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Two ames tests were conducted on nitrilotriacetic acid trisodium salt (NTA Na3) with negative results reported (Zeiger, 1992 & Loprieno, 1985) both in the presence and absence of a metabolic activation system. As a further weight of evidence a bacterial (E.Coli PQ37) SOS Chromotest (no OECD guideline available for this study type) was also conducted with NTA Na3 up to a concentration of 1000 µg/test with negative results. These three studies taken together are considered sufficient to address in vitro gene mutation in bacteria.

Robbiano (1999) reported on an in vitro mammalian micronucleus study in rat and human kidney cells of NTA tested up to a concentration of 5.6 mM. 24 h after application 250 mg/kg folic acid was injected for stimulation of cell proliferation. Test concentrations were selected up to a level where a 30 % reduction in cell viability was observed. NTA induced micronuclei in primary cultures of rat and human kidney cells with approximately two-fold increases observed at the 1.8 mM and three-fold increases observe at the 5.6 mM dose levels. This study was evaluated as Klimisch 3 due to significant methodological dificiencies.

Modesti (1995) reported a positive result on the chromosome aberration effect of NTA Na3 in terms of micronuclei in interphase cells, acentric fragments, chromatin bridges and lagging chromosomes at ana-telophase. NTA treatments of Chinese Hamster (CI-1) cells increased the frequencies of micronuclei after 24 or 48 h exposures. The induction of micronuclei was statistically significant at 2 mM NTA and 3 mM NTA in the 24 h fixation time and at 3 mM NTA in the 48 h fixation time (P < 0.01). However, the study exert significant methodological deficiencies).

Further chromosome aberration studies were reported by Montaldi (1988) and Loveday (1989). In the first of these two studies Montaldi (1988) reported on a chromosome aberration assessment of NTA Na3 across a wide range of experimental conditions. In this study human lymphocyte cultures were exposed to high doses of NTA Na3(10-2M) for long exposure times (up to 5 days) without metabolic activation and examined for chromosome aberrations. NTA Na3 did not significantly increase the frequency of chromosomal aberrations in any of the different experimental conditions adopted. It is worth noting that the highest doses of NTA Na3 tested (10-2 M in experiment A and 7.5× 10-3 in experiment B) were toxic and so at these dose levels no mutagenicity results were obtained.

A further negative mutagenicity result was reported by Loveday (1989). NTA was tested for chromosome aberrations up to its limit of solubility (5 µg/ml) in Chinese Hamster Ovary cells with and without an S9 metabolic activation system. All cellular categories of chromosome aberrations were combined for the statistical analysis of the results, which was based on the percentage of total cells with aberrations. At these dose levels no evidence of increased chromosome aberration was reported above controls and the pH of the medium was reported to have remained constant. Within the same report an assessment of sister chromatid exchanges (SCE’s) was also made. Both low-dose and high-dose positive controls (low and high positive control concentrations were as follows: experiments with S9 used CP at 0.4-0.5 and 2.5 µg/ml; experiments without S9 used MMC at 0.0015-0.002 and 0.010 µg/ml) were included in this SCE assessment protocol. The response with the low-dose positive control had to exceed 20 % over the solvent control for a negative experiment to be acceptable. A clear negative response was reported for NTA with and without the S9 metabolic activation system. There was no evidence of increased levels of SCE’s above controls.

A SCE assay in human peripheral blood lymphocytes with NTA and hamster (CHO) cell lines with NTA Na3 was conducted (Brat 1994). NTA did not increase the SCE frequency in human cells following a 72-h exposure period. NTA Na3 did not increase the frequency of SCE’s in CHO cells following a 27-h exposure period. A metabolic activation system was not included in the experiment al protocol and consequently the study has been rated as Klimisch 3 (significant methodological deficiencies).

It is important to note that the SCE assessments presented in Loveday (1989) and Brat (1994) detail studies conducted with NTA and NTA Na3 respectively. The consistency of the results across these two studies is important in supporting the assumption that both NTA and NTA Na3 demonstrate the same physiological behaviour.

