Phosphinylidynetrimethanol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Bacterial reverse mutation assay / Ames test (OECD 471; publication): negative with and without activation in Salmonella typhimurium strains (TA 98, 100, 1535, 1537); an Ames test with the missing 5^th strain (Escherichia coli WP2uvrA or Salmonella typhimurium TA102) is scientifically not necessary due to structural considerations;

- Mammalian cell micronucleus test (OECD 487; GLP): negative with and without activation in Chinese hamster lung cells (V79);

- Mammalian cell gene mutation test using the hprt gene (OECD 476; GLP): negative with and without activation in Chinese hamster ovary cells (CHO).

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:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Remarks:

Only four strains of S. typhimurium (TA1535, TA1537, TA98, TA100) were used, data on E.coli WP2 strains or S. typhimurium TA102 are lacking. However, since these strains were mainly included in the recent version of OECD 471 because the four formerly only recommended S. typhimurium strains TA1535, TA1537, TA98 and TA100 may not detect certain oxidising mutagens, cross-linking agents and hydrazines, and this mode of action is not likely to occur based on the chemical structure of the test item, this restriction is considered to be negligible.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

only four strains of S. typhimurium (TA1535, TA1537, TA98, TA100) were used

Deviations:

no

Principles of method if other than guideline:

Only four strains of S. typhimurium (TA1535, TA1537, TA98, TA100) were used, data on E.coli WP2 strains or S. typhimurium TA102 are lacking. However, since these strains were mainly included in the recent version of OECD 471 because the four formerly only recommended S. typhimurium strains TA1535, TA1537, TA98 and TA100 may not detect certain oxidising mutagens, cross-linking agents and hydrazines, and this mode of action is not likely to occur based on the chemical structure of the test item, this restriction is considered to be negligible.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

no details given

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

rat liver oxidative enzyme metabolizing system (S-9 mix) with Aroclor-1254-induced rat liver

Test concentrations with justification for top dose:

1000, 100, 10, 0 µg/plate

Vehicle / solvent:

none

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Aflatoxin B1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Aflatoxin B1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: N-methyl1-N-nitro-N-nitrosoguanidine (MNNG)

Details on test system and experimental conditions:

Water was used to dissolve the test item THPO.
Liver homogenate was employed at a level of 0.1 ml per 1.0 ml of S-9 mix.

Rationale for test conditions:

no details given

Evaluation criteria:

no details given

Statistics:

Control values for each strain, both without and with the S-9 mix, were found not to differ significantly from the normal distribution by the Kalmagorov-Smimov goodness-of-fit test (P> 0.2 in each case). Differences between each replicate control value and the mean control value from each individual experiment were also normally distributed, with a better fit in each case than that of the absolute control values. Least significant differences (LSD) of experimental values from concurrently determined control values were based on the variance between sets of replicate control values summed over each set of experiments by strain and S-9 condition as follows: LSD = t√MS, where t is the t-statistic value at a given confidence level with degrees of freedom equal to the number of control data pairs, and MS is the overall variance of individual replicate differences from the mean of each individual experiment summed over all experiments. Within the range of control values, the variance was not dependent on the mean in any strain either without or with the S-9 mix.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

none

Conclusions:

Tris(hydroxymethyl)phosphine oxide (THPO) did not exhibit significant mutagenic activity under the conditions employed.

Executive summary:

The mutageniy activity of the test item Tris(hydroxymethyl)phosphine oxide (THPO) was tested in S. typhimurium strains TA100, TA98, TA1535, and TA1537 with and without metabolic activation using the quantitative top agar overlay technique.

Tris(hydroxymethyl)phosphine oxide (THPO) did not exhibit significant mutagenic activity under the conditions employed.

