N,N',N'',N'''-tetrakis(4,6-bis(butyl-(N-methyl-2,2,6,6-tetramethylpiperidin-4-yl)amino)triazin-2-yl)-4,7-diazadecane-1,10-diamine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

N,N',N'',N'''-tetrakis(4,6-bis(butyl-(N-methyl-2,2,6,6-tetramethylpiperidin-4-yl)amino)triazin-2-yl)-4,7-diazadecane-1,10-diamine

EC Number:

401-990-0

EC Name:

N,N',N'',N'''-tetrakis(4,6-bis(butyl-(N-methyl-2,2,6,6-tetramethylpiperidin-4-yl)amino)triazin-2-yl)-4,7-diazadecane-1,10-diamine

Cas Number:

106990-43-6

Molecular formula:

C132 H250 N32

IUPAC Name:

N2-[2-({4,6-bis[butyl(1,2,2,6,6-pentamethylpiperidin-4-yl)amino]-1,3,5-triazin-2-yl}[3-({4,6-bis[butyl(1,2,2,6,6-pentamethylpiperidin-4-yl)amino]-1,3,5-triazin-2-yl}amino)propyl]amino)ethyl]-N2-[3-({4,6-bis[butyl(1,2,2,6,6-pentamethylpiperidin-4-yl)amino]-1,3,5-triazin-2-yl}amino)propyl]-N4,N6-dibutyl-N4,N6-bis(1,2,2,6,6-pentamethylpiperidin-4-yl)-1,3,5-triazine-2,4,6-triamine

Method

Species / strain

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: human lymphocytes
Main Assay1: human whole blood (BioIVT (UK))
Main Assay2, 3: human whole blood (Biopredic International (France)), two batches (pooled)
- Normal cell cycle time (negative control): 1.5-2.0 cell cycle length

For lymphocytes:
- Sex, age and number of blood donors:
Main Assay 1 -1 donor: Female, Age 27 years old, (ERBC Code Number 2020/36)
Main Assay 2 - 2 donors: Male, Age 24 years old, (ERBC Code Number 2020/63), Female, Age 33 years old (ERBC Code Number 2020/64)
Main Assay 3 - 2 donors: Male, Age 23 years old, (ERBC Code Number 2021/11), Female, Age 23 years old (ERBC Code Number 2021/12)
- Whether whole blood or separated lymphocytes were used: whole blood
- Whether blood from different donors were pooled or not: pooled
- Mitogen used for lymphocytes: phytohaemagglutinin (PHA)

CULTURE MEDIUM
- RPMI 1640 1x (Dutch modification): 500mL
- Foetal Calf Serum: 100mL
- L-Glutamine (200mM): 6.25mL
- Antibiotic solution: 1.25mL

- foetal calf serum was heat-inactivated at 56°C for 20 minutes before use.
- For the initiation of the cultures, medium with the addition of phytohaemagglutin (PHA) was used
in the following proportion: 10mL of PHA was added to 500mL of medium.

Cytokinesis block (if used):

Cytochalasin B

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital – 5,6-Benzoflavone

Test concentrations with justification for top dose:

Details on the test concentrations are given in table in "Any other information on materials and methods incl. tables"

Main Assay 1: steep decline of cytotoxicity and no concentration tested showed an adequate cytotoxicity.
Main Assay 2: short treatment series were not consistent with those of Main Assay 1. Following the continuous treatment in the absence of S9 metabolism treatment, marked and moderate cytotoxicity was seen at the two highest dose levels tested of 25.3 and 22.0 μg/mL (64% and 41%, respectively).
Main Assay 3: following treatment in the absence of S9 metabolism, severe cytotoxicity was observed from 18.5 μg/mL onward where few cells or no cells were recovered onto slides.Following treatment in the presence of S9 metabolism, severe cytotoxicity was observed from 27.8 μg/mL onward where no cells were recovered onto slides.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: The solvent proved to be the best solvent and a clear solution was obtained

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

ethanol (1%)

True negative controls:

no

Positive controls:

yes

Positive control substance:

colchicine

cyclophosphamide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: with S9 mix: 3h; without S9 mix: 3h and 31h
- Expression time (cells in growth medium): 31h (continous treatment, -S9), 32.5h (short treatment, +/-S9)
- Fixation time (start of exposure up to fixation or harvest of cells): not specified

STAIN (for cytogenetic assays): Acridine Orange (0.1 mg/mL in PBS)

NUMBER OF REPLICATIONS:
Two replicate cell cultures were prepared at each test point.