As a consequence of positive in vitro results highlighted in studies reported by Robbiano (1989) and Modesti (1995) further in vivo investigation of these endpoints had to be assessed in accordance with REACH annex IX and X. Engelhardt (2000) reported on an OECD 474 and GLP compliant mouse micronucleus study. 5 males/dose were treated orally per gavage twice within 24 hours with Trilon A92R (92.4 % NTA Na3) at doses of 0, 500, 1000, 2000  mg/kg bw. The two oral administration of the test item did not lead to any increase in the number of polychromatic erythrocytes containing either small or large micronuclei. The test item does not demonstrate any clastogenic potential and there was no evidence of chromosome distribution impairment during mitosis in bone marrow cells in vivo.

In vivo germ cell tests reported by Zordan (1990) indicate that there is evidence that ip dosing of 275 mg/kg has the potential for induction of aneuploidy in mouse spermatocytes. Spermatocytes were sampled 6 h after treatment (3 h after administration of 0.3 ml of 0.5 % colchicine).The incidence of hyperhaploid secondary spermatocytes on a total of 100 haploid/hyperhaploid cells per animal was used as an index of non-disjunctional events in the germ cell line. SCE cell frequency in bone marrow was assessed in 25 well differentiated second generation metaphases per animal. In this study in 3 male test animals 3.3 % hyperhaploid spermatocytes were found against a 0.4 % hyperhaploid negative control level. The reliability and relevance of this test system is questionable. As a consequence of the level of documentation available, this study is reported as Klimisch 3. In addition the relevance of the route of administration is in doubt. Intra-peritoneal dosing is not a relevant route of human exposure. In addition, the gametes of test animals are in physiologically close proximity to the point at which ip dosing occurs. Consequently it is reasonable to suggest that following ip dosing, gamete exposure to the test item departs from a level which is likely to occur in the course of normal physiological exposure scenarios. Consequently this is finding that is highly questionable both from a reliability and relevance to human exposure and standpoint.

Further in vivo assessment of the mutagenicity of NTA Na3 was conducted by BASF (2002). In this non-guideline GLP compliant study the ability of NTANa3 to induce aneuploidy in secondary spermatocytes after oral treatment was assessed. The results of this study show that 100, 300 and 1000 mg/kg bw of NTA Na3 did not significantly increase the frequency of hyperhaploid spermatocytes as compared to vehicle control groups. It should be noted, however, that the frequency of hyperhaploid cells in the positive control group (Diethylstilbestrol 200 mg/kg bw i.p. dose) was not as enhanced as expected. The negative results from this study are in contrast with the results from Zordan (1990) and provide more evidence that the route of exposure and test system may be of limited relevance.

In summary, results derived from this test battery are sufficient to cover the data requirement for in vitro and in vivo mutagenicity under REACH. Multiple genotoxicity studies exist for NTA Na3however the majority of the data is deemed unreliable. A careful assessment of the available information in accordance with the data points detailed under REACH indicate that Nitrilotriacetic acid trisodium salt is considered not mutagenic, a conclusion supported by the draft EU risk assessment report for NTA Na3 (2008).

Short description of key information:

- Two negative Ames tests (Zeiger, 1992; Loprieno, 1985).

- One negative bacterial SOS chromotest (Venier, 1989).

- One negative L5178Y mouse lymphoma cell mutagenisis assay (Myhr, 1988).

- One negative human lymphoma chromosome aberration study (Montaldi, 1988).

- One positive in vitro micronuclei study in primary cultures of rat and human kidney cells (Robbiano, 1999 [CAS 139-13-9 (considered to be equivalent to CAS 5064-31-8 upon solubilisation]).

- One positive in vitro mammalian cell micronucleus test in Cl-1 cells (Modesti, 1995).

- Two negative in vitro sister chromatid exchange studies (Loveday, 1989; Brat, 1994).

- One negative in vitro chromosome aberration result (Loveday, 1989).

- One negative in vivo micronucleus assay (Engelhardt, 2004).

- One negative in vivo chromosome aberration study in spermatocytes (Völkner, 2000).

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Results derived from this test battery are sufficient to cover the data requirement for in vitro and in vivo mutagenicity under REACH. NTA itself does not appear to exert genotoxic effects. In certain target tissues (e.g. kidney) high NTA concentrations may form Zn NTA, for which an aneuploidigenic effect may not be excluded. However, NTA as such is considered non-mutagenic.