N-[4-[(9,10-dihydro-4-hydroxy-9,10-dioxo-1-anthryl)amino]phenyl]acetamide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27-10-2004 - 19-11-2004

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:

2005

Report date:

2005

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: EC Directive 2000/32/EC, L 136, Annex 4D, B.13/B.14

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Japanese Substance Control Law (JSCL) Test Guideline III. 1 Gene Mutation Test With Bacteria

Principles of method if other than guideline:

Salmonella/microsome mutagenicity test (Ames test) (plate incorporation) was performed to assess the mutagenic nature of the test chemical.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

N-[4-[(9,10-dihydro-4-hydroxy-9,10-dioxo-1-anthryl)amino]phenyl]acetamide

EC Number:

267-636-0

EC Name:

N-[4-[(9,10-dihydro-4-hydroxy-9,10-dioxo-1-anthryl)amino]phenyl]acetamide

Cas Number:

67905-17-3

Molecular formula:

C22H16N2O4

IUPAC Name:

N-[4-[(9,10-dihydro-4-hydroxy-9,10-dioxo-1-anthryl)amino]phenyl]acetamide

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material : Polysynthrene Blue R
- Molecular formula): C22H16N2O4
- Molecular weight : 372.37 g/mol
- Smiles notation : O=C(Nc1ccc(Nc2c3c(c(O)cc2)C(=O)c2c(cccc2)C3=O)cc1)C
- InChl: 1S/C22H16N2O4/c1-12(25)23-13-6-8-14(9-7-13)24-17-10-11-18(26)20-19(17)21(27)15-4-2-3-5-16(15)22(20)28/h2-11,24,26H,1H3,(H,23,25)
- Substance type: Organic
- Physical state: Blue Powder
- Content: 83.5% by HPLC
14.3% organic by-productes by HPLC
2.1% Water by Karl-Fischer Titration
0.1% inorganic impurities by sulphated ash

- Name of test material : N-[4-[(9,10-dihydro-4-hydroxy-9,10-dioxo-1-anthryl)amino]phenyl]acetamide- Molecular formula): C22H16N2O4- Molecular weight : 372.378 g/mol- Smiles notation : O=C(Nc1ccc(Nc2c3c(c(O)cc2)C(=O)c2c(cccc2)C3=O)cc1)C- InChl: 1S/C22H16N2O4/c1-12(25)23-13-6-8-14(9-7-13)24-17-10-11-18(26)20-19(17)21(27)15-4-2-3-5-16(15)22(20)28/h2-11,24,26H,1H3,(H,23,25)- Substance type: Organic- Physical state: Powder

Method

Target gene:

Histidine for Salmonella typhimurium strains and tryptophan for E.coli strains

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

other: Salmonella typhimurium strains TA98: hisD3052 rfa uvr B+R TA100: hisG46 rfa uvr B+R Ta1535: hisG46 rfa uvrB TA1537: hisC3076 rfa uvrB Escherichia coli WP2uvrA: pKM101

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

other: pKM101

Metabolic activation:

with and without

Metabolic activation system:

S9 metabolic activation system was prepared from liver of male Sprague Dawley rat homogenate

Test concentrations with justification for top dose:

Plate incorporation assay:
With: 0, 50, 160, 500, 1600 or 5000 µg/plate
Without: 0, 50, 160, 500, 1600 or 5000 µg/plate

Vehicle / solvent:

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

Controls

Untreated negative controls:

yes

Remarks:

Untreated control

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

sodium azide

other: 2-nitrofluorene (TA98, -S9), 2-aminoanthracene (All strains, +S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
- Cell density at seeding (if applicable): No data

DURATION
- Preincubation period: No data
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

SELECTION AGENT (mutation assays): No data

SPINDLE INHIBITOR (cytogenetic assays): No data

STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: Triplicate

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: No data

NUMBER OF CELLS EVALUATED: No data

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): No data

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: Yes
- Any supplementary information relevant to cytotoxicity: Toxicity was assessed after microscopic thinning of the bacterial lawn and/or reduction of the number of spontaneously occuring mutants compared to the corresponding solvent control value

