4-benzoylbenzene-1,2,3-triyl tris(6-diazo-5,6-dihydro-5-oxonaphthalene-1-sulphonate)

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:

1982

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

No historical control range data given

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1982

Report date:

1982

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted in 1997

Deviations:

yes

Remarks:

No historical control range data given

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

4-benzoylbenzene-1,2,3-triyl tris(6-diazo-5,6-dihydro-5-oxonaphthalene-1-sulphonate)

EC Number:

227-030-9

EC Name:

4-benzoylbenzene-1,2,3-triyl tris(6-diazo-5,6-dihydro-5-oxonaphthalene-1-sulphonate)

Cas Number:

5610-94-6

Molecular formula:

C₄₃H₂₂N₆O₁₃S₃

IUPAC Name:

4-benzoylbenzene-1,2,3-triyl tris(6-diazo-5,6-dihydro-5-oxonaphthalene-1-sulphonate)

Test material form:

solid

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: protected from light

Method

Target gene:

his operon, trp operon

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1538

Species / strain 3

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

cofactor-supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male rats, treated with Aroclor 1254

Test concentrations with justification for top dose:

Based on a range-finding study (= Experiment 1, performed in all tester strains; doses applied: 4 - 10 000 μg/mL), the following concentrations were used in the main experiment (= Experiment 2): 0.8, 4, 20, 100, 500 and 2500 μg/plate with and without metabolic activation.

The top dose was selected based on precipitation observed at ≥ 2500 μg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

other: 2-aminoanthracene (2AA), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) (range-finding study (Experiment 1) and main experiment (Experiment 2))

DURATION
- Exposure duration: 48 - 72 h

NUMBER OF REPLICATIONS: triplicates

DETERMINATION OF CYTOTOXICITY
- Method: revertant colony number and microscopical inspection of the bacterial background lawn

The results of both experiments (exp.1 and 2) were used for determining the mutagenic properties of the test substance.

Evaluation criteria:

A chemical is considered to have a positive response, if it shows a significant increase in the number of induced revertants and/or shows dose-dependent effect.

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, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: yes, observed at ≥ 2500 μg/plate with and without S9 mix in the plates of all tested strains

Any other information on results incl. tables

Table 1: Summary of test results (Experiment 1; dose-finding assay (Plate Incorporation Method)

With or without S9 Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates)
		Frameshift type			Base-pair substitution type
		TA 98	TA 1537	TA 1538	TA 100	TA 1535	WP2 uvr A
–	Solvent control (DMSO)	27	8	27	121	27	36
	4	30	11	12	90	33	33
	20	25	9	13	107	27	40
	100	37	9	20	120	31	37
	500	26	5	22	102	26	30
	2500	24 P	7 P	14 P	115 P	27 P	35 P
	10000	21 P	5 P	5 P	83 P	23 P	41 P
	Positive controls (µg/plate)	2NF (5)	9AA (50)	2NF (5)	SA (1)	SA (1)	MNNG (10)
	Mean (No. of colonies/plate)	204	81	319	833	617	1610
+	Solvent control (DMSO)	39	3	24	99	11	33
	4	35	14	8	115	11	38
	20	40	5	24	164	15	62
	100	36	4	18	125	5	41
	500	41	5	21	119*	13	44
	2500	40 P	9 P	17 P	135 P	8 P	47 P
	10000	33 P	8 P	15 P	125 P	9 P	40 P
	Positive controls (µg/plate)	2AA (1)					2AA (10)
	Mean (No. of colonies/plate)	917	56	786	894	113	241
	Positive controls (µg/plate)	B(a)P (10)
	Mean (No. of colonies/plate)	605	121	243	607	19	93

2AA = 2-aminoanthracene

2NF = 2-nitrofluorene

9AA = 9-aminoacridine

B(a)P = benzo(a)pyrene

MNNG = N-methyl-N'-nitro-N-nitrosoguanidine

P = precipitate

SA = sodium azide

 

Table 2: Summary of test results (Experiment 2, main assay (Plate Incorporation Method))

With or without S9 Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates)
		Frameshift type			Base-pair substitution type
		TA 98	TA 1537	TA 1538	TA 100	TA 1535	WP2 uvr A
–	Solvent control (DMSO)	25	10	13	120	29	32
	0.8	28	8	13	133	23	23
	4	20	7	12	128	23	26
	20	28	10	10	137	23	24
	100	30	8	13	127	24	26
	500	28	9	11	131	21	21
	2500	27 P	8 P	14 P	125 P	23 P	26 P
	Positive controls (µg/plate)	2NF (5)	9AA (50)	2NF (5)	SA (1)	SA (1)	MNNG (10)
	Mean (No. of colonies/plate)	204	81	319	833	617	1610
+	Solvent control (DMSO)	31	14	17	125	13	24
	0.8	41	11	17	111	10	27
	4	34	12	20	111	13	32
	20	39	10	21	126	11	30
	100	34	8	19	123	15	25
	500	37	11	19	123	13	37
	2500	30 P	10 P	20 P	110 P	12 P	35 P
	Positive controls (µg/plate)	2AA (1)					2AA (10)
	Mean (No. of colonies/plate)	917	56	786	894	113	241
	Positive controls (µg/plate)	B(a)P (10)
	Mean (No. of colonies/plate)	605	121	243	607	19	93

2AA = 2-aminoanthracene

2NF = 2-nitrofluorene

9AA = 9-aminoacridine

B(a)P = benzo(a)pyrene

MNNG = N-methyl-N'-nitro-N-nitrosoguanidine

P = precipitate

SA = sodium azide

Applicant's summary and conclusion

Conclusions:

It is concluded that the test substance is not mutagenic in these bacterial test systems neither in the absence nor in the presence of an exogenous metabolizing system. 

Executive summary:

The test item was tested for mutagenicity with Salmonella typhimurium strains TA 100, TA 1535, TA 1537, TA 1538, TA 98 and E. coli WP2uvrA with and without the addition of a metabolic activation system according to OECD Guideline 471. The test compound was tested at doses of 0.4 to 10000 µg/plate and proved to be not toxic. Visible precipitation on the plates has been observed at 2500 µg/plate. For mutagenicity testing 2500 µg/plate was chosen as the highest dose. 

The test compound did not cause a significant increase in the number of colonies with any of the tester strains either in the absence or presence of S9 mix. No dose dependent effect was obtained.

It is concluded that the test substance is not mutagenic in these bacterial test systems neither in the absence nor in the presence of an exogenous metabolizing system.