Tris(4-chlorophenyl)sulfonium 5-benzoyl-4-hydroxy- 2-methoxybenzenesulfonate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 May - 15 Jun 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP- Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted Jul 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Reference

Name:

Unnamed

Type:

Constituent

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): PM-5927
- Physical state: off-white powder
- Lot/batch No.: 07-02
- Expiration date of the lot/batch: 05 Feb 2008
- Storage condition of test material: at room temperature protected from light

Method

Target gene:

his operon (S. typhimurium strains), trp operon (E. coli strains)

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:

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 rats treated with phenobarbital (80 mg/kg bw) and beta-naphthoflavone (100 mg/kg bw)

Test concentrations with justification for top dose:

Dose range finding test: 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate for TA 100 and E. coli WP2 uvr A (with and without metabolic activation)
Experiment 1: 33, 100, 333, 1000 and 3300 µg/plate for TA 98, TA 1535 and TA 1537 (with and without metabolic activation)
Experiment 2: 10, 33, 100, 333, 1000 and 2000 μg/plate for all strains (with and without metabolic activation)

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:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene

Remarks:

See 'Any other information on materials and methods incl. tables' for details

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: triplicates each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: reduction of background bacterial lawn

Evaluation criteria:

The assays were considered acceptable if they met the following criteria:
a) the negative control data (number of spontaneous revertants per plate) should be within the laboratory historical range for each tester strain.
b) the positive control chemicals should produce responses in all tester strains, which are within the laboratory historical range documented for each positive control substance. Furthermore, the mean plate count should be at least 3 times the concurrent vehicle control group.
c) the selected dose range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.

A test substance is considered negative (not mutagenic) in the test if:
a) the total number of revertants in tester strain TA 100 is not greater than 2 times the concurrent control, and the total number of revertants in tester strains TA 1535, TA 1537, TA 98 or WP2 uvr A is not greater than 3 times the concurrent control.
b) the negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive (mutagenic) in the test if:
a) the total number of revertants in tester strain TA 100 is greater than 2 times the concurrent control, and the total number of revertants in tester strains TA 1535, TA 1537, TA 98 or WP2 uvr A is greater than 3 times the concurrent control.
b) in case a positive response will be repeated, the positive response should be reproducible in at least one independently repeated experiment.
The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.

Statistics:

Mean values and standard deviation were calculated.

Results and discussion

Test results

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no precipitation was observed in the dose-range finding test at the start or at the end of the incubation period in both test strains (TA 100, E. coli WP2 uvrA)

RANGE-FINDING/SCREENING STUDIES:
A range-finding study was performed with TA 100 and E. coli WP2 uvr A, using 8 concentrations ranging from 33-5000 μg/plate (with and without metabolic activation). The highest concentration of the test substance used in the subsequent mutation assay was the level at which the test substance showed limited cytotoxicity (2000 µg/plate). The results were presented as part of experiment 1.

COMPARISON WITH HISTORICAL CONTROL DATA: yes; the results fall within the historical data range. See Table 3 under 'Any other information on results incl. tables'

ADDITIONAL INFORMATION ON CYTOTOXICITY: In the range-finding study, a reduction in bacterial background lawn and/or number of revertant colonies was observed from concentrations of 1000 µg/plate in TA 100 and from concentrations of 3330 µg/plate in WP2 uvrA, with and without metabolic activation.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1. Test results of experiment 1 (plate incorporation method)

 

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates ± Standard deviation)
		Base-pair substitution type			Frameshift type
		TA 100	TA1535	WP2 uvR	TA98	TA1537
–	DMSO	118 ± 13	12 ± 4	10 ± 3	24 ± 4	13 ± 4
–	3	108 ± 13	-	13 ± 3	-	-
–	10	112 ± 4	7 ± 2	12 ± 1	21 ± 5	10 ± 3
–	33	99 ± 9	8 ± 2	13 ± 6	18 ± 4	12 ± 1
–	100	124 ± 4	7 ± 2	10 ± 4	16 ± 2	11 ± 3
–	333	98 ± 5	8 ± 1	11 ± 3	19 ± 3	7 ± 3
–	1000	92 ± 19*	4 ± 1*	10 ± 2	13 ± 3	8 ± 1
–	2000	-	MC	-	1 ± 1*	MC*
–	3330	0 ± 0*	-	MC*	-	-
–	5000	0 ± 0*	-	0 ± 0*	-	-
Positive controls, –S9	Name	MMS	SA	4-NQO	NF	9AC
	Concentrations (μg/plate)	650	5	10	10	60
	Mean No. of colonies/plate (average of 3 ± SD)	1062 ± 40	966 ± 23	696 ± 40	1047 ± 7	271 ± 49
+	DMSO	90 ± 7	10 ± 1	26 ± 4	31 ± 2	13 ± 4
+	3	98 ± 8	-	25 ± 6	-	-
+	10	104 ± 13	6 ± 2	24 ± 2	23 ± 5	12 ± 5
+	33	96 ± 11	6 ± 3	29 ± 9	21 ± 5	9 ± 3
+	100	97 ± 7	8 ± 2	29 ± 5	26 ± 3	10 ± 2
+	333	90 ± 4	5 ± 2	22 ± 2	22 ± 5	10 ± 3
+	1000	66 ± 13*	6 ± 2*	22 ± 3	17 ± 3	6 ± 3
+	2000	-	0 ± 0*	-	2 ± 2*	0 ± 0*
+	3330	MC*	-	10 ± 3*	-	-
+	5000	0 ± 0*	-	5 ± 2*	-	-
Positive controls, +S9	Name	2AA	2AA	2AA	2AA	2AA
	Concentrations (μg/plate)	1	1	10	1	2.5
	Mean No. of colonies/plate (average of 3 ± SD)	996 ± 40	221 ± 23	311 ± 8	830 ± 48	459 ± 16