Finally, an Ames test with the 5th missing strain is not required due to structural considerations: THPO is not a highly reactive agent and is therefore not expected to be a cross-linking agent, has no oxidizing properties and is no hydrazine.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 01, 2020 - July 13, 2020 (experimental phase)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

under GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

adopted 29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

n/a

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

V79: Chinese hamster lung male, established after spontaneous transformation of cells isolated from the lung of a normal Chinese hamster (male).
Supplier: ECACC (European Collection of Authenticated Cell Cultures)
The V79 cell line is well established in toxicology studies; stability of karyotype and morphology makes them suitable for gene toxicity assays with low background aberrations.
These cells are chosen because of their small number of chromosomes (diploid number, 2n=22) and because of the high proliferation rate (doubling time 12-14 h). The V79 cell line was established after spontaneous transformation of cells isolated from the lung of a normal Chinese hamster (male). This cell line was purchased from ECACC. The cell stocks are kept in liquid nitrogen. Checking of mycoplasma infections is carried out before freezing. Trypsin-EDTA (0.25 % Trypsin, 1 mM EDTA × 4 Na) solution is used for cell detachment to subculture. The laboratory cultures are maintained in 75 cm² plastic flasks at 37°C in a humidified atmosphere containing 5 % CO2. The V79 cells for this study are grown in DME (Dulbecco’s Modified Eagle’s) medium supplemented with L-glutamine (2 mM) and 1 % of Antibiotic-antimycotic solution (containing 10000 NE/mL penicillin, 10 mg/mL streptomycin and 25 μg/mL amphotericin-B) and heat-inactivated foetal bovine serum (final concentration 10 %). During the 4 and 24-hour treatments with test item, solvent (negative control) and positive controls, the serum content is reduced to 5 %.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix of phenobarbital and β-naphthoflavone induced rat liver

Type and composition of metabolic activation system:
- source of S9 : Trinova Biochem GmbH (Rathenau Strasse 2; D-35394 Giessen, Germany; Manufacturer: MOLTOX INC., P.O. BOX 1189; BOONE, NC 28607 USA)
- method of preparation of S9 mix : n/a
- concentration or volume of S9 mix and S9 in the final culture medium
The complete S9-mix was freshly prepared containing components with the following ratios:
S9 fraction 3 mL*
HEPES** 20 mM 2 mL
KCl 330 mM 1 mL
MgCl2 50 mM 1 mL
NADP*** 40 mM 1 mL
Glucose-6-phosphate 50 mM 1 mL
DME medium 1 mL
*= 1.5% in the treatment medium
**= N-2-Hydroxyethylpiperazine-N-2-Ethane Sulphonic Acid
***= β-Nicotinamide Adenine Dinucleotide Phosphate
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability)
A certificate of analysis was obtained from the supplier and documented with the raw data.

Test concentrations with justification for top dose:

350, 750 and 1400 µg/mL
In this In Vitro Mammalian Cell Micronucleus Test, the concentration levels were chosen mainly based on the maximum recommended concentration. Since the test item has a low toxicity and a low molecular weight for this the 10mM was chosen as the highest dose (based on the updated OECD Guideline 487 (2016).

Vehicle / solvent:

DME (Dulbecco’s Modified Eagle’s) medium

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DME (Dulbecco’s Modified Eagle’s) medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

colchicine

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate) : duplicate
- Number of independent experiments : 3 treatment groups with altering conditions

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): Cells were seeded into 92 × 17 mm dishes (diameter x height, for tissue cultures in TC sterile quality) at 5 × 10exp5 cells each and were incubated for 24 hours in 10 mL of DME medium containing 10 % foetal bovine serum
- Test substance added in medium (DME)

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 24h to allow attachment
- Exposure duration/duration of treatment: 4h or 24h

