N,N'-[1,4-phenylenebis(methylene)]bis{N,N-dimethyl-3-[(prop-2-enoyl)amino]propan-1-aminium} dichloride

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 Oct 2019 - 27 Feb 2020

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Only 2-aminoanthracene was used to test the efficacy of the S9-mix. Additional mutagens requiring metabolic activation such as benzo(a)pyrene or dimethylbenzanthracene were not used to characterise the S9-mix.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Adopted in 1997

Deviations:

yes

Remarks:

Only 2-aminoanthracene was used to test the efficacy of the S9-mix. Additional mutagens requiring metabolic activation such as benzo(a)pyrene or dimethylbenzanthracene were not used to characterise the S9-mix.

GLP compliance:

yes (incl. QA statement)

Remarks:

Slovak National Accreditation Service, Bratislava, Slovak Republic

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

{[4-({dimethyl[3-(prop-2-enamido)propyl]azaniumyl}methyl)phenyl]methyl}dimethyl[3-(prop-2-enamido)propyl]azanium dichloride

EC Number:

812-548-5

Cas Number:

1621424-03-0

Molecular formula:

C24H40Cl2N4O2

IUPAC Name:

{[4-({dimethyl[3-(prop-2-enamido)propyl]azaniumyl}methyl)phenyl]methyl}dimethyl[3-(prop-2-enamido)propyl]azanium dichloride

Method

Target gene:

his operon for S. typhimurium strains and trp operon for the E. coli strain

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 97

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 98

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 100

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 1535

Species / strain 5

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : The mammalian liver post-mitochondrial fraction (S9) used for metabolic activation was prepared from Sprague Dawley male rats (Charles River, Velaz, Czech Republic). Animals were pre-treated with 20-methylcholanthrene (administered i.p. at 80 mg/kg bw) 5 days prior to killing.
- method of preparation of S9 mix: The S9 homogenate was diluted with co-factors: 33 mM KCl, 8 mM MgCl2, 5 mM, glucose-6-phosphate, 4 mM NADP, and 100 mM phosphate buffer (pH = 7.4) to obtain the S9-mix.
- concentration or volume of S9 mix and S9 in the final culture medium: The S9 concentration in the S9-mix was 10%. The volume of S9-mix in the final culture medium was 0.5 mL.
- quality controls of S9: The activity of S9 fraction was determined in bacterial reverse gene mutation test on Salmonella typhimurium strains TA98 and TA100.

Test concentrations with justification for top dose:

Range-finding experiment: 50, 150, 500, 1500, 2500, and 5000 µg/plate, without metabolic activation, TA 100, TA 98
Main experiment: 50, 150, 500, 1500, 2500, and 5000 µg/plate, with and without metabolic activation, all strains
5000 µg/plate was selected as the highest test concentration based on the results of a range-finding experiment in which no cytotoxicity was observed up to and including the highest concentration.

Vehicle / solvent:

- Vehicle used: sterile deionised (purified) water

- Justification for choice of vehicle: The vehicle used did not affect the spontaneous mutation level and it is recommended for use in this test.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-aminoanthracene

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: single experiment

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 1E+09 cells/mL
- Test substance added in agar (plate incorporation).

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 48 – 72 h

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition

METHODS FOR MEASUREMENTS OF GENOTOXICIY
After the incubation period, the number of revertant colonies per plate was counted by hand. The mutation frequency at each dose concentration level of the test substance was compared to the one observed in negative and positive controls.

Evaluation criteria:

Considering biological relevance the test substance is considered positive if the assay is valid and the following conditions are met:
- Concentration-related increase over the tested range and reproducible increase at one or more concentrations in the number of revertant colonies per plate in at least one strain with or without metabolic activation.
- Mutation factor > 2.
A positive result indicates that the test substance induces mutations in Salmonella typhimurium or E.coli.
The test substance for which results do not meet the above criteria is considered non-mutagenic in this test.
Negative results indicate that under the reported test conditions, the test substance does not produce mutations in Salmonella typhimurium and E.coli.

Statistics:

Mean values and standard deviation were calculated.
The mutation frequency (MF) at each tested concentration of the test substance was compared to the MF observed in negative and positive controls.
The statistical analysis was carried out using unpaired T-test.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 97

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDY
A range-finding assay to determine the optimal non-toxic test concentrations of FV-1295 was performed. Six concentrations of the test item (50, 150, 500, 1500, 2500, and 5000 µg/plate) were tested using Salmonella typhimurium test strains TA 100 and TA 98 without metabolic activation. No decrease in the number of revertant colonies was observed up to the highest tested concentration.

STUDY RESULTS
Ames test:
- Signs of toxicity: No signs of toxicity were noted in any strain up to and included the highest concentration tested, i.e 5000 µg/plate.
- Mean number of revertant colonies per plate and standard deviation: the mean number of revertant colonies was not significantly increased for any strains at any concentration tested. Please see Table 3 under "Any other information on results incl. tables".

HISTORICAL CONTROL DATA
In general, the results of the present study fall within the historical control data (HCD) range (Table 1 under "Any other information on results incl. tables"). For TA 100, with metabolic activation, mean levels of spontaneous mutations were slightly above the HCD range (252 revertants/plate, HCD range: 120 - 231 revertants/plate). For TA 98, with metabolic activation, mean levels of mutations induced by the positive control were above the HCD range (2417 revertants/plate, HCD range: 150 - 1644 revertants/plate). For WP2uvrA, with and without metabolic activation, mean levels of mutations induced by the positive control were above the HCD range (without S9: 1033 revertants/plate, HCD range: 82 - 912 revertants/plate; with S9: 855 revertants/plate, HCD range: 194 - 648 revertants/plate). Although the mean numbers of revertants per plate were increased, no increase was observed after treatment with the test substance. Therefore, these differences are not considered to affect the final outcome of the study.

Any other information on results incl. tables

 Table 1: Historical negative and positive control values 2015 - 2019

Historical control data
Revertants per plate
		Metabolic activation
		without				with
		Mean	SD	Min	Max	Mean	SD	Min	Max
TA 100	Neg	189	37	112	272	177	30	120	231
TA 100	Pos	691	170	440	1148	1025	434	398	2888
TA 1535	Neg	22	8	6	43	23	8	11	43
TA 1535	Pos	615	171	336	964	193	127	34	604
TA 97	Neg	161	30	114	227	193	36	127	286
TA 97	Pos	1088	525	400	2776	870	367	370	1616
TA 98	Neg	24	7	12	43	35	11	19	85
TA 98	Pos	261	144	117	680	754	446	150	1644
WP2uvrA	Neg	36	11	18	68	44	9	31	70
WP2uvrA	Pos	377	268	82	912	387	136	194	648

SD = Standard deviation

Min = minimum value

Max = maximum value

Neg = negative control

Pos = positive control

Table 2: Results of dose range finding assay without metabolic activation

Without (-) S9-Mix	Test substance concentration	Mean number of revertant colonies per plate
	(μg/plate)	(average of 3 plates ± Standard deviation)
		Base-pair substitution type	Frameshift type
		TA 100	TA 98
-	vehicle (water)	369 ± 24	19 ± 5
-	50	370 ± 12	22 ± 5
-	150	359 ± 9	20 ± 6
-	500	345 ± 12	20 ± 4
-	1500	371 ± 21	19 ± 2
-	2500	369 ± 17	27 ± 2
-	5000	373 ± 11	22 ± 2
Positive controls, -S9	Name	Na-azide	2-NF
	Concentrations (μg/plate)	1.5	3
	Mean No. of colonies/plate (average of 3 ± SD)	1165* ± 84	117* ± 8

Na-azide = Sodium azide

2-NF = 2-nitrofluorene

* = p < 0.05

Table 3: Results of Ames test with FV-1295 (plate incorporation)

With (+) or without (-) S9-Mix	Test substance concentration	Mean number of revertant colonies per plate
	(μg/plate)	(average of 3 plates ± Standard deviation)
		Base-pair substitution type			Frameshift type
		TA 100	TA1535	WP2uvrA	TA 98	TA 97
-	vehicle (water)	257 ± 11	22 ± 1	40 ± 3	18 ± 2	173 ± 20
-	50	255 ± 10	25 ± 8	43 ± 5	21 ± 3	168 ± 25
-	150	243 ± 13	20 ± 2	41 ± 7	19 ± 2	179 ± 34
-	500	236 ± 11	24* ± 2	42 ± 7	18 ± 2	180 ± 25
-	1500	251 ± 3	19 ± 3	40 ± 4	21 ± 3	174 ± 9
-	2500	254 ± 15	18* ± 2	45 ± 5	19 ± 1	188 ± 22
-	5000	247 ± 20	23 ± 4	42 ± 6	20 ± 5	185 ± 21
Positive controls, -S9	Name	Na-azide	Na-azide	4-NQO	2-NF	9-AA
	Concentrations (μg/plate)	1.5	1.5	5	3	75
	Mean No. of colonies/plate (average of 3 ± SD)	891* ± 47	947* ± 113	1033* ± 70	177* ± 17	987* ± 49
+	vehicle (water)	252 ± 9	19 ± 3	46 ± 3	20 ± 2	188 ± 30
+	50	258 ± 4	19 ± 1	52 ± 3	19 ± 1	189 ± 5
+	150	253 ± 7	18 ± 4	51 ± 2	22 ± 3	185 ± 9
+	500	255 ± 5	20 ± 4	51 ± 6	21 ± 2	188 ± 4
+	1500	256 ± 14	19 ± 2	50 ± 3	18 ± 1	180 ± 16
+	2500	253 ± 7	18 ± 2	49 ± 1	22 ± 3	175 ± 12
+	5000	257 ± 7	19 ± 4	52 ± 3	23 ± 4	177 ± 19
Positive controls, +S9	Name	2-AA	2-AA	2-AA	2-AA	2-AA
	Concentrations (μg/plate)	1	5	5	5	5
	Mean No. of colonies/plate (average of 3 ± SD)	2692* ± 60	178* ± 15	855* ± 87	2417* ± 293	1007* ± 44

Na-azide = Sodium azide

4-NQO = 4-nitroquinoline-N-oxide

2-NF = 2-nitrofluorene

9-AA = 9-aminoacridine

2-AA = 2-Aminoanthracene

* = p < 0.05

Applicant's summary and conclusion

Conclusions:

Under the conditions of the present Ames test, the test substance FV-1295 was negative for genotoxicity with and without metabolic activation.