2,2-bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate]

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:

6 July 2006 - 11 July 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed in accordance with OECD & EU test guidelines in compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

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 (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2,2-bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate]

EC Number:

249-720-9

EC Name:

2,2-bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate]

Cas Number:

29598-76-3

Molecular formula:

C65H124O8S4

IUPAC Name:

2,2-bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate]

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Sponsor's identification: ADEKA STAB AO-412S
Description: White powder
Chemical name: Propanoic acid, 3-(dodecylthio)-,2,2-bis[[3-dodecylthio)-1-oxopropoxy]methyl]-1,3-propanediyl ester
CAS No.: 29598-76-3
Purity: >99%
Batch number: 30216
Date received: 05 June 2006
Storage conditions: Room temperature in the dark

Method

Target gene:

uvrB- (salmonella typhimurium)
WP2urvA- (Escherichia coli)

Species / strain

Species / strain 1

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not specified

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9-mixture

Test concentrations with justification for top dose:

In the prelimary toxicity test the concentrations tested were 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate.
Mutation Test experiment 1 & 2, five concentrations of the test material (50, 150, 500m 1500 and 500 µg/plate) were assayed in triplicate agains eachtester strain.

Vehicle / solvent:

The test material was insoluble in sterile distilled water and dimethyl sulphoxide at 50 mg/ml but was fully soluble in acetone at the same concentration in solubility checks performed in-house. Acetone was therefore selected as the vehicle.

Controls

Controls 1

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:

3µg/plate for TA100, 5 µg/plate for TA1535 and 2 µg/plate for WP2uvrA-

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

80µg/plate for TA1537

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

0.2µg/plate for TA98

Details on test system and experimental conditions:

S9-Mix and Agar
The S9-mix was prepared immediately before using sterilised co0factors and maintained on ice for the duration of the test
S9 5.0 mL
1.5 M KCL/0.4 M MgCl2 1.0 mL
0.1 M Glucose-6-phosphate 2.5 mL
0.1 M NADPH 2.0 mL
0. M NADH 2.0 mL
0.2 M Sodium phsopahate buffer (pH 7.4) 25.0 mL
Sterile distilled water 12.5 mL
A 0.5 mL aliquot of S9-mix and 2mL of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile Vogel-Bonner Minimal agar plate in order to assess the sterility of the S9-mix .
Top agag was prepared using 0.% Difco Bacto agar (lot no. 2102667 01/10) and 0.5% sodium chloride with 5 mL of 1.0 mM histidine and 1.0 mM biotin or 1.0 mM tryptophan solution added to each 100mL of top agar.

Preliminary Toxicity Test
In order to select appropriate dose levels for use in the main test, a preliminary test was carried out to determine the toxicity of the test material. The concentrations tested were 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 j.lg/plate. The test was performed by mixing 0.1 ml of bacterial culture (TA100 or WP2uvrA), 2 ml of molten, trace histidine or tryptophan supplemented, top agar, 0.1 ml of test material formulation and 0.5 ml of S9-mix or phosphate buffer and overlaying onto sterile plates of Vogel-Bonner Minimal agar (30 ml/plate ). Ten concentrations of the test material formulation and a vehicle control (acetone) were tested. In addition, 0.1 ml of the maximum concentration of the test material and 2 ml of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile Nutrient agar plate in order to assess the sterility of the test material. After approximately 48 hours incubation at 37°C the plates were assessed for numbers of revertant colonies using a Domino colony counter and examined for effects on the growth of the bacterial background lawn. Manual counts were performed at 5000 µg/plate because of excessive test material precipitation.

Mutation Test - Experiment 1
Five concentrations of the test material (50, 150, 500, 1500 and 5000 µg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.
Measured aliquots (0.1 ml) of one of the bacterial cultures were dispensed into sets of test tubes followed by 2.0 ml of molten, trace histidine or tryptophan supplemented, top agar, 0.1 ml of the test material formulation, vehicle or positive control and either 0.5 ml of S9-mix or phosphate buffer. The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test material both with and without S9-mix.
All of the plates were incubated at 3 7°C for approximately 48 hours and the frequency of revertant colonies assessed using a Domino colony counter. Manual counts were performed at 5000 µg/plate because of excessive test material precipitation.

Mutation Test - Experiment 2
The second experiment was performed using methodology as described for Experiment 1, using fresh bacterial cultures, test material and control solutions. The test material dose range was the same as Experiment 1 (50 to 5000 µg/plate).

Evaluation criteria:

There are several criteria for determining a positive results, such as a dose-related increase in revertant frequency over the dose range tested and/or reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS(%) can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response.
A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

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:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. A pale particulate precipitate was observed at and above 1500 µg/plate this did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the strains of bacteria, at any dose level either with or withoutmetabolic activation.

All of the positive control chemical used in the test induced marked increase in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

 The test material was non-toxic to the strains of bacterial used (TA100 and WP2uvr A-). The test material formulation S9-Mix used in this experiment were both shown to be sterile.

The number of revertant colonies for the toxicity assay were:

With (+) or without (-) S9-mix	Strain	Dose (µg/plate)
		0	0.15	0.5	1.5	5	15	50	150	500	1500	5000
-	TA100	104	106	104	91	86	99	100	80	98	91P	108P
+	TA100	96	76	64	78	84	80	66	85	75	91P	101P
-	Wp2uvrA-	23	20	18	24	22	16	21	20	19	24P	22P
+	Wp2uvrA-	31	19	29	36	29	21	31	27	26	34P	31P

P - Precipitate

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Test material was considered to be non-mutagenic under the conditions of this study.

Executive summary:

The aim of the study was to assess the mutagenic potential of the test material Propanoic acid, 3-(dodecylthio)-,2,2-bis[[3-dodecylthio)-1-oxopropoxy]methyl]-1,3-propanediyl ester using a bacterial/microsome test system.

The study was based on the in vitro technique Ames test which mutagenic activity was assess by exposing histidine auxotrophs of Salmonella typhimurium and tryptophan auxotrophs of E-coli to various concentrations of the test material. This was done in accordance to the following guidelines:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

EU Method B13/14 (Mutagenicity – Reverse Mutation Test Using Bacteria).

The test material was considered to be non-mutagenic under the conditions of the study.