NUMBER OF CELLS EVALUATED:
- selected doses, untreated, solvent controls, positive control (Cyclophosphamide): 1000 binucleated cells per culture were scored.
- cultures treated with colchicine: 1000 mononucleated cells per cell culture were scored.

DETERMINATION OF CYTOTOXICITY
CBPI was used to measure the cytotoxic effect.
CBPI = (mononucleated + 2xbinucleated + 3x multinucleated)/ total number of cells counted
%Cytotoxicity = 100-100(CBPIt -1/CBPIc-1), t= test item treated culture, c= solvent control culture


POSITIVE CONTROLS
- Without metabolic activation: Colchicine (batch no. SLBX1712, obtained from Sigma).
- With metabolic activation: Cyclophosphamide (labelled as: Cyclophosphamide monohydrate, batch no. MKCG5464, obtained from Sigma)
- All positive control items were dissolved immediately prior to use in sterile water of injectable grade (batch no. 1901341 obtained from Galenica Senese).

Evaluation criteria:

The test item is considered as clearly positive if the following criteria are met:
– Significant increases in the proportion of micronucleated cells over the concurrent controls occur at one or more concentrations.
– The proportion of micronucleated cells at such data points exceeds the normal range based on historical control values (95% control limits).
– There is a significant dose effect relationship.

The test item is considered clearly negative if the following criteria are met:
– None of the dose levels shows a statistically significant increase in the incidence of micronucleated cells.
– There is no concentration related increase when evaluated with the Cochran-Armitage trend test.
– All the results are inside the distribution of the historical control data (95% control limits)

Statistics:

For the statistical analysis, a modified χ2 test was used to compare the number of cells with
micronuclei in control and treated cultures.
Cochran-Armitage Trend Test (one-sided) was performed to aid determination of concentration response relationship.

Results and discussion

Test results

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Refer to explanation for the third experiment, where dose levels spaced by a narrow interval were used, which ranged from the higher and lower doses tested in the previous experiments. (+ S9, highest dose: 100 µg/mL, -S9, highest dose 75 µg/mL).

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: no effect
- Data on osmolality: no effect
- Possibility of evaporation from medium:
- Water solubility: low (see preliminary study)
- Precipitation and time of the determination: no precipitation or opacity of the medium visible by eye was noticed at the beginning or by the end of treatment at any dose level.
- Definition of acceptable cells for analysis:
> The micronucleus diameter was less than 1/3 of the nucleus diameter.
> The micronucleus diameter was greater than 1/16 of the nucleus diameter.
> No overlapping with the nucleus was observed.
> Micronuclei were non-refractile and had the same staining intensity as the main nuclei.

PRELIMINARY STUDY:
This study was performed due to the low solubility of the test item in aqueous solvents. It was performed using DMSO, acetone and ethanol, with ethanol proved to be the best solvent and a clear solution was obtained at the concentration of 112 mg/mL corresponding to 100 mg/mL as active ingredient.

COMPARISON WITH HISTORICAL CONTROL DATA:
Following treatment with the test item, all incidences of micronucleated cells were within the distribution of historical negative control (95% confidence limits), no statistically significant increase over the concurrent solvent control value was observed at any concentration tested nor dose effect relationship was seen.

Any other information on results incl. tables

Incidence of micronucleated cells (%) after treatment with the test item in comparison to historical control data

Main Assay	S9	Treatment time (hours)	Harvest Time (hours)	Dose level (µg/mL)	Cytotoxicity (%)	Presence of precipitation/ opacity
2	-	31	31	Untreated solvent 22.0 19.1 16.6	0.45 0.40 0.60 0.50 0.50	- - NS NS NS
				Linear trend                                               NS Historical control data (95% confidence limits)                0.00-1.00
3	-	3	32.5	Untreated solvent 14.8 9.88 6.60	0.30 0.30 0.55 0.20 0.15	- - NS NS NS
				Linear trend                                               NS Historical control data (95% confidence limits)                0.00-0.77
	+			Untreated solvent 22.2 14.8 9.88	0.30 0.20 0.40 0.15 0.50	-   NS NS NS
				Linear trend                                               NS Historical control data (95% confidence limits)                0.00-0.95

Applicant's summary and conclusion

Conclusions:

On the basis of the results obtained, it is concluded that the test item does not induce micronuclei in human lymphocytes after in vitro treatment, under the reported experimental conditions.

