2,5-dimethoxyaniline

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:

2,5-dimethoxyaniline

EC Number:

203-040-9

EC Name:

2,5-dimethoxyaniline

Cas Number:

102-56-7

Molecular formula:

C8H11NO2

IUPAC Name:

2,5-dimethoxyaniline

Details on test material:

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

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
experiment II: 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 and 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:

negative

Cytotoxicity / choice of top concentrations:

other: No toxic effects, with the exception of strain WP2 uvrA in experiment I, where a minor toxic effect was observed at 5000 µg/plate in the absence of metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

- Precipitation: No precipitation was observed

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

    Summary of Results Pre-Experiment and Experiment I

Study Name: 1280500	Study Code: Harlan-CCR 1280500
Experiment: 1280500 VV Plate	Date Plated: 27/07/2009
Assay Conditions:	Date Counted: 30/07/2009

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			12 ± 2	10 ± 3	33 ± 2	131 ± 4	61 ± 5
	Untreated			14 ± 2	10 ± 3	31 ± 7	138 ± 5	57 ± 5
	2,5-Dimethoxyanilin	3 µg		12 ± 1	7 ± 3	35 ± 2	133 ± 19	54 ± 7
		10 µg		14 ± 2	10 ± 2	28 ± 8	135 ± 12	57 ± 8
		33 µg		13 ± 2	13 ± 1	28 ± 4	123 ± 8	56 ± 8
		100 µg		13 ± 2	9 ± 3	33 ± 6	139 ± 7	60 ± 5
		333 µg		11 ± 2	10 ± 3	37 ± 9	132 ± 10	52 ± 7
		1000 µg		13 ± 1	10 ± 0	29 ± 2	130 ± 4	56 ± 2
		2500 µg		13 ± 1	9 ± 1	28 ± 2	141 ± 12	38 ± 2
		5000 µg		11 ± 3	8 ± 2	27 ± 1	143 ± 9	35 ± 6
	NaN3	10 µg		1830 ± 67			1848 ± 184
	4-NOPD	10 µg				268 ± 15
	4-NOPD	50 µg			79 ± 8
	MMS	3.0 µL						1103 ± 36
With Activation	DMSO			15 ± 2	17 ± 3	41 ± 3	161 ± 16	70 ± 4
	Untreated			16 ± 3	17 ± 5	31 ± 4	177 ± 14	64 ± 13
	2,5-Dimethoxyanilin	3 µg		15 ± 5	16 ± 3	44 ± 13	152 ± 15	70 ± 7
		10 µg		15 ± 3	16 ± 2	35 ± 5	150 ± 2	69 ± 9
		33 µg		15 ± 5	18 ± 1	40 ± 8	143 ± 10	63 ± 10
		100 µg		17 ± 3	16 ± 3	46 ± 4	159 ± 10	67 ± 12
		333 µg		15 ± 3	16 ± 1	44 ± 2	155 ± 12	63 ± 13
		1000 µg		15 ± 1	14 ± 1	50 ± 8	155 ± 6	65 ± 9
		2500 µg		13 ± 1	13 ± 6	40 ± 5	156 ± 8	42 ± 2
		5000 µg		16 ± 1	17 ± 3	45 ± 4	143 ± 10	29 ± 6
	2-AA	2.5 µg		349 ± 32	353 ± 13	1891 ± 362	2294 ± 340
	2-AA	10.0 µg						275 ± 13

Key to Positive Controls
NaN3 2-AA 4-NOPD MMS	sodium azide 2-aminoanthracene 4-nitro-o-phenylene-diamine methyl methane sulfonate

 Summary of Results Experiment II

Study Name: 1280500	Study Code: Harlan-CCR 1280500
Experiment: 1280500 HV2 Pre	Date Plated: 13/08/2009
Assay Conditions:	Date Counted: 19/08/2009

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			17 ± 2	14 ± 1	35 ± 4	133 ± 19	55 ± 10
	Untreated			24 ± 2	9 ± 3	35 ± 7	160 ± 4	52 ± 10
	2,5-Dimethoxyanilin	33 µg		16 ± 3	13 ± 3	33 ± 3	137 ± 11	54 ± 10
		100 µg		17 ± 3	12 ± 2	34 ± 6	124 ± 5	50 ± 3
		333 µg		17 ± 3	12 ± 3	31 ± 10	138 ± 3	52 ± 3
		1000 µg		17 ± 4	11 ± 3	29 ± 0	140 ± 11	43 ± 3
		2500 µg		18 ± 3	15 ± 1	33 ± 3	132 ± 12	48 ± 3
		5000 µg		16 ± 4	13 ± 4	32 ± 4	141 ± 6	44 ± 4
	NaN3	10 µg		1701 ± 48			1732 ± 79
	4-NOPD	10 µg				395 ± 12
	4-NOPD	50 µg			105 ± 4
	MMS	3.0 µL						356 ± 20
With Activation	DMSO			24 ± 4	23 ± 2	38 ± 7	129 ± 3	62 ± 5
	Untreated			25 ± 4	19 ± 4	42 ± 8	164 ± 15	64 ± 8
	2,5-Dimethoxyanilin	33 µg		24 ± 2	22 ± 2	46 ± 5	148 ± 12	67 ± 5
		100 µg		24 ± 2	22 ± 5	46 ± 5	139 ± 17	56 ± 7
		333 µg		23 ± 1	22 ± 2	49 ± 9	136 ± 23	58 ± 2
		1000 µg		24 ± 3	21 ± 2	39 ± 5	142 ± 20	53 ± 7
		2500 µg		23 ± 2	18 ± 5	40 ± 9	133 ± 14	55 ± 11
		5000 µg		24 ± 3	20 ± 1	40 ± 5	126 ± 16	47 ± 3
	2-AA	2.5 µg		261 ± 30	158 ± 18	1091 ± 77	1623 ± 49
	2-AA	10.0 µg						292 ± 36

Key to Positive Controls
NaN3 2-AA 4-NOPD MMS	sodium azide 2-aminoanthracene 4-nitro-o-phenylene-diamine methyl methane sulfonate

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

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:

The test item 2,5-Dimethoxyanilin was assessed for its potential to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA.

The assay was performed in two independent experiments both 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

Experiment II:                                   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 metabolic activation in both independent 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 with the exception of strain WP2 uvrA in experiment I, where a minor toxic effect was observed at 5000 µg/plate in the absence of metabolic activation

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with 2,5-Dimethoxyanilin at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

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