Molybdenum trioxide, reaction products with bis[O,O-bis(2-ethylhexyl)] hydrogen dithiophosphate

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:

11 July 2017 to ****

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

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:

Molybdenum, bis[O,O-bis(2-ethylhexyl) phosphorodithioato-.kappa.S,.kappa.S']dioxodi-.mu.-thioxodi-, (Mo-Mo)

EC Number:

615-708-0

Cas Number:

72030-25-2

Molecular formula:

N/A

IUPAC Name:

Molybdenum, bis[O,O-bis(2-ethylhexyl) phosphorodithioato-.kappa.S,.kappa.S']dioxodi-.mu.-thioxodi-, (Mo-Mo)

Constituent 2

Reference substance name:

Molybdenum, bis[O,O-bis(2-ethylhexyl) phosphorodithioato-.kappa.S,.kappa.S']-.mu.-oxodioxo-.mu.-thioxodi-

Cas Number:

153128-45-1

Molecular formula:

N/A

IUPAC Name:

Molybdenum, bis[O,O-bis(2-ethylhexyl) phosphorodithioato-.kappa.S,.kappa.S']-.mu.-oxodioxo-.mu.-thioxodi-

Constituent 3

Reference substance name:

Molybdenum, bis[O,O-bis(2-ethylhexyl) phosphorodithioato-.kappa.S,.kappa.S']oxodi-.mu.-thioxothioxodi-

Molecular formula:

N/A

IUPAC Name:

Molybdenum, bis[O,O-bis(2-ethylhexyl) phosphorodithioato-.kappa.S,.kappa.S']oxodi-.mu.-thioxothioxodi-

Constituent 4

Reference substance name:

Unspecified 2-ethylhexyl thiophosphate esters

Molecular formula:

N/A

IUPAC Name:

Unspecified 2-ethylhexyl thiophosphate esters

Constituent 5

Reference substance name:

Unspecified 2-ethylhexyl phosphate esters

Molecular formula:

N/A

IUPAC Name:

Unspecified 2-ethylhexyl phosphate esters

Constituent 6

Reference substance name:

Unspecified 2-ethylhexyl dithiophosphate esters

Molecular formula:

N/A

IUPAC Name:

Unspecified 2-ethylhexyl dithiophosphate esters

Test material form:

liquid

Specific details on test material used for the study:

Description: Blackish blue-green opaque liquid
Purity: 100% (nominal); UVCB (provided by Sponsor)

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 Aroclor^TM 1254

Test concentrations with justification for top dose:

Mutagenicity Assay: 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 6.67, 10.0, 33.3, 66.7, 100, 333, 667, 1000, 3333 and 5000 µg per plate in ethanol.

Vehicle / solvent:

Ethanol

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2 aminoanthracene

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 0.3x10^9 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: TA1537, TA98, TA1535, TA100, and E. coli WP2 uvrA (pKM101)

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 maximum dose of 5000 µg per plate was achieved using a concentration of 100 mg/mL and a 50.0 µL plating aliquot.
Precipitate was observed beginning at 3333 or at 5000 µg per plate with all conditions. Toxicity was observed beginning at 3333 µg per plate with tester strain WP2 uvrA in the absence of S9 activation.

Mutagenicity Assay
No toxicity was observed. Precipitate was observed beginning at 1500 µg per plate with all conditions.
No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation.

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. Ethanol was used as the vehicle.

 

In the preliminary toxicity assay, the dose levels tested were 6.67, 10.0, 33.3, 66.7, 100, 333, 667, 1000, 3333 and 5000 µg per plate. Precipitate was observed beginning at 3333 or at 5000 µg per plate with all conditions. Toxicity was observed beginning at 3333 µg per plate with tester strain WP2 uvrA in the absence of S9 activation. 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. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 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.