Molybdenum, bis(dibutylcarbamodithioato)di-μ-oxodioxodi-, sulfurized

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:

13 May 2016 to 16 April 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry and Fisheries. Test Data for Registration of Agricultural Chemicals, 12 Nohsan No. 8147, Guideline 2-1-19-1, Agricultural Production Bureau, November 24, 2000.

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:

Molybdenum, bis(dibutylcarbamodithioato)di-μ-oxodioxodi-, sulfurized

EC Number:

270-180-5

EC Name:

Molybdenum, bis(dibutylcarbamodithioato)di-μ-oxodioxodi-, sulfurized

Cas Number:

68412-26-0

Molecular formula:

C18H36N2Mo2O2S4X2 Where X = O or S

IUPAC Name:

Molybdenum oxide sulfide dibutyldithiocarbamate complex

Test material form:

solid: particulate/powder

Details on test material:

- Identification: Molybdenum, bis (dibutylcarbamodithioato)
- CAS Number: 68412-26-0

Specific details on test material used for the study:

CAS RN: 68412-26-0
Purity: This substance has an Unknown or Variable composition, is a Complex reaction product, or a Biological material (UVCB)
Physical state/Appearance: Yellow powder

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 pKM 101

Metabolic activation:

with and without

Metabolic activation system:

S9 Rat Liver, induced with phenobarbitone/β-naphthoflavone

Test concentrations with justification for top dose:

Test 1 and Test 2: 5, 15, 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.

Vehicle / solvent:

Dimethyl sulfoxide (DMSO)

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

other: 4-Nitroquinoline-1-oxide (in the absence of S9 Mix), 2-Aminoanthracene (in the Presence of S9 Mix)

Details on test system and experimental conditions:

Strains used to detect base changes and frameshift mutations:
Base change mutagens: S. typhimurium TA1535 and TA100, and E. coli WP2 uvrA (pKM101).
Frameshift mutagens: S. typhimurium TA1537 and TA98.

The strains of S. typhimurium and E. coli were obtained from established commercial source and were stored at ca -80°C as aliquots of nutrient broth cultures. Each batch of frozen strain was tested for amino acid requirement and, where applicable, for cell membrane permeability (rfa mutation), deficiency in DNA excision repair system (uvrA/uvrB mutation), and the pKM101 plasmid that confers resistance to antibiotics. The responses of the strains to a series of reference mutagens were also assessed.

For use in tests, an aliquot of frozen culture was added to 25 mL of nutrient broth and incubated, with shaking, at 37ºC for 10 hours.

Preparation of S9 Fraction
S9 fraction was purchased from a commercial source and was prepared from male Sprague-Dawley derived rats, dosed with phenobarbital/5,6-benzoflavone to stimulate mixed-function oxidases in the liver and stored at approximately -80°C.

Preparation of S9 Mix
The S9 mix contained: S9 fraction (10% - Test 1; 20% v/v – Test 2), MgCl2 (8 mM), KCl (33 mM), sodium phosphate buffer pH 7.4 (100 mM), glucose-6-phosphate (5 mM) and NADP (4 mM) in water.

First Test (Test 1)
Aliquots of the test item solutions, or vehicle control, or positive control were placed in glass tubes. S9 mix (0.5 mL) or 0.1 M pH 7.4 sodium phosphate buffer (0.5 mL) was added, followed by 0.1 mL of a 10-hour bacterial culture and 2 mL of agar containing histidine (0.05 mM), biotin (0.05 mM) and tryptophan (0.05 mM). The mixture was thoroughly shaken and overlaid onto previously prepared Petri dishes containing 25 mL minimal agar. Each Petri dish was individually labelled with a unique code, identifying the contents of the dish. Three Petri dishes were used for each treatment. Plates were also prepared without the addition of bacteria in order to assess the sterility of the test item, S9 mix and sodium phosphate buffer. All plates were incubated at approximately 37ºC for between 48 and 72 hours. After this period, the appearance of the background bacterial lawn was examined and revertant colonies counted using an automated colony counter (Perceptive Instruments Sorcerer). Some plates were scored manually because of the presence of precipitate.
Any toxic effects of the test item may be detected by a substantial reduction in mean revertant colony counts (≤ 50% reduction), by a sparse or absent background bacterial lawn, or both.

Second Test (Test 2)
As a clear negative response was obtained in the first test, a variation to the test procedure was used for the second test. The pre-incubation procedure is not suitable for DMSO when added at a volume of 0.2 mL, which is toxic under such conditions. The variation used was, therefore, an increase in the S9 content of the S9 mix from 10% v/v to 20% v/v. The maximum concentration chosen was again 5000 µg/plate.

Evaluation criteria:

If exposure to a test item produces a reproducible increase in mean revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537) that of the concurrent vehicle controls, with some evidence of a positive concentration-response relationship, it is considered to exhibit mutagenic activity in this test system. If exposure to a test item does not produce a reproducible increase in mean revertant colony numbers, it is considered to show no evidence of mutagenic activity in this test system. No statistical analysis is performed. If the results obtained fail to satisfy the criteria for a clear “positive” or “negative” response, even after additional testing, the test data may be subjected to analysis to determine the statistical significance of any increases in revertant colony numbers. The statistical procedures used are those described by Mahon et al (1989) and are usually Dunnett’s test followed, if appropriate, by trend analysis. Biological importance will be considered along with statistical significance. In general, treatment-associated increases in mean revertant colony numbers below two or three times those of the vehicle controls (as described above) are not considered biologically important. It should be noted that it is acceptable to conclude an equivocal response if no clear results can be obtained.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

It was concluded that the test item showed no evidence of mutagenic activity in this bacterial system under the test conditions employed.

Executive summary:

Histidine-dependent auxotrophic mutants of Salmonella typhimurium, strains TA1535, TA1537, TA98 and TA100, and a tryptophan-dependent mutant of Escherichia coli, strain WP2 uvrA (pKM101), were exposed to the test item diluted in dimethyl sulfoxide (DMSO). DMSO was also used as a vehicle control.

Two independent mutation tests were performed as standard plate incorporation assays at 5, 15, 50, 150, 500, 1500 and 5000 µg/platein the presence and absence of liver preparations (S9 mix) from rats treated with phenobarbital and 5, 6-benzoflavone. 

Toxicity, observed as a reduction in revertant colony numbers, was seen following exposure to the test item at 5000 µg/plate in strain TA98 and at 500 µg/plate in strain TA1537 in the absence of S9 mix in the first test, and in strain TA98 at 1500 and 5000 µg/plate in the absence of S9 mix and in strain TA1537 at 50, 150 and 1500 µg/plate in the presence of S9 mix in the second test.

A coloration and precipitate was observed on all plates containing the test item at 1500 µg/plate and above in both tests.

No evidence of mutagenic activity was seen at any concentration of the test item in either mutation test.

The concurrent positive controls verified the sensitivity of the assay and the metabolizing activity of the liver preparations. The mean revertant colony counts for the vehicle controls were within or close to the current historical control range for the laboratory.

It was concluded that the test item showed no evidence of mutagenic activity in this bacterial system under the test conditions employed.