Lecithins, acetylated

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In Vitro Mutagenic effects - Bacterial. OECD 471. Not mutagenic. Not classified as a mutagen. Reliability = 1.
In Vitro Clastogenic effects - Mammalian. OECD 473. Not clastogenic. Not classified as a clastogen. Reliability = 1.
In Vitro Mutagenic effects - Mammalian. OECD 476. Not mutagenic. Not classified as a mutagen. Reliability = 1.

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

other: EC Commission Directive 2000/32/EC Annex 4D–B.13/14 Number L 136

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium, other: TA98, TA100, TA1535, and TA1537

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

-For the toxicity-mutation test: 33.3, 66.7, 100, 333, 667, 1000, 3333, and 5000 µg/plate
-For the mutagenicity test: 333, 667, 1000, 3333, and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Hexane
- Justification for choice of solvent/vehicle: Based on the solubility of the test substance and compatibility with the target cells.

Negative solvent / vehicle controls:

yes

Remarks:

Hexane

Positive controls:

yes

Positive control substance:

other: benzo[a]pyrene (TA98 +S9); 4-nitroquinoline N-oxide (WP2uvrA -S9); acridine mutagen ICR-191 (TA1537 -S9); sodium azide (TA100 and TA1535 -S9); 2-aminoanthracene (TA100, TA1535, TA1537, and WP2uvrA +S9); 2-nitrofluorene (TA98 -S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: The plate incorporation method was applied. In the non-activated assays, 0.5 mL of sham mix and 100 µL of vehicle, test substance dilution, or positive control were added to pre-heated (45–48°C) glass culture tubes containing 2 mL of selective top agar, followed by 100 µL of tester strain. The test substance dilutions and controls were allowed to flash off in the pre-heated tubes for 10 minutes prior to the addition of the bacterial tester strains. In the S9-activated assays, 100 µL of the vehicle, test substance dilution, or positive control were added to pre-heated (45–48°C) glass culture tubes containing 2 mL of selective top agar, followed by 100 µL of tester strain and 0.5 mL of S9 mix. The test substance dilutions and controls were allowed to flash off in the pre-heated tubes for 10 minutes prior to the addition of the bacterial tester strains. All mixtures were vortexed and overlaid onto the surface of minimum glucose agar plates. After the overlay solidified, the plates were inverted and incubated for approximately 49-50.5 hours at 37 ± 2°C. Plates that were not evaluated immediately following incubation were stored at approximately 4°C. All toxicity-mutation test dose preparations of negative (vehicle) controls, test substance, and positive controls were plated in duplicate. All mutagenicity test dose preparations of negative (vehicle) controls, test substance, and positive controls were plated in triplicate.

DURATION
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: 2 trials with 3 treatments per concentration

SCORING
The appearance of the bacterial background lawn was assessed microscopically for test substance toxicity and precipitation. Toxicity was scored relative to the concurrent tester strain specific negative control, and evaluated as a decrease in the mean number of revertant bacterial colonies per plate. In addition, the thinning or disappearance of the bacterial background lawn was considered as signs of toxicity. Revertant colonies were counted with an automated colony counter. Plates that could not be accurately counted automatically were counted manually.

Evaluation criteria:

-Tester Strain Integrity: To demonstrate the presence of the rfa mutation, all S. typhimurium tester strain cultures must exhibit sensitivity to crystal violet. To demonstrate the presence of the uvrA and uvrB mutations, all tester strains cultures must exhibit sensitivity to ultraviolet light. To demonstrate the presence of the pKM101 plasmid, tester strain cultures of TA98 and TA100 must exhibit resistance to ampicillin.
-Tester Strain Culture Density: To ensure that appropriate numbers of bacteria are plated, all tester strain culture densities must be approximately 1E9 cells per millilitre.
-Negative Control Values : The tester strain cultures must exhibit a characteristic mean number of spontaneous revertants per plate when plated along with the negative (vehicle) control under selective conditions. The acceptable ranges for the mean values of negative controls are as follows:TA98(8-60), TA100(60-240), TA1535(4-45), TA1537(2-25), WP2uvrA( 5-60).
-Positive Control Values: Each mean positive control value must exhibit at least a 3.0-fold increase over the respective mean negative (vehicle) control value for each tester strain.
-Toxicity: A minimum of 3 non-toxic scorable dose levels were required to validate the study. A dose level was considered toxic if it caused: A >50% reduction in the mean number of revertants per plate relative to the mean negative control value and exhibited a dose-dependent drop in the revertant count, or a reduction in the background lawn. In the event that less than 3 non-toxic dose levels were achieved, the affected portion of the test was repeated with an appropriate change in dose levels.

Statistics:

For each tester strain, the mean of the number of revertants and the standard deviations were calculated.

