3-(benzothiazol-2-yl)-7-(diethylamino)-2-oxo-2H-1-benzopyran-4-carbonitrile

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:

Experimental start date: 05 July 2017 Experimental completion date: 18 September 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

3-(benzothiazol-2-yl)-7-(diethylamino)-2-oxo-2H-1-benzopyran-4-carbonitrile

EC Number:

274-668-9

EC Name:

3-(benzothiazol-2-yl)-7-(diethylamino)-2-oxo-2H-1-benzopyran-4-carbonitrile

Cas Number:

70546-25-7

Molecular formula:

C21H17N3O2S

IUPAC Name:

3-(1,3-benzothiazol-2-yl)-7-(diethylamino)-2-oxo-2H-chromene-4-carbonitrile

impurity 1

Reference substance name:

unknown impurities

IUPAC Name:

unknown impurities

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

Appearance: Olive green powder
Storage Conditions: Room temperature, in the dark
Expiry Date: 24 April 2020
Formulated concentrations were adjusted to allow for the stated water/impurity content of the test item.

Method

Target gene:

Histidine locus in S. typhimurium and tryptophan locus in E.coli.

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

a liver microsomal preparation (S9-mix) prepared from rats

Test concentrations with justification for top dose:

Experiment 1 - Plate Incorporation Method
The maximum concentration was 5000 µg/plate (the maximum recommended dose level).
Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate) were tested

Vehicle / solvent:

The test item was insoluble in sterile distilled water, dimethyl sulphoxide, dimethyl formamide and acetonitrile at 50 mg/mL, acetone at 100 mg/mL and tetrahydrofuran at 200 mg/mL in solubility checks performed in–house. The test item formed the best doseable suspension in dimethyl sulphoxide, therefore, this solvent was selected as the vehicle.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

2 µg/plate for WP2uvrA, 3 µg/plate for TA100, 5 µg/plate for TA1535

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Absence of S9-mix

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

80 µg/plate for TA1537

Positive control substance:

9-aminoacridine

Remarks:

Absence of S9-mix

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

0.2 µg/plate for TA98

Positive control substance:

other: 4-Nitroquinoline-1-oxide

Remarks:

Absence of S9-mix

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

1 µg/plate for TA100, 2 µg/plate for TA1535 and TA1537, 10 µg/plate WP2uvrA

Positive control substance:

other: 2-Aminoanthracene

Remarks:

Presence of S9-mix

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

5 µg/plate for TA98

Positive control substance:

benzo(a)pyrene

Remarks:

Presence of S9-mix

Details on test system and experimental conditions:

Experimental Design and Study Conduct
Test Item Preparation and Analysis
The test item was accurately weighed and, on the day of the experiment, approximate half-log dilutions prepared in dimethyl sulphoxide by mixing on a vortex and sonication for 20 minutes at 40 °C. Formulated concentrations were adjusted to allow for the stated water/impurity content (2.4%) of the test item. All formulations were used within four hours of preparation and were assumed to be stable for this period. Homogeneity/stability analysis was performed outside the confines of this study under Envigo Research Limited project number WX02TH for a low dose (0.1 mg/mL) and a high dose (200 mg/mL). Analysis for concentration of the test item formulations is not a requirement of the test guidelines and was, therefore, not determined. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Test for Mutagenicity: Experiment 1 - Plate Incorporation Method
Eight concentrations of the test item were assayed in triplicate against each tester strain, using the direct plate incorporation method.

Without Metabolic Activation
0.1 mL of the appropriate concentration of test item, solvent vehicle or appropriate positive control was added to 2 mL of molten, trace amino-acid supplemented media containing 0.1 mL of one of the bacterial strain cultures and 0.5 mL of phosphate buffer. These were then mixed and overlayed onto a Vogel Bonner agar plate. Negative (untreated) controls were also performed on the same day as the mutation test. Each concentration of the test item, appropriate positive, vehicle and negative controls, and each bacterial strain, was assayed using triplicate plates.

With Metabolic Activation
The procedure was the same as described previously except that following the addition of the test item formulation and bacterial culture, 0.5 mL of S9 mix was added to the molten, trace amino-acid supplemented media instead of phosphate buffer.

Incubation and Scoring
All of the plates were incubated at 37 ± 3 ºC for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). Several manual counts were required due to revertant colonies spreading slightly, thus distorting the actual plate count.

Test for Mutagenicity: Experiment 2
The second experiment was not performed because the OECD 471 test guideline permits non repetition of the experiment when a clear, positive response is obtained in the first experiment. Therefore, a second, confirmatory experiment was not required.

