1,4-dimethoxy-2-nitrobenzene

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: well performed GLP and OECD guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

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-dimethoxy-2-nitrobenzene

EC Number:

201-903-4

EC Name:

1,4-dimethoxy-2-nitrobenzene

Cas Number:

89-39-4

Molecular formula:

C8H9NO4

IUPAC Name:

1,4-dimethoxy-2-nitrobenzene

Details on test material:

- Name of test material (as cited in study report): 2,5- Dimethoxynitrobenzol

Method

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-Naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

pre-experiment/experiment I: 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

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 test

DURATION
- Exposure: After solidification the plates were incubated upside down for at least 48 hours at 37 °C in the dark.

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

Test results 1

Species / strain:

other: TA 1535, TA 1537, WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

other: postive at toxic concentrations without activation

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No
- Other confounding effects:
COMPARISON WITH HISTORICAL CONTROL DATA: performed
ADDITIONAL INFORMATION ON CYTOTOXICITY:

The plates incubated with the test item showed reduced background growth at the following concentrations (µg/plate):

Experiment I
Strain: without S9 mix / with S9 mix
TA 1535: 2500 - 5000 / 1000 - 5000
TA 1537: 1000 - 5000 / 2500 - 5000
TA 98: 2500 - 5000 / 2500 - 5000
TA 100: 2500 - 5000 / 2500 - 5000
WP2 uvrA: 5000 / 2500 - 5000

Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in strains TA 1535, TA 1537 at 5000 µg/plate and in strain WP2 uvrA from 2500 µg/plate up to 5000 µg/plate in the presence of metabolic activation.

Any other information on results incl. tables

  Summary of Results Pre-Experiment and Experiment I

Study Name: 1261801	Study Code: Harlan-CCR 12614801
Experiment: 1261801 VV plate	Date Plated: 20/04/2009
Assay Conditions:	Date Counted: 23/04/2009

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			11 ± 2	9 ± 1	26 ± 6	145 ± 16	51 ± 0
	Untreated			14 ± 1	7 ± 1	26 ± 5	167 ± 15	54 ± 9
	2,5-	3 µg		13 ± 1	9 ± 4	26 ± 7	142 ± 23	50 ± 12
	Dimethoxynitrobenzol	10 µg		16 ± 4	6 ± 4	25 ± 4	139 ± 2	53 ± 11
		33 µg		11 ± 5	8 ± 1	24 ± 7	162 ± 4	55 ± 4
		100 µg		20 ± 7	10 ± 5	28 ± 7	180 ± 11	53 ± 5
		333 µg		17 ± 2	9 ± 1	30 ± 4	228 ± 6	48 ± 3
		1000 µg		23 ± 3	17 ± 7^R	36 ± 6	370 ± 7	42 ± 3
		2500 µg		28 ± 3^R	9 ± 3^R	59 ± 11^R	847 ± 68^R	50 ± 7
		5000 µg		14 ± 8^R	17 ± 2^R	69 ± 13^R	657 ± 35^R	36 ± 14^R
	NaN3	10 µg		1978 ± 80			2297 ± 65
	4-NOPD	10 µg				354 ± 22
	4-NOPD	50 µg			79 ± 3
	MMS	3.0 µL						1307 ± 64
With Activation	DMSO			16 ± 3	11 ± 1	34 ± 3	146 ± 15	63 ± 6
	Untreated			18 ± 2	13 ± 6	34 ± 9	167 ± 24	59 ± 12
	2,5-	3 µg		19 ± 3	17 ± 7	28 ± 7	134 ± 15	58 ± 1
	Dimethoxynitrobenzol	10 µg		16 ± 5	12 ± 2	32 ± 4	149 ± 7	53 ± 5
		33 µg		18 ± 2	8 ± 4	28 ± 6	165 ± 12	65 ± 6
		100 µg		16 ± 4	8 ± 1	32 ± 3	214 ± 13	63 ± 4
		333 µg		18 ± 5	13 ± 1	37 ± 5	307 ± 31	60 ± 11
		1000 µg		34 ± 6^R	17 ± 5	51 ± 3	657 ± 35	51 ± 13
		2500 µg		36 ± 6^R	12 ± 2^R	57 ± 1^R	951 ± 179^R	24 ± 6^R
		5000 µg		0 ± 0^R	4 ± 2^R	67 ± 24^R	140 ± 26^R	13 ± 5^R
	2-AA	2.5 µg		311 ± 4	275 ± 2	2066 ± 205	2487 ± 70
	2-AA	10.0 µg						319 ± 30

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	R	Reduced background growth

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
positive

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did induce gene mutations in the genome of the strains TA 98 and TA 100.
Therefore, 2,5-Dimethoxynitrobenzol is considered to be mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

The test item 2,5-Dimethoxynitrobenzol was assessed for its potential to induce gene mutations in the plate incorporation test (experi­ment I) using Salmo­nella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escheri­chia coli strain 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:            3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

The plates incubated with the test item showed reduced background growth at the following concentrations (µg/plate):

Strain	Experiment I
	without S9 mix	with S9 mix
TA 1535	2500 - 5000	1000 - 5000
TA 1537	1000 - 5000	2500 - 5000
TA 98	2500 - 5000	2500 - 5000
TA 100	2500 - 5000	2500 - 5000
WP2 uvrA	5000	2500 - 5000

No precipitation of the test item occurred up to the highest investigated dose.

Substantial and dose dependent increases in revertant colony numbers were observed following treatment with 2,5-Dimethoxynitrobenzol in strains TA 98 and TA 100. The number of colonies exceeded the threshold of twice in strain TA 98 at 2500 µg/plate and above in the absence of metabolic activation at concentrations showing reduced background growth. The number of colonies exceeded the threshold of twice in strain TA 100 at 1000 µg/plate and above in the absence of metabolic activation and at 333 µg/plate and above in the presence of metabolic activation.

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