Species / strain:

S. typhimurium, other: TA98, TA100, TA1535, TA1537

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

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

Additional information on results:

Toxicity- Mutation Test
In the toxicity-mutation test, the maximum dose evaluated was 5000 µg/plate for tester strains TA98, TA100, TA1535, TA1537, and WP2uvrA in the absence and presence of S9 metabolic activation. This dose was achieved using a concentration of 50 mg/mL and a 100 µL plating aliquot. The dose levels used in this test were 33.3, 66.7, 100, 333, 667, 1000, 3333, and 5000 µg/plate. The plate incorporation method was employed. No positive mutagenic responses were observed at any dose level in any tester strain in the absence or presence of S9 metabolic activation. No toxicity was observed at any dose level with any tester strain in either the absence or presence of S9. A >50% reduction in mean number of revertants was observed at 1000 and 66.7 µg/plate for TA1537 with S9 activation; however, this reduction occurred at intermediate dose levels with no dose related correlation. Test substance precipitation was observed starting at 3333 µg/plate in both the non-activated and activated testing system.
Mutagenicity Test
Based on the toxicity-mutation test, the maximum dose evaluated in the mutagenicity test was 5000 µg/plate for tester strains TA98, TA100, TA1535, TA1537, and WP2uvrA in the absence and presence of S9 metabolic activation. This dose was achieved using a concentration of 50 mg/mL and a 100 µL plating aliquot. The dose levels used in this test were 333, 667, 1000, 3333, and 5000 µg/plate for all tester strains. The plate incorporation method was employed. No positive mutagenic responses were observed at any dose level or with any tester strain in either the absence or presence of S9 metabolic activation. No toxicity was observed at any dose level with any tester strain in either the absence or presence of S9. Test substance precipitation was observed starting at 1000 µg/plate in the non-activated testing system in 2/3 plates with tester strain TA100 and 1/3 plates with tester strain TA1537. For all remaining in all tester strains both with and without S9 activation the test substance precipitation was observed starting at 3333 µg/plate.

Conclusions:

All criteria for a valid study were met. Under the conditions of this study, the test substance showed no evidence of mutagenicity in the Bacterial Reverse Mutation Test either in the absence or presence of Aroclor-induced rat liver S9. It was concluded that the test substance was negative in this in vitro test.

Executive summary:

The test substance, was evaluated for mutagenicity in the Bacterial Reverse Mutation Test using the plate incorporation method.  Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and Escherichia coli strain WP2uvrA were tested in the absence and presence of an exogenous metabolic activation system (Aroclor-induced rat liver S9). The test was performed in 2 phases. The first phase was the toxicity-mutation test, which established the dose range for the mutagenicity test, and provided a preliminary mutagenicity evaluation. The second phase was the mutagenicity test, which evaluated and confirmed the mutagenic potential of the test substance. Hexane was chosen as the dosing vehicle based on the solubility of the test substance and compatibility with the target cells. The test substance formed a hazy amber solution in hexane at 50 mg/mL, the highest stock concentration that was prepared for use on this study. In the toxicity-mutation test, the maximum dose evaluated was 5000 µg/plate for tester strains TA98, TA100, TA1535, TA1537, and WP2uvrA in the absence and presence of S9 metabolic activation. This dose was achieved using a concentration of 50 mg/mL and a 100 µL plating aliquot. The dose levels used in this test were 33.3, 66.7, 100, 333, 667, 1000, 3333, and 5000 µg/plate. The plate incorporation method was employed. No positive mutagenic responses were observed at any dose level in any tester strain in the absence or presence of S9 metabolic activation. No toxicity was observed at any dose level with any tester strain in either the absence or presence of S9. Test substance precipitation was observed starting at 3333 µg/plate in both the non-activated and activated testing system. Based on the toxicity-mutation test, the maximum dose evaluated in the mutagenicity test was 5000 µg/plate for tester strains TA98, TA100, TA1535, TA1537, and WP2uvrA in the absence and presence of S9 metabolic activation. This dose was achieved using a concentration of 50 mg/mL and a 100 µL plating aliquot. The dose levels used in this test were 333, 667, 1000, 3333, and 5000 µg/plate for all tester strains. The plate incorporation method was employed. No positive mutagenic responses were observed at any dose level or with any tester strain in either the absence or presence of S9 metabolic activation. No toxicity was observed at any dose level with any tester strain in either the absence or presence of S9. Test substance precipitation was observed starting at 1000 and 3333 µg/plate. All criteria for a valid study were met. Under the conditions of this study, the test substance showed no evidence of mutagenicity in the Bacterial Reverse Mutation Test either in the absence or presence of Aroclor-induced rat liver S9. It was concluded that the test substance was negative in this in vitro test.

Reference 2

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:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Qualifier:

according to guideline

Guideline:

other: EC Commission Directive 2000/32/EC Annex 4A-B10 Number L 136 (2000)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: McCoy's 5A medium supplemented with 10% foetal bovine serum (FBS), 2 mM L glutamine, 100 units penicillin/mL and 100 µg streptomycin/mL
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes; This test system has been demonstrated to be sensitive to the clastogenic activity of a variety of chemicals.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver (S9)

Test concentrations with justification for top dose:

100, 250, 500, 1000, 2500 and 5000 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: hexane
- Justification for choice of solvent/vehicle: Hexane was determined to be the solvent of choice based on the solubility of the test substance and compatibility with the target cells. The test substance formed a cloudy brown solution in the vehicle at the highest stock concentration prepared, 500 mg/mL. Precipitation was observed in the treatment media at every concentration tested in all testing conditions in both the preliminary and chromosome aberration assays.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: mitomycin C (MMC), cyclophosphamide (CP)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium - McCoy's 5A medium supplemented with 10% foetal bovine serum (FBS), 2 mM L glutamine, 100 units penicillin/mL and 100 µg streptomycin/mL

DURATION
- Preincubation period: 16-24 hours
- Exposure duration: 4 hours in the absence and presence of S9 metabolic activation, and for 20 hours in the absence of metabolic activation
- Expression time (cells in growth medium): After completion of the 4 hour exposure periods only, the cells were collected by centrifugation, washed once with phosphate buffered saline, fed with complete medium, and incubated until cell harvest. The incubations were conducted at 37 ± 2°C in a humidified atmosphere of 5 ± 2% CO2 in air.
- Fixation time (start of exposure up to fixation or harvest of cells): no data
Exponentially growing CHO K1 cells were seeded in labelled, sterile flasks. Approximately 5E5 cells/25 cm² flask were inoculated in complete medium. Cultures were incubated at 37 ± 2°C in a humidified atmosphere of 5 ± 2% CO2 in air

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 5E5 cells/25 cm²

DETERMINATION OF CYTOTOXICITY
- Method: cells were harvested by trypsinization and counted by an automatic cell counter; cell viability data were obtained, but not reported; cell counts were used to determine cell growth inhibition relative to the solvent control

OTHER EXAMINATIONS:
- Other: The osmolality and pH of the vehicle control, as well as the highest soluble test substance concentration in the culture media, were determined.

Evaluation criteria:

The following conditions were used as a guide to determine a positive response: A statistically significant increase (p <0.05, Fisher’s exact test) in the percentage of cells with structural aberrations was seen in one or more treatment groups relative to the vehicle control response; the observed increase frequencies were accompanied by a concentration-related increase; a statistically significant increase was observed at the highest dose only (NOTE: Statistically significant values that did not exceed the historical control range for the negative/vehicle control may be judged as not being biologically significant.

The following condition was used as a guide to determine an equivocal response: Results observed in any of the assays resulted in statistically significant elevations in structural chromosome aberrations at more than one test concentration level, except the highest dose, without demonstrating a dose-responsive trend.

The test substance was judged negative if the following condition was met: There was no statistically significant increase in the percentage of cells with structural aberrations in any treatment group relative to the vehicle control group.

Statistics:

The clastogenic potential of the test substance was assessed based on its ability to induce structural chromosome aberrations. The experimental unit is the cell; therefore the percentage of cells with structural aberrations was used for the assessment. Data were evaluated using scientific judgment. Statistical analysis was used as a guide to determine whether or not the test substance induced a positive response. Interpretation of the statistical analysis also relied on additional considerations including the magnitude of the observed test substance response relative to the vehicle control response and the presence of a dose responsive trend. Statistical analysis consisted of a Fisher’s exact test to compare the percentage of cells with structural or numerical aberrations (or the percentage of cells with more than one aberration, if required) in the test substance treated groups with the vehicle control response. A Cochran-Armitage test for dose responsiveness was conducted only on values that were statistically significant based on the Fisher’s exact test. Statistical analyses was conducted on the percentage of cells with numerical aberrations as well. The following conditions were used as a guide to determine a positive response: 1) a statistically significant increase (p < 0.05, Fisher’s exact test) in the percentage of cells with structural aberrations was seen in one or more treatment groups relative to the vehicle control response, 2) the observed increased frequencies were accompanied by a concentration-related increase, 3) a statistically significant increase was observed at the highest dose only.

Note: Statistically significant values that did not exceed the historical control range for the negative/vehicle control may be judged as not being biologically significant.

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:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation was observed in the treatment media at every concentration tested in all testing conditions in both the preliminary and chromosome aberration assays.
- Other confounding effects: The test substance formed a cloudy brown solution in the vehicle at the highest stock concentration prepared, 500 mg/mL.

RANGE-FINDING/SCREENING STUDIES: Concentrations for the chromosome aberration assay were selected based on the results from a preliminary toxicity test assessing the cell growth inhibition relative to the vehicle control. The cultures were microscopically inspected for the extent of monolayer confluency relative to the vehicle control. In the preliminary toxicity assay CHO cells were exposed to a total of 9 concentrations of the test substance ranging from 10 to 5000 µg/mL as well as a vehicle control and an untreated solvent control. The cells were exposed for 4 hours in both the absence and presence of an exogenous metabolic activation system (Aroclor-induced S9), or for 20 hours in the absence of S9 activation. Precipitation was observed in the treatment medium at every concentration tested. Precipitation ranged from a thin film at lower concentrations to large aggregations at higher concentrations. Osmolality and pH measurements were taken from the highest test substance concentration (5000 µg/mL), a middle test substance concentration (500 µg/mL) and the vehicle control media. Based on visual inspection of the pH-sensitive treatment medium at the beginning and end of the treatment periods, the pH of the test substance concentrations in media were similar to the pH of the vehicle controls and the pH did not change during the treatment period. In the non-activated test system, the measured pH for 5000 and 500 µg/mL in media was 7.55 and 7.63 respectively, compared to 7.59 for the vehicle control. In the S9-activated test system, the measured pH for 5000 and 500 µg/mL in media was 7.21 and 7.27 respectively, compared to 7.29 for the vehicle control. The osmolality of the highest test substance concentration, 5000 µg/mL, tested in treatment media was 294 and 308 mmol/kg in the non-activated and S9-activated test conditions, respectively. The osmolality of the 500 µg/mL test substance concentration in treatment media was 291 and 308 mmol/kg in the non-activated and S9-activated test conditions, respectively. The osmolality of the vehicle in the treatment medium was 294 and 307 mmol/kg in the non-activated and S9-activated test condition, respectively. The observed changes in osmolality were ≤ 20% and were not considered significant.

