4-[2-[4-[benzylmethyl(ethyl)amino]phenyl]vinyl]-1-(2-hydroxyethyl)pyridinium acetate

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:

07 June 2017 to 24 July 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: forward mutation assay in mammalian cells

Test material

Test material information

Constituent 1

Reference substance name:

4-[2-[4-[benzylmethyl(ethyl)amino]phenyl]vinyl]-1-(2-hydroxyethyl)pyridinium acetate

EC Number:

281-468-5

EC Name:

4-[2-[4-[benzylmethyl(ethyl)amino]phenyl]vinyl]-1-(2-hydroxyethyl)pyridinium acetate

Cas Number:

83950-14-5

Molecular formula:

C24H27N2O.C2H3O2

IUPAC Name:

4-[2-[4-[benzylmethyl(ethyl)amino]phenyl]vinyl]-1-(2-hydroxyethyl)pyridinium acetate

Test material form:

liquid

Method

Target gene:

TK

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Eurofins Munich stock culture
- Cell cycle doubling time: 10-12 hours
- Modal number of chromosomes: near diploid karyotype (40 ± 2 chromosomes)
- Normal (negative control) cell cycle time: 10-12
- Cloning Efficiency: usually more than 50%.

MEDIA USED
- Type and identity of media : RPMI 1640 complete medium
- Properly maintained: yes, cleansed and stored over liquid nitrogen (after thawing subcultured three times per week.)
- Periodically checked for Mycoplasma contamination: yes

Metabolic activation:

with and without

Metabolic activation system:

S9 liver microsomal fraction was prepared at Eurofins Munich. Male Wistar rats were induced with phenobarbital (80 mg/kg bw) and ß-naphthoflavone (100 mg/kg bw) for three consecutive days by oral route.

Test concentrations with justification for top dose:

without metabolic activation: 10, 25, 55, 60, 65, 70 and 90 µg/mL
with metabolic activation: 25, 40, 60, 70, 75, 80 and 90 µg/mL

Concentrations based on toxicity pre-test showing reduction of suspension growth at 50 µg/mL (RSG <10%) without metabolic activation and 150 µg/mL (RSG < 10%) with metabolic activation

Vehicle / solvent:

1% DMSO v/v

Controls

Untreated negative controls:

yes

Remarks:

treatment medium

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

ethylmethanesulphonate

methylmethanesulfonate

Remarks:

for the positive controls the number of mutants was > GEF (126)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding for cloning efficiency 1.6 cell/well
- Cell density at seeding for mutant determination: 2000 cells on 200 uL/well

DURATION
- Exposure time: 4 hours with and without metabolic activation in 11 mL RPMI medium with 5% horse serum (25 cm2 flasks) (10E7 cells)
- Expression time (cells in growth medium): 2 days at 37 °C in 5% CO2/95% humidified air in RPMI medium
- Selection time: incubationwith TFT during 12 days at 37 °C in 5% CO2/95% humidified air

NUMBER OF REPLICATIONS: 2 for cloning efficiency; 4 for mutagenicity

DETERMINATION OF CYTOTOXICITY
- Method: relative suspension growth/relative total growth

Rationale for test conditions:

accoding to OECD 490

Evaluation criteria:

Positive (mutagenetic) result:
- The induced mutant frequency meets or exceeds the Global Evaluation factor (GEF) of 126 mutants per 106 cells and
- dose-dependent increase in mutant frequency is detected.

Besides, combined with a positive effect in the mutant frequency, an increased occurrence of small colonies (≥40% of total colonies) is an indication for potential clastogenic effects and/or chromosomal aberrations.
According to the OECD guideline, the biological relevance is considered first for the interpretation of results. Statistical methods might be used as an aid in evaluation of the test result.

A test item is considered to be negative if the induced mutant frequency is below the GEF and the trend of the test is negative.

Results and discussion

Test results

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

RTG 11.5% at 90 ug/mL without metabolic activation and 11% at 90 ug/mL with metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks:

MF [mutants / 106 cells] > 600; % small colonies > 40%

Any other information on results incl. tables

Main Experiment without metabolic activation

	Test Group	Conc.	RCE^a[%]	RTG^b[%]	MF^c[mutants/ 10^6cells]	IMF^d[mutants/ 10^6cells]	GEF^eexceeded	Statistical Significant Increase^f	Precipitate
		[µg/mL]
Exp without S9	C1	0	90.0	99.1	69.7	/	/	/	-
	C2		95.8	110.4		/	/	/	-
	S1	0	100.0	100.0	57.2	/	/	/	-
	S2					/	/	/	-
	2	10	95.8	96.9	50.4	-6.8	-	-	-
	3	25	109.4	90.2	45.4	-11.8	-	-	-
	6	55	98.9	55.0	67.7	10.6	-	-	-
	7	60	87.3	36.7	52.0	-5.2	-	-	-
	8	65	102.2	29.2	55.6	-1.5	-	-	-
	9	70	91.4	16.4	43.7	-13.5	-	-	-
	13	90	95.8	11.5	44.6	-12.6	-	-	-
	EMS	300	82.2	82.2	634.7	577.6	+	+	-
	MMS	10	61.6	60.1	676.6	619.4	+	+	-
Main Experiment with metabolic activation
Exp with S9	C1	0	88.7	93.0	64.7	/	/	/	-
	C2		97.4	100.4		/	/	/	-
	S1	0	100.0	100.0	65.0	/	/	/	-
	S2					/	/	/	-
	3	25	104.0	89.4	37.6	-27.4	-	-	-
	4	40	104.0	66.2	52.7	-12.3	-	-	-
	7	60	81.1	38.6	91.5	26.5	-	-	-
	9	70	87.4	29.8	79.3	14.3	-	+	-
	10	75	79.9	17.2	81.3	16.4	-	-	-
	11	80	100.6	19.6	56.2	-8.8	-	-	-
	13	90	76.5	11.0	87.3	22.3	-	-	-
	B[a]P	2.5	72.2	48.5	955.9	890.9	+	+	-

 

C: Negative Controls

S: Solvent Controls

a: Relative Cloning Efficiency, RCE = [(CE_{dose group}/ CE_{of corresponding controls}) x 100]

Cloning Efficiency, CE = ((-LN (((96 - (mean P1,P2)) / 96)) / 1.6) x 100)

b: Relative Total Growth, RTG = (RSG x RCE)/100

c: Mutant Frequency, MF = {-ln [negative cultures/total wells (selective medium)] / -ln [negative cultures/total wells (non selective medium)]}x800

d: Induced Mutant Frequency, IMF = mutant frequency sample – mean value mutant frequency corresponding controls

e: Global Evaluation Factor, GEF (126); +: GEF exceeded, -: GEF not exceeded

f: statistical significant increase in mutant frequency compared to solvent controls (Mann Whitney test , p<0.05).
+: significant; -not significant

EMS Ethylmethanesulfonate; MMS:Methylmethanesulfonate; B[a]P:Benzo[a]pyrene

Applicant's summary and conclusion

Conclusions:

The substance is considered to be non-mutagenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells.

Executive summary:

In an vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells according to OECD 490, cells were exposed to the substance at concentrations between 1 and 90 ug/mL for 4 hours (with and without metabolic activation). The concentrations were based on cytotoxicity (as RGF). No increase in mutant colonies compared to vehicle and medium controls was observed. Therefore it can be concluded that the substance is non-mutagenic in this assay.