Molybdenum, N,N-bis(C11-14-branched and linear alkyl)carbamodithioate oxo thioxo complexes

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:

30 January 1997 to 6 March 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

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:

Bis(C11-14-branched and linear alkyl)dithiocarbamic acid

Molecular formula:

N/A

IUPAC Name:

Bis(C11-14-branched and linear alkyl)dithiocarbamic acid

Constituent 2

Reference substance name:

Molybdenum, bis(C11-14 branched and linear alkyl)carbamodithioato-.kappa.S,.kappa.S')oxodi-thioxothioxodi-

Molecular formula:

N/A

IUPAC Name:

Molybdenum, bis(C11-14 branched and linear alkyl)carbamodithioato-.kappa.S,.kappa.S')oxodi-thioxothioxodi-

Constituent 3

Reference substance name:

Molybdenum, bis(C11-14 branched and linear alkyl)carbamodithioato-.kappa.S,.kappa.S')dioxodi-thioxodi-

Molecular formula:

N/A

IUPAC Name:

Molybdenum, bis(C11-14 branched and linear alkyl)carbamodithioato-.kappa.S,.kappa.S')dioxodi-thioxodi-

Constituent 4

Reference substance name:

Molybdenum, bis(N,N-di-C11-14 branched and linear alkyl)carbamodithioato-.kappa.S,.kappa.S')-oxodioxo-thioxodi-

Molecular formula:

N/A

IUPAC Name:

Molybdenum, bis(N,N-di-C11-14 branched and linear alkyl)carbamodithioato-.kappa.S,.kappa.S')-oxodioxo-thioxodi-

Test material form:

liquid

Specific details on test material used for the study:

- Purity: 100%
- Description: Brown viscous liquid

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

Test concentrations with justification for top dose:

Test 1 and Test 2: 5, 15, 50, 150, 500, 1500, 5000 µg.

5000 µg is the standard top dose recommended in the guidelines. Cytotoxicity was observed at 5000 µg in some cases and is noted in the results.

Controls

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

Details on test system and experimental conditions:

The Salmonella typhimurium strains were obtained from the University of California at Berkeley on culture discs on 4 August 1995 whilst the Escherichia coli strain WP2uvrA was obtained from the British Industrial Biological Research Association on 17 August 1987; all of the strains were stored at -196 °C in liquid nitrogen.
Prior to the master strains being used, characterization checks were carried out to determine the amino-acid requirement, presence of rfa, R factors, uvrB mutation and the spontaneous reversion rate.

Preparation of S9 Fraction
S9 was prepared in-house on 29 January 1997 from the liver of male Sprague Dawley rats weighing approximately 200 g, which had each received a single i.p. injection of Aroclor 1254 at 500 mg/kg, five days before S9 preparation. The S9 was stored at -196 °C in liquid nitrogen and prior to use, all batches of S9 were checked for suitability using a recognized mutagenic compound (2AA).

Preparation of S9 Mix
The S9 mix contained: S9 fraction (10 %), MgCl2 (0.4 M), KCl (1.65 M), sodium phosphate buffer pH 7.4 (0.2 M), glucose-6-phosphate (0.1 M) and NADP (0.1 M) in water.

Test 1 (Range-finding Study)
Up to six concentrations of the test material were assayed in triplicate against each rester strain, using the direct plate incorporation method in accordance with the standard methods for mutagenicity tests using bacteria.

Test Material and Vehicle Controls
Known aliquots (0.1 mL) of one of the bacterial suspensions were dispensed into sets of sterile test tubes followed by 2.0 mL of molten trace histidine/tryptophan supplemented top agar at 45 °C, 0.1 mL of the appropriately diluted test material or vehicle control and either 0.5 mL of the S9 liver microsome 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 with and without S9-mix.

Positive Controls
- Without Activation: A known aliquot (0.1 mL) of one of the positive control solutions was added to a test tube containing 2.0 mL of molten, trace histidine/tryptophan supplemented top agar and 0.1 mL of the appropriate bacterial suspension. Finally, 0.5 mL of phosphate buffer was added to the tube, the contents mixed and poured over the surface of a Vogel-Bonner Minimal agar plate. This procedure was then repeated, in triplicate, for each tester strain.

- With Activation: A known aliquot (0. 1 mL) of 2AA solution was added to a test tube containing 2.0 mL of molten, trace histidine/tryptophan supplemented top agar and 0.1 mL of the appropriate bacterial suspension. Finally, 0.5 mL of S9-mix was added to the tube, the contents mixed and poured over the surface of a Vogel-Bonner Minimal agar plate. This procedure was then repeated, in triplicate, for each tester strain.

Incubation and Assessment of Plates
All of the plates were incubated at 37 °C for approximately 48 hours and the frequency of revertant colonies assessed using a Domino colony counter. Plates were scored manually at the maximum recommended dose due to precipitate.

Test 2 (Main Study)
The second test was performed using methodology as described for Test 1, using fresh bacterial cultures, up to six concentrations of the test material and control solutions in triplicate.

Evaluation criteria:

For a substance to be considered positive in this test system, it should have induced a dose-related and statistically significant increase in mutation rate (of at least twice the spontaneous reversion rate) in one or more strains of bacteria in the presence and/or absence of the S9 microsomal enzymes in both experiments at sub-toxic dose levels.
In the event of the two experiments giving conflicting or equivocal results, then a third experiment may be performed to confirm the correct response. To be considered negative, the number of induced revertants compared to spontaneous revertants should be less than twofold at each dose level employed, the intervals of which should be between two and five fold and extend to the limits imposed by toxicity, solubility or up to the maximum recommended dose of 5000 µg/plate.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

5000µg only

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

5000µg only

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

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:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 98

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:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

5000µg only

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

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

Executive summary:

A reverse mutation assay was performed to the standardized guideline OECD 471 under GLP conditions. Salmonella typhimurium strains TA1535, TA1537, TA98 and TA 100 and Escherichia coli Strain WP2 uvrA were treated with the test material using the Ames plate incorporation method at up to six dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors).

 

The dose range was determined in a preliminary toxicity assay and ranged between 15 and 5000 µg/plate in the first test. The test was repeated on a separate day using a similar dose range to Test 1, fresh cultures of the bacterial strains and fresh test material formulations. Extra dose levels were inserted into both tests to allow for test material induced toxicity. The vehicle (acetone) control plates produced counts of revertant colonies within the normal range.

 

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without the metabolic system.

 

The test material caused a visible reduction to the growth of the bacterial background lawn to the majority of the tester strains, both with and without metabolic activation initially at 1500 µg/plate. It was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate, this was, in some cases, also the toxic limit. A precipitate was observed 5000 µg/plate; this did not interfere with the scoring of revertant.

 

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.

 

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