Executive summary:

The test item was assayed for the ability to induce micronuclei in human lymphocytes, following in vitro treatment in the presence and absence of S9 metabolic activation. Three treatment conditions were performed. A short term treatment, where the cells were treated for 3 hours, was performed in the absence and presence of S9 metabolism. The harvest time of 32.5 hours, corresponding to approximately two cell cycle lengths, was used. A long term (continuous) treatment was also performed only in the absence of S9 metabolism, until harvest at 31 hours. Solutions of the test item were prepared in ethanol. Based on the solubility of the test item, a Main Assay 1 was performed where treatments were as follows:

Main Assay	S9	Treatment time (hours)	Harvest Time (hours)	Dose level (µg/mL)
1	-	3	32.5	250, 167, 111, 74.1, 49.4, 32.9, 21.9, 14.6 and 9.75
	+
	-	31	31	250, 167, 111, 74.1, 49.4, 32.9, 21.9, 14.6, 9.75 and 6.50

 

Since for all treatment series, a steep decline of cytotoxicity was observed and no concentration tested showed an adequate cytotoxicity to assess the frequency of micronucleated cells, a Main Assay 2 was performed where treatments were spaced by a narrower interval (1.15). Treatments were performed as follows:

Main Assay	S9	Treatment time (hours)	Harvest Time (hours)	Dose level (µg/mL)
2	-	3	32.5	25.2, 21.9, 19.0, 16.5, 14.4, 12.5, 10.9, 9.46, 8.22 and 7.15
	+			33.3, 28.9, 25.2, 21.9, 19.0, 16.5, 14.4 and 12.5
	-	31	31	25.3, 22.0, 19.1, 16.6, 14.5, 12.6, 10.9, 9.51, 8.27 and 7.19

Possibly due to the properties of the test item, no reproducible results of cytotoxcity results were obtained with the short exposure both in the absence and presence of S9 metabolism. In agreement with the Sponsor, a further experiment (Main Assay 3), was performed by using dose levels spaced by a narrow interval and ranging from the higher and lower doses tested in the previous experiments. Treatments were as follows:

Main Assay	S9	Treatment time (hours)	Harvest Time (hours)	Dose level (µg/mL)
3	-	3	32.5	75.0, 50.0, 33.3, 27.8, 22.2, 18.5, 14.8, 9.88 and 6.60
	+			100, 75.0, 50.0, 41.7, 33.3, 27.8, 22.2, 14.8, 9.88 and 6.60

 

Each experiment included appropriate negative and positive controls. Two replicate cell cultures were prepared at each test point. The actin polymerisation inhibitor Cytochalasin B was added prior to the targeted mitosis to allow the selective analysis of micronucleus frequency in binucleated cells. The cytokinesisblock proliferation index CBPI was calculated in order to evaluate cytotoxicity. Dose levels for the scoring of micronuclei were selected with the aim to evaluate the test item concentrations at adequate levels of cytotoxicity, covering a range from a moderate to slight or no toxicity. Due to the steep decline of cytotoxicity observed after the short exposure both in the absence and presence of S9, the highest treatment level with no cytotoxicity obtained in Main Assay 3, was selected as the highest dose level for scoring. The next higher dose levels (18.5µg/mL and 27.8µg/mL respectively) could not be evaluated due to excessive cytotoxicity (very few or no cells). Based on the results obtained, the following concentrations were selected for the scoring of micronuclei:

 

Main Assay	S9	Treatment time (hours)	Harvest Time (hours)	Dose level (µg/mL)	Cytotoxicity (%)	Presence of precipitation/ opacity
2	-	31	31	0.00 22.0 19.1 16.6	- 41 8 5	- Np No No
3	-	3	32.5	0.00 14.8 9.88 6.60	- 8 10 9	- Np No No
	+			0.00 22.2 14.8 9.88	- 11 6 8	- Np No No

One thousand binucleated cells per culture were scored to assess the frequency of micronucleated cells. Adequate cell proliferation was observed in untreated and solvent control cultures and the appropriate number of doses and cells was analysed. Statistically significant increases in the incidence of micronucleated cells were observed following treatments with the positive controls Cyclophosphamide and Colchicine and the responses were compatible with those generated in our historical control database, indicating the correct functioning of the test system. The study was accepted as valid. Following treatment with the test item, all incidences of micronucleated cells were within the distribution of historical negative control (95% confidence limits), no statistically significant increase over the concurrent solvent control value was observed at any concentration tested nor dose effect relationship was seen.

It is concluded that the test item does not induce micronuclei in human lymphocytes after in vitro treatment, under the reported experimental conditions.