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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial gene mutation test

Key Study:

In a study performed to the standardized guideline OECD 471, under GLP conditions the test material was considered to be non-mutagenic under the test conditions of this test (Safepharm Laboratories Ltd, 1997).

In vitro micronucleus test:

Key Study:

In a study performed to the standardized guideline OECD 487, under GLP conditions significant or dose dependent increases in micronuclei induction were observed in treatment groups with or without S9; therefore under the conditions of the study, these results indicate the test substance was negative for the induction of micronuclei in the presence and absence of the exogenous metabolic activation system (BioReliance, 2018).

In vitro gene mutation in mammalian cells test:

Key Study:

In a study performed to the standardised guideline OECD 490 under GLP conditions, the test material was negative for the ability to induce forward mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells, in the presence and absence of an exogenous metabolic activation system (BioReliance, 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:

30 January 1997 to 6 March 1997

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

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Purity: 100%
- Description: Brown viscous liquid

Species / strain / cell type:

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

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.

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.

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

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

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

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

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

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.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 August 2017 to ****

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

- Purity: >90%
- Description: Opague black viscous liquid

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

Peripheral blood lymphocytes were obtained from a healthy non-smoking, 26 year old female and were cultured in complete medium (RPMI 1640 containing 15% fetal bovine serum, 2 mM L glutamine, 100 units penicillin, 100 µg/mL streptomycin) by adding 0.5 mL heparinized blood to a centrifuge tube containing 5 mL of complete medium with 2% phytohemagglutinin. The cultures were then incubated at 37 ± 1 °C in a humidified atmosphere of 5 ± 1% CO2 in air for 44 to 48 hours.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Based on the results of the preliminary toxicity test, the doses selected for testing in the micronucleus assay were as follows:
- Non-activated: 1, 2, 4, 8, 15 µg/mL, for 4 and 20 hrs,
- Non-activated: 1, 2, 4, 8, 15 µg/mL, for 4 and 24 hrs, and
- S9-activated: 1, 2, 4, 8, 15 µg/mL, for 4 and 20 hrs.

Precipitation of the test substance dosing solution in the treatment medium was determined using unaided eye at the beginning and conclusion of treatment. The highest dose evaluated for the micronuclei was selected based on the following:
- 4-hour (-S9): Visible precipitate at the end of treatment period;
- 4-hour (+S9): Visible precipitate at the end of treatment period, and
- 24-hour (-S9): Visible precipitate at the end of treatment period.

Vehicle / solvent:

Acetone

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

other:

Details on test system and experimental conditions:

Details on mammalian cell lines
Human peripheral blood lymphocytes were obtained from a healthy non-smoking individual (26 years of age) and were cultured in complete medium (RPMI 1640 containing 15% fetal bovine serum, 2 mM L glutamine, 100 units penicillin, 100 µg/mL streptomycin) by adding 0.5 mL heparinized blood to a centrifuge tube containing complete medium with 2% phytohemagglutinin. The cultures were incubated at 37 ± 1°C in a humidified atmosphere of 5 ± 1% CO2 in air for 44-48 hours.

Exogenous Metabolic Activation System
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 final concentration of its components in serum-free RPMI-1640 medium were: S9 (20 µL/mL), MgCl2 (2 mM), KCl (6 mM), glucose-6-phosphate (1 mM), and NADP (sodium salt; 1 mM).

