Phosphonic acid, calcium salt (2:1)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

In the assessment of calcium dihydrogenphosphite (Ca(H2PO3)2, CAS 13780-04-6), a read-across approach is followed based on the information available for potassium phosphonate (KH2PO3/K2HPO3 EC 915-179-9). This read-across strategy is based on the hypothesis that the phosphite anion is the driver for the ecotoxicological and toxicological effects of both salts.The read-across hypothesis is justified by the immediate dissociation of calcium dihydrogenphosphate and potassium phosphonate upon dissolution in aqueous media. Both phosphite salts are highly soluble (>200 g/L) and are only present in their dissociated form in solution, i.e. the calcium or potassium cation and the phosphite anion. The transformation of the salts into the ions is rapid and complete in relevant environmental and physiological media and therefore no systemic exposure to the salts as such occurs. Exposure to the non-common cations (Ca2+ and K+) does not influence the prediction of the (eco)-toxicity because both elements are abundantly present in natural environments and emissions from these salts do not significantly increase the exposure concentration for calcium and potassium. Moreover, calcium and potassium are major essential element for living organisms.Further information is included as attachment in section 13 of IUCLID.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

not specified

Key result

Species / strain:

E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

not specified

Conclusions:

Interpretation of results:negativeIt can be concluded that the test item KH2PO3/K2HPO3 does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism, under the reported experimental conditions. Under normal physiological conditions, it is expected that potassium phosphonate dissociates in the potassium cation and phospite anion. It is assumed that the phosphite anion is the active ingredient reposonsible for the effects and therefore, results for the potassium phosphonate multicomponent can also be used for assessing the genetic toxicity of calcium dihydrogenphosphite (Ca(H2PO3)2).

Executive summary:

KH2PO3/K2HPO3 was examined for the ability to induce gene mutations in tester strains of Salmonella typhimurium and Escherichia coli, as measured by reversion of auxotrophic strains to prototrophy. Under normal physiological conditions, it is expected that potassium phosphonate dissociates in the potassium cation and phospite anion. It is assumed that the phosphite anion is the active ingredient reposonsible for the effects and therefore, results for the potassium phosphonate multicomponent can also be used for assessing the genetic toxicity of calcium dihydrogenphosphite (Ca(H2PO3)2).

The five tester strains TA1535, TA1537, TA98, TA100 and WP2 uvrA were used. Experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with phenobarbitone and betanaphthoflavone.

The test item was used as a solution in sterile water for injection. Dose levels are expressed as active constituents.

The test item KH2PO3/K2HPO3 was assayed in the toxicity test at a maximum concentration of 5000 µg/plate and at four lower concentrations spaced at approximately half-log intervals: 1580, 500, 158 and 50.0 µg/plate. No precipitation of the test item was observed at the end of the incubation period at any concentration. No toxicity was observed with any tester strain at any dose level, in the absence or presence of S9 metabolism. Under the assumption that the phosphite anion is the driver for the effects, these ranges correspond to 4206 - 42 µg Ca(H2PO3)2 / plate, based on equimolar concentrations of the phosphite anion.

In Main Assay I, using the plate incorporation method, the test item KH2PO3/K2HPO3 was assayed at the maximum dose level of 5000 µg/plate and at four lower dose levels spaced by two-fold dilutions: 2500, 1250, 625 and 313 µg/plate, corresponding to 4206, 2103, 1052, 526 and 263 µg Ca(H2PO3)2 / plate under equimolar concentrations for the phosphite anion. No toxicity was observed with any tester strain at any dose level, in the absence or presence of S9 metabolism.

As no relevant increases in revertant numbers were observed at any concentration tested, a pre-incubation step was included for all treatments of Main Assay II. The test item was assayed at the same dose levels used in Main Assay I. No toxicity was observed with any tester strain at any dose level, in the absence or presence of S9 metabolism.

No precipitation of the test item was observed at the end of the incubation period, at any concentration in any experiment.

The test item did not induce two-fold increases in the number of revertant colonies in the plate incorporation or pre-incubation assay, at any dose level, in any tester strain, in the absence or presence of S9 metabolism.

