3-[(3-sulfanylbutanoyl)oxy]-2,2-bis{[(3-sulfanylbutanoyl)oxy]methyl}propyl 3-sulfanylbutanoate

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:

08 Jan - 05 Mar 2009

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

All experiments for the test substance and for the positive controls were performed only in duplicates. All experiments for the solvent control were performed in triplicates, but no standard deviations were given. It should be noted that the study report is a translation from Japanese to English.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Experiments for the test substance and for the positive controls were performed only in duplicates, for the solvent control in triplicates, but no standard deviations were given. Only 2-Aminoanthracene was used as positive control with S9-mix.

Principles of method if other than guideline:

This study was conducted in accordance with "Standards for Toxicity Investiagations" (Ministry of Labor, Notification No. 77, September 1, 1988 and Notification No. 67, June 2, 1997) "Procedures of Mutagenicity Test Using Microorganisms and Evaluation of Test Results" (Ministry of Labor, Offical Notification, February 8, 1999), "III Mutagenicity test" of "Reverse-Mutation Assay in Bacteria" prescribed in "Testing Methods Relating to the New Chemical Substances" (Notification No. 1121002 of the Pharmaceutical and Food Safety Bureau, MHLW, No.2 (2003.11.13) of the Manufacturing Industries Bureau, METI & No. 031121002 of the Environmental Health Department, MOE (November 21, 2003)).

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

3-{[(3R)-3-sulfanylbutanoyl]oxy}-2,2-bis({[(3R)-3-sulfanylbutanoyl]oxy}methyl)propyl (3R)-3-sulfanylbutanoate; 3-{[(3R)-3-sulfanylbutanoyl]oxy}-2,2-bis({[(3S)-3-sulfanylbutanoyl]oxy}methyl)propyl (3R)-3-sulfanylbutanoate; 3-{[(3R)-3-sulfanylbutanoyl]oxy}-2-({[(3R)-3-sulfanylbutanoyl]oxy}methyl)-2-({[(3S)-3-sulfanylbutanoyl]oxy}methyl)propyl (3R)-3-sulfanylbutanoate; 3-{[(3S)-3-sulfanylbutanoyl]oxy}-2,2-bis({[(3S)-3-sulfanylbutanoyl]oxy}methyl)propyl (3R)-3-sulfanylbutanoate; 3-{[(3S)-3-sulfanylbutanoyl]oxy}-2,2-bis({[(3S)-3-sulfanylbutanoyl]oxy}methyl)propyl (3S)-3-sulfanylbutanoate

EC Number:

700-255-4

Cas Number:

31775-89-0

Molecular formula:

C21H36O8S4

IUPAC Name:

3-{[(3R)-3-sulfanylbutanoyl]oxy}-2,2-bis({[(3R)-3-sulfanylbutanoyl]oxy}methyl)propyl (3R)-3-sulfanylbutanoate; 3-{[(3R)-3-sulfanylbutanoyl]oxy}-2,2-bis({[(3S)-3-sulfanylbutanoyl]oxy}methyl)propyl (3R)-3-sulfanylbutanoate; 3-{[(3R)-3-sulfanylbutanoyl]oxy}-2-({[(3R)-3-sulfanylbutanoyl]oxy}methyl)-2-({[(3S)-3-sulfanylbutanoyl]oxy}methyl)propyl (3R)-3-sulfanylbutanoate; 3-{[(3S)-3-sulfanylbutanoyl]oxy}-2,2-bis({[(3S)-3-sulfanylbutanoyl]oxy}methyl)propyl (3R)-3-sulfanylbutanoate; 3-{[(3S)-3-sulfanylbutanoyl]oxy}-2,2-bis({[(3S)-3-sulfanylbutanoyl]oxy}methyl)propyl (3S)-3-sulfanylbutanoate

Method

Target gene:

his operon for the S. typhimurium strains
trp operon for the E. coli strain

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 and 5,6-benzoflavon

Test concentrations with justification for top dose:

Main test:
313, 625, 1250, 2500 and 5000 µg/plate with and without metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (Lot No. WF032, 100.0% in purity, spectrophotometric grade, DOJINDO Laboratories) was used.
- Justification for choice of solvent/vehicle:
The test substance is insoluble in distilled water (information provided by the sponsor), but soluble in DMSO (solubility ≥ 500 mg/mL).

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene (0.5 µg/plate in water, TA98; 1 µg/plate in water, TA100: 2 µg/plate in water, TA1535, TA1537; 20 µg/plate in water, WP2uvrA)

Remarks:

with S9 mix

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (0.01 µg/plate in DMSO, TA100, WP2uvrA; 0.1 µg/plate in DMSO, TA98); sodium azide (0.5 µg/plate in water,TA1535); 2-methoxy-6-chloro-9-[3-(2-chloroethyl)aminopropylamino]acridine*2HCl (0.5 µg/plate in DMSO,TA1537)

Remarks:

without S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: All experiments with the negative control were performed in triplicates. All experiments with the positive controls and with the test substance were performed in duplicates.

DETERMINATION OF CYTOTOXICITY
- Method: Inspection of reduction in the number of revertant colonies

Evaluation criteria:

The test substance may be considered positive in this test system if the following criteria are met: The test substance is considered mutagenic, if two-fold (or more) increases in mean revertant numbers are observed at two consecutive dose levels or at the highest practicable dose level only. In addition, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose levels. All cultures must demonstrate the characteristic mean number of spontaneous revertants in the vehicle controls.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

for TA1537 with S9 mix at concentrations ≥ 2500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test substance is insoluble in distilled water (information provided by the sponsor), but soluble in DMSO (solubility ≥ 500 mg/mL).
- Precipitation: Precipitation of the test substance was observed at test concentrations ≥ 1250 µg/plate without metabolic activation for all tester strains (see Table 2).

