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Genetic toxicity in vitro

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Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to OECD TG 476, EC B.17 and US EPA OPPTS 870.5300 and in acordance with the Principles of Good Laboratory Practice (GLP).
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Principles of method if other than guideline:
not applicable
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Target gene:
The in vitro mammalian cell gene mutation tests is used to detect gene mutations induced by chemical substances. Suitable cell lines include L5178Y mouse lymphoma cells, the CHO, AS52 and V79 lines of Chinese hamster cells, and TK6 huan lymphoblastoid cells. In these cell lines the most commonly-used genetic enpoints measure mutation at thymidine kinase (TK) and hypoxanthine-guanine phosphoribosyl transferase (HPRT), and a transgene of xanthineguanine phosphoribosyl transferase (XPRT). The TK, HPRT, and XPRT mutation tests detect different spectra of genetic events. The autosomal location of TK and XPRT allows the detection of genetic events (e.g. large deletions) not detected at the HPRT locus on the X-chromosomes.
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: Ham's F-12 medium (GIBCO, Grand Island, New York) supplemented with 5% (v/v) heat-inactivated (56°C, 30 minutes), dialyzed fetal bovine serum (GIBCO), antibiotics and antimycotics (penicillin G, 100 units/ml; streptomycin sulfate, 0.1 mg/ml; fungizone, 0.25 µg/ml; GIBCO), and an additional 2 mM L-glutamine (GIBCO)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 12545
Test concentrations with justification for top dose:
Preliminary Toxicity Assay - 0.6, 1.2, 2.3, 4.7, 9.4, 18.8, 37.5, 75.0, and 150.0 µg/ml
Initial Mutagenicity Assay - 1.5, 4.74, 15.0, 47.4, and 150 µg/ml
Confirmatory Gene Mutation Assay - 1.5, 4.74, 15.0, 47.4, and 150 µg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: 1% Ethanol
- Justification for choice of solvent/vehicle: Test material solubiility
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
The solvent ethanol selected for dissolving the test material was used as the negative control treatment.
True negative controls:
yes
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
other: 20-methylcholantherene
Remarks:
Ethyl methanesulfonate (EMS, CAS No. 62-50-0) at 621ug/ml and 20-methylcholanthrene (20-MCA, CAS No. 56-49-5) at 4 and 8 ug/ml.
Details on test system and experimental conditions:
The cell line CHO-K1-BH4, originally obtained from Dr. Abraham Hsie, Oak Ridge National Laboratory, Oak Ridge, Tennessee, was used in this study. The CHO-K1-BH4 cell line was selected as the test system for this study because it is sensitive to mutagens, has a low background mutant frequency, and is readily available. Stock cultures were stored at about -80°C or below. The cultures were periodically checked for mycoplasma contamination (American Type Culture Collection, Manassas, Virginia). The cells were grown as monolayer cultures in plastic disposable tissue culture labware under standard conditions of approximately 5% CO2 in air at 37C in a humidified incubator.

The test material was found to be soluble in ethanol (ethanol; CAS No. 64-17-5) up to 62.65 mg/ml. Therefore, ethanol was selected as the solvent used to dissolve the test material and was used as the vehicle control treatment. Ethyl methanesulfonate (EMS, CAS No. 62-50-0) was used as the positive control for the non-activation system (without S9 factor) at a final concentration of 621 g/ml. The positive control for assays performed with S9 (activation system) was 20-methylcholanthrene (20-MCA, CAS No. 56-49-5) at concentrations of 4 and 8 g/ml. The dose levels of EMS and 20-MCA were based upon our unpublished findings.

All test material solutions were prepared fresh on the day of treatment and used within one hour of preparation. The test material was first dissolved in ethanol and further diluted (1:100) with the treatment medium to obtain the desired concentrations. This technique has been shown to be an effective method for detecting various chemical mutagens in this test system (Hsie et al, 1981). EMS was dissolved in treatment medium. 20-MCA was dissolved first in dimethyl sulfoxide and further diluted in the culture medium. All dosing units were expressed in µg/ml.

Cells in logarithmic growth phase were trypsinized and placed in medium containing 5% serum at a standard density of 3.0 x 106 cells/T-75 flask approximately 24 hours prior to treatment. At the time of treatment, the culture medium was replaced with serum-free medium, S9 mix (when applicable), and the test chemical, the vehicle control, or the positive control chemical. The cells were treated for approximately 4 hours at 37°C and the exposure was terminated by washing the cells with phosphate buffered saline (Ca++ and Mg++ free).