Substantial toxicity (greater than a 50% reduction in cell growth relative to the vehicle control) was observed in several cultures in the 4 hour non-activated test condition. Although there were test substance treated cultures with substantial toxicity, it was determined through comparison with the confluency assessment that the toxicity was variable between cultures and did not appear dose-related. Based on the findings from the preliminary toxicity assay, the highest concentrations chosen for the chromosome aberration assay was 5000 µg/mL for each test condition.

The highest concentration selected for microscopic analysis was based on the limit dose for the assay, 5000 µg/mL.

  

At the highest test concentration evaluated microscopically for chromosome aberrations, 5000 µg/mL, a 7% growth inhibition and a 38% mitotic inhibition in relation to the vehicle control was observed. The percentage of cells with structural or numerical aberrations in the test substance-treated group was not significantly increased above that of the vehicle control at any concentration (p ≥ 0.05, Fisher's exact test). The percentage of cells with structurally damaged chromosomes in the MMC (positive control) treatment group (14.5%) was statistically significant (p < 0.05, Fisher's exact test).

  

In S9-activated cells, at the highest test concentration evaluated microscopically for chromosome aberrations, 5000 µg/mL, a 16% growth increase and an 11% mitotic increase in relation to the vehicle control was observed. The percentage of cells with structural aberrations in the test substance-treated group was not significantly increased above that of the vehicle control at any concentration (p ≥ 0.05, Fisher's exact test). The percentage of cells with numerical aberrations in the test substance-treated groups was increased above that of the vehicle control group at 1000, and 5000 µg/mL (10.5% and 12.5% respectively) (p < 0.05, Fisher's exact test) in which the main proportion of numerical aberrations was endoreduplication. The percentage of cells with structurally damaged chromosomes in the CP (positive control) treatment group (26.0%) was statistically significant (p < 0.05, Fisher's exact test).

  

In non-S9-activated cells, at the highest test concentration evaluated microscopically for chromosome aberrations, 5000 µg/mL, a 26% mitotic increase in relation to the vehicle control was observed. The cell growth index could not be observed due to interfering precipitate. The percentage of cells with numerical aberrations in the test substance-treated groups was not increased above that of the vehicle control group at any concentration (p ≥ 0.05, Fisher's exact test). The percentage of cells with structural aberrations in the test substance-treated groups significantly increased above that of the vehicle control group at 5000 µg/mL (p < 0.05, Fisher's exact test) in accordance with a dose responsive trend (p < 0.05, Cochran-Armitage trend test). However, this finding was not considered biologically relevant because the percentage of cells with structural aberrations was within the laboratory negative control range. The percentage of cells with structurally damaged chromosomes in the MMC (positive control) treatment group (14.5%) was statistically significant (p < 0.05, Fisher's exact test).

Conclusions:

Interpretation of results: Negative

All criteria for a valid study were met. Under the conditions of this study, the test substance was not found to induce structural chromosome aberrations in the in vitro mammalian chromosome aberration test in Chinese hamster ovary cells in either the non-activated or S9-activated test systems. Based on this it was concluded that the test substance was negative in this in vitro test. It should be noted that the test substance was found to induce numerical chromosome aberrations in the S9-activated test system only. However, this assay was not designed to evaluate this endpoint. therefore, the assay was negative.

Executive summary:

The test substance was evaluated for its ability to induce chromosome aberrations in vitro in Chinese hamster ovary (CHO) cells in both the absence and presence of an exogenous S9 metabolic activation system (Aroclor-induced rat liver S9). Numerical aberrations were also recorded. The study was conducted according to OECD guideline 473. The concentrations chosen for the chromosome aberration assay were 100, 250, 500, 1000, 2500 and 5000 µg/mL for each test condition.

   

The percentage of cells with structural aberrations was not increased above that of the vehicle control at any concentration tested in the 4 hour S9-activated and non-activated test condition. A statistically significant increase in the percentage of cells with structural aberrations was observed in the 20 hour non-activated test condition in accordance with a dose responsive trend. However, this finding was not considered biologically relevant because the percentage of cells with structural aberrations was within the laboratory negative control range.The test substance induced a statistically significant increase in numerical aberrations (with endoreduplication) in the S9-activated test systemin accordance with a dose responsive trend. This response was outside the negative historical control range with the highest response occurring in the highest dose tested, 5000 µg/mL.

   

Under the conditions of this study, the test substance was not found to induce structural chromosome aberrations in the in vitro mammalian chromosome aberration test in Chinese hamster ovary cells in either the non-activated or S9-activated test systems. Based on this it was concluded that the test substance was negative in this in vitro test. It should be noted that the test substance was found to induce numerical chromosome aberrations in the S9-activated test system only. However, this assay was not designed to evaluate this endpoint. Therefore, the assay was negative.