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): No data

- OTHER: No data

Rationale for test conditions:

No data

Evaluation criteria:

The test chemical was considered positive if-
- it produces atleast 2-fold increase in the mean number of revertants/plate of atleast one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control at complete bacterial background lawn
- it induces a dose related increase in the mean number of revertants per plate of at least one of the test strains over the mean number of revertants per plate of the appropriate vehicle in atleast two or three concentrations of the test item at complete bacterial background lawn

If the aboce critera is not achieved, it is considered to show no mutagenic activity in the system

Statistics:

No data

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

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
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: Visible precipitation was observed on plates at 160 µg/plate and above
- Definition of acceptable cells for analysis: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: No data

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: No data

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: No data
- Indication whether binucleate or mononucleate where appropriate: No data

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: No data
- Negative (solvent/vehicle) historical control data: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: No data
- Other observations when applicable: No data

Remarks on result:

other: No mutagenic potential

Any other information on results incl. tables

Table: Results of the mutagenicity test for the test chemical

TA100
S9	Dose levelsµg/plate	Number of revertants/plate			Mean	SD	Ratio dose/control	p
		Plate 1	Plate 1	Plate 3
+	2-Aminoanthracene	1703	1512	1460	1558.3	128.0	12.7
+	Negative control	124	128	119	123.7	4.5	-
+	DMSO	111	134	122	122.3	11.5	-
+	50	135	116	126	125.7	9.5	1.0
+	160	137	130	151	139.3	10.7	1.1	P
+	500	135	125	127	129.0	5.3	1.1	P
+	1600	129	127	139	131.7	6.4	1.1	P
+	5000	122	104	110	112.0	9.2	0.9	p
-	Sodium azide	586	539	620	581.7	40.7	5.4
-	Negative control	123	115	119	119.0	4.0	-
-	DMSO	109	119	98	108.7	10.5	-
-	50	109	109	104	107.3	2.9	1.0
-	160	109	103	130	114.0	14.2	1.0	P
-	500	109	98	109	105.3	6.4	1.0	P
-	1600	103	108	115	108.7	6.0	1.0	P
-	5000	109	98	79	95.3	15.2	0.9	P

P: Precipitated

TA1535
S9	Dose levelsµg/plate	Number of revertants/plate			Mean	SD	Ratio dose/control	p
		Plate 1	Plate 1	Plate 3
+	2-Aminoanthracene	440	439	456	445.0	9.5	47.7
+	Negative control	4	7	10	7.0	3.0	-
+	DMSO	10	9	9	9.3	0.6	-
+	50	8	8	4	6.7	2.3	0.7
+	160	11	7	5	7.7	3.1	0.8	P
+	500	11	5	4	6.7	3.8	0.7	P
+	1600	7	10	8	8.3	1.5	0.9	P
+	5000	12	6	7	8.3	3.2	0.9	p
-	Sodium azide	193	1889	143	175.0	37.8	32.8
-	Negative control	13	6	11	10.0	3.6	-
-	DMSO	6	4	6	5.3	1.2	-
-	50	7	4	5	5.3	1.5	1.0
-	160	6	7	5	6.0	1.0	1.1	P
-	500	10	7	3	6.7	3.5	1.3	P
-	1600	6	8	5	6.3	1.5	1.2	P
-	5000	8	5	10	7.7	2.5	1.4	P

P: Precipitated

 

TA1537
S9	Dose levelsµg/plate	Number of revertants/plate			Mean	SD	Ratio dose/control	p
		Plate 1	Plate 1	Plate 3
+	2-Aminoanthracene	99	102	93	98.0	4.6	5.9
+	Negative control	19	17	12	16.0	3.6	-
+	DMSO	18	16	16	16.7	1.2	-
+	50	28	22	17	22.3	5.5	1.3
+	160	20	23	26	23.0	3.0	1.4	P
+	500	24	24	20	22.7	2.3	1.4	P
+	1600	19	23	24	22.0	2.6	1.3	P
+	5000	14	17	27	19.3	6.8	1.2	p
-	9-aminoacridine	270	222	153	215.0	58.8	23.0
-	Negative control	12	15	8	11.7	3.5	-
-	DMSO	7	9	12	9.3	2.5	-
-	50	13	11	16	13.3	2.5	1.4
-	160	9	11	8	9.3	1.5	1.0	P
-	500	8	7	12	9.0	2.6	1.0	P
-	1600	15	13	10	12.7	2.5	1.4	P
-	5000	10	13	11	11.3	1.5	1.2	P

