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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial gene mutation test

Key Study:

A study was conducted to the standardized OECD 471 guideline, under GLP conditions. The test item was negative for the ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system (BioReliance Corporation, 2018).

In vitro micronucleus test:

Key Study:

A study was conducted to the standardized OECD 487 guideline, under GLP conditions. The test item was negative for the induction of micronuclei in the presence and absence of the exogenous metabolic activation system

(BioReliance Corporation, 2018).

In vitro gene mutation in mammalian cells test:

Key Study:

A study was conducted to the standardized OECD 490 guideline, under GLP conditions. The test item 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 Corporation, 2018).

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 July 2017 to ****
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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:
Description: Blackish blue-green opaque liquid
Purity: 100% (nominal); UVCB (provided by Sponsor)
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^TM 1254
Test concentrations with justification for top dose:
Mutagenicity Assay: 50, 150, 500, 1500 and 5000 µg.
5000 µg is the standard top dose recommended in the regulatory guidelines that this assay follows. Other concentrations used were a series of ca half-log10 dilutions of the highest concentration.

Preliminary Toxicity Assay: Conducted at dose levels of 6.67, 10.0, 33.3, 66.7, 100, 333, 667, 1000, 3333 and 5000 µg per plate in ethanol.
Vehicle / solvent:
Ethanol
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: 2 aminoanthracene
Details on test system and experimental conditions:
Preparation of Tester Strain
Overnight cultures were prepared from the appropriate frozen permanent stock. Following inoculation, each flask was placed in a shaker/incubator and incubated at 37±2°C for approximately 12 hours before the anticipated time of harvest. Each culture was monitored spectrophotometrically for turbidity and was harvested at a percent transmittance yielding a titer of greater than or equal to 0.3x10^9 cells per milliliter. The actual titers were determined by viable count assays on nutrient agar plates.

Exogenous Metabolic Activation
The S9 metabolic activation system was purchased commercially from MolTox (Boone, NC) and stored at 60°C or colder until use. It was prepared from male Sprague-Dawley rats that were injected intraperitoneally with Aroclor™ 1254. Each bulk preparation was assayed for its ability to metabolize benzo(a)pyrene and 2 aminoanthracene to forms mutagenic to Salmonella typhimurium TA100.
The S9 mix was prepared on the day of use and contained: S9 (10%), sodium phosphate buffer (pH 7.4; 100 mM), MgCl2 (8 mM), KCl (33 mM), glucose-6-phosphate (5 mM), and β-nicotinamide-adenine dinucleotide phosphate (4 mM). The Sham mix, containing 100 mM phosphate buffer at pH 7.4, was also prepared on the day of use.

Frequency and Route of Administration
The test system was exposed to the test substance via the plate incorporation methodology originally described by Ames et al. (1975) and updated by Maron and Ames (1983).

Preliminary Toxicity Assay to Select Dose Levels
The preliminary toxicity assay was used to establish the dose range over which the test substance would be assayed. TA98, TA100, TA1535, TA1537 and WP2 uvrA were exposed to the vehicle alone and ten dose levels of the test substance, with a single plate/condition, on selective minimal agar in the presence and absence of S9 mix. Dose levels for the mutagenicity assay were based upon post-treatment toxicity.

Mutagenicity Assay
TA98, TA100, TA1535, TA1537 and WP2 uvrA were exposed to the vehicle alone, positive controls and at least five dose levels of test substance, in triplicate, in the presence and absence of S9 mix.
To confirm the sterility of the S9, Sham mixes, test substance and the vehicle, each was plated on selective agar with an aliquot volume equal to that used in the assay and incubated under the same conditions as the assay.
One half (0.5) milliliter of S9 or Sham mix, 100 µL of tester strain (cells seeded) and 50.0 µL of vehicle, positive control, or test substance dilution were added to 2.0 mL of molten selective top agar at 45 ± 2 °C, vortexed, and overlaid onto minimal bottom agar. After the overlay solidified, the plates were inverted and incubated for 48 to 72 hours at 37 ± 2 °C. Plates that were not counted immediately following the incubation period were stored at 2 8 °C until colony counting could be conducted.

Scoring
The condition of the bacterial background lawn was evaluated for evidence of test substance toxicity by using a dissecting microscope. Precipitate was evaluated after the incubation period by visual examination without magnification. Toxicity and degree of precipitation were scored relative to the vehicle control plate. Colonies were enumerated either by hand or by machine.

Tester Strain Verification
On the day of use in each assay, all tester strain cultures were checked for the appropriate genetic markers.

