: 6.7, 10, 33, 67, 100, 333, 667, 1000, 3333, 5000, with and without metabolic activation
Experiment 1: 100, 333, 1000, 3333, 5000, with and without metabolic activation
Experiment 2: 33, 100, 333, 1000, 5000, with and without metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

- Justification for choice of solvent/vehicle: Sponsor indicated that the test article is stable in DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

All salmonella strains (with activation)

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 activation)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA 100, TA 1535 (without activation)

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 activation)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

WP2 uvrA (without activation)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar ; preincubation


DURATION
- Preincubation period: 60 minutes

- Expression time (cells in growth medium): 48 - 72 hours

NUMBER OF REPLICATIONS: 3 plates for each test concentration

DETERMINATION OF CYTOTOXICITY
- Method: Background lawn assessment, revertant colony counts

Evaluation criteria:

For a test substance to be considered positive, it must cause at least a doubling in the mean revertants per plate of at least one tester strain with a minimum of two increasing concentrations of test article.

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 WP2 uvrA and 3-fold of the solvent control for TA 1535 and TA 1537.

Cytotoxicity is defined as a reduction in the number of colonies by >50% compared with the solvent control and/or at least a moderate reduction in
the background lawn (background code 3,4 or 5).

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:

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:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

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:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

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:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

3333 - 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

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:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS

- Precipitation: Interfering precipitate noted at 5000 µg/plate in TA 98 and TA 1535 (+/-MA), TA 1537 (-MA) and WP2 uvrA (-MA) 1000 - 5000 (+MA)

COMPARISON WITH HISTORICAL CONTROL DATA: Results were within range of historical control data

Remarks on result:

other: No mutagenic potential

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

	TA 100			WP2 uvrA
Concentration (µg/Plate)	Plate 1 + MA	Plate 2 - MA	Cytotoxic (Yes/No)	Plate 1 + MA	Plate 2 - MA	Cytotoxic (Yes/No)
0	133	98	No	16	23	No
6.7	128	110	No	13	8	No
10	123	95	No	28	17	No
33	134	98	No	13	9	No
67	150	84	No	19	19	No
100	147	111	No	17	15	No
333	146	107	No	11	20	No
667	134	104	No	22	26	No
1000	148	123	No	32	16	No
3333	146	116	No	15	18	No
5000	168	113	No	24	16	No

*solvent control with DMSO

Table 3: Experiment 1 Mutagenicity Assay Number of revertants per plate (mean of 3 plates)

	TA98			TA100			TA1535
Conc. (µg/plate)	— MA	+ MA	Cytotoxic (yes/no)	— MA	+ MA	Cytotoxic (yes/no)	— MA	+ MA	Cytotoxic (yes/no)
0*	16	27	No	134	161	No	11	12	No
100	21	30	No	131	156	No	11	11	No
333	11	24	No	134	163	No	10	14	No
1000	20	24	No	129	164	No	12	13	No
3333	20	32	No	141	167	No	9	11	No
5000	15	28	No	126	172	No	10	15	No
Positive control	367	873	No	720	1035	No	637	129	No

*solvent control with DMSO

Table 3: Experiment 1 Mutagenicity Assay Number of revertants per plate (mean of 3 plates)

	TA1537			WP2uvrA
Conc. (µg/plate)	— MA	+  MA	Cytotoxic (yes/no)	— MA	+  MA	Cytotoxic (yes/no)
0*	5	12	No	17	19	No
100	4	8	No	14	18	No
333	6	8	No	17	13	No
1000	4	9	No	10	16	No
3333	6	8	Yes	16	15	No
5000	6	6	Yes	17	14	No
Positive control	1070	119	No	292	108	No

*solvent control with DMSO

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

	TA98			TA100			TA1535
Conc. (µg/plate)	— MA	+ MA	Cytotoxic (yes/no)	— MA	+ MA	Cytotoxic (yes/no)	— MA	+ MA	Cytotoxic (yes/no)
0*	26	44	No	106	129	No	10	12	No
33	26	39	No	121	131	No	11	11	No
100	24	46	No	136	153	No	9	16	No
333	26	35	No	117	142	No	11	14	No
1000	25	39	No	119	166	No	11	11	No
5000	19	38	No	138	167	No	11	10	No
Positive control	292	901	No	534	945	No	446	106	No

*solvent control with DMSO

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

	TA1537			WP2 uvrA
Conc. (µg/plate)	— MA	+ MA	Cytotoxic (yes/no)	— MA	+ MA	Cytotoxic (yes/no)
0*	6	5	No	21	14	No
33	5	6	No	16	14	No
100	5	7	No	21	15	No
333	4	6	No	18	15	No
1000	5	9	No	19	18	No
5000	3	7	Yes	22	15	No
Positive control	598	97	No	430	118	No

*solvent control with DMSO

Conclusions:

Under test conditions, no mutagenic effect was observed for the test substance tested up to cytotoxic concentration in any of the test strains in two independent experiments without and with metabolic activation. The test substance is non-mutagenic in test strains used.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 April 2018 to 10 October 2018

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:

2016

Deviations:

yes

Remarks:

Fewer than 300 cells were counted without activation at some concentration because of insufficient cells

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro mammalian cell chromosome aberration assay

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature under a nitrogen headspace which is to be replenished each time the container is opened; protected from light
- Stability under test conditions: unstable after repeated contact with moisture in the air
- Solubility and stability of the test substance in the solvent/vehicle: According to the Sponsor’s recommendations a solubility test was performed with tetrahydrofuran (THF). Based on the results THF was used as best suitable solvent up to the maximum recommended concentration of 2 mg/mL.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not applicable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was diluted shortly before treatment. From the highest test item stock solution separate dosing solutions were prepared for each of the concentrations by serial dilution with the solvent and each dosing solution was added directly to the cell culture medium.
- Preliminary purification step (if any): none

FORM AS APPLIED IN THE TEST (if different from that of starting material) : liquid

OTHER SPECIFICS:
- measurement of pH, osmolality, and precipitate in the culture medium to which the test chemical is added: The pH was within the physiological range of 7.0 ± 0.4 and for osmolality 345 mOsmol/kg was measured at a test item concentration of 2000 µg/mL (solvent control: 340 mOsmol/kg).