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- If cytokinesis blocked method was used for micronucleus assay: indicate the identity of cytokinesis blocking substance (e.g. cytoB), its concentration, and duration and period of cell exposure. - none used
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays):
The cell pellets were kept in the latest fixative overnight at 1-10°C before slides were prepared. The slides were prepared the day after the harvest, when the cells were centrifuged and resuspended in a minimal amount of fresh fixative. Several drops of suspension were gently spread onto multiple clean, dry microscope slides. After the slides had dried the cells were stained for 25 minutes in filtered 10 % (v/v) Giemsa pH 6.8 buffer. The slides were rinsed, dried and mounted with coverslips.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored):
One thousand cells from each culture (2000 per concentration) were analysed for micronuclei. This was achieved by scoring 1000 cells from each of the duplicate test item or positive and negative control treated cultures.
- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification):
The number of cells containing micronuclei on each slide was noted. Observations were recorded on raw data sheets. A micronucleus was only recorded if it met the following criteria:
1) The micronucleus has the same staining characteristics and a similar morphology to the main nucleus, and
2) Any micronucleus present are separate in the cytoplasm or only just touching a main nucleus, and
3) Micronuclei is smooth edged and smaller than approximately one third the diameter of the main nuclei.
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): n/a
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): n/a
- Determination of polyploidy: n/a
- Determination of endoreplication: n/a

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: relative increase in cell count (RICC)
- Any supplementary information relevant to cytotoxicity: n/a

METHODS FOR MEASUREMENTS OF GENOTOXICITY
micronuclei frequency

Rationale for test conditions:

The objective of this study was to evaluate the clastogenic and aneugenic potential of Tris(hydroxymethyl)phosphine oxide (THPO) by its effects on the frequency of micronuclei in cultured Chinese hamster lung cell line treated in the absence and presence of a rat liver metabolising system.
The in vitro micronucleus assay is a genotoxicity test system used for the detection of micronuclei in the cytoplasm of interphase cells. These micronuclei may originate from acentric fragments (chromosome fragments lacking a centromere) or whole chromosomes that are unable to migrate with the rest of the chromosomes during the anaphase of cell division. The assay detects the activity of both clastogenic and aneugenic chemicals in cells.
This study is an requiremnt under REACH Annex VIII and performance details are described in OECD TG 487.

Evaluation criteria:

Evaluation Criteria of the Assay

Providing that all acceptability criteria are fulfilled, a test item is considered to be clearly positive if, in any of the experimental conditions examined:
• at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
• any of the results are outside the distribution of the laboratory historical negative control data (based 95% control limit),
• the increase of mutant frequency is concentration-related when evaluated with an appropriate trend test.

When all of these criteria are met, the test item is then considered able to induce chromosome breaks and/or gain or loss in this test system.

Providing that all acceptability criteria are fulfilled, a test item is considered clearly negative if, in all experimental conditions examined:
• none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
• there is no concentration-related increase when evaluated with an appropriate trend test,
• all results are inside the distribution of the historical negative control data (based 95% control limit).

The test item is then considered unable to induce chromosome breaks and/or gain or loss in this test system.

Statistics:

CHI² test

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: no impact
- Data on osmolality: no impact
- Possibility of evaporation from medium: no
- Water solubility: sufficiently soluble
- Precipitation and time of the determination: none stated

RANGE-FINDING/SCREENING STUDIES (if applicable): no limitations due to cytotoxicity or precipitation

STUDY RESULTS
- Concurrent vehicle negative and positive control data
see attached tables in overall remarks

For all test methods and criteria for data analysis and interpretation:
none relevant

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
see attached tables in overall remarks

Conclusions:

The study was conducted according to OECD TG 487 under GLP. Hence, the results can be considered reliable. The test item was tested up to limit concentrations according to the guideline, as no cytotoxicity or precipitation was noted. Therefore, Tris(hydroxymethyl)phosphine oxide (THPO) is considered as non-genotoxic with the micronucleus test.

Executive summary:

This study according to OECD TG 487 under GLP was performed to evaluate the clastogenic and aneugenic potential of the test itemTris(hydroxymethyl)phosphine oxide (THPO), by its effects on the frequency of micronuclei in the cultured Chinese hamster lung cell line V79 treated in the absence and presence of a rat liver metabolizing system.

