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Additional information

In accordance with Article 13(1) of Regulation (EC) No 1907/2006, information on intrinsic properties of substances may be generated by means other than tests, provided that the conditions set out in Annex XI are met. This includes the use of alternative methods, for example, in vitro methods or qualitative or quantitative structure-activity relationship models or from information from structurally related substances (grouping or read-across).

In accordance with Annex XI, Section 1.5, substances whose physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity may be considered as a group, or ‘category’ of substances. Application of the group concept requires that physicochemical properties, human health effects and environmental effects or environmental fate may be predicted from data for reference substance(s) within the group by interpolation to other substances in the group (read-across approach).

The similarities may be based on:

(1) a common functional group;

(2) the common precursors and/or the likelihood of common breakdown products via physical and biological processes, which result in structurally similar chemicals; or

(3) a constant pattern in the changing of the potency of the properties across the category.

There are no or no adequate and reliable studies available in which the potential in vitro cytogenicity and in vitro gene mutation in mammalian cells of Aluminium dihydrogen triphosphate have been investigated. Therefore, in order to fulfil the standard information requirements, assessment of these endpoints/properties is conducted by means of read-across from the following reference substance:

-           Aluminium orthophosphate (EC 232-056-9)

The similarity between the source and target substance is based on:

(1) common functional groups: aluminium orthophosphate and aluminium dihydrogen triphosphate are both inorganic salts of aluminium (Al3+) cations and orthophosphate (PO43-) or dihydrogen triphosphate (H2P3O103-) anions, respectively; the latter anion being composed of orthophosphate moieties.

(2) common precursors and/or the likelihood of common breakdown products via physical and biological processes, which result in structurally similar chemicals: both the source and target substance are ionic compounds resulting from the neutralization reaction of an aluminium base and orthophosphoric acid. Condensation of orthophosphate to diphosphate, triphosphate, etc. is achieved by heat-induced removal of molecular water. The reverse reactions, i.e. chemical or biological hydrolysis of condensated phosphates into orthophosphate and dissolution of the ionic bonds, lead to the common breakdown products aluminium (Al3+) cations and orthophosphate (PO43-) anions.

(3) constant pattern in the changing of the potency of the properties across the category: in general, independently of the cation under consideration, the water solubility of phosphates decreases with increasing degree of phosphate condensation (orthophosphate > diphosphate > triphosphate > polyphosphate).

In accordance with the provisions set out in Annex XI, Section 1.5, the results of the studies used for assessment and read-across are adequate for the purpose of classification and labelling and/or risk assessment; have adequate and reliable coverage of the key parameters addressed in the corresponding test method; cover an exposure duration comparable to or longer than the corresponding test method; and adequate and reliable documentation of the applied method is provided in the technical dossier.

A detailed read-across justification is provided in Section 13 of the technical dossier.

In vitro gene mutation in bacteria

Aluminium dihydrogen triphosphate was tested for induction of gene mutations in bacteria in an Ames Test conducted in accordance with OECD Guideline 471 and under GLP conditions (Enomoto, 2002). Triplicate cultures of Salmonella typhimurium tester strains TA 98, TA 100, TA 1535 and TA 1537 and Escherichia coli tester strain WP2 uvrA pKM 101 were exposed to the test material at up to 5000 µg/plate (with and without metabolic activation) in two independent experiments following the pre-incubation method. Appropriate vehicle (DMSO) and positive controls (with and without metabolic activation) were concurrently tested.

The test material was insoluble in DMSO (and water), and was therefore applied as suspension. No signs of cytotoxicity (growth inhibition) were observed. A precipitate was seen at 1250 µg/plate or more in the absence of S9 mix and at 625 µg/plate or more in the presence of S9 mix. No increase in the number of revertant colonies was noted in any strain relative to the corresponding vehicle control, irrespective of the presence or absence of metabolic activation. Positive control cultures yielded the expected results.

Under the conditions of this study, the test material did not induce reverse gene mutations in Salmonella typhimurium (TA 98, TA 100, TA 1535 and TA 1537) or Escherichia coli (WP2 uvrA pKM101) tester strains, both in the presence and absence of metabolic activation. Therefore, the test material is considered to be not mutagenic in bacteria.

In support of this notion, the available data on Aluminium orthophosphate is taken into account for assessment of in vitro gene mutation in bacteria.

In a GLP-compliant OECD Guideline 471 study, Aluminium orthophosphate (suspended in DMSO) was tested negative for induction of gene mutations in S. typhimurium TA 98, TA 100, TA 1535 and TA 1537 and E. coli WP2 uvr A at up to 5000 µg/plate with and without metabolic activation (Thompson, 2010).

