4H-Pyrazino[2,1-a]isoquinolin-4-one, 2-acetyl-1,2,3,6,7,11b-hexahydro-

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:

2004-12-21 to 2005-05-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

470-180-7

EC Name:

-

Cas Number:

61196-40-5

Molecular formula:

C14H16N2O2

IUPAC Name:

2-acetyl-1H,2H,3H,4H,6H,7H,11bH-pyrazino[2,1-a]isoquinolin-4-one

Method

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-induced rat liver S-9 mix.

Test concentrations with justification for top dose:

Concentration range in the 1st main test (with and without metabolic activation): 5.00, 15.8, 50.0, 158, 500, 1580 and 5000 µg/plate
Concentration range in the 2nd main test (with and without metabolic activation): 50.0, 158, 500, 1580 and 5000 µg/plate

Vehicle / solvent:

Solvent: DMSO

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

benzo(a)pyrene

cumene hydroperoxide

other: Daunomycin and N-ethyl-N'-nitro-N-nitrosoguanidine in the absence of S9 mix. 2-Aminoanthracene with S9 mix.

Rationale for test conditions:

According to the guideline

Evaluation criteria:

A test material is defined as non-mutagenic in this assay if:
- "no" or "weak increases" occur in the first and second series of the main experiment. ("Weak increases" randomly occur due to experimental variation.)

A test material is defined as mutagenic in this assay if:
- a dose-related (over at least two test material concentrations) increase in the number of revertants is induced, the maximal effect is a "clear increase", and the effects are reproduced at similar concentration levels in the same test system;
- "clear increases" occur at least at one test material concentration, higher concentrations show strong precipitation or cytotoxicity, and the effects are reproduced at the same concentration level in the same test system.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 6

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Any other information on results incl. tables

Table 1 Summary of mean revertant numbers, run 1

Test Material	Concentration [µg/plate]	+/- S9 Mix	Mean revertant colonies /
			TA 98	TA 100	TA 102
Solvent control	0	-	17	139	237
Test item	5	-	20	127	237
	15.8	-	23	127	221
	50	-	19	130	211
	158	-	20	133	266
	500	-	25	124	220
	1580	-	21	136	197
	5000	-	18	110	150
Solvent control	0	+	32	162	310
Test item	5	+	28	157	305
	15.8	+	32	159	307
	50	+	23	145	313
	158	+	28	149	318
	500	+	31	162	281
	1580	+	31	153	146
	5000	+	23	140	211
Positive controls	Name	-	DAUN	ENNG	CUM
	Conc [µg/plate]		4	5	200
	Revert. Colonies/plate		271	548	1126
	Name	+	2-AA	2-AA	B(a)P
	Conc [µg/plate]		2	2	10
	Revert. Colonies/plate		401	614	1659

Test Material	Concentration [µg/plate]	+/- S9 Mix	Mean revertant colonies /
			1535	1537	E. coli WP2 uvrA
Solvent control	0	-	18	6	43
Test item	5	-	24	3	41
	15.8	-	27	6	46
	50	-	27	5	44
	158	-	20	3	44
	500	-	17	4	46
	1580	-	24	7	43
	5000	-	19	11	23
Solvent control	0	+	24	8	47
Test item	5	+	25	7	55
	15.8	+	25	8	53
	50	+	23	8	44
	158	+	22	6	46
	500	+	21	6	49
	1580	+	23	7	43
	5000	+	16	2	28
Positive controls	Name	-	ENNG	9-AA	ENNG
	Conc [µg/plate]		10	50	5
	Revert. Colonies/plate		312	347	1027
	Name	+	2-AA	2-AA	2-AA
	Conc [µg/plate]		2	5	10
	Revert. Colonies/plate		215	265	407

Table 2 Summary of mean revertant numbers, run 2

Test Material	Concentration [µg/plate]	+/- S9 Mix	Mean revertant colonies /
			TA 98	TA 100	TA 102
Solvent control	0	-	17	132	211
Test item	50	-	20	130	222
	158	-	16	123	241
	500	-	16	125	221
	1580	-	16	126	187
	5000	-	11	105	154
Solvent control	0	+	27	189	207
Test item	50	+	27	184	194
	158	+	31	197	193
	500	+	23	191	189
	1580	+	21	182	184
	5000	+	21	161	170
Positive controls	Name	-	DAUN	ENNG	CUM
	Conc [µg/plate]		4	5	200
	Revert. Colonies/plate		522	597	1077
	Name	+	2-AA	2-AA	B(a)P
	Conc [µg/plate]		2	2	10
	Revert. Colonies/plate		113	180	568

Test Material	Concentration [µg/plate]	+/- S9 Mix	Mean revertant colonies /
			1535	1537	E. coli WP2 uvrA
Solvent control	0	-	19	7	47
Test item	50	-	20	5	38
	158	-	16	8	44
	500	-	18	9	37
	1580	-	21	6	37
	5000	-	15	12	31
Solvent control	0	+	23	10	50
Test item	50	+	23	11	53
	158	+	28	12	49
	500	+	23	9	41
	1580	+	23	7	42
	5000	+	16	7	32
Positive controls	Name	-	ENNG	9-AA	ENNG
	Conc [µg/plate]		10	50	5
	Revert. Colonies/plate		436	391	910
	Name	+	2-AA	2-AA	2-AA
	Conc [µg/plate]		2	10	10
	Revert. Colonies/plate		120	209	152

DAUN: Daunomycin

ENNG: N-Ethyl-N'-nitro-N-nitroso-guanidine

CUM: Cumene hydroperoxide

2-AA: 2-Aminoanthracene

B(a)p: Benzo(a)pyrene

9-AA: 9-Aminoacridine

Applicant's summary and conclusion

Conclusions:

Under the conditions of this assay the test item was not mutagenic to bacterial cells.

Executive summary:

The investigations for the mutagenic potential of the test material were performed using Salmonella typhimurium tester strains TA 98, TA 100, TA 102, TA 1535 and TA 1537, and Escherichia coli WP2 uvrA according to OECD TG 471. The plate incorporation test with and without addition of liver S9 mix from Aroclor 1254-pretreated rats was used. Two independent experimental series were performed. In the two series with S9 mix, 10 or 30 % S9 in the S9 mix were used in the 1st and 2nd series, respectively. The test item was dissolved in dimethyl sulfoxide (DMSO) and tested at concentrations ranging from 5.00 to 5000 µg/plate. Precipitation of the test material on the agar plates did not occur. Toxicity to the bacteria was not observed. Daunomycin, N-ethyl-N'-nitro-N-nitrosoguanidine, 9-aminoacridine and cumene hydroperoxide served as strain specific positive control test materials in the absence of S9 mix. 2-Aminoanthracene and benzo[a]pyrene were used for testing the bacteria and the activity of the S9 mix. Each treatment with the test materials used as positive controls led to a clear increase in revertant colonies, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used. With and without addition of S9 mix as the external metabolizing system, the test material was not mutagenic under the experimental conditions described.