The test item was dissolved in DME (Dulbecco’s Modified Eagle’s) medium and the following concentrations were selected on the basis of cytotoxicity investigations made in a preliminary study (with and without metabolic activation using S9 mix of phenobarbital and β-naphthoflavone induced rat liver). In this experiment (both run in duplicate with concurrent negative and positive controls) at least 2000 cells were analyzed for micronucleis at concentrations and treatment (exposure)/sampling (expression) intervals given below:

 

4/24 h treatment/sampling time

without S9-mix: 350, 750 and 1400g/mL test item

24/24 h treatment/sampling time

without S9-mix: 350, 750 and 1400g/mL test item

4/24 h treatment/sampling time

with S9-mix: 350, 750 and 1400g/mL test item

 

In this In Vitro Mammalian Cell Micronucleus Test, the concentration levels were chosen mainly based on the maximum recommended concentration. Since the test item has a low toxicity and a low molecular weight for this the 10mM was chosen as the highest dose (based on the updated OECD Guideline 487 (2016).

 

In the absence and presence of metabolic activation no cytotoxicity was observed up to the highest applied concentration of 1400 µg/mL THPO (main constituent).

 

In this In Vitro Mammalian Cell Micronucleus Test, the frequency of the cells with micronuclei did not show biologically and statistically significant increases compared to the concurrent and historical controls whenTris(hydroxymethyl)phosphine oxide (THPO)was examined in the absence and in the presence of metabolic activation, up to the1400 µg/mLconcentration.All values were within the range of the laboratory historical control data.

 

There was no precipitation of the test item at any dose level tested. No biologically relevant changes in pH or osmolality of the test system were noted at the different dose levels tested.

 

In the concurrent negative control groups the percentage of cells with micronuclei was in the historical control range. The positive controls in the absence ofmetabolic activationMitomycin C(0.8 µg/mL/ 4 and 24 hours treatment) and Colchicine (0.15 µg/mL 4-hour treatment and 0.03 µg/mL 24-hour treatment) and in the presence ofmetabolic activationCyclophosphamide (6.25 µg/mL 4-hor treatment) caused expected biologically relevant increases of cells with micronuclei compared to the solvent and were compatible with the historical positive control data.

 

 

Tris(hydroxymethyl)phosphine oxide (THPO) testedup to the maximum recommended concentration (1400 µg/mL),both with and without metabolic activation, did not induce breakage and / or chromosomal loss in Chinese Hamster lung cells under the test conditions. Therefore,Tris(hydroxymethyl)phosphine oxide (THPO)is considered as non-genotoxic with the micronucleus test.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental phase: November 10, 2020 - November 26, 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)

Version / remarks:

adopted 29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Target gene:

The method is based on the detection of mutations (either induced or spontaneously generated) in the hypoxanthine-guanine phosphoribosyl transferase enzyme locus (hprt) located on the X chromosome.

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Sub-line (K1) of Chinese hamster ovary cell line CHO (CHO K1) supplied by ECACC (European Collection of Authenticated Cell Cultures)
- Suitability of cells: one of the most commonly used cells.

For cell lines:
- Absence of Mycoplasma contamination: absence of mycoplasma contamination was tested by Central Agricultural Office, National Animal Health Institute, Budapest, Hungary;
- Cell cycle length, doubling time or proliferation index: doubling time of cells is about 22 hours.


