N-(2,3-dihydro-2-oxo-1H-benzimidazol-5-yl)-3-hydroxy-4-[[2,5-dimethoxy-4-[(methylamino)sulphonyl]phenyl]azo]naphthalene-2-carboxamide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guidelline study (OECD TG 471), performed under GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

N-(2,3-dihydro-2-oxo-1H-benzimidazol-5-yl)-3-hydroxy-4-[[2,5-dimethoxy-4-[(methylamino)sulphonyl]phenyl]azo]naphthalene-2-carboxamide

EC Number:

235-426-8

EC Name:

N-(2,3-dihydro-2-oxo-1H-benzimidazol-5-yl)-3-hydroxy-4-[[2,5-dimethoxy-4-[(methylamino)sulphonyl]phenyl]azo]naphthalene-2-carboxamide

Cas Number:

12225-08-0

Molecular formula:

C27H24N6O7S

IUPAC Name:

4-{[2,5-dimethoxy-4-(methylsulfamoyl)phenyl]diazenyl}-3-hydroxy-N-(2-oxo-2,3-dihydro-1H-benzimidazol-5-yl)-2-naphthamide

Test material form:

solid: nanoform, no surface treatment

Method

Species / strain

Species / strain / cell type:

other: Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100, and Escherichia coli WP2 uvrA

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 (induced with phenobarbital/beta-naphtoflavone; experiment I); hamster liver S9 (non-induced; experiment II)

Test concentrations with justification for top dose:

Pre-experiment/Experiment I and II: 3, 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide (TA 1535 and TA 100), 4-Nitro-o-phenylene-diamine (TA 1537 and TA 98), methyl methane sulfonate (WP2 uvrA)

Remarks:

without metabolic activation

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (for all strains)

Remarks:

with metabolic activation (rat liver S9)

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (TA 1535, TA 100, TA 1537, WP2 uvrA), congo red (TA 98)

Remarks:

with metabolic activation (hamster liver S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar,
Experiment I: plate incorporation; with and without metabolic activation (induced rat liver S9-mix)
Experiment II: preincubation; with and without metabolic activation (uninduced hamster liver S9-mix)

DURATION
- Preincubation period (expeiment II): 30 minutes
- Exposure duration: at least 48 hours

NUMBER OF REPLICATIONS: 3 plates

DETERMINATION OF CYTOTOXICITY
A reduction in the number of spontaneous revertants (below the induction factor of 0.5) or a clearing of the bacterial background lawn.

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test results

Key result

Species / strain:

other: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

minor effects: experiment I: TA 98 at 5000 µg/plate with S9 mix; experiment II: TA 1535 from 1000 - 5000 µg/plate and WP2 uvrA at 5000 µg/plate with S9 mix

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:
The test item precipitated in the overlay agar in the test tubes from 1000 to 5000 µg/plate. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 333 to 5000 µg/plate with and without S9 mix in experiment I and with S9 mix in experiment II, and from 1000 - 5000 µg/plate without S9 mix in experiment II . The undissolved particles had no influence on the data recording.

COMPARISON WITH HISTORICAL CONTROL DATA: performed

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Minor toxic effects, evident as a reduction in the number of revertants, were observed in strain TA 98 at 5000 µg/plate with S9 mix in experiment I. In experiment II, minor toxic effects were observed in strain TA 1535 from 1000 - 5000 µg/plate and in strain WP2 uvrA at 5000 µg/plate with S9 mix.

Any other information on results incl. tables

Pre -Experiment and Experiment I:

Study Name: 1485302	Study Code: Harlan CCR1485302
Experiment: 1485302 VV Plate	Date Plated: 13/06/2012
Assay Conditions:	Date Counted: 19/06/2012

 

Metabolic Activation	Test Group	Dose Level (per plate)		Revertant Colony Counts (Mean ±SD)
				TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Without Activation	DMSO			17 ± 4	18 ± 3	25 ± 5	128 ± 3	49 ± 12
	Untreated			16 ± 8	20 ± 1	30 ± 3	142 ± 8	52 ± 8
	test item	3 µg		15 ± 5	18 ± 4	22 ± 3	118 ± 4	56 ± 5
		10 µg		14 ± 1	19 ± 2	25 ± 2	144 ± 5	45 ± 11
		33 µg		20 ± 3	18 ± 1	22 ± 3	137 ± 6	45 ± 7
		100 µg		17 ± 4	20 ± 3	24 ± 4	123 ± 10	51 ± 8
		333 µg		15 ± 4^P	15 ± 1^P	28 ± 6^P	116 ± 3^P	42 ± 2^P
		1000 µg		14 ± 1^P	13 ± 1^P	21 ± 6^P	102 ± 14^P	46 ± 9^P
		2500 µg		13 ± 2^P M	12 ± 3^P M	18 ± 2^P M	103 ± 11^P M	51 ± 4^P M
		5000 µg		11 ± 3^P M	9 ± 2^P M	14 ± 3^P M	87 ± 6^P M	40 ± 9^P M
	NaN3	10 µg		2054 ± 71			2146 ± 47
	4-NOPD	10 µg				330 ± 19
	4-NOPD	50 µg			70 ± 2
	MMS	3.0 µL						1196 ± 58
With Activation	DMSO			23 ± 5	27 ± 0	44 ± 12	159 ± 22	65 ± 15
	Untreated			19 ± 8	22 ± 2	42 ± 1	165 ± 11	64 ± 8
	Test item	3 µg		24 ± 5	29 ± 7	44 ± 3	152 ± 16	64 ± 3
		10 µg		30 ± 4	22 ± 6	44 ± 8	153 ± 17	60 ± 3
		33 µg		25 ± 3	30 ± 4	46 ± 10	154 ± 19	60 ± 4
		100 µg		25 ± 5	27 ± 3	38 ± 7	173 ± 19	66 ± 5
		333 µg		23 ± 3^P	28 ± 3^P	41 ± 9^P	154 ± 15^P	64 ± 6^P
		1000 µg		27 ± 6^P	24 ± 6^P	33 ± 8^P	143 ± 31^P	65 ± 14^P
		2500 µg		23 ± 3^P M	17 ± 3^P M	34 ± 2^P M	143 ± 9^P M	50 ± 3^P M
		5000 µg		15 ± 5^P M	14 ± 3^P M	20 ± 2^P M	115 ± 12^P M	48 ± 6^P M
	2-AA	2.5 µg		336 ± 20	297 ± 3	2303 ± 97	2406 ± 101
	2-AA	10.0 µg						326 ± 12