Species / strain / cell type:

lymphocytes: peripheral human

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: healthy and non-smoking donors with no known recent exposure to genotoxic chemicals and radiation
- Suitability of cells: suitable for assay
- Cell cycle length, doubling time or proliferation index: proliferation index: 1.00-1.58
- Sex, age and number of blood donors if applicable: in each experiment blood was collected only from a single donor to reduce inter-individual variability.
- Whether whole blood or separated lymphocytes were used if applicable: Whole blood samples treated with an anti-coagulant (e. g. heparin) were pre-cultured in the presence of mitogen (phyto-haematogglutinin, PHA).
- Number of passages if applicable: no information
- Methods for maintenance in cell culture if applicable: not applicable
- Modal number of chromosomes: no information
- Normal (negative control) cell cycle time: 1.49/1.58

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
Complete Culture Medium: RPMI 1640 medium supplemented with 15% foetal bovine serum (FBS); 100 U/100 µg/mL penicillin/streptomycin solution: 0.24 g/mL PHA-L. Also used for the long-term treatment and the post incubation.
Treatment Medium (short-term exposure): Complete culture medium without FBS.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with

Metabolic activation system:

phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg) activated rat liver S9

Test concentrations with justification for top dose:

Experiment I: 500, 1500 and 1700 µg/mL (-MA), 1500, 1700 and 2000 µg/mL (+MA);
Experiment II: 10, 20, 50 and 100 µg/mL (-MA)
Concentrations were based on the results of the pre-experiment for cytotoxicity.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: tetrahydrofuran (THF)
- Justification for choice of solvent/vehicle: based on sponsor's recommendation

Untreated negative controls:

yes

Remarks:

Treatment medium

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

ACTIVATION
The S9 liver microsomal fraction was prepared at Eurofins Munich. Male Wistar rats were induced with phenobarbital (80 mg/kg bw) and ß-naphthoflavone (100 mg/kg bw) for three consecutive days by oral route. The preparation was performed according to Ames et al..
The following quality control determinations were performed:
a) Biological activity in the Salmonella typhimurium assay using 2-aminoanthracene
b) Sterility Test
A stock of the supernatant containing the microsomes was frozen in aliquots of 2 and 4 mL and stored at +/- -75 °C.
The protein concentration in the S9 preparation (Lot: 020218) was 39.5 mg/mL.

An appropriate quantity of the S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.75 mg/ml in the cultures. The final percentage of S9 mix in cell culture medium was 5% (v/v).
Cofactors were added to the S9 mix to reach the concentrations below:
8 mM MgCl2
33 mM KCl
5 mM Glucose-6-phosphate
5 mM NADP
in 100 mM sodium-phosphate-buffer pH 7.4. During the experiment the S9 mix was stored on ice.


METHOD OF APPLICATION: in medium;

DURATION
- Preincubation period: not applicable
- Exposure duration: Experiment 1: 4 hours, experiment 2: 24 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hours

SPINDLE INHIBITOR (cytogenetic assays): least 2 h before harvesting, colcemid was added to the cultures (final concentration 0.2 µg/mLl)

STAIN (for cytogenetic assays): Giesma

NUMBER OF REPLICATIONS: Duplicate cultures at each concentration, exposure without metabolic activation was repeated with 24-hour exposure time.

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: The cultures were harvested by centrifugation 24 h after beginning of treatment. The supernatant was discarded and the cells were resuspended in approximately 5 mL hypotonic solution (0.4% KCl). The cell suspension was incubated at room temperature for 20 min. After removal of the hypotonic solution by centrifugation the cells were fixed with 3+1 methanol + glacial acetic acid. The fixation procedure was repeated twice. Slides were prepared by dropping the cell suspension onto a clean microscopic slide. The cells were stained with giemsa and according to the Fluorescent plus Giemsa technique, respectively. The slides were coverslipped using 2-3 drops of Eukitt(R). Afterwards they were air dried.

NUMBER OF CELLS EVALUATED: 300 cells were evaluated for cytogenicity where available except where a clear positive response was observed in the positive control.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE: 300 cells (cytogenicity), 1000 cells (cytotoxicity)

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index;

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

Rationale for test conditions:

Guideline test conditions

Evaluation criteria:

Acceptability criteria:
- the number of aberrations found in the negative and/or solvent controls falls within the range of historical laboratory control data / is considered acceptable for addition to the laboratory historical negative control database.
- concurrent positive controls should induce responses that are compatible with those generated in the historical positive control data base and produce a statistically significant increase compared with the concurrent negative control
- the proliferation criteria in the solvent control should be similar to the corresponding negative control (where applicable)
- All three experimental conditions were tested unless one resulted in positive results
- Adequate number of cells and concentrations are analysable
Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined:
a) at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) the increase is dose-related when evaluated with an appropriate trend test,
c) any of the results are outside the 95% control limits of the historical negative control data.

Providing that all acceptability criteria are fulfilled, a test chemical is considered clearly negative if, in all experimental conditions examined
a) none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) there is no concentration-related increase when evaluated with an appropriate trend test,
c) all results are inside the 95% control limits of the historical negative control data.

