Reaction products of N6,N6'-hexane-1,6-diylbis[N2,N4-dibutyl-N2,N4,N6-tris(2,2,6,6-tetramethylpiperidin-4-yl)-1,3,5-triazine-2,4,6-triamine] and allylbromide, subsequently reacted with ethaneperoxoic acid, hydrogenated

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

26 Sep 2014

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

1,3,5-Triazine-2,4,6-triamine, N2,N2’-1,6-hexanediylbis[N4,N6-dibutyl-N2,N4,N6-tris(2,2,6,6-tetramethyl-4-piperidinyl)-, N-allyl derivs., oxidized, hydrogenated

EC Number:

812-927-5

Cas Number:

1902936-62-2

Molecular formula:

not availabale

IUPAC Name:

1,3,5-Triazine-2,4,6-triamine, N2,N2’-1,6-hexanediylbis[N4,N6-dibutyl-N2,N4,N6-tris(2,2,6,6-tetramethyl-4-piperidinyl)-, N-allyl derivs., oxidized, hydrogenated

Test material form:

solid

Details on test material:

Appearance: - physical state: solid; - color: rose

Method

Target gene:

not applicable

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Suitability of cells: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

liver S9 from rats induced with phenobarbital i.p. and β-naphthoflavone orally

Test concentrations with justification for top dose:

1st Experiment

4-hour exposure, 18-hour sampling time, without S9 mix
0; (6.25; 12.5; 25;) 50; 100; 200 μg/mL

4-hour exposure, 18-hour sampling time, with S9 mix
0; (6.25;) 12.5; 25; 50 (100; 200) μg/mL

2nd Experiment
18-hour exposure, 18-hour sampling time, without S9 mix
0; (6.25; 12.5; 25;) 50; 100; 200 μg/mL

4-hour exposure, 28-hour sampling time, with S9 mix
0; (6.25; 12.5; 25;) 50; 100; 200μg/mL

Concentrations in parenthesis were not scored.

The top dose was determined by precipitation and cytotoxicity as determined for V79 cells during the HPRT test.

Vehicle / solvent:

DMSO

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 24 - 30 hours
- Exposure duration: 4h or 18h
- Expression time (cells in growth medium): 10, 14 or 24 hours

- Fixation time (start of exposure up to fixation or harvest of cells): 28h


SPINDLE INHIBITOR (cytogenetic assays): colcemide
STAIN (for cytogenetic assays): 7.5% (v/v) Giemsa/Titrisol solution pH 7.2

NUMBER OF REPLICATIONS:2

NUMBER OF CELLS EVALUATED: 300 metaphases per dose group (150 per replicate)

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

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

Rationale for test conditions:

Concentrations were chosen based on range-finding studies.

Evaluation criteria:

The V79 in vitro cytogenetic assay is considered valid if the following criteria are met:
• The quality of the slides must allow the identification and evaluation of a sufficient number of analyzable metaphases.
• The numbers of cells with structural/numerical aberrations in the negative control has to be within the range of the historical negative control data.
• The positive control substances both with and without S9 mix have to induce a distinct increase of structural chromosome aberrations.

The test substance is considered as “positive” if the following criteria are met:
• A statistically significant, dose-related and reproducible increase in the number of cells
with structural chromosome aberrations (excl. gaps).
• The number of aberrant cells (excl. gaps) exceeds both the concurrent negative/vehicle
control value and the historical negative control data range.
A test substance generally is considered as “negative” if the following criteria are met:
• The number of cells with structural aberrations (excl. gaps) in the dose groups is not
statistically significant increased above the concurrent negative/vehicle control value and
is within the historical negative control data range.

Statistics:

The statistical evaluation of the data was carried out using the MUCHAN program system (BASF SE). The proportion of metaphases with structural aberrations was calculated for each group. A comparison of each dose group with the negative control group was carried out using Fisher's exact test for the hypothesis of equal proportions. This test was Bonferroni- Holm corrected versus the dose groups separately for each time and was performed one-sided. If the results of this test are statistically significant compared with the respective vehicle control, labels (* p ≤ 0.05, ** p ≤ 0.01) are printed in the tables.

Results and discussion

Test results

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Effects of osmolality: none
- Evaporation from medium: non volatile
- Water solubility: poorly soluble
- Precipitation: The highest applied stock solution of 270 mg/mL (Test group: 2700 μg/mL) was a homogeneous suspension in the most appropriate solvent DMSO. Test substance
suspensions in DMSO were obtained from 8.4 mg/mL (Test group: 84.4 μg/mL) onward. The test substance precipitation (macroscopically) in culture medium was observed at
84.4 μg/mL and above after 4 hours in the absence and presence of S9 mix.