P: Precipitated

 

TA98
S9	Dose levelsµg/plate	Number of revertants/plate			Mean	SD	Ratio dose/control	p
		Plate 1	Plate 1	Plate 3
+	2-Aminoanthracene	1054	1120	1076	1083.3	33.6	54.2
+	Negative control	18	23	23	21.3	2.9	-
+	DMSO	19	21	20	20.0	1.0	-
+	50	21	18	14	17.7	3.5	0.9
+	160	28	27	21	25.3	3.9	1.3	P
+	500	30	21	28	26.3	4.7	1.3	P
+	1600	32	26	34	30.7	4.2	1.5	P
+	5000	20	34	34	29.3	8.1	1.5	p
-	2-nitrofluorene	301	299	301	300.3	1.2	26.5
-	Negative control	12	14	9	11.7	2.5	-
-	DMSO	14	13	7	11.3	3.8	-
-	50	14	14	8	12.0	3.5	1.1
-	160	8	14	13	11.7	3.2	1.0	P
-	500	16	13	12	13.7	2.1	1.2	P
-	1600	13	4	12	9.7	4.9	0.9	P
-	5000	7	11	10	9.3	2.1	0.8	P

P: Precipitated

 

E.coli WP2uvrA
S9	Dose levelsµg/plate	Number of revertants/plate			Mean	SD	Ratio dose/control	p
		Plate 1	Plate 1	Plate 3
+	2-Aminoanthracene	125	97	88	103.3	19.3	7.0
+	Negative control	15	18	11	14.7	3.5	-
+	DMSO	16	11	17	14.7	3.2	-
+	50	17	19	17	17.7	1.2	1.2
+	160	16	15	15	15.3	0.6	1.0	P
+	500	15	11	18	14.7	3.5	1.0	P
+	1600	14	16	14	14.7	1.2	1.0	P
+	5000	16	13	12	13.7	2.1	0.9	p
-	9-NQO	529	578	572	559.7	26.7	31.7
-	Negative control	17	17	23	19.0	3.5	-
-	DMSO	26	14	13	17.7	7.2	-
-	50	21	15	15	17.0	3.5	1.0
-	160	18	14	17	16.3	2.1	0.9	P
-	500	15	20	20	18.3	2.9	1.0	P
-	1600	19	17	15	17.0	2.0	1.0	P
-	5000	12	19	18	16.3	3.8	0.9	P

P: Precipitated

 

Applicant's summary and conclusion

Conclusions:

The registered substance was tested non-mutagenic (negative) both in the presence and absence of S9 metabolic activation in Salmonella typhimurium TA1535, TA1537, TA98 and TA100 and Escherichia coli WP2uvrA tester strains. The test was performed according to OECD TG 471 and in compliance with OECD Principles of Good Laboratory Practice.

Executive summary:

Salmonella/microsome mutagenicity test (Ames test) was performed to assess the mutagenic nature of theregistered substance. The study was conducted according to the plate incorporation method and in the presence and absence of S9 metabolic activation system using Salmonella typhimurium TA1535, TA1537, TA98 and TA100 and Escherichia coli WP2uvrA tester strains. The test chemical was dissolved in DMSO and used at concentrationsof 0, 50, 160, 500, 1600 or 5000 µg/plate. The plates were incubated for 48 hrs and observed for revertant colonies. Concurrent solvent(Dimethyl sulfoxide),negative controland positive control substances were also included in the study. The test chemical did not induce a relevant or dose-dependent increase in the number of revertants colonies at any concentrations tested either in the presence or absence of S9 metabolic activation system using Salmonella typhimurium TA1535, TA1537, TA98 and TA100 and Escherichia coli WP2uvrA tester strains.