Key result
Species / strain:
other: TA1537, TA98, TA1535, TA100, and E. coli WP2 uvrA (pKM101)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Preliminary Toxicity Assay
The maximum dose of 5000 µg per plate was achieved using a concentration of 100 mg/mL and a 50.0 µL plating aliquot.
Precipitate was observed beginning at 3333 or at 5000 µg per plate with all conditions. Toxicity was observed beginning at 3333 µg per plate with tester strain WP2 uvrA in the absence of S9 activation.

Mutagenicity Assay
No toxicity was observed. Precipitate was observed beginning at 1500 µg per plate with all conditions.
No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation.
Conclusions:
The test item was negative for the ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system.
Executive summary:

The test item was tested to evaluate its mutagenic potential by measuring its ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system. Ethanol was used as the vehicle.

 

In the preliminary toxicity assay, the dose levels tested were 6.67, 10.0, 33.3, 66.7, 100, 333, 667, 1000, 3333 and 5000 µg per plate. Precipitate was observed beginning at 3333 or at 5000 µg per plate with all conditions. Toxicity was observed beginning at 3333 µg per plate with tester strain WP2 uvrA in the absence of S9 activation. Based upon these results, the maximum dose tested in the mutagenicity assay was 5000 µg per plate.

 

In the mutagenicity assay, the dose levels tested were 50.0, 150, 500, 1500 and 5000 µg per plate. No toxicity was observed. Precipitate was observed beginning at 1500 µg per plate with all conditions. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation.

 

These results indicate the test item was negative for the ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 June 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:
Description: Blackish blue-green opaque liquid
Purity: 100% (nominal); UVCB (provided by Sponsor)
Species / strain / cell type:
lymphocytes:
Remarks:
human
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: 7.5, 13, 25, 50, 105 µg/mL, for 4 and 24 hrs, and
- S9-activated: 25, 50, 100, 150, 200, 250, 300 µg/mL, for 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 55 ± 5% cytotoxicity (CBPI relative to the vehicle control), and
- 24-hour (-S9): Visible precipitate at the end of treatment period.
Vehicle / solvent:
Ethanol
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
other: Vinblastine (VB)
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
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid

Preliminary Toxicity Test

Visible precipitate and hemolysis were observed in treatment medium at the following doses:

 

Treatment

Condition

Treatment

Time

Visible precipitate

Hemolysis at the conclusion of Treatment period

At the beginning of Treatment period

At the conclusion of Treatment period

Non-activated

4 hr

³ 35 µg/mL

³ 105 µg/mL

³ 1050 µg/mL

24 hr

³ 35 µg/mL

³ 105 µg/mL

³ 350 µg/mL

S9-activated

4 hr

³ 35 µg/mL

³ 350 µg/mL

³ 350 µg/mL

 

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

³ 25 µg/mL

³ 50 µg/mL

24 hr

³ 25 µg/mL

105 µg/mL

S9-activated

4 hr

³ 25 µg/mL

³ 150 µg/mL

 

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

50

6

24 hr

105

24

S9-activated

4 hr

150

53

 

The osmolality of the test substance doses in treatment medium was considered acceptable. The pH of the highest dose of test substance in treatment medium was 7.0. Cytotoxicity ( ≥ 50% cytokinesis-blocked proliferation index (CBPI) relative to the vehicle control0 was observed at doses ≥ 1050 µg/mL in the non activated 4-hour exposure group; and at doses ≥ 350 µg/mL in the S9 activated 4-hour and the non-activated 24-hour exposure groups.

Conclusions:
The test item was negative for the induction of micronuclei in the presence and absence of the exogenous metabolic activation system. 
Executive summary:

The test item 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. HPBL were treated for 4 hours in the absence and presence of S9, and for 24 hours in the absence of S9. Ethanol was used as the vehicle.

 

In the preliminary toxicity assay, the doses tested ranged from 0.35 to 3500 µg/mL, which was based on the solubility of the test item in the vehicle. Cytotoxicity (≥ 50% cytokinesis-blocked proliferation index (CBPI) relative to the vehicle contro) was observed at doses ≥ 1050 µg/mL in the non-activated 4-hour exposure group; and at doses ≥ 350 µg/mL in the S9 activated 4-hour and the non-activated 24-hour exposure groups. At the conclusion of the treatment period, visible precipitate was observed at doses ≥ 105 µg/mL in the non-activated 4 and 24-hour exposure groups, and at doses ≥ 350 µg/mL in the S9 activated 4-hour exposure group. Based upon these results, the doses chosen for the micronucleus assay ranged from 7.5 to 105 µg/mL for the non-activated 4 and 24-hour exposure groups and from 25 to 300 µg/mL for the S9-activated 4-hour exposure group.

