Carbonotrithioic acid, bis(phenylmethyl) ester

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

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

Test material information

Constituent 1

Reference substance name:

-

EC Number:

460-390-7

EC Name:

-

Cas Number:

26504-29-0

Molecular formula:

C15H14S3

IUPAC Name:

bis(benzylsulfanyl)methanethione

Details on test material:

- Name of test material (as cited in study report): DIBENZYL TRITHIOCARBONATE
- Physical state: yellow orange-colored solid
- Analytical purity: 96.2
- Purity test date: 2003-10-27
- Lot/batch No.: 1 UG 114
- Expiration date of the lot/batch: November 2004
- Storage condition of test material: at room temperature and protected from light

Method

Target gene:

Histidine operon

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Liver S9 homogenate was prepared from rats that have been induced with Arochlor 1254

Test concentrations with justification for top dose:

31.3, 62.5, 125, 250 and 500 µg/plate, for both mutagenicity experiments without S9
62.5, 125, 250, 500 and 1000 µg/plate, for the TA 1535, TA 1537 and TA 98 strains in the first experiment with S9.
31.3, 62.5, 125, 250 and 500 µg/plate, for the TA 100 and TA 102 strains in the first experiment with S9 as for all the strains in the second experiment with S9

Vehicle / solvent:

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

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: - S9 mix: Sodium azide (NAN3), TA 1535 & TA 100; 9-Aminoacridine (9AA), TA 1537; 2-Nitrofluorene (2NF), TA 98 strain; Mitomycin C (MMC), TA 102 strain. + S9 mix: 2-Anthramine (2AM), TA 1535; TA 1537; TA 98; TA 100 strain and 2AM, TA 102 strains

Details on test system and experimental conditions:

DETERMINATION OF CYTOTOXICITY (preliminary range-finding test)
- Test: in TA 98, TA 100 and TA 102 strains, with or without S9 mix ; 6 dose-levels (one plate/dose level)
- Method: relative total growth (decrease in the number of revertant colonies and/or a thinning of the bacterial lawn);

EXPERIMENTS
Number of independent experiments: 2.

METHOD OF APPLICATION:
* Direct plate incorporation method: for preliminary test and first experiment
* Preincubation: for the second experiment

DURATION
- Preincubation period: 60 min
- Exposure duration: 48-72H

NUMBER OF REPLICATIONS: triplicates

Evaluation criteria:

Reproducible increase in the number of revertant colonies (2-fold for TA98/TA100 and TA102, 3-fold for TA 1535/TA 1537) compared with vehicle controls in any strain at any dose-level and/or evidence of a dose-relationship.
Reference to historical data and consideration to biological relevance may also be taken into account.

Statistics:

Not applicable

Results and discussion

Test results

Test results 1

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

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

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

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

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

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

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

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 102

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

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING STUDY:
- Results on solubility: freely soluble in the vehicle (DMSO) at 50 mg/mL.
- Results on cytotoxicity:
A moderate to marked toxicity was noted at dose-levels = 1000 µg/plate in the TA 98 and TA 102 strains without S9 mix and in the TA 100 strain with and without S9 mix. A moderate to marked toxicity was observed at dose-levels = 2500 µg/plate in the TA 98 and TA 102 strains with S9 mix.
- Precipitation:
A moderate to marked precipitate was observed in the Petri plates when scoring the revertants generally at dose-levels = 500 µg/plate.

MAIN STUDY:
A moderate to marked precipitate was observed in the Petri plates when scoring the revertants mainly at dose-levels = 250 µg/plate without S9 and = 500 µg/plate with S9

The test item did not induce any noteworthy increase in the number of revertants, with and without S9 mix, in any of the five strains.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