Preliminary Toxicity Test for Selection of Dose Levels
Lymphocytes were exposed to vehicle alone and to nine concentrations of test substance for 4 hours in the absence and presence of S9, and for 24 hours in the absence of S9 using single cultures. Precipitation of test substance in the treatment medium was determined using unaided eye at the beginning and conclusion of treatment. The osmolality in treatment medium of the vehicle, the highest dose, lowest precipitating dose, and the highest soluble dose was measured. The pH of the highest dose in the treatment medium was measured using test tape. Dose levels for the micronucleus assay were based upon post-treatment toxicity (cytokinesis-blocked proliferation index (CBPI) relative to the vehicle control) or visible precipitate in the treatment medium at the conclusion of the treatment period. .After the 4 hour treatment in the non-activated and the S9-activated studies, the cells were centrifuged, the treatment medium was aspirated, the cells were washed with calcium and magnesium free phosphate buffered saline (CMF-PBS), re-fed with complete medium containing cytochalasin B at 6.0 µg/mL and returned to the incubator under standard conditions. For the 24 hour treatment in the non-activated study, cytochalasin B (6.0 µg/mL) was added at the beginning of the treatment.

Micronucleus Assay
Lymphocytes were exposed to vehicle alone and to a minimum of five concentrations of test substance for 4 hours in the absence and presence of S9, and for 24 hours in the absence of S9 using duplicate cultures. Precipitation of test substance in the treatment medium was determined using unaided eye at the beginning and conclusion of treatment.

Collection of Cells (Preliminary Toxicity Test and Micronucleus Assay)
Cells were collected by centrifugation, swollen with 0.075M KCl, washed with fixative (methanol: glacial acetic acid, 25:1 v/v), and the suspension of fixed cells was applied to glass microscope slides and air-dried. The slides were stained with acridine orange.

Cell Cycle Kinetics Scoring (Preliminary Toxicity Test and Micronucleus Assay)
For the preliminary toxicity test, at least 500 cells, if possible, were evaluated to determine the CBPI at each dose level and the control. For the micronucleus assay, at least 1,000 cells (500 cells per culture), if possible, were evaluated to determine the CBPI at each dose level and the control. The CBPI was determined using the following formula:

CBPI = 1X Mononucleated cells + 2 x Binucleated cells + 3 x Multinucleated cells / Total number of cells scored

% Cytostasis (cytotoxicity) = 100 -100 {(CBPItest-1) /(CBPIvehicle-1)}

Micronucleus Scoring
The slides from at least three test substance concentrations were coded and a minimum of 2000 binucleated cells from each concentration (if possible, 1000 binucleated cells from each culture) were examined and scored for the presence of micronuclei. Micronuclei in a binucleated cell (MN-BN) were recorded if they met the following criteria:
• the micronucleus should have the same staining characteristics as the main nucleus
• the micronuclei should be separate from the main nuclei or just touching (no cytoplasmic bridges)
• the micronuclei should be of regular shape and approximately 1/3 or less than the diameter of the main nucleus.

Statistics:

Statistical analysis was performed using the Fisher's exact test (p ≤ 0.05) for a pairwise comparison of the percentage of micronucleated cells in each treatment group with that of the vehicle control. The Cochran-Armitage trend test was used to assess dose-responsiveness.

Key result

Species / strain:

lymphocytes: Human peripheral blood

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

highest dose evaluated was 15 ug/L

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Micronucleus Assay

Visible precipitate was observed in treatment medium at the following doses:

Treatment Condition	Treatment Time	Visible precipitate
		At the beginning of Treatment period	At the conclusion of Treatment period
Non-activated	4 hr	15 µg/mL	15 µg/mL
	24 hr	15 µg/mL	15 µg/mL
S9-activated	4 hr	15 µg/mL	15 µg/mL

 

The pH of the highest dose of test substance in treatment medium was 7.5. Cytotoxicity (CBPI relative to the vehicle control) was observed as follows:

 

Treatment Condition	Treatment Time	Highest Evaluated Dose (µg/mL)	Cytotoxicity (%)
Non-activated	4 hr	15	7
	24 hr	15	25
S9-activated	4 hr	15	11

 

No significant or dose‑dependent increases in micronuclei induction were observed in treatment groups with or without S9 (p > 0.05; Fisher’s Exact and Cochran-Armitage tests).

The results for the positive and vehicle controls indicate that all criteria for a valid assay were met.

Conclusions:

No significant or dose dependent increases in micronuclei induction were observed in treatment groups with or without S9; therefore under the conditions of the study, these results indicate the test substance was negative for the induction of micronuclei in the presence and absence of the exogenous metabolic activation system.

Executive summary:

The test substance was tested to evaluate the potential to induce micronuclei in human peripheral blood lymphocytes (HPBL) in both the absence and presence of an exogenous metabolic activation system. The study has been performed to the standardized guideline OECD 487, under GLP conditions. HPBL cells were treated for 4 hours in the absence and presence of S9, and for 24 hours in the absence of S9. Acetone was used as the vehicle.

 

In the preliminary toxicity assay, the doses tested ranged from 0.5 to 5000 µg/mL, which was the limit dose for this assay. Cytotoxicity [≥ 50% cytokinesis-blocked proliferation index (CBPI) relative to the vehicle control] was observed at doses ≥ 150 µg/mL in the non-activated 4-hour exposure group; at doses ≥ 500 µg/mL in the S9-activated 4-hour exposure group; and at doses ≥ 50 µg/mL in the non-activated 24-hour exposure group. At the conclusion of the treatment period, visible precipitate was observed at doses ≥ 15 µg/mL in all three exposure groups. Based upon these results, the doses chosen for the micronucleus assay ranged from 1 to 15 µg/mL for all three exposure groups.

 

In the micronucleus assay, cytotoxicity (≥ 50% CBPI relative to the vehicle control) was not observed at any dose in any of the three exposure groups. At the conclusion of the treatment period, visible precipitate was observed at 15 µg/mL in all three exposure groups. The doses selected for evaluation of micronuclei were 1, 2, and 15 µg/mL for the non-activated and S9-activated 4-hour exposure groups; and 2, 8, and 15 µg/mL for the non activated 24-hour exposure group.

 

No significant or dose dependent increases in micronuclei induction were observed in treatment groups with or without S9 (p > 0.05; Fisher’s Exact and Cochran-Armitage tests).

 

The results indicate the test substance was negative for the induction of micronuclei in the presence and absence of the exogenous metabolic activation system.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 October 2017 to ****

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

GLP compliance:

yes

Type of assay:

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

Specific details on test material used for the study:

- Purity: >90%
- Description: Opague black viscous liquid

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):

L5178Y/TK+/- cells, clone 3.7.2C, obtained from the American Type Culture Collection (repository number CRL-9518), Manassas, VA. Each batch of frozen cells was tested and found to be free of mycoplasma contamination.

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

In the preliminary toxicity assay, the concentrations tested were 9.77, 19.5, 39.1, 78.1, 156, 313, 625, 1250 and 2500 µg/mL.
The maximum concentration evaluated was based on solubility limitations of the test substance in the vehicle. Visible precipitate was observed at concentrations ≥78.1 µg/mL at the beginning and end of treatment. Relative suspension growth (RSG) was 24, 17 and 16% at concentrations of 1250 µg/mL (4-hour treatment with S9), 156 µg/mL (4-hour treatment without S9) and 19.5 µg/mL (24-hour treatment without S9), respectively. RSG was zero to nine percent at all higher concentrations using all treatment conditions. Based upon these results, the concentrations chosen for the definitive mutagenicity assay were 10, 20, 40, 80 and 100 µg/mL (4-hour treatment with S9), 5, 10, 20, 40, 80 and 100 µg/mL (4-hour treatment without S9) and 2.5, 5, 10, 25, 35 and 80 µg/mL (24-hour treatment without S9).

Vehicle / solvent:

Tetrahydrofuran (THF)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

methylmethanesulfonate

Details on test system and experimental conditions:

Solubility Determination
Tetrahydrofuran (THF) was the vehicle of choice based on the solubility of the test substance and compatibility with the target cells. The test substance formed workable suspensions in THF at concentrations of approximately 50 to 500 mg/mL in the solubility test conducted.

