Trialuminium bismuth hexaoxide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Three key studies are available assessing the potential clastogenicity and mutagenicity of the substance. These in vitro tests include a reverse mutation assay (Ames test), a chromosome aberration assay and a mouse lymphoma assay all conducted to OECD guidelines and GLP.

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:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

other: Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix prepared from the livers of rats treated with phenobarbital and B-Naphtha flavone

Test concentrations with justification for top dose:

Experiment 1 (with and without S9 mix): 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment 2 (with and without S9 mix): 15, 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Sterile distilled water
- Justification for choice of solvent/vehicle: The test item was insoluble in sterile distilled water, dimethyl sulphoxide, dimethyl formamide and acetonitrile at 50 mg/mL, acetone at 100 mg/mL and tetrahydrofuran at 200 mg/mL in solubility checks performed in–house. The test item formed the best doseable suspension in sterile distilled water, therefore, this solvent was selected as the vehicle.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-Aminoanthracene (AA) for TA100, TA1535, TA1537 and WP2 uvrAin the presence of metabolic activation

Details on test system and experimental conditions:

Dosing:
The test item was accurately weighed and, on the day of each experiment, approximate half-log dilutions prepared in sterile distilled water by mixing on a vortex mixer and sonication for 10 minutes at 40 °C. No correction for purity was required. All formulations were used within four hours of preparation and were assumed to be stable for this period.

Experiment 1 (plate incorporation method):
An aliquot of the appropriate concentration of test item formulation, solvent vehicle or appropriate positive control was added together with an aliquot of the bacterial strain cultures and phosphate buffer to molten, trace amino-acid supplemented media. These were then mixed and overlayed onto a Vogel-Bonner agar plate. Negative (untreated) controls were also performed on the same day as the mutation test. Each concentration of the test item, appropriate positive, vehicle and negative controls, and each bacterial strain, were assayed using triplicate plates. The procedure was the same as described above with metabolic activation except that following the addition of the test item formulation and bacterial culture, an aliquot of S9-mix was added to the molten, trace amino-acid supplemented media instead of phosphate buffer.

All of the plates were incubated at 37 ± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).

Experiment 2 (pre-incubation method):
As the result of Experiment 1 was deemed negative, Experiment 2 was performed using the pre-incubation method in the presence and absence of metabolic activation. Six test item dose levels per bacterial strain were selected in the second mutation test in order to achieve both a minimum of four non-toxic dose levels and the potential toxic limit of the test item following the change in test methodology from plate incorporation to pre-incubation.

An aliquot of the appropriate bacterial strain culture, phosphate buffer and appropriate concentration of test item formulation, solvent vehicle or appropriate positive control were incubated at 37 ± 3 °C for 20 minutes (with shaking) prior to addition of molten, trace amino-acid supplemented media and subsequent plating onto Vogel-Bonner plates. Negative (untreated) controls were also performed on the same day as the mutation test employing the plate incorporation method. All testing for this experiment was performed in triplicate. The procedure was the same as described above with metabolic activation except that following the addition of the test item formulation and bacterial strain culture, S9-mix was added to the tube instead of phosphate buffer, prior to incubation at 37 ± 3 °C for 20 minutes (with shaking) and addition of molten, trace amino-acid supplemented media. All testing for this experiment was performed in triplicate.

All of the plates were incubated at 37 ± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). Manual counts were performed at 5000 μg/plate because of a test item film. Further manual counts were also required due to revertant colonies spreading slightly, thus distorting the actual plate count.

Rationale for test conditions:

Standard as per OECD guidelines

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested.
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS.
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response).

A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met. Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.

Statistics:

Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control. Values that the program concluded as statistically significant but were within the in-house historical profile were not reported.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

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

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

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

Species / strain:

E. coli WP2 uvr A

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:

Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile. The test item formulation was also shown to be sterile.

The vehicle (sterile distilled water) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method). Consequently, the same maximum dose level was used as the maximum dose in the second mutation test (pre-incubation method). Similarly, there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the second mutation test, although small reductions in colony frequency were noted, particularly for TA1535, at the upper dose levels.

A brown test item induced colouration was noted from 150 μg/plate (caused staining of the revertant colonies) with an opaque test item film observed at 5000 μg/plate. Neither of these observations prevented the scoring of revertant colonies.

