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Additional information

Read-across data of the components of the submission substance or similar substances is available for genetic toxicity:

Gene mutations in bacteria:

Triple Superphosphate:

The test item was assessed for its potential to induce gene mutations in the plate incorporation test according to OECD TG 471 in Salmonella typhimurium strains TA 1535, TA 1537, TA98 and TA100 as well as the Escherichia coli strain WP2 uvrA with and without metabolic activation (Aroclor 1254-induced rat liver S9-mix from male Sprague-Dawley rats) at concentrations of 125, 250, 375, 500, 625, 1250, 2500 and 5000 microg/plate in the initial toxicity-mutation assay and at concentrations of 50, 150, 500, 1250, and 3125 microg/plate for Salmonella and 150, 500, 1250, 3125, and 5000 microg/plate for E. coli in the confirmatory mutagenicity assay. All criteria for a valid study were met as described in the protocol. The results indicate that, under the conditions of this study, the test substance did not cause any positive response in the presence and absence of Aroclor-induced rat liver S9. The test item did not reveal any mutagenic activity. The appropriate reference mutagenes showed distinct positive mutagenic effects.

Calcium sulfate:

Calcium sulfate dihydrate was tested in an OECD 471 study with Salmonella typh. strains TA 98, TA100, TA 1535 and TA 1537 and with the Escherichia coli WP2 uvrA with and without metabolic activation for reverse mutations. No mutagenic activity was observed in tested concentrations up to 3000 µg/plate. The test substance did not induce mutations in bacteria under the conditions of this study.

Humic acid, potassium salt:

The est substance was assayed for the mutagenicity by the Bacterial Reverse Mutation Test according to EU method B.13/14 with Salmonella  typhimurium strains TA 98, TA 100, TA 1535 and TA 1537 and Escherichia coliWP2 uvrA. The test substance was dissolved in water and assayed in doses of 50-5000mg which were applied to plates in amount of 0.1 or 0.2 mL.

Two series of experiments were performed with each strain - without metabolic activation and with a supernatant of rat liver and a mixture of cofactors. No mutagenic effects were observed in any of the tester strains with and without metabolic activation.

Gene mutations in mammalian cells

Complexed Superphosphate-TSP (CSP-TSP):

The analogue substance CSP-TSP (same substance without high content of Ca sulfate) was tested for induction of mutations at the TK (Thymidine Kinase) locus in L5178Y TK+/-mouse lymphoma cells. After a preliminary toxicity test, the aqueous extract of the test item was tested in two independent experiments, with and without a metabolic activation system (S9 mix) in cells cultured for 3 or 24 hours. The aqueous extract was tested because none of the applicable solvents was able to solve the test substance completely. Elemental analysis of the insoluble part of the test substance confirmed that it consisted of inert, poorly soluble inorganic materials (dicalcium phosphate, tricalcium phosphate, silica and traces of metal oxides (ferrous oxides, aluminium oxides).

Experiments without S9 mix: Dose-levels were 312.5, 625, 1250, 2500, 3750 and 5000 µg/mL for the first (3-hour treatment) and second experiment (24‑hour treatment). Precipitation was noted in the culture medium at dose-levels ≥ 625 µg/mL and ≥ 1250 µg/mL at the end of the 24- and 3-hour treatment periods, respectively. Cytotoxicity: Following the 3-hour treatment, a moderate to severe toxicity was induced at dose-levels ≥ 3750 µg/mL, (58 to 98% decrease in Adj. RTG), whilst for the 24-hour treatment, a severe toxicity was induced at dose‑levels ≥ 1250 µg/mL, (81 to 100% decrease in Adj. RTG). Mutagenicity: Following the 3- and 24-hour treatments, no noteworthy increase in the mutation frequency or dose response was noted in comparison to the vehicle control. These results met the criteria for a negative response. Experiments with S9 mix: Dose-levels were 312.5, 625, 1250, 2500, 3750 and 5000 µg/mL for the first and second experiments (3-hour treatment). Precipitation was noted in the culture medium at dose‑levels ≥ 1250 µg/mL. Cytotoxicity: In the first experiment, a severe toxicity was induced at dose-levels ≥ 3750 µg/mL (82-100% decrease in Adj. RTG) whilst in the second experiment,a moderate to severe toxicity was induced at dose-levels ≥ 3750 µg/mL (50 to 100% decrease in Adj. RTG). Mutagenicity: No noteworthy increase in the mutation frequency was observed in comparison to the vehicle control at dose-levels up to 3750 µg/mL in either experiment. Under the experimental conditions of this study, the aqueous extract of the test item did not show any mutagenic activity in the mouse lymphoma assay, in the presence or in the absence of a rat metabolizing system.

Monoammonium phosphate:

The substance was tested in an OECD 476 / EU B.17 study. In the absence of S9-mix, Ammonium dihydrogenorthophosphate did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the duration of treatment time. In the presence of S9-mix, Ammonium dihydrogenorthophosphate did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the concentration of the S9 for metabolic activation. It is concluded that Ammonium dihydrogenorthophosphate is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.

Calcium sulfate:

Calcium sulfate dihydrate was tested for induction of gene mutatations in a study according to OECD Guideline 476 / EU method B.17. The test material did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and is therefore considered to be non mutagenic under the conditions of the test.

Chromosomal abberations/micronuclei in mammalian cells:

Single Superphosphate (SSP):

The potential of the test substance to cause structural chromosome aberrations was tested in cultured peripheral human lymphocytes (with repeat experiment) according to OECD TG 473. The test substance was suspended in DMSO. The metabolic activation system was Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone. Precipitation in the exposure medium was observed at dose levels of 333 µg/ml and above. No toxicity was observed up to and including the highest precipitating tested dose. The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. The test substance did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently repeated experiments. It is concluded that this test is valid and that the test substance does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations and is therefore not clastogenic in human lymphocytes under the experimental conditions described in this report.

Humic acid, potassium salt:

The clastogenicity potential of the test substance was determined in an In Vitro Mammalian Chromosome Aberration Test.acocording to OECD Guideline 473. The test was carried out in human peripheral blood lymphocytes with and without metabolic activation system in two separate assays. Three concentrations - 0.5, 1.0 and 2.5 mg/mL of test substance were used in the main test. A total of 200 well-spread metaphases were examined per concentration on coded slides. Concurrent positive (Cyclophosphamide, Thiotepa) and negative (water) controls were included in each experiment. Under the test conditions the test substance did not induce an increase of structural chromosome aberrations in cultured human peripheral blood lymphocytes.

Calcium sulfate:

Calcium sulfate dihydrate was tested for induction of micronuclei in an in vivo study according to OECD 474 in mice. Male mice received the test substance in doses up to 5000 mg/kg bw in carboxymethyl cellulose in single applications. At least 2000 polychromatic erythrocytes per animal were scored for the incidence of micronuclei. No significant induction of micronuclei was observed in any of the dose groups.

In conclusion, the data described above provide evidence for all components of the submission substance and all relevant endpoints (gene mutation in bacteria, gene mutations in mammalian cells, chromosomal aberrations), that the submission substance is not expected to show genetic toxicity.


Short description of key information:
There is experimental evidence for all components of the submission substance and for all relevant endpoints (gene mutation in bacteria, gene mutations in mammalian cells, chromosomal aberrations), which demonstrates absence of mutagenic effects.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the data described above, the submission substance does not have to be classified for genetic toxicity according to Regulation (EC) No 1272/2008.