2,3-Quinoxalinedione, 1,4-dihydro-6-methoxy-7-nitro-

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:

2011 -08-01 till 2011-08-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline-conform study under GLP without deviations

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

1,4-Dihydro-6-methoxy-7-nitroquinoxaline-2,3-dione

EC Number:

614-960-9

Cas Number:

69399-57-1

Molecular formula:

C9H7N3O5

IUPAC Name:

1,4-Dihydro-6-methoxy-7-nitroquinoxaline-2,3-dione

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): Nitro-C-Dion trocken (NMQD)
- Physical state: Yellow powder
- Analytical purity: 91.0 % (w/w); (dose calculation not adjusted to purity)
- Impurities (identity and concentrations):
- Stability under test conditions: > 72 h in DMSO at room temperature
- Storage condition of test material: At room temperature

Method

Species / strain

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-Naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

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

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: better than other

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide; 4-nitro-o-phenylene-diamine; methyl methane sulfonate, 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation; preincubation;


DURATION
- Preincubation period: 1 hour
- Exposure duration: 72 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

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

in strain TA 1537 and TA 98 without S9 mix

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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 and on the incubated agar plates from 1000 to 5000 µg/plate. The undissolved particles had no influence on the data recording.
- Other confounding effects:
COMPARISON WITH HISTORICAL CONTROL DATA: performed
ADDITIONAL INFORMATION ON CYTOTOXICITY: no cytotoxic effects

Remarks on result:

other: other: reverse mutation assay

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

     Summary of Results Pre-Experiment and Experiment I

 

Study Name: 1431901	Study Code: Harlan CCR 1431901
Experiment: 1431901 VV Plate	Date Plated: 01/08/2011
Assay Conditions:	Date Counted: 04/08/2011

 

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			14 ± 5	13 ± 4	33 ± 11	138 ± 9	48 ± 6
	Untreated			18 ± 4	19 ± 2	36 ± 4	134 ± 9	50 ± 6
	Nitro-C-Dion	3 µg		14 ± 7	17 ± 5	33 ± 5	133 ± 9	46 ± 5
	trocken (NMQD)	10 µg		12 ± 4	14 ± 2	33 ± 8	132 ± 3	52 ± 3
		33 µg		14 ± 2	13 ± 2	46 ± 3	130 ± 13	54 ± 3
		100 µg		13 ± 3	19 ± 3	56 ± 14	146 ± 12	53 ± 10
		333 µg		16 ± 3	22 ± 1	55 ± 12	123 ± 7	41 ± 11
		1000 µg		16 ± 3^P	27 ± 1^P	60 ± 11^P M	145 ± 10^P	40 ± 8^P
		2500 µg		7 ± 1^P M	31 ± 4^P M	52 ± 10^P M	141 ± 28^P	46 ± 6^P
		5000 µg		12 ± 2^P M	41 ± 4^P M	62 ± 8^P M	150 ± 3^P M	38 ± 4^P M
	NaN3	10 µg		1892 ± 120			2097 ± 22
	4-NOPD	10 µg				338 ± 34
	4-NOPD	50 µg			94 ± 14
	MMS	3.0 µL						1021 ± 41
With Activation	DMSO			18 ± 5	18 ± 3	37 ± 7	140 ± 6	61 ± 5
	Untreated			16 ± 5	19 ± 9	45 ± 6	156 ± 3	56 ± 13
	Nitro-C-Dion	3 µg		18 ± 5	13 ± 2	42 ± 14	158 ± 19	58 ± 5
	trocken (NMQD)	10 µg		21 ± 4	15 ± 5	38 ± 4	137 ± 7	58 ± 12
		33 µg		16 ± 1	17 ± 2	43 ± 7	144 ± 14	53 ± 3
		100 µg		19 ± 4	16 ± 6	45 ± 3	144 ± 19	62 ± 3
		333 µg		20 ± 6	20 ± 8	57 ± 1	151 ± 12	50 ± 2
		1000 µg		18 ± 8^P	21 ± 6^P	44 ± 4^P M	169 ± 16^P	53 ± 2^P
		2500 µg		19 ± 6^P M	18 ± 5^P M	44 ± 6^P M	168 ± 15^P M	40 ± 5^P M
		5000 µg		11 ± 4^P M	18 ± 5^P M	43 ± 6^P M	153 ± 13^P M	48 ± 4^P M
	2-AA	2.5 µg		358 ± 39	283 ± 12	2027 ± 170	2284 ± 166
	2-AA	10.0 µg						362 ± 18

 

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

    Summary of Results Experiment II

 