Statistics:

Statistical significance at the 5% level (p < 0.05) was evaluated by the Fischer´s exact test. The p value was used as a limit in judging for significance levels in comparison with the corresponding solvent control. Only aberrant cells without gaps were used for the calculation.
Statistical significance at the 5% level (p < 0.05) was evaluated by the ¿² test for trend. The p value was used as a limit in judging for significance levels.

Species / strain:

lymphocytes: peripheral human

Metabolic activation:

without

Genotoxicity:

other: equivocal

Remarks:

The response in the absence of metabolic activation was considered to be equivocal

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

1000 µg/ml (-MA, 4-hour exposure); 50 µg/ml -MA, 24-hour exposure

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

lymphocytes: peripheral human

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Remarks:

The mitotic index was 70% at 2000 µg/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: 7.0 ± 0.4
- Effects of osmolality: 2000 µg/ml: 345 mOsmol/kg; solvent control: 340 mOsmol/kg
- Evaporation from medium: no information, not considered to be a factor
- Water solubility: water is an unsuitable solvent
- Precipitation: no precipitation observed
- Definition of acceptable cells for analysis: no information
- Other confounding effects: none recorded

RANGE-FINDING/SCREENING STUDIES:

A range-finding study for cytotoxicity was carried out.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: without metabolic activation: 6.3-54.7 aberrant cells, excluding gaps; with metabolic activation: 8.0-37.3 aberrant cells, excluding gaps.
- Negative (solvent/vehicle) historical control data: 4 hour treatment: 0.26% - 3.84% aberrant cells without metabolic activation (4 and 24 hour treatment); -0.21% - 3.94 % aberrant cells with metabolic activation. 24 hour treatment: -0.33% - 3.60% aberrant cells without metabolic activation

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: mitotic index. Cell cycle delay was evaluated by
- Other observations when applicable: the number of cells available for analysis was less than 300 in the solvent control and at the highest concentration evaluated in experiment I, and at the two highest concentrations without metabolic activation in experiment II.

Table 1 Test for Cytotoxicity

Dose Group	Metabolic activation	Concentration [µg/ml]	Mitotic Index Culture 1	Mitotic Index Relative [%]	Precipitate (+/-)
C	-	0	21	81	-
S	-	0	26	100	-
1	-	5	n.d	-	-
2	-	10	n.d	-	-
3	-	25	n.d	-	-
4	-	50	n.d	-	-
5	-	100	n.d	-	-
6	-	250	37	142	-
7	-	500	25	96	-
8	-	1000	19	73	-
9	-	1500	24	92	-
10	-	2000	26	100	-
C	+	0	72	288	-
S	+	0	25	100	-
1	+	5	n.d	-	-
2	+	10	n.d	-	-
3	+	25	n.d	-	-
4	+	50	n.d	-	-
5	+	100	n.d	-	-
6		250	55	220	-
7		500	64	256	-
8		1000	28	112	-
9		1500	54	216	-
10		2000	14	56	-

Table 2 Summary of results

Dose group	Concentration [µg/ml]	Metabolic activation	Treatment (hours)	Relative Mitotic Index [%]	Proliferation Index	Mean % Aberrant Cells incl. Gaps	Mean % Aberrant Cells excl. Gaps	Precipitation^a	Statistical Significance^b
C	0	-	4	149	1.58	1.7	1.7	-	-
S	0	-	4	100	1.45	2.7	2.0	-	/
1	500	-	4	92	/	6.7	3.7	-	-
3	1500	-	4	38	/	3.5	3.5	-	-
5	1700	-	4	43	1.00	4.7	3.7	-	-
EMS	600	-	4	92	/	14.0	12.0	-	+
C	0	+	4	111	1.08	3.3	2.7	-	-
S	0	+	4	100	1.47	2.3	2.0	-	/
4	1500	+	4	93	/	3.7	1.7	-	-
6	1700	+	4	80	/	4.0	2.7	-	-
8	2000	+	4	70	1.21	5.0	3.3	-	-
CPA	5	+	4	105	/	17.1	16.4	-	+
C	0	+	24	138	1.49	3.0	1.7	-	-
S	0	+	24	100	1.03	3.7	2.7	-	/
1	10	+	24	43	/	6.7	3.7	-	-
2	20	+	24	78	/	5.3	2.3	-	-
3	50	+	24	53	1.02	10.0	6.4	-	+
4	100	+	24	34	1.00	8.9	7.2	-	+
EMS	400	+	24	41	/	34.4	34.4	-	+

a: - without precipitation. + with precipitation

b: statistical significant increase compared to solvent controls (Fisher’s exact test, p< 0.05), +: significant; -not significant

The mitotic index was determined in 1000 cells per culture of each test group. The relative values of the mitotic index are related to the solvent controls.

4-hour treatment: 300 cells evaluated for each concentration, except for the positive controls (EMS: 150 cells, CPA: 140 cells) due to a clearly positive increase in chromosomal aberrations. In the experiment without metabolic activation, fewer than 300 cells were evaluated for chromosome aberrations in the solvent control (293 cells) and the concentration 1500 µg/mL (257 cells) as not enough cells were present one the slides.

24-hour treatment: 300 cells evaluated for each concentration, except for the positive control (EMS: 90 cells) due to a clearly positive increase in chromosomal aberrations. Fewer than 300 cells were evaluated for chromosome aberrations for the concentrations 50 µg/ml (250 cells) and 100 µg/ml (291 cells) due to few cells present on the slides.