MMS: methylmethanesulfonate

SA : sodium azide

4-NQO: 4-nitroquinoline N-oxide

NF: 2-nitrofluorene

9AC: 9-aminoacridine

2-AA: 2 -amino-anthracene

*: slight/moderate/extreme reduction in bacterial background lawn

MC: microcolonies

-: not tested

 

 

Table 2. Test results of experiment 2 (plate incorporation method)

 

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates ± Standard deviation)
		Base-pair substitution type			Frameshift type
		TA 100	TA1535	WP2 uvR	TA98	TA1537
–	DMSO	79 ± 9	6 ± 3	14 ± 3	13 ± 1	6 ± 1
–	10	76 ± 6	5 ± 2	15 ± 2	24 ± 8	9 ± 4
–	33	78 ± 8	5 ± 0	20 ± 4	22 ± 5	7 ± 2
–	100	75 ± 3	6 ± 2	19 ± 3	24 ± 2	7 ± 4
–	333	92 ± 11	8 ± 4	21 ± 2	14 ± 4	4 ± 1
–	1000	38 ± 70*	6 ± 1*	14 ± 4	18 ± 4	3 ± 2
–	2000	MC*	MC*	11 ± 1*	9 ± 1*	MC*
Positive controls, –S9	Name	MMS	SA	4-NQO	NF	9AC
	Concentrations (μg/plate)	650	5	10	10	60
	Mean No. of colonies/plate (average of 3 ± SD)	1013 ± 88	871 ± 17	1170 ± 10	978 ± 45	373 ± 90
+	DMSO	69 ± 3	5 ± 2	15 ± 1	17 ± 3	7 ± 1
+	10	65 ± 2	4 ± 1	15 ± 1	14 ± 1	6 ± 1
+	33	50 ± 8	5 ± 1	16 ± 1	15 ± 4	5 ± 2
+	100	47 ± 5	4 ± 2	19 ± 5	13 ± 2	8 ± 3
+	333	57 ± 4	4 ± 2	14 ± 2	15 ± 4	6 ± 2
+	1000	44 ± 12*	2 ± 1*	17 ± 5	15 ± 4	4 ± 3*
+	2000	MC*	4 ± 2*	12 ± 3*	3 ± 3*	MC*
Positive controls, +S9	Name	2AA	2AA	2AA	2AA	2AA
	Concentrations (μg/plate)	2.5	1	10	1	5
	Mean No. of colonies/plate (average of 3 ± SD)	786 ± 30	150 ± 4	294 ± 10	381 ± 17	216 ± 51

MMS: methylmethanesulfonate

SA : sodium azide

4-NQO: 4-nitroquinoline N-oxide

NF: 2-nitrofluorene

9AC: 9-aminoacridine

2-AA: 2 -amino-anthracene

*: slight/moderate/extreme reduction in bacterial background lawn

MC: microcolonies

-: not tested

 

 

Table 3. Historical controls

 

Strain	Control	without S9-mix	with S9-mix
		Mean ± 3 x SD	Mean ± 3 x SD
TA100	Solvent	126 ± 79	116 ± 83
TA100	Positive	1038 ± 596	1094 ± 996
TA1535	Solvent	13 ± 14	12 ± 14
TA1535	Positive	1145 ± 819	183 ± 233
WP2 uvR	Solvent	14 ± 17	15 ± 8
WP2 uvR	Positive	643 ± 565	254 ± 343
TA98	Solvent	20 ± 19	25 ± 21
TA98	Positive	1053 ± 748	648 ± 166
TA1537	Solvent	6 ± 8	6 ± 9
TA1537	Positive	315 ± 415	334 ± 433

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative