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Toxicological information

Genetic toxicity: in vitro

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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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
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:
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 / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not specified
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.
Controlsopen allclose all
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-
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
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.