3,6-bis(ethylamino)-9-[2-(methoxycarbonyl)phenyl]-2,7-dimethylxanthylium 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:

11 July 2016 to 22 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 reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

3,6-bis(ethylamino)-9-[2-(methoxycarbonyl)phenyl]-2,7-dimethylxanthylium chloride

EC Number:

221-326-1

EC Name:

3,6-bis(ethylamino)-9-[2-(methoxycarbonyl)phenyl]-2,7-dimethylxanthylium chloride

Cas Number:

3068-39-1

Molecular formula:

C27H29N2O3.Cl

IUPAC Name:

3,6-bis(ethylamino)-9-[2-(methoxycarbonyl)phenyl]-2,7-dimethylxanthylium chloride

Test material form:

solid: particulate/powder

Details on test material:

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

Specific details on test material used for the study:

No correction factor required.

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 nutrient broth) 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 nutrient broth) 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): 0.54, 1.7, 5.4, 17, 52 and 164 μg/plate (absence of S9-mix) and 1.7, 5.4, 17, 52, 164 and 275 μg/plate (presence of S9-mix)
- Experiment 1A: TA1535, TA1537 and TA98 5.4, 17, 52, 164 and 512 µg/plate (absence of S9-mix) and TA98 5.4, 17, 52, 164 and 512 µg/plate (presence of S9-mix)
- Experiment 2 (all strains): 15, 27, 48, 86, 154 and 275 μg/plate (absence and presence of S9-mix)
- Experiment 2A (TA 98 and WP2uvrA): 17, 52, 164, 512 and 1000 µg/plate (presence of S9-mix)

Vehicle / solvent:

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

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191 and 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 ethanol 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.

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.
- Dose range finding test: Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in both tester strains in the absence and presence of S9-mix.
- First mutation experiment: Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in tester strains TA1535 and TA1537 in the presence of S9-mix. In strains TA1537 (absence of S9-mix) and TA98 (presence of S9-mix), fluctuations in the number of revertant colonies below the laboratory historical control data range were observed. However, since no dose-relationship was observed, these reductions are not considered to be caused by toxicity of the test material. It is more likely these reductions are caused by an incidental fluctuation in the number of revertant colonies.
- Mutagenicity: In tester strain TA1535, up to 5.8- and 3.0-fold increases were observed in the absence and presence of S9-mix, respectively. In the other tester strains, no increase in the number of revertants was observed upon treatment with the test material under all conditions tested.

MUTATION EXPERIMENT 1A
Since insufficient toxicity without precipitate on the plates was observed in the tester strains TA1535, TA1537 and TA98 in the absence of S9- mix and TA98 in the presence of S9-mix, an additional experiment was performed.
- 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.
- Toxicity: Cytotoxicity, as evidenced by a decrease in the number of revertants was observed in all three tester strains.
- Mutagenicity: In tester strain TA1535, an up to 3.1-fold increase was observed in the absence of S9-mix. In the other tester strains, no increase in the number of revertants was observed upon treatment with the test material under all conditions tested.

MUTATION EXPERIMENT 2
To obtain more information about the possible mutagenicity of the test material, a second mutation experiment was performed in the absence and presence of 10 % (v/v) S9-mix.
- 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, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix, except in tester strains TA98 and WP2uvrA in the presence of S9-mix. In strain TA98 (absence of S9-mix), a fluctuation in the number of revertant colonies below the laboratory historical control data range was observed at the lowest dose of 15 μg/plate. However, since no dose-relationship was observed, this reduction is not considered to be caused by toxicity of the test material. It is more likely this reduction is caused by an incidental fluctuation in the number of revertant colonies.
- Mutagenicity: In the second mutation assay, no biologically relevant increase in the number of revertants was observed upon treatment with the test material under all conditions tested.

MUTATION EXPERIMENT 2A
Since insufficient toxicity without precipitate on the plates was observed in the tester strains TA98 and WP2uvrA in the presence of S9-mix, an additional experiment was performed.
- 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.
- Toxicity: Cytotoxicity, as evidenced by a decrease in the number of revertants was observed in both tester strains TA1535 and TA1537 in presence of S9-mix.
- Mutagenicity: No increase in the number of revertants was observed upon treatment with the test material under all conditions tested.

DISCUSSION
In tester strain TA1535, increases in the number of revertant colonies more than three-times the concurrent vehicle control were observed. Although the increases observed in the absence of S9-mix were in two out of three experiments, the increases were not dose related, in the first experiment caused by one outlier and within the historical control data range, therefore these increases are considered to be not biologically relevant. The increase observed in the presence of S9-mix was in one experiment only, not dose related and within the historical control data range, therefore this increase is considered to be not biologically relevant.
The other four tester strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants in any of the experiments performed.
The negative control values were within the laboratory historical control data ranges.
The 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, except the response for TA100 in the second experiment (presence of S9-mix). In the second mutation assay, the mean plate count of the positive control of TA100 in the presence of S9-mix was not within the acceptability criteria. The value of 391 revertant colonies was outside the historical control data range. The purpose of the positive control is as a reference for the test system, where a positive response is required to check if the test system functions correctly. Since the value was more than 3 times greater than the concurrent solvent control values, this deviation in the mean plate count of the positive control had no effect on the results of the study.
Based on the results of this study it is concluded that the test material is not mutagenic.