 

C:       Negative Control (Culture Medium)

S:        Solvent Control (0.5% THF; v/v)

EMS:  Positive Control (without metabolic activation: Ethylmethanesulfonate)

CPA:  Positive Control (with metabolic activation: Cyclophosphamide)

Table 3 Aberration data, 24 hour exposure without metabolic activation

Dose Group	C	S	1	2	3	4	EMS
Concentration µg/ml	0	0	10	20	50	100	400
Number of cells scored	300	300	300	300	250	291	90
Polyploid cells	1	0	1	0	0	1	1
Aberrant cells including gaps	9	11	20	16	25	26	31
Aberrant cells excluding gaps	5	8	11	7	16	21	31
Gaps	4	3	12	9	12	7	4
Iso-gaps	0	0	1	0	2	1	1
Chromatid breaks	4	4	10	4	16	13	34
Chromatid fragments	0	0	0	2	0	0	0
Chromatid deletions	0	0	0	0	1	2	1
Chromatid exchanges	0	1	0	0	0	1	9
Chromosome iso-breaks	0	0	0	0	1	1	1
Chromosome iso-fragments	1	3	1	1	1	3	4
Chromosome iso-deletions	0	0	0	0	0	0	0
Chromosomal exchanges	0	0	0	0	0	1	0
Multiple aberrations	0	0	0	0	0	0	1
Chromosomal disintegration	0	0	0	0	0	0	0

C:       Negative Control (Culture Medium)

S:        Solvent Control (0.5% THF; v/v)

EMS:  Positive Control (without metabolic activation: Ethylmethanesulfonate)

Conclusions:

Tris[3-(trimethoxysilyl)propyl]-1,3,5-triazinane-2,4,6-trione has been tested in a valid in vitro chromosome aberration assay conducted according to OECD TG 473 and in compliance with GLP using peripheral human lymphocytes. No increase in the percentage of cells with chromosome aberrations was observed when cells were exposed for four hours to the test substance up to the limit concentration with metabolic actvation or to cytotoxic concentrations without metabolic activation. After continuous exposure for 24 hours without metabolic activation, a biologically-relevant increase of the aberration rates was noted after treatment at cytotoxic concentrations. These increases were statistically significant and concentration-dependent, however, they may be artefacts as they were observed in the higher end of the cytotoxicity range. Fewer than 300 cells per concentration were available for evaluation, so it was not possible to count additional cells to verify the result. It is concluded that the test substance is equivocal for the induction of chromosome aberration under the conditions of the test.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study will be available 30/06/2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

2016

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro gene mutation study in mammalian cells

Target gene:

Thymidine kinase operon

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Eurofins Munich stock cultures
- Suitability of cells: recommended by guideline
- Cell cycle length, doubling time or proliferation index: 10-12 hour doubling time
- Methods for maintenance in cell culture if applicable: To prevent high backgrounds arising from spontaneous mutation, cells lacking TK can be eliminated by culturing cells in RPMI 1640 supplemented with: 9.0 µg/ml hypoxanthine; 15.0 µg/ml thymidine; 22.5 µg/ml glycine; 0.1 µg/ml methotrexate.
The cells are resuspended in medium without methotrexate but with thymidine, hypoxanthine and glycine for 1 - 3 days.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically 'cleansed' against high spontaneous background: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital (80 mg/kg bw) and ß-naphthoflavone (100 mg/kg bw) activated rat liver S9

Test concentrations with justification for top dose:

without metabolic activation: 50, 75, 200, 500, 1000 and 1500 µg/ml
with metabolic activation: 25, 50, 75, 200, 500, 1000 and 2000 µg/ml
The selection of the concentrations used in the main experiment was based on data from the pre-experiment according to OECD TG 490. In the experiment without metabolic activation the highest selected concentration (1500 µg/ml) was based on cytotoxicity (RTG was 13.5%). In the experiment with metabolic activation the highest recommended concentration (2000 µg/ml) was tested; precipitation occurred at this concentration.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: tetrahydrofuran (THF).
- Justification for choice of solvent/vehicle: Sponsor's recommendation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

ethylmethanesulphonate

methylmethanesulfonate

Details on test system and experimental conditions:

ACTIVATION
An appropriate quantity of the S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.75 mg/mL in the cultures. Cofactors were added to the S9 mix to reach the concentrations below:
8 mM MgCl2
33 mM KCl
5 mM Glucose-6-phosphate
5 mM NADP
in 100 mM sodium-phosphate-buffer pH 7.4. During the experiment the S9 mix was stored on ice.

METHOD OF APPLICATION: in medium;
- Cell density at seeding (if applicable): 1E+7
DURATION
- Preincubation period: No
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 12 days

SELECTION AGENT (mutation assays): trifluorothymidine (TFT). Cells from each experimental group were seeded in four 96-well plates at a density of approximately 2000 cells/well in 200 µL selective medium with TFT. The plates were scored after an incubation period of about 12 days at 37 °C in 5% CO2/95% humidified air.

NUMBER OF REPLICATIONS: Duplicate cultures

NUMBER OF CELLS EVALUATED:
- Cloning efficiency: 1.6 cells/well in two 96 well plates.
- Mutant frequency 2000 cells/well in four 96 well plates


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; relative total growth.
- Any supplementary information relevant to cytotoxicity: The relative total growth (RTG) is the product of the relative suspension growth (RSG; calculated by comparing the SG of the dose groups with the SG of the control) and the relative cloning efficiency (RCE) for each culture: RTG = RSG x RCE /100

- OTHER: colony sizing was performed for the highest concentrations of the test item and for the negative and positive controls.