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out of historical range response (Cariello and Piegorsch, 1996)).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.

Statistics:

Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

at 5000 µg/plate

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

from 1500 µg/plate

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 6

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

from 5 µg/plate

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 7

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 8

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

from 500 µg/plate

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 9

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile. The test item formulation was also shown to be sterile. These data are not given in the report.
Results for the negative controls (spontaneous mutation rates) were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.
The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method).
A red test item induced colouration was initially noted at 15 µg/plate becoming dark green/grey at 5000 µg/plate. A test item particulate precipitate was also noted at 5000 µg/plate. None of these observations prevented the scoring of revertant colonies.
There were no biologically significant increases in revertant colony frequency noted to any of the bacterial strains in the absence of S9-mix, however the test item induced statistically significant and dose-related increases in the frequency of TA100, TA98 and TA1537 revertant colonies from 5 µg/plate (TA98), 500 µg/plate (TA100) and 1500 µg/plate (TA1537). Smaller increases were also noted for TA1535 at 5000 µg/plate. The increases observed in the presence of S9-mix for TA100 and TA98 at the upper test item dose levels were particularly large and very much in excess of the maxima untreated/vehicle control in house historical ranges for the relevant bacterial strains with a maximum increase of 31.2 times over the concurrent vehicle control noted for TA98 at 5000 µg/plate. Any excursions outside the maxima ranges, particularly when a dose-response relationship is apparent, must be considered to be evidence of a biological response. A weaker statistically significant response was also noted to TA100 dosed in the absence of S9-mix at 50 µg/plate, however these counts were within the in-house untreated/vehicle historical control profile for the strain and showed no evidence of a dose-response relationship.

Any other information on results incl. tables

Spontaneous Mutation Rates (Concurrent Negative Controls)

Experiment 1

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA		TA98		TA1537
111		15		24		36		10
103	(104)	21	(21)	17	(20)	30	(37)	19	(14)
98		26		19		45		12

 

Test Results: Experiment 1 – Without Metabolic Activation (Plate Incorporation)

Test Period		From: 15 September 2017					To: 18 September 2017
S9-Mix (-)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (DMSO)	82 81 90	(84) 4.9#	23 20 26	(23) 3.0	20 21 25		(22) 2.6	27 36 44	(36) 8.5	18 19 22	(20) 2.1
	1.5 µg	88 82 79	(83) 4.6	23 26 24	(24) 1.5	17 29 19		(22) 6.4	42 37 29	(36) 6.6	12 16 15	(14) 2.1
	5 µg	92 109 97	(99) 8.7	27 17 24	(23) 5.1	17 20 21		(19) 2.1	29 39 38	(35) 5.5	15 9 16	(13) 3.8
	15 µg	94 96 88	(93) 4.2	22 26 32	(27) 5.0	18 21 18		(19) 1.7	39 37 33	(36) 3.1	18 19 22	(20) 2.1
	50 µg	115 87 119	* (107) 17.4	35 28 31	(31) 3.5	18 20 17		(18) 1.5	44 32 29	(35) 7.9	22 11 21	(18) 6.1
	150 µg	89 107 96	(97) 9.1	30 30 35	(32) 2.9	18 16 14		(16) 2.0	29 33 38	(33) 4.5	18 14 20	(17) 3.1
	500 µg	99 92 119	(103) 14.0	34 35 23	(31) 6.7	20 19 19		(19) 0.6	38 33 29	(33) 4.5	24 17 8	(16) 8.0
	1500 µg	114 82 89	(95) 16.8	19 31 20	(23) 6.7	16 21 13		(17) 4.0	29 30 30	(30) 0.6	20 17 18	(18) 1.5
	5000 µg	95 P 83 P 88 P	(89) 6.0	29 P 24 P 31 P	(28) 3.6	13 P 16 P 18 P		(16) 2.5	32 P 32 P 33 P	(32) 0.6	15 P 16 P 15 P	(15) 0.6
Positive controls S9-Mix (-)	Name Dose Level No. of Revertants	ENNG		ENNG		ENNG			4NQO		9AA
		3 µg		5 µg		2 µg			0.2 µg		80 µg
		561 584 657	(601) 50.1	677 722 709	(703) 23.2	772 781 867		(807) 52.4	202 195 225	(207) 15.7	281 364 389	(345) 56.5

ENNG     N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO      4-Nitroquinoline-1-oxide

9AA        9-Aminoacridine

P                    Test item precipitate

*                    p ≤ 0.05

#                    Standard deviation

Test Results: Experiment 1 – With Metabolic Activation (Plate Incorporation)

