Tetrakis(hydroxymethyl)phosphonium chloride, oligomeric reaction products with urea

Endpoint summary

Additional information

Gene mutation potential:

One mammalian cell gene mutation assay is available (Wollny, 2010) and considered as the key study based on its reliability 1 according to the Klimisch cotation criteria. This study was performed according to the OECD 476 and in compliance with GLP.

Chinese hamster lung fibroblasts cells (V79) cultured in vitro were exposed to Tetrakis (hydroxymethyl) phosphonium chloride, oligomeric reaction products with urea (THPC-urea) at concentration ranging from 5.2 to 83.2 µg AI/mL in the presence and absence of mammalian metabolic activation (S9 mix induced by Phenobarbital/Beta-Naphthoflavone) in two independent experiments. A pre-test was performed in order to determine the concentration range for the mutagenicity experiments. No precipitation of the test item was observed up to the maximum concentration of both main experiments. In the first main experiment, cytotoxic effects were observed at 41.6 µg AI/mL without metabolic activation and at 83.2 µg AI/mL and above with metabolic activation. In the second main experiment, cytotoxic affects occurred at 32.5 µg AI/mL and above without and at 65.0 µg AI/mL and above with metabolic activation. No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments, neither with nor without metabolic activation. The positive controls EMS and DMBA had a marked mutagenic effect, as was seen by a biologically relevant increase in mutant frequencies as compared to the corresponding untreated controls and thus demonstrated the sensitivity of the test system and the activity of the used S9 mix. In conclusion it can be stated that the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, Tetrakis(hydroxymethyl) phosphonium chloride, oligomeric reaction products with urea is considered to be non-mutagenic.

One bacterial cell gene mutation assay is available (Sire, 2014) and considered as the key study based on its reliability 1 according to the Klimisch cotation criteria. This study was performed according to the OECD 471 and in compliance with GLP.

Bacterial strains S. Typhimurium cultured in vitro were exposed to Tetrakis (hydroxymethyl) phosphonium chloride, oligomeric reaction products with urea (THPC-urea) at concentration ranging from 3.91 to 1000 µg AI/plate in the presence and absence of mammalian metabolic activation (S9 mix induced by Arochlor 1254) in two independent experiments. A pre-test was performed in order to determine the concentration range for the mutagenicity experiments.

Experiments without S9 mix

The selected treatment-levels were ranged from 3.91 to 1000 µg/plate.

No precipitate was observed in the Petri plates when scoring the revertants at any dose-levels.

A moderate to strong toxicity (decrease in the number of revertants and/or thinning of the bacterial lawn) was noted at dose-levels >62.5 µg/plate in the TA 1537 strain, >125 µg/plate in the TA 1535 and TA 100 strains and >250 µg/plate in the TA 98 and TA 102 strains.

Slight increases in the number of revertants were noted in the TA 98 strain in the first experiment. These increases seemed dose-related and exceeded the positive threshold of 2-fold the vehicle control value. Nevertheless the mean number of revertants remained within the vehicle control historical range (up to 39 revertants/plate versus [12-39] for the vehicle control historical data). Furthermore, no similar results were noted either in the second or in the third experiment. The slight increases noted in the first experiment were therefore considered to be not biologically relevant.

 

The test item did not induce any other noteworthy increase in the number of revertants, in any other strains or test conditions.

 

Experiments with S9 mix

The selected treatment-levels were ranged from 3.91 to 1000 µg/plate.

 

No precipitate was observed in the Petri plates when scoring the revertants at any dose-levels.

 

A moderate to strong toxicity (decrease in the number of revertants and/or thinning of the bacterial lawn) was noted at dose-levels >125 µg/plate in the TA 1535, TA 1537, TA 98 and TA 100 strains and > 250 µg/plate in the TA 102 strain.

 

Slight increases in the number of revertants were noted in the TA 98 strain in the second experiment (pre‑incubation method). These increases seemed dose-related, they exceeded the positive threshold of 2-fold the vehicle control value and the mean number of revertants exceeded the vehicle control historical range (up to 67 revertants/plateversus[16-44] for the vehicle control historical data). Nevertheless, no similar effect was observed either in the third experiment using the pre-incubation method, despite the use of a narrower range of dose-levels, or in the first and third experiments using the direct plate incorporation method. Since the slight increases noted in the second experiment were not reproducible they were considered to be not biologically relevant.

