N,N'-dibutyl-N,N'-bis(1,2,2,6,6-pentamethylpiperidin-4-yl)-6-(pyrrolidin-1-yl)-1,3,5-triazine-2,4-diamine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016 / 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: HPRT Locus assay

Test material

Test material information

Constituent 1

Reference substance name:

N,N'-dibutyl-N,N'-bis(1,2,2,6,6-pentamethylpiperidin-4-yl)-6-(pyrrolidin-1-yl)-1,3,5-triazine-2,4-diamine

EC Number:

940-393-4

Cas Number:

1702355-94-9

Molecular formula:

C35 H66 N8

IUPAC Name:

N,N'-dibutyl-N,N'-bis(1,2,2,6,6-pentamethylpiperidin-4-yl)-6-(pyrrolidin-1-yl)-1,3,5-triazine-2,4-diamine

Test material form:

solid: bulk

Details on test material:

- State of aggregation: solid

Method

Target gene:

HPRT locus

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: derived from the Chinese hamster
- Modal number of chromosomes: 20
- Normal (negative control) cell cycle time: 12-16h

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Ham's F12 medium containing stable glutamine and hypoxanthine (PAN Biotech; Cat. No. P04-15500) supplemented with 10% (v/v) fetal calf serum (FCS).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital and β-naphthoflavone induced rat liver S9 mix

Test concentrations with justification for top dose:

Based on the data and the observations from the pretest and taking into account the current guidelines, the following doses were selected in this study.
First Experiment:
Without S9 mix, 4-hour exposure: 0.78 - 50 ug/ml
With S9 mix, 4-hour exposure: 0.78 - 50 ug/ml

Second Experiment:
Without S9 mix, 4-hour exposure: 2.50 - 40 ug/ml
With S9 mix, 4-hour exposure: 2.50 - 40 ug/ml

Third Experiment:
With S9 mix, 4-hour exposure: 2.5 - 40 ug /ml

Vehicle / solvent:

In comparison to other commonly used vehicles (e.g. DMSO, acetone etc.) tetrahydrofurane (THF) is the most suitable. Therefore, THF was selected as the vehicle, which had been demonstrated to be suitable in the CHO/HPRT assay. The final concentration of the vehicle THF in culture medium was 0.5% (v/v).

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): in 300 cm² flasks, 20x106 cells in 40 mL)

DURATION
- Exposure duration: 4h
- Expression time (cells in growth medium): 3d incl. 2 passages
- Fixation time (start of exposure up to fixation or harvest of cells): days 7-9 Drying, fixation, staining and counting of the cloning efficiency 1 3rd passage of the treated cells; addition of selection medium ("TG" medium); and seeding of the cloning efficiency 2 (viability), from day 16 Drying, fixation, staining and counting of the selected colonies and cloning efficiency 2

NUMBER OF REPLICATIONS: each dose in triplicate, three independent experiments

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: medium was removed and the remaining colonies were fixed with methanol, stained with Giemsa and counted

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

OTHERS:

Changes in pH were recorded by a change in the indicator color of the culture medium (phenol red: no color change from pH 6.7 - 8.3). The pH was measured for the top concentrations and for the vehicle controls with and without S9 mix.

Osmolality was measured in the top concentrations and the vehicle controls with and without S9 mix.

Test substance precipitation was assessed immediately after dosing the test cultures and at the end of treatment.

Cell morphology: The test cultures of all test substance concentrations were examined microscopically for cell morphology and cellular attachment at the end of the exposure period, which is a further indication for cytotoxicity.

Evaluation criteria:

A test substance is considered to be clearly positive if all following criteria are met:
• A statistically significant increase in mutant frequencies is obtained.
• A dose-related increase in mutant frequencies is observed.
• The corrected mutation frequencies (MFcorr.) exceeds both the concurrent negative/vehicle control value and the range of our laboratory’s historical negative control data (95% control limit).
Isolated increases of mutant frequencies above our historical negative control range or isolated statistically significant increases without a dose-response relationship may indicate a biological effect but are not regarded as sufficient evidence of mutagenicity.
A test substance is considered to be clearly negative if the following criteria are met:
• Neither a statistically significant nor dose-related increase in the corrected mutation frequencies is observed under any experimental condition.
• The corrected mutation frequencies in all treated test groups is close to the concurrent vehicle control value and within the range of our laboratory’s historical negative control data (95% control limit).

Statistics:

An appropriate statistical trend test (MS EXCEL function RGP) was performed to assess a possible dose-related increase of mutant frequencies. The used model is one of the proposed models of the International Workshop on Genotoxicity Test procedures Workgroup Report (6). The dependent variable was the corrected mutant frequency and the independent variable was the concentration. The trend was judged as statistically significant whenever the one-sided p-value (probability value) was below 0.05 and the slope was greater than 0. In addition, a pair-wise comparison of each test group with the vehicle control group was carried out using one-sided Fisher's exact test with Bonferroni-Holm correction (7, 8). The calculation was performed using R (9).
If the results of these tests were statistically significant compared with the respective vehicle control, labels (s p ≤ 0.05) are printed in the tables. However, both, biological and statistical significance are considered together.

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

CYTOTOXICITY
Cytotoxic effects, as indicated by clearly reduced cloning efficiencies of about or below 20% of the respective negative control values were observed in the 2nd Experiment in the absence of S9 mix and in the 1st and 2nd Experiment in the presence of S9 mix at the highest applied concentrations. Clearly reduced cell densities in the 2nd passage were obtained from about 40.00 μg/mL onward. Those test groups were discontinued. In addition, a clearly reduced cloning efficiency of below 20% of the respective vehicle control value was observed in the 1st Experiment in the presence of S9 mix at 50.00 μg/mL (CE1 relative: 8.1%).

CELL MORPHOLOGY
After 4 hours treatment, the cell morphology and attachment of the cells was not adversely influenced (grade > 2) at the highest applied concentrations in the 1st and 2nd Experiment in the absence of S9 mix in the 2nd Experiment in the presence of S9 mix and.

TREATMENT CONDITIONS
Osmolality and pH values were not influenced by test substance treatment. In this study, test substance precipitation was observed macroscopically in culture medium at the end of treatment from about 15.00 μg/mL onward under all experimental conditions

Any other information on results incl. tables

Summary tables for the three experiments are attached

Applicant's summary and conclusion

Conclusions:

In this study, in the 1st Experiment in the absence of metabolic activation and in the 1st and 2nd Experiment in the presence of metabolic activation the highest concentrations tested were clearly cytotoxic. In all experimental parts, the highest concentrations evaluated for gene mutations showed strong precipitation in culture medium. Two single statistically significant increased mutant frequencies as well as a positive trend in mutant colonies in the 1st Experiment in the presence of S9 mix were considered as not biological relevant.
Based on the results of the present study, the test substance did not cause any biologically relevant increase in the mutant frequencies either without S9 mix or after the addition of a metabolizing system in three experiments performed independently of each other.
Thus, under the experimental conditions of this study, the test substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.