RANGE-FINDING/SCREENING STUDIES: yes

HISTORICAL CONTROL DATA
- Negative (solvent/vehicle): negative control data range (0.0% - 4.7% aberrant metaphases, excl. gaps)
ADDITIONAL INFORMATION ON CYTOTOXICITY:
After 4 hours treatment in the absence and presence of S9 mix, no cytotoxicity was observed as indicated by a reduced relative cloning efficiency of about or below 20% relative survival upthe highest applied concentration of 2700 μg/mL.

Any other information on results incl. tables

Table 1: Results

					Genotoxicity				Cytotoxicity**
	Schedule				Aberrant cells [%]
Exp.	Exposure/ mix Aberrant cells [%] Polyploid RPD Mitotic preparation period	Test groups	S9 mix	precipitation	incl. gaps#	excl. gaps#	rel. [%]	Polyploid cells [%]	RPD number [%]	Mitotic index [%]
1	4/18 hrs	Vehicle control1	-	-	9.0	4.3	0.7	1.3	100.0	100.0
		6.25 µg/mL	-	-	n.d.	n.d.	n.d.	n.d.	100.9	n.d.
		12.50 µg/mL	-	-	n.d.	n.d.	n.d.	n.d.	104.3	n.d.
		25.00 µg/mL	-	-	n.d.	n.d.	n.d.	n.d.	103.3	n.d.
		50.00 µg/mL	-	-	6.3	4.7	1.0	0.0	109.7	113.6
		100.00 µg/mL	-	-	6.7	4.0	0.3	0.3	109.5	113.6
		200.00 µg/mL	-	+	9.3	5.3	0.3	1.3	99.4	109.5
		Positive control2	-	-	12.3	9.0s	2.3	0.3	n.t.	92.8
2	18/18 hrs	Vehicle control1	-	-	12.7	3.7	0.7	0.0	100.0	100.0
		6.25 µg/mL	-	-	n.d.	n.d.	n.d.	n.d.	99.2	n.d.
		12.50 µg/mL	-	-	n.d.	n.d.	n.d.	n.d.	102.6	n.d.
		25.00 µg/mL	-	-	n.d.	n.d.	n.d.	n.d.	100.4	n.d.
		50.00 µg/mL	-	-	10.7	4.3	0.7	1.3	104.6	110.4
		100.00 µg/mL	-	-	10.0	3.0	0.0	0.3	107.3	110.4
		200.00 µg/mL	-	+	8.7	3.3	0.3	2.7	106.6	102.6
		Positive control2	-	-	46.3	34.7s	17.3	1.0	n.t.	78.3
1	4/18 hrs	Vehicle control1	+	-	2.7	0.3	0.0	1.0	100.0	100.0
		6.25 µg/mL	+	-	n.d.	n.d.	n.d.	n.d.	107.5	n.d.
		12.50 µg/mL	+	-	9.3	3.0s	1.3	1.7	117.1	106.9
		25.00 µg/mL	+	-	10.0	3.3s	0.7	1.0	117.7	113.2
		50.00 µg/mL	+	+	9.0	3.7s	0.7	0.7	123.8	128.6
		100.00 µg/mL	+	+	n.d.	n.d.	n.d.	n.d.	113.6	n.d.
		200.00 µg/mL	+	+	n.d.	n.d.	n.d.	n.d.	104.2	n.d.
		Positive control2	+	-	37.0	33.3s	16.0	0.0	n.t.	94.7
2	4/28 hrs	Vehicle control1	+	-	13.3	4.7	1.0	1.3	100.0	100.0
		6.25 µg/mL	+	-	n.d.	n.d.	n.d.	n.d.	98.8	n.d.
		12.50 µg/mL	+	-	n.d.	n.d.	n.d.	n.d.	102.9	n.d.
		25.00 µg/mL	+	-	n.d.	n.d.	n.d.	n.d.	102.6	n.d.
		50.00 µg/mL	+	-	10.3	5.3	0.7	1.7	102.4	90.1
		100.00 µg/mL	+	-	9.0	2.7	0.3	0.0	93.2	90.1
		200.00 µg/mL	+	+	12.7	5.7	1.0	0.7	95.7	68.6
		Positive control2	+	-	29.7	24.0s	9.7	0.0	n.t.	66.0

* Precipitation occured at the end of exposure period

** Relative values compared with the respective vehicle control

# Inclusive cells carrying exchanges

n.d. Not determined

n.t. Not tested

S Aberration frequency statistically significant higher than corresponding control values

1 DMSO 1% (v/v)

2 CPP 0.5 μg/mL

Applicant's summary and conclusion

Conclusions:

The substance is not clastogenic in mammalian cells in vitro.