Trihydrogen trifluoro[phosphato(3-)-O]borate(3-)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From 2010-01-25 to 2010-03-01

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Well documented GLP study performed according to the UK Environmental Mutagen Society which is based on the OECD Guideline 476 and EU Method B.17. There was only a minor deviation reported: no analysis was performed on homogeneity, concentration or stability of the test material formulation, however this is considered not to affect the purpose or integrity of the study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: United Kingdom Environmental Mutagen Society

Deviations:

yes

Remarks:

no analysis on homogeneity, concentration or stability of test material formulation, however this is considered not to affect the purpose or integrity of the study

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

no analysis on homogeneity, concentration or stability of test material formulation, however this is considered not to affect the purpose or integrity of the study

Test guideline 3

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

no analysis on homogeneity, concentration or stability of test material formulation, however this is considered not to affect the purpose or integrity of the study

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Orthophosphoric acid

EC Number:

231-633-2

EC Name:

Orthophosphoric acid

Cas Number:

7664-38-2

IUPAC Name:

phosphoric acid

Details on test material:

- Name of test material: phosphoric acid 85%
- Substance type: inorganic
- Physical state: clear colourless liquid
- Analytical purity: 85.6%
- Purity test date: 2010-01-06
- Lot/batch No.: 209
- Storage condition of test material: at room temperature in the dark

Method

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: 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.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: no
- 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 -/-) 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.

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitol/beta-naphthoflavone S9 from livers of male Sprague-Dawley rats

Test concentrations with justification for top dose:

0, 61.25, 122.5, 245, 490, 735 and 980 µg/mL

Vehicle / solvent:

- Solvent(s) used: R0 medium

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

in the absence of metabolic activation

Migrated to IUCLID6: Sigma batch 142314732109252 at 400 µg/mL and 150 µ/mL for the 4 - and 24-hour exposures respectively

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

in the presence of metabolic activation

Migrated to IUCLID6: Acros batch A0164185 at 2 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: not applicable
- Exposure duration: 4-hour exposure with and without metabolic activation; 24-hour exposure without metabolic activation
- Expression time (cells in growth medium): 2 days (dilution of 2 x 1E05 cells/mL)
- Selection time (if incubation with a selection agent): 10 to 14 days of incubation
- Fixation time (start of exposure up to fixation or harvest of cells): 3 hours (0.025 mL of MTT solution, 2.5 mg/mL in PBS)

SELECTION AGENT (mutation assays): selective medium containing 4 µg/mL 5-trifluorothymidine (TFT in 96-well microtitre plates
SPINDLE INHIBITOR (cytogenetic assays): not applicable
STAIN (for cytogenetic assays): not applicable

NUMBER OF REPLICATIONS: duplicates

NUMBER OF CELLS EVALUATED: At the end of the treatment period, for each experiment, the cells were washed twice using the R10 medium then resuspended in R20 medium at a cell density of 2 x 1E05 cells/mL. The cultures were incubated and subcultured every 24 hours for the expression period of two days, by counting and dilution to 2 x 1E05 cells/mL. On day 2 of the experiment, the cells were counted, diluted to 1E04 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.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency, viability, relative suspension growth and relative total growth

OTHER EXAMINATIONS:
- Determination of polyploidy: no
- Determination of endoreplication: no
- Other: no

Evaluation criteria:

The normal range for mutant frequency per survivor is 50-200 x 1E-06 for the TK +/- locus in L5178Y cells at Harlan Laboratories Ltd. Vehicle controls results should ideally be within this range, although minor errors in cell counting and dilution or exposure to the metabolic activation system may cause this to be slightly elevated. Experiments where the vehicle control values are markedly greater than 250 x 1E-06 mutant frequency per survivor are not normally acceptable and will be repeated. Positive control chemicals should induce at least three to five fold increases in mutant frequency greater than the corresponding vehicle control. For a test material to demonstrate a mutagenic response it must produce a statistically significant increase in the induced mutant frequency (IMF) over the concurrent vehicle mutant frequency value. The IMF must exceed 126 x 1E-06 for the microwell method based on the global background MF (Moore et al 2003). Therefore any test material dose level that has a mutation frequency value that is greater than the corresponding vehicle control by the GEF of 126 x 1E-06 will be considered positive. However, if a test material produces a modest increase in mutant frequency, which only marginally exceeds the GEF value and is not reproducible or part of a dose-related response, then it may be considered to have no toxicological significance. Conversely, when a test material induces modest reproducible increases in the mutation frequencies that do not exceed the GEF value then scientific judgement will be applied. If the reproducible responses are significantly dose-related and include increases in the absolute numbers of mutant colonies then they may be considered to be toxicological significant.