There were no increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no biologically relevant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (pre-incubation method). A small, statistically significant increase in TA100 revertant colony frequency was observed in the absence of S9-mix at 500 μg/plate in the second mutation test, however this response was within the in-house historical vehicle/untreated control values for the strain and was, therefore considered of no biological relevance.

Conclusions:

The test substance was considered to be non-mutagenic under the conditions of the conducted reverse mutation assay.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

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

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Source:
The L5178Y TK+/- 3.7.2c mouse lymphoma cell line was obtained from Dr. J. Cole of the MRC Cell Mutation Unit at the University of Sussex, Brighton, UK. The cells were originally obtained from Dr. D. Clive of Burroughs Wellcome (USA) in October 1978 and were frozen in liquid nitrogen at that time.

Cell culture:
The stocks of cells are stored in liquid nitrogen at approximately -196 °C. Cells were routinely cultured in RPMI 1640 medium with Glutamax-1 and HEPES buffer supplemented with Penicillin, Streptomycin, Sodium pyruvate, Amphotericin B and 10% donor horse serum (giving R10 media) at 37 °C with 5% CO2 in air. The cells have a generation time of approximately 12 hours and were subcultured accordingly. RPMI 1640 with 20% donor horse serum (R20), 10% donor horse serum (R10), and without serum (R0), are used during the course of the study. Master stocks of cells were tested and found to be free of mycoplasma.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix prepared from the livers of male rats dosed with Phenobarbital and B-Naphtha flavone

Test concentrations with justification for top dose:

Preliminary toxicity test: 0, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 µg/mL
Main test 4 hours without metabolic activation: 0, 0.06, 0.13, 0.25, 0.5, 1, 2, 4 and 8 µg/mL
Main test 4 hours with metabolic activation: 0, 0.015, 0.03, 0.06, 0.13, 0.25, 0.5, 1 and 2 µg/mL
Main test 24 hours without metabolic activation: 0, 0.03, 0.06, 0.13, 0.25, 0.5, 1, 2 and 4 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test item was insoluble in culture medium at 20 mg/mL, acetone and tetrahydrofuran at 200 mg/mL but was suspendable in dimethyl sulphoxide (DMSO) at 200 mg/mL in solubility checks performed in-house.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

Preliminary toxicity test:
A preliminary toxicity test was performed on cell cultures at 5 x 10^5 cells/mL, using a 4 hour exposure period both with and without metabolic activation (S9), and at 1.5 x 10^5 cells/mL using a 24-hour exposure period without S9. The dose range used in the preliminary toxicity test was 0.25 to 64 μg/mL for all three of the exposure groups. These dose levels were selected to avoid the excessive precipitate that was observed in the solubility test. Following the exposure periods the cells were washed twice with R10, resuspended in R20 medium, counted and then serially diluted to 2 x 10^5 cells/mL, unless the mean cell count was less than 3 x 10^5 cells/mL in which case all the cells were maintained. The cultures were incubated at 37 °C with 5% CO2 in air and sub-cultured after 24 hours by counting and diluting to 2 x 10^5 cells/mL, unless the mean cell count was less than 3 x 10^5 cells/mL in which case all the cells were maintained. After a further 24 hours the cultures were counted and then discarded. The cell counts were then used to calculate Suspension Growth (SG) values. The SG values were then adjusted to account for immediate post exposure toxicity, and a comparison of each exposure SG value to the concurrent vehicle control performed to give a percentage Relative Suspension Growth (%RSG) value. Results from the preliminary toxicity test were used to set the test item dose levels for the mutagenicity test.

Mutagenicity test:
Several days before starting the experiment, an exponentially growing stock culture of cells was set up so as to provide an excess of cells on the morning of the experiment. The cells were counted and processed to give 1 x 10^6 cells/mL in 10 mL aliquots in R10 medium in sterile plastic universals for the 4-hour exposure groups in both the absence and presence of metabolic activation, and 0.3 x 10^6 cells/mL in 10 mL cultures were established in 25 cm2 tissue culture flasks for the 24-hour exposure group in the absence of metabolic activation. The exposures were performed in duplicate (A + B), both with and without metabolic activation (2% S9 final concentration) at eight dose levels of the test item, vehicle and positive controls. To each universal was added S9 mix if required, exposure dilutions, the positive controls, and sufficient R0 medium to bring the total volume to 20 mL (R10 was used for the 24-hour exposure group). The exposure vessels were incubated at 37 °C for 4 or 24 hours with continuous shaking using an orbital shaker within an incubated hood.

Measurements of survival, viability and mutant frequency:
At the end of the exposure periods, the cells were washed twice using R10 medium then resuspended in R20 medium at a cell density of 2 x 10^5 cells/mL. The cultures were incubated at 37 °C with 5% CO2 in air and subcultured every 24 hours for the expression period of two days, by counting and dilution to 2 x 10^5 cells/mL, unless the mean cell count was less than 3 x 10^5 cells/mL in which case all the cells were maintained. On Day 2 of the experiment, the cells were counted, diluted to 10^4 cells/mL and plated for mutant frequency (2000 cells/well) in selective medium containing 4 μg/mL 5-trifluorothymidine (TFT) in 96-well microtitre plates. Cells were also diluted to 10 cells/mL and plated (2 cells/well) for viability (%V) in non-selective medium. The daily cell counts were used to obtain a Relative Suspension Growth (%RSG) value that gives an indication of post exposure toxicity during the expression period as a comparison to the vehicle control, and when combined with the Viability (%V) data, a Relative Total Growth (RTG) value.

Plate scoring:
Microtitre plates were scored using a magnifying mirror box after ten to twelve days incubation at 37 °C with 5% CO2 in air. The number of positive wells (wells with colonies) was recorded together with the total number of scorable wells (normally 96 per plate). The numbers of small and large colonies seen in the TFT mutation plates were also recorded as the additional information may contribute to an understanding of the mechanism of action of the test item. Colonies are scored manually by eye using qualitative judgment. Large colonies are defined as those that cover approximately ¼ to ¾ of the surface of the well and are generally no more than one or two cells thick. In general, all colonies less than 25% of the average area of the large colonies are scored as small colonies. Small colonies are normally observed to be more than two cells thick. To assist the scoring of the TFT mutant colonies 0.025 mL of thiazolyl blue tetrazolium bromide (MTT) solution, 2.5 mg/mL in phosphate buffered saline (PBS), was added to each well of the mutation plates. The plates were incubated for two hours. MTT is a vital stain that is taken up by viable cells and metabolized to give a brown/black color, thus aiding the visualization of the mutant colonies, particularly the small colonies.

Rationale for test conditions:

Standard as per OECD guidelines

Evaluation criteria:

An approach for defining positive and negative responses is recommended to assure that the increased mutation frequency (MF) is biologically relevant. In place of statistical analysis generally used for other tests, it relies on the use of a predefined induced mutant frequency (i.e. increase in MF above the concurrent control), designated the Global Evaluation Factor (GEF) of 126 x 10^-6, which is based on the analysis of the distribution of the vehicle control MF data from participating laboratories.
Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined, the increase in MF above the concurrent background exceeds the GEF and the increase is concentration related (e.g., using a trend test). The test chemical is then considered able to induce mutation in this test system.

Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly negative if, in all experimental conditions examined there is no concentration related response or, if there is an increase in MF, it does not exceed the GEF. The test chemical is then considered unable to induce mutations in this test system.

Statistics:

No statistical analysis conducted

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

not applicable

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Test substance confounding effects:
There was no marked change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm.

Preliminary cytotoxicity test:
There was evidence of slight reductions in the Relative Suspension Growth (%RSG) of cells treated with the test item in all of the three exposure groups when compared to the concurrent vehicle control groups. Precipitate of the test item was observed at and above 4 μg/mL in the 4-hour –S9 exposure group, at and above 0.5 μg/mL in the 4-hour + S9 exposure group, and at and above 2 μg/mL in the 24-hour –S9 exposure group. Therefore, the dose levels selected for the main test were based on precipitate.