All test cultures were identified using self adhesive labels containing a code system that identified the test material, experiment number, treatment, and replicate.

The selected concentrations of the test material in the stock dosing solutions used for treatment in the initial gene mutation assay (Assay B1) were verified by the Analytical Chemistry Laboratory, Toxicology and Environmental Research and Consulting, The Dow Chemical Company, Midland, Michigan. Samples were diluted in acetonitrile and analyzed by high performance liquid chromatography with ultraviolet detection (HPLC/UV).

S9 liver homogenates prepared from Aroclor 1254-induced male Sprague-Dawley rats were purchased from a commercial source and stored at approximately -80°C or below. Thawed S9 was reconstituted at a final concentration of 10% (v/v) in a “mix” (O’Neill et al., 1982). The S9 mix consisted of the following co-factors: 10 mM MgCl2•6H2O, 5 mM glucose-6-phosphate, 4 mM nicotinamide adenine dinucleotide phosphate, 10 mM CaCl2, 30 mM KCl, and 50 mM sodium phosphate (pH 8.0). The reconstituted mix was added to the culture medium to obtain the desired final concentration of S9 in the culture, i.e., 2% v/v. Hence, the final concentration of the co-factors in the culture medium is 1/5th of the concentrations stated above.

Toxicity Assay - The cytotoxicity of the test material was assessed by determining the ability of the treated cells to form colonies. This assay was conducted for selecting concentrations of the test material to be used in the gene mutation assay. Cells were seeded into T-25 flasks (1.0 x 106 cells/flask) approximately 24 hours prior to treatment. Treatment was for approximately 4 hours with various concentrations of the test material with one replicate per dose in the presence and absence of S9. After termination of treatment, the cells were trypsinized and replated at a density of 200 cells/dish into 60 mm dishes (3/dose) and the dishes incubated for 7 days to allow colony formation. The colonies were then fixed/stained with methanol/crystal violet. The number of colonies/dish was counted and the mean colonies/treatment were expressed relative to the vehicle control value. The highest dose level of the test material (150 µg/ml) was limited by the solubility of the test material in the treatment medium. The other concentrations tested were separated by a factor 2.

Gene Mutation Assay - Based upon the toxicity assay, precipitating dose levels in the absence and presence of S9 were selected as the top concentrations for each system. At least four additional concentrations of the test material, the lowest of which was expected to give cell survival comparable to vehicle control, were also tested in the gene mutation assay. The concentrations were usually spaced apart by a factor of 3.2. The cytotoxicity of the selected concentrations was determined concurrent with the gene mutation assay.Each dose level was set up in duplicate from the time of treatment until the completion of the assay. The cultures were trypsinized at the end of the treatment and replated at a density of 1 x 106 cells/100 mm dish (at least two dishes/replicate) for phenotypic expression (O'Neill et al., 1977a; O'Neill and Hsie, 1979). In addition, 200 cells/60 mm dish (three dishes/replicate) were also plated to determine the toxicity and incubated for approximately 7 days to permit colony formation. During the phenotypic expression period (8 days), cells in the larger petri dishes were subcultured every 2-3 days and plated (at least two dishes/replicate) at a density of about 1 x 106 cells/100 mm petri dish. At each subculture, cells from various dishes within each replicate were pooled prior to replating. At the end of the expression period, the cultures were trypsinized and plated at a density of 2 x 105 cells/100 mm dish (a total of 10 dishes/treatment) in the selection media (Ham's F-12 without hypoxanthine and with 6-thioguanine) for the determination of Hgprt- mutants and 200 cells/60 mm dish (three dishes/treatment) in Ham's F-12 medium without hypoxanthine for determination of cloning efficiency. The dishes were incubated for about 8 days and the colonies were fixed/stained with methanol/crystal violet. The mutant frequency (expressed as mutants per 106 clonable cells) for each replicate were calculated by the following formula (Kirkland, 1989):
MF = K x (m / c)

Where:
K = Pc x 106 / Pm

Pc = the number of cells plated for the survival plates
Pm = the number of cells plated for the mutation plates
c = the mean colonies per plate for survival plates for each replicate
m = the mean colonies per plate for mutation plates for each replicate