MEDIA USED
The CHO K1 cells for this study were grown in Ham's F12 medium (F12-10) supplemented with 1 % Antibiotic-antimycotic solution (containing 10000 U/mL penicillin, 10 mg/mL streptomycin and 25 µg/mL amphotericin-B) and heat-inactivated bovine serum (final concentration 10 %). During the treatments with the test item, solvent (negative control) and positive controls, the serum content was reduced to 5 % (F12-5). The selection medium (F12*-SEL) for 6-TG resistant mutants contained 3.4 µg/mL 6-thioguanine (6-TG) (EX-CELL® CD CHO Serum-Free Medium for CHO Cells-SEL).
For each experiment the cells were thawed rapidly, diluted in Ham's F12 medium containing 10 % foetal bovine serum and incubated at 37°C in a humidified atmosphere of 5 % CO2 in air

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: S9 was provided by Trinova Biochem GmbH, Giessen, Germany (manufacturer: Moltox Inc., USA).
- method of preparation of S9 mix: S9 fraction of phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver
- concentration or volume of S9 mix and S9 in the final culture medium: 40 µL/mL
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): A certificate of analysis was obtained from the supplier and documented with the raw data.

Test concentrations with justification for top dose:

Based on the result of the preliminary cytotoxicity assay, the concentration levels for the performed Mutation Assay were chosen according to the maximum recommended. Since the test item has a low toxicity and a low molecular weight for this the 10 mM was chosen as the highest dose (based on the updated OECD Guideline 476 (2016).
The Mutation Assay was performed with and without metabolic activation system at the concentrations given below (5-hour treatment period):
175, 350, 700 and 1400 µg/mL.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used:
a) negative controls: Ham's F12 medium = solvent of test item and positive control (EMS); Dimethyl sulfoxide (DMSO) = solvent of positive control (DMBA);
b) positive controls:
- without metabolic activation: Ethyl methanesulfonate (EMS), dissolved in Ham's F12 medium at a final concentration of 1.0 µL/mL;
- With metabolic activation: 7,12-Dimethyl benzanthracene (DMBA), dissolved in DMSO at a final concentration of 20 µg/mL.

- Justification for choice of solvent/vehicle: The test item was dissolved in Ham's F12 medium, whereby a clear solution was obtained up to a concentration of 200 mg/mL. This solvent is compatible with the survival of the CHO cells and the S9 activity and was chosen based on the results of the preliminary solubility test. The suitability of the solvent was confirmed with the available laboratory’s historical database.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: 5 replicate s of 2 cultures
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 5 x10^6 cells were each placed in sterile dishes and incubated for approximately 24 hours before treatment
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 5-hour treatment
- Harvest time after the end of treatment (sampling/recovery times): cells were harvested after 19-hour incubation period

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): After the treatment period, cells were cultured to allow expression of the mutant phenotype. During the expression period, cells were regularly sub-cultured to maintain them in exponential growth until day 8.
- Selection time (if incubation with a selective agent): The selection period was 8 days.
- If a selective agent is used (e.g., 6-thioguanine or trifluorothymidine), indicate its identity, its concentration and, duration and period of cell exposure:
Cells were cultured in selection medium (hypoxanthine Ham's F12-SEL medium) containing 3.4 µg/mL of 6-thioguanine (6-TG, Sigma-Aldrich Chemie GmbH, Germany, Lot: SLBZ6113).
- Number of cells seeded and method to enumerate numbers of viable and mutants cells:
2 x 10^5 cells / dish in 4 x five dishes per treatment, yielding 4x10^6 cells per treatment were cultured for selection.
The mutation frequency was calculated by dividing the total number of mutant colonies by the number of cells selected (4x10^6 cells: 4 x 5 dishes at 2x10^5 cells/dish), corrected for the cloning efficiency of cells prior to mutant selection (viability), and was expressed as 6-TG resistant mutants per 10^6 clonable cells


METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: cloning efficiency; relative survival (RS), other: relative population growth
- Any supplementary information relevant to cytotoxicity:
Determination of "Relative Survival to Treatment" is performed by the following calculation:
Relative Survival (%) = (Average number of colonies per treated culture / Average number of colonies per Solvent (vehicle) control dish) x 100.

The assay parameter "Relative Population Growth" shows the cumulative growth of the treated cells, relative to the control, over the expression period and prior to mutant selection:
Relative Population Growth (%) = (Treated culture population increase over the expression period / Solvent (Vehicle) control culture population increase over the expression period) x 100.