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
-	Positive Control Ethanol 0.54 1.7 5.4 17 52 164 512 1600 5000	1038 119 - 123 123 119 106 0 n e MC 0 0	870 5 11 9 15 14 29 13 n NP - - -	1751 29 - 19 20 17 17 9 2 n 0 a 0 NP a	1372 15 29 23 28 13 28 9 nNP - - -	832 3 5 5 3 6 0 5 n NP - - -
+	Positive Control Ethanol 1.7 5.4 17 52 164 275 512 1600 5000	1264 124 140 142 137 95 n 17 s - 0 e 0 a 0 NP a	241 9 15 11* 27 12 6 n 4 s NP - - -	382 29 31 27 29 22 18 - 10 0 n 0 NP a	1176 20 8 14 27 21 23 15 n NP - - -	443 7 15 6 7 5 n 0 s E NP MC - - -

Mean number of revertant colonies/3 replicate plates

MC = Microcolonies

NP = No precipitate

a = Bacterial background lawn absent

e = Bacterial background lawn extremely reduced

n = Normal bacterial background lawn

s = Bacterial background lawn slightly reduced

*Mean of two plates, one plate could not be determined due to a technical error

 

Table 2: Experiment 1A (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
-	Positive Control Ethanol 5.4 17 52 164 512	- - - - - - -	934 9 12 27 28 12 0 n NP	- - - - - - -	1476 19 16 16 17 13 0 n NP	951 4 3 6 3 0 0 n NP
+	Positive Control Ethanol 5.4 17 52 164 512	- - - - - - -	- - - - - - -	- - - - - - -	1155 23 28 34 32 20 1 n NP	- - - - - - -

Mean number of revertant colonies/3 replicate plates

NP = No precipitate

n = Normal bacterial background lawn

 

Table 3: 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
-	Positive Control Ethanol 15 27 48 86 154 275	814 99 100 129 98 32 n 4 s 5 s NP	823 9 12 15 21 6 4 n 1 s NP	1277 18 29 18 16 16 13 3 n NP	1397 14 7 10 13 6 5 2 n NP	997 6 7 3 3 n 0 s e MC a NP
+	Positive Control Ethanol 15 27 48 86 154 275	391 73 94 95 88 60 n 17 s 8 m NP	90 10 13 12 8 5 5 e NP MC	293 28 24 30 33 23 16 15 n NP	491 24 28 23 28 21 16 11 n NP	410 5 5 4 n 4 s 1 s 0 a 0 a NP

Mean number of revertant colonies/3 replicate plates

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

 

Table 4: 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
+	Positive Control Ethanol 17 52 164 512 1000	- - - - - - -	- - - - - - -	293 22 22 18 19 8 1 n NP	501 31 31 21 24 5 5 n NP	- - - - - - -

Mean number of revertant colonies/3 replicate plates

NP = No precipitate

n = Normal bacterial background lawn

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 evaluated in four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and in 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) using ethanol as the vehicle. Additional experiments were performed to complete the data of the first and second experiment.

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 a reduction of the bacterial background lawn, was observed in both tester strains in the absence and presence of S9-mix. 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 the concentration ranges of 0.54 to 164 μg/plate in the absence of S9-mix and 1.7 to 275 μg/plate in the presence of 5 % (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. Cytotoxicity, as evidenced by a decrease in the number of revertants and/or a reduction of the bacterial background lawn, was only observed in TA1535 and TA1537 in the presence of S9-mix.

Since insufficient toxicity without precipitate on the plates was observed in the tester strains TA1535, TA1537 and TA98 in the absence of S9- mix and TA98 in the presence of S9-mix, an additional experiment was performed (experiment 1A) in which the test material was tested at the concentration range of 5.4 to 512 μg/plate. Toxicity was observed in all tester strains.

In a follow-up experiment of the assay with additional parameters, the test material was tested at a concentration range of 15 to 275 μg/plate in the absence and presence of 10 % (v/v) S9-mix in all tester strains. Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix, except in the tester strains TA98 and WP2uvrA in the presence of S9-mix.

Since insufficient toxicity without precipitate on the plates was observed in the tester strains TA98 and WP2uvrA in the presence of S9-mix, an additional experiment was performed (experiment 2A) in which the test material was tested at the concentration range of 17 to 1000 μg/plate. Toxicity was observed in both tester strains.

In tester strain TA1535, increases in the number of revertant colonies more than three-times the concurrent vehicle control were observed. Although the increases observed in the absence of S9-mix were in two out of three experiments, the increases were not dose related, in the first experiment caused by one outlier and within the historical control data range, therefore these increases are considered to be not biologically relevant. The increase observed in the presence of S9-mix was in one experiment only, not dose related and within the historical control data range, therefore this increase is considered to be not biologically relevant.

The other four tester strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants in any of the experiments performed.

In this study, acceptable responses were obtained for the negative and strain-specific positive control materials 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.