Mutagenicity test:
There was no evidence of marked dose related toxicity following exposure to the test item in all of the three exposure groups, as indicated by the %RSG and RTG values. There was no evidence of any marked reductions in viability (%V) in either of the three exposure groups, indicating that residual toxicity had not occurred. Acceptable levels of toxicity were seen with the positive control substances. The concentrations of 4 and 8 μg/mL in the 4-hour –S9 exposure group, 1 and 2 μg/mL in the 4-hour +S9 exposure group were not plated out for 5-TFT resistance and viability due to excessive precipitate.

Precipitate of the test item was observed at and above 2 μg/mL in the 4-hour –S9 exposure group. Precipitate of the test item was observed at and above 0.5 μg/mL in the 4-hour +S9 exposure group. Precipitate of the test item was observed at 4 μg/mL in the 24-hour –S9 exposure group. The presence of one analyzable precipitating dose level in in all three exposure groups satisfies the requirements of the OECD guideline.

The vehicle controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive controls produced marked increases in the mutant frequency per viable cell achieving the acceptability criterion, indicating that the test system was operating satisfactorily, and that the metabolic activation system was functional.

The test item did not induce any toxicologically significant or dose related increases in the mutant frequency x 10^-6 per viable cell at any of the dose levels, in any of the three exposure groups.

Main experiment results:

 

Treatment (µg/mL)	4 hours no S9
	%RSG	RTG	MF
0	100	1.00	120.34
0.06	84	0.94	112.66
0.13	89	1.04	94.73
0.25	96	1.07	96.79
0.5	112	1.14	101.71
1	101	1.12	117.40
2	108	1.21	93.11
	99
	100
MF threshold for a positive response = 246.34
EMS
400	58	0.36	1320.11

Treatment (µg/mL)	4 hours with S9
	%RSG	RTG	%RSG
0	100	1.00	121.81
0.015	99	1.02	116.36
0.03	101	1.03	113.11
0.06	95	0.99	117.74
0.13	108	1.05	120.76
0.25	96	1.01	114.29
0.5	100	1.03	121.64
1	110
2	110
MF threshold for a positive response = 247.81
CP
1.5	85	0.69	752.27

 

Treatment (µg/mL)	24 hours no S9
	%RSG	RTG	%RSG
0	100	1.00	144.63
0.03	90
0.06	96
0.13	103	0.96	115.32
0.25	89	1.01	139.64
0.5	87	0.91	131.27
1	78	0.98	126.31
2	72	0.92	152.79
4	71	0.83	111.73
MF threshold for a positive response = 270.63
EMS
150	37	0.33	1120.42

Conclusions:

The test item, did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the Global Evaluation Factor (GEF) of 126 x 10^-6, consequently it is considered to be non-mutagenic in this assay.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

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)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: Human

Details on mammalian cell type (if applicable):

CELLS USED
For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non-smoking volunteer (aged 18-35) who had been previously screened for suitability. The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. Based on over 20 years in-house data for cell cycle times for lymphocytes using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells to calculate the average generation time (AGT) for human lymphocytes it is considered to be approximately 16 hours. Therefore using this average the in-house exposure time for the experiments for 1.5 x AGT is 24 hours.
The details of the donors used are:
Preliminary toxicity test and main experiment: female, aged 22 years

MEDIA USED
Cells (whole blood cultures) were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10 % foetal bovine serum (FBS), at approximately 37 ºC with 5 % CO2 in humidified air. The lymphocytes of fresh heparinized whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix prepared from the livers of male rats treated with Phenobarbital and B-Naphtha flavone

Test concentrations with justification for top dose:

Dose range finder: 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 µg/mL
4 hour exposure with metabolic activation: 0, 1, 2, 4, 8, 16, 32 and 64 µg/mL
4 hour exposure without metabolic activation: 0, 1, 2, 4, 8, 16, 32 and 64 µg/mL
24 hour exposure without metabolic activation: 0, 1, 2, 4, 8, 16, 32 and 64 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test item was insoluble in culture medium at 20 mg/mL, acetone and tetrahydrofuran at 200 mg/mL but was suspendable in dimethyl sulphoxide (DMSO) at 200 mg/mL in solubility checks performed in-house.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

Test item preparation:
The molecular weight of the test item was given as 385.94, therefore, the maximum dose level was 2000 μg/mL, the maximum recommended dose level. The test item was treated as being 100% pure and no purity correction was required. Prior to each experiment, the test item was accurately weighed, formulated in DMSO and appropriate serial dilutions prepared. There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm. The test item was formulated within two hours of it being applied to the test system; the test item formulations were assumed to be stable.

4 hour exposure with and without metabolic activation:
After approximately 48 hours incubation at approximately 37 ºC, 5% CO2 in humidified air, the cultures were transferred to tubes and centrifuged. Aliquots of the culture medium was removed, reserved, and replaced with the required volume of MEM (including serum) and an aliquot of the appropriate solution of vehicle control or test item was added to each culture. For the positive control, and aliquot of the appropriate solution was added to the cultures. 20% S9-mix (i.e. 2% final concentration of S9 in standard co-factors) was added to the cultures of the Preliminary Toxicity Test and Main Experiment for those requireing metabolic activation. After 4 hours at approximately 37 ºC, 5% CO2 in humidified air, the cultures were centrifuged, the treatment medium removed by suction and replaced with a wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the original culture medium. The cells were then re-incubated for a further 20 hours at approximately 37 ºC in 5% CO2 in humidified air.

24 hour exposure without metabolic activation:
As the exposure was continuous the cultures were established, at a nominal volume of 9.9 mL. After approximately 48 hours incubation the cultures were removed from the incubator and dosed with vehicle control, test item dose solution or positive control solution. The nominal final volume of each culture was 10 mL. The cultures were then incubated at approximately 37 ºC, 5% CO2 in humidified air for 24 hours. The preliminary toxicity test was performed using all three of the exposure conditions as described for the Main Experiment but using single cultures only.

For the preliminary test, parallel flasks, containing culture medium without whole blood, were established for the three exposure conditions so that test item precipitate observations could be made. Precipitate observations were recorded at the beginning and end of the exposure periods.
Using a qualitative microscopic evaluation of the microscope slide preparations from each treatment culture, appropriate dose levels were selected for mitotic index evaluation. Mitotic index data was used to estimate test item toxicity and for selection of the dose levels for the main test. For the main tests parallel flasks, containing culture medium without whole blood, were established for the three exposure conditions so that test item precipitate observations could be made. Precipitate observations were recorded at the beginning and end of the exposure periods.

Cell harvest:
Mitosis was arrested by addition of demecolcine (Colcemid 0.1 μg/mL) 2.5 hours before the required harvest time. After incubation with demecolcine, the cells were centrifuged, the culture medium was drawn off and discarded, and the cells re-suspended in hypotonic KCl. After approximately fourteen minutes (including centrifugation), most of the hypotonic solution was drawn off and discarded. The cells were re-suspended and then fixed by dropping the KCl cell suspension into fresh methanol/glacial acetic acid. The fixative was changed at least three times and the cells stored at approximately 4 ºC to ensure complete fixation prior to slide preparation.

Preparation of metaphase spreads:
The lymphocytes were re-suspended in several mL of fresh fixative before centrifugation and re-suspension in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry. Each slide was permanently labeled with the appropriate identification data.

Staining:
When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.

Qualitative assessment:
The slides were checked microscopically to determine the quality of the metaphases and also the toxicity and extent of precipitation, if any, of the test item. These observations were used to select the dose levels for mitotic index evaluation. Slides were coded to ensure no bias. A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.

Scoring:
Where possible, 300 consecutive well-spread metaphases from each concentration were counted (150 per duplicate), where there were at least 15 cells with aberrations (excluding gaps), slide evaluation was terminated. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system recommended in the 1983 UKEMS guidelines for mutagenicity testing and the International System for Human Cytogenetic Nomenclature (ISCN). Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides. In addition, cells with 69 chromosomes or more were scored as polyploid cells and the incidence of polyploid cells (%) (including the incidence of cells with endoreduplicated chromosomes) is also reported. Many experiments with human lymphocytes have established a range of aberration frequencies acceptable for control cultures in normal volunteer donors.

Rationale for test conditions:

Standard as per OECD guidelines

Evaluation criteria:

A test item is considered clearly negative if:
1) The number of cells with structural aberrations in all evaluated dose groups should be within the range of the laboratory historical control data.
2) No toxicologically or statistically significant increase of the number of cells with structural chromosome aberrations is observed following statistical analysis.
3) There is no concentration-related increase at any dose level.

A test item can be classified as genotoxic if:
1) The number of cells with structural chromosome aberrations is outside the range of the laboratory historical control data.
2) At least one concentration exhibits a statistically significant increase in the number of cells with structural chromosome aberrations compared to the concurrent negative control.
3) The observed increase in the frequency of cells with structural aberrations is considered to be dose-related.

Statistics:

The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.

A toxicologically significant response is recorded when the p value calculated from the statistical analysis of the frequency of cells with aberrations excluding gaps is less than 0.05 when compared to its concurrent control and there is a dose-related increase in the frequency of cells with aberrations which is reproducible. Incidences where marked statistically significant increases are observed only with gap-type aberrations will be assessed on a case by case basis.

Species / strain:

lymphocytes: Human

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:

Preliminary test:
A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure, at and above 31.25 μg/mL, in the 4(20)-hour exposure group in the absence of S9, at and above 7.81 μg/mL in the presence of S9 and at and above 125 μg/mL in the continuous exposure group. Precipitate was also noted in the blood cultures at the end of the exposure period, at and above 7.81 μg/mL in both the 4(20)-hour exposure groups and at and above 15.63 μg/mL in the 24-hour exposure groups. Precipitate was carried through onto the slides and was seen on the slides at and above 62.5 μg/mL in all three exposure groups.
Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 2000 μg/mL in all three exposure groups. The dose levels selected for mitotic index evaluation were the lowest dose levels where precipitate occurred. The test item demonstrated no marked evidence of toxicity in any of the exposure groups at the dose levels selected for mitotic index. The selection of the maximum dose level for the Main Experiment was based on the lowest precipitating dose level and was 64 μg/mL for all three exposure groups.

Main experiments:
The qualitative assessment of the slides determined that there was no marked toxicity as was in the preliminary toxicity test and there were metaphases suitable for scoring present up to 64 μg/mL in all three exposure groups. Precipitate observations were made at the end of exposure in blood-free cultures and precipitate was noted at and above 32 μg/mL in the 4(20)-hour exposure group in the absence of S9 and in the 24-hour exposure group, and at and above 16 μg/mL in the presence of S9. Precipitate was seen in the blood cultures at the end of exposure at and above 16 μg/mL in all three exposure groups. Precipitate was seen on the 4(20)-hour exposure group slides at 64 μg/mL and on the 24-hour exposure group slides at and above 32 μg/mL. Based on the above observations it was considered that the lowest precipitating dose level was 16 μg/mL for all three exposure groups.

The mitotic index data confirms the qualitative observations in that no dose-related inhibition of mitotic index was observed in the 4(20)-hour exposure groups in the absence or presence of S9. The 24-hour exposure groups did demonstrate a plateau in toxicity with 46%, 52% and 54% mitotic inhibition at 4, 8 and 16 μg/mL respectively. The maximum dose level selected for metaphase analysis was the lowest precipitating dose level (16 μg/mL).

The assay was considered valid as it met all of the following criteria:
The frequency of cells with chromosome aberrations (excluding gaps) in the vehicle control cultures were within the current historical control data range.
All the positive control chemicals induced a demonstrable positive response (p≤0.01) and confirmed the validity and sensitivity of the assay and the integrity of the S9-mix.
The study was performed using all three exposure conditions using a top concentration which meets the requirements of the current testing guideline.
The required number of cells and concentrations were analyzed.
The test item did not induce any statistically significant increases in the frequency of cells with aberrations either in the absence or presence of metabolic activation.
The test item did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in any of the three exposure groups.

Conclusions:

The test item did not induce any statistically significant increases in the frequency of cells with chromosome aberrations, in either the absence or presence of a liver enzyme metabolizing system. The test item was, therefore, considered to be non-clastogenic to human lymphocytes in vitro under the conditions employed.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

No in vivo testing for genotoxicity endpoints are required based on the full dataset of in vitro tests available.

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

No indication of clastogenicity or mutagenicity was noted in the available in vitro mutagenicity and clastogenicity tests and the available results were all negative. Accordingly classification of the substance for mutagenic effects is not deemed warranted in accordance with the CLP Regulation (EC No. 1272/2008).