Tetrakis(hydroxymethyl)phosphonium chloride

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro DNA damage and/or repair study

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

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

Adequacy of study:

key study

Study period:

August 1989 to april 1990

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study with no major deficiencies.

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1990

Report date:

1990

Reference 2

Reference Type:

other: supplemental information to the study IRI 741343

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)

Deviations:

yes

Remarks:

Evaluation criteria for a positive result were not analysed for statistical significance. Test substance stability was not tested

GLP compliance:

yes (incl. QA statement)

Type of assay:

DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro

Test material

Test material information

Constituent 1

Reference substance name:

Tetrakis(hydroxymethyl)phosphonium sulphate(2:1)

EC Number:

259-709-0

EC Name:

Tetrakis(hydroxymethyl)phosphonium sulphate(2:1)

Cas Number:

55566-30-8

Molecular formula:

C4H12O4P.1/2O4S

IUPAC Name:

bis[tetrakis(hydroxymethyl)phosphonium] sulphate

Method

Target gene:

not applicable

Species / strain

Species / strain / cell type:

hepatocytes: primary cultures (rat)

Metabolic activation:

without

Metabolic activation system:

not required

Test concentrations with justification for top dose:

0.6, 1.2, 2.4, 4.7, 9.7, 18.9, 37.8, 75.5 µg  main ingredient/mL 
(0.78, 1.56, 3.13, 6.25, 12.5, 25, 50.0 and 100.0 µg active substance/mL).  

Vehicle / solvent:

sterile ultra pure water

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Remarks:

vehicle control; water

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

Remarks:

Migrated to IUCLID6: for indirect acting, at 0.5 and 2.0 µg/mL

Controls 2

Positive controls:

yes

Positive control substance:

other: 4,4'-bis-dimethylaminobenzophenone (Michler's ketone) for direct acting, at 2 and 8 µg/mL

Details on test system and experimental conditions:

Cultures were established with hepatocytes derived from the liver of a young adult male Fischer 344 rat, according to the modified
procedures of Williams et al., 1982. The rat was killed by overdose of halothane followed by cervical dislocation.
THPS-75 and 10 µCi/mL tritiated thymidine ([6-3H]-TdR) were added together to the culture. THPS was tested in quadruplicate wells.
The hepatocyte cultures with test or control solution and tritiated thymidine were incubated at 37°C for a total of 18-20 hours, then washed and treated with 1% sodium citrate for 10 minutes to allow the nuclei to swell (to permit better quantification of nuclear grains).
After 7 days, autoradiographs were developed and slides stained, dried and covered for microscopic examination.
UDS was measured by silver grain counts in the photographic emulsion in triplicate coded cultures at each test level, in two separate
experiments.
Both positive controls (2-AAF and Michler's ketone) and negative controls (ultra pure water) were tested in parallel with the test sets.

DURATION: 18-20 hours in two separate examinations as required for autoradiographic determinations in OECD Guidelines 482

STAIN (for cytogenetic assays):slides were stained in methyl-green/ pyronin Y solution

NUMBER OF CELLS EVALUATED: Mean net nuclear grain count was determined from triplicate cover slips (150 total nuclei)
for each treatment in two separate experiments

DETERMINATION OF CYTOTOXICITY
- Method: count of the nuclear grains and substract the average number of cytoplasmic grains in 3 muclear sized areas adjacent to each
nucleus (background count). This value was reffred to as the nuclear grain count. The cover-slpis were coded to prevent bias while grain
counting. The data were recorded as the average net grain counts for 3 cultures.

Evaluation criteria:

Criteria for assessment of a test substance as positive in this study were:
1. An increase in mean net nuclear grain count to at least 6 grains per nucleus in excess of concurrent vehicle control value,
And/or
2. Percentage nuclei with 6 or more net grains to increase above 10% of the examined population, in excess of the concurrent vehicle
control value,
And/or
3. Percentage nuclei with 20 or more net grains to reach or exceed 2% of the examined population.
A population average between 0 and 6 net grains would be considered a marginal response.

Statistics:

No statistical analysis for significance of results was carried out.

Results and discussion

Test results

Species / strain:

hepatocytes: Rat hepatocyte primary cultures

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from 50 µg/mL

Vehicle controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Toxicity of  THPS-75 was observed in the range of 37.7-75.5 µg/mL as main ingredient.
Both direct and indirect acting positive controls demonstrated the sensitivity of the test system.
The test substance was assessed not to have met any of the criteria for a clearly positive result in either experiment.  In the second experiment,  

mean net grains per nucleus of up to 3.13 at the highest concentrations  could be considered as marginal result. 

