6'-(dibutylamino)-3'-methyl-2'-(phenylamino)spiro[isobenzofuran-1(3H),9-(9H)-xanthen]-3-one

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:

1988-02-12 to 1988-02-28

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1988

Report date:

1988

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

yes

Remarks:

Only 4 tester strains were used.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

6'-(dibutylamino)-3'-methyl-2'-(phenylamino)spiro[isobenzofuran-1(3H),9-(9H)-xanthen]-3-one

EC Number:

403-830-5

EC Name:

6'-(dibutylamino)-3'-methyl-2'-(phenylamino)spiro[isobenzofuran-1(3H),9-(9H)-xanthen]-3-one

Cas Number:

89331-94-2

Molecular formula:

C35H36N2O3

IUPAC Name:

6'-(dibutylamino)-3'-methyl-2'-(phenylamino)-3H-spiro[2-benzofuran-1,9'-xanthen]-3-one

Method

Species / strain

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor induced rat liver (S9 mix)

Test concentrations with justification for top dose:

DRF: 5, 50, 500, 5000 µg/plate with or without metabolic activation
Main test: 50, 150, 500, 1500, 5000 µg/plate with or without metabolic activation

Vehicle / solvent:

- Vehicle used: DMSO

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene, 2-AA

Remarks:

with S9 mix; 2 µg/plate for strains TA 1535 and TA 1537; 0.5 µ/plate for strain TA 98; 1 µg/plate for strain TA 100

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

1 µg/plate for strain TA 98, without S9 mix

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

80 µg/plate for strain TA 1537, without S9 mix

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without S9 mix; 5 µg/plate for strain TA 1535; 3 µg/plate for strain TA 100.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
- Cell density at seeding: approximately 2 x 10^9 organisms per mL which was assessed photometrically.

DURATION
- Exposure duration: 72 h

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: reduction in revertant colony counts or by the absence of a complete background bacterial lawn.

Rationale for test conditions:

According to the guideline

Evaluation criteria:

The mean number of revertant colonies for all treatment groups is compared with those obtained for negative and positive control groups. The effect of metabolic activation is assessed by comparing the results obtained both in the presence and absence of the liver microsomal fraction for each treatment group. A Compound is deemed to provide evidence of mutagenic potential if a statistically significant dose-related increase in the number of revertant colonies of at least twice the concurrent solvent control is obtained in two separate experiments.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1538

Remarks:

Please refer to study entry of YKG26 for further information

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:

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:

valid

Positive controls validity:

valid

Test results 3

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:

valid

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:

valid

Positive controls validity:

valid

Test results 5

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:

valid

Positive controls validity:

valid

Additional information on results:

Concentration of the test substance resulting in precipitation: 1500 μg/plate

Any other information on results incl. tables

Table 1: Summary of the Results, Test 1

Strain		Dose Level (µg/plate)	S9 mix	Mean revertant colony count	SD	Individual revertant colony count
TA 1535		5000	-	7	0.6	7P,8P,7P
		1500	-	10	1.2	11P,9P,11P
		500	-	13	2.9	15,15,10
		150	-	10	1.0	11,9,10
		50	-	16	1.5	14,16,17
		0	-	12	2.1	10,11,14
		Solvent	-	14	2.3	15,11,15
		5000	+	7	1.5	6P,7P,9P
		1500	+	9	3.1	12P,10P,6P
		500	+	10	1.2	11,11,9
		150	+	12	6.1	9,19,8
		50	+	10	4.0	6,14,11
		0	+	10	1.5	12,9,10
		Solvent	+	12	4.2	9,11,17
TA 1537		5000	-	7	2.6	6P,5P,10P
		1500	-	8	1.2	7P,9P,9P
		500	-	14	2.1	16,12,15
		150	-	8	1.5	10,7,8
		50	-	8	2.1	6,9,10
		0	-	14	1.5	16,14,13
		Solvent	-	16	3.5	18,12,18
		5000	+	8	1.5	6P,9P,8P
		1500	+	9	2.0	9P,11P,7P
		500	+	11	1.5	9,11,12
		150	+	14	5.1	8,18,15
		50	+	11	3.1	8,12,14
		0	+	10	1.2	11,11,9
		Solvent	+	11	1.5	11,10,13
TA 98		5000	-	20	2.0	20P,22P,18P
		1500	-	30	5.7	35P,32P,24P
		500	-	27	12.5	23,41,17
		150	-	28	3.1	25,31,29
		50	-	32	7.5	33,39,24
		0	-	35	5.8	38,28,38
		Solvent	-	30	4.0	34,31,26
		5000	+	11	1.0	12P,10P,11P
		1500	+	19	5.3	13P,21P,23P
		500	+	21	6.1	22,14,26
		150	+	21	5.3	25,23,15
		50	+	18	2.6	20,19,15
		0	+	19	1.0	18,20,19
		Solvent	+	19	1.2	20,20,18
TA 100		5000	-	81	2.5	81P,78P,83P
		1500	-	104	15.7	97P,122P,93P
		500	-	117	13.1	103,129,119
		150	-	122	16.5	141,113,112
		50	-	119	8.6	111,128,117
		0	-	120	4.5	120,116,125
		Solvent	-	113	10.0	103,114,123
		5000	+	91	11.5	79P,92P,102P
		1500	+	103	4.6	98P,104P,107P
		500	+	135	6.1	140,128,136
		150	+	129	8.5	132,119,135
		50	+	125	15.8	139,129,108
		0	+	132	9.1	128,142,125
		Solvent	+	121	3.1	122,124,118
Positive Control
TA 1535	ENNG	5	-	771	38.4	740,759,814
TA 1537	9-AA	80	-	X	-	X, X, X,
TA 98	NF	1	-	107	11.4	110,116,94
TA 100	ENNG	3	-	378	25.0	361,367,407
TA 1535	2-AA	2	+	100	10.6	96,92,112
TA 1537	2-AA	2	+	74	9.1	81,64,78
TA 98	2-AA	0.5	+	135	17.1	133,119,153
TA 100	2-AA	1	+	348	32.1	351,378,314
P Precipitation SD Standard deviation X Too many colonies for accurate counting ENNG N-ethyl-N'-nitro-N-nitrosoguanidine 9 AC 9-aminoacridine 2-AA 2-aminoanthracene NF 2-nitrofluorene