The colony forming ability of cells is measured by "Absolute Cloning Efficiency"(CE) at the time of mutant selection:
Absolute Cloning Efficiency (%) = (Average number of viable colonies per dish / 200) x100.

Rationale for test conditions:

Based on the result of the preliminary cytotoxicity assay (absence of cytotoxicity), the concentration levels for the performed Mutation Assay were chosen according to the maximum recommended for lower- cytotoxic substances in OECD Guideline 476 as well as the number of cells analysed.

Evaluation criteria:

Providing that all acceptability criteria are fulfilled, a test item is considered to be clearly positive if, in any of the experimental conditions examined:
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- any of the results are outside the distribution of the laboratory historical negative control data (based 95 % control limit),
- the increase of mutant frequency is concentration-related when evaluated with an appropriate trend test.
Providing that all acceptability criteria are fulfilled, a test item is considered clearly negative if, in all experimental conditions examined:
- none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- there is no concentration-related increase when evaluated with an appropriate trend test,
- all results are inside the distribution of the historical negative control data (based 95 % control limit).

Statistics:

Statistical Analysis was performed with SPSS PC+ software for the following data:
- mutant frequency between the negative (solvent) control group and the test item or positive control item treated groups.
- mutant frequency between the laboratory historical negative (solvent) control group and concurrent negative (solvent) control, the test item or positive control item treated groups.
- The lower and upper 95 % confidence intervals of historical control were calculated with C-chart.
- The data were checked for a linear trend in mutant frequency with treatment dose using the adequate regression analysis by Microsoft Excel software.

Species / strain:

Chinese hamster Ovary (CHO)

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

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: The pH values of test item solutions did not show any significant alterations compared to the concurrent control groups
- Data on osmolality: In the Main Mutation Assay the osmolality values of treatment solutions were similar compared to the concurrent control.
- Water solubility: 1 g /mL, predicted with WSKOWWIN 1.42 (US EPA)
- Precipitation and time of the determination: There was no precipitation of the test item at any dose level tested.

RANGE-FINDING/SCREENING STUDIES:
Treatment concentrations for the mutation assay were selected on the basis of the result of a pre-test on toxicity. In the pre-test on toxicity, precipitations were not observed at the applied concentrations. In addition, pH and osmolality was considered for dose level selection. Based on the result of the preliminary cytotoxicity assay, the concentration levels for the performed Mutation Assay were chosen according to the maximum recommended for lower- cytotoxic substances in OECD Guideline 476 (updated 2016). Four concentrations were selected for the treatment in the main mutation assay with and without metabolic activation.

STUDY RESULTS
- Concurrent vehicle negative and positive control data : see 'Attached background material'

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship: not observed
- Statistical analysis: positive controls showed statistically significant increase in mutant freqencies (p < 0.01), test item showed no statistically significant increase in mutant frequencies

Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements:
o Relative total growth (RTG) or relative survival (RS) and cloning efficiency: no significant decrease in relative population growth, relative survival or cloning efficiency observed for test item treated cultures

- Genotoxicity results:
o Number of cells treated and sub-cultures for each cultures: Approximately 5x10^6 cells/concentration were used and incubated for 24 hours before treatment and approximately 20x10^6 cells / concentration were treated.
o Number of cells plated in selective medium: 2x10^5 cells /dish for mutant selection (4x 5 dishes, yielding 4x10^6 cells per treatment)
o Number of resistant colonies in selective medium, and related mutant frequency: see 'Attached background material'

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95 % control limits for the distribution as well as the number of data)
- Positive historical control data: see 'Attached background material'
- Negative (solvent/vehicle) historical control data: see 'Attached background material'

Conclusions:

- negative with and without activation in Chinese hamster ovary cells: Tris(hydroxymethyl)phosphine oxide (THPO) is not mutagenic;
- no cytotoxicity up to the highest applied concentration (with and without activation).