Evaluation criteria:
For an assay to be acceptable, the mutant frequency in positive controls should have been significantly higher than the vehicle controls. An additional criterion was that the mutant frequency in the vehicle controls should have been within reasonable limits of the laboratory historical control values and literature values. The test chemical was considered positive if it induced a statistically significant, dose-related, reproducible increase in mutant frequency. The final interpretation of the data took into consideration such factors as the mutant frequency and cloning efficiencies in the vehicle controls.
Statistics:
The frequency of mutants per 106 clonable cells was statistically evaluated using a weighted analysis of variance; weights were derived from the inverse of the mutant frequency variance. The actual plate counts are assumed to follow a Poisson distribution; therefore the mean plate count was used as an estimate of variance (Kirkland, 1989).
If the analysis of variance was significant at alpha = 0.05, a Dunnett's t-test was conducted (Winer, 1971), comparing each treated group and the positive control to the vehicle control (alpha = 0.05, one-sided). Linear dose-related trend tests were performed if any of the pairwise comparisons of test material with the vehicle control yielded significant differences.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
pH and Osmolality
The pH and osmolality of treatment medium containing approximately 157 µg/ml of the test material and medium containing 1% ethanol were determined using a Denver Basic pH meter (Denver Instrument Co., Arvada, Colorado) and an OSMETTE A freezing point osmometer (Precision Systems, Inc., Natick, Massachusetts). Alterations in the pH and osmolality of the culture medium have been shown to induce false positive responses in in vitro genotoxicity assays (Thilagar et al., 1984; Galloway et al., 1985; Cifone, 1985). There was no appreciable change in either the pH or osmolality at this concentration as compared to the culture medium with solvent alone (culture medium with the test material, pH = 7.36, osmolality = 492 mOsm/kgH2O; culture medium with 1% ethanol, pH = 7.32, osmolality = 492 mOsm/kgH2O).

Assay A1 – Preliminary Toxicity Assay
In a preliminary toxicity assay, the test material was tested at concentrations of 0 (vehicle control), 0.6, 1.2, 2.3, 4.7, 9.4, 18.8, 37.5, 75.0, and 150.0 µg/ml in the absence and presence of an externally supplied metabolic activation system (S9). The highest three concentrations (37.5, 75.0, and 150.0 µg/ml) precipitated in the treatment medium. The treated cultures in the absence and presence of S9 activation showed little to no toxicity with the relative cell survival (RCS) values ranging from 112.9 to 146.5% in the absence of S9 and 90.2 to 165.0% in the presence of S9. Based upon the results of this assay, target concentrations of 0 (vehicle control) 1.5, 4.74, 15.0, 47.4, and 150 µg/ml of the test material were selected for the initial gene mutation assay in the absence of S9 and presence of S9.

Assay B1 – Initial Mutagenicity Assay
In the initial mutagenicity assay (Assay B1), the highest two concentrations (47.4 and 150 µg/ml) precipitated in the treatment medium. In the absence of S9, little to no toxicity was observed with RCS values ranging from 103.1 to 127.4%. In the presence of S9, little to no toxicity was observed with RCS values ranging from 103.5 to 139.6%. The mutant frequencies observed in cultures treated with the test material in the absence and presence of S9 were not significantly different from the concurrent vehicle control values. All mutant frequencies were within a reasonable range of historical background values.

Assay C1 – Confirmatory Mutagenicity Assay
Based upon the previous results, targeted concentrations of 0 (vehicle control), 1.5, 4.74, 15, 47.4, and 150 µg/ml in the absence and presence of S9 were selected for the confirmatory gene mutation assay. In this assay, the highest two concentrations precipitated in the treatment medium. There was little to no toxicity observed, as indicated by RCS values, in the absence of S9 (103.4 to 131.3%). In the presence of S9, RCS values indicated little to no toxicity with RCS values ranging from 76.9 to 122.5%. The mutant frequencies observed in cultures treated with the test material in the absence of S9 and presence of S9 were not significantly different from the concurrent vehicle control values and were within the range of the laboratory historical background.
In both the initial and confirmatory mutagenicity assays, the positive control chemicals induced significant increases in mutant frequencies and this data confirmed the adequacy of the experimental conditions for detecting induced mutations.
The analytically determined concentrations of the test material in the stock dosing solutions in Assay B1 ranged from 87.5 to 94.7% of target and verified that concentrations used for treatment were within acceptable range.
Remarks on result:
other: other: CHO-K1-BH4
Remarks:
Migrated from field 'Test system'.