Table 2: Summary of the Results, Test 2

Strain		Dose Level (µg/plate)	S9 mix	Mean revertant colony count	SD	Individual revertant colony count
TA 1535		5000	_	7	0.6	7P,8P,7P
		1500	-	10	3.0	10P,7P,13P
		500	-	11	1.0	12,11,10
		150	-	9	3.5	13,7,7
		50	-	13	2.6	10,15,14
		0	-	15	1.5	16,15,13
		Solvent	-	14	2.5	17,14,12
		5000	+	5	1.5	5P,7P,4P
		1500	+	11	2.0	9P,11P,13P
		500	10	7	2.1	5,8,9
		150	+	14	4.2	15,9,17
		50	+	7	2.5	7,4,9
		0	+	13	3.6	9,16,14
		Solvent	+	14	4.6	18,15,9
TA 1537		5000	-	6	2.1	5P,4P,8P
		1500	-	9	1.5	9P,10P,7P
		500	-	12	1.5	14,11,12
		150	-	9	2.5	7,12,9
		50	-	9	3.2	8,13,7
		0	-	12	3.6	16,11,9
		Solvent	-	14	0.6	14,13,14
		5000	+	6	1.0	7P,6P,5P
		1500	+	10	0.0	10P,10P,10P
		500	+	11	2.6	16,11,9
		150	+	10	2.1	12,8,9
		50	+	11	0.6	11,11,10
		0	+	13	2.0	11,13,15
		Solvent	+	15	1.5	13,15,16
TA 98		5000	-	24	6.0	30P,25P,18P
		1500	-	23	1.7	24P,24P,21P
		500	-	34	4.0	34,30,38
		150	-	29	8.3	36,20,32
		50	-	31	7.0	32,24,38
		0	-	37	4.4	32,39,40
		Solvent	-	36	8.0	28,37,44
		5000	+	13	1.5	15P,13P,12P
		1500	+	17	3.1	20P,16P,14P
		500	+	22	6.1	15,23,27
		150	+	20	5.3	26,18,16
		50	+	17	2.5	17,20,15
		0	+	19	2.1	17,21,18
		Solvent	+	20	1.5	19,20,22
TA 100		5000	-	89	13.9	85P,77P,104P
		1500	-	99	6.7	95P,107P,96P
		500	-	135	17.0	118,134,152
		150	-	133	13.5	133,120,147
		50	-	138	2.1	137,140,136
		0	-	142	9.1	146,132,149
		Solvent	-	127	18.8	117,149,116
		5000	+	107	9.0	116P,98P,108P
		1500	+	109	19.1	102P,95P,131P
		500	+	139	7.8	141,130,145
		150	+	137	3.5	139,133,139
		50	+	133	7.8	137,138,124
		0	+	148	10.1	153,136,154
		Solvent	+	142	13.3	157,134,134
Positive Control
TA 1535	ENNG	5	-	231	70.6	158,235,299
TA 1537	9-AA	80	-	2348	55.5	2292,2403,2350
TA 98	NF	1	-	127	4.7	131,129,122
TA 100	ENNG	3	-	357	12.1	368,344,358
TA 1535	2-AA	2	+	124	9.5	131,127,113
TA 1537	2-AA	2	+	76	7.1	77,68,82
TA 98	2-AA	0.5	+	129	7.0	122,136,129
TA 100	2-AA	1	+	432	48.6	377,450,469
P Precipitation SD Standard deviation X Too many colonies for accurate counting ENNG N-ethyl-N'-nitro-N-nitrosoguanidine 9 AC 9-aminoacridine 2-AA 2-aminoanthracene NF 2-nitrofluorene

Applicant's summary and conclusion

Conclusions:

The test substance did not show any mutagenic effects in any of the tester strains.

Executive summary:

In a reverse mutation assay in bacteria according to EU method B.13/14 (Huntingdon, 1988), four strains (TA98, TA100, TA1535, TA1537) of S. typhimurium were exposed to the test substance dissolved in DMSO at concentrations of 50 to 5000 µg/plate in the presence and absence of metabolic activation. The test substance was tested up to a limit concentration of 5000 µg/plate. Precipitation was observed at concentrations of 1500 µg/plate and above. There was no evidence or a concentration related positive response of induced mutant colonies over background. Thus, the test substance is not regarded as mutagenic.