Study Name: 1431901	Study Code: Harlan CCR 1431901
Experiment: 1431901 HV2 Pre	Date Plated: 09/08/2011
Assay Conditions:	Date Counted: 12/08/2011

 

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			13 ± 1	14 ± 1	25 ± 3	99 ± 1	45 ± 8
	Untreated			10 ± 5	19 ± 6	35 ± 1	131 ± 12	48 ± 6
	Nitro-C-Dion	3 µg		15 ± 6	16 ± 1	31 ± 7	99 ± 7	58 ± 8
	trocken (NMQD)	10 µg		10 ± 3	17 ± 6	40 ± 4	102 ± 12	52 ± 6
		33 µg		15 ± 2	24 ± 5	46 ± 4	112 ± 12	59 ± 9
		100 µg		15 ± 1	22 ± 7	70 ± 1	109 ± 14	52 ± 8
		333 µg		17 ± 3	17 ± 5	70 ± 4	115 ± 11	58 ± 6
		1000 µg		12 ± 4^P	25 ± 4^P	63 ± 10^P	113 ± 1^P	48 ± 11^P
		2500 µg		10 ± 2^P M	25 ± 2^P M	53 ± 7^P M	124 ± 4^P M	46 ± 6^P M
		5000 µg		13 ± 1^P M	47 ± 6^P M	70 ± 7^P M	121 ± 6^P M	52 ± 5^P M
	NaN3	10 µg		1886 ± 174			2037 ± 110
	4-NOPD	10 µg				300 ± 2
	4-NOPD	50 µg			89 ± 15
	MMS	3.0 µL						539 ± 59
With Activation	DMSO			18 ± 5	21 ± 8	41 ± 9	142 ± 9	65 ± 6
	Untreated			18 ± 3	22 ± 5	44 ± 6	150 ± 13	60 ± 11
	Nitro-C-Dion	3 µg		22 ± 5	23 ± 2	38 ± 11	141 ± 21	66 ± 7
	trocken (NMQD)	10 µg		18 ± 5	19 ± 4	45 ± 14	126 ± 17	61 ± 3
		33 µg		21 ± 9	22 ± 4	44 ± 9	135 ± 14	64 ± 7
		100 µg		20 ± 4	22 ± 9	62 ± 4	118 ± 21	60 ± 3
		333 µg		18 ± 2	24 ± 3	55 ± 1	130 ± 15	46 ± 10
		1000 µg		23 ± 7^P	23 ± 4^P	63 ± 6^P	143 ± 11^P	67 ± 18^P
		2500 µg		18 ± 3^P M	19 ± 4^P M	59 ± 2^P M	141 ± 6^P M	67 ± 7^P M
		5000 µg		16 ± 6^P M	20 ± 5^P M	63 ± 21^P M	135 ± 26^P M	65 ± 14^P M
	2-AA	2.5 µg		311 ± 8	230 ± 6	1639 ± 86	1356 ± 122
	2-AA	10.0 µg						399 ± 99

 

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

 

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
positive without S9 mix in strains TA 1537 and TA 98

In conclusion, it can be stated that during the described mutage¬nicity test and under the experimental conditions reported, the test item did induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Executive summary:

The test item Nitro-C-Dion trocken (NMQD) was assessed for its potential to induce gene mutations in the plate incorporation test (experi­ment I) and the pre-incubation test (experiment II) usingSalmo­nella typhimuriumstrains TA 1535, TA 1537, TA 98, and TA 100, and theEscheri­chia colistrain WP2 uvrA.

The assay was performed with and without liver microsomal activation. Each concentration and the controls were tested in triplicate. The test item was tested at the following concentrations:

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

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in both experiments.

No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabol­ic activation.

Substantial and dose dependent increases in revertant colony numbers were observed following treatment with Nitro-C-Dion trocken (NMQD) in strains TA 98 and TA 1537 in the absence of metabolic activation (S9 mix). The number of colonies exceeded the threshold of trice the number of the corresponding solvent control in strain TA 1537 at 5000 µg/plate. A slight and dose dependent increase in the number of revertants was also detected in strain TA 98 without metabolic activation but the threshold of two was not quite reached. To verify the weak positive response of the first experiment, a second experiment was performed using the more sensitive pre-incubation method. In experiment II without S9 mix the number of colonies exceeded the threshold of trice the number of the corresponding solvent control in strain TA 1537 at 5000 µg/plate and of twice the number of the corresponding solvent control in strain TA 98 from 100 µg/plate up to 5000 µg/plate.

Appropriate reference mutagens were used as positive controls. They showed a distinct in­crease of induced revertant colonies.