Reference 3

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

according to guideline

Guideline:

other: ICH S2A document April 24, 1996

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Qualifier:

according to guideline

Guideline:

other: EC Commission Directive 2000/32/EC, Annex 4E No. L136

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

Preliminary Toxicity Assay: 0.5, 1.5, 5, 15, 50, 150, 500, 1500, 5000 µg/mL
Initial Mutagenesis Assay (cloning): 50, 100, 125, 250, 500 µg/mL (without activation); 50, 100, 125, 250, 500 µg/mL (with activation)
Extended Treatment Assay (cloning): 25, 50, 100, 125, 250 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Hexane
- Justification for choice of solvent/vehicle: Hexane was selected by the Sponsor as the solvent for the test substance. The test substance was soluble in hexane at 1000 mg/mL the maximum concentration prepared for the preliminary toxicity assay.

Negative solvent / vehicle controls:

yes

Remarks:

Hexane

Positive controls:

yes

Positive control substance:

other: Methyl methanesulfonate (MMS) and 7,12-dimethyl-benz(a)anthracene (DMBA)

Details on test system and experimental conditions:

METHOD OF APPLICATION: Treatment was carried out in conical tubes containing cells, F0P medium or S9 activation mixture, and test or control substance. Treatment tubes were gassed with 5±1% CO2 in air, capped tightly, and incubated with mechanical mixing for 4 (-S9, +S9) or 24 (-S9) hours at 37±1°C. The preparation and addition of test substance dosing solutions were carried out under amber lighting and the cells were incubated in the dark during the exposure period. After the treatment period, the cells were washed twice with F0P or F0P supplemented with horse serum, L-glutamine, penicillin, and streptomycin (F10P). After the second wash, the cells were resuspended in F10P, gassed with 5±1% CO2 in air, and placed on the droller drum apparatus at 37±1°C.

DURATION
- Exposure duration: 4 or 24 hours
- Expression time (cells in growth medium): 2 days for 4-hour exposure; 3 days for 24-hour exposure
- Selection time (if incubation with a selection agent): 10-14 days

SELECTION AGENT (mutation assays): Trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 3E5 cells/mL (at end of treatment)

DETERMINATION OF CYTOTOXICITY
- Method: Exposure in the absence and presence of S9 activation for 4 hours, and without activation for 24 hours. For the 4-hour exposure, the cell population density was determined 24 and 48 hours after the exposure; the cultures were adjusted to 3E5 cells/mL after 24 hours only. For the 24-hour exposure, cell population density was determined 24, 48, and 72 hour after exposure. The cell population was adjusted to 3E5 cells/mL immediately after test substance removal and 24 hours after test substance removal. Cultures with <3E5 cells/mL were not adjusted. Toxicity was measured as suspension growth of the treated cultures relative to the growth of the solvent control cultures after 48 hours.

OTHER: Scoring Procedures: After the incubation, the VC (viable count) plates were counted for the total number of colonies per plate and the total relative growth determined. The TFT-resistant colonies were then counted for each culture with ≥20% total relative growth (including at least one concentration with ≥10% but ≤20% total growth). The diameters of the TFT-resistant colonies for the positive and solvent controls and, in the case of a positive response, the test-substance-treated cultures were determined over a range of approximately 0.2 to 1.1 mm.

Evaluation criteria:

The following criteria must be met for the mutagenesis assay to be considered valid:
Negative Controls
The average spontaneous mutant frequency of the solvent (or vehicle) control cultures must be within 35 to 140 TFT-resistant mutants per 10E6 surviving cells. Low spontaneous mutant frequencies, i.e., 20 to 34 mutants per 10E6 surviving cells, are considered acceptable if small colony recovery is demonstrated . The average cloning efficiency of the solvent (or vehicle) controls must be between 65% and 120% and the total suspension growth between 8-32 for the 4-hour exposure and 20-180 for the 24-hour exposure.
Positive Controls
The mutant frequency for at least one dose of the positive controls must meet the criteria for a positive response and induce a increase in small colony mutants according to the following criteria: Induced Mutant Frequency (IMF) positive control ≥ 300E-6 mutants with 40% small colonies or small colony IMF for positive control ≥ 150E-6 .
Test Article-Treated Cultures
Cultures treated with a minimum of four concentrations of test substance must be attained and their mutant frequencies reported. The highest test substance concentration must produce 80% to 90% toxicity unless limited by solubility or the maximum required concentration. In the case of a test substance with a steep toxicity curve (no concentrations with 10-20% survival), the results may be considered acceptable if a concentration spacing of ≤ 2-fold is used and the highest concentration tested showed <20% survival or total kill . For example, the test is considered acceptable if the highest concentration cloned for mutant selection exhibits >20% survival and the next highest concentration, which is ≤ 2 times the cloned concentration, is too toxic to clone.