 

The test item did not induce any other noteworthy increase in the number of revertants, in any other strains or test conditions.

for the vehicle control historical data). Furthermore, no similar results were noted either in the second or in the third experiment. The slight increases noted in the first experiment were therefore considered to be not biologically relevant.

 

The test item did not induce any other noteworthy increase in the number of revertants, in any other strains or test conditions.

 

Chromosomal aberration potential:

One cytogenetic study in Human Lymphocyes cells (SPL, 1992) was available and considered as the key study (Kr:2). This study was performed according to the OECD 473 and in compliance with GLP. Human Lymphocytes cells were exposed to Tetrakis(hydroxymethyl) phosphonium chloride, oligomeric reaction products with urea within two tests. In the first chromosome aberration test, in both presence and absence of metabolic activation, the treatment harvesting time was 20 hours and the concentrations selected were 0, 7, 13 and 26 µg AI/mL without metabolic activation and 0, 26, 53 and 103 µg AI/mL with S9 -mix. In the second chromosomal aberration test, the treatment harvesting time was 20/44 hours and the concentrations selected were 0, 7, 13 and 26 µg AI/mL without S9 mix and at 0, 7, 13 and 26 µg AI/mL with S9 -mix.

Positive controls (cyclophosphamide with S9 and ethylmethanesulphonate without S9) induced the appropriate positive response while negative control (solvent) showed no chromosomal aberrations.

In the first test, THPC-urea did not induced a statistically significant increases in chromosomal aberrations at all dose levels with and without metabolic activation. In the second test, THPC-urea induced a statistically significant increases in chromosomal aberrations at 13 and 26 µg AI/mL with and without metabolic activation for a harvest time of 20 hours but not after 44 hours of harvest.

Under the test conditions of this study, the Authors concluded that the test material is clastogenic to human lymphocytes in vitro.

But given the results described above, the report study was reviewed by an independent Toxicologist from Central Toxicology Laboratory of Zeneca Limited (Bailey, 1995). He considered that given the lack of reproductibility of the results and the absence of the clastogenic effects after 44 hours of harvest (+/-S9), the test is ambiguous and a third experiment in the same conditions is necessary.

In order to clarify these ambiguous results, a micronucleus test in mouse was adopted (SPL, 1995). This study was performed according to the OECD 474 and in compliance with GLP and considered as the key study (Kr:1). Animals were treated by gavage with THPC-urea at doses of 140, 280 and 561 mg AI/kg b.w. Bone marrow cells were harvested at 24 hours post-treatment for all doses tested groups and at 48 hours for the highest dose group.There was no statistically significant change in the PCE/NCE ratio in any of the test material dose groups when compared to their concurrent vehicle (water) control groups, however, a dose-related reduction was observed in the 24 -hour groups, this together with the presence of clinical signs and premature deaths would indicate systemic absorption had occured. The positive control (cyclophosphamide) induced the appropriate response. There was not a significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any treatment time. Therefore, THPC-urea was considered as not clastogenic.

Conclusion:

Based on all these results, Tetrakis (hydroxymethyl) phosphonium chloride, oligomeric reaction products with urea was considered as not genotoxic.

Justification for selection of genetic toxicity endpoint
No study was selected, since all in vitro and in vivo studies were negative.

Short description of key information:
- Mutagenicity: negative (OECD 476, Kr:1)
- Mutagenicity: negative (OECD 471, Kr:1)
- Clastogenicity/Aneugenicity: Ambiguous (In vitro test (OECD 473, Kr:2) but negative in in vivo test (OECD 474, Kr:1)).

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on absence of mutagenicity in mammalian and bacterial cells and absence of clastogenicity in chromosome aberration study in vivo,Tetrakis (hydroxymethyl) phosphonium chloride, oligomeric reaction products with urea was considered as not genotoxic. Therefore no classification is required according to the EU legislation (Directive 67/548/EEC and CLP regulation (1272/2008)).