1,3,5-Triazine-2,4-diamine, N2-[(1R,2S)-2,3-dihydro-2,6-dimethyl-1H-inden-1-yl]-6-(1-fluoroethyl)-

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Test material

Test material information

Constituent 1

Reference substance name:

N-[(1R,2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl]-6-[(1R)-1-fluoroethyl]-1,3,5-triazine-2,4-diamine

EC Number:

619-749-5

Cas Number:

730979-19-8

Molecular formula:

C16H20FN5

IUPAC Name:

N-[(1R,2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl]-6-[(1R)-1-fluoroethyl]-1,3,5-triazine-2,4-diamine

Method

Target gene:

HPRT-locus (hypoxanthine-guanine phosphoribosyl transferase locus)

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: PPA Ready Mix, commercially available by PAA, Paching, Austria, consisting of Eagle's minimal essential medium (MEM, Earle) with 10% FCS and supplements. During treatment with AE 1170437 technical, PAA Ready Mix with 2% FCS was used. For selection of mutants, culture medium containing 10 µg/mL of 6-thioguanine (6-TG) was used.
- Properly maintained: yes, V79 cell stocks are stored in liquid nitrogen. Laboratory cultures were maintained in plastic tissue culture vessels at 37 °C in a humidified atmosphere containing approximately 5% CO2. Exponential growth of cell cultures was maintained by subculturing at least twice a week. For cell detachment in order to subculture, an adequate dilution (ranging between 1:2.5 and 1:5) in phosphate buffered saline (PBS) of a commercially available PBS solution consisting of 0.5% trypsin and 0.2% EDTA (ethylenediamine- N,N,N',N'-tetraacetic acid) has been employed.
- Periodically checked for Mycoplasma contamination: yes, using a DNA-Staining Kit (Biochrom) according to method provided by supplier
- Periodically checked for karyotype stability: yes, utilising a modified protocol of the method by Moorhead et al. (1960)
- Periodically "cleansed" against high spontaneous background: yes, by subcloning due to plating of about 1000 cells per culture vessel at least every 2 weeks. If necessary, the spontaneous frequency of HPRT-mutants was additionally reduced by supplementing the culture medium with thymidine (9 µg/mL), hypoxanthine (10 µg/mL), glycine (22.5 µg/mL) and methotrexate (0.3 µg/mL). A 6-TG sensitive subclone was then used for the HPRT-test.

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9-mix

Test concentrations with justification for top dose:

Pretest: 2.5, 5, 10, 20, 40, 80, 160, 320 µg/mL (± S9)
Main test: 15, 30, 60, 90, 120, 150 µg/mL (-S9); 10, 20, 40, 80, 160, 320 µg/mL (+S9)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (1% v/v)
- Justification for choice of solvent/vehicle: solubility of test substance in this vehicle up to 250 mg/mL

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

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

DURATION
- Exposure duration: 5 hours
- Expression time (cells in growth medium): normally 6 days
- Selection time (if incubation with a selection agent): 6-8 days
- Fixation time (start of exposure up to fixation or harvest of cells): on day 12 to 14, depending on selection period

SELECTION AGENT (mutation assays): 10 µg/mL 6-thioguanine (6-TG)
STAIN: Giemsa

NUMBER OF REPLICATIONS: 2 x 8 plates per concentration per trial, independently repeated

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency, relative total growth

OTHER:
Pretest: Determination of cytotoxicity after 5 hours exposure. Cells were plated out to Petri dishes (3 dishes, 200 cells each), and the colonies formed were counted. Colonies in treated cultures were compared to those in vehicle control cultures (relative cloning efficiency). Based on these findings concentrations were chosen for the main test.

Main test:
The method is based on the publication of Myhr and DiPaolo (1978). Exponentially growing V79 cells (4x10e6) were plated in duplicate flasks and exposed to various test substance concentrations after attachment (16-24 hours) for 5 hours in 20 mL culture medium. The cell monolayers were washed, trypsinised, and 1.5x10e6 cells were replated in new flasks. Additionally, 3 Petri dishes per culture were plated with 200 cells each for determination of colony development and cytotoxicity associated with test substance directly after treatment. The dishes were incubated for 6 days.