Test Period		From: 15 September 2017					To: 18 September 2017
S9-Mix (+)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (DMSO)	89 75 80	(81) 7.1#	21 33 21	(25) 6.9	32 33 37		(34) 2.6	37 37 37	(37) 0.0	22 9 16	(16) 6.5
	1.5 µg	87 72 92	(84) 10.4	25 23 25	(24) 1.2	39 25 29		(31) 7.2	39 58 54	(50) 10.0	6 24 9	(13) 9.6
	5 µg	82 92 82	(85) 5.8	26 34 29	(30) 4.0	18 26 25		(23) 4.4	61 79 57	*** (66) 11.7	15 17 16	(16) 1.0
	15 µg	83 93 96	(91) 6.8	28 34 24	(29) 5.0	30 30 21		(27) 5.2	57 62 57	** (59) 2.9	15 10 21	(15) 5.5
	50 µg	104 77 80	(87) 14.8	34 27 34	(32) 4.0	19 24 26		(23) 3.6	81 63 73	*** (72) 9.0	25 16 31	(24) 7.5
	150 µg	95 94 110	(100) 9.0	30 34 36	(33) 3.1	28 20 22		(23) 4.2	100 100 96	*** (99) 2.3	14 26 21	(20) 6.0
	500 µg	112 101 113	** (109) 6.7	29 31 26	(29) 2.5	27 22 29		(26) 3.6	211 204 201	*** (205) 5.1	30 28 28	(29) 1.2
	1500 µg	167 194 199	*** (187) 17.2	32 40 27	(33) 6.6	32 26 31		(30) 3.2	635 644 673	*** (651) 19.9	33 45 33	** (37) 6.9
	5000 µg	346 P 339 P 361 P	*** (349) 11.2	44 P 34 P 38 P	** (39) 5.0	21 P 25 P 25 P		(24) 2.3	1198 P 1161 P 1102 P	*** (1154) 48.4	46 P 42 P 51 P	*** (46) 4.5
Positive controls S9-Mix (+)	Name Dose Level No. of Revertants	2AA		2AA		2AA			BP		2AA
		1 µg		2 µg		10 µg			5 µg		2 µg
		2345 2457 2297	(2366) 82.1	219 361 387	(322) 90.4	544 569 589		(567) 22.5	386 393 393	(391) 4.0	586 648 571	(602) 40.8

2AA 2-Aminoanthracene

BP          Benzo(a)pyrene

P            Precipitate

**           p ≤ 0.01

***         p ≤ 0.001

#            Standard deviation

Applicant's summary and conclusion

Conclusions:

Macrolex Fluoreszenzrot G was considered to be mutagenic under the conditions of this test.

Executive summary:

Introduction

The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA OCSPP harmonized guideline - Bacterial Reverse Mutation Test.

Methods

Salmonella typhimuriumstrains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using the Ames plate incorporation method at eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 was predetermined and was 1.5 to 5000 mg/plate.

The OECD 471 test guideline permits non-repetition of the experiment when a clear positive response is obtained in the first experiment, therefore, with the Sponsor’s approval, testing was suspended at the end of Experiment 1.

Results

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method).

A red test item induced colouration was initially noted at 15 µg/plate becoming dark green/grey at 5000 µg/plate. A test item particulate precipitate was also noted at 5000 µg/plate. None of these observations prevented the scoring of revertant colonies. 

There were no biologically significant increases in revertant colony frequency noted to any of the bacterial strains in the absence of S9-mix, however the test item induced statistically significant and dose-related increases in the frequency of TA100, TA98 and TA1537 revertant colonies from 5 µg/plate (TA98), 500 µg/plate (TA100) and 1500 µg/plate (TA1537). Smaller increases were also noted for TA1535 at 5000 µg/plate. The increases observed in the presence of S9-mix for TA100 and TA98 at the upper test item dose levels were particularly large and very much in excess of the maxima untreated/vehicle control in‑house historical ranges for the relevant bacterial strains with a maximum increase of 31.2 times over the concurrent vehicle control noted for TA98 at 5000 µg/plate. Any excursions outside the maxima ranges, particularly when a dose-response relationship is apparent, must be considered to be evidence of a biological response. A weaker statistically significant response was also noted to TA100 dosed in the absence of S9-mix at 50 µg/plate, however these counts were within the in-house untreated/vehicle historical control profile for the strain and showed no evidence of a dose-response relationship.

Conclusion

Macrolex Fluoreszenzrot G was considered to be mutagenic under the conditions of this test.