None

Conclusions:
Interpretation of results (migrated information):
negative

The results of the in vitro Chinese Hamster Ovary cell/hypoxanthine-guanine-phosphoribosyl transferase (CHO/HGPRT) forward gene mutation assay with ACCUTRACE™ S10 Fuel Marker indicate that under the conditions of this study, the test article was non-mutagenic when evaluated in the absence or presence of an externally supplied metabolic activation (S9) system.
Executive summary:

Accutrace™ S10 Fuel Marker (1-(Decyloxy)-2-(1-methylpropyl)-4-(triphenylmethyl) benzene) was evaluated in the in vitro Chinese Hamster Ovary cell/hypoxanthine-guanine-phosphoribosyl transferase (CHO/HGPRT) forward gene mutation assay. The genotoxic potential of the test material was assessed in two independent assays in the absence and presence of an externally supplied metabolic activation (S9) system. The concentrations ranged from 1.5 to 150 µg/ml in the absence and presence of S9. The highest concentration was based on the limit of solubility of the test material in the treatment medium. The analytically-determined concentrations of Accutrace™ S10 Fuel Marker in the dose preparations ranged from 87.5 to 94.7% of the targeted values. The adequacy of the experimental conditions for detection of induced mutation was confirmed by employing positive control chemicals, ethyl methanesulfonate for assays in the absence of S9 and 20-methylcholanthrene for assays in the presence of S9. Vehicle control cultures were treated with the solvent used to dissolve the test material (i.e. ethanol). There were no statistically significant treatment-related increases in the mutant frequency in the test material-treated cultures compared to the vehicle control cultures in either the absence or presence of S9. The results of the CHO/HGPRT forward gene mutation assay with ACCUTRACE™ S10 Fuel Marker indicate that under the conditions of this study, the test article was non-mutagenic when evaluated in the absence or presence of an externally supplied metabolic activation (S9) system.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP/Guideline Study
Justification for type of information:
Read across to an analogue based on structural similarity. An analogue justification is attached to section 13 of the dataset
Reason / purpose:
read-across: supporting information
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
Each S. typhimurium tester strain contains, in addition to a mutation in the histidine
operon, additional mutations that enhance sensitivity to some mutagens. The rfa
mutation results in a cell wall deficiency that increases the permeability of the cell to
certain classes of chemicals such as those containing large ring systems that would
otherwise be excluded. The deletion in the uvrB gene results in a deficient DNA
excision-repair system. Tester strains TA98 and TA100 also contain the pKM101
plasmid (carrying the R-factor). It has been suggested that the plasmid increases
sensitivity to mutagens by modifying an existing bacterial DNA repair polymerase
complex involved with the mismatch-repair process.
TA98 and TA1537 are reverted from histidine dependence (auxotrophy) to histidine
independence (prototrophy) by frameshift mutagens. TA100 is reverted by both
frameshift and base substitution mutagens and TA1535 is reverted only by mutagens
that cause base substitutions.
The E. coli tester strain has an AT base pair at the critical mutation site within the trpE
gene. Tester strain WP2uvrA (pKM101) has a deletion in the
uvrA gene resulting in a deficient DNA excision-repair system. Tryptophan revertants
can arise due to a base change at the originally mutated site or by a base change
elsewhere in the chromosome causing the original mutation to be suppressed. Thus,
the specificity of the reversion mechanism is sensitive to base substitution mutations.
Additional strain / cell type characteristics:
other: rfa mutation, uvrB deletion
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver S-9 homogenate
Test concentrations with justification for top dose:
100, 266, 707, 1880, and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl formamide (DMF)
- Justification for choice of solvent/vehicle: Solubility test was conducted to determine the highest soluble concentration of the test
substance in vehicles compatible with this test system in the order of preference,
sterile water, DMSO, Ethanol, Acetone and DMF at 50 mg/mL. Though Acetone is one of the organic vehicles compatible with this test system, since
Advinus did not have any historical control data on using Acetone as the vehicle, in
consultation with the sponsor, it was decided not to use Acetone as vehicle.
DMF is also one of the organic vehicles compatible with this test system. Therefore,
based on the results of the solubility test and in consultation with the Sponsor, DMF
was selected as the vehicle of choice to prepare the stock and dilutions of the test
substance as well as the positive controls.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
other: 2-Aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: 48-72 hours
- Exposure duration: 67 hours

