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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial gene mutation test

Key Study:

In this in vitro assessment of the mutagenic potential of the test item, 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. It was concluded that the test item showed no evidence of mutagenic activity in this bacterial system under the test conditions employed (Envigo Research Limited, Ltd, 2018).

 

In vitro micronucleus test:

Key Study:

This study was designed to assess the potential of the test item to cause an increase in the induction of micronuclei in cultured human peripheral blood lymphocytes in vitro. Under the conditions of the study, it was concluded that the test item did not show evidence of causing an increase in the induction of micronuclei in cultured human lymphocytes, in this in vitro test system under the experimental conditions described (Envigo Research Limited, Ltd, 2018).

 

In vitro gene mutation in mammalian cells test:

Key Study:

The test item was tested for mutagenic potential in an in vitro mammalian cell mutation assay. This test system is based on detection and quantitation of forward mutation in the subline 3.7.2c of mouse lymphoma L5178Y cells, from the heterozygous condition at the thymidine kinase locus (TK+/-) to the thymidine kinase deficient genotype (TK-/-). Under the conditions of the study, it was concluded that the test item did not demonstrate mutagenic potential in this in vitro cell mutation assay (Envigo Research Limited, Ltd, 2018).

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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

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

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

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)

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.

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

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

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

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

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

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.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 May 2017 to 08 March 2018

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

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

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

Culture of Lymphocytes
Human blood was collected aseptically from two healthy, non-smoking, adult (between 18-35 years of age) donors, pooled in equal volumes from each donor and were stimulated to undergo cell division by the addition of phytohaemagglutinin (PHA). All cultures were then incubated at 34 to 39°C.

Media
HML Media: RPMI 1640, supplemented with 10% fetal calf serum, 0.2 IU/mL sodium heparin, 20 IU/mL penicillin / 20 µg/mL streptomycin and 2.0 mM L glutamine.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Vehicle / solvent:

DMSO

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: colchicine

Details on test system and experimental conditions:

POSITIVE CONTROLS
In the absence of S9 mix
- Identity: Mitomycin C
- Exposure concentrations: 0.3 µg/mL (3-hour treatment), 0.1 µg/mL (20-hour treatment)

- Identity: Colchicine
- Exposure concentrations: 0.07 µg/mL (3-hour treatment), 0.015 µg/mL (20-hour treatment)

In the presence of S9 mix
- Identity: Cyclophosphamide
- Exposure concentration: 5 µg/mL (3-hour treatment)

S9 METABOLIZING SYSTEM
- S9 Fraction: Prepared from livers of male Sprague-Dawley derived rat dosed with phenobarbital and 5,6-benzoflavone, and stored at -90 to -70°C.

- Preparation of S9 Mix: S9 fraction (10% v/v), MgCl2 (8 mM), KCl (33 mM), sodium phosphate buffer pH 7.4 (100 mM), glucose-6-phosphate (5 mM), NADP (4 mM).

METHOD OF APPLICATION: In medium

DURATION
- Exposure duration:
> In the absence of S9 mix: 3 hours and 20 hours
> In the presence of S9 mix: 3 hours

TEST CONCENTRATIONS
- Preliminary toxicity test: -S9 and +S9 mix: 0.07, 0.14, 0.29, 0.58, 1.16, 2.31, 4.63, 9.25, 18.5 and 37 µg/mL
- Main tests:
-S9 mix (3 hours); 4.63, 9.25, 18.5 and 37 µg/mL
+S9 mix (3 hours); 4.63, 9.25, 18.5 and 37 µg/mL
-S9 mix (20 hours); 4.63, 9.25, 18.5 and 37 µg/mL
- Cultures analyzed for micronucleus frequency:
-S9 mix (3 hours); 9.25, 18.5 and 37 µg/mL
+S9 mix (3 hours); 9.25, 18.5 and 37 µg/mL
-S9 mix (20 hours); 9.25, 18.5 and 37 µg/mL

MAIN TEST PROCEDURES
3-Hour Treatment in the Absence and Presence of S9 Mix and 20-Hour Treatment in the Absence of S9 Mix:
- positive control cultures included
- duplicate cultures prepared at each treatment level and positive control
- quadruplicate cultures were prepared for vehicle controls
- two slides were prepared from each culture

MICROSCOPIC EXAMINATION
- examined by fluorescence microscopy
- incidences of mononucleate, binucleate and polynucleate cells were assessed per 500 cells per culture
- presence of an unusual number of, for example, cells undergoing mitosis, polyploid cells, necrotic cells and debris, if any, was also noted
- incidence of micronucleated cells per 1000 binucleate cells per culture were scored where possible.

Evaluation criteria:

Acceptance Criteria
The following criteria were applied for assessment of assay acceptability:
The concurrent vehicle control must be considered acceptable for addition to the laboratories historical vehicle control database (lie below or close to the upper control limit).
Concurrent positive controls must induce responses that are compatible with the laboratories historical positive control database and produce statistically significant increases compared with the concurrent vehicle control.
The criteria for selection of the top dose concentration are consistent with those outlined in the study plan.
Tests that did not fulfill the required criteria were rejected and therefore are not reported.

Key result

Species / strain:

lymphocytes: Human

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

 Preliminary Toxicity Test

The highest concentration tested was 37 µg/mL and precipitate was observed by eye at the end of treatment at 37 µg/mL.

No reduction in CBPI was observed at any concentration tested under any test condition when compared with the vehicle controls. These data were used to select concentrations for the main test.

 

Main Test

 3-Hour Treatment in the Absence of S9 Mix:

- Cytotoxicity

Concentrations of the test item used for the main micronucleus test were 4.63, 9.25, 18.5 and 37 µg/mL. Precipitate was observed by eye at the end of treatment at 37 µg/mL. No reduction in CBPI compared to vehicle control values was observed at any concentration tested. Concentrations of the test item selected for micronucleus analysis were 9.25, 18.5 and 37 µg/mL.

- Micronucleus Analysis

The test item did not cause any statistically significant increases in the number of binucleate cells containing micronuclei when compared with the vehicle controls.

Mean micronucleus induction in the vehicle control was below the upper control limit of the laboratory historical control database.

The positive control compounds (mitomycin C and colchicine) caused statistically significant increases in the number of binucleate cells containing micronuclei, demonstrating the sensitivity of the test system.

  

3-Hour Treatment in the Presence of S9 Mix:

- Cytotoxicity

Concentrations of the test item used for the main micronucleus test were 4.63, 9.25, 18.5 and 37 µg/mL. Precipitate was observed by eye at the end of treatment at 37 µg/mL. No reduction in CBPI compared to vehicle control values was observed at any concentration tested. Concentrations of the test item selected for micronucleus analysis were 9.25, 18.5 and 37 µg/mL.

- Micronucleus Analysis

The test item did not cause any statistically significant increases in the number of binucleate cells containing micronuclei when compared with the vehicle controls.

Mean micronucleus induction in the vehicle control was below the upper control limit of the laboratory historical control database.

The positive control compound (cyclophosphamide) caused a statistically significant increase in the number of binucleate cells containing micronuclei, demonstrating the efficacy of the S9 mix and the sensitivity of the test system.

 

20-Hour Treatment in the Absence of S9 Mix:

- Cytotoxicity

Concentrations of the test item used for the main micronucleus test were 4.63, 9.25, 18.5 and 37 µg/mL. Precipitate was observed by eye at the end of treatment at 37 µg/mL. No reduction in CBPI compared with vehicle control values, was observed at any concentration tested. Concentrations of the test item selected for micronucleus analysis were 9.25, 18.5 and 37 µg/mL.

- Micronucleus Analysis

The test item did not cause any statistically significant increases in the number of binucleate cells containing micronuclei when compared with the vehicle controls.

Mean micronucleus induction in the vehicle control was below the upper control limit of the laboratory historical control database.

The positive control compounds (mitomycin C and colchicine) caused statistically significant increases in the number of binucleate cells containing micronuclei, demonstrating the sensitivity of the test system.

Conclusions:

The test item did not show evidence of causing an increase in the induction of micronuclei in cultured human lymphocytes in this in vitro test system under the experimental conditions described.