 

Key to Positive Controls		Key to Plate Postfix Codes
NaN3 2-AA 4-NOPD MMS	sodium azide 2-aminoanthracene 4-nitro-o-phenylene-diamine methyl methane sulfonate	P M	Precipitate Manual count

Experiment II:

Study Name: 1485302	Study Code: Harlan CCR1485302
Experiment: 1485302 HV2 Pre	Date Plated: 03/07/2012
Assay Conditions:	Date Counted: 06/07/2012

 

Metabolic Activation	Test Group	Dose Level (per plate)		Revertant Colony Counts (Mean ±SD)
				TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Without Activation	DMSO			16 ± 2	25 ± 1	28 ± 3	140 ± 5	54 ± 8
	Untreated			19 ± 8	22 ± 7	31 ± 4	169 ± 15	52 ± 7
	Test item	3 µg		16 ± 6	23 ± 4	31 ± 6	141 ± 12	54 ± 6
		10 µg		18 ± 7	25 ± 4	31 ± 2	145 ± 9	57 ± 4
		33 µg		16 ± 1	27 ± 11	24 ± 2	144 ± 13	58 ± 7
		100 µg		12 ± 3	27 ± 1	28 ± 3	133 ± 5	45 ± 4
		333 µg		10 ± 1	21 ± 1	25 ± 2	129 ± 10	47 ± 4
		1000 µg		9 ± 1^P	22 ± 2^P	15 ± 2^P	126 ± 4^P	50 ± 2^P
		2500 µg		10 ± 3^P M	20 ± 3^P M	23 ± 6^P M	127 ± 15^P M	42 ± 4^P M
		5000 µg		9 ± 2^P M	13 ± 4^P M	15 ± 3^P M	108 ± 6^P M	32 ± 4^P M
	NaN3	10 µg		1931 ± 56			1977 ± 45
	4-NOPD	10 µg				271 ± 7
	4-NOPD	50 µg			83 ± 6
	MMS	3 µL						917 ± 25
With Activation	DMSO			25 ± 9	29 ± 1	43 ± 4	135 ± 26	73 ± 3
	Untreated			18 ± 3	31 ± 2	50 ± 4	138 ± 5	53 ± 9
	Test item	3 µg		24 ± 5	31 ± 4	50 ± 1	152 ± 5	68 ± 14
		10 µg		27 ± 5	37 ± 8	54 ± 6	139 ± 15	66 ± 9
		33 µg		21 ± 2	26 ± 2	43 ± 1	138 ± 5	72 ± 11
		100 µg		20 ± 8	26 ± 2	44 ± 12	138 ± 9	56 ± 2
		333 µg		18 ± 2^P	35 ± 3^P	39 ± 3^P	126 ± 1^P	48 ± 3^P
		1000 µg		11 ± 5^P M	29 ± 2^P M	38 ± 4^P M	123 ± 6^P M	41 ± 4^P M
		2500 µg		10 ± 2^P M	26 ± 8^P M	23 ± 7^P M	115 ± 7^P M	34 ± 8^P M
		5000 µg		6 ± 2^P M	23 ± 3^P M	19 ± 4^P M	106 ± 6^P M	26 ± 4^P M
	2-AA	2.5 µg					1865 ± 3
	2-AA	2.5 µg		702 ± 14	154 ± 14
	2-AA	10 µg						320 ± 80
	Congo red	500 µg				1143 ± 39

 

Key to Positive Controls		Key to Plate Postfix Codes
NaN3 2-AA 4-NOPD Congo red MMS	sodium azide 2-aminoanthracene 4-nitro-o-phenylene-diamine Congo red methyl methane sulfonate	P M	Precipitate Manual count

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative with and without metabolic activation

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Executive summary:

Mutagenic activity of the test item was investigated in Salmonella typhimurium strains TA 1535, TA 1537, TA98 and TA100 as well as Escherichia coli strain WP2 uvrA with (induced rat liver S9 mix) and without metabolic activation at concentrations of 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate using the plate incorporation assay (experiment I). Due to the test items characteristic as an azo-dye the test was also conducted using the Prival modification, i.e. testing the above mentioned bacterial strains in the preincubation assay without and with uninduced hamster liver S9 mix for metabolic activation (experiment II). This test was performed using the concentrations 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate.

Minor toxic effects, evident as a reduction in the number of revertants, were observed in strain TA 98 at 5000 µg/plate with S9 mix in experiment I. In experiment II, minor toxic effects were observed in strain TA 1535 from 1000 - 5000 µg/plate and in strain WP2 uvrA at 5000 µg/plate with S9 mix.

The test item did not reveal any mutagenic activity under the conditions tested. There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

The appropriate reference mutagenes showed distinct positive mutagenic effects.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.