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Toxicological information

Genetic toxicity: in vitro

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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

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
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
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.