Executive summary:

This study was designed to assess the potential of the test item to cause an increase in the induction of micronuclei in cultured human peripheral blood lymphocytes in vitro. The study consisted of a preliminary toxicity test and a main micronucleus test. Human lymphocytes in whole blood culture, stimulated to divide by addition of phytohaemagglutinin (PHA) 48 hours prior to treatment, were exposed to the test item for 3 hours in both the absence and presence of exogenous metabolic activation (S9 mix) and for 20 hours in the absence of S9 mix. The maximum final concentration to which the cells were exposed was 37 µg/mL, to test up to the maximum solubility of the test item. Vehicle (dimethyl sulphoxide [DMSO]) and positive control cultures were included in all appropriate test conditions. 

Three test item concentrations were assessed for induction of micronuclei. The highest concentration selected (37 µg/mL) showed a visible precipitate by eye at the end of the treatment period. Following 3-hour and 20-hour treatment in the absence of S9 mix and 3-hour treatment in the presence of S9 mix, no reduction in the cytokinesis-block proliferative index (CBPI) was observed at any concentration tested. Concentrations of the test item selected for micronucleus analysis for all three exposures were 9.25, 18.5 and 37 µg/mL, based upon the assessment of 500 cells per culture for the incidences of mononucleate, binucleate and polynucleate cells.

In both the absence and presence of S9 mix, following 3-hour treatment, and in the absence of S9 mix, following 20-hour treatment, the test item did not cause any statistically significant increases in the number of binucleate cells containing micronuclei when compared with the vehicle controls, based upon the examination by fluorescent microscopy of 1000 binucleate cells per culture.

The positive control compounds (mitomycin C, colchicine and cyclophosphamide) caused statistically significant increases in the number of binucleate cells containing micronuclei under appropriate conditions, demonstrating the efficacy of the S9 mix and the sensitivity of the test system.

It was concluded that the test item did not show evidence of causing an increase in the induction of micronuclei in cultured human lymphocytes, in this in vitro test system under the experimental conditions described.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 August 2017 to 16 April 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Health and Welfare. Evaluation and Licensing Division, Pharmaceutical and Medical Safety Bureau, Notification No. 1604, 1 November 1999.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Specific details on test material used for the study:

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

Target gene:

TK +/-, locus of the L5178Y mouse lymphoma cell line

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS
L5178Y mouse lymphoma (3.7.2c) cells (Clive and Spector, 1975); these cells are heterozygous at the thymidine kinase locus, TK +/-. Spontaneous thymidine kinase deficient mutants, TK -/-, were eliminated from the cultures by a 24-hour incubation in the presence of methotrexate, thymidine, hypoxanthine and glycine two days prior to storage at -196 to -150°C, in heat-inactivated donor horse serum (HiDHS) containing 10% DMSO. Cultures were used within ten days of recovery from frozen stock. Cell stocks were periodically checked for freedom from mycoplasma contamination.

MEDIA
The following media, obtained from a suitable supplier, were used:
R0: RPMI 1640, buffered with 2 mg/mL sodium bicarbonate, supplemented with 2.0 mM L-glutamine and 50 µg/mL gentamicin.
R10p: R0, supplemented with 0.1% v/v Synperonic F68, 1.0 mM sodium pyruvate and HiDHS at 10% v/v. (R10p medium was used for cell culture unless otherwise specified). Selective medium consisted of R10p containing 4 µg/mL trifluorothymidine (TFT).

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Preliminary toxicity test:
0.14, 0.29, 0.58, 1.16, 2.31, 4.63, 9.25, 18.5, 37.0 and 74.0 µg/mL

Mutation tests:
-S9 mix (3 hours): 4.63, 9.25, 18.5, 37 and 74 µg/mL
+S9 mix (3 hours): 4.63, 9.25, 18.5, 37 and 74 µg/mL
-S9 mix (24 hours): 4.63, 9.25, 18.5, 37 and 74 µg/mL

The concentrations assessed for determination of mutant frequency in the main test were based upon the preliminary toxicity test data, the objective being to test up to the maximum solubility using a suitable vehicle.

The solubility of the test item was assessed in dimethyl sulphoxide (DMSO), water, acetone and ethanol as part of a separate study. The vehicle which afforded the highest solubility was DMSO, in which the test item dissolved at 7.4 mg/mL, the test item was practically insoluble in water, acetone and ethanol.

The osmolality of the test item in medium was tested at 74 µg/mL; no fluctuations in osmolality of the medium of more than 50 mOsm/kg were observed compared with the vehicle control. No fluctuations in pH of the medium were observed at 74 µg/mL of more than 1.0 unit compared with the vehicle control. The maximum final concentration tested in the preliminary toxicity test was 74 µg/mL.

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION
In medium

S9 METABOLIZING SYSTEM
- S9 Fraction: S9 fraction, prepared from male Sprague-Dawley derived rats dosed with phenobarbital and 5,6-benzoflavone to stimulate mixed-function oxidases in the liver (stored at -90 to -70°C).

- Preparation of S9 Mix: S9 mix contains: S9 fraction (5% v/v), glucose-6-phosphate (6.9 mM), NADP (1.4 mM) in R0. The co factors were prepared, neutralised with 1N NaOH and filter sterilised before use.

FORMULATION OF TEST ITEM
The test item was formulated and diluted in DMSO (shortly before dosing). The final volume added to the cultures was 1% v/v.

MAIN TEST
- Cultures:
For 3-hour exposures, cultures contained a total of 6 x 10^6 cells. The final volume of the cultures was 10 mL and the final concentration of the S9 fraction was 2% v/v, if present. For 24-hour exposures, cultures contained a total of 3 x 10^6 cells in a total volume of 10 mL. One culture was prepared for each concentration of the test item for each test condition. Vehicle controls were tested in duplicate for each test condition.
The test item was formulated and serially diluted in the vehicle. Aliquots of 100 µL of test item dilution (at 100 times the desired final concentration) or vehicle were added to each culture prior to incubation for 3 hours (continuous shaking at 34 to 39 ºC) or 24 hours (static incubator, at 34 to 39 ºC, 5% (v/v) CO2). At the end of the 3-hour exposure period, the cells were washed once, re-suspended in R10p to nominally 2 x 10^5 cells/mL (assuming no cell loss), incubated (at 34 to 39 ºC, 5% (v/v) CO2) and sampled after 24 and 48 hours to assess growth in suspension. After sampling at 24 hours the cell density was readjusted to 2 x 10^5 cells/mL with R10p where necessary. At the end of the 24-hour exposure period, the cells were washed once, re-suspended in 10 mL R10p and counted, to ascertain cell density and treatment growth. The cultures were then diluted to 2 x 10^5 cells/mL with R10p as appropriate, incubated and sampled after 24 and 48 hours to assess growth in suspension. After sampling at 24 hours the cell density was readjusted to 2 x 10^5 cells/mL with R10p where necessary.
The relative suspension growth (RSG) was used to determine the concentrations of test item used in the main test; ideally the maximum concentration should reduce relative total growth (RTG) to approximately 10 to 20% of the concurrent vehicle control value. There was no evidence of toxicity in the preliminary toxicity test, so the maximum concentration tested in the main mutation test in all exposure conditions was 74 µg/mL. The formulations were added at 1% final volume in medium.

- 3-hour Treatment in the Absence and Presence of S9 Mix:
> Cultures contained a total of 1.2 x 10^7 cells in a final volume of 20 mL
> Final concentration of the S9 fraction was 2% v/v, if present
> Duplicate cultures prepared for each test item concentration and positive control
> Quadruplicate cultures prepared for vehicle controls
> Aliquots of 200 µL of test item dilution (100 times the desired final concentration), vehicle or positive control added and then all cultures incubated with continuous shaking for 3 hours at 34 to 39 ºC
> Cells were washed once and re-suspended in R10p to nominally 2 x 10^5 cells/mL incubated for a further 48 hours
> Cultures were sampled after 24 and 48 hours to assess growth in suspension; after 24 hours the cell density was readjusted to 2 x 10^5 cells/mL with R10p where necessary; after 48 hours cultures with a density of more than 1 x 10^5 cells/mL were assessed for cloning efficiency (viability) and mutant potential by plating in 96-well plates
> Cloning efficiency assessed by plating 1.6 cells/well, two plates prepared per culture
> Mutant potential assessed by plating 2 x 10^3 cells/well in selective medium, two plates prepared per culture
> Plates were placed in a humidified incubator at 34 to 39 ºC in an atmosphere of 5% CO2 in air

- 24-hour Treatment in the Absence of S9 Mix:
> Duplicate 20 mL cultures containing 6 x 10^6 cells were treated for 24 hours with 200 µL of test item or positive control
> Quadruplicate cultures were prepared for vehicle controls
> At end of the exposure period, the cells washed once, re-suspended in 20 mL R10p and counted to ascertain treatment growth
> Cultures were then diluted to 2 x 10^5 cells/mL with R10p as appropriate, incubated and sampled after 24 and 48 hours to assess growth in suspension
> After sampling at 24 hours the cell density was readjusted to 2 x 10^5 cells/mL with R10p where necessary
> Following the above, the procedure was the same as in the 3-hour treatment. The maximum concentration assessed for mutant frequency in the main test was 74 µg/mL

Key result

Species / strain:

mouse lymphoma L5178Y cells

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

Table 1: Main mutation test – 3-hour treatment in the absence of S9 mix

Treatment / Concentration		Cell Concentration (x10^5/mL)		Viability Plate Count^a		Mutant Plate Count^a		Mean RTG	Mean MF
(µg/mL)	Replicate	24 h	48 h	Day 2		Day 2		(%)	(x10^-6)
	ID
Vehicle Control^b	A	4.48	11.43	42	(192)	163	(192)	100	86
	B	5.14	10.32	40	(192)	160	(192)
	C	4.80	12.42	43	(192)	163	(192)
	D	5.54	9.92	41	(192)	166	(192)
Molybdenum	A	4.64	10.65	38	(192)	165	(192)	90	91
4.63	B	4.87	10.63	48	(192)	159	(192)
Molybdenum	A	4.66	10.18	39	(192)	162	(192)	79	88
9.25	B	3.39	11.86	47	(192)	164	(192)
Molybdenum	A	4.49	11.06	49	(192)	161	(192)	67	103
18.5	B	2.77	12.30	53	(192)	163	(192)
Molybdenum	A	4.55	11.30	44	(192)	165	(192)	89	82
37	B	4.83	11.15	49	(192)	167	(192)
Molybdenum	A	4.20	10.88	46	(192)	163	(192)	83	86
74	B	5.10	10.05	45	(192)	166	(192)
MMS	A	4.47	11.33	51	(192)	77	(192)	75	556
10	B	4.30	10.81	55	(192)	80	(192)
a. Number of non-colony bearing wells (total number of wells)
b. Vehicle control = DMSO (1% v/v)
MMS - Methyl methanesulphonate
RTG - Relative Total Growth
MF - Mutant Frequency

 

Table 2: Main mutation test – 3-hour treatment in the presence of S9 mix

 

Treatment / Concentration		Cell Concentration (x10^5/mL)		Viability Plate Count^a		Mutant Plate Count^a		Mean RTG	Mean MF
(µg/mL)	Replicate	24 h	48 h	Day 2		Day 2		(%)	(x10^-6)
	ID
Vehicle Control^b	A	6.77	10.32	36	(192)	168	(192)	100	78
	B	6.43	10.22	34	(192)	165	(192)
	C	5.79	10.31	38	(192)	160	(192)
	D	5.83	10.74	41	(192)	161	(192)
Molybdenum	A	5.71	10.92	34	(192)	163	(192)	110	71
4.63	B	5.45	11.19	25	(192)	162	(192)
Molybdenum	A	5.74	11.66	24	(192)	165	(192)	118	65
9.25	B	5.20	10.63	26	(192)	160	(192)
Molybdenum	A	5.02	11.31	24	(192)	165	(192)	110	61
18.5	B	5.43	10.59	26	(192)	164	(192)
Molybdenum	A	5.76	10.19	33	(192)	156	(192)	99	81
37	B	5.58	10.43	31	(192)	164	(192)
Molybdenum	A	6.75	10.65	32	(192)	161	(192)	110	93
74	B	6.09	10.17	36	(192)	153	(192)
BaP	A	4.94	9.46	46	(192)	45	(192)	58	850
1.5	B	4.55	9.04	50	(192)	43	(192)
a. Number of non-colony bearing wells (total number of wells)
b. Vehicle control = DMSO (1% v/v)
BaP - Benzo[a]pyrene
RTG - Relative Total Growth
MF - Mutant Frequency   Table 3: Main mutation test – 24-hour treatment in the absence of S9 mix Treatment / Concentration Cell Concentration (x10^5/mL) Viability Plate Count^a Mutant Plate Count^a Mean RTG Mean MF (µg/mL) Replicate ID 0 h 24 h 48 h Day 2 Day 2 (%) (x10^-6) Vehicle Control^b A 13.23 4.31 14.24 38 (192) 160 (192) 100 74   B 13.41 4.79 13.32 37 (192) 168 (192)       C 12.73 5.29 13.14 46 (192) 170 (192)       D 12.60 4.86 12.07 37 (192) 166 (192)     Molybdenum A 11.56 4.88 13.06 49 (192) 164 (192) 75 85 4.63 B 10.94 5.20 12.59 51 (192) 169 (192)     Molybdenum A 11.24 4.64 13.90 39 (192) 168 (192) 88 73 9.25 B 11.16 4.95 13.42 42 (192) 165 (192)     Molybdenum A 10.12 5.47 12.68 43 (192) 166 (192) 76 79 18.5 B 10.82 4.27 14.66 48 (192) 167 (192)     Molybdenum A 10.96 4.52 13.35 47 (192) 167 (192) 71 71 37 B 10.63 4.59 14.63 54 (192) 174 (192)     Molybdenum A 11.11 4.97 14.26 50 (192) 163 (192) 71 100 74 B 10.70 4.04 14.71 56 (192) 164 (192)     MMS A 10.88 4.31 11.64 71 (192) 53 (192) 46 1060 5 B 10.95 4.68 11.92 66 (192) 45 (192)     a. Number of non-colony bearing wells (total number of wells) b. Vehicle control = DMSO (1% v/v)   MMS - Methyl methanesulphonate RTG - Relative Total Growth MF - Mutant Frequency

 

Conclusions:

The test item did not demonstrate mutagenic potential in this in vitro cell mutation assay.

Executive summary:

The test item was tested for mutagenic potential in an in vitro mammalian cell mutation assay. This test system is based on detection and quantitation of forward mutation in the subline 3.7.2c of mouse lymphoma L5178Y cells, from the heterozygous condition at the thymidine kinase locus (TK+/-) to the thymidine kinase deficient genotype (TK-/-).

The study consisted of a preliminary toxicity test and three independent mutagenicity assays. The cells were exposed for either 3 hours or 24 hours in the absence of exogenous metabolic activation (S9 mix) or 3 hours in the presence of S9 mix.

The test item was soluble at 7.4 mg/mL in dimethyl sulphoxide (DMSO). A final concentration of 74 µg/mL, dosed at 1% v/v, was used as the maximum concentration in the preliminary toxicity test, in order to test up to the limit of solubility using a suitable vehicle. 

Following exposure to the test item for 3 hours or 24 hours at concentrations from 0.14 to 74 µg/mL, there were no significant reductions in relative suspension growth (RSG) in any exposure condition. The concentrations assessed for determination of mutant frequency in the main test were based upon these data, the objective being to test up the maximum solubility using a suitable vehicle.

Following 3-hour treatment in the absence and presence of S9 mix, there were no increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor (GEF), within acceptable levels of toxicity. The maximum concentration assessed for mutant frequency in the 3-hour treatment in both the absence and presence of S9 mix was 74 µg/mL. In the absence and presence of S9 mix there were no significant reductions in relative total growth (RTG).

In the 24-hour treatment, the maximum concentration assessed for mutant frequency was 74 µg/mL. No increase in mutant frequency exceeded the sum of the mean concurrent vehicle control mutant frequency and the GEF. There were no significant reductions in RTG.

In all tests the concurrent vehicle and positive control values were within acceptable ranges.

It was concluded that the test item did not demonstrate mutagenic potential in this in vitro cell mutation assay, under the experimental conditions described.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)