First assay

Strain	Compound	Dose level per plate	S-9 mix	Mean revertant colony counts	SD	Ratio treated / solvent		Individual revertant colony counts
TA 1535	DMSO		-	10	3			7,13,10
	TEST ITEM	31.3 µg	-	5	1	0.5		6, 4, 6
		62.5 µg	-	15	3	1.5		12,16,18
		125µg	-	9	4	0.9		4,10,12
		250µg	-	6	2	0.6		8,7,4
		500µg	-	10	2	1.0		12 Sp, 8 Sp, 11 Sp
	NAN3	1 µg	-	396	49	39.6		430, 419, 340
	DMSO		+	12	6			11, 6,18
	TEST ITEM	62.5 µg	+	16	2	1.4		18,14,17
		125µg	+	15	4	1.3		19,11,14
		250µg	+	18	2	1.5		19, 19, 16
		500µg	+	18	10	1.5		8 Mp, 17 Mp, 28 Mp
		1000µg	+	11	1	0.9		12 Sp, 11 Sp, 10 Sp
	2AM	2 µg	+	204	18	17.5		184, 217, 211
TA 1537	DMSO		-	7	0			7, 7, 7
	TEST ITEM	31.3 µg	-	5	1	0.7		5, 4, 6
		62.5µg	-	4	3	0.6		7,2,4
		125µg	-	5	1	0.7		5,6,4
		250µg	-	3	2	0.5		6,2,2
		500µg	-	2	2	0.3		1 Sp,4 Sp,2 Sp
	9AA	50 µg	-	647	190	92.4		709, 798, 434
	DMSO		+	8	3			6, 6,12
	TEST ITEM	62.5 µg	+	11	1	1.3		11,
		125µg	+	7	2	0.8		4,8,8
		250µg	+	12	7	1.5		5,13,18
		500 µg	+	10	6	1.2		16 Mp, 7 Mp, 6 Mp
		1000µg	+	6	2	0.8		6 Sp,8 Sp,4 Sp
	2AM	2µg	+	91	10	11.4		81, 101, 91
TA 98	DMSO		-	28	9			36, 29, 19
	TEST ITEM	31.3 µg	-	23	5	0.8		26, 26,17
		62.5 µg	-	20	4	0.7		24,19,16
		125 µg	-	25	5	0.9		25, 20, 29
		250 µg	-	24	5	0.8		28 Mp, 24 Mp, 19 Mp
		500µg	-	25	14	0.9		8 Sp,32 Sp,34 Sp
	2NF	0.5 µg	-	141	16	5.0		131,160,133
	DMSO		+	32	3			32, 29, 35
	TEST ITEM	62.5 µg	+	50	5	1.6		48, 47, 56
		125 µg	+	59	19	1.8		67, 37, 72
		250 µg	+	41	6	1.3		46, 43, 35
		500 µg	+	42	13	1.3		30 Mp, 41 Mp, 55 Mp
		1000 µg	+	48	30	1.5		79 Sp, 46 Sp, 20 Sp
	2AM	2 µg	+	1460	290	45.6		1571,1677,1131
TA 100	DMSO		-	99	25		127, 92, 79
	TEST ITEM	31.3µg	-	77	19	0.8	98, 67, 65
		62.5µg	-	86	12	0.9	77, 81, 99
		125µg	-	95	7	1.0	92, 103,90
		250µg	-	71	11	0.7	81 Mp, 60 Mp, 73 Mp
		500µg	-	58	30	0.6	24 Sp, 78 Sp, 73 Sp
	NAN3	1µg	-	423	48	4.3	442, 458, 368
	DMSO		+	95	19		115, 78, 91
	TEST ITEM	31.3µg	+	66	3	0.7	67, 63, 69
		62.5µg	+	77	4	0.8	77, 73,81
		125µg	+	77	15	0.8	61, 90, 80
		250µg	+	82	22	0.9	59,85, 102
		500µg	+	70	5	0.7	66 Mp, 75 Mp, 68 Mp
	2AM	2 µg	+	651	49	6.9	678, 680, 595
TA 102	DMSO		-	370	17		361, 389, 359
	TEST ITEM	31.3µg	-	418	25	1.1	398, 410, 446
		62.5µg	-	404	19	1.1	387, 425, 399
		125µg	-	499	119	1.3	366, 535, 595
		250µg	-	432	38	1.2	468 Mp, 436 Mp, 392 Mp
		500µg	-	499	131	1.3	635 Sp, 487 Sp, 374 Sp
	MMC	0.5µg	-	1599	156	4.3	1422, 1714, 1662
	DMSO		+	425	54		363, 459, 454
	TEST ITEM	31.3µg	+	508	47	1.2	558, 466, 499
		62.5µg	+	550	136	1.3	497, 448, 704
		125µg	+	635	137	1.5	792, 569, 543
		250µg	+	471	15	1.1	480, 479, 453
		500µg	+	720	201	1.7	916 Mp, 729 Mp, 515 Mp
	2AM	10µg	+	2162	773	5.1	2635,2581, 1270

SD: Standard deviation

Mp: Moderate precipitate

Sp:Strong precipitate

- : Absence of S-9

+: Presence ofS-9

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

DIBENZYL TRITHIOCARBONATE did not show mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium

Executive summary:

The potential of DIBENZYL TRITHIOCARBONATE to induce reverse mutation in Salmonella typhimurium (strains: TA 1535, TA 1537, TA 98, TA 100 and TA 102) was evaluated in accordance with the international guidelines (OECD 471, Commission Directive No. B13/14) in compliance with the Principles of Good Laboratory Practice.

DIBENZYL TRITHIOCARBONATE was tested in two independent experiments, with and without a metabolic activation system, both performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the preincubation method (60 minutes, 37°C). Bacterias were exposed to DIBENZYL TRITHIOCARBONATE at six dose-levels (three plates/dose-level) selected from a preliminary toxicity test: 31.3 to 500 µg/plate without S9 and 62.5 to 1000 µg/plate for the TA 1535, TA 1537 and TA 98 strains in the first experiment with S9 or 31.3 to 500 µg/plate for the TA 100 and TA 102 strains in the first experiment as for all the strains in the second with S9. After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored.

The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid. DIBENZYL TRITHIOCARBONATE did not induce any noteworthy increase in the number of revertants, both with and without S9 mix, in any of the five strains.

Under these experimental conditions, DIBENZYL TRITHIOCARBONATE did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.