Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

For HEDP sodium salts, information is available from in vitro (two Ames tests, one mouse lymphoma assay, one micronucleus test) and in vivo tests (micronucleus test, dominant-lethal-assay). None of the studies gave any indication of genotoxic potential. A combined chronic toxicity / carcinogenicity study performed with the disodium salt of HEDP gave no rise to concern regarding genotoxic / carcinogenic effects.

Gene mutation (Bacterial reverse mutation assay / Ames test): negative with and without activation in all strains tested (similar to OECD TG 471). Cytogenicity in mammalian cells: not required: in vivo cytogenicity result is available Mutagenicity in mammalian cells: negative with and without activation in L5178Y mouse lymphoma cells (OECD TG 476).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Micronucleus assay in mouse (intraperitoneal administration): negative (similar to OECD TG 474)

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

For the induction of genotoxicity, two principle mechanistic actions can be distinguished: the potential to induce either mainly gene- or chromosome mutations. For the conclusion on the mutagenic potential of a substance, both modes of action have to be investigated with suitable test systems.

 

Concerning the induction of gene mutations, two Ames tests (Klimisch 2) (for HEDP-3Na salt and HEDP-H acid) and one mouse lymphoma study (Klimisch 1) (for HEDP-2Na salt) are available. The Ames tests followed the principles of the OECD guideline 471, but were conducted in the 1970s, where GLP principles have not yet been established. Furthermore, they included neither the Salmonella typhimurium tester strain TA 102 nor an equivalent Escherichia coli strain to evaluate the induction of DNA crosslinking. Therefore, another Ames test according to OECD guideline 471 and GLP principles was conducted with the strain TA 102 to address this specific question. No indication for a genotoxic potential was observed in this test either. The hypothesis that HEDP salts or acid do not induce gene mutations is not only supported by the two already available Ames tests, but also by a fully OECD- and GLP-compliant mouse lymphoma assay conducted in 2010, that gave no indication for mutagenicity.

 

With regard to the induction of chromosome aberrations, information from two in vitro studies (mouse lymphoma assay, Klimisch 1; micronucleus test, Klimisch 1) and two in vivo tests (dominant-lethal-assay, Klimisch 2; micronucleus test, Klimisch 4) are available.

Though the main focus of the mouse lymphoma assay lies on the identification of gene mutations, it is also suited to detect chromosome aberrations. According to the OECD guideline 476, these could be differentiated from the gene mutations by their smaller colony size. As no dose-related, reproducible increase in the mutant frequency occurred at all in the mouse lymphoma test with HEDP-2Na, it can be concluded that the substance did neither induce gene-, nor chromosome mutations under the conditions of the test.

The potential to induce micronuclei was investigated in an in vitro study with human lymphocytes following OECD guideline 487. In two independent experiments, the substance did not induce a relevant increase of micronucleated cells in the absence (exposure period: 4 and 20 h) and presence of S9 mix (exposure period: 4 h). The only observed significant induction of micronuclei occurred at the lowest concentration in experiment I with S9 mix, but the value was within the historical control and thus considered as biologically irrelevant. Neither precipitation nor cytotoxicity were detected up to the highest dose level.

The in vivo micronucleus test included several deviations from current OECD standards (2 instead of 3 doses; highest dose tested lower than MTD; 4 instead of 5 animals per dose; bone marrow samples collected after 6 h, not after 18-24 h). Though the sampling time was shorter than recommended in the guideline, the positive control induced a clear effect.

Another relevant in vivo study for the evaluation of chromosome mutations is the dominant-lethal-assay. The study reviews the chromosomal aberration potential in germ cells of male mice by observation of pre-and post-implantation losses. The formation of chromosomal alterations in germ cells is basically the same as that for somatic cells (e.g., deletions, inversions, translocations). As stated in the OECD Guideline 478, dominant lethals are generally accepted to be the result of chromosomal aberrations (structural and numerical anomalies), although intrauterine deaths may also be caused by gene mutations and reproduction toxic effects. Though the study was conducted prior to the adaption of the respective OECD and GLP guideline, it met the principles laid down in the current OECD guideline 478. However, a concurrent positive control was not included in the assay, but might according to the OECD guideline be omitted if positive control data are available from another dominant-lethal-assay in the same laboratory within the last 12 months. In the dominant-lethal assay, mice were dosed by gavage up to 1000 mg of HEDP-2Na/kg bw/d for 5 days. No statistically significant chromosomal aberrations were observed in any of the treatments. Under the conditions of the dominant-lethal-assay, HEDP-2Na did not induce cytogenic effects.

Taking into consideration the lack of effects seen in the two micronucleus assays and the in vitro mouse lymphoma test, it can be concluded that sodium salts or acid of HEDP are not suspected to induce cytogenetic effects in vitro or in vivo. This is supported by negative results in the carcinogenicity study and the dominant lethal assay,

 

In conclusion, no potential to induce gene or chromosome mutations was observed in any of the in vitro- and in vivo genetic toxicity assays or the carcinogenicity study.

 

Justification for grouping:

See CSR Annex I or IUCLID section 13.



Justification for classification or non-classification

The available information on read across substances indicates that, when tested in vitro, HEDP sodium salt does not cause mutagenicity in mammalian cells; this is supported by evidence for lack of bacterial mutagenicity from disodium HEDP. In addition, no evidence for cytogenicity was shown when disodium HEDP was tested in vitro or in vivo. Therefore it is concluded that classification for mutagenicity is not required for sodium salts of HEDP according to Regulation (EC) No 1272/2008.