NUMBER OF REPLICATIONS: 3

NUMBER OF CELLS EVALUATED: 1-2x109 Colony Forming Units (CFU)/mL

DETERMINATION OF CYTOTOXICITY
- Method: Number of revertants per plate
The revertant colonies will be counted manually and the plates will be examined for bacterial
background lawn. The condition of the bacterial background lawn will be evaluated for evidence
of test substance toxicity and precipitate. Evidence of toxicity will be scored relative to the vehicle
control plate and recorded along with the revertant count for that plate. Toxicity will be evaluated
as a decrease in the number of revertant colonies per plate and/or a thinning or disappearance of
the bacterial background lawn. Precipitation will be evaluated after the incubation period by visual
examination without magnification.
Evaluation criteria:
The Salmonella typhimurium and Escherichia coli reverse mutation test is considered acceptable if
it meets the following criteria:
· Tester strain integrity
All Salmonella typhimurium tester strains must exhibit sensitivity to crystal violet and to
ultraviolet light to demonstrate the presence of rfa mutation and uvrB mutation,
respectively.
The Escherichia coli tester strain must exhibit sensitivity to ultraviolet light to
demonstrate the presence of uvrA mutation.
Salmonella typhimurium strains TA98 and TA100 and Escherichia coli strain WP2uvrA
(pKM101) must exhibit resistance to ampicillin to demonstrate the presence of the
plasmid R-factor.
· The spontaneous reversion rates in the solvent/vehicle control must be in the range of
in-house historical data
· All tester strain culture titers must be in the range of 1-2x109 cells/mL to ensure that
appropriate numbers of bacteria are used for plating.
· Each mean, positive control value must exhibit at least a 3.0-fold increase over the
respective mean vehicle control value for each tester strain.
· Toxicity: A minimum of three non-toxic dose levels will be required to evaluate assay
data. A dose level is considered toxic if it causes a >50% reduction in the mean number
of revertants per plate relative to the mean vehicle control value (this reduction must be
accompanied by an abrupt dose-dependent drop in the revertant count) or a reduction in
the background lawn. In the event that less than three non-toxic dose levels are achieved,
the affected portion of the assay will be repeated with an appropriate change in dose
levels.
Statistics:
none
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and 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
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

Under the conditions of the current study, the test substance, XU-18838.00 was negative
(non-mutagenic) in this Salmonella-Escherichia coli/Mammalian-Microsome Reverse
Mutation Assay.
Executive summary:

XU-18838.00 was tested for its mutagenic potential in the bacterial reverse mutation assay. The study was conducted using TA98, TA100, TA1535 and TA1537 strains of Salmonella typhimurium and WP2uvrA (pKM101) strain of Escherichia coli in two phases. In the first phase, an initial toxicity-mutation test was performed. The second phase was an independent confirmatory mutation test. The bacterial tester strains were exposed to the test substance in the presence and absence of a metabolic activation system (S-9 fraction prepared from Aroclor 1254 induced rat liver) using a preincubation procedure. XU-18838.00 was found to be soluble in Dimethyl formamide (DMF) at 50 mg/mL. In the initial toxicity-mutation assay, XU-18838.00 was exposed in duplicate at 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate test doses along with the vehicle and appropriate positive controls. The mean and standard deviation of revertant colonies were calculated for each test dose and the controls for all the tester strains. There was a moderate precipitation of the test substance on the basal agar plates at the top dose of 5000 µg/plate. No toxicity was observed up to 1500 XU-18838.00 was tested for its mutagenic potential in the bacterial reverse mutation assay. The study was conducted using TA98, TA100, TA1535 and TA1537 strains of Salmonella typhimurium and WP2uvrA (pKM101) strain of Escherichia coli in two phases. In the first phase, an initial toxicity-mutation test was performed. The second phase was an independent confirmatory mutation test. The bacterial tester strains were exposed to the test substance in the presence and absence of a metabolic activation system (S-9 fraction prepared from Aroclor 1254 induced rat liver) using a preincubation procedure. XU-18838.00 was found to be soluble in Dimethyl formamide (DMF) at 50 mg/mL. In the initial toxicity-mutation assay, XU-18838.00 was exposed in duplicate at 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate test doses along with the vehicle and appropriate positive controls. The mean and standard deviation of revertant colonies were calculated for each test dose and the controls for all the tester strains. There was a moderate precipitation of the test substance on the basal agar plates at the top dose of 5000 µg/plate. No toxicity was observed up to 1500 µg/plate, either in the presence or absence of metabolic activation as the intensity of the bacterial back-ground lawn was comparable to respective vehicle control plates. However, at the top dose of 5000 µg/plate, a slight thinning of the bacterial background lawn was noticed in the presence and absence of metabolic activation. There was no positive mutagenic response observed in any of the tester strains in any of the tested doses either in the presence or in the absence of metabolic activation.g/plate, either in the presence or absence of metabolic activation as the intensity of the bacterial back-ground lawn was comparable to respective vehicle control plates. However, at the top dose of 5000 µg/plate, a slight thinning of the bacterial background lawn was noticed in the presence and absence of metabolic activation. There was no positive mutagenic response observed in any of the tester strains in any of the tested doses either in the presence or in the absence of metabolic activation.

Based on these initial findings, in the confirmatory mutation assay, XU-18838.00 was exposed in triplicate at 100, 266, 707, 1880, and 5000 µg/plate test doses along with the vehicle and appropriate positive controls. The mean and standard deviation of revertant colonies were calculated for each test dose and the controls for all the tester strains. There was a moderate precipitation of the test substance on the basal agar plates at the top dose of 5000 µg/plate. No toxicity was observed up to 1880 µg/plate, either in the presence or absence of metabolic activation as the intensity of the bacterial back-ground lawn was comparable to respective vehicle control plates. However, at the top dose of 5000 µg/plate, a slight thinning of the bacterial background lawn was noticed in the presence and absence of metabolic activation. There was no positive mutagenic response observed in any of the strains in any of the tested doses either in the presence or in the absence of metabolic activation. In this study, there was a more than 3-fold increase in the mean numbers of revertant colonies in the positive controls, demonstrating the sensitivity of the assay.

All criteria for a valid study were met as described in the protocol. Under the conditions of the current study, the test substance, XU-18838.00 was negative (non-mutagenic) in this Salmonella-Escherichia coli/Mammalian-Microsome Reverse Mutation Assay.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD Guideline GLP Study
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Remarks:
The only exception was full GLP characterization of the test material was performed concurrently with the in-life phase of the study. Additionally, concentration checks and stability were not performed for the control substances used in this study.
Qualifier:
according to
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Deviations:
no
Remarks:
same as above
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Remarks:
same as above
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
Remarks:
same as above
Principles of method if other than guideline:
not applicable
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
not applicable
Species / strain / cell type:
lymphocytes: Rat
Details on mammalian cell type (if applicable):
Blood samples were collected by cardiac puncture, following euthanasia with carbon dioxide. In the assays, blood samples from individual rats were pooled and whole blood cultures were set up in RPMI 1640 medium (with 25 mM HEPES, GIBCO, Grand Island, New York) supplemented with 10% heat-inactivated fetal bovine serum (GIBCO), antibiotics and antimycotics (Fungizone 0.25 µg/ml; penicillin G, 100 u/ml; and streptomycin sulfate, 0.1 mg/ml; GIBCO), 30 µg/ml PHA-P (HA16, Remel Europe Ltd., Dartford, England), and an additional 2 mM L-glutamine (GIBCO). Cultures were initiated by inoculating approximately 0.5 ml of whole blood/5 ml of culture medium. Cultures were set up in duplicate at each dose level in T-25 plastic tissue culture flasks and incubated at 37°C.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
In the initial assay, cultures were treated with the test material in the absence and presence of S9 activation for 4 hours at concentrations of 0 (vehicle control), 2.3, 4.7, 9.4, 18.8, 37.5, 75.0, and 150 µg/ml. Cultures were also treated continuously for 24 hours in the absence of S9 with the above concentrations plus an additional lower concentration of 1.2 µg/ml.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle: recommended by various guidelines and regulatory agencies.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
The solvent ethanol selected for dissolving the test material was used as the negative control treatment.
True negative controls:
yes
Positive controls:
yes
Positive control substance:
other: Mitomycin C and cyclophosphamide monohydrate
Remarks:
Cyclophosphamide monohydrate (CP, Sigma, CAS No. 6055-19-2) was the positive control for the activation assay at final concentrations of 2 and 4 μg/ml.
Details on test system and experimental conditions:
Treatment Procedure without Metabolic Activation
Approximately Forty-eight hours (+/- 1 hour) after initiation of the cultures, the cell suspension was dispensed into 15 ml sterile disposable centrifuge tubes (5.5 ml/tube, two cultures per dose level). The cells were sedimented by centrifugation and the culture medium removed and saved. The cells were exposed to 5 ml of treatment medium (RPMI 1640, HEPES, and antibiotics) containing the test or positive or vehicle control treatments for 4 hours (+/- 30 minutes) at 37°C and the exposure was terminated by washing the cells with 5 ml of culture medium. The cells were then placed in individual sterile disposable tissue culture flasks (T-25) along with approximately 4.5 ml of the original culture medium until the time of harvest. The cultures were harvested 24 hours (+/- 1 hour) after treatment initiation (i.e., 20 hours, +/- 1 hour after treatment termination). Cultures were treated continuously with the test material for approximately 1.5X normal cell cycle length. The vehicle control, positive control, and test material were added directly to the culture flasks 48 hours (+/- 1 hour) after initiation of the cultures and the cultures were harvested 24 hours (+/- 1 hour) later.

Treatment Procedure using Metabolic Activation
Only the short treatment (4 hours +/- 30 minutes) procedure was used with S9. Approximately Forty-eight hours (+/- 1 hour) after initiation of the cultures, the cell suspension was dispensed into 15 ml sterile disposable centrifuge tubes (5.5 ml/tube, two cultures per dose level). The cells were sedimented by centrifugation and the culture medium removed and saved. The cells were exposed to 5 ml of treatment medium (RPMI 1640, HEPES, antibiotics, and the S9 mix) containing the test or positive or vehicle control treatments for 4 hours (+/- 30 minutes) at 37°C and the exposure was terminated by washing the cells twice with 5 ml of culture medium. The cells were then placed in individual sterile disposable tissue culture flasks (T-25) along with 4.5 ml of the original culture medium until the time of harvest. The cultures were harvested at 24 hours (+/- 1 hour) after treatment initiation (i.e., 20 hours, +/- 1 hour after treatment termination).
Evaluation criteria:
Colcemid (1 µg/culture) was added 2-3 hours prior to harvest. The cells were swollen by hypotonic treatment (0.075 M KCl), fixed with methanol:acetic acid (3:1), dropped on microscope slides, and stained in Giemsa. Mitotic indices were determined as the number of cells in metaphase among 1000 lymphocytes/replicate and expressed as percentages. Slides from the vehicle controls, positive controls, and three concentrations of the test material were selected for cytogenetic analysis. Initially, slides from the short treatment (with and without S9) were evaluated for cytogenetic analysis. Slides from the continuous treatment without S9 were evaluated only when the results with short treatment yielded negative findings.
One hundred metaphases/replicate were examined from coded slides for structural abnormalities. The number of cells examined for structural abnormalities was reduced to 50 metaphases per replicate when high numbers of aberrations were observed (approximately 20%). The microscopic coordinates of metaphases containing aberrations were recorded. Only those metaphases that contained 42 + 2 centromeres were scored with the exception of cells with multiple aberrations, in which case accurate counts of the centromeres would not be possible. Structural chromosomal abnormalities counted include chromatid and chromosome gaps, chromatid breaks and exchanges, chromosome breaks and exchanges, and miscellaneous (chromosomal disintegration, chromosomal pulverization, etc.). Those cells having five or more aberrations/cell were classified as a cell with multiple aberrations. Chromatid gaps and chromosome gaps were not included in calculations of total cytogenetic aberrations. In addition, a total of 100 metaphases/replicate were examined for incidence of polyploidy.
Statistics:
Standard statistical methods were employed.
Species / strain:
lymphocytes: rat
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Assay A1 -
In the initial assay, cultures were treated with the test material in the absence and presence of S9 activation for 4 hours at concentrations of 0 (vehicle control), 2.3, 4.7, 9.4, 18.8, 37.5, 75.0, and 150 µg/ml. Cultures were also treated continuously for 24 hours in the absence of S9 with the above concentrations plus an additional lower concentration of 1.2 µg/ml. The test material precipitated in the treatment medium at the top three concentrations (i.e., 37.5, 75.0, and 150 µg/ml) in all treatment conditions. Analytically detected concentrations of the test material in the stock solutions (Assay A1) varied from 96.1 to 103.0% of the target and verified that concentrations used for treatment were within an acceptable range.

Short Treatment:
In the absence of S9, the cultures displayed little to no toxicity with relative mitotic indices ranging from 81.9 to 121.3% compared to the vehicle control values. In the presence of S9, the mitotic indices of the treated cultures ranged from 101.7 to 139.0% as compared to the vehicle control values. Based upon these results, cultures treated with targeted concentrations of 0 (vehicle control), 2.3, 18.8, and 150 µg/ml were chosen for the determination of chromosomal aberration frequencies and incidence of polyploidy both in the absence and presence of S9 activation.

Among the cultures treated with the positive control chemicals for 4 hour, 0.5 µg/ml of MMC and 2 µg/ml of CP were selected for evaluation of aberrations in the absence and presence of S9, respectively. There were no significant increases in the incidence of polyploid cells in any of the test material treated cultures as compared to the vehicle control values.