 

In the micronucleus assay, cytotoxicity (≥ 50% CBPI relative to the vehicle control) was not observed at any dose in the non-activated 4 and 24 hour exposure groups. Cytotoxicity was observed at doses ≥ 150 µg/mL in the S9 activated 4-hour exposure group. At the conclusion of the treatment period, visible precipitate was observed at doses ≥ 50 µg/mL in the non-activated 4-hour exposure group; at doses ≥ 150 µg/mL in the S9 activated 4-hour exposure group; and at 105 µg/mL in the non-activated 24-hour exposure group. The doses selected for evaluation of micronuclei were 13, 25, and 50 µg/mL for the non-activated 4 hour exposure group; 50, 100, and 150 µg/mL for the S9-activated 4-hour exposure group; and 13, 25, and 105 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).

 

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

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31 August 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:
Description: Blackish blue-green opaque liquid
Purity: 100% (nominal); 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):
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:
S9 mix
Test concentrations with justification for top dose:
In the preliminary toxicity assay, the concentrations tested were 4.88, 9.77, 19.5, 39.1, 78.1, 156, 313, 625 and 1250 µg/mL. 
The maximum concentration evaluated was based on solubility limitations of the test item in the vehicle. Visible precipitate was observed at concentrations ≥ 78.1 µg/mL by the end of treatment. Relative suspension growth (RSG) was 73% at a concentration of 78.1 µg/mL (4-hour treatment both with and without S9) and 61% at a concentration of 19.5 µg/mL (24-hour treatment without S9). RSG was 0% at all higher concentrations under each treatment conditions. 
Based upon these results, the concentrations chosen for the definitive mutagenicity assay were 5, 10, 20, 40 and 80 µg/mL (4 hour treatment with and without S9) and 2.5, 5, 10, 20, 30 and 40 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. Following sonication at 35.9°C for 30 minutes, the test substance formed workable suspensions in THF at concentrations of approximately 50 to 200 mg/mL.

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
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Conclusions:
The test item 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 item was evaluated 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. An untreated control was included in the study in order to compare the data with the vehicle control.

 

In the preliminary toxicity assay, the concentrations tested were 4.88, 9.77, 19.5, 39.1, 78.1, 156, 313, 625 and 1250 µg/mL. The maximum concentration evaluated was based on solubility limitations of the test item in the vehicle.  Visible precipitate was observed at concentrations ≥ 78.1 µg/mL by the end of treatment. Relative suspension growth (RSG) was 73% at a concentration of 78.1 µg/mL (4-hour treatment both with and without S9) and 61% at a concentration of 19.5 µg/mL (24-hour treatment without S9). RSG was 0% at all higher concentrations under each treatment conditions. Based upon these results, the concentrations chosen for the definitive mutagenicity assay were 5, 10, 20, 40 and 80 µg/mL (4 hour treatment with and without S9) and 2.5, 5, 10, 20, 30 and 40 µg/mL (24-hour treatment without S9).

 

In the definitive mutagenicity assay visible precipitate was observed at a concentration of 80 µg/mL by the end of treatment. Cultures treated at all concentrations using a 4-hour treatment with and without S9 and at concentrations of 2.5, 5, 10 and 20 µg/mL using a 24-hour treatment without S9 exhibited 18 to 96% (4-hour treatment with S9), 62 to 104% (4 hour treatment without S9) and 10 to 102% RSG (24-hour treatment without S9) and were cloned. In the 24-hour treatment without S9, one cloned culture at a concentration of 20 µg/mL was excessively toxic. However, at the Sponsor’s request, the plates for this culture were scored to provide mutagenicity data. Cultures treated at higher concentrations in the 24-hour treatment without S9 were discarded prior to cloning due to excessive toxicity. Relative total growth of the cloned cultures ranged from 13 to 95% (4 hour treatment with S9), 57 to 111% (4-hour treatment without S9) and 7 to 99% (24 hour treatment without S9). No increases in induced mutant frequency ≥90 mutants/10^6 clonable cells were observed under any treatment condition. Due to the excessive toxicity observed in the 24-hour treatment without S9, where at least four concentrations in duplicate cultures were not available for mutagenicity evaluation, this test condition was retested with an adjustment in concentrations. The concentrations chosen for the retest of the definitive mutagenicity assay were 1.25, 2.5, 5, 10, 20, 25 and 35 µg/mL.

 

In the retest of the definitive mutagenicity assay, no visible precipitate was observed at the beginning or end of treatment. Cultures treated at concentrations above 10 µg/mL were discarded prior to cloning due to excessive toxicity. Relative total growth of the cloned cultures ranged from 77 to 133%. No increases in induced mutant frequency ≥90 mutants/10^6 clonable cells were observed. Although the total solvent suspension growth was below the acceptable range due to toxicity of the vehicle to the test system, acceptable data were collected for the untreated control, and therefore, a valid assay was achieved. 

 

These results indicate that the test item 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)

Additional information

Justification for classification or non-classification