The cells in flasks were subcultured after 3 days by reseeding 1.5x10e6 cells into new flasks which were cultured for another 3 days (6 days expression period in total). At the end of the expression period cultures were reseeded in 8 Petri dishes per culture at 3x10e5 cells per dish in selection medium containing 10 µg/mL 6-TG. In addition, 200 cells per dish were seeded into 3 Petri dishes per culture in culture medium without 6-TG to determine absolute cloning efficiency for each concentration. After incubation for 6-8 days colonies were fixed, stained with Giemsa, and 6-TG resistant colonies were counted. At the same time the number of colonies in the cloning efficiency dishes was counted.

The activation assay was performed independently. The procedure was identical to the non-activation assay except for the replacement of 1 mL of culture medium in the flask by 1 mL of S9-mix during the exposure period.

Two trials were performed each with and without activation.

Evaluation criteria:

- The average cloning efficiency of the negative and vehicle controls should be at least 50%.
- The average of mutant frequency of the vehicle controls should not exceed 25x10e-6 cells.
- The mutant frequency of the two cultures of the vehicle and/or the negative control should differ only to an acceptable extent. As a rule of thumb, the difference of mutant frequencies should not be greater than 5x10e-6.
- The positive control should induce an average mutant frequency of at least three times that of the vehicle control.
- If not limited by the solubility of the test substance in the vehicle the highest concentration should induce cytotoxicity of about 80 to 90% or should be a concentration where precipitation occurs in the medium. The survival at the lowest concentration should be in the range of the negative control.
- For the calculation of an acceptable mutant frequency at least 5 dishes per culture should be available and relative survival to treatment, relative population growth and absolute cloning efficiency should be 10% or greater.

Statistics:

Mutant frequencies are submitted to a weighted analysis of variance as well as to a weighted recursive regression, both with Poisson derived weights (Hsie et al., 1981; Arlett et al., 1989).
According to the acceptance criteria mutant frequencies based on less than 5 plates and/or on a relative survival to treatment and/or a relative population growth and/or an absolute cloning efficiency below 10% are not included in the statistical analysis.
The two mutant frequency values obtained per group are considered as independent measurements thus increasing the power of the statistical tests applied. Since the protocol of the HPRT assay requires at least two independent trials, the overall analysis without respectively with activation is the most important one for classifying substances into mutagens and non-mutagens. However, separate analyses were run for each trial in order to examine the consistency of the results.
All acceptable groups are included in the weighted analysis of variance followed by pairwise comparisons to the vehicle control on a nominal significance level of α = 0.05 using the Dunnett test (Dunnett, 1955). The regression analysis part is performed on the basis of the actual concentrations thereby omitting the positive, negative and vehicle controls. If there is a significant concentration related increase of the mutant frequency (α = 0.05) in the main analysis the highest concentration will be dropped and the analysis will be repeated. This procedure will be repeated until p > 0.05. In that way eliminated concentrations are flagged correspondingly.

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:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Concentrations of up to 320 µg/ml AE 1170437 technical did not change the pH in the medium of the pre-test.
- Effects of osmolality: The osmolality in the medium of the pre-test was not changed by concentrations of up to 320 µg/mL AE 1170437 technical.
- Water solubility: The test substance was not soluble in water, therefore, the respective amounts were dissolved in DMSO which did not exceed a final concentration of 1% (v/v) in the medium.
- Precipitation: In the absence of S9-mix substance precipitation occurred in the medium at the concentration 150 µg/mL. With S9-mix substance precipitation was noted at 160 µg/mL and above.


RANGE-FINDING/SCREENING STUDIES:
Precipitation of AE 1170437 technical in the culture medium started at 160 µg/mL. Without S9 mix cytotoxic effects of AE 1170437 technical were observed at 80 µg/mL and above. With S9 mix cytotoxicity was evident at 160 µg/mL and above. Due to these findings, AE 1170437 technical was tested in the respective first mutation experiment in concentrations ranging from 15 µg/mL to 150 µg/mL without metabolic activation and from 10 µg/mL to 320 µg/mL with metabolic activation. The same concentrations were used for the independent repeats.

COMPARISON WITH HISTORICAL CONTROL DATA
The results of the control groups were well comparable with the historic control data provided in the report.

Any other information on results incl. tables

The means of the absolute cloning efficiency for the vehicle controls in the mutation experiments were 69.6% and 98.7% in the experiments without activation. In experiments with metabolic activation 64.2% and 65.2% were observed. These results demonstrate good cloning conditions for the experiments.