It is concluded that the test item KH2PO3/K2HPO3 and Ca(H2PO3)2 do not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism, under the reported experimental conditions.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

In the assessment of calcium dihydrogenphosphite (Ca(H2PO3)2, CAS 13780-04-6), a read-across approach is followed based on the information available for potassium phosphonate (KH2PO3/K2HPO3 EC 915-179-9). This read-across strategy is based on the hypothesis that the phosphite anion is the driver for the ecotoxicological and toxicological effects of both salts.The read-across hypothesis is justified by the immediate dissociation of calcium dihydrogenphosphate and potassium phosphonate upon dissolution in aqueous media. Both phosphite salts are highly soluble (>200 g/L) and are only present in their dissociated form in solution, i.e. the calcium or potassium cation and the phosphite anion. The transformation of the salts into the ions is rapid and complete in relevant environmental and physiological media and therefore no systemic exposure to the salts as such occurs. Exposure to the non-common cations (Ca2+ and K+) does not influence the prediction of the (eco)-toxicity because both elements are abundantly present in natural environments and emissions from these salts do not significantly increase the exposure concentration for calcium and potassium. Moreover, calcium and potassium are major essential element for living organisms.Further information is included as attachment in section 13 of IUCLID.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Evaluation criteria:

I

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Conclusions:

Interpretation of results:positive without metabolic activationnegative with metabolic activationIt can be concluded that Potassium phosphonate – Multicomponent induces micronuclei in Chinese hamster V79 cells after in vitro treatment in the absence of S9 metabolic activation, under the reported experimental conditions. Under normal physiological conditions, it is expected that potassium phosphonate dissociates in the potassium cation and phospite anion. It is assumed that the phosphite anion is the active ingredient reposonsible for the effects and therefore, results for the potassium phosphonate multicomponent can also be used for assessing the genetic toxicity of calcium dihydrogenphosphite (Ca(H2PO3)2).

Executive summary:

Potassium Phosphonate multicomponent (K2HPO3/KH2PO3) was assayed for the ability to induce micronuclei in Chinese hamster V79 cells, following in vitro treatment in the presence and absence of S9 metabolic activation.

Under normal physiological conditions, it is expected that potassium phosphonate dissociates in the potassium cation and phospite anion. It is assumed that the phosphite anion is the active ingredient reposonsible for the effects and therefore, results for the potassium phosphonate multicomponent can also be used for assessing the genetic toxicity of calcium dihydrogenphosphite (Ca(H2PO3)2).

Two main experiments were performed.

In the first experiment, the cells were treated for 3 hours in the presence and absence of S9 metabolism, respectively. The harvest time of 27 hours corresponding to approximately 2.0 cell cycle lengths was used.

As negative results were obtained, a second experiment was performed in the absence of S9 metabolism using approximately the same harvest time. A continuous treatment until harvest at 26 hours was used.

For the first main experiment, the maximum dose level for treatment was selected in agreement with the Study Protocol.

Dose levels of 5000, 2500, 1250, 625, 313, 156, 78.1 39.1 µg/mL were used both in the absence and presence of S9 metabolism.

Based on the cytotoxicity observed in the first experiment, dose levels of 4000, 3300, 2780, 2310, 1930, 1610, 1340, 1120, 930 and 775 µg/mL were selected for the second main experiment. Under the assumption that the phosphite anion is the driver for the effects, these ranges correspond to 4206 - 32.9 µg Ca(H2PO3)2/mL and 3365 - 652 µg Ca(H2PO3)2/mL for the first and second main experiment, respectively, based on equimolar concentrations of the phosphite anion.

Test item solutions were prepared in sterile water for injection.

Each experiment included appropriate negative and positive controls. Two cell cultures were prepared at each test point. The actin polymerisation inhibitor Cytochalasin-B was added prior to the targeted mitosis to allow the selective analysis of micronucleus frequency in binucleated cells.

Dose levels were selected for the scoring of micronucleated cells on the basis of the cytotoxicity of the test item treatments calculated by thecytokinesis-block proliferation index (CBPI).

In the first experiment, the cells were treated for 3 hours in the presence and absence of S9 metabolism, respectively. The harvest time of 27 hours corresponding to approximately 2.0 cell cycle lengths was used.

As negative results were obtained, a second experiment was performed in the absence of S9 metabolism using approximately the same harvest time. A continuous treatment until harvest at 26 hours was used.

