Dibutyl [(dipropoxyphosphinothioyl)thio]succinate

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:

09 December 1996 to 20 February 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Dibutyl 2-[(dipropoxyphosphorothioyl)sulfanyl]succinate

Cas Number:

68413-47-8

Molecular formula:

C18H35O6PS2

IUPAC Name:

Dibutyl 2-[(dipropoxyphosphorothioyl)sulfanyl]succinate

Test material form:

liquid

Specific details on test material used for the study:

- Description: Colourless liquid
- Storage: Ambient <25°C under artificial light

Method

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:

S9 Rat Liver, induced with S9-mix

Test concentrations with justification for top dose:

Mutagenicity Assay: 0, 50, 150, 500, 1500 and 5000 μg.
5000 μg is the standard top dose recommended in the regulatory guidelines that this assay follows. Other concentrations used were a series of ca half-log10 dilutions of the highest concentration.

Preliminary Toxicity Assay: Conducted at dose levels of : 0, 50, 150, 500, 1500 and 5000 μg per plate.

Vehicle / solvent:

dimethyl sulfoxide (DMSO)

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

other: 2 aminoanthracene (in the presence of S9 Mix)

Details on test system and experimental conditions:

Preparation of Tester Strain
Overnight cultures were prepared from the appropriate frozen permanent stock. Following inoculation, each flask was placed in a shaker/incubator and incubated at 37±2°C for approximately 12 hours before the anticipated time of harvest. Each culture was monitored spectrophotometrically for turbidity and was harvested at a percent transmittance yielding a titer of greater than or equal to 0.3x109 cells per milliliter. The actual titers were determined by viable count assays on nutrient agar plates.

Exogenous Metabolic Activation
The S9 metabolic activation system was purchased commercially from MolTox (Boone, NC) and stored at 60°C or colder until use. It was prepared from male Sprague-Dawley rats that were injected intraperitoneally with Aroclor™ 1254. Each bulk preparation was assayed for its ability to metabolize benzo(a)pyrene and 2 aminoanthracene to forms mutagenic to Salmonella typhimurium TA100.

The S9 mix was prepared on the day of use and contained: S9 (10%), sodium phosphate buffer (pH 7.4; 100 mM), MgCl2 (8 mM), KCl (33 mM), glucose-6-phosphate (5 mM), and β-nicotinamide-adenine dinucleotide phosphate (4 mM). The Sham mix, containing 100 mM phosphate buffer at pH 7.4, was also prepared on the day of use.

Frequency and Route of Administration
The test system was exposed to the test substance via the plate incorporation methodology originally described by Ames et al. (1975) and updated by Maron and Ames (1983).

Preliminary Toxicity Assay to Select Dose Levels
The preliminary toxicity assay was used to establish the dose range over which the test substance would be assayed. TA98, TA100, TA1535, TA1537 and WP2 uvrA were exposed to the vehicle alone and ten dose levels of the test substance, with a single plate/condition, on selective minimal agar in the presence and absence of S9 mix. Dose levels for the mutagenicity assay were based upon post-treatment toxicity.

Mutagenicity Assay
TA98, TA100, TA1535, TA1537 and WP2 uvrA were exposed to the vehicle alone, positive controls and at least five dose levels of test substance, in triplicate, in the presence and absence of S9 mix.
To confirm the sterility of the S9, Sham mixes, test substance and the vehicle, each was plated on selective agar with an aliquot volume equal to that used in the assay and incubated under the same conditions as the assay.

One half (0.5) milliliter of S9 or Sham mix, 100 µL of tester strain (cells seeded) and 50.0 µL of vehicle, positive control, or test substance dilution were added to 2.0 mL of molten selective top agar at 45±2°C, vortexed, and overlaid onto minimal bottom agar. After the overlay solidified, the plates were inverted and incubated for 48 to 72 hours at 37±2C. Plates that were not counted immediately following the incubation period were stored at 2 8C until colony counting could be conducted.

Scoring
The condition of the bacterial background lawn was evaluated for evidence of test substance toxicity by using a dissecting microscope. Precipitate was evaluated after the incubation period by visual examination without magnification. Toxicity and degree of precipitation were scored relative to the vehicle control plate. Colonies were enumerated either by hand or by machine.

Tester Strain Verification
On the day of use in each assay, all tester strain cultures were checked for the appropriate genetic markers.

Results and discussion

Test results

Key result

Species / strain:

other: other: TA1537, TA98, TA1535, TA100, and E. coli WP2 uvrA

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

Positive controls validity:

valid

Additional information on results:

Preliminary Toxicity Assay
The dose range used in the preliminary toxicity study was 0, 50, 150, 500, 1500 and 5000 μg per plate. The test material was non-toxic to the strains of bacteria used (Ta100 and WP2uvrA).

Mutagenicity Assay
Precipitate was observed at and above 1500 μg per plate; this did not interfere with the scoring of revertant colonies.

No significant increase in the frequency of revertant colonies was recorded for any of the bacterial strains with any dose of the test material, either with or without metabolic activation.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies and the activity of the S9 fraction were found to be satisfactory.

Applicant's summary and conclusion

Conclusions:

The test item was negative for the ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system.

Executive summary:

The test item was tested to evaluate its mutagenic potential by measuring its ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system. DMSO    was used as the vehicle.

 

In the preliminary toxicity assay, the dose levels tested were 0, 50, 150, 500, 1500 and 5000 μg per plate. Based upon these results, the maximum dose tested in the mutagenicity assay was 5000 μg per plate.

 

In the mutagenicity assay, the dose levels tested were 50.0, 150, 500, 1500 and 5000 μg per plate. No toxicity was observed. Precipitate was observed beginning at 1500 μg per plate with all conditions.

 

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies and the activity of the S9 fraction were found to be satisfactory.

 

No significant increase in the frequency of revertant colonies was recorded for any of the bacterial strains with any dose of the test material, either with or without metabolic activation.

 

These results indicate the test item was negative for the ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system.