In the 4 hour non-activation assay, the frequency of cells with aberrations in the vehicle control was 0.5% and the corresponding values at treatment levels of 2.3, 18.8, and 150 µg/ml were 0.5, 0.0, and 0.0%, respectively. In the activation assay, cultures treated with the test material at concentrations of 2.3, 18.8, and 150 µg/ml had aberrant cell frequencies of 0.5, 0.5, and 0.0%, respectively as compared to the vehicle control value of 1.0%. Statistical analyses of these data did not identify significant differences between the vehicle control and any of the treated cultures without or with S9 activation. The frequencies of aberrant cells observed in the test material treated cultures were within the laboratory historical background range.

Significant increases in the frequency of cells with aberrations were observed in cultures treated with the positive control chemicals. Aberrant cell frequencies in MMC (-S9, 4 hour treatment), and CP (+S9, 4 hour treatment) cultures were both 32.0%.

A second assay with treatment of cultures in the presence of S9 was not considered necessary since the results of the initial test yielded clearly negative results.

Continuous Treatment:
Based upon the negative findings in the 4-hour treatment in the absence of metabolic activation, slides from the continuous 24-hour treatment were evaluated. Cultures treated continuously for 24 hours in the absence of S9 activation had little to no toxicity with relative mitotic indices ranging from 88.4 to 127.9% relative to the vehicle control value. Based upon these results, cultures treated with targeted concentrations of 0 (vehicle control), 2.3, 18.8, and 150 µg/ml were chosen for the determination of chromosomal aberration frequencies and incidence of polyploidy. Cultures treated with 0.075 µg/ml MMC were selected to serve as the positive control.

There were no significant increases in the incidence of polyploid cells in any of the test material-treated cultures as compared to the vehicle control values.

The frequency of aberrant cells in the vehicle control was 0.5% and the corresponding values at concentration levels of 2.3, 18.8, and 150 µg/ml were 1.0, 1.0, and 1.0%, respectively. There were no statistically significant differences between the test material treated cultures and the vehicle control values and all values were within the laboratory historical background range.

A significant increase in the frequency of cells with aberrations was observed in cultures treated with the positive control chemical. Aberrant cell frequency in MMC treated cultures was 15.5%
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

None

Conclusions:
Interpretation of results (migrated information):
negative

It was concluded that under the experimental conditions used, ACCUTRACE™ S10 Fuel Marker was non-genotoxic in this in vitro chromosomal aberration test.
Executive summary:

ACCUTRACE™ S10 Fuel Marking System (1-(Decyloxy)-2-(1-methylpropyl)-4-(triphenylmethyl) benzene) was evaluated in an in vitro chromosomal aberration assay utilizing rat lymphocytes. Approximately 48 hours after the initiation of whole blood cultures, cells were treated either in the absence or presence of S9 activation with concentrations ranging from 0 (vehicle control) to 150 ug ACCUTRACE™ S10 Fuel Marking System per ml of culture medium. The highest concentration was based on the limit of solubility of the test material in the treatment medium. The duration of treatment was 4 or 24 hours without S9 and 4 hours with S9. The analytically determined concentrations of ACCUTRACE™ S10 Fuel Marking System in the dose preparations ranged from 96.1 to 103.0% of the targeted values. Selection of concentrations for the determination of the incidence of chromosomal aberrations was based upon precipitation of the test material and the mitotic indices. In this study cultures treated with targeted concentrations of 0 (vehicle control), 2.3, 18.8, and 150 ug/ml in the absence of S9 (4- and 24-hour treatment) and in the presence of S9 (4-hour treatment) were analyzed.

There were no significant increases in the frequency of cells with aberrations administered ACCUTRACE™ S10 Fuel Marking System in either the absence or presence of S9 activation. Cultures treated with the positive control chemicals (i.e., mitomycin C without S9 and cyclophosphamide with S9) had significantly higher incidences of aberrant cells in all assays. Based upon these results, ACCUTRACE™ S10 Fuel Marking System was considered to be non-genotoxic in this in vitro chromosomal aberration assay utilizing rat lymphocytes.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Bacterial mutagenicity data are not available for the registered substance, therefore read across of data from a structural analogue, ((3,4-bis(Hexyloxy)phenyl)methanetriyl) tribenzene, was used to address this data requirement. The justification for the use of read across is attached at section 13 of this dataset.

OECD GLP genotoxicity assays were negative on bacterial (analogue substance) and mammal cell gene mutation and on chromosome aberration in rat lymphocytes (registered substance). Hence, there is no concern for genotoxicity with the registered substance.


Justification for classification or non-classification

No genotoxicity detected in any of the assays.