For the first main experiment, the maximum dose level for treatment was selected in agreement with the Study Protocol.

Dose levels of 5000, 2500, 1250, 625, 313, 156, 78.1 39.1 µg Potassium Phosphonate multicomponent (K2HPO3/KH2PO3) /mL were used both in the absence and presence of S9 metabolism.

Based on the cytotoxicity observed in the first experiment, dose levels of 4000, 3300, 2780, 2310, 1930, 1610, 1340, 1120, 930 and 775 µg Potassium Phosphonate multicomponent (K2HPO3/KH2PO3) /mL were selected for the second main experiment.

Test item solutions were prepared in sterile water for injection.

Each experiment included appropriate negative and positive controls. Two cell cultures were prepared at each test point. The actin polymerisation inhibitor Cytochalasin-B was added prior to the targeted mitosis to allow the selective analysis of micronucleus frequency in binucleated cells.

Dose levels were selected for the scoring of micro-nucleated cells on the basis of the cytotoxicity of the test item treatments calculated by thecytokinesis-block proliferation index (CBPI).

               S9           Treatment time (hours)      Harvest time (hours)        Dose level (µg K2HPO3/KH2PO3/mL)

                                                                                                                                                                              

1             -/+                    3                                           27                            5000, 2500 and 1250

2             -                     26                                           26                            4000, 3300 and 2780

One thousand binucleated cells per culture were scored to assess the frequency of micronucleated cells.

Following treatment with the test item, no statistically significant increase in the incidence of micronucleated cells over the concurrent negative control value was observed at any dose level in the first main experiment.

In the second main experiment, statistically significant increases in the incidence of micronucleated cells over the concurrent negative control value were observed at the high and intermediate dose levels selected for scoring. In addition, a dose-effect relationship was indicated and at the highest dose level, incidences exceeded the range of RTC historical values for negative controls in both replicate cultures.

Statistically significant increases in the incidence of micronucleated cells were observed following treatments with the positive controls Cyclophosphamide, Mitomycin-C and Colchicine, indicating the correct functioning of the test system.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

In the assessment of calcium dihydrogenphosphite (Ca(H2PO3)2, CAS 13780-04-6), a read-across approach is followed based on the information available for potassium phosphonate (KH2PO3/K2HPO3 EC 915-179-9). This read-across strategy is based on the hypothesis that the phosphite anion is the driver for the ecotoxicological and toxicological effects of both salts.The read-across hypothesis is justified by the immediate dissociation of calcium dihydrogenphosphate and potassium phosphonate upon dissolution in aqueous media. Both phosphite salts are highly soluble (>200 g/L) and are only present in their dissociated form in solution, i.e. the calcium or potassium cation and the phosphite anion. The transformation of the salts into the ions is rapid and complete in relevant environmental and physiological media and therefore no systemic exposure to the salts as such occurs. Exposure to the non-common cations (Ca2+ and K+) does not influence the prediction of the (eco)-toxicity because both elements are abundantly present in natural environments and emissions from these salts do not significantly increase the exposure concentration for calcium and potassium. Moreover, calcium and potassium are major essential element for living organisms.Further information is included as attachment in section 13 of IUCLID.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

other: no increases in mutant frequency were observed in the absence or presence of S9 metabolic activation, at any concentration level. A statistically significant increase in mutant frequency highest dose level (lower than GEF, not biologically relevant)

Cytotoxicity / choice of top concentrations:

other: No relevant toxicity at 3 hrs, in absence and presence of S9 metabolic activation, at any concentration tested. Following 24 hour treatment, marked toxicity was observed at 5000 µg/mL, while no relevant toxicity was observed over the remaining doses.

Vehicle controls validity:

other: mutant frequencies fell within the normal range

Untreated negative controls validity:

other: The cloning efficiencies at day 2 fell within the range of 65-120%

Positive controls validity:

other: positive control induced clear increases in mutant frequency

Remarks on result:

other: all strains/cell types tested

Conclusions:

Interpretation of results:negativeIt can be concluded that Potassium phosphonate does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation, under the reported experimental conditions. Under normal physiological conditions, it is expected that potassium phosphonate dissociates in the potassium cation and phospite anion. It is assumed that the phosphite anion is the active ingredient reposonsible for the effects and therefore, results for the potassium phosphonate multicomponent can also be used for assessing the genetic toxicity of calcium dihydrogenphosphite (Ca(H2PO3)2).