Two trials were performed with and without metabolic activation. The mutant frequencies of the negative controls and of the vehicle controls were all within the normal range. The positive controls EMS and DMBA induced clear mutagenic and statistically significant effects in all trials with and without metabolic activation, respectively. All controls were well comparable to the historical data provided in the report.

Without S9-mix clear cytotoxic effects were induced; concentration-related decreases in relative survival to treatment and/or in relative population growth were observed for test substance-treated cultures in the absence of metabolic activation, whereas

no such decreases were found in the presence of metabolic activation. However, precipitation of the test substance in the medium was observed at 150 µg/mL without S9-mix and at 160 and 320 µg/mL with S9-mix. The test substance induced no relevant increases in mutant frequencies. In addition, the overall statistical analysis reveals no statistically significant increases, as well.

V79/HPRT Mutagenesis assay – examples for results (trials with highest mutation frequencies):

Concentration (µg/mL)	Survival to treatment		Relative population growth (% vehicle control)	Total mutant colonies (8 plates)	Absolute cloning efficiency (%) ± SD	Mutant frequency x10e-6
	Mean colony number ± SD	% Vehicle control
Without activation
negative	192.0±25.2	106.3	62.6	0	70.8±4.7	0.0
negative	237.0±9.5	97.9	207.4	3	131.3±21.2	1.0
vehicle	180.7±11.2	100.0	100.0	2	118.0±11.5	0.7
vehicle	242.0±14.7	100.0	100.0	4	79.3±20.7	2.1
EMS900 µg/mL	165.3±6.7	91.5	53.4	192	104.3±8.8	76.7
EMS900 µg/mL	156.7±8.5	64.7	82.2	208	52.7±7.3	164.6
15	231.3±23.7	128.0	80.2	1	64.8±12.1	0.6
15	195.3±4.0	80.7	155.4	6	82.0±9.6	3.0
30	223.7±7.5	123.8	48.0	2	65.8±19.8	1.3
30	190.0±12.5	78.5	108.4	0	83.2±10.0	0.0
60	210.7±20.6	116.6	51.5	4	82.7±5.3	2.0
60	189.3±8.7	78.2	151.5	12	88.7±5.5	5.6
90	206.0±13.0	114.0	64.3	4	108.8±16.6	1.5
90	255.0±16.5	105.4	82.2	3	78.8±5.0	1.6
120	176.0±11.5	97.4	95.8	2	114.7±10.4	0.7
120	212.3±5.1	87.7	40.7	0	80.2±4.3	0.0
150 (P)	228.0±20.7	126.2	42.0	2	112.0±7.1	0.7
150 (P)	197.3±21.1	81.5	45.5	0	81.5±6.9	0.0
With activation
negative	148.3±20.5	89.2	111.2	10	78.3±7.9	5.3
negative	198.7±8.6	134.2	106.6	6	56.3±0.8	4.4
vehicle	166.3±21.6	100.0	100.0	6	58.7±9.5	4.3
vehicle	148.0±12.2	100.0	100.0	5	69.7±3.0	3.0
DMBA 20 µg/mL	82.0±15.4	49.3	14.7	58	69.8±6.0	34.6
DMBA 20 µg/mL	64.3±6.1	43.5	17.6	57	55.3±9.5	42.9
10	168.3±4.0	101.2	143.1	14	52.8±0.8	11.0
10	172.7±13.8	116.7	76.1	5	61.0±4.9	3.4
20	171.0±2.6	102.8	176.5	10	59.0±6.3	7.1
20	175.0±14.1	118.2	87.9	13	70.3±4.6	7.7
40	141.7±9.5	85.2	152.2	6	66.3±4.0	3.8
40	110.0±1.7	74.3	85.2	1	53.3±8.0	0.8
80	153.0±15.5	92.0	109.0	12	67.5±2.0	7.4
80	199.0±19.1	134.5	54.8	12	55.8±3.3	9.0
160 (P)	159.7±11.9	96.0	108.5	10	50.5±10.8	8.3
160 (P)	166.3±8.1	112.4	39.2	13	66.5±3.5	8.1
320 (P)	158.0±18.0	95.0	69.1	4	70.2±3.8	2.4
320 (P)	149.7±16.6	101.1	57.5	9	58.2±1.2	6.4

 P: precipitation

CONCLUSION:

The test substance was evaluated as non-mutagenic with and without metabolic activation under the conditions chosen.