Preparation of Cells
L5178Y/TK+/- cells, clone 3.7.2C, were obtained from the American Type Culture Collection (repository number CRL-9518), Manassas, VA. Each batch of frozen cells was tested and found to be free of mycoplasma contamination. Prior to use in the assay, L5178Y/TK+/- cells were cultured for 24 hours in the presence of thymidine, hypoxanthine, methotrexate and glycine to poison the TK-/- cells. L5178Y/TK+/- cells were prepared in 50% conditioned F0P supplemented with 10% horse serum and 2 mM L-glutamine (F10P) and 50% Fischer's Media for Leukemic Cells of Mice with 0.1% Pluronics F 68 (F0P). All media contained antibiotics.

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 lot of S9 was assayed for sterility and its ability to metabolize at least two pro-mutagens to forms mutagenic to Salmonella typhimurium TA100.
The S9 mix was prepared on the day of use and final concentrations of its components in the test system were: DL-isocitric acid (17.4 mM), NADP (sodium salt; 3.0 mM), and S9 (10 µL/mL).

Preliminary Toxicity Test for Selection of Dose Levels
L5178Y/TK+/- cells were exposed to the vehicle alone in duplicate cultures and nine concentrations of test substance using single cultures for 4 hours in the presence and absence of S9, and for 24 hours in the absence of S9. The maximum concentration evaluated was based on solubility limitations of the test substance in the vehicle; precipitation of the test substance in the treatment medium was determined with the unaided eye at the beginning and end of treatment. The pH of the treatment medium was measured and adjusted if necessary to maintain neutral pH. Osmolality in treatment medium of the vehicle control, the highest concentration, the lowest precipitating concentration and the highest soluble concentration also was measured. Dose levels for the definitive assay were based on post-treatment cytotoxicity (growth inhibition relative to the vehicle control) or solubility limitations of the test substance in the treatment medium.

For the preliminary toxicity assay only, after a 4-hour treatment in the presence and absence of S9, cells were washed with culture medium and cultured in suspension for two days post treatment, with cell concentration adjustment on the first day. After a 24 hour treatment in the absence of S9, cells were washed with culture medium and immediately readjusted to 3 x 10^5 cells/mL. Cells were then cultured in suspension for an additional two days post-treatment with cell concentration adjustment on the first day.

Mouse Lymphoma Assays
L5178Y/TK+/- cells were exposed to the vehicle alone and five to seven concentrations of test substance at appropriate dose intervals using duplicate cultures for 4 hours in the presence and absence of S9, and for 24 hours in the absence of S9. Precipitation was determined with the unaided eye at the beginning and end of treatment.

Treatment of Target Cells
The preparation and addition of the test substance to the test system was carried out under filtered lighting. A mixture containing 50 µL of test substance or vehicle control formulation or 100 µL of positive control formulation were added to treatment medium. All pH adjustments were performed prior to adding S9 or target cells. Either medium or S9 mix (as appropriate) and 6 x 106 L5178Y/TK+/- cells were added, cultures were capped tightly, and incubated with mechanical mixing at 37 ± 1°C for 4 or 24 hours.
For the definitive assay only, at the end of the exposure period, the cells were washed with culture medium, resuspended in F10P, and incubated at 37 ± 1°C for two days following treatment. Cell population adjustments to 3 x 10^5 cells/mL were made as follows:
• 4 hour treatment – 1 and 2 days after treatment.
• 24 hour treatment – immediately after test substance removal; 2 and 3 days after treatment.

Selection of Mutant Phenotype
Cells from selected dose levels were cultured in triplicate with 2-4 μg TFT/mL at a density of 1 x 10^6 cells/100 mm plate in cloning medium containing 0.22 to 0.24% agar. For estimation of cloning efficiency at the time of selection, 200 cells/100 mm plate were cultured in triplicate in cloning medium without TFT (viable cell (VC) plate). Cultures were incubated under standard conditions (37 ± 1°C in a humidified atmosphere of 5 ± 1% CO2 in air) for 10 or 11 days. The plates were stored under refrigerated conditions (2-8°C) for 8 days prior to scoring.