Executive summary:

Potassium phosphonate was examined for mutagenic activity by assaying for the induction of 5 trifluorothymidine resistant mutants in mouse lymphoma L5178Y cells after in vitro treatment, in the absence and presence of S9 metabolic activation, using a fluctuation method. Under normal physiological conditions, it is expected that potassium phosphonate dissociates in the potassium cation and phospite anion. It is assumed that the phosphite anion is the active ingredient reposonsible for the effects and therefore, results for the potassium phosphonate multicomponent can also be used for assessing the genetic toxicity of calcium dihydrogenphosphite (Ca(H2PO3)2).

A solubility trial indicated that the maximum practicable concentration of the test item in the final treatment medium was 5000 µg/mL (of active constituent Potassium phosphonate) using Complete Medium (10%) as solvent. On the basis of this result, a cytotoxicity assay was performed. Both in the absence and presence of S9 metabolic activation, the test item was assayed at a maximum dose level of 5000 µg/mL and at a wide range of lower concentrations: 2500, 1250, 625, 313, 156, 78.1, 39.1 and 19.5 µg/mL. Under the assumption that the phosphite anion is the driver for the effects, these ranges correspond to 4206 - 16.4 µg Ca(H2PO3)2 / mL, based on equimolar concentrations of the phosphite anion.

Using the short treatment time (3 hours), both in the absence and presence of S9 metabolic activation, no relevant toxicity was observed, at any concentration tested.

Using the 24 hour treatment time, the test item yielded marked toxicity at 5000 µg/mL, reducing RS to 14%. No relevant toxicity was observed over the remaining dose levels tested.

Based on the results obtained in the preliminary trial, two independent assays for mutation to trifluorothymidine resistance were performed using the dose levels described in the following table:

 S9        Treatment       time (hours)         Dose levels µg/mL (potassium phosphonate).

1                    -                     3                    5000, 2500, 1250, 625 and 313

1                    +                    3                    5000, 2500, 1250, 625 and 313

2                    -                     24                 5000, 3570, 2550, 1820, 1300 and 930

2                    +                    3                    5000, 3570, 2550, 1820, 1300 and 930

 

Adequate levels of cytotoxicity, covering a range from the maximum to slight or no toxicity, were observed in all treatment series. No increases in mutant frequencies were observed following the short treatment with the test item, in the absence or presence of S9 metabolism. A statistically significant increase in mutant frequency was observed after 24 hour treatment at the highest concentrations tested. However, this increase was lower than the global evaluation factor.

Negative and positive control treatments were included in each mutation experiment in the absence and presence of S9 metabolism. The mutant frequencies in the solvent control cultures fell within the normal range. Marked increases were obtained with the positive control treatments indicating the correct functioning of the assay system.

It can be concluded that Potassium phosphonate and calcium dihydrogenphosphite do not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation, under the reported experimental conditions.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Experimental in vitro data are available for the substance potassium phosphonate. Under normal physiological conditions, it is expected that potassium phosphonate dissociates in the potassium cation and phospite anion in aqueous media. It is assumed that the phosphite anion is the active ingredient reposonsible for the effects and therefore, results for the potassium phosphonate multicomponent can also be used for assessing the genetic toxicity of calcium dihydrogenphosphite (Ca(H₂PO₃)₂).

The substance Potassium Phosphonate did not induce reverse mutation in Salmonella typhimurium or Escherichia coli (Scarcella, 2013) and not induce mutation at the TK locus of L5178Y mouse lymphoma cells (Bisini, 2013).

Statistically significant increases in the incidence of micronucleated cells were observed in Chinese hamster V79 cells after in vitro treatment in the absence of S9 metabolic activation. Since the positive result is obtained from the test without methabolic activation it is reasonable to state that an in vivo study is not to be proposed in regards of the animal welfare, because methabolism prevent to express the suspected mutagenic potential.

 

Further considerations need to be made based on the DAR, volume 3, Annex B, B.6, April 2005 in the framework of the assesment of the substance for PPP application.

