3,6-bis(diethylamino)-9-[2-(ethoxycarbonyl)phenyl]xanthylium chloride

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:

14 July 2016 to 01 August 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial forward mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

3,6-bis(diethylamino)-9-[2-(ethoxycarbonyl)phenyl]xanthylium chloride

EC Number:

219-233-6

EC Name:

3,6-bis(diethylamino)-9-[2-(ethoxycarbonyl)phenyl]xanthylium chloride

Cas Number:

2390-63-8

Molecular formula:

C30H35N2O3.Cl

IUPAC Name:

3,6-bis(diethylamino)-9-[2-(ethoxycarbonyl)phenyl]xanthylium chloride

Test material form:

solid: particulate/powder

Details on test material:

- Appearance: Greenish brown crystalline powder
- Storage conditions of test material: At room temperature
- Stable until: 10 June 2021 (expiry date)

Method

Target gene:

- Histidine requirement in the Salmonella typhimurium strains (Histidine operon).
- Tryptophan requirement in the Escherichia coli strain (Tryptophan operon).

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

- Type and identity of media: Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth (Oxoid) and incubated in a shaking incubator (37 ± 1 °C, 150 rpm), until the cultures reached an optical density of 1.0 ± 0.1 at 700 nm (10^9 cells/mL). Freshly grown cultures of each strain were used for testing.
- Properly maintained: Yes. The Salmonella typhimurium strains are regularly checked to confirm their histidine requirement, crystal violet sensitivity, ampicillin resistance (TA98 and TA100), UV sensitivity and the number of spontaneous revertants. Stock cultures of the strains were stored in liquid nitrogen (-196 °C).

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

- Type and identity of media: Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth (Oxoid) and incubated in a shaking incubator (37 ± 1 °C, 150 rpm), until the cultures reached an optical density of 1.0 ± 0.1 at 700 nm (10^9 cells/mL). Freshly grown cultures of each strain were used for testing.
- Properly maintained: Yes. The strain is regularly checked to confirm the tryptophan requirement, UV-sensitivity and the number of spontaneous revertants. Stock cultures were stored in liquid nitrogen (-196 °C).

Metabolic activation:

with and without

Metabolic activation system:

S9-mix (rat liver S9-mix induced by Aroclor 1254)

Test concentrations with justification for top dose:

- Dose range finding study (TA100 and WP2uvrA only): 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate (absence and presence of S9-mix)
- Experiment 1 (TA1535, TA1537 and TA98): 0.55, 1.7, 5.4, 17, 52 and 164 μg/plate (absence and presence of S9-mix)
- Experiment 2 (all strains): 5, 10, 25, 50, 100 and 164 μg/plate (absence and presence of S9-mix)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide (DMSO)
- Justification for choice of solvent/vehicle: Chosen following a solubility test

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191, 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DOSE RANGE FINDING TEST/ MUTATION ASSAY
Selection of an adequate range of doses was based on a dose range finding test with the strains TA100 and WP2uvrA, both with and without 5 % (v/v) S9-mix and reported as part of the first mutation experiment. The highest concentration of test material used in the subsequent mutation assay was the level at which the test material inhibited bacterial growth.

MUTATION ASSAY
At least five different doses (increasing with approximately half-log steps) of the test material were tested in each strain both in the absence and presence of 5 % (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. In a follow-up experiment with additional parameters, the test material was tested both in the absence and presence of 10 % (v/v) S9-mix in all tester strains.
Top agar in top agar tubes was melted by heating to 45 ± 2 °C. The following solutions were successively added to 3 mL molten top agar: 0.1 mL of a fresh bacterial culture (10^9 cells/mL) of one of the tester strains, 0.1 mL of a dilution of the test material in DMSO and either 0.5 mL S9-mix (in case of activation assays) or 0.5 mL 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0 °C for 48 ± 4 h. After this period revertant colonies (histidine independent (His+) for Salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli) were counted.

NUMBER OF REPLICATIONS: Testing was performed in triplicate

COLONY COUNTING
The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test material precipitate to interfere with automated colony counting were counted manually. Evidence of test material precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope.

DETERMINATION OF CYTOTOXICITY
- Method: To determine the toxicity of the test material, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined.

Evaluation criteria:

ACCEPTABILITY OF THE ASSAY
The assay is considered acceptable if it meets the following criteria:
a) The vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at the testing facility.
b) The selected dose range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
c) No more than 5 % of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.

DATA EVALUATION
In addition to the criteria stated below, any increase in the total number of revertants should be evaluated for its biological relevance including a comparison of the results with the historical control data range.
A test material is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent vehicle control.
b) The negative response should be reproducible in at least one follow-up experiment.
A test material is considered positive (mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 is greater than three (3) times the concurrent vehicle control.
b) In case a follow up experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

Statistics:

No formal hypothesis testing was done..

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium, other: TA1535, TA1537, TA98 and TA100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

DOSE RANGE FINDING TEST/FIRST MUTATION EXPERIMENT
- Precipitate: Precipitation of the test material on the plates was not observed at the start or at the end of the incubation period in any tester strain. Due to the colour of the test material the number of revertants and bacterial background lawn could not be determined at dose levels of 512 to 5000 μg/plate.
- Toxicity: Cytotoxicity, as evidenced by a decrease in the number of revertants and/or reduction of the bacterial background lawn, was observed I the range finding test in TA100 (absence and presence of S9-mix) and WP2uvrA (absence of S9-mix). Cytotoxicity was observed in the first mutation experiment in all three tester strains in the absence and presence of S9-mix.
- Mutagenicity: No increase in the number of revertants was observed upon treatment with the test material under all conditions tested.

MUTATION EXPERIMENT 2
- Precipitate: Precipitation of the test material on the plates was not observed at the start or at the end of the incubation period.
- Toxicity: Cytotoxicity, as evidenced by a decrease in the number of revertants and/or reduction of the bacterial background lawn, was observed in all tester strains in the absence and presence of S9-mix.
- Mutagenicity: In the second mutation assay, no increase in the number of revertants was observed upon treatment with the test material under all conditions tested.
In strain TA100, a fluctuation in the mean number of revertant colonies above the laboratory historical control data range was observed in the absence of S9-mix. However, since the increase was not two-fold (a maximum of 1.2-fold was reached), this increase was not considered to be relevant.

DISCUSSION
All bacterial strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants in two experiments.
The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Any other information on results incl. tables

Table 1: Dose Range-finder and Experiment 1 (Plate incorporation assay 5 % S9)

+/- S9 Mix	Concentration (µg/plate)	Mean number of colonies/plate
		Base-pair Substitution Type		Frameshift Type
		TA100	TA1535	WP2uvrA	TA98	TA1537
-	PC DMSO 0.55 1.7 5.4 17 52 164 512 1600 5000	1135 117 - 98 123 89 n 15 s e NP MC ND ND ND	866 10 11 15 22 25 11 n 5 m NP - - -	1242 21 - 19 18 23 17 4 n NP ND ND ND	1377 22 22 23 31 27 19 n 1 s NP - - -	755 8 5 7 18 5 n 1 m 1 a NP - - -
+	PC DMSO 0.55 1.7 5.4 17 52 164 512 1600 5000	1230 99 - 96 140 126 n 94 s e NP MC ND ND ND	263 10 10 13 21 17 n 13 s 6 m NP - - -	323 22 - 24 16 28 24 12 n NP ND ND ND	1028 29 29 28 51 33 34 n 1 s NP - - -	400 10 13 9 12 14 n 4 m 1 a NP - - -

Mean number of revertant colonies/3 replicate plates

PC = Positive control

MC = Microcolonies

NP = No precipitate

ND = Not determined - the number of revertants and the bacterial background lawn could not be determined due to the colour of the test material

a = Bacterial background lawn absent

e = Bacterial background lawn extremely reduced

m = Bacterial background lawn moderately reduced

n = Normal bacterial background lawn

s = Bacterial background lawn slightly reduced

 

Table 2: Experiment 2 (Plate incorporation assay 10 % S9)

+/- S9 Mix	Concentration (µg/plate)	Mean number of colonies/plate
		Base-pair Substitution Type		Frameshift Type
		TA100	TA1535	WP2uvrA	TA98	TA1537
-	PC DMSO 5 10 25 50 100 164	922 131 131 120 154 n 26 s 5 m a NP MC	843 10 11 12 16 7 n 7 s a NP MC	1516 19 24 18 11 9 1 n 0 a NP	1252 23 16 20 19 15 6 n 2 s NP	870 7 10 5 3 n 4 m 0 a 0 a NP
+	PC DMSO 5 10 25 50 100 164	1155 109 114 119 155 127 n 48 s e NP MC	136 13 17 13 11 10 n 4 s e NP MC	483 36 32 28 20 24 12 n 0 e NP	556 32 22 37 29 23 23 4 n NP	499 12 9 11 18 n 8 s 4 m a NP MC

Mean number of revertant colonies/3 replicate plates

PC = Positive control

MC = Microcolonies

NP = No precipitate

a = Bacterial background lawn absent

e = Bacterial background lawn extremely reduced

m = Bacterial background lawn moderately reduced

n = Normal bacterial background lawn

s = Bacterial background lawn slightly reduced

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, it is concluded that the test material is not mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

The potential of the test material to cause mutagenic effects in bacteria was assessed in accordance with the standardised guidelines OECD 471 and EU Method B.13/14 under GLP conditions.

The test material was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in two independent experiments in the presence and absence of S9-mix (rat liver S9-mix induced by Aroclor 1254).

In the dose range finding test, the test material was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. The test material did not precipitate on the plates at this dose level. Cytotoxicity, as evidenced by a decrease in the number of revertants and/or reduction of the bacterial background lawn was observed in TA100 (absence and presence of S9-mix) and WP2uvrA (absence of S9-mix). Due to the colour of the test material the number of revertants and bacterial background lawn could not be determined at dose levels of 512 to 5000 μg/plate. Results were reported as part of the first mutation assay.

Based on the results of the dose range finding test, the test material was tested in the first mutation assay at a concentration range of 0.55 to 164 μg/plate in the absence and presence of 5 % (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. Cytotoxicity was observed in all three tester strains in the absence and presence of S9-mix.

In a follow-up experiment of the assay with additional parameters, the test material was tested at a concentration range of 5 to 164 μg/plate in the absence and presence of 10 % (v/v) S9-mix in all strains. Cytotoxicity was observed in all tester strains in the absence and presence of S9-mix.

The test material did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains TA1535, TA1537, TA98 and TA100 and in the number of revertant (Trp+) colonies in the tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment. The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Under the conditions of this study, it is concluded that the test material is not mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assay.