Statistics:

The cytotoxic effects of each treatment condition were expressed relative to the solvent-treated control for suspension growth over 2 days post-treatment and for total growth (suspension growth corrected for plating efficiency at the time of selection). The mutant frequency (number of mutants per 10E6 surviving cells) for each treatment condition was determined by dividing the average number of colonies in the three TFT (trifluorothymidine) plates by the average number of colonies in the three corresponding VC plates and multiplying by the dilution factor (2E-4) then multiplying by 10E6. For simplicity, this is described as (Average # TFT colonies/average # VC colonies)x200.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

-Preliminary Toxicity Assay
The maximum concentration tested in the preliminary toxicity assay was 5000 µg/mL. Visible precipitate was present at concentrations ≥ 500 µg/mL in treatment medium at the beginning and end of the 4-hour exposure. Visible precipitate was present at concentrations ≥ 500 µg/mL at the beginning of treatment and ≥ 150 µg/mL at the end of the 24-hour exposure. The osmolality of the solvent control was 309 mmol/kg and the osmolality of the highest soluble concentration, 150 µg/mL at the beginning of treatment, was 292 mmol/kg. Suspension growth relative to the solvent controls at 5000 µg/mL was 72% without activation and 0% with S9 activation with a 4-hour exposure. Suspension growth relative to the solvent controls at 1500 and 5000 µg/mL was 0% without activation a with a 24-hour exposure. Based on the results of the toxicity test, the concentrations tested in the mutagenesis assay ranged from 5.0 to 500 µg/mL for non-activated and S9-activated cultures with a 4-hour exposure and from 2.5 to 250 µg/mL for the non-activated cultures with a 24-hour exposure.
-Mutagenesis Assays
Visible precipitate was present at 500 µg/mL in treatment medium at the end of treatment. In the non-activated system, cultures treated with concentrations of 50, 100, 125, 250, and 500 µg/mL were cloned and produced a range in suspension growth from 90% to 127%. In the S9-activated system, cultures treated with the same concentrations were cloned and produced a range in suspension growth from 70% to 100%. No cloned cultures exhibited mutant frequencies ≥ 90 mutants per 1E6 clonable cells over that of the solvent control. No concentration-related increase in mutant frequency was observed. The total growth ranged from 88% to 112% for the non-activated cultures at concentrations from 50 to 500 µg/mL and 67% to 129% for the S9-activated cultures at concentrations from 50 to 500 µg/mL. The results of the initial assay were negative in the absence and presence of S9 activation. Because no unique metabolic requirements were known about the test substance, only an extended treatment assay in the absence of S9 for a 24-hour exposure period was performed.
-The Extended Treatment Assay
Visible precipitate was present at 250 µg/mL in treatment medium at the end of treatment. Cultures treated with concentrations of 25, 50, 100, 125, and 250 µg/mL were cloned and produced a range in suspension growth from 75% to 93%. No cloned cultures exhibited mutant frequencies ≥ 90 mutants per 1E6 clonable cells over that of the solvent control. No concentration-related increase in mutant frequency was observed. The total growth ranged from 64% to 82% for non-activated cultures with a 24-hour exposure at concentrations from 25 to 250 µg/mL. The trifluorothymidine-resistant colonies for the positive and solvent control cultures from both assays were sized according to diameter over a range from approximately 0.2 to 1.1 mm. The colony sizing for the MMS positive controls yielded the expected increase in small colonies (verifying the adequacy of the methods used to detect small colony mutants) and large colonies

Conclusions:

Interpretation of results (migrated information):
negative

Under the conditions of this study, the test substance was concluded to be negative in the absence and presence of S9 metabolic activation in the L5178Y/TK+/- Mouse Lymphoma Mutagenesis Assay. The assay was negative.

The study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability, repeatability).

Executive summary:

The test substance was tested in the L5178Y/TK^+/-Mouse Lymphoma Mutagenesis Assay in the absence and presence of Aroclor-induced rat liver S9. The preliminary toxicity assay was used to establish the concentration range for the mutagenesis assay. The mutagenesis assay was used to evaluate the mutagenic potential of the test substance. Hexane was selected by the Sponsor as the solvent for the test substance. The test substance was soluble in hexane at 1000 mg/mL the maximum concentration prepared for the preliminary toxicity assay. In the preliminary toxicity assay, the maximum concentration of the test substance in treatment medium was 5000 µg/mL. Visible precipitate was present at concentrations ≥ 500 µg/mL in treatment medium at the end of the 4-hour exposure and ≥ 150 µg/mL at the end of the 24-hour exposure. Selection of concentrations for the mutation assay was based on reduction of suspension growth relative to the solvent control and precipitation profile. Substantial toxicity, i.e., suspension growth of 50% of the solvent control, was observed at concentrations ≥ 1500 µg/mL with S9 activation with a 4-hour exposure and without activation with a 24-hour exposure. Based on the results of the preliminary toxicity assay, the concentrations tested in the initial mutagenesis assay ranged from 5.0 to 500 µg/mL for both the non-activated and S9-activated cultures with a 4-hour exposure. Visible precipitate was present at 500 µg/mL in treatment medium at the end of treatment. The concentrations chosen for cloning were 50, 100, 125, 250, and 500 µg/mL with and without S9 activation. Untreated cultures, with and without S9 activation, were tested concurrently. No cloned cultures exhibited mutant frequencies ≥ 90 mutants per 1E6 clonable cells over that of the solvent control. There was no concentration-related increase in mutant frequency. Based on the results of the preliminary toxicity and initial mutation assays, the concentrations tested in the extended treatment assay ranged from 2.5 to 250 µg/mL for non-activated cultures with a 24-hour exposure. Visible precipitate was present at 250 µg/mL in treatment medium at the end of treatment. Untreated cultures, with and without S9 activation, were tested concurrently. The concentrations chosen for cloning were 25, 50, 100, 125, and 250 µg/mL. No cloned cultures exhibited mutant frequencies ≥ 90 mutants per 1E6 clonable cells over that of the solvent control. There was no concentration-related increase in mutant frequency. The trifluorothymidine-resistant colonies for the positive and solvent control cultures from both assays were sized according to diameter over a range from approximately 0.2 to 1.1 mm. The colony sizing for the MMS positive controls yielded the expected increase in small colonies (verifying the adequacy of the methods used to detect small colony mutants) and large colonies. All criteria for a valid test were met. Under the conditions of this study, the test substance was concluded to be negative in the presence and absence of S9 metabolic activation in the L5178Y/TK^+/-Mouse Lymphoma Mutagenesis Assay. The assay was negative.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In Vivo Mammalian Bone Marrow Chromosome Aberration Test. OECD Guideline 475. Negative. Reliability = 1.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

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:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5385 (In Vivo Mammalian Cytogenetics Tests: Bone Marrow Chromosomal Analysis)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Qualifier:

according to guideline

Guideline:

other: ICH S2A (1995)

Deviations:

no

Remarks:

The study was conducted according to the guidelines in effect at the time of study conduct

GLP compliance:

yes

Type of assay:

other: Rat bone marrow chromosome aberration/micronucleus test

Species:

rat

Strain:

other: Sprague-Dawley SD® (Hsd:SD®)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: approximately59 days old
- Weight at main study start: Males weighed 228-275 g and females weighed 164-195 g
- Assigned to test groups randomly: Yes
- Fasting period before study: No
- Housing: Socially housed 2 or 3 animals of the same sex within a group in stainless steel perforated floor cages
- Diet (e.g. ad libitum): ad libitum except during designated procedures
- Water (e.g. ad libitum): ad libitum except during designated procedures
- Acclimation period: At least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25°C
- Humidity (%): 30-70%
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): 12 h light and 12 h dark

Route of administration:

oral: gavage

Vehicle:

Vehicle: Corn Oil NF

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

The dose formulations were prepared up to one day prior to dosing. The required amount of test substance was weighed using a positive displacement pipette and transferred to an appropriate 50 mL plastic tube. Approximately 20 mL of the vehicle was added and the formulation was vortexed until mixed. A measuring cylinder was pre-rinsed with the vehicle and the content of the plastic tube was transferred into the cylinder. The plastic tube was rinsed with a small amount of vehicle. The cylinder was inverted to mix then the contents sonicated 5-10 minutes to de-foam the suspension. The formulation was then brought to final volume with corn oil and inverted to mix. The dose formulations were maintained on stir plates throughout the dosing procedure.

The positive control solution was freshly prepared on the day of use.

Duration of treatment / exposure:

Animals were given a single oral dose via intragastric gavage.

Frequency of treatment:

Once

Post exposure period:

None

Remarks:

Doses / Concentrations:
0, 500, 1000, 2000 mg/kg
Basis:

No. of animals per sex per dose:

Vehicle: 5/sex (sampling time 18 hours); 5/sex (sampling time 42 hours)
500 mg/kg: 5/sex (sampling time 18 hours)
1000 mg/kg: 5/sex (sampling time 18 hours)
2000 mg/kg: 5/sex (sampling time 18 hours); 5/sex (sampling time 42 hours)

Positive control: 3/sex (sampling time 18 hours)

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide was given via gavage at a dose of 20 mg/kg.

Details of tissue and slide preparation:

Bone marrow prep for micronucleus test: After centrifugation, each resulting cell pellet was resuspended in 2 mL of filtered foetal bovine serum and re centrifuged. The cell pellet was resuspended in a small volume of foetal bovine serum to facilitate smearing in the conventional manner on glass microscope slides. Several smears were prepared from each animal and fixed in methanol for at least 10 minutes.

Bone marrow prep for chromosome aberration test: After centrifugation, each resulting cell pellet was resuspended in 10 mL aqueous 0.075M potassium chloride (hypotonic solution) and incubated for approximately 12 minutes at ca. 37°C, before addition of 2 mL of fixative (3 vol methanol:1 vol acetic acid) with mixing. Following centrifugation, the supernatant was discarded and the cells treated with 3 changes of neat fixative. After the third change of fixative, the cell pellet was collected by centrifugation and resuspended in fixative at an appropriate density for slide preparation. The fixed cells were dropped onto clean slides and air-dried before staining. At least two slides were prepared from each animal. Fixed cells not used for slide preparation were discarded after completion of the experimental phase of the study.

Slide staining for micronucleus test: The slides for examination were encoded to minimize potential operator bias and then mounted temporarily in aqueous Acridine Orange.

Slide staining for chromosome aberration test: The slides were washed with 3 changes of purified water (approximately 1 minute per wash) then stained with 10% (v/v) Giemsa for 15 minutes, rinsed in purified water then washed in running tap water for approximately 5 minutes, air-dried then mounted with coverslips using synthetic mountant.