From the DAR in fact the following considerations need to be taken into account:

The salt dissociates in water to potassium and phosphite, which is readily excreted via urine and faeces.

However, there is evidence for lacking in vivo mutagenicity of phosphite from available information on the comparable active substance Fosety 1-Al: According to the U.S. EPA Registration Eligibility Document for Fosetyl-Al, eight mutagenicity tests performed with Fosetyl-Al were negative (US EPA OPP 1990). The tests included two micronucleous test in Swiss mice and CD1 mice. Fosety I-Al did not produce a mutagenic effect in any of the test

A review is available for the micronucleus test that was conducted according to OECD Guideline 474 with CD1 mice. The study was done at doses ranging from 0.6 to 3.6 g/kg (which is 75% of its LD50 value) (FELKNER 1982). A more detailed review of the same study is available from the Netherlands RIVM, Centre for Substances and Risk Assessment (BAARS 1998):

Groups of CDI mice (5 mice/sex/dose) were given orally 1 or 2 doses of 0, 600, 1200, 2400 and 3600 mg Fosetyl-Al/kg, separated by hour interval. The vehicle was 10% aqueous acacia. No increase in the incidence of polychromatic erythrocytes with micronuclei was observed in the bone marrow of any of the treatment groups, 30 hours after the single dose or 24 hours after the second dose.

Remark: The dose levels given corresponded to 12.5, 25, 50 and 75% of the acute oral LD5o (4800 mg Fosetyl-Al /kg). All animals that received 1 or 2 doses of 3600 mg Fosetyl-Al/kg per oral and 10% of those given a single dose of 2400 mg Fosetyl-Al/kg died. At the dose level of 2400 mg Fosetyl-Al /kg at 30 and 48 hours slight cytotoxicity (a decrease in polychromatic erythrocytes and nucleated cells) was observed. Thus, only mice given the 3 lower dose levels were evaluated.

Since Fosetyl Al has been shown to release phosphite anions, the results of these studies are applicable to Potassium phosphite. It is, therefore, reasonable to conclude that potassium phosphite is not mutagenic. If it were, the effect would be reflected in the tests for Fosetyl-Al.

Severalvitroandin vivomutagenicity studies were performed with Fosetyl-Al. Some of these are briefly discussed by the U.S. EPA (US EPA OPP 1990), and some, in more depth, by BAARS et al. (1998). The tests included two Ames tests with S.typhimirium,two phage induction tests using E. coli, two micronucleus tests in Swiss mice and CD-1 mice, one DNA repair test using E.colt,and onesaccaromicae cerevisiaeassay. BAARS et al. (1998) described a chromosome aberration test on mammalian cells, in which no increase was found in the number of cells with chromosome aberrations, following three hours of exposure of the cells to doses of 3 - 100 mg Fosety I-Al/ml culture medium.

Based on consistent negative results of eight studies, the U.S. EPA concluded that Fosetyl-Al is not a mutagen. Due to the fact that Fosetyl-Al releases phosphite, this conclusion is applicable to Potassium Phosphite. Each molecule of Fosetyl-Al releases 3 molecules of phosphite. Therefore, if phosphite was a mutagen, this would be reflected in the results of the studies with Fosetyl-Al. Potassium ion is not mutagenic.

 

Furthermore, an oncogenicity feeding study with monosodium phosphite was conducted in rats. The study is applicable to Potassium Phosphite. According to GREGER et al. (1991) sodium and potassium salts have the same toxicokinetics effects. Ingestion of inorganic anions, increased urinary excretion of anions, whether the anions were ingested as sodium or as potassium salts. Thus, informationon studies with sodium phosphite can be used to make inferences to Potassium Phosphite

 

Based on all above considerations no proposal for genotoxicity is made for calcium dihydrogenphosphite.

Justification for selection of genetic toxicity endpoint

No selection can be made, since the assessment is made on a Weight of evidence approach

Short description of key information:

Non mutagen

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The substance is not classified genetic mutagen because it doesn't meet the classification criteria of the CLP regulation n. 1272/2008:

- Category 1 (1A; 1B): substances known to induce heritable mutations or to be regarded as if they induce heritable mutations in the germ cells of humans. Substances known to induce heritable mutations in the germ cells of humans;

- Category 2: substances which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans.