tetrakis(2,6-dimethylphenyl)-m-phenylene biphosphate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 April 2009 and 01 June 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection:19/08/08 Date of signature: 04/03/09

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

432-770-2

EC Name:

-

Cas Number:

139189-30-3

Molecular formula:

C38 H40 O8 P2

IUPAC Name:

3-{[bis(2,6-dimethylphenoxy)phosphoryl]oxy}phenyl bis(2,6-dimethylphenyl) phosphate

Details on test material:

Sponsor's identification:PX-200Chemical name:Tetrakis(2,6-dimethylphenyl)m-phenylene bisphosphatePurity: 96.40%Description: White solidBatch number: F10204Date received: 27 March 2009Storage conditions:Room temperature in the dark

Method

Target gene:

Thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Peniciliin (100 units/ml), Streptomycin (100 µg/ml), sodium pyruvate (1 mM), Amphotericin B (2.5 µg/ml) and 10% donor horse serum (giving R10 media) at 37°C with 5% carbon dioxide in air.- Properly maintained: yes- Periodically checked for Mycoplasma contamination:yes- Periodically checked for karyotype stability: no- Periodically "cleansed" against high spontaneous background: yes. Before the stocks of cells were frozen they were cleansed of homozygous (TK -/-) nutants by culuting in THMG medium for 24 hours. This medium contained Thmidine (9 µg/ml), hypoxanthine (15 µg/ml) Mehotrexate (0.3 µg/ml) and glycine (22.5 µg/ml). For the following 24 hours the cells were cultured in THG medium (i.e. THMG without Methotrexate) before being returned to R10 medium.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital and beta-naphthoflavone induced rat liver, S9

Test concentrations with justification for top dose:

The dose range used in the preliminary toxicity test was 19.53 to 5000 µg/ml for all three exposure groups.Experiment 1: Six dose levels ranging from 78.13 to 2500 µg/ml.Experiment 2: 9.77 to 1250 µg/ml in the absence of metabolic actiavtion, 39.06 to 1250 µg/ml in the presence of metabolic activation.Vehicle and positive controls were used in parallel with the test material. Solvent (Acetone) treatment groups were used as the vehicle controls. Ethylmethanesulphonate (EMS) Sigma batch 126K0758 at 400 µg/ml and 150 µg/ml for Experiment 1 and Experiment 2 respectively, was used as the positive control in the absence of metabolic activation. Cyclophosphamide (CP) Acros batch A0164185 at 2 µg/ml was used as the positive control in the presence of metabolic activation.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Solvent (Acetone) treatment groups were used as the vehicle controls. - Justification for choice of solvent/vehicle: Not stated

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Solvent (Acetone) treatment groups were used as the vehicle controls.

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Solvent (Acetone) treatment groups were used as the vehicle controls.

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Without metabolic activation

Details on test system and experimental conditions:

-Cell Culture:The stocks of cells are stored in liquid nitrogen at -196°C. Cells were routinely cultured in RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/ml), Streptomycin (100 µg/ml), Sodium pyruvate (1 mM), Amphotericin B (2.5 µg/ml) 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) and without serum (R0) are used during the course of the study.-Cell Cleansing:The TK +/- heterozygote cells grown in suspension spontaneously mutate at a low but significant rate. Before the stocks of cells were frozen they were cleansed of homozygous (TK -/-) mutants by culturing in THMG medium for 24 hours. This medium contained Thymidine (9 µg/ml), Hypoxanthine (15 µg/ml), Methotrexate (0.3 µg/ml) and Glycine (22.5 µg/ml). For the following 24 hours the cells were cultured in THG medium (i.e. THMG without Methotrexate) before being returned to R10 medium.-Preparation of Test and Control Material:The test material was accurately weighed and dissolved in Acetone before the appropriate dilutions were made. The molecular weight of the test material was supplied by the sponsor as 686.67, therefore the maximum dose level investigated in the preliminary toxicity test was 5000 µg/ml, the maximum recommended dose level. The purity of the test material was 96.4% and was accounted for in the formulations. There was no marked change in pH when the test material was dosed into media and the osmolality did not increase by more than 50 mOsm. Analysis for concentration, homogeneity and stability of the test material preparations were not a requirement of the test method and were therefore not performed.Vehicle and positive controls were used in parallel with the test material. Solvent (Acetone) treatment groups were used as the vehicle controls. Ethylmethanesulphonate (EMS) Sigma batch 126K0758 at 400 µg/ml and 150 µg/ml for Experiment 1 and Experiment 2 respectively, was used as the positive control in the absence of metabolic activation. Cyclophosphamide (CP) Acros batch A0164185 at 2 µg/ml was used as the positive control in the presence of metabolic activation.-Microsomal Enzyme Fractions:PB/ßNF S9 was prepared in-house on 23 November 2008 and 05 April 2009 from the livers of male Wistar HanTM rats weighing ~200g. These had each received, orally, three consecutive daily doses of phenobarbital/ß-naphthoflavone (80/100 mg per kg per day) prior to S9 preparation on the fourth day.The S9 was stored at -196°C in a liquid nitrogen freezer.S9-mix was prepared by mixing S9, NADP (5 mM), G-6-P (5 mM), KCl (33 mM) and MgCl2 (8 mM) in R0.20% S9-mix (i.e. 2% final concentration of S9) was added to the cultures of the Preliminary Toxicity Test and of Experiment 1. In Experiment 2, 10% S9-mix (i.e. 1% final concentration of S9), was added.-Preliminary Toxicity Test:A preliminary toxicity test was performed on cell cultures at 5E+05 cells/ml, using a 4 hour exposure time both with and without metabolic activation (S9), and at 1.5E+05 cells/ml using a 24-hour exposure without S9. The dose range used in the preliminary toxicity test was 19.53 to 5000 µg/ml for all three exposure groups. Following the exposure period the cells were washed twice with R10, resuspended in R20 medium, counted and then serially diluted to 2E5 cells/ml.The cultures were incubated and sub-cultured after 24 hours by counting and diluting to 2E5 cells/ml. After a further 24 hours the cultures were counted and then discarded. The cell counts were then used to calculate Suspension Growth values (SG). The SG values were then adjusted to account for immediate post treatment toxicity, and a comparison of each treatment SG value to the concurrent vehicle control performed to give a % Relative Suspension Growth Value (%RSG).Results from the preliminary toxicity test were used to set the test material dose levels for the mutagenicity experiments. Maximum dose levels were selected using the following criteria:i)Maximum recommended dose level, 5000 µg/ml or 10 mM.ii)The presence of excessive precipitate where no test material-induced toxicity was observed.iii)Test material-induced toxicity, where the maximum dose level used should produce 10 to 20% survival (the maximum level of toxicity required). This optimum upper level of toxicity was confirmed by an IWGT meeting in New Orleans, USA (Moore et al 2002).-Mutagenicity Test:-Experiment 1: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 1E+06 cells/ml in 10 ml aliquots in R10 medium in sterile plastic universals. The treatments were performed in duplicate (A + B), both with and without metabolic activation (S9-mix) at six dose levels of the test material (78.13 to 2500 µg/ml), vehicle and positive controls. To each universal was added 2 ml of S9-mix if required, 0.2 ml of the treatment dilutions, (0.2 ml for the positive control) and sufficient R0 medium to bring the total volume to 20 ml.The treatment vessels were incubated at 37°C for 4 hours with continuous shaking using an orbital shaker within an incubated hood.-Experiment 2:As in Experiment 1, an exponentially growing stock culture of cells was established. The cells were counted and processed to give 1E+06 cells/ml in 10 ml duplicate cultures in R10 medium for the 4-hour treatment with metabolic activation cultures. In the absence of metabolic activation the exposure period was extended to 24 hours therefore 0.3E+06 cells/ml in 10 ml duplicate cultures were established in 25 cm2 tissue culture flasks. To each culture 2 ml of S9 mix was added if required, 0.2 ml of the treatment dilutions (0.2 ml for the positive control) and sufficient R0 medium to give a final volume of 20 ml (R10 is used for the 24-hour exposure group). The dose range of the test material was 9.77 to 1250 µg/ml in the absence of metabolic activation and 39.06 to 1250 µg/ml in the presence of metabolic activation. The treatment vessels were incubated at 37°C with continuous shaking using an orbital shaker for 24 hours in the absence of metabolic activation and 4 hours in the presence of metabolic activation.At the end of the treatment period, for each experiment, the cells were washed twice using R10 medium then resuspended in R20 medium at a cell density of 2E+05 cells/ml. The cultures were incubated and subcultured every 24 hours for the expression period of two days, by counting and dilution to 2E+05 cells/ml. On Day 2 of the experiment, the cells were counted, diluted to 10E+04 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 treatment 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.

Evaluation criteria:

See remarks section.

Statistics:

See remarks section.

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

non-mutagenic

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS- Effects of pH: none stated in report- Effects of osmolality: none stated in report- Evaporation from medium: none stated in report- Water solubility: none stated in report- Precipitation: Preliminary toxicity test: In the 4-hour exposure group in the absence of metabolic activation (S9), there was evidence of modest reductions in the Relative Suspension Growth (%RSG) of cells treated with the test material when compared to the concurrent vehicle controls. However, marked reductions in %RSG values were observed in the 4-hour exposure group in the presence of metabolic activation and the 24-hour exposure group in the absence of metanolic actiavtion. A precipitate of the test material was observed at and above 78.13 µg/ml. Based on the %RSG values, maximum exposure of the test material to the cells was considered to occur at approximately 1250 to 2500 µg/ml in the 4-hour exposure groups, both in the absence and presence of metabolic activation, and at approximately 625 to 1250 µg/ml in the 24-hour exposure group in the absence of metabolic activation. It was therefore considered that the presence of precipitate was effectively reducing the exposure of the test material to the cells. therefore, the maximum dose level in the mutagenicity test was initially set ar 2500 µg/ml.- Other confounding effects: None stated.RANGE-FINDING/SCREENING STUDIES: -Preliminary Toxicity Test:The dose range of the test material used in the preliminary toxicity test was 19.53 to 5000 µg/ml. The results for the Relative Suspension Growth (%RSG) were as follows:Dose % RSG (-S9) % RSG (+S9) % RSG (-S9)(µg/ml) 4-Hour Exposure 4-Hour Exposure 24-Hour Exposure0100 100 10019.53113 100 3339.06128 96 3378.13118 104 34156.25119 101 36312.5114 96 38625128 110 22125066 30 25250066 34 345000127 95 58In the 4-hour exposure group in the absence of metabolic activation (S9), there was evidence of modest reductions in the Relative Suspension Growth (%RSG) of cells treated with the test material when compared to the concurrent vehicle controls. However, marked reductions in %RSG values were observed in the 4-hour exposure group in the presence of metabolic activation and the 24-hour exposure group in the absence of metabolic activation. A precipitate of the test material was observed at and above 78.13 µg/ml. Based on the %RSG values, maximum exposure of the test material to the cells was considered to occur at approximately 1250 to 2500 µg/ml in the 4-hour exposure groups, both in the absence and presence of metabolic activation, and at approximately 625 to 1250 µg/ml in the 24-hour exposure group in the absence of metabolic activation. It was therefore considered that the presence of precipitate was effectively reducing the exposure of the test material to the cells. Therefore, the maximum dose level in the mutagenicity test was initially set at 2500 µg/ml.COMPARISON WITH HISTORICAL CONTROL DATA: Not applicable.ADDITIONAL INFORMATION ON CYTOTOXICITY:None stated.

Remarks on result:

other: strain/cell type:

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

 Experiment 1.

The results of the microtitre plate counts and their analysis are presented in Tables 2 to 7.

There was evidence of toxicity following exposure to the test material in both the absence and presence of metabolic activation, as indicated by the %and RTG values. The most marked reductions in %and RTG values were observed at 1250 µg/ml in both the absence and presence of metabolic activation (Tables 3 and 6). This was taken to confirm that maximum exposure of the test material to the cells had been achieved at this dose level in both the absence and presence of metabolic activation. Therefore, the maximum dose level for the 4-hour exposure group in the presence of metabolic activation in the second experiment was set at 1250 µg/ml. There was no evidence of any dose related reductions in (%V) viability, therefore indicating that no residual toxicity had occurred in either of the exposure groups. Acceptable levels of toxicity were seen with both positive control substances (Tables 3 and 6).

Neither of the vehicle control mutant frequency values were outside the range of 50 to 200 x 10^-6viable cells that is acceptable for L5178Y cells at Harlan Laboratories Ltd,,. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional (Tables 3 and 6).

The test material did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10^-6per viable cell in either the absence or presence of metabolic activation (Tables 3 and 6). A precipitate of test material was observed at and above 156.25 µg/ml.

The numbers of small and large colonies and their analysis are presented in Tables 4 and 7.

Experiment 2.

The results of the microtitre plate counts and their analysis are presented in Tables 8 to 13.

As was seen previously there was evidence of dose-related reductions in %and RTG values in cultures dosed with the test material in both the absence and presence of metabolic activation. The most marked reduction in %and RTG values in the 24‑hour exposure group in the absence of metabolic activation was observed at 625 µg/ml. This was taken to confirm that maximum exposure of the test material to the cells in this exposure group had been achieved at this dose level. There was once again no evidence of any dose related reductions in (%V) viability, therefore indicating that no residual toxicity occurred in either the absence or presence of metabolic activation. Near optimum levels of test material-induced toxicity were achieved in the absence of metabolic activation. Both positive controls induced acceptable levels of toxicity (Tables 9 and 12).

The 24-hour exposure without metabolic activation treatment, demonstrated that the extended time point had a marked effect on the toxicity of the test material.

Neither of the vehicle control mutant frequency values were outside the acceptable range of 50 to 200 x 10^-6viable cells. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional (Tables 9 and 12).

The test material did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency in either the absence or presence of metabolic activation (Tables 9 and 12). Heterogeneity (poor correlation between A and B plates) was observed at 1250 µg/ml in the 4-hour exposure group in the presence of metabolic activation. However, with no evidence of any responses in either the first or second experiment, the data was considered acceptable for the purpose of this study. A precipitate of test material was observed at and above 156.25 µg/ml.

The numbers of small and large colonies and their analysis are presented in Tables 10 and 13.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):other: Non-mutagenicThe test material did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and is therefore considered to be non mutagenic under the conditions of the test.

Executive summary:

Introduction. 

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test material on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The test method used meets the requirements of the OECD (476) of Commission Regulation (EC) No. 440/2008 of.

Methods.

Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test material at six dose levels, in duplicate, together with vehicle (solvent) and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9). In Experiment 2, the cells were treated with the test material at up to eight dose levels using a 4‑hour exposure group in the presence of metabolic activation (1% S9) and a 24‑hour exposure group in the absence of metabolic activation.

The dose range of test material was selected following the results of a preliminary toxicity test and was 78.13 to 2500 µg/ml for the first experiment. For the second experiment the dose range was 9.77 to 1250 µg/ml in the absence of metabolic activation and 39.06 to 1250 µg/ml in the presence of metabolic activation.

Results.

The maximum dose level used in the mutagenicity tests was limited by the presence of precipitate effectively reducing the exposure of the test material to the cells in the preliminary toxicity test, as indicated by the %values. A precipitate of test material was observed at and above 156.25 µg/ml in the mutagenicity tests. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control materials induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test material did not induce any statistically significant or dose-related increases in the mutant frequency at any of the dose levels, either with or without metabolic activation, in either the first or the second experiment using a dose range where maximum exposure of the test material to the cells had been achieved.

Conclusion. 

The test material was considered to be non-mutagenic to L5178Y cells under the conditions of the test.