RANGE-FINDING/SCREENING STUDIES: The test results of the dose finding study are shown in Table 1. No increases in the number of the revertant colonies of any tester strain were observed with or without metabolic activation. Cytotoxicity was detected by a reduction in the number of revertant colonies for the tester strain TA1537 at a test concentrations of 5000 µg/plate with S9 mix. Precipitation of the test substance was observed at a test concentrations ≥ 1250 µg/plate without metabolic activation for all tester strains.

COMPARISON WITH HISTORICAL CONTROL DATA: The values for the positive and the negative controls are in the range of the historical control data.

ADDITIONAL INFORMATION ON CYTOTOXICITY: Cytotoxicity was detected by a reduction in the number of revertant colonies for the tester strain TA1537 at test concentrations ≥ 2500 µg/plate with S9 mix (see Table 2).

Any other information on results incl. tables

Table 1. Test results of the dose finding study (preincubation method).

	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 2 or 3 plates*)
		Base-pair substitution type			Frameshift type
		TA 100	TA1535	WP2uvrA	TA98	TA1537
– S9 -mix	0	97	13	29	22	15
– S9 -mix	4.88	116	7	35	21	13
– S9 -mix	19.5	100	9	29	17	14
– S9 -mix	78.1	99	11	26	21	19
– S9 -mix	313	117	13	26	21	17
– S9 -mix	1250	101 P	8 P	32 P	20 P	8 P
– S9 -mix	5000	105 P	6 P	38 P	22 P	7 P
Positive controls, – S9 -mix	Name	AF-2	NaN3	AF-2	AF-2	ICR-191
	Concentrations (μg/plate)	0.01	0.5	0.01	0.1	0.5
	Mean No. of colonies/plate (average of 2 plates)	603	468	311	517	1580
+ S9 -mix	0	112	13	33	39	30
+ S9 -mix	4.88	102	11	33	23	18
+ S9 -mix	19.5	100	10	39	25	21
+ S9 -mix	78.1	104	8	33	28	20
+ S9 -mix	313	127	12	30	31	25
+ S9 -mix	1250	118	13	43	29	11
+ S9 -mix	5000	104	6	34	31	7
Positive controls, + S9 -mix	Name	2AA	2AA	2AA	2AA	2AA
	Concentrations (μg/plate)	1	2	10	0.5	2
	Mean No. of colonies/plate (average of 2 plates)	962	246	634	278	234

 * All experiments of the solvent control were performed in triplicates. All experiments for the positive controls and for the test substance were performed in duplicates.

AF-2 = 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide

NaN₃= Sodium azide

ICR-191 = 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)aminopropylamino]acridine*2HCl

2AA = 2-Aminoanthracene

P = Precipitate

 

Table 2. Test results of the main study (preincubation method).

	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 2 or 3 plates*)
		Base-pair substitution type			Frameshift type
		TA 100	TA1535	WP2uvrA	TA98	TA1537
– S9 -mix	0	103	10	28	25	10
– S9 -mix	313	117	7	26	21	9
– S9 -mix	625	108	9	31	23	5
– S9 -mix	1250	104 P	8 P	32 P	26 P	6 P
– S9 -mix	2500	110 P	12 P	32 P	15 P	6 P
– S9 -mix	5000	108 P	8 P	26 P	24 P	6 P
Positive controls, –S9 -mix	Name	AF-2	NaN3	AF-2	AF-2	ICR-191
	Concentrations (μg/plate)	0.01	0.5	0.01	0.1	0.5
	Mean No. of colonies/plate (average of 2 plates)	674	344	377	516	1441
+ S9 -mix	0	114	7	33	32	24
+ S9 -mix	313	105	7	30	28	16
+ S9 -mix	625	140	7	29	22	11
+ S9 -mix	1250	113	8	28	30	14
+ S9 -mix	2500	112	6	30	37	7
+ S9 -mix	5000	117	11	35	31	7
Positive controls, +S9 -mix	Name	2AA	2AA	2AA	2AA	2AA
	Concentrations (μg/plate)	1	2	10	0.5	2
	Mean No. of colonies/plate (average of 2 plates)	1039	202	657	371	284

 * All experiments with the solvent control were performed in triplicates. All experiments with the positive controls and with the test substance were performed in duplicates.

AF-2 = 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide

NaN₃= Sodium azide

ICR-191 = 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)aminopropylamino]acridine*2HCl

2AA = 2-Aminoanthracene

P = Precipitate

The test result of the main test are shown in Table 2. No increases in the number of the revertant colonies of any test strain were observed with or without metabolic activation. Cytotoxicity was detected by a reduction in the number of revertant colonies for the tester strain TA1537 at a test concentrations ≥ 2500 µg/plate with S9 mix. Precipitation of the test substance was observed at test concentrations ≥ 1250 µg/plate without metabolic activation for all tester strains.

Applicant's summary and conclusion

Conclusions:

Under the conditions of the Ames test the test substance was not mutagenic in any of the five strains (TA 1535, TA 1537, TA 98, TA 100 and WP2 uvr A) tested with and without metabolic activation.