Rationale for test conditions:

Based on guideline

Evaluation criteria:

- The induced mutant frequency meets or exceeds the Global Evaluation factor (GEF) of 126 mutants per 106 cells and
- a dose-dependent increase in mutant frequency is detected.
Besides, combined with a positive effect in the mutant frequency, an increased occurrence of small colonies (=40% of total colonies) is an indication for potential clastogenic effects and/or chromosomal aberrations.
According to the OECD guideline, the biological relevance is considered first for the interpretation of results. Statistical methods might be used as an aid in evaluation of the test result.
A test item is considered to be negative if the induced mutant frequency is below the GEF and the trend of the test is negative.

Statistics:

Statistical significance at the 5% level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Growth inhibition was observed in the experiment without metabolic activation: The relative total growth (RTG) was 13.5% for the highest concentration evaluated.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The pH-value detected with the test item was within the physiological range.
- Effects of osmolality: The osmolality were within the physiological range.
- Evaporation from medium: no information
- Water solubility: no information
- Precipitation: Precipitation of the test item was noted in the main experiment without metabolic activation at concentrations of 1500 µg/ml and in the experiment with metabolic activation at concentration of 2000 µg/ml.
- Definition of acceptable cells for analysis:
A mutation assay is considered acceptable if:
- At least three out of four 96-well plates from the TFT resistance-testing portion of the experiment are scorable
- The cloning efficiency of the negative and/or solvent controls is in the range 65% - 120%.
- The spontaneous mutant frequency in the negative and/or solvent controls is in the range
50 - 170 mutants per 106 cells
- The cell number of the negative/solvent controls should undergo 8 - 32-fold increase during a 2-day growth period (short-term treatment)
- The clastogenic positive controls (MMS and B[a]P) have to produce an induced mutant frequency (total mutant frequency minus concurrent negative control mutant frequency) of at least 300 mutants per 106 cells with at least 40% of the colonies being small colonies or with an induced small colony mutant frequency of at least 150 mutants per 106 cells.
- The RTG must be greater than 10%.

- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES:

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells:

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture:
- Indication whether binucleate or mononucleate where appropriate:

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
- Negative (solvent/vehicle) historical control data:

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: [complete, e.g. CBPI or RI in the case of the cytokinesis-block method; RICC, RPD or PI when cytokinesis block is not used]
- Other observations when applicable: [complete, e.g. confluency, apoptosis, necrosis, metaphase counting, frequency of binucleated cells]

Summary: Main Experiment, without and with metabolic activation

Test Group	Conc. [µg/ml]	Metabolic activation	RCE^a[%]	RTG^b[%]		MF^c[mutants/ 10^6cells]	IMF^d[mutants/ 10^6cells]	GEF^eexceeded	Statistical Significant Increase^f	Precipitate
C1	0	-	88.3	94.3		80.7	/	/	/	-
C2	0	-	86.9	92.0		80.7	/	/	/	-
S1		-	100.0	100.0		87.5	/	/	/	-
S2		-	100.0	100.0		87.5	/	/	/	-
1	50	-	100.9	114.6		83.2	-4.3	-	-	-
2	75	-	89.7	90.3		83.9	-3.7	-	-	-
3	200	-	74.6	81.7		97.7	10.2	-	-	-
4	500	-	76.9	89.0		101.4	13.9	-	-	-
5	1000	-	100.9	88.9		56.0	-31.5	-	-	-
6	1500	-	71.4	13.5		90.0	2.5	-	-	+
EMS	300	-	62.7	56.2		1017.9	930.4	+	+	-
MMS	10	-	66.4	53.4		606.8	519.2	+	+	-
C1	0	+	90.0	94.7		153.2	/	/	/	-
C2	0	+	94.3	103.9		153.2	/	/	/	-
S1	0	+	100.0		100.0	134.9	/	/	/	-
S2	0	+	100.0		100.0	134.9	/	/	/	-
2	25	+	95.8	109.5		163.4	28.5	-	-	-
3	50	+	90.0	96.4		115.2	-19.7	-	-	-
4	75	+	87.3	99.6		119.7	-15.2	-	-	-
5	200	+	88.6	103.7		135.0	0.1	-	-	-
6	500	+	90.0	111.0		123.8	-11.2	-	-	-
7	1000	+	100.6	95.8		125.0	-10.0	-	-	-
8	2000	+	107.5	74.7		114.4	-20.5	-	-	+
B[a]P	1.5	+	73.1	36.8		825.1	690.2	+	+	-

C:       Negative Controls

S:         Solvent control (0.25% THF; v/v)

a:        Relative Cloning Efficiency, RCE = [(CE_{dose group}/ CE_{of corresponding controls}) x 100]

            Cloning Efficiency, CE = ((-LN (((96 - (mean P1,P2)) / 96)) / 1.6) x 100)

b:        Relative Total Growth, RTG = (RSG x RCE)/100

c:        Mutant Frequency,

            MF = {-ln [negative cultures/total wells (selective medium)] / -ln [negative cultures/total wells (non-selective medium)]}x800

d:        Induced Mutant Frequency, IMF = mutant frequency sample – mean value mutant frequency corresponding controls

e:        Global Evaluation Factor, GEF (126); +: GEF exceeded, -: GEF not exceeded

f:         statistical significant increase in mutant frequency compared to solvent controls (Mann Whitney test, p<0.05). +: significant; -not significant

EMS:   Ethylmethanesulfonate

MMS:  Methylmethanesulfonate

B[a]P:  Benzo[a]pyrene

Conclusions:

Tris[3-(trimethoxysilyl)propyl]-1,3,5-triazinane-2,4,6-trione has been tested in a valid in vitro mammalian cell gene mutation assay, conducted according to OECD TG 490 and in compliance with GLP, using mouse lymphoma L5178Y cells and the microwell method. No biologically relevant increase in the number of mutants was found after treatment with the test item at limit concentration with metabolic activation or cytotoxic concentration without metabolic activation. The Global Evaluation Factor was not exceeded by the induced mutant frequency at any concentration. Appropriate negative (growth medium), solvent (tetrahydrofuran) and positive controls were included and gave expected results. It is concluded that the test substance is not mutagenic to mammalian cells under the conditions of the test.

Reference 4

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 March 2019 to 5 August 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
at room temperature under a nitrogen or argon after opening; protected from light
- Solubility and stability of the test substance in the solvent/vehicle:
according to the Sponsor’s recommendations THF was used as best suitable solvent up to the maximum recommended concentration of 2 mg/mL (0.5% THF, v/v final concentration)


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
The test item was diluted shortly before treatment and each dosing solution was added directly to the cell culture medium.
- Preliminary purification step (if any): none

OTHER SPECIFICS:
- measurement of pH, osmolality, and precipitate in the culture medium to which the test chemical is added:
The pH was within the physiological range of 7.0 ± 0.4 and for osmolality a range of 331-338 mOsmol/kg was measured at a test item concentration of 2000 µg/mL (solvent control: 348-342 mOsmol/kg).

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Human peripheral blood lymphocytes from healthy and non-smoking donors with no known recent exposure to genotoxic chemicals and radiation.
- Suitability of cells: recommended in guideline
- Cell cycle length, doubling time or proliferation index: not specified
- Sex, age and number of blood donors: one healthy non-smoking donor with no known recent exposure to genotoxic chemicals and radiation
- Whether whole blood or separated lymphocytes were used: whole blood
- Whether blood from different donors were pooled or not: Blood was collected only from a single donor to reduce inter-individual variability
- Mitogen used for lymphocytes: whole blood samples treated with an anti-coagulant (e. g. heparin) were pre-cultured in the presence of mitogen (phyto-haematogglutinin, PHA)

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
Complete Culture Medium: RPMI 1640 medium supplemented with 15% foetal bovine serum (FBS); 100 U/100 µg/mL penicillin/streptomycin solution: 2.4 µg/mL PHA.
Treatment Medium (short-term exposure): Complete culture medium without FBS.
After Treatment medium/ Treatment medium: Complete culture medium with 15 % FBS and 6 µg/mL cytochalasin B.

Cytokinesis block (if used):

Actin polymerisation inhibitor Cytochalasin B

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg) activated rat liver S9

Test concentrations with justification for top dose:

Experiment I: 500, 1000, 1500 and 2000 µg/mL (-MA), 1500, 1700 and 2000 µg/mL (+MA);
Experiment II: 500, 1000, 1700 and 1750 µg/mL (-MA)
Concentrations were based on the results of the pre-experiment for cytotoxicity.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: tetrahydrofuran (THF)
- Justification for choice of solvent/vehicle: based on sponsor's recommendation

Untreated negative controls:

yes

Remarks:

cell culture medium

Negative solvent / vehicle controls:

yes

Remarks:

cell culture medium with 0.5% THF

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

other: colchicine (migrated information)

Details on test system and experimental conditions:

ACTIVATION
The S9 liver microsomal fraction was prepared at Eurofins Munich. Male Wistar rats were induced with phenobarbital (80 mg/kg bw) and ß-naphthoflavone (100 mg/kg bw) for three consecutive days by oral route. The preparation was performed according to Ames et al..
The following quality control determinations were performed:
a) Biological activity in the Salmonella typhimurium assay using 2-aminoanthracene and benzo[a]pyrene
b) Sterility Test
A stock of the supernatant containing the microsomes was frozen in aliquots of 2 and 4 mL and stored at +/- -75 °C.
The protein concentration in the S9 preparation (Lot: 210918) was 35.7 mg/mL.

An appropriate quantity of the S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.75 mg/ml in the cultures. The final percentage of S9 mix in cell culture medium was 5% (v/v).
Cofactors were added to the S9 mix to reach the concentrations below:
8 mM MgCl2
33 mM KCl
5 mM Glucose-6-phosphate
5 mM NADP
in 100 mM sodium-phosphate-buffer pH 7.4. During the experiment the S9 mix was stored on ice. The final concentration of S9 mix in the cultures was 5%.


METHOD OF APPLICATION: in medium;

DURATION
- Pre-experiment: 4h incubation
- Exposure duration: Experiment 1: 4 hours, Experiment 2: 44 hours
- Actin polymerisation inhibitor exposure: 6 µg/mL cytochalasin B added after treatment and prior to the targeted mitosis for 40 h to 42 h at 37°C and 5% CO2 during Experiment 1; 6 µg/mL cytochalasin B added after treatment and prior to the targeted mitosis for 43 h at 37°C


NUMBER OF REPLICATIONS: Duplicate cultures at each concentration except for the pre-experiment

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: At the end of the cultivation, the complete culture medium was removed. Subsequently, the cells were treated with cold hypotonic solution (0.075 M KCl) for 30 minutes at room temperature and immediately centrifuged. The pellet was resuspended with a solution which consisted of fixation solution and NaCl 0.9% (1+1) and was centrifuged. After that the cells were fixed with methanol and glacial acetic acid (3+1). The cells were resuspended gently and the suspension was dropped onto clean glass slides. Consecutively, the cells were dried on a heating plate. The cells were stained with acridine orange solution.

SPINDLE INHIBITOR (cytogenetic assays): cytochalasin B.

STAIN (for cytogenetic assays): acridine orange solution

NUMBER OF CELLS EVALUATED: 2000 binucleated cells per concentration (1000 binucleated cells per slide)

CRITERIA FOR SCORING AND SELECTION OF ANALYSABLE CELLS: Criteria of Fenech i.e. clearly surrounded by a nuclear membrane, having an area of less than one-third of that of the main nucleus, being located within the cytoplasm of the cell and not linked to the main nucleus via nucleoplasmic bridges. Mononucleated and multinucleated cells and cells with more than six micronuclei were not considered


DETERMINATION OF CYTOTOXICITY
- Method: performed by determining cell proliferation in both treated and control cultures. The proliferation rate was determined by calculation of the cytokinesis-block proliferation index (CBPI) which was used to calculat cytostasis.

OTHER EXAMINATIONS:
- Determination of polyploidy: no
- Determination of endoreplication: no

Rationale for test conditions:

Guideline test conditions

Evaluation criteria:

Acceptability of the assay:
A mutation assay is considered acceptable if it meets the following criteria:
- The concurrent negative/solvent control is considered acceptable for addition to the laboratory historical negative/solvent control database.
- Concurrent positive controls should induce responses that are compatible with those generated in the laboratory’s historical positive control data base and produce a statistically significant increase compared with the concurrent negative/solvent control.
- Cell proliferation criteria in the negative/solvent control should be fulfilled.
- All experimental conditions are tested unless one resulted in positive results.
- Adequate number of cells and concentrations are analysable.
Criteria for the selection of top concentration are fulfilled.
Evaluation of results:
A test item is considered to be clearly positive if, in any of the experimental conditions examined:
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative/solvent control
- the increase is concentration-related in at least one experimental condition when evaluated with an appropriate trend test
- any of the results are outside the distribution of the historical negative/solvent control data (e.g. Poisson-based 95% control limits).
When all of these criteria are met, the test item is considered able to induce chromosome breaks and/or gain or loss in this test system.
A test item is considered to be clearly negative if in all experimental conditions examined none of the criteria mentioned above are met.

Statistics:

Statistical significance: Statistical significance at the 5% level (p < 0.05) was evaluated by the non-parametric ¿² test. The p value was used as a limit in judging for significance levels in comparison with the concurrent solvent control.
Trend test: Statistical significance at the 5% level (p < 0.05) was evaluated by the ¿² test. The p value was used as a limit in judging for significance levels

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

without metabolic activation, 4-hour exposure: 2000 µg/mL, cytostasis 34%; with metabolic activation, 4-hour exposure: cytostasis below 30%; without metabolic activation, 44-hour treatment: cytostasis below 30% at 1750 µg/mL, no cells at higher level.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: 7.0 ± 0.4
- Effects of osmolality: 2000 µg/ml: 338 mOsmol/kg at Exp I, 331 mOsmol/kg at Exp II ; solvent control: 348 mOsmol/kg at Exp I, 342 mOsmol/kg at Exp II
- Precipitation: precipitation of the test item was noted in experiment I with metabolic activation, at the concentration of 2000 µg/mL after 4 h treatment and in experiment II without metabolic activation for the concentration of 1700 µg/mL and higher after 44 h treatment.
- Other confounding effects: none recorded

RANGE-FINDING/SCREENING STUDIES:

A pre-experiment was conducted under identical conditions as described for the main experiment I (4 h incubation) in order to determine the toxicity of the test item. The CBPI was used to calculate cytostasis for the quantification of cytotoxicity. The following concentrations were tested without and with S9 mix:
7.8, 15.6, 31.3, 62.5, 125, 250, 500, 1000, 1500, 2000 µg/mL. The concentration of 2000 mg/mL was considered to be the highest test concentration to be used in this test system following the recommendation of the corresponding OECD testing guideline 487. Cytostasis was between 0 and 24%, so 2000 µg/mL was used as the top concentration in the main experiment.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: for MMS without metabolic activation : 0.46% - 6.19% micronucleated cells; for Colchicine without metabolic activation : 0.00-8.12%; for CPA with metabolic activation: 1.18-4.28%
- Negative historical control data: without metabolic activation : 0.29% - 1.16% micronucleated cells ; with metabolic activation : 0.28-1.26%
- Solvent historical control data: without metabolic activation : 0.06% - 1.58% micronucleated cells ; with metabolic activation : 0.19-1.38%

Table 1:  Summary: Experiment I, without metabolic activation, 4-hour treatment, 44-hour fixation interval

Dose Group	Concentration [µg/mL]	Number of cells evaluated	Cytostasis [%]	Relative Cell Growth [%]	Micro-nucleated Cells Frequency [%]	P	Statistical Significant Increase^a
C	0	3000	23	77	1.50	-	/
S	0	2000	0	100	0.50	-	/
1	500	2000	5	95	0.40	-	-
2	1000	2000	12	88	0.50	-	-
3	1500	2000	16	84	0.85	-	-
7	2000	2155	35	65	1.44	-	+
MMS	50 µg/mL	2000	51	49	5.80	-	+
Colc	0.4 µg/mL	2000	48	52	6.05	+	+

 

Micronucleated cell frequency historical control limit, negative control: 0.06% - 1.58%

Table2: Summary: Experiment II, without metabolic activation, 44-hour treatment, 44-hour fixation interval

