1,4-bis(p-tolylamino)anthraquinone

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is from Study Report

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

The experiments were performed to assess the potential of the test item to induce gene mutations by means of two independent Salmonella typhimurium and Escherichia coli reverse mutation assays. Experiment I was performed as a plate incorporation assay. Since a negative result was obtained in this experiment, experiment II was performed as a pre-incubation assay.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

1,4-bis(p-tolylamino)anthraquinone

EC Number:

204-909-5

EC Name:

1,4-bis(p-tolylamino)anthraquinone

Cas Number:

128-80-3

Molecular formula:

C28H22N2O2

IUPAC Name:

1,4-bis[(4-methylphenyl)amino]-9,10-anthraquinone

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Test Item Identity: SANDOPLAST GREEN GSB (1,4-bis(p-tolylamino)anthraquinone)
- Lot/batch No.of test material: FRAA016121
- Expiration date of the lot/batch: October 01, 2003
- Purity test date: no data

RADIOLABELLING INFORMATION (Not applicable)
- Radiochemical purity: N/A
- Specific activity: N/A
- Locations of the label: N/A
- Expiration date of radiochemical substance: N/A

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: no data
- Solubility and stability of the test substance in the solvent/vehicle: 10 days in water, saline, polyethylene glycol and CMC
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:no data

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: On the day of the experiment, the test item SANDOPLAST GREEN GSB was suspended in DMSO.
- Preliminary purification step (if any): no data
- Final dilution of a dissolved solid, stock liquid or gel: no data
- Final preparation of a solid: no data

Method

Target gene:

Histidine

Species / strain

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

No data

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

Rat Liver S9

Test concentrations with justification for top dose:

In the pre-experiment the concentration range of the test item was 3 – 5000 μg/plate. The pre-experiment is reported as experiment I since no relevant toxic effects were observed and 5000 μg/plate were chosen as maximal concentration.
The concentration range included two logarithmic decades. The following concentrations were tested:
0; 33, 100; 333; 1000; 2500; and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was soluble in DMSO

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

For strains TA 1535, TA 100 without metabolic activation

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine, 4-NOPD

Remarks:

For strains TA 1537, TA 98 without metabolic activation

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

For strains WP2 uvrA without metabolic activation

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene, 2-AA

Remarks:

For strains TA 1535, TA 1537, TA 98, TA100, WP2 uvrA with metabolic activation.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar

DURATION
- Preincubation period: 1 h
- Exposure duration:48 h

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: other: evaluation of background lawn

Rationale for test conditions:

No data

Evaluation criteria:

The Salmonella typhimurium and Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
- regular background growth in the negative and solvent control
- the spontaneous reversion rates in the negative and solvent control are in the range of our historical data
- the positive control substances should produce a significant increase in mutant colony frequencies

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:

No statistical evaluation of the data is required.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The test item was assessed for its potential to induce gene mutations according to 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:
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 (below the factor of 0.5), evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation.
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment 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. In experiment II, with metabolic activation, the number of colonies in strain TA 98 (negative control) was slightly above the range of our historical data. Since this deviation is rather small, this effect is judged to be based upon statistical fluctuations and has no detrimental impact on the outcome of the study.The historical range of positive controls was exceeded in strains TA 1535 (experiment I and II) and in strain TA 100 (experiment I) without metabolic activation. This effect indicates the sensitivity of the strains rather than compromising the assay. Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.
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.

Remarks on result:

other: No mutagenic potential

Any other information on results incl. tables

The following concentrations were tested for toxicity and mutation induction with each 3 plates.

Substance	Concentration /plate (µg)	Revertants per plate (mean of 3 plates)
		TA 1535		TA 1537		TA 98		TA 100		WP2 uvrA
S-9		-	+	-	+	-	+	-	+	-	+
Negative control		19	16	9	13	35	32	128	168	53	56
Solvent control		16	14	6	11	34	36	128	132	55	54
4-NOPD	50			65
4-NOPD	100					272
MMS	4 (µl)									859
NaN3	10	1216						931
2-AA	2,5		220		115		995		1039
2-AA	10										200
Test item	3	15	12	7	14	33	36	131	145	51	53
	10	18	13	8	13	30	37	126	145	47	51
	33	16	10	7	13	26	35	128	149	45	48
	100	14	11	6	12	25	33	127	140	47	49
	333	13	12	5	13	23	31	128	139	48	48
	1000	15	11	6	13	23	32	112	137	46	48
	2500	12	9	5	10	19	27	117	132	48	48
	5000	8	8	6	10	17	27	109	125	48	47

 

Applicant's summary and conclusion

Conclusions:

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. Therefore, the given test chemical is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutati on assay.

Executive summary:

This study was performed to investigate the potential of the given test chemical to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the 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 (S9 mix). Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations: 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 (below the factor of 0.5), evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with test chemical 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. 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. Therefore, the given test chemical is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.