Microscopic Examination - Micronucleus Test: The slides were randomized and encoded to minimize potential operator bias and then examined by fluorescence microscopy using a blue excitation filter and a yellow barrier filter. A total of 2000 immature erythrocytes per animal were examined for the presence of micronuclei. Usually only one smear was examined per animal, the remaining smears were held temporarily as reserves in case of technical problems with the first smear.

In addition, the proportion of immature erythrocytes was assessed by examination of a total of at least 1000 erythrocytes per animal. The incidence of any micronucleated mature erythrocytes observed during this assessment was recorded as a check for potential micronucleus like artifacts.

Microscopic Examination - Chromosome Aberration Test:

Mitotic Index (MI) - Slides were randomized then encoded to minimize potential operator bias. They were examined by light microscopy, and the mitotic index was determined by examination of at least 1000 cells per animal. The relative mitotic index (RMI) was calculated as a percentage ratio compared with the concurrent vehicle control group.

Detailed Examination for Chromosome Aberrations - Slides were examined by light microscopy, and (where practical) a total of 100 readable metaphases per animal was examined for the presence of chromosome aberrations using oil immersion optics.

Evaluation criteria:

Micronucleus Test - A positive response is normally indicated by a statistically significant dose related (where appropriate) increase in the incidence of micronucleated immature erythrocytes. A negative result is indicated when there is no dose related increase in the incidence of micronucleated immature erythrocytes and when individual and group mean values fall within (or close to) the historical control range. An equivocal response is obtained when the results do not meet the criteria specified for a positive or negative response.

Chromosome Aberration Test - Where statistical analysis is performed, a positive response is normally indicated by a statistically significant (dose related, if applicable) increase in the incidence of aberrant cells for the treatment group compared with the concurrent control group. A negative result is indicated where group mean incidences of aberrant metaphase cells for the group treated with the test substance are not significantly greater than incidences for the concurrent control group and where these values fall within or close to the historical control range. An equivocal response is obtained when the results do not meet the criteria specified for a positive or negative response.

Statistics:

See additional information below for statistical analyses.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No significant clinical signs of reaction to treatment or mortalities were observed for any of the treatment groups.

Conclusions:

Interpretation of results: Negative
Animals treated with the test substance did not show any significant increases in the proportion of micronucleated immature erythrocytes or aberrant metaphases at either sampling time. It is therefore concluded that the test substance did not show any evidence of genotoxicity in the combined rat bone marrow chromosome aberration/micronucleus test, when tested in accordance with regulatory guidelines.

Executive summary:

The purpose of this study was to evaluate the genotoxicity of the test substance using the combined rat bone marrow chromosome aberration/micronucleus test. Young adult rats were treated with a single administration of the control article (corn oil), positive control (cyclophosphamide) or the test substance (500, 1000, or 2000 mg/kg), orally by intragastric gavage. No significant clinical signs of reaction to treatment or mortalities were observed for any of the groups.

 

Animals were euthanized 18 or 42 hours after treatment (3 hours after colchicine administration) and bone marrow from the femurs was collected. For the micronucleus test, bone marrow smears prepared (from the right femur) were fixed, temporarily stained with Acridine orange, and examined under code using fluorescence microscopy. A total of 2000 immature erythrocytes per animal were examined for the presence of micronuclei indicative of chromosome damage. In addition, the proportion of immature erythrocytes was assessed for each animal as a measure of potential bone marrow toxicity. For the chromosome aberration test, bone marrow cells from the left femur were treated with hypotonic solution then fixed, dropped onto clean slides, air-dried, stained with Giemsa and examined under codeusing light microscopy. The mitotic index was determined by examination of at least 1000 cells per animal. The relative mitotic index was calculated as a percentage ratio compared with the concurrent vehicle control group. A total of 100 readable metaphases per animal were examined for the presence of structural and numerical chromosome aberrations.

 

Animals treated with the test substance did not show any statistically significant increases in the incidence of micronucleated immature erythrocytes or any significant decreases in the proportion of immature erythrocytes. In addition, the incidences of micronucleated immature erythrocytes all fell within the laboratory historical control range. Animals treated with the test substance did not show any statistically significant increases in the proportion of aberrant metaphases at any experimental point. In addition, the proportion of aberrant metaphases for vehicle and test article groups was within or close to the laboratory historical control range. The positive control agent caused substantial increases in the proportions of micronucleated immature erythrocytes and aberrant metaphases for each individual animal confirming the sensitivity of the test.

 

Animals treated with the test substance did not show any significant increases in the proportion of micronucleated immature erythrocytes or aberrant metaphases at either sampling time. It is therefore concluded that the test substance did not show any evidence of genotoxicity in the combined rat bone marrow chromosome aberration/micronucleus test, when tested in accordance with regulatory guidelines.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The test substance did not produce mutagenicity when evaluated in three in vitro assays. In the chromosome aberration study, an increase in numerical, but not structural, aberrations was observed at the 1000 and 5000 µg/mL levels, in the S9-activated test system only. This is not considered a positive finding in this assay. However, this finding was evaluated in an in vivo assay and found to be negative.

Justification for classification or non-classification

The substance did not produce mutagenicity when evaluated in cell culture or laboratory animals. The substance does not need to be classified for mutagenicity according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.