Dose Group	Concentration [µg/mL]	Number of cells evaluated	Cytostasis [%]	Relative Cell Growth [%]	Micro-nucleated Cells Frequency [%]	P	Statistical Significant Increasea
C	0	2000	0	103	0.45	-	/
S	0	2000	0	100	0.35	-	/
2	500	2000	2	98	0.90	-	+
3	1000	3000	0	105	0.70	-	-
6	1700	2000	0	127	0.90	+	+
7	1750	2000	0	113	0.90	+	+
MMS	50 µg/mL	2000	0	100	3.25	-	+
Colc	0.4 µg/mL	2000	87	13	3.90	-	+

Micronucleated cell frequency historical control limit, negative control: 0.06% - 1.58%

Table 3: Summary: Experiment I, with metabolic activation

Dose Group	Concentration [µg/mL]	Number  of cells evaluated	Cytostasis [%]	Relative Cell Growth [%]	Micro- Nucleated Cells Frequency [%]	P	Statistical Significant Increase^a
C	0	2000	0	100	1.30	-	/
S	0	3000	0	100	1.03	-	/
4	1500	3000	17	83	1.03	-	-
5	1750	3000	2	98	1.60	-	-
6	2000	2000	0	121	1.25	+	-
CPA	15 µg/mL	2000	41	59	3.55	-	+

Micronucleated cell frequency historical control limit, negative control: 0.19% - 1.38%

C:                                         Negative Control (Culture medium)

S:                                           Solvent Control (THF 0.5% v/v in culture medium)

P:                                           Precipitation (+: precipitation, -: no precipitation)

a:                                          statistically significant increase compared to solvent control (c² test , p<0.05). +: significant; -: not significant

MMS:                                    Methylmethanesulfonate, Positive Control (without metabolic activation)

Colc.:                                    Colchicine, Positive Control (without metabolic activation)

CPA:                                     Cyclophosphamide, Positive Control (with metabolic activation)

 

Relative Cell Growth:                 100 x ((CBPI_{Test conc}– 1) / (CBPI_control -1))

 

Cytostasis [%] = 100- Relative Cell Growth [%]

^*: the cytostasis is defined 0, when the relative cell growth exceeds 100%

Conclusions:

2,4,6-tris[3-(trimethoxysilyl)propyl) isocyanurate (CAS 26115-70-8) has been tested in an in vitro mammalian cell micronucleus study conducted according to OECD TG 487 and in compliance with GLP. No induction of structural or numerical chromosomal damage in human lymphocytes was observed. Therefore, the test item is considered to be non-mutagenic with respect to clastogenicity and aneugenicity under the conditions of the test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

1,3,5 -Tris[3-(trimethoxysilyl)propyl]-1,3,5-triazinane-2,4,6-trione has been tested in a valid bacterial reverse mutation assay (Microbiological Associates, 1996), conducted according to OECD TG 471, and in compliance with GLP, using Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 or E. coli WP2 uvr A. No increase in the number of revertants was observed in any test strain, with or without metabolic activation when tested up to limit concentration. Appropriate positive and solvent controls were added and gave expected results. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test.

A supporting study was also available (BRRC, 1991), conducted according to OECD TG 471, with acceptable restrictions, and in compliance with GLP, using Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 or TA 1538. No increase in the number of revertants was observed in any test strain, with or without metabolic activation when tested up to limit concentration. Appropriate positive and solvent controls were added and gave expected results. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test.

1,3,5 -Tris[3-(trimethoxysilyl)propyl]-1,3,5-triazinane-2,4,6-trione has been tested in a valid in vitro chromosome aberration assay conducted according to OECD TG 473 and in compliance with GLP using peripheral human lymphocytes (Eurofins, 2018a). No increase in the percentage of cells with chromosome aberrations was observed when cells were exposed for four hours to the test substance up to the limit concentration with metabolic actvation or to cytotoxic concentrations without metabolic activation. After continuous exposure for 24 hours without metabolic activation, a biologically-relevant increase of the aberration rates was noted after treatment at cytotoxic concentrations. These increases were statistically significant and concentration-dependent, however, they may be artefacts as they were observed in the higher end of the cytotoxicity range. Fewer than 300 cells per concentration were available for evaluation, so it was not possible to count additional cells to verify the result. It is concluded that the test substance is equivocal for the induction of chromosome aberration under the conditions of the test.

1,3,5 -Tris[3-(trimethoxysilyl)propyl]-1,3,5-triazinane-2,4,6-trione has been tested in a valid in vitro mammalian cell gene mutation assay (Eurofins, 2018b), conducted according to OECD TG 490 and in compliance with GLP, using mouse lymphoma L5178Y cells and the microwell method. No biologically relevant increase in the number of mutants was found after treatment with the test item at limit concentration with metabolic activation or cytotoxic concentration without metabolic activation. The Global Evaluation Factor was not exceeded by the induced mutant frequency at any concentration. Appropriate negative (growth medium), solvent (tetrahydrofuran) and positive controls were included and gave expected results. It is concluded that the test substance is not mutagenic to mammalian cells under the conditions of the test.

Due to the equivocal result of the OECD 473 chromosome aberration test, a further in vitro micronucleus test according to OECD TG 487 has been carried out.

1,3,5 -Tris[3-(trimethoxysilyl)propyl]-1,3,5-triazinane-2,4,6-trione has been tested in a valid in vitro micronucleus test conducted according to OECD TG 487 and in compliance with GLP using peripheral human lymphocytes (Eurofins, 2019). No induction of structural or numerical chromosomal damage in human lymphocytes was observed. Therefore, the test item is considered to be non-mutagenic with respect to clastogenicity and aneugenicity under the conditions of the test.

Justification for classification or non-classification

Based on the available data for 1,3,5-tris[3-(trimethoxysilyl)propyl]-1,3,5-triazinane-2,4,6-trione, no classification is required for genetic toxicity according to Regulation (EC) No. 1272/2008.