The total number of colonies per culture was determined for the VC plates and the total relative growth calculated. The total number of colonies per TFT plate was then determined for those cultures with ≥10% total growth (including at least one concentration between 10 and 20% total growth, if possible). Colonies were counted and the diameter of the TFT colonies from the positive control and vehicle control cultures were determined over a range from 0.2 to 1.1 mm.

Extended Treatment and/or Confirmatory Assay
Verification of a clear positive response was not required (OECD Guideline 490). For negative results without activation, an extended treatment assay was performed in which cultures were continuously exposed to the test substance for 24 hours without S9 activation. The extended treatment assay was performed concurrently with the initial assay. For negative results with S9 activation, a confirmatory assay was not required unless the test substance was known to have specific requirements of metabolism.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Cultures treated at concentration up to 15 µg/mL. No increases in induced mutant frequency ≥90 mutants/10^6 clonable cells were observed.

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

The results indicate the test material was negative for the ability to induce forward mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells, in the presence and absence of an exogenous metabolic activation system.

Executive summary:

The test substance was evaluated in a study performed to the standardized guideline OECD 490 under GLP conditions for its ability to induce forward mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells in the presence and absence of an exogenous metabolic activation system. Tetrahydrofuran (THF) was used as the vehicle.

 

In the preliminary toxicity assay, the concentrations tested were 9.77, 19.5, 39.1, 78.1, 156, 313, 625, 1250 and 2500 µg/mL. The maximum concentration evaluated was based on solubility limitations of the test substance in the vehicle. Visible precipitate was observed at concentrations ≥78.1 µg/mL at the beginning and end of treatment. Relative suspension growth (RSG) was 24, 17 and 16% at concentrations of 1250 µg/mL (4-hour treatment with S9), 156 µg/mL (4-hour treatment without S9) and 19.5 µg/mL (24-hour treatment without S9), respectively. RSG was or approximated 0% at all higher concentrations using all treatment conditions. Based upon these results, the concentrations chosen for the definitive mutagenicity assay were 10, 20, 40, 80 and 100 µg/mL (4-hour treatment with S9), 5, 10, 20, 40, 80 and 100 µg/mL (4-hour treatment without S9) and 2.5, 5, 10, 25, 35 and 80 µg/mL (24-hour treatment without S9).

 

In the initial definitive mutagenicity assay, visible precipitate was observed at concentrations ≥80 µg/mL at the beginning of treatment and end of treatment. Cultures treated at concentrations of 10, 20, 40 and 80 µg/mL (4-hour treatment with S9) and 5, 10, 20, 40 and 80 µg/mL (4-hour treatment without S9) exhibited 85 to 116% and 48 to 93% RSG, respectively, and were cloned. The 24-hour treatment without S9 was not cloned as all dose levels were excessively toxic. This condition was repeated. Relative total growth of the cloned cultures ranged from 34 to 106% (4‑hour treatment with S9) and 60 to 108% (4-hour treatment without S9). No increases in induced mutant frequency ≥90 mutants/10^6 clonable cells were observed under any treatment condition. 

 

In the retest definitive mutagenicity assay, the concentrations tested were 1.25, 2.5, 5, 10, 15, 20, 25, 30 and 40 µg/mL. No visible precipitate was observed at the beginning or end of treatment. Cultures treated at concentrations of 1.25, 2.5, 5, 10 and 15 µg/mL (24-hour treatment without S9) exhibited 12 to 87% RSG, and were cloned. Relative total growth of the cloned cultures ranged from 9 to 72% (24‑hour treatment without S9). No increases in induced mutant frequency ≥90 mutants/10^6 clonable cells were observed.

 

These results indicate the test material was negative for the ability to induce forward mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells, in the presence and absence of an exogenous metabolic activation system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available