2-[7-(diethylamino)-2-oxo-2H-1-benzopyran-3-yl]benzoxazole-5-sulphonamide

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:

17 July 2016 to 03 August 2016

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

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:

2-[7-(diethylamino)-2-oxo-2H-1-benzopyran-3-yl]benzoxazole-5-sulphonamide

EC Number:

270-393-3

EC Name:

2-[7-(diethylamino)-2-oxo-2H-1-benzopyran-3-yl]benzoxazole-5-sulphonamide

Cas Number:

68427-35-0

Molecular formula:

C20H19N3O5S

IUPAC Name:

2-[7-(diethylamino)-2-oxo-2H-chromen-3-yl]-1,3-benzoxazole-5-sulfonamide

Test material form:

solid: particulate/powder

Details on test material:

- Appearance: Yellowish orange powder
- Storage conditions of test material: At room temperature
- Stable under storage conditions until: 30 November 2020 (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 LTD, Hampshire, England) 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 LTD, Hampshire, England) 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): 5.4, 17, 52, 164, 512 and 1600 μg/plate (absence and presence of S9-mix)
- Experiment 2 (all strains): 86, 154, 275, 492, 878 and 1568 μg/plate (absence and presence of S9-mix)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: A solubility test was performed. The test material could not be dissolved in water. A homogeneous suspension of 50 mg/mL was obtained in dimethyl sulfoxide. At concentrations of 16 mg/mL and lower the test material was dissolved in dimethyl sulfoxide. The stock solution was treated with ultrasonic waves to obtain an homogeneous suspension (Dose range finding test) or until the test material had completely dissolved (mutation experiments). The lower test concentrations were prepared by subsequent dilutions in DMSO.

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.

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 TA 98

Metabolic activation:

with

Genotoxicity:

positive

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:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium, other: TA1535, TA1537 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 4

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: In the dose range finding test, precipitation of the test material on the plates was observed at the start of the incubation period at concentrations of 512 μg/plate and upwards. At the end of the incubation period, precipitation was observed at 164 μg/plate and above (absence of S9-mix) and at 512 and 1600 μg/plate and upwards (TA100 and WP2uvrA, respectively). In the first experiment, precipitation of the test material on the plates was observed at the start and at the end of the incubation period at concentrations of 512 μg/plate and upwards.
- Toxicity: No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed. 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 level tested. 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 presence of S9-mix, the test material induced an up to 3.8-dose related increase in tester strain TA98. In all 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
- Precipitate: Precipitation of the test material on the plates was observed at the start of the incubation period at the concentration of 492 μg/plate. At the end of the incubation period, precipitation was observed at 1568 μg/plate in the absence of S9-mix, except in tester strain TA100. In the presence of S9-mix, precipitation was only observed in tester strain TA1535 at 1568 μg/plate.
- Toxicity: The bacterial background lawn was reduced in all tester strains in the presence of S9-mix at the highest tested concentration. In addition, a reduction in the bacterial background lawn was observed in tester strain TA100 in the absence of S9-mix and a biologically relevant decrease in the number of revertants was observed in tester strain TA1537 in the absence of S9-mix.
- Mutagenicity: In the presence of S9-mix, the test material induced an up to 4.4-dose related increase in tester strain TA98. In all other tester strains, no increase in the number of revertants was observed upon treatment with the test material under all conditions tested.

DISCUSSION
In the presence of S9-mix, the test material induced dose related increases in tester strain TA98. The increases observed were above the laboratory historical control data range, in two independently repeated experiments and were up to 4.4-fold the concurrent vehicle controls and therefore considered to be biologically relevant.
In the other tester strains (TA1535, TA1537, TA100 and WP2uvrA), the test material did not induce a significant dose-related increase in the number of revertant colonies in any 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.

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 1.7 5.4 17 52 164 512 1600 5000	711 76 83 68 77 92 NP 90 SP 88 SP 71 MP 68 n MP	815 9 - 6 7 12 15 NP 10 SP 10 n MP -	1367 24 25 24 28 25 NP 26 SP 19 SP 21 MP 13 n MP	1227 10 - 6 14 10 13 NP 11 SP 16 n MP -	1187 4 - 4 4 6 7 NP 6 SP 4 n MP -
+	PC DMSO 1.7 5.4 17 52 164 512 1600 5000	1003 75 60 73 71 80 79 NP 82 SP 79 MP 72 n MP	201 12 - 8 7 5 11 NP 14 SP 9 n MP -	390 32 34 32 38 33 33 34 NP 43 SP 19 n MP	743 22 - 14 18 23 48 NP 58 SP 84 n MP -	362 6 - 5 11 7 12 NP 7 SP 14 n MP -

Mean number of revertant colonies/3 replicate plates

PC = Positive control

MP = Moderate Precipitate

NP = No precipitate

SP = Slight Precipitate

n = Normal bacterial background lawn

 

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 86 154 275 492 878 1568	1049 98 128 115 133 105 103 n 122 s NP	947 18 17 11 14 18 13 NP 12 n SP	1494 23 20 22 28 21 23 NP 21 n SP	1466 16 15 17 21 14 14 NP 16 n SP	1029 5 7 5 3 4 4 NP 1 n SP
+	PC DMSO 86 154 275 492 878 1568	1062 97 111 114 117 112 108 n 111 s NP	138 14 12 16 16 18 16 n NP 12 s SP	435 32 30 29 26 22 n 37 s 33 s NP	475 20 24 38 34 66 n 70 s 88 s NP	480 10 7 9 6 6 12 n 13 s NP

Mean number of revertant colonies/3 replicate plates

PC = Positive control

NP = No precipitate

SP = Slight Precipitate

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 mutagenic in the Salmonella typhimurium reverse mutation assay and not mutagenic in the Escherichia coli reverse mutation assay. The mutagenicity was confined only to incubations with metabolic activation in one strain.

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 was tested up to or beyond a precipitating dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. Results of this dose range finding test 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 5.4 to 1600 μg/plate in the absence and presence of 5 % (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. The test material was precipitated on the plates at concentrations of 512 and 1600 μg/plate. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

In a follow-up experiment of the assay with additional parameters, the test material was tested at a concentration range of 86 to 1568 μg/plate in the absence and presence of 10 % (v/v) S9-mix in all strains. Precipitation of the test material on the plates was observed at 1568 μg/plate in the absence of S9-mix, except in tester strain TA100 and in the presence of S9-mix in tester strain TA1535 only. In the presence of S9-mix, the bacterial background lawn was reduced in all tester strains at the highest tested concentration. In the absence of S9-mix, a reduction in the bacterial background lawn was observed in tester strain TA100 and a biologically relevant decrease in the number of revertants was observed in tester strain TA1537.

In the presence of S9-mix, the test material induced dose related increases in tester strain TA98. The increases observed were above the laboratory historical control data range, in two independently repeated experiments and were up to 4.4-fold the concurrent vehicle controls and therefore considered to be biologically relevant. In the other tester strains (TA1535, TA1537, TA100 and WP2uvrA), the test material did not induce a significant dose-related increase in the number of revertant colonies in any 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 the study, it is concluded that the test material is mutagenic in the Salmonella typhimurium reverse mutation assay and not mutagenic in the Escherichia coli reverse mutation assay. The mutagenicity was confined only to incubations with metabolic activation in one strain.