Executive summary:

The test item Tris(hydroxymethyl)phosphine oxide (THPO) was tested in a Mammalian Cell Gene Mutation Test (HPRT test) in CHO-K1 cells in accordance with OECD Guideline 476 and under GLP compliance.

The purpose of this study was to determine whether the test item or its metabolites can induce forward mutation at the hypoxanthine-guanine phosphoribosyl transferase enzyme locus (hprt) in cultured Chinese hamster cells. The test item was dissolved in Ham's F12 medium and the concentrations of the main test were selected on the basis of cytotoxicity investigations made in a preliminary study without and with metabolic activation using S9 mix of phenobarbital and β-naphthoflavone induced rat liver.

In this Mammalian Cell Gene Mutation Test (HPRT test), the concentration levels were chosen mainly based on the maximum recommended concentration. Since the test item has a low toxicity and a low molecular weight, 10 mM was chosen as the highest dose (based on the updated OECD Guideline 476 (2016).

The Mutation Assay was performed at the concentrations and treatment intervals given below:

- Mutation Assay 5-hour treatment period without S9-mix: 175, 350, 700 and 1400 µg/mL

- Mutation Assay 5-hour treatment period with S9-mix: 175, 350, 700 and 1400 µg/mL

Following treatment and 19 hours incubation period, phenotypic expression was allowed for 8 days followed by a mutant selection period for another 8 days.

In the absence and in the presence of metabolic activation no cytotoxicity was observed up to the highest applied concentration of 1400 µg/mL.

In this In Vitro Mammalian Cell Gene Mutation Test, the frequency of the cells with mutations did not show biologically and statistically significant increases compared to the concurrent and historical controls when Tris(hydroxymethyl)phosphine oxide (THPO) was examined in the absence and in the presence of metabolic activation. All values were within the range of the laboratory historical control data and no dose-response relationships were noted.

No biologically relevant changes in pH or osmolality of the test system were noted at the different dose levels tested.

The mutation frequency found in the solvent controls was well within the range of historical laboratory control data. The concurrent positive controls Ethyl methanesulfonate (1.0 µL/mL) and 7, 12-Dimethyl benzanthracene (20 µg/mL) caused the expected biologically relevant and statistically significant increase of cells with mutation frequency as compared to solvent controls and were compatible with the historical positive control data. Thus, the study is considered valid.

In conclusion, Tris(hydroxymethyl)phosphine oxide (THPO) tested up to the maximum recommended concentration (1400 µg/mL) without and with metabolic activation system over a 5-hour treatment period did not induce statistically and biologically significant increases in mutant frequency compared to the solvent control.

Thus, it is concluded that the test item, Tris(hydroxymethyl)phosphine oxide (THPO), was not mutagenic in this In Vitro Mammalian Cell Gene Mutation Test performed with Chinese hamster ovary cells.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

No definitive human relevance framework can be described due to the lack of any test item related genotoxic and mutagenic activity in vitro. Despite the fact that no mode of action analysis can be performed, no data gap was identified here. The tonnage-driven data requirements under REACH were fully met, and the database is of good quality, sufficient to exclude that any risk with regard to genotoxic effects may arise for humans from Tris(hydroxymethyl)phosphine oxide (THPO).

Additional information

The substance Tris(hydroxymethyl)phosphine oxide (THPO) did not show genotoxic potential in an Ames test, in a Mammalian Cell Micronucleus Test and in a Mammalian Cell Gene Mutation Test.

Justification for classification or non-classification

The substance Tris(hydroxymethyl)phosphine oxide (THPO) did not show genotoxic potential in an Ames test, in a Mammalian Cell Micronucleus Test and in a Mammalian Cell Gene Mutation Test.

Based on these in vitro results, THPO is considered to be not genotoxic and does not require classification according to Regulation (EC) No. 1272/2008.