Statistics:

The experimental data were analysed using a dedicated computer program which follows the statistical guidelines recommended by the UKEMS. Dose levels that have survival values less than 10% are excluded from any statistical analysis, as any response they give would be considered to have no biological or toxicological relevance.

Results and discussion

Test results

Test results 1

Species / strain:

other: L5178Y TK+/- 3.7.2c mouse lymphoma cell line

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

4-hour exposure with and without metabolic activation

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Very modest dose related toxicity, no evidence of any reductions in viability (%V), therefore no residual toxicity occurred

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

other: L5178Y TK+/- 3.7.2c mouse lymphoma cell line

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

24-hour exposure without metabolic activation

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Modest dose related toxicity following exposure to the test material. There was no evidence of any reductions in viability (%V), therefore indicating that no residual toxicity had occurred.

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: no data on effects, however it was mentioned that there was no marked change in pH when the test material was dosed into media
- Effects of osmolality: no data on effects, however it was mentioned that the osmolality did not increase by more than 50 mOsm
- Precipitation: no precipitate of the test material was observed at any of the dose levels tested in the preliminary toxicity test (0, 3.83, 7.66, 15.31, 30.63, 61.25, 122.5, 245, 490, 980)

RANGE-FINDING/SCREENING STUDIES: In the three exposure groups (4-h exposure -S9, 4-hour exposure +S9 and 24-hour exposure -S9) there were no marked dose related reductions in the Relative Suspension Growth (%RSG) of treated cells with the test material when compared to the concurrent vehicle controls. However, a modest reduction was observed at the 10 mM limit dose in all three of the exposure groups with the greatest reduction observed in the 24-hour exposure group in the absence of metabolic activation. No precipitate of the test material was observed at any of the dose levels. In the subsequent mutagenicity test, the maximum dose level for all three of the exposure groups was the 10 mM limit dose.

COMPARISON WITH HISTORICAL CONTROL DATA: The mutation frequency is situated within the range of historical control values for the positive controls and solvent control.

ADDITIONAL INFORMATION ON CYTOTOXICITY: no data

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Results for 4 -hour exposure (-S9):

%RSG for treatment: ranged from 110 to 75 (from dose 61.25 to 980 µg/mL)

%V for treatment: ranged from 84.40 to 90.38 (from dose 61.25 to 980 µg/mL)

RTG for treatment: ranged from 1.10 to 0.80 (from dose 61.25 to 980 µg/mL)

MF for treatment: ranged from 97.01 to 82.17 (from dose 61.25 to 980 µg/mL)

The values for the solvent control were 100 (%RSG), 84.40 (%V), 1.00 (RTG) and 84.42 (MF)

The values for the positive control EMS were 76 (%RSG), 88.81 (%V), 0.80 (RTG) and 688.67 (MF)

Results for 4 -hour exposure (+S9):

%RSG for treatment: ranged from 94 to 81 (from dose 61.25 to 980 µg/mL)

%V for treatment: ranged from 95.38 to 96.26 (from dose 61.25 to 980 µg/mL)

RTG for treatment: ranged from 1.07 to 0.92 (from dose 61.25 to 980 µg/mL)

MF for treatment: ranged from 107.17 to 86.65 (from dose 61.25 to 980 µg/mL)

The values for the solvent control were 100 (%RSG), 84.40 (%V), 1.00 (RTG) and 115.46 (MF)

The values for the positive control CP were 53 (%RSG), 39.58 (%V), 0.25 (RTG) and 1311.27 (MF)

Results for 24 -hour exposure (-S9):

%RSG for treatment: ranged from 104 to 53 (from dose 61.25 to 980 µg/mL)

%V for treatment: ranged from 113.09 to 97.17 (from dose 61.25 to 980 µg/mL)

RTG for treatment: ranged from 1.28 to 0.57 (from dose 61.25 to 980 µg/mL)

MF for treatment: ranged from 55.11 to 70.25 (from dose 61.25 to 980 µg/mL)

The values for the solvent control were 100 (%RSG), 91.99 (%V), 1.00 (RTG) and 79.09 (MF)

The values for the positive control EMS were 55 (%RSG), 85.83 (%V), 0.52 (RTG) and 692.36 (MF)