There were some evidences of a dose response relationship in both mean net grains per nucleus and % nuclei with =6 net  grains 

in this experiment only, and mainly at toxic concentrations where a reduced number of cells was scored.
Overall, THPS-75 is concluded not to induce unscheduled DNA synthesis.

Table 1: Unscheduled DNA synthesis in cultured rat hepatocytes: Experiment 1

Compound (µg/mL)		Cells scored	Mean net grains per nucleus	% nuclei with ≥6 net grains	% nuclei with ≥20 net grains
Water (10µL added) Solvent 1 Solvent 2		150 150	-1.63 -0.05	0.67 5.33	0.00 0.00
Michler’s ketone 2.0 8.0		150 100	7.78 7.89	66.0 63.0	0.00 2.00
2-AAF 0.5 2.0		150 150	7.17 7.76	60.67 64.67	0.00 0.67
THPS-75		150 100 100 100 100 150 50 75	-0.65 -0.22 -0.29 -0.37 0.71 -0.41 1.91 1.12	3.33 0.00 4.00 5.00 5.00 0.00 14.00 1.33	0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
Active substance	Main ingredient
0.78	0.6
1.56	1.2
3.13	2.4
6.25	4.7
12.5	9.4
25	18.9
50	37.8
100	75.5

Table 2: Unscheduled DNA synthesis in cultured rat hepatocytes: Experiment 2

Compound (µg/mL)		Cells scored	Mean net grains per nucleus	% nuclei with ≥6 net grains	% nuclei with ≥20 net grains
Water (10µL added) Solvent 1 Solvent 2		50 100	1.15 1.80	6.00 18.00	0.00 0.00
Michler’s ketone 2.0 8.0		30 20	53.44 50.95	100.0 100.0	100.00 100.00
2-AAF 0.5 2.0		20 10	53.04 57.20	100.0 100.0	100.00 100.00
THPS-75		150 100 100 150 100 150 50 50	-0.33 -0.94 -0.13 -2.26 0.08 2.27 2.83 3.13	4.67 7.00 4.00 1.33 9.00 18.67 14.00 22.00	0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
Active substance	Main ingredient
0.78	0.6
1.56	1.2
3.13	2.4
6.25	4.7
12.5	9.4
25	18.9
50	37.8
100	75.5

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

THPS is concluded not to induce unscheduled DNA synthesis in this system (rat hepatocytes primary culture)

Executive summary:

THPS 75%, with a main ingredient concentration of 75.5%, was tested for its potential for unscheduled DNA synthesis according to OECD Guidelines 482.

Testing was carried out at concentration of 0.78,1.56,3.13,6.25,12.5, 25, 50.0 and 100.0 µg/mL as active substance, based on cytotoxicity at 333 µg/mL as active substance (251.4 µg/mL as main ingredient) in an Ames test (see "Ames_Dillon_1990" in this section). No metabolic activation was required for freshly prepared hepatocytes.

Cultures were established with hepatocytes derived from the collagenase-perfused liver of a young adult male Fischer 344 rat.

THPS-75 and 10µCi/mLtritiated thymidine ([6-³H]-TdR) were added together to the culture.

Both positive controls (2-AAF and Michler’s ketone) and negative controls (ultra pure water) were tested in parallel with the test sets.

The hepatocyte cultures with test or control solution and tritiated thymidine were incubated at 37°C for a total of 18-20 hours, then washed and treated with 1% sodium citrate for 10 minutes to allow the nuclei to swell (to permit better quantification of nuclear grains.

After 7 days, autoradiographs were developed and slides stained, dried and covered for microscopic examination. UDS was measured by silver grain counts in the photographic emulsion in triplicate coded cultures at each test level, in two separate experiments.

Toxicity of THPS-75 was observed in the range of 37.7-75.5 µg/mL as main ingredient.

Both direct and indirect acting positive controls demonstrated the sensitivity of the test system.

The test substance was assessed not to have met any of the criteria for a clearly positive result in either experiment. In the second experiment, mean net grains per nucleus of up to 3.13 at the highest concentrations could be considered a marginal result. There was some evidence of a dose response in both mean net grains per nucleus and % nuclei with ≥ 6 net grains in this experiment only, and mainly at toxic concentrations where a reduced number of cells was scored.

Overall, THPS is concluded not to induce unscheduled DNA synthesis.