In vitro cytogenicity

Aluminium dihydrogen triphosphate was tested for induction of structural and/or numerical chromosome aberrations in a GLP-compliant study following OECD Guideline 473 (Nakagawa, 2002). Based on the results of a preliminary cytotoxicity study, duplicate cultures of Chinese hamster CHL/IU cells were exposed for 6 h (short-term exposure) to the test material at 6.25, 12.5, 25, 50, 100 µg/mL in the absence, and at 250, 500, 1000, 1500, 2000 µg/mL in the presence of metabolic activation (S9 mix). Concurrent vehicle (DMSO) and positive control (-S9: Mitomycin C; +S9: Benzo(a)pyrene) were included and yielded the expected results.

In the absence of metabolic activation, the test material did not induce any (relevant) increase in the incidence of cells with structural or numerical aberrations up to the highest concentration tested (100 µg/mL). No cytotoxicity, precipitate or pH effects were noted.

In the presence of metabolic activation, the test material did not induce any (relevant) increase in the incidence of cells with structural or numerical aberrations up to 1500 µg/mL. The incidence of cells with structural aberrations was increased to 33 cells/100 observed cells in one of the duplicate cultures at 2000 µg/mL; the other culture was discarded due to high cytotoxicity, leading to less than 50 metaphase cells for evaluation. Cytotoxicity was observed at 1000 µg/mL and above. Precipitate and suspended test material were observed at all test concentrations. At start and end of exposure, a significant and concentration-dependent decrease in pH (down to pH 5.8) was noted at 1500 and 2000 µg/mL.

Based on the increased incidence of cells with structural aberrations at 2000 µg/mL, a confirmatory study was conducted at 1600, 1800 and 2000 µg/mL in the presence of metabolic activation. No increase in the incidence of aberrant cells was observed at 1600 µg/mL, and the incidence of cells with structural aberrations was increased to 4.5 and 11 cells/100 cells at 1800 and 2000 µg/mL, respectively. Cytotoxicity was observed at 1800 µg/mL and above. Precipitate and suspended test material were observed at all test concentrations. At start and end of exposure, a significant and concentration-dependent decrease in pH (down to pH 6.2) was noted at all test concentrations.

According to the study report, the above increase in cells with structural chromosome aberrations were interpreted as positive (clastogenic) effects of the test material, and the long-term (24 h) treatment was therefore not conducted. However, these effects correlated with a dose-dependent and significant increase in cytotoxicity and decrease in pH of the culture medium, and are thus considered to be not biologically relevant.

Taken together, under the conditions of this study, the test material did not induce an increase in the incidence of Chinese hamster CHL/IU cells with structural or numerical chromosome aberrations after short-term (6 h) treatment up to 100 µg/mL in the absence and up to 1800 µg/mL in the presence of metabolic activation. An increased incidence of cells with structural (but not numerical) chromosome aberrations was observed at 2000 µg/mL in the presence of metabolic activation. However, this effect correlated with a dose-dependent and significant increase in cytotoxicity and decrease in pH, and thus considered to be not biologically relevant.

No long-term (24 h) treatment was performed. Therefore, despite the negative results in the short-term treatment studies with and without metabolic activation, the overall results of this study are considered to be inconclusive with regard to the potential clastogenic effects of the test material.

Due to the lack of a fully reliable study, the available data on Aluminium orthophosphate is considered as key information for assessment of in vitro cytogenicity by means of read-across.

A GLP-compliant in vitro chromosome aberration study was conducted with Aluminium orthophosphate in accordance with OECD Guideline 473 (Bowles, 2010). Duplicate cultures of human lymphocytes, treated with the test material (4 h with and with metabolic activation; 24 h without metabolic activation), were evaluated for chromosome aberrations at three dose levels (80, 160 and 320 µg/mL), together with vehicle (MEM culture medium) and positive controls (-S9: Mitomycin C; +S9: Cyclophosphamide).

All vehicle (solvent) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control materials induced statistically significant increases in the frequency of cells with aberrations indicating the sensitivity of the assay and the efficacy of the metabolising system.

The test material was non-toxic and did not induce any statistically significant increases in the frequency of cells with aberrations, in any of the exposure conditions, using a dose range that was limited by precipitate.

Under the conditions of this study, the test material did not induce any statistically significant increase in the frequency of human lymphocytes with chromosome aberrations with and without metabolic activation. Therefore, the test material is considered to be not clastogenic to human lymphocytes in vitro.

In vitro gene mutation in mammalian cells

There are no studies available, in which Aluminium dihydrogen triphosphate has been investigated for induction of gene mutations in mammalian cells. Therefore, the assessment of this endpoint is conducted by means of read-across to the available data on Aluminium orthophosphate.

The potential mutagenicity of Aluminium orthophosphate on thethymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line was assessed in a GLP-compliant study performed according to OECD Guideline 476 (Flanders, 2010). One main experiment was performed. In this main experiment, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test material at six dose levels , in duplicate, together with vehicle (R0 medium) and positive controls. The exposure groups used were as follows: 4-hour exposures both with and without metabolic activation, and 24 hours without metabolic activation. The dose range of test material was selected following the results of a preliminary toxicity test and was 20 to 320 μg/mL for all three of the exposure groups.

With no evidence of any marked toxicity in the preliminary toxicity test, the maximum dose level in the mutagenicity test was limited by the presence of excessive precipitate. A precipitate of the test material was observed at and above 20 μg/mL at the end of the exposure period in all three of the exposure groups in the mutagenicity test. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control materials induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

Under the conditions of this study, the test material did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in any of the three exposure groups. Therefore, the test material is considered to be not mutagenic to L5178Y cells.

In vivo micronucleus study

An in vivo micronucleus test was conducted with Aluminium dihydrogen triphosphate in a study conducted in accordance with OECD Guideline 474 and under GLP conditions (MHLW, 2008). In a preliminary study, groups of Crlj: CD1(ICR) mice (3 per sex, dose level and collection time point) were given the test material per oral gavage at 250, 500, 1000 and 2000 mg/kg bw. Groups of 3 animals per sex and dose level were sacrificed at 24, 48 and 72 h post-administration. At each time point, bone marrow cells were collected from the femurs and analysed for the ratio of immature erythrocytes (polychromatic erythrocytes, PCE) to mature erythrocytes (normochromatic erythrocytes, NCE), and for the occurrence of micronuclei in PCE (MNPCE).

In this preliminary study, no mortality occurred and no signs of systemic toxicity or changes in body weight were observed. There was no decrease in the number of PCE per 200 erythrocytes evaluated at any dose level. Likewise, no increase in the number of MNPCE was observed in any dose group at any collection time point.

Based on these results, in the main study, groups of 6 male mice were given the test material at 500, 1000 and 2000 mg/kg bw per oral gavage. Concurrent vehicle (0.5% w/v caboxymethyl cellulose sodium salt in water) and positive (Mitomycin C, 1 mg/kg bw) controls were included. The positive control substance was applied as intraperitoneal injection. Animals were sacrificed at 24 h post-administration and cell analyses were performed as above.

There were no mortalities, clinical signs or changes in body weight. There was no decrease in the number of PCE per 200 erythrocytes evaluated, and no increase in the frequency of micronucleated immature erythrocytes (MNPCE) was observed at any dose level compared to the vehicle control group.

Positive control animals showed a statistically significant increase in MNPCE compared with the vehicle control.

Under the conditions of this study, the test material did not induce any increase in the frequency of immature erythrocytes with micronuclei in Crlj: CD1(ICR) mice following oral exposure at up to 2000 mg/kg bw. Therefore, the test material is considered to be not clastogenic in vivo.


Justification for selection of genetic toxicity endpoint
No study was selected, since endpoint conclusions are based on the results of three in vitro and one in vivo studies (OECD guideline and GLP), which shall be considered jointly for the purpose of hazard assessment and in accordance with Annex VII-VIII, Section 8.4, of Regulation (EC) no 1907/2006 (REACH).

Short description of key information:
In vitro gene mutation in bacteria: negative with and without metabolic activation (OECD 471, GLP)
In vitro cytogenicity: negative with and without metabolic activation (OECD 473, GLP; read-across from Aluminium orthophosphate EC 232-056-9)
In vitro gene mutation in mammalian cells: negative with and without metabolic activation (OECD 476, GLP; read-across from Aluminium orthophosphate EC 232-056-9)
In vivo micronucleus study: negative in male/female mice up to 2000 mg/kg bw (OECD 474, GLP)

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The available data indicate that the substance does not meet the classification criteria in accordance with Regulation (EC) No 1272/2008 (CLP) and the Globally Harmonized System of Classification and Labelling of Chemicals (GHS).

CLP

